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<front>
<journal-meta>
<journal-id journal-id-type="publisher-id">Front. Immunol.</journal-id>
<journal-title>Frontiers in Immunology</journal-title>
<abbrev-journal-title abbrev-type="pubmed">Front. Immunol.</abbrev-journal-title>
<issn pub-type="epub">1664-3224</issn>
<publisher>
<publisher-name>Frontiers Media S.A.</publisher-name>
</publisher>
</journal-meta>
<article-meta>
<article-id pub-id-type="doi">10.3389/fimmu.2024.1302163</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Immunology</subject>
<subj-group>
<subject>Original Research</subject>
</subj-group>
</subj-group>
</article-categories>
<title-group>
<article-title>VapC12 ribonuclease toxin modulates host immune response during <italic>Mycobacterium tuberculosis</italic> infection</article-title>
</title-group>
<contrib-group>
<contrib contrib-type="author">
<name>
<surname>Tyagi</surname>
<given-names>Shaifali</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
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</contrib>
<contrib contrib-type="author">
<name>
<surname>Sadhu</surname>
<given-names>Srikanth</given-names>
</name>
<xref ref-type="aff" rid="aff3">
<sup>3</sup>
</xref>
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<contrib contrib-type="author">
<name>
<surname>Sharma</surname>
<given-names>Taruna</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
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<contrib contrib-type="author">
<name>
<surname>Paul</surname>
<given-names>Abhijit</given-names>
</name>
<xref ref-type="aff" rid="aff4">
<sup>4</sup>
</xref>
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<contrib contrib-type="author">
<name>
<surname>Pandey</surname>
<given-names>Manitosh</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
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<contrib contrib-type="author">
<name>
<surname>Nain</surname>
<given-names>Vaibhav Kumar</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
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<contrib contrib-type="author">
<name>
<surname>Rathore</surname>
<given-names>Deepak Kumar</given-names>
</name>
<xref ref-type="aff" rid="aff3">
<sup>3</sup>
</xref>
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<contrib contrib-type="author">
<name>
<surname>Chatterjee</surname>
<given-names>Samrat</given-names>
</name>
<xref ref-type="aff" rid="aff4">
<sup>4</sup>
</xref>
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<contrib contrib-type="author" corresp="yes">
<name>
<surname>Awasthi</surname>
<given-names>Amit</given-names>
</name>
<xref ref-type="aff" rid="aff3">
<sup>3</sup>
</xref>
<xref ref-type="author-notes" rid="fn001">
<sup>*</sup>
</xref>
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<contrib contrib-type="author" corresp="yes">
<name>
<surname>Pandey</surname>
<given-names>Amit Kumar</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="author-notes" rid="fn001">
<sup>*</sup>
</xref>
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<aff id="aff1">
<sup>1</sup>
<institution>Mycobacterial Pathogenesis Laboratory, Translational Health Science and Technology Institute</institution>, <addr-line>Faridabad, Haryana</addr-line>, <country>India</country>
</aff>
<aff id="aff2">
<sup>2</sup>
<institution>Special Centre for Molecular Medicine, Jawaharlal Nehru University</institution>, <addr-line>New Delhi</addr-line>, <country>India</country>
</aff>
<aff id="aff3">
<sup>3</sup>
<institution>Immunobiology Laboratory, Translational Health Science and Technology Institute</institution>, <addr-line>Faridabad, Haryana</addr-line>, <country>India</country>
</aff>
<aff id="aff4">
<sup>4</sup>
<institution>Complex Analysis Laboratory, Translational Health Science and Technology Institute</institution>, <addr-line>Faridabad, Haryana</addr-line>, <country>India</country>
</aff>
<author-notes>
<fn fn-type="edited-by">
<p>Edited by: Rajiv Kumar, Banaras Hindu University, India</p>
</fn>
<fn fn-type="edited-by">
<p>Reviewed by: Sadiya Parveen, Johns Hopkins University, United States</p>
<p>Mohd Saqib, Albany Medical College, United States</p>
</fn>
<fn fn-type="corresp" id="fn001">
<p>*Correspondence: Amit Awasthi, <email xlink:href="mailto:aawasthi@thsti.res.in">aawasthi@thsti.res.in</email>; Amit Kumar Pandey, <email xlink:href="mailto:amitpandey@thsti.res.in">amitpandey@thsti.res.in</email>
</p>
</fn>
</author-notes>
<pub-date pub-type="epub">
<day>07</day>
<month>03</month>
<year>2024</year>
</pub-date>
<pub-date pub-type="collection">
<year>2024</year>
</pub-date>
<volume>15</volume>
<elocation-id>1302163</elocation-id>
<history>
<date date-type="received">
<day>26</day>
<month>09</month>
<year>2023</year>
</date>
<date date-type="accepted">
<day>01</day>
<month>02</month>
<year>2024</year>
</date>
</history>
<permissions>
<copyright-statement>Copyright &#xa9; 2024 Tyagi, Sadhu, Sharma, Paul, Pandey, Nain, Rathore, Chatterjee, Awasthi and Pandey</copyright-statement>
<copyright-year>2024</copyright-year>
<copyright-holder>Tyagi, Sadhu, Sharma, Paul, Pandey, Nain, Rathore, Chatterjee, Awasthi and Pandey</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/">
<p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</p>
</license>
</permissions>
<abstract>
<p>Mechanistic understanding of antibiotic persistence is a prerequisite in controlling the emergence of MDR cases in Tuberculosis (TB). We have reported that the cholesterol-induced activation of VapC12 ribonuclease is critical for disease persistence in TB. In this study, we observed that relative to the wild type, mice infected with &#x394;<italic>vapC12</italic> induced a pro-inflammatory response, had a higher pathogen load, and responded better to the anti-TB treatment. In a high-dose infection model, all the mice infected with &#x394;<italic>vapC12</italic> succumbed early to the disease. Finally, we reported that the above phenotype of &#x394;<italic>vapC12</italic> was dependent on the presence of the TLR4 receptor. Overall, the data suggests that failure of a timely resolution of the early inflammation by the &#x394;<italic>vapC12</italic> infected mice led to hyperinflammation, altered T-cell response and high bacterial load. In conclusion, our findings suggest the role of the VapC12 toxin in modulating the innate immune response of the host in ways that favor the long-term survival of the pathogen inside the host.</p>
</abstract>
<kwd-group>
<kwd>tuberculosis</kwd>
<kwd>toxin-antitoxin</kwd>
<kwd>virulence</kwd>
<kwd>neutrophils</kwd>
<kwd>persistence</kwd>
<kwd>and inflammation</kwd>
</kwd-group>
<contract-sponsor id="cn001">Translational Health Science and Technology Institute<named-content content-type="fundref-id">10.13039/501100020622</named-content>
</contract-sponsor>
<counts>
<fig-count count="8"/>
<table-count count="0"/>
<equation-count count="0"/>
<ref-count count="72"/>
<page-count count="14"/>
<word-count count="8129"/>
</counts>
<custom-meta-wrap>
<custom-meta>
<meta-name>section-in-acceptance</meta-name>
<meta-value>Microbial Immunology</meta-value>
</custom-meta>
</custom-meta-wrap>
</article-meta>
</front>
<body>
<sec id="s1" sec-type="intro">
<title>Introduction</title>
<p>COVID-19 has augmented the tuberculosis (TB) burden by disrupting the existing healthcare systems resulting in a delay in diagnosis and treatment (<xref ref-type="bibr" rid="B1">1</xref>). In spite of the availability of anti-tubercular therapy, its eradication still remains obscure (<xref ref-type="bibr" rid="B2">2</xref>). In addition, non-compliance to a prolonged treatment regimen fuels the development of multidrug and extensively drug-resistant TB posing blockades to global TB control (<xref ref-type="bibr" rid="B3">3</xref>). The virulence of <italic>Mycobacterium tuberculosis (M. tuberculosis</italic>) is highly transmissible primarily through the aerosolization of the respiratory secretions from the infected individuals. This together with a relatively low virulence has allowed this pathogen to successfully co-evolve with humans for centuries without causing any symptoms or death (<xref ref-type="bibr" rid="B4">4</xref>). We believe that the complex network of host-pathogen interaction, thus developed during evolution, defines TB in terms of its presentation either as an asymptomatic infection or an acute infection presented with clear symptoms. Nevertheless, both the pathogen and the host have gradually evolved mechanisms that would identify and neutralize any mutually directed threats.</p>
<p>Innate immunity is an indispensable arm of the host immune system mediated by phagocytes such as macrophages; bona fide host cells harboring the mycobacteria (<xref ref-type="bibr" rid="B5">5</xref>). The role of neutrophils which are highly motile innate immune cells has been shown in experimental models of TB pathogenesis (<xref ref-type="bibr" rid="B6">6</xref>&#x2013;<xref ref-type="bibr" rid="B9">9</xref>). Following the <italic>M. tuberculosis</italic> infection, these are the primary cells to percolate the lungs and have an extremely intricate role in TB pathology (<xref ref-type="bibr" rid="B10">10</xref>). Neutrophils mediate their phagocytic activity by producing proteases, reactive oxygen and nitrogen intermediates and antimicrobial peptides (<xref ref-type="bibr" rid="B11">11</xref>). These immune cells are the most common cell type present in the sputum, bronchoalveolar fluid and cavities of active TB patients (<xref ref-type="bibr" rid="B10">10</xref>, <xref ref-type="bibr" rid="B12">12</xref>). There is a negative correlation between their abundance in the blood of <italic>M. tuberculosis-</italic>infected patients and disease severity (<xref ref-type="bibr" rid="B13">13</xref>&#x2013;<xref ref-type="bibr" rid="B16">16</xref>). On the contrary, some neutrophil subpopulations are also known to aid in providing a permissive replicating niche and nutrient reservoir for <italic>M. tuberculosis</italic>, indicating their role in promoting mycobacterial persistence (<xref ref-type="bibr" rid="B17">17</xref>, <xref ref-type="bibr" rid="B18">18</xref>). The same has also been reported in <italic>M. tuberculosis</italic> infected rhesus macaques (<xref ref-type="bibr" rid="B19">19</xref>) and zebrafish (<xref ref-type="bibr" rid="B20">20</xref>). Growing evidence suggests the phenotypic and functional diversities of neutrophils which extend well beyond their conventional role as antimicrobial agent. Timely resolution of neutrophils at the site of infection dictates their fate as either protective or dysregulated immune cells and is crucial for the effective restoration of the excessive inflammatory response to homeostasis. The ineffective clearance of neutrophils leads to exacerbation of inflammatory response and perpetuation of tissue damage (<xref ref-type="bibr" rid="B21">21</xref>). Furthermore, the role of mycobacterial genes in shaping an appropriate neutrophil-mediated innate immune response and its timely resolution that affects the downstream adaptive immunity, remains largely unknown.</p>
<p>Previously, using the guinea pig model, we demonstrated that both lungs and spleen isolated from guinea pigs infected with <italic>M. tuberculosis</italic> lacking the VapC12 ribonuclease toxin were presented with higher bacterial burden and increased lung pathology relative to the wild type (WT) (<xref ref-type="bibr" rid="B22">22</xref>). We hypothesize that the VapC12 toxin in <italic>M. tuberculosis</italic> is critical for modulating the host immune response and hence the absence of this toxin in <italic>M. tuberculosis</italic> resulted in a severe form of the disease. To further our understanding of the role of the host immune system in imparting the above phenotype to the <italic>vapC12</italic> deleted strain, in the current study, we performed comprehensive immune-profiling studies in mice infected with WT, &#x394;<italic>vapC12</italic> and the complemented strains to identify host correlates of disease persistence. We found that in comparison to WT, the lungs of mice infected with the mutant strain were presented with higher bacterial load and enhanced pathology. Additionally, these mice showed an excess of macrophages, neutrophils and myeloid-derived suppressor cells (MDSCs) in the lungs with an alleviated T-cell immune response. In the survival study, performed with a high-dose infection model, while all the mice infected with WT strain survived none of the mice from the &#x25b5;<italic>vapC12</italic> group survived 18 weeks post-infection. Additionally, lung transcriptome analysis revealed that the observed neutrophil accumulation was mirrored by increased <italic>S100A8</italic> and <italic>S100A9</italic> mRNA levels in the lungs infected with &#x394;<italic>vapC12</italic> when compared with the WT strain. Interestingly, we found that the enhanced pathogenesis observed in the mice infected with &#x394;<italic>vapC12</italic> was dependent on the presence of TLR-4 receptors. These results suggest that vapC12 ribonuclease mediated regulation of expression of pathogen-associated molecular patterns (PAMPs) in <italic>M. tuberculosis</italic> modulates host immune response which is essential for the long-term survival of the pathogen inside the host. A better understanding of the mechanism of generation and maintenance of antibiotic persistence in the context of host-pathogen interaction will go a long way in designing novel intervention strategies against tuberculosis. Host-directed therapies (HDT) targeting such pathways in combination with the existing anti-TB treatment can increase the efficacy of the current TB regimen.</p>
</sec>
<sec id="s2">
<title>Methods</title>
<sec id="s2_1">
<title>Bacterial strains and culture conditions</title>
<p>All mycobacterial strains were grown and maintained in Middlebrook 7H9 broth and 7H11 agar (Difco Middlebrook 7H9 broth [catalog no. 271310] and 7H11 agar [catalog no. 283810]) and supplemented with 10% OADS (bovine serum albumin, oleic acid, and dextrose); 0.05% Tween was added in 7H9 broth to enhance bacterial growth. Bacterial enumeration by CFU was examined on 7H11 agar plates. The concentrations of antibiotics used were as follows: hygromycin B (50&#x2009;&#xb5;g/mL) and kanamycin (25&#x2009;&#xb5;g/mL).</p>
</sec>
<sec id="s2_2">
<title>Mouse infection study</title>
<p>Animal experiment protocols were reviewed and approved by the animal ethics committee of Translational Health Science and Technology Institute India [(IAEC/THSTI/183), (IAEC/THSTI/167)], Faridabad, India. Animal experiments were performed in accordance with guidelines provided by the Committee for the Control and Supervision of Experiments on Animals (CCSEA). Pathogen-free C57BL/6 and C3H/HeJ mice were obtained from Experimental Animal Facility, THSTI. As described earlier, briefly, prior to infection, mid-log phase cultures (O.D =0.8-1) of H37Rv (WT), &#x394;<italic>vapC12</italic> and &#x394;<italic>vapC12:vapBC12 M. tuberculosis</italic> strains were harvested, washed and single-cell suspensions were prepared (<xref ref-type="bibr" rid="B23">23</xref>). Six to eight-week old mice were infected by aerosol exposure in the Glas-Col airborne infection apparatus with approximately 50-100 CFUs of different strains. Mice from each group (N&#x2009;=&#x2009;5) were sacrificed at different time points as indicated in the experiments (Day 0, 2, 4 and 8 weeks post infection). The bacillary load was determined by removing lungs and spleen of infected mice and the serial dilution of the organ homogenates was plated on 7H11 agar plates supplemented with 10% OADS for bacterial enumeration. The lung sections were treated with 10% formalin and hematoxylin and eosin stained were used for histopathological analysis. A blind assessment strategy was employed for scoring the granulomas. For drug clearance study, after establishing bacterial infection till week 4, mice were treated with 10mg/kg RIF through oral gavaging till week 8. Also, a separate untreated group was kept as a control. Bacillary enumeration was done on week 4 and 8 by aseptically removing organs and homogenizing them. The homogenized organs were then serially diluted and plated on 7H11 agar (containing Polymyxin A, amphotericin B, vancomycin, trimethoprim, carbenicillin and cycloheximide mix).</p>
</sec>
<sec id="s2_3">
<title>Flow cytometry analysis</title>
<p>Mice were sacrificed at 2 and 4 week post-infection, and their lungs, spleen and LN (draining lymph node) were isolated. Single-cell suspension was prepared and stained (1 x 10<sup>6</sup> cells) with fluorescently labelled anti-mouse CD11b (eBioscience Invitrogen, USA), anti-mouse Gr1 (eBioscience Invitrogen, USA), anti-mouse F4/80-FITC (eBioscience Invitrogen, USA), anti-mouse CD68 (eBioscience Invitrogen, USA), anti-mouse CD206 (eBioscience Invitrogen, USA), anti-mouse CD80 (eBioscience Invitrogen, USA), anti-mouse NK1.1 (eBioscience Invitrogen, USA), anti-mouse CD3 (BD Biosciences USA), anti-mouse CD4 (BD Biosciences USA), anti-mouse CD8 (eBioscience Invitrogen, USA) and anti-mouse &#x3b3;&#x3b4;-TCR (eBioscience Invitrogen, USA). Briefly, for surface staining, cells were stained for 20 minutes at room temperature, and were washed twice with PBS. Stained samples were then fixed for 20 minutes using 4% paraformaldehyde. Data was acquired on FACS Canto (BD Biosciences) and analyzed using FlowJo software (Tree Star, Ashland, OR, USA) (<xref ref-type="bibr" rid="B24">24</xref>, <xref ref-type="bibr" rid="B25">25</xref>).</p>
</sec>
<sec id="s2_4">
<title>RNA sequencing</title>
<p>For transcriptome analysis, lung tissues of mice infected with different strains (4-week infected WT, &#x25b5;<italic>vapC12</italic> and &#x394;<italic>vapC12:vapBC12</italic>) were homogenized by bead beating, and total RNA was isolated using RNeasy mini kit according to the manufacturers protocol (Qiagen catalog no. 74104). RNA was eluted in nuclease-free water, subjected to DNA removal using TURBO DNase. NEB Ultra II directional RNA-sequencing Library Prep kit protocol was used to prepare libraries for total RNA-Seq (NEB, catalog no. E7760L). An initial concentration of 500ng of total RNA was taken for the assay. mRNA molecules are captured using magnetic Poly(T) beads (NEB, catalog no. E7490L) following purification, the enriched mRNA was fragmented using divalent cations under elevated temperatures. The cleaved RNA fragments were copied into first-strand cDNA using reverse transcriptase. Second strand cDNA synthesis was performed using DNA Polymerase I and RNase H enzyme. The cDNA fragments were then subjected to a series of enzymatic steps that repair the ends, tails the 3 end with a single &#x2018;A base, followed by ligation of the adapters. The adapter-ligated products were then purified and enriched using the following thermal conditions: initial denaturation 98&#xb0;C for 30sec; 12 cycles of - 98&#xb0;C for 10sec, 65&#xb0;C for 75sec; final extension of 65&#xb0;C for 5mins. PCR products are then purified and checked for fragment size distribution on Fragment Analyzer using HS NGS Fragment Kit (1-6000bp) (Agilent, catalog no. DNF-474-1000). Genes with low expression value (mean FPKM value&lt;0.5) were treated as background (i.e. &#x2018;noise) (<xref ref-type="bibr" rid="B26">26</xref>) and hence excluded from further analysis. After the noise removal, the gene expression values for each experimental condition were merged, and the final data comprised the FPKM value of 13796 genes. To assess the variation in the gene expression data, we performed the principal component analysis (PCA) using the <italic>prcomp</italic> package in R, which applies the singular value decomposition to the data matrix. For the comparison between gene expression profiles from mice infected lungs with WT and &#x25b5;<italic>vapC12</italic> strain of <italic>M. tuberculosis</italic>, genes with a fold change of 2 and false discovery rate (FDR)-adjusted p-value &lt;0.05 were considered differentially expressed. The Benjamin-Hochberg multiple hypothesis testing (<xref ref-type="bibr" rid="B27">27</xref>) was applied to obtain the FDR-adjusted p-value. Subsequently, the analyses were performed on the MATLAB and R software. The <italic>mafdr</italic> function in MATLAB was applied to obtain the FDR-adjusted p-value. PCA plot, volcano plot and dot plot were generated in R using <italic>ggplot2</italic>, <italic>enrichplot</italic> and <italic>gplots</italic> packages. Heatmaps were built using heatmap.2. Pathway enrichment analysis was performed in R using the <italic>clusterProfiler</italic> package.</p>
</sec>
<sec id="s2_5">
<title>Quantitative RT-PCR</title>
<p>Total RNAs from lungs were isolated using RNeasy Micro kit (Qiagen), and the cDNAs were synthesized by reverse transcriptase (RT) using the high capacity cDNA archive random priming kit (Applied Biosystems) according to the manufacturers recommended protocol. Real-time PCR was performed with 2 &#xd7; SYBR mix (TaKaRa) in a QuantStudio-6 Flex Real-Time PCR System (Applied Biosystems) using the default run program. The relative expression of the target genes was calculated using the target threshold cycle value (Ct) and the 2<sup>&#x2212;&#x394;&#x394;</sup>
<italic>
<sup>Ct</sup>
</italic> method with GAPDH as the internal loading control (<xref ref-type="bibr" rid="B28">28</xref>). The primer sequences used for real time PCR are listed in <xref ref-type="supplementary-material" rid="ST1">
<bold>Supplementary Table S1</bold>
</xref>.</p>
</sec>
<sec id="s2_6">
<title>Depletion of neutrophils</title>
<p>Neutrophils were depleted using 200&#x3bc;g anti-mouse Ly6G (Bio X Cell) or isotype IgG (Bio X Cell). Briefly, in acute infections (less than 21 dpi), mice were given 200&#x3bc;g of anti-mouse Ly6G intra- peritoneally every other day between 10 and 20 dpi.</p>
</sec>
<sec id="s2_7">
<title>Statistical analysis</title>
<p>Each experiment was repeated at least three times independently and three biological replicates were used. Data were plotted as mean &#xb1; SEM. Students t-test (Mann-Whitney), two-way ANOVA and one-way ANOVA (Kruskal-Wallis) were performed wherever applicable by using Graph Pad Prism 7.0 software. For the survival curve, Kaplan Meier statistical analysis was performed. P-value &lt; 0.05 was considered to be statistically significant.</p>
</sec>
</sec>
<sec id="s3" sec-type="results">
<title>Results</title>
<sec id="s3_1">
<title>VapC12 ribonuclease toxin modulates growth, virulence and drug tolerance in <italic>M. tuberculosis</italic>
</title>
<p>To replicate our earlier findings of the study performed on guinea pigs (<xref ref-type="bibr" rid="B22">22</xref>), we infected C57BL/6 mice with WT, <italic>vapC12</italic> null mutant and complemented <italic>M. tuberculosis</italic> strains. We found a significantly higher bacterial load in the lungs and spleen of mice infected with <italic>vapC12</italic> null strain as compared to the WT and complemented strains both at 4 and 8 weeks post-infection (<xref ref-type="fig" rid="f1">
<bold>Figures&#xa0;1A, B</bold>
</xref>). In comparison to the WT, gross examination of the lungs infected with the mutant strain revealed higher inflammation and enhanced tissue damage. Histological examination of the lungs infected with the WT strain at 8 weeks post-infection revealed a typical necrotic granuloma in the centre of the lung (40X) with epithelioid cells and lymphocytes in the granuloma (100X) (<xref ref-type="fig" rid="f1">
<bold>Figure&#xa0;1C</bold>
</xref> and <xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure&#xa0;1A</bold>
</xref>). In contrast, at a similar time point post infection, lung tissues of mice infected with the <italic>vapC12</italic> null strain displayed a significantly higher number of granulomas (<xref ref-type="fig" rid="f1">
<bold>Figure&#xa0;1D</bold>
</xref>). Mice infected with complemented strain had a phenotype similar to those infected with WT strain (<xref ref-type="fig" rid="f1">
<bold>Figures&#xa0;1C, D</bold>
</xref>). Previously, using kill-curve experiments, we have demonstrated that the absence of the <italic>vapC12</italic> gene in <italic>M. tuberculosis</italic> reduced the frequency of generation of drug-tolerant population in presence of rifampicin under <italic>in-vitro</italic> growth conditions (<xref ref-type="bibr" rid="B22">22</xref>). To replicate the above findings under disease conditions, we performed a drug clearance study using C57BL/6 mice infection model. We infected a group of mice with either WT and &#x25b5;<italic>vapC12</italic> strain and estimated the rate of pathogen clearance by estimating bacterial load in infected organs at different time points (<xref ref-type="fig" rid="f1">
<bold>Figure&#xa0;1E</bold>
</xref>). As expected, 4 week post infection, the organs infected with &#x25b5;<italic>vapC12</italic> had high bacterial load in comparison with the organs isolated from the WT infected mice (<xref ref-type="fig" rid="f1">
<bold>Figures&#xa0;1F, G</bold>
</xref>). Importantly, upon treatment with RIF and INH, the rate of decline in the bacterial load was higher in mice infected with &#x25b5;<italic>vapC12</italic> strain relative to WT at 8 weeks post infection (<xref ref-type="fig" rid="f1">
<bold>Figures&#xa0;1F, G</bold>
</xref>). To account for the differences in the initial bacterial load (week 0), we had to normalize the data relative to week 0 between H37Rv and <italic>vapC12</italic> mutant strains (<xref ref-type="fig" rid="f1">
<bold>Figures&#xa0;1F, G</bold>
</xref>
<bold>).</bold> Importantly, upon treatment with RIF we observed a ~2 fold and ~7 fold increase in the killing of the pathogen in the lungs and spleen respectively in the mice infected with &#x25b5;<italic>vapC12</italic> as compared to mice infected with the H37Rv (<xref ref-type="fig" rid="f1">
<bold>Figures&#xa0;1F, G</bold>
</xref>) whereas treatment with INH resulted in ~10 fold better killing in the lungs and spleen of the mice infected with &#x25b5;<italic>vapC12</italic> relative of the H37Rv. These results show that the absence of <italic>vapC12</italic> gene in <italic>M. tuberculosis</italic> decreased the frequency of generation of drug-tolerant population potentiating the efficiency of RIF and INH treatment in infected mice (<xref ref-type="fig" rid="f1">
<bold>Figures&#xa0;1F, G</bold>
</xref>). Since <italic>vapC12</italic> deleted strain of <italic>M. tuberculosis</italic> demonstrated enhanced pathogenicity, we measured the estimated time of survival of WT and <italic>&#x25b5;vapC12</italic> infected mice in a high dose infection model (500-1000 bacteria/lung) (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure&#xa0;1B</bold>
</xref>). Interestingly, we found that 16 weeks post-infection, all the mice (n=10) infected with the WT strain survived while none of the mice infected with the &#x25b5;<italic>vapC12</italic> survive (n=10) survived (<xref ref-type="fig" rid="f1">
<bold>Figure 1H</bold>
</xref>). Collectively, these data suggested that absence of <italic>vapC12</italic> imparted hypervirulence phenotype and reduces the frequency of generation of persisters in mice infected with <italic>M. tuberculosis</italic>.</p>
<fig id="f1" position="float">
<label>Figure&#xa0;1</label>
<caption>
<p>
<italic>vapC12</italic> mutant strain of <italic>M. tuberculosis</italic> modulates growth and drug tolerance. <bold>(A, B)</bold> Bacterial load in the <bold>(A)</bold> lungs or <bold>(B)</bold> spleen of mice infected with H37Rv, <italic>&#x394;vapC12</italic>, and <italic>&#x394;vapC12</italic>:<italic>vapBC12</italic> strains of <italic>M. tuberculosis</italic> at designated time points, the lungs were homogenized in 2 ml of saline, and ten-fold serial dilutions of homogenates were plated on 7H11+OADC plates. Each group constituted five mice per time point. Data plotted represent the mean &#xb1; SEM. Significant differences observed between groups are indicated. Data were analyzed using the MannWhitney test with **P &lt; 0.01 and *P &lt;0.05. <bold>(C)</bold> Photomicrographs of H&amp;E-stained (40&#xd7; and 100&#xd7;) and whole section image of lung sections from mice infected with different strains of <italic>M. tuberculosis</italic> at 8 weeks post infection. G=Granuloma, As=Alveolar Space, EC=Epithelioid Cells and L=Lymphocytes <bold>(D)</bold> Granuloma fraction of the lung tissue samples of mice infected with different strains of <italic>M. tuberculosis</italic>, based on the semi-quantitative estimation of the fraction of the lung tissue covered with granuloma. Data were analyzed using the MannWhitney test with *P &lt; 0.05 and **P &lt; 0.01. <bold>(E)</bold> Schematic representation of mice infection experiment. Mice (n&#x2009;=&#x2009;5 per group at each time point) were infected with H37Rv and &#x394;<italic>vapC12</italic>. After the establishment of infection for 4&#x2009;weeks, antibiotics (INH or RIF) were orally administered to the mice for the next 4&#x2009;weeks. A parallel group of mice was left untreated (control). Bacterial loads were enumerated in the lungs <bold>(F)</bold> and spleens <bold>(G)</bold> at different time points post-infection as depicted by scatter plot. Each data point represents the log10 CFU of an infected animal in individual organs, and the error bar depicts the SEM for each group. Bar graph represents the percent killing of the <italic>vapC12</italic> mutant strain with respect to WT at week 8 post-infection in the <bold>(F)</bold> lungs and <bold>(G)</bold> spleen. Data was normalized with the untreated group and plotted with respect to H37Rv (100%) on the y-axis. Data were analyzed using the MannWhitney test with **P &lt; 0.01 and *P &lt;0.05. <bold>(H)</bold> Percent survival plot of mice (n=20) infected with H37Rv and <italic>&#x394;vapC12</italic> strains for 18 weeks post infection. Data were analyzed using the log-rank test with ***P value 0.0005.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fimmu-15-1302163-g001.tif"/>
</fig>
</sec>
<sec id="s3_2">
<title>VapC12 toxin modulates host immune response during <italic>M. tuberculosis</italic> infection</title>
<p>To understand the <italic>vapC12</italic> mediated differences in the host immune response, we performed detailed immune-profiling studies on organs isolated from mice infected with WT, &#x25b5;<italic>vapC12</italic> and complemented <bold>s</bold>trains of <italic>M. tuberculosis</italic> at week 4 post-infection (<xref ref-type="fig" rid="f2">
<bold>Figure&#xa0;2A</bold>
</xref>). We estimated the innate immune cell population; monocytes, macrophages, MDSCs, neutrophils, and Natural Killer-T (NKT) cells in lungs and spleen isolated from infected mice (<xref ref-type="fig" rid="f2">
<bold>Figures&#xa0;2B, C</bold>
</xref> and <xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure&#xa0;2A</bold>
</xref>). A set of uninfected (UI) mice was included as a control group in all the experiments. We found that relative to the WT, both lungs and spleen isolated from mice infected with &#x25b5;<italic>vapC12</italic> had a significantly lower percentage (~2%) of monocytes population (<xref ref-type="fig" rid="f2">
<bold>Figures&#xa0;2B, C</bold>
</xref>). In contrast, relative to the WT, organs isolated from the mice infected with the <italic>vapC12</italic> mutant strain had a significantly higher percentages of macrophage, neutrophil and MDSCs (<xref ref-type="fig" rid="f2">
<bold>Figures&#xa0;2B, C</bold>
</xref>). The restoration of the mutant specific phenotype to the WT levels in the group of mice infected with the complemented strain further confirms the role of VapC12 toxin in modulating the host immunity. We also observed that the MDSCs population in the lungs was significantly elevated by ~3-fold in the mutant strain in comparison to the WT infected group (<xref ref-type="fig" rid="f2">
<bold>Figures&#xa0;2B, C</bold>
</xref>). Our findings corroborated the previous studies as the accumulation of MDSCs parallels disease progression in lung parenchyma and exacerbate disease by suppressing T-cell functions (<xref ref-type="bibr" rid="B29">29</xref>&#x2013;<xref ref-type="bibr" rid="B32">32</xref>). Neutrophil infiltration in the lungs; which is a common feature of disease severity in TB patients (<xref ref-type="bibr" rid="B10">10</xref>) and susceptible mice (<xref ref-type="bibr" rid="B18">18</xref>, <xref ref-type="bibr" rid="B33">33</xref>, <xref ref-type="bibr" rid="B34">34</xref>) was found to be ~3-fold higher in the <italic>vapC12</italic> mutant strain as compared to the WT and complemented strain (<xref ref-type="fig" rid="f2">
<bold>Figures&#xa0;2B, C</bold>
</xref>). Furthermore, the neutrophil population in the spleen isolated from <italic>vapC12</italic> infected mice was also observed to be ~3 fold higher than in the WT (<xref ref-type="fig" rid="f2">
<bold>Figure&#xa0;2C</bold>
</xref>
<bold>).</bold> Surprisingly, we found that the percentage of NKT cells in lungs and spleen isolated from <italic>vapC12</italic> mutant infected mice were 2-fold and 3-fold elevated as compared to the organs isolated from WT infected mice (<xref ref-type="fig" rid="f2">
<bold>Figures&#xa0;2D, E</bold>
</xref>). It has been reported that individuals with pulmonary TB have lower number of NKT cells as compared to the healthy controls, indicating that they may have a protective role in TB infection (<xref ref-type="bibr" rid="B35">35</xref>, <xref ref-type="bibr" rid="B36">36</xref>). Together, these data indicated that <italic>vapC12</italic> mutant induces hyperinflammation by promoting the infiltration of myeloid cells in the lungs.</p>
<fig id="f2" position="float">
<label>Figure&#xa0;2</label>
<caption>
<p>VapC12 toxin modulates host immune response during <italic>M. tuberculosis</italic> infection. <bold>(A)</bold> Schematic representation of the murine infection experiment. Mice (<italic>n</italic>&#x2009;=5 per group at each time point) were infected with H37Rv, <italic>&#x394;vapC12</italic>, and <italic>&#x394;vapC12</italic>:<italic>vapBC12</italic>. Immuno-profiling of lung infiltrated cells and splenocytes harvested at 4 weeks post infection. <bold>(B, C)</bold> Zebra plots and its representative bar graphs indicating the percentage frequency of monocytes (Gr1-CD11b+F4/80-), macrophages (Gr1-CD11b+F4/80+), MDSCs (CD11b+ Gr1+<sup>int</sup>) and neutrophils (CD11b+Gr-1<sup>high</sup>) cells harvested from <bold>(B)</bold> lungs and <bold>(C)</bold> spleen. Data are shown as mean &#xb1; SEM (n=5 animals per group). Data were analyzed using the one-way ANOVA followed by Tukeys multiple comparison test (*P &lt;0.05, **P &lt; 0.01, ***P&lt;0.0005, ****P&lt;0.0001). <bold>(D, E)</bold> Zebra plots and its representative bar graphs showing the percentage frequency of NKT (NK1.1+CD3+) cells harvested from <bold>(D)</bold> lungs and <bold>(E)</bold> spleen. Data are shown as mean &#xb1; SEM (n=5 animals per group). Data were analyzed using the one-way ANOVA followed by Tukeys multiple comparison test (*P &lt;0.05, **P &lt; 0.01, ***P&lt;0.0005, ****P&lt;0.0001).</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fimmu-15-1302163-g002.tif"/>
</fig>
</sec>
<sec id="s3_3">
<title>VapC12 toxin regulates the polarization of macrophages during <italic>M. tuberculosis</italic> infection</title>
<p>The choice of effector mechanism selected by the host to clear infection is regulated by the type of cytokine response dictated by the initial innate immune response. The fate of Th1 and Th2 polarisation, ideal for intracellular and extracellular pathogens respectively, is effectively decided by the initial differentiation of the macrophages to either classical or alternate types. As an effective host immune escape strategy, the pathogen tries to manipulate the macrophage differentiation process in its own favor by subverting the host cell metabolic pathways (<xref ref-type="bibr" rid="B37">37</xref>). To investigate role of the <italic>vapC12</italic> gene in manipulating macrophage differentiation we used antibodies against CD80 and CD206, classical markers for M1 and M2 respectively to quantify the relative M1/M2 population in the lungs of mice infected with different <italic>M. tuberculosis</italic> strains. We found a ~1.5-fold decrease in the frequency of M1 (CD80+) macrophages in lungs isolated from mice infected with <italic>vapC12</italic> mutant as compared to the WT. On the contrary, we found higher frequency of M2 expressing macrophages in the lung tissue infected with &#x25b5;<italic>vapC12</italic> (<xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3A</bold>
</xref> and <xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure&#xa0;3A</bold>
</xref>). Furthermore, the <italic>vapC12</italic> mutant strain demonstrated a ~10% increase in M1 macrophages and a corresponding ~10% decline in M2 macrophages as compared to the WT (<xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3A</bold>
</xref>).The same was further validated by quantifying the transcripts of markers specific to either M1 or M2 macrophages. We estimated the transcripts levels of M1 [CD80 and CD86] and M2 [Arginase1 (Arg1), YM1 and Fizz1) specific markers by isolating RNA from the lungs of WT, <italic>vapC12</italic> mutant infected and uninfected (UI) mice. As expected, we found ~6-fold (Fizz1), ~10-fold (YM1) and ~35-fold (Arg1) increase in the expression of M2 markers in the <italic>vapC12</italic> mutant strain as compared to the WT strain (<xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3B</bold>
</xref>). In concordance with elevated levels of M2 markers, a clear upregulation in the gene expression levels of the Th2 related cytokines; IL 6 (****P&lt;.0.0001) and IL10 (*P&lt;.0.05) was also observed (<xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3C</bold>
</xref>). Surprisingly, the transcription signature associated with M1 and Th1 markers CD80, CD86, IFN-&#x3b3; and TNF-&#x3b1; showed non-significant changes in their gene expression levels and a clear reduction in Th1-induced secretion of IL-2 was noted (<xref ref-type="fig" rid="f3">
<bold>Figures&#xa0;3B</bold>
</xref>, <xref ref-type="fig" rid="f3">
<bold>C</bold>
</xref>). In context to T cell differentiation, we observed that the <italic>vapC12</italic> mutant demonstrated an increased percentage of CD4+ T cells producing IFN-&#x3b3; in both the lungs and spleen when compared to the WT. Conversely, there was a notable reduction in the IL17-producing T-cell population in the spleen of the <italic>vapC12</italic> mutant compared to the WT, with no discernible impact observed in the lung (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure&#xa0;3B</bold>
</xref>
<bold>).</bold> Overall, the data convincingly demonstrates that the ratio of M1/M2 (Th1/Th2) is significantly lower in the mice infected with &#x25b5;<italic>vapC12</italic> as compared to the WT strain. In sum, these data suggest that levels of VapC12 ribonuclease help <italic>M. tuberculosis</italic> control the M1/M2 polarization modulating the Th1/Th2 pathway critical for regulating the disease outcome.</p>
<fig id="f3" position="float">
<label>Figure&#xa0;3</label>
<caption>
<p>VapC12 toxin regulates the polarization of macrophages during <italic>M. tuberculosis</italic> infection. <bold>(A)</bold> Left panel M1: shows the histogram and bar graph of CD80 and right panel M2: shows the histogram and bar graph of CD206 in Gr1-CD11b+ F4/80+ cells by flow cytometry and bar graph represents MFI (Mean fluorescent index) and frequency (%). Data were analyzed using FlowJo software (tree star) and statistical calculation was presented as mean &#xb1; SEM, n = 5. ***P&lt;0.0002, ****P &lt; 0.0001. <bold>(B)</bold> Bar graph for mRNA expression of M1 (CD80 and CD86) and M2 (Arg1, YM1 and Fizz1) macrophage markers in the lung cells, data representative of triplicates *P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001, ****P &lt; 0.0001 <bold>(C)</bold> Bar graph for mRNA expression of TH1 (IFN-&#x3b3;, TNF and IL-2) and TH2 (IL-4, IL-10 and IL-6) in the lung cells, data representative of triplicates *P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001, ****P &lt; 0.0001.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fimmu-15-1302163-g003.tif"/>
</fig>
</sec>
<sec id="s3_4">
<title>
<italic>VapC12</italic> is critical for induction of adequate T cell immune response during <italic>M. tuberculosis</italic> infection</title>
<p>In addition to myeloid cells, CD4<sup>+</sup> and CD8<sup>+</sup> T lymphocytes are the key players in the establishment of protective immunity against TB in mice. The changes in cellular phenotype coincided with the significant decrease in the adaptive immune markers i.e. CD4<sup>+</sup> and CD8<sup>+</sup> T lymphocytes in the organs isolated from the <italic>&#x25b5;vapC12</italic> infected mice relative to the WT. In lungs harvested from mice infected with <italic>vapC12</italic> mutant strain, we observed a significant 3-fold and 2-fold reduction in the percentage of CD4<sup>+</sup> and CD8<sup>+</sup> T-cells respectively, both with p&lt;0.05 as compared to the WT infected mice (<xref ref-type="fig" rid="f4">
<bold>Figure&#xa0;4A</bold>
</xref> and <xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure&#xa0;4A</bold>
</xref>). Surprisingly, while the CD4<sup>+</sup> T-cell profile in the spleen for both groups were identical to the lungs, the CD8<sup>+</sup> T-cell remained unaltered (<xref ref-type="fig" rid="f4">
<bold>Figure&#xa0;4B</bold>
</xref>). As shown in <xref ref-type="fig" rid="f4">
<bold>Figure&#xa0;4C</bold>
</xref>, both CD4<sup>+</sup> and CD8<sup>+</sup> T-cell percentages was suppressed in the lymph nodes of <italic>vapC12</italic> infected mice. Interestingly, in comparison to the WT, the secondary lymphoid organs isolated from the mice infected with <italic>&#x25b5;vapC12</italic> harbored significantly higher percentage of &#x3b3;&#x3b4;-T cell 4 weeks post-infection (<xref ref-type="fig" rid="f4">
<bold>Figures&#xa0;4B, C</bold>
</xref>) with no such differences observed in the lungs (<xref ref-type="fig" rid="f4">
<bold>Figure&#xa0;4A</bold>
</xref>). This data corroborates the previous findings which suggest the role of &#x3b3;&#x3b4;-T cells in TB pathogenesis by inducing inflammation and granuloma formation (<xref ref-type="bibr" rid="B38">38</xref>). Thus, we conclude that the absence of VapC12 toxin in <italic>M. tuberculosis</italic> prevents the induction of adequate T-cell response in the lungs and lymphoid organs (lymph nodes and spleen) of infected mice.</p>
<fig id="f4" position="float">
<label>Figure&#xa0;4</label>
<caption>
<p>
<italic>vapC12</italic> is critical for induction of adequate T cell immune response during <italic>M. tuberculosis</italic> infection. <bold>(A-C)</bold> Zebra plots and its representative bar graphs indicating percentage frequency of CD4, CD8 and &#x3b3;&#x3b4;+T cells in the <bold>(A)</bold> lung <bold>(B)</bold> spleen and <bold>(C)</bold> Lymph nodes. Data were analyzed using FlowJo and shown as mean &#xb1; SEM (n=5 animals per group). Data were analyzed using the one-way Anova test followed by Tukeys multiple comparison test (*P &lt;0.05, **P &lt; 0.01, ***p&lt;0.0005, ****p&lt;0.0001).</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fimmu-15-1302163-g004.tif"/>
</fig>
</sec>
<sec id="s3_5">
<title>VapC12 is essential for the resolution of neutrophil-mediated inflammation in tuberculosis</title>
<p>To further delineate the role of host factors associated with the hypervirulent phenotype of the mutant strain, we analysed the RNA sequencing data of host genes from lung tissues of mice infected with WT, &#x25b5;<italic>vapC12</italic> and complemented strain. The same was subjected to a detailed transcriptome analysis (<xref ref-type="fig" rid="f5">
<bold>Figure&#xa0;5A</bold>
</xref>). Initially, a principal component analysis (PCA) was performed to visualize the variations in the obtained expression profiles. Data displayed in the first two principal components (PCs) suggest that all three gene clusters are well separated from each other (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure&#xa0;5A</bold>
</xref>). To study the VapC12-mediated differential expression of the host genes, we compared the gene expression profiles of lung tissues from mice infected with the &#x25b5;<italic>vapC12</italic> and the WT strains (<xref ref-type="fig" rid="f5">
<bold>Figure&#xa0;5B</bold>
</xref>). In total, 372 differentially expressed genes (DEGs) were obtained, out of which 82 and 290 genes were up- and down-regulated, respectively (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure&#xa0;5B</bold>
</xref> and <xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary File S1</bold>
</xref>). Pathway enrichment analysis of the upregulated genes revealed an enrichment in the processes related to neutrophil functions like neutrophil migration and neutrophil-mediated immunity (<xref ref-type="fig" rid="f5">
<bold>Figure&#xa0;5C</bold>
</xref>). We found that in comparison to the WT, infection with the &#x25b5;<italic>vapC12</italic> strain enhanced the expression of genes that encode proteins implicated in neutrophil-mediated immunity in the host (Cxcl2, Trem1, S100A8/A9 and Ccl3) (<xref ref-type="fig" rid="f5">
<bold>Figure&#xa0;5D</bold>
</xref>). S100A8 and S100A9; cytosolic proteins of neutrophils are important for the recruitment of neutrophils to the granulomas as demonstrated in human and animal models of TB (<xref ref-type="bibr" rid="B39">39</xref>). A ~2.56 fold (log<sub>10</sub>) increase in the transcript levels of genes encoding <italic>S100a8</italic> and <italic>S100a9</italic> by the host infected with the <italic>vapC12</italic> mutant strongly suggests an increase in neutrophil recruitment and accumulation in the lung tissues. Genes belonging to the inflammation-associated markers like matrix metalloproteases (Mmp8 and Mmp9), secreted by neutrophils that are known to be critical for the formation of granuloma and inducing inflammation during the acute stage of infection (<xref ref-type="bibr" rid="B7">7</xref>), were found to be significantly induced in the lung tissues of mice infected with the mutant strain. We also found an increase in the expression of <italic>Cxcr2</italic> (22-fold)<italic>, Cxcl1</italic> (18-fold) and <italic>Cxcl2</italic> (177-fold) genes which suggests enhanced neutrophil recruitment in lungs of mice infected with <italic>vapC12</italic> mutant relative to the WT (<xref ref-type="bibr" rid="B40">40</xref>) (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary File S2</bold>
</xref>). Additionally, IL1a (30-fold) and IL1b (19-fold), the pro-inflammatory cytokine markers associated with TB (<xref ref-type="bibr" rid="B41">41</xref>) were also found to be significantly upregulated in the tissue isolated from mice infected with mutant strain as compared to the WT and complemented strain (<xref ref-type="fig" rid="f5">
<bold>Figure&#xa0;5E</bold>
</xref>). Interestingly, in comparison to the WT, the expression of cystatins such as Cstdc4 [3.2-fold (log<sub>10</sub>)], Stfa2l1 [2.8-fold (log<sub>10</sub>)] and Csta2 [2.26-fold (log<sub>10</sub>)] which are the endogenous inhibitors of the host antimicrobial proteins cathepsins (<xref ref-type="bibr" rid="B42">42</xref>)were also increased in lungs of mutant strain infected animals (<xref ref-type="fig" rid="f5">
<bold>Figure&#xa0;5F</bold>
</xref>). Simultaneously, peptidases which play an important role in pathogen killing by activating the antimicrobial peptides were found to be significantly reduced in the <italic>&#x25b5;vapC12</italic> infected mice lungs (<xref ref-type="fig" rid="f5">
<bold>Figures&#xa0;5G, H</bold>
</xref>), setting off an unrestricted growth of <italic>M. tuberculosis</italic> leading to excessive inflammatory responses. Additionally, the observed reduction in the transcript level of genes associated with adaptive immune response correlates with our earlier findings that the <italic>&#x25b5;vapC12</italic> infected mice fail to induce an effective adaptive immune response (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure&#xa0;5C</bold>
</xref>). Together, these observations suggest that <italic>&#x25b5;vapC12</italic> proliferates and induces a proinflammatory response by modulating the transcript level of proteins that enhances neutrophil recruitment, IL-1 production, endopeptidase inhibitors and by suppressing the antimicrobial proteases and appropriate adaptive immune response inside the host.</p>
<fig id="f5" position="float">
<label>Figure&#xa0;5</label>
<caption>
<p>VapC12 is essential for the resolution of neutrophil-mediated inflammation in tuberculosis. <bold>(A)</bold> Pictorial representation of the process of animal experiment with mice. RNA isolated from lung tissues of mice (n=3) infected with WT, &#x25b5;<italic>vapC12</italic> and complemented strain after 4 weeks of infection. <bold>(B)</bold> The volcano plot showing comparison of gene expression profiles from mice infected with parental (WT) and &#x25b5;<italic>vapC12</italic> strain of <italic>M. tuberculosis</italic>. Differentially expressed genes (DEGs) were selected using the fold change cut-off value of 2 and FDR-adjusted p-value &lt; 0.05. The up- and down-regulated genes are highlighted here in red and blue, respectively. <bold>(C)</bold> Dot plot of the gene ontology (GO) pathway enriched in upregulated genes. The x-axis represents the number of obtained upregulated genes for each pathway. <bold>(D)</bold> Heatmap representing the expression levels of the 21 upregulated genes involved in the neutrophil associated function. Data were Z-score transformed and the name of the genes are displayed on the right. The names of the corresponding infected strains for each sample are mentioned at the bottom of the figure. <bold>(E)</bold> Heatmap representing the expression levels of the 9 upregulated genes involved in the IL-1-mediated signaling. <bold>(F)</bold> Heatmap representing the expression levels of the 10 upregulated genes involved in the peptidase inhibitor activity. <bold>(G)</bold> Dot plot of the gene ontology (GO) pathway enriched in down-regulated genes. The x-axis represents the number of obtained upregulated genes for each pathway. <bold>(H)</bold> Heatmap representing the expression levels of the 15 downregulated genes involved in the peptidase regulator activity.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fimmu-15-1302163-g005.tif"/>
</fig>
</sec>
<sec id="s3_6">
<title>Depletion of neutrophils failed to restore the <italic>vapC12</italic> mutant phenotype</title>
<p>As neutrophils are an abundant component of the innate immune system, their highly inflammatory actions have potentially severe consequences for host tissues (<xref ref-type="bibr" rid="B43">43</xref>). When the regulatory mechanisms responsible for clearing apoptotic neutrophils are compromised or if the underlying infection cannot be resolved, the actions of neutrophils may turn detrimental to the host (<xref ref-type="bibr" rid="B44">44</xref>, <xref ref-type="bibr" rid="B45">45</xref>). As previously reported, neutrophils depletion can be achieved by the administration of 1A8 antibody (<xref ref-type="bibr" rid="B45">45</xref>). To confirm the role of neutrophils in &#x25b5;<italic>vapC12</italic> induced hypervirulent phenotype we infected groups of C57BL/6 mice, with either 1A8 antibody or their isotype control (<xref ref-type="fig" rid="f6">
<bold>Figure&#xa0;6A</bold>
</xref>
<bold>).</bold> Lungs treated with 1A8 antibody in <italic>vapC12</italic> mutant resulted in significant decrease in the neutrophils population relative to the isotype group (<xref ref-type="fig" rid="f6">
<bold>Figure&#xa0;6B</bold>
</xref>). Additionally, 1A8 antibody treatment resulted in a significant decrease in the monocytes (~4%) and macrophages population (~5%) in the lungs (<xref ref-type="fig" rid="f6">
<bold>Figure&#xa0;6B</bold>
</xref>). Treatment with 1A8 antibody also resulted in a significant reduction (5%) in the population of CD4<sup>+</sup> and CD8<sup>+</sup> T cells in the lungs and spleen harvested from antibody-treated <italic>vapC12</italic> mutant mice as compared to WT (<xref ref-type="fig" rid="f6">
<bold>Figure&#xa0;6C</bold>
</xref>). Surprisingly, we did not find any difference in the overall lung and spleen bacillary burden in mice infected with <italic>vapC12</italic> mutant strain that were either treated with 1A8 antibody or the isotype control (<xref ref-type="fig" rid="f6">
<bold>Figure&#xa0;6D</bold>
</xref>). These data collectively suggest that the &#x25b5;<italic>vapC12</italic> induced hypervirulence phenotype is independent of the increase in the infiltration of neutrophil observed in the lungs of &#x25b5;<italic>vapC12</italic> infected mice.</p>
<fig id="f6" position="float">
<label>Figure&#xa0;6</label>
<caption>
<p>Neutrophil depletion failed to restore the <italic>vapC12</italic> mutant phenotype to the wild type levels. <bold>(A)</bold> Schematic representation of antibody depletion experiment in mice. <italic>&#x394;vapC12</italic> infected mice (n=5) were either treated with neutrophil depleting Anti-Ly6G or IgG control antibody every alternative day from day 10 to day 21 post-infection. Mice were sacrificed on day 21 for immunophenotyping &amp; bacterial enumeration. <bold>(B)</bold> Zebra plots and its representative bar graph shows the percentage frequency of monocytes (Gr1-CD11b+F4/80-), macrophages (Gr1-CD11b+F4/80+),neutrophils (CD11b+Gr-1<sup>high</sup>) cells in the lungs. Data are shown as mean &#xb1; SEM (n=5 animals per group). Data were analyzed using the one-way ANOVA followed by Tukeys multiple comparison test (*P &lt;0.05, **P &lt; 0.01, ***P&lt;0.0005, ****P&lt;0.0001). <bold>(C)</bold> Zebra plots and its representative bar graphs indicating percentage frequency of CD4<sup>+</sup> and CD8<sup>+</sup> T cells in the lung and spleen. Data were analyzed using FlowJo and shown as mean &#xb1; SEM (n=5 animals per group). Data were analyzed using the one-way ANOVA test followed by Tukeys multiple comparison test (*P &lt;0.05, **P &lt; 0.01, ***p&lt;0.0005, ****p&lt;0.0001). <bold>(D)</bold> Scatter plot showing bacterial load in the lungs and spleen of mice infected with <italic>&#x394;vapC12</italic> at designated time points, the lungs were homogenized in 2 ml of saline, and ten-fold serial dilutions of homogenates were plated on 7H11+OADC plates. Each group constituted five mice per time point. Data plotted represent the mean &#xb1; SEM. Significant differences observed between groups are indicated. Data were analyzed using the MannWhitney test with **P &lt; 0.01 and *P &lt;0.05.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fimmu-15-1302163-g006.tif"/>
</fig>
</sec>
<sec id="s3_7">
<title>
<italic>VapC12</italic> deletion induced hypervirulence in <italic>M. tuberculosis</italic> is dependent on TLR4</title>
<p>Neutrophil associated proteins, S100A8/A9 function as DAMPs (danger-associated molecular pattern) and can act as a ligand of TLR4 thus activating the innate immune responses (<xref ref-type="bibr" rid="B46">46</xref>). Since RNA-sequencing data showed that both S100A8/A9 neutrophil-associated markers were elevated in the lungs of v<italic>apC12</italic> mutant, we hypothesized that TLR4 signaling could play an important role in &#x25b5;<italic>vapC12</italic> mediated hypervirulent phenotype. To study the role of TLR4 signaling, we infected C3H/HeJ mice (carrying mutated TLR4) with both the WT and &#x25b5;<italic>vapC12</italic> strains (<xref ref-type="fig" rid="f7">
<bold>Figure&#xa0;7A</bold>
</xref>). In parallel, we included a C57BL/6 mice group infected with WT and <italic>vapC12</italic> mutant as a hypervirulent phenotype control. As expected, relative to the WT, we observed an increase in the bacterial load in the organs isolated from C57BL/6 mice infected with the &#x25b5;<italic>vapC12</italic> strain (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure&#xa0;6</bold>
</xref>). Surprisingly, the above phenotype observed in the C57BL/6 mice was completely reversed in C3H/HeJ mice (<xref ref-type="fig" rid="f7">
<bold>Figures&#xa0;7B, C</bold>
</xref>
<bold>).</bold> In comparison to the C57/BL6, C3H/HeJ mice infected with &#x25b5;<italic>vapC12</italic> were presented with a ~10-fold &amp; ~40-fold reduction in bacterial burden in the lungs &amp; spleen respectively (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure&#xa0;6A, B</bold>
</xref>
<bold>).</bold> Interestingly, the reduction in the bacterial burden observed in C3H/HeJ mice coincided with a significant reduction in the population of neutrophils in these organs (p &lt; 0.05). Additionally, we also observed that the percentage of MDSCs and macrophage population from the lungs of &#x25b5;<italic>vapC12</italic> infected TLR4 defective mice were restored to the WT levels (<xref ref-type="fig" rid="f7">
<bold>Figure&#xa0;7D</bold>
</xref>). Surprisingly, in comparison to the lungs, restoration of the adaptive immune response in &#x25b5;<italic>vapC12</italic> infected TLR4 defective mice were specifically observed in the spleen (<xref ref-type="fig" rid="f7">
<bold>Figure&#xa0;7E</bold>
</xref>). These findings were in line with our bacterial enumeration data suggesting that TLR4 signaling pathway could play an important role in &#x25b5;<italic>vapC12</italic> mediated hypervirulent phenotype observed in the mice.</p>
<fig id="f7" position="float">
<label>Figure&#xa0;7</label>
<caption>
<p>
<italic>vapC12</italic> deletion induced hypervirulence in <italic>M. tuberculosis</italic> is dependent on TLR4. <bold>(A)</bold> Schematic representation of the animal experiment. C3H/HeJ mice were aerosol infected with H37Rv and <italic>&#x394;vapC12</italic>. Mice were sacrificed on indicated time point for bacterial enumeration and immunophenotyping. <bold>(B, C)</bold> Scatter plot showing bacterial load in the <bold>(B)</bold> lungs and <bold>(C)</bold> spleen of mice infected with H37Rv and <italic>&#x394;vapC12</italic> at indicated time points, the lungs were homogenized in 2 ml of saline, and ten-fold serial dilutions of homogenates were plated on 7H11+OADC plates. Each group constituted five mice per time point. Data plotted represent the mean &#xb1; SEM. Significant differences observed between groups are indicated. Data were analyzed using the MannWhitney test with **P &lt; 0.01 and *P &lt;0.05. <bold>(D)</bold> Zebra plot and its representative bar graphs showing the percentage frequency of monocytes (Gr1-CD11b+F4/80-), macrophages (Gr1-CD11b+F4/80+), MDSCs (CD11b+ Gr1+<sup>int</sup>) and neutrophils (CD11b+Gr-1<sup>high</sup>) cells in the lung. Data are shown as mean &#xb1; SEM (n=5 animals per group). Data were analyzed using the one-way ANOVA followed by Tukeys multiple comparison test (*P &lt;0.05, **P &lt; 0.01, ***P&lt;0.0005, ****P&lt;0.0001). <bold>(E)</bold> Zebra plots and its representative bar graphs indicating the percentage frequency of CD4<sup>+</sup> and CD8<sup>+</sup> T cells in the lung and spleen. Data were analyzed using FlowJo and shown as mean &#xb1; SEM (n=5 animals per group). Data were analyzed using the one-way ANOVA test followed by Tukeys multiple comparison test (*P &lt;0.05, **P &lt; 0.01, ***p&lt;0.0005, ****p&lt;0.0001).</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fimmu-15-1302163-g007.tif"/>
</fig>
</sec>
</sec>
<sec id="s4" sec-type="discussion">
<title>Discussion</title>
<p>Earlier we reported that the vapC12 ribonuclease toxin targets proT tRNA and modulates the levels of various proline-rich PE-PGRS proteins (<xref ref-type="bibr" rid="B22">22</xref>). In the same study, we further demonstrated that organs isolated from guinea pigs infected with <italic>M. tuberculosis</italic> strain lacking <italic>vapC12</italic> gene had higher bacterial load and were found to be more pathogenic measured in terms of the degree of inflammation and pathological lesions as compared to the WT strain (<xref ref-type="bibr" rid="B22">22</xref>). The current study was undertaken to further our understanding of the hypothesis that <italic>vapC12</italic> mediated differential expression of antigenic proteins modulates host immune response. Due to easy availability of good genetic and immunological toolbox, we in the current study have decided to use mice as a surrogate host. Similar to the phenotype observed in guinea we found that the <italic>vapC12</italic> deficient strain of <italic>M. tuberculosis</italic> demonstrated a hypervirulent phenotype marked by an increase in bacterial burden and enhanced pathological lesions. Subsequently we have also demonstrated that &#x25b5;<italic>vapC12</italic> promoted the influx of pro-inflammatory myeloid cells at the site of infection resulting in robust transcription of S100A8/A9 proteins providing enhanced growth benefit to the mutant strain as compared to the WT. By inhibiting the S100A8/9-TLR4 axis we were able to mitigate the bacterial growth in <italic>vapC12</italic> mutant by reducing the detrimental inflammatory immune response.</p>
<p>The killing of pathogens by macrophages is known to be the primary defence mechanism against intracellular pathogens. Like T-lymphocytes, macrophages also differentiate into M1 and M2 phenotypes depending on their microenvironment and amino acid metabolic shift (<xref ref-type="bibr" rid="B37">37</xref>). Arginine catabolism via iNOS leads to the production of L-citrulline and nitric oxide (NO), playing a predominant role in providing resistance against parasitic and bacterial infections. On the contrary, use of arginine as a substrate by Arg1 results in the generation of L-ornithine and urea and is known to promote disease activity (<xref ref-type="bibr" rid="B37">37</xref>). During <italic>M. tuberculosis</italic> infection, Arg1 stimulation <italic>in-vitro</italic> in macrophages through a TLR-dependent and STAT6-independent pathway leads to the inhibition of NO production. Conversely, lack of Arg1 was connected to elevated NO expression and improved control of mycobacterial infection (<xref ref-type="bibr" rid="B47">47</xref>). Arginine metabolism has been linked to infection as high arginase activity has been reported in lesions of leishmaniasis patients and human TB granulomas (<xref ref-type="bibr" rid="B48">48</xref>&#x2013;<xref ref-type="bibr" rid="B50">50</xref>). Most of the available piece of evidence suggests that pathogens responsible for acute as well as chronic infections manipulate STAT6-PPAR&#x3b3;/&#x3b4; pathways to avoid M1 polarization (<xref ref-type="bibr" rid="B51">51</xref>&#x2013;<xref ref-type="bibr" rid="B55">55</xref>). While the WT <italic>M. tuberculosis</italic> induced polarization of macrophage towards M1, a skewed polarization towards M2 macrophages observed in mice infected with <italic>vapC12</italic> deficient <italic>M. tuberculosis</italic> strain suggests that a <italic>vapC12</italic>-dependent protein expression actively influences the macrophage polarization in the host. As a consequence, there is an altered T-cell response activated in terms of Th1/Th2 by the host. This implies that <italic>M. tuberculosis</italic> actively modulates host immunity by differentially expressing its proteins and the resulting spatiotemporal expression of <italic>M. tuberculosis</italic> antigens critically regulate the host immune pathways which ultimately affects the disease progression and outcome. Contrary to the WT, failure of &#x25b5;<italic>vapC12</italic> to prevent the accumulation of excessive neutrophils at the site of infection results in enhanced inflammation, M2 polarization and a Th2 type response which together creates an environment conducive for the growth of <italic>M. tuberculosis</italic> inside the host. In agreement with our observations, several reports have established that excessive neutrophil infiltration leads to tissue damage and exacerbation of inflammation which is detrimental to the host (<xref ref-type="bibr" rid="B56">56</xref>&#x2013;<xref ref-type="bibr" rid="B59">59</xref>). Additionally, upregulation of the IL-1 cytokine signaling genes suggest increased levels of proinflammatory IL-1&#x3b1; and IL-1&#x3b2; cytokines in the lungs infected with <italic>vapC12</italic> deficient <italic>M. tuberculosis</italic> strain. The dysregulation of the levels of IL-1 and IL-1 receptor antagonists cannot be ruled out.</p>
<p>Cathepsins carry out efficient innate and adaptive antibacterial functions, either directly by taking part in the death of invasive bacteria or by processing and presenting the antigen of infected bacteria to lymphocytes, which escalates the removal of the pathogen and triggers the development of long-lasting immunity (<xref ref-type="bibr" rid="B60">60</xref>). However, bacteria have the ability to manipulate cathepsin expression and proteolytic activity in order to benefit their own intracellular survival in macrophages and escape the host-mediated immune surveillance (<xref ref-type="bibr" rid="B60">60</xref>). <italic>M. tuberculosis</italic> avoids contact with cathepsins by modulating phagosome maturation, impairing the delivery of lysosomes to phagosomes in NF-&#x3ba;B dependent manner and by escaping from the phagosomes to the cytosol, thus facilitating its replication and survival (<xref ref-type="bibr" rid="B61">61</xref>&#x2013;<xref ref-type="bibr" rid="B63">63</xref>). Cystatins, which are the inhibitors of cathepsins; the proteolytic enzymes involved in the control of <italic>M. tuberculosis</italic> infection were increased at the transcript level in the lungs of &#x25b5;<italic>vapC12</italic> infected mice (<xref ref-type="bibr" rid="B42">42</xref>). In support of increased peptidase inhibitor activity, we also observed suppressed levels of peptidases in lung homogenates of &#x25b5;<italic>vapC12</italic> infected mice. Our findings are also in concordance with previous reports that cathepsins play a central role in the immune system and hence its necessary to regulate its activity to maintain homeostasis within cells or tissues (<xref ref-type="bibr" rid="B64">64</xref>). In recent years, MDSCs which are a heterogeneous population of immunocytes from myeloid origin have also been found to be a safe harbor for <italic>M. tuberculosis</italic> proliferation (<xref ref-type="bibr" rid="B65">65</xref>, <xref ref-type="bibr" rid="B66">66</xref>). As reported in recent studies, we have identified that the increase in MDSCs population mirrored alleviating protective T-cell response in the spleen of mice infected with the <italic>vapC12</italic> mutant as compared to the WT strain infected mice (<xref ref-type="bibr" rid="B30">30</xref>, <xref ref-type="bibr" rid="B67">67</xref>, <xref ref-type="bibr" rid="B68">68</xref>). These results implicate that the influx of neutrophils along with MDSCs, IL-1 cytokines and endopeptidase inhibitors might be responsible for increased bacillary load and lung pathology in the mice infected with &#x25b5;<italic>vapC12</italic> mutant.</p>
<p>S100A8/A9 proteins, members of the S100 family promote the recruitment of myeloid cells, specifically neutrophils, dendritic cells and monocytes to the lungs through the induction of inflammatory cytokines (<xref ref-type="bibr" rid="B39">39</xref>). A recent study has mechanistically demonstrated that the expression of these calprotectin is needed for the upregulation of CD11b integrin on neutrophils resulting in their accumulation during chronic TB infection (<xref ref-type="bibr" rid="B69">69</xref>). Indeed, our study revealed that S100A8/A9 levels corroborated accumulation of myeloid-derived inflammatory cells in the lungs of mice infected with <italic>vapC12</italic> strain during TB disease progression. The RNA-sequencing data also showed increased expression of the marker gene Cxcr2 suggesting increased recruitment of myeloid cells at week 4 post-infection in the <italic>vapC12</italic> mutant infected mice. These findings are consistent with the literature available on the function of S100A8/A9 in host defence mechanisms against bacterial and viral infections (<xref ref-type="bibr" rid="B39">39</xref>, <xref ref-type="bibr" rid="B70">70</xref>).</p>
<p>S100A8/A9 plays a decisive role in innate immunity by binding to receptors such as TLR4 (<xref ref-type="bibr" rid="B46">46</xref>) and receptor for advanced glycation end products (RAGE), which activates multiple inflammatory pathways mediated by these pattern-recognition receptors (PRRs) (<xref ref-type="bibr" rid="B46">46</xref>, <xref ref-type="bibr" rid="B71">71</xref>, <xref ref-type="bibr" rid="B72">72</xref>). Finally, we also investigated the role of TLR4 in inducing S100A8/A9 mediated hyper inflammation in <italic>vapC12</italic> mutant. By blocking the TLR4 signaling (C3He/HeJ mice) the bacillary burden in the lungs of <italic>vapC12</italic> mice became comparable to that of the WT strain. Based on these findings, we believe that the absence of TLR4 in C3He/HeJ prevented the induction of PAMP-mediated inflammatory response. This resulted in the restoration of the growth of <italic>vapC12</italic> mutant to that of the WT levels. The exact mechanism of S100A8/A9 overexpression and TLR4 signal activation induced in <italic>vapC12</italic> mutant requires further investigation. Characterizing the differentially expressed secretory proteins of <italic>vapC12</italic> mutant would further give us a better insight into mechanistically understanding the downstream processes.</p>
<p>In conclusion, we have studied how the absence of the ribonuclease toxin in <italic>M. tuberculosis</italic> greatly impacted the ability of the pathogen to dampen the early innate immune response. The uninterrupted migration of granulocytes and continuous expression of proinflammatory cytokines resulted in enhanced pathology. The resulting M2 mediated Th-2 response, generated in the mice infected with &#x25b5;<italic>vapC12</italic> favored the replication and growth of <italic>M. tuberculosi</italic>s. Overall our data suggest that VapC12 ribonuclease toxin-mediated differential expression of <italic>M. tuberculosis</italic> proteins help in the timely resolution of TLR4-mediated inflammation which is critical for disease persistence in TB (<xref ref-type="fig" rid="f8">
<bold>Figure&#xa0;8</bold>
</xref>).</p>
<fig id="f8" position="float">
<label>Figure&#xa0;8</label>
<caption>
<p>Model depicting the VapC12 toxin-mediated host immunomodulation during <italic>M. tuberculosis</italic> infection. Our findings demonstrate that the absence of ribonuclease toxin VapC12 favors unrestricted bacterial growth and exacerbated pathology. Additionally, <italic>vapC12</italic> mutant hinders the ability of <italic>M. tuberculosis</italic> to control the initial influx of myeloid cells to the site of infection leading to continuous inflammation and blunted T-cell response. Further, myeloid cells mediated hyper-inflammation resulted in over activation of S100A8/A9-TLR4 signaling. Finally, <italic>vapC12</italic> mediated expression of <italic>M. tuberculosis</italic> proteins is critical for disease persistence in tuberculosis. Illustrated using Biorender (<uri xlink:href="https://www.biorender.com">www.biorender.com</uri>).</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fimmu-15-1302163-g008.tif"/>
</fig>
</sec>
<sec id="s5" sec-type="data-availability">
<title>Data availability statement</title>
<p>The datasets presented in this study can be found in online repository - India Nucleotide Data Archive (INDA) maintained at Indian Biological Data Centre (IBDC), India. The accession number(s) can be found below: <uri xlink:href="https://inda.rcb.ac.in/viaaccessionIDPRJEB63988">https://inda.rcb.ac.in/viaaccessionIDPRJEB63988</uri>.</p>
</sec>
<sec id="s6" sec-type="ethics-statement">
<title>Ethics statement</title>
<p>The animal study was approved by Institutional Animal Ethics Committee (IAEC), THSTI. The study was conducted in accordance with the local legislation and institutional requirements.</p>
</sec>
<sec id="s7" sec-type="author-contributions">
<title>Author contributions</title>
<p>ST: Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Writing &#x2013; original draft, Writing &#x2013; review &amp; editing. SS: Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Writing &#x2013; review &amp; editing. TS: Data curation, Investigation, Methodology, Writing &#x2013; review &amp; editing. AP: Formal analysis, Methodology, Software, Writing &#x2013; review &amp; editing. MP: Investigation, Methodology, Writing &#x2013; review &amp; editing. VN: Investigation, Writing &#x2013; review &amp; editing. DR: Methodology, Software, Writing &#x2013; review &amp; editing. SC: Methodology, Software, Writing &#x2013; review &amp; editing. AA: Project administration, Resources, Writing &#x2013; review &amp; editing. AKP: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing &#x2013; original draft, Writing &#x2013; review &amp; editing.</p>
</sec>
</body>
<back>
<sec id="s8" sec-type="funding-information">
<title>Funding</title>
<p>The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This study was supported by the India-Singapore grant by the Department of Science and Technology (DST), India, to AKP. (INT/Sin/P-08/2015). DST-SERB grant (CRG/2019/004250) from the Department of Science and Technology, Government of India, and intramural funding by THSTI to AKP are acknowledged. ST was supported by fellowship from UGC [19/06/2016(i)EU-V].</p>
</sec>
<ack>
<title>Acknowledgments</title>
<p>We are thankful to EAF and IDRF at THSTI for their support. We thank ILBS, New Delhi and histopathology facility at THSTI for histopathological analysis and Surjeet Yadav for lab maintenance. THSTI is acknowledged for providing all support for equipment and other infrastructure.</p>
</ack>
<sec id="s9" sec-type="COI-statement">
<title>Conflict of interest</title>
<p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>
<p>The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.</p>
</sec>
<sec id="s10" sec-type="disclaimer">
<title>Publisher&#x2019;s note</title>
<p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p>
</sec>
<sec id="s11" sec-type="supplementary-material">
<title>Supplementary material</title>
<p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type="uri" xlink:href="https://www.frontiersin.org/articles/10.3389/fimmu.2024.1302163/full#supplementary-material">https://www.frontiersin.org/articles/10.3389/fimmu.2024.1302163/full#supplementary-material</ext-link>
</p>
<supplementary-material xlink:href="Table_1.xlsx" id="ST1" mimetype="application/vnd.openxmlformats-officedocument.spreadsheetml.sheet"/>
<supplementary-material xlink:href="Table_2.xlsx" id="ST2" mimetype="application/vnd.openxmlformats-officedocument.spreadsheetml.sheet"/>
<supplementary-material xlink:href="Presentation_1.pdf" id="SM1" mimetype="application/pdf"/>
</sec>
<ref-list>
<title>References</title>
<ref id="B1">
<label>1</label>
<citation citation-type="book">
<person-group person-group-type="author">
<collab>WHO</collab>
</person-group>. <source>Global TB report</source>. (<year>2021</year>). <publisher-loc>Geneva</publisher-loc>: <publisher-name>World Health Organization</publisher-name>.</citation>
</ref>
<ref id="B2">
<label>2</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Adams</surname> <given-names>KN</given-names>
</name>
<name>
<surname>Takaki</surname> <given-names>K</given-names>
</name>
<name>
<surname>Connolly</surname> <given-names>LE</given-names>
</name>
<name>
<surname>Wiedenhoft</surname> <given-names>H</given-names>
</name>
<name>
<surname>Winglee</surname> <given-names>K</given-names>
</name>
<name>
<surname>Humbert</surname> <given-names>O</given-names>
</name>
<etal/>
</person-group>. <article-title>Drug tolerance in replicating mycobacteria mediated by a macrophage-induced efflux mechanism</article-title>. <source>Cell</source>. (<year>2011</year>) <volume>145</volume>:<fpage>39</fpage>&#x2013;<lpage>53</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1016/j.cell.2011.02.022</pub-id>.</citation>
</ref>
<ref id="B3">
<label>3</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Dartois</surname> <given-names>VA</given-names>
</name>
<name>
<surname>Rubin</surname> <given-names>EJ</given-names>
</name>
</person-group>. <article-title>Anti-tuberculosis treatment strategies and drug development: challenges and priorities</article-title>. <source>Nat Rev Microbiol</source>. (<year>2022</year>) <volume>20</volume>:<fpage>685</fpage>&#x2013;<lpage>701</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1038/s41579-022-00731-y</pub-id>.</citation>
</ref>
<ref id="B4">
<label>4</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Ehrt</surname> <given-names>S</given-names>
</name>
<name>
<surname>Schnappinger</surname> <given-names>D</given-names>
</name>
<name>
<surname>Rhee</surname> <given-names>KY</given-names>
</name>
</person-group>. <article-title>Metabolic principles of persistence and pathogenicity in Mycobacterium tuberculosis</article-title>. <source>Nat Rev Microbiol</source>. (<year>2018</year>) <volume>16</volume>:<fpage>496</fpage>&#x2013;<lpage>507</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1038/s41579-018-0013-4</pub-id>.</citation>
</ref>
<ref id="B5">
<label>5</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Bussi</surname> <given-names>C</given-names>
</name>
<name>
<surname>Gutierrez</surname> <given-names>MG</given-names>
</name>
</person-group>. <article-title>Mycobacterium tuberculosis infection of host cells in space and time</article-title>. <source>FEMS Microbiol Rev</source>. (<year>2019</year>) <volume>43</volume>:<page-range>341&#x2013;61</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1093/femsre/fuz006</pub-id>.</citation>
</ref>
<ref id="B6">
<label>6</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Lombard</surname> <given-names>R</given-names>
</name>
<name>
<surname>Doz</surname> <given-names>E</given-names>
</name>
<name>
<surname>Carreras</surname> <given-names>F</given-names>
</name>
<name>
<surname>Epardaud</surname> <given-names>M</given-names>
</name>
<name>
<surname>Le Vern</surname> <given-names>Y</given-names>
</name>
<name>
<surname>Buzoni-Gatel</surname> <given-names>D</given-names>
</name>
<etal/>
</person-group>. <article-title>IL-17RA in non-hematopoietic cells controls CXCL-1 and 5 critical to recruit neutrophils to the lung of mycobacteria-infected mice during the adaptive immune response</article-title>. <source>PloS One</source>. (<year>2016</year>) <volume>11</volume>:<elocation-id>e0149455</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.1371/journal.pone.0149455</pub-id>.</citation>
</ref>
<ref id="B7">
<label>7</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Kang</surname> <given-names>DD</given-names>
</name>
<name>
<surname>Lin</surname> <given-names>Y</given-names>
</name>
<name>
<surname>Moreno</surname> <given-names>J-R</given-names>
</name>
<name>
<surname>Randall</surname> <given-names>TD</given-names>
</name>
<name>
<surname>Khader</surname> <given-names>SA</given-names>
</name>
</person-group>. <article-title>Profiling early lung immune responses in the mouse model of tuberculosis</article-title>. <source>PloS One</source>. (<year>2011</year>) <volume>6</volume>:<elocation-id>e16161</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.1371/journal.pone.0016161</pub-id>.</citation>
</ref>
<ref id="B8">
<label>8</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Capuano</surname> <given-names>SV</given-names>
<suffix>III</suffix>
</name>
<name>
<surname>Croix</surname> <given-names>DA</given-names>
</name>
<name>
<surname>Pawar</surname> <given-names>S</given-names>
</name>
<name>
<surname>Zinovik</surname> <given-names>A</given-names>
</name>
<name>
<surname>Myers</surname> <given-names>A</given-names>
</name>
<name>
<surname>Lin</surname> <given-names>PL</given-names>
</name>
<etal/>
</person-group>. <article-title>Experimental Mycobacterium tuberculosis infection of cynomolgus macaques closely resembles the various manifestations of human M</article-title>. <source>tuberculosis infection. Infection Immun</source>. (<year>2003</year>) <volume>71</volume>:<page-range>5831&#x2013;44</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1128/IAI.71.10.5831-5844.2003</pub-id>.</citation>
</ref>
<ref id="B9">
<label>9</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Palmer</surname> <given-names>MV</given-names>
</name>
<name>
<surname>Wiarda</surname> <given-names>J</given-names>
</name>
<name>
<surname>Kanipe</surname> <given-names>C</given-names>
</name>
<name>
<surname>Thacker</surname> <given-names>TC</given-names>
</name>
</person-group>. <article-title>Early pulmonary lesions in cattle infected via aerosolized Mycobacterium bovis</article-title>. <source>Veterinary Pathol</source>. (<year>2019</year>) <volume>56</volume>:<page-range>544&#x2013;54</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1177/0300985819833454</pub-id>.</citation>
</ref>
<ref id="B10">
<label>10</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Eum</surname> <given-names>S-Y</given-names>
</name>
<name>
<surname>Kong</surname> <given-names>J-H</given-names>
</name>
<name>
<surname>Hong</surname> <given-names>M-S</given-names>
</name>
<name>
<surname>Lee</surname> <given-names>Y-J</given-names>
</name>
<name>
<surname>Kim</surname> <given-names>J-H</given-names>
</name>
<name>
<surname>Hwang</surname> <given-names>S-H</given-names>
</name>
<etal/>
</person-group>. <article-title>via LE, Barry III CE: Neutrophils are the predominant infected phagocytic cells in the airways of patients with active pulmonary TB</article-title>. <source>Chest</source>. (<year>2010</year>) <volume>137</volume>:<page-range>122&#x2013;8</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1378/chest.09-0903</pub-id>.</citation>
</ref>
<ref id="B11">
<label>11</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Gideon</surname> <given-names>HP</given-names>
</name>
<name>
<surname>Phuah</surname> <given-names>J</given-names>
</name>
<name>
<surname>Junecko</surname> <given-names>BA</given-names>
</name>
<name>
<surname>Mattila</surname> <given-names>JT</given-names>
</name>
</person-group>. <article-title>Neutrophils express pro-and anti-inflammatory cytokines in granulomas from Mycobacterium tuberculosis-infected cynomolgus macaques</article-title>. <source>Mucosal Immunol</source>. (<year>2019</year>) <volume>12</volume>:<page-range>1370&#x2013;81</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1038/s41385-019-0195-8</pub-id>.</citation>
</ref>
<ref id="B12">
<label>12</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Ozaki</surname> <given-names>T</given-names>
</name>
<name>
<surname>Nakahira</surname> <given-names>S</given-names>
</name>
<name>
<surname>Tani</surname> <given-names>K</given-names>
</name>
<name>
<surname>Ogushi</surname> <given-names>F</given-names>
</name>
<name>
<surname>Yasuoka</surname> <given-names>S</given-names>
</name>
<name>
<surname>Ogura</surname> <given-names>T</given-names>
</name>
</person-group>. <article-title>Differential cell analysis in bronchoalveolar lavage fluid from pulmonary lesions of patients with tuberculosis</article-title>. <source>Chest</source>. (<year>1992</year>) <volume>102</volume>:<page-range>54&#x2013;9</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1378/chest.102.1.54</pub-id>.</citation>
</ref>
<ref id="B13">
<label>13</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Berry</surname> <given-names>MP</given-names>
</name>
<name>
<surname>Graham</surname> <given-names>CM</given-names>
</name>
<name>
<surname>McNab</surname> <given-names>FW</given-names>
</name>
<name>
<surname>Xu</surname> <given-names>Z</given-names>
</name>
<name>
<surname>Bloch</surname> <given-names>SA</given-names>
</name>
<name>
<surname>Oni</surname> <given-names>T</given-names>
</name>
<etal/>
</person-group>. <article-title>An interferon-inducible neutrophil-driven blood transcriptional signature in human tuberculosis</article-title>. <source>Nature</source>. (<year>2010</year>) <volume>466</volume>:<page-range>973&#x2013;7</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1038/nature09247</pub-id>.</citation>
</ref>
<ref id="B14">
<label>14</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Liu</surname> <given-names>Q</given-names>
</name>
<name>
<surname>Pan</surname> <given-names>L</given-names>
</name>
<name>
<surname>Han</surname> <given-names>F</given-names>
</name>
<name>
<surname>Luo</surname> <given-names>B</given-names>
</name>
<name>
<surname>Jia</surname> <given-names>H</given-names>
</name>
<name>
<surname>Xing</surname> <given-names>A</given-names>
</name>
<etal/>
</person-group>. <article-title>Proteomic profiling for plasma biomarkers of tuberculosis progression</article-title>. <source>Mol Med Rep</source>. (<year>2018</year>) <volume>18</volume>:<page-range>1551&#x2013;9</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.3892/mmr</pub-id>.</citation>
</ref>
<ref id="B15">
<label>15</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Lowe</surname> <given-names>DM</given-names>
</name>
<name>
<surname>Bandara</surname> <given-names>AK</given-names>
</name>
<name>
<surname>Packe</surname> <given-names>GE</given-names>
</name>
<name>
<surname>Barker</surname> <given-names>RD</given-names>
</name>
<name>
<surname>Wilkinson</surname> <given-names>RJ</given-names>
</name>
<name>
<surname>Griffiths</surname> <given-names>CJ</given-names>
</name>
<etal/>
</person-group>. <article-title>Neutrophilia independently predicts death in tuberculosis</article-title>. <source>Eur Respir J</source>. (<year>2013</year>) <volume>42</volume>:<page-range>1752&#x2013;7</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1183/09031936.00140913</pub-id>.</citation>
</ref>
<ref id="B16">
<label>16</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Martineau</surname> <given-names>AR</given-names>
</name>
<name>
<surname>Newton</surname> <given-names>SM</given-names>
</name>
<name>
<surname>Wilkinson</surname> <given-names>KA</given-names>
</name>
<name>
<surname>Kampmann</surname> <given-names>B</given-names>
</name>
<name>
<surname>Hall</surname> <given-names>BM</given-names>
</name>
<name>
<surname>Nawroly</surname> <given-names>N</given-names>
</name>
<etal/>
</person-group>. <article-title>Neutrophil-mediated innate immune resistance to mycobacteria</article-title>. <source>J Clin Invest</source>. (<year>2007</year>) <volume>117</volume>:<page-range>1988&#x2013;94</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1172/JCI31097</pub-id>.</citation>
</ref>
<ref id="B17">
<label>17</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Lovewell</surname> <given-names>RR</given-names>
</name>
<name>
<surname>Baer</surname> <given-names>CE</given-names>
</name>
<name>
<surname>Mishra</surname> <given-names>BB</given-names>
</name>
<name>
<surname>Smith</surname> <given-names>CM</given-names>
</name>
<name>
<surname>Sassetti</surname> <given-names>CM</given-names>
</name>
</person-group>. <article-title>Granulocytes act as a niche for Mycobacterium tuberculosis growth</article-title>. <source>Mucosal Immunol</source>. (<year>2021</year>) <volume>14</volume>:<page-range>229&#x2013;41</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1038/s41385-020-0300-z</pub-id>.</citation>
</ref>
<ref id="B18">
<label>18</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Mishra</surname> <given-names>BB</given-names>
</name>
<name>
<surname>Lovewell</surname> <given-names>RR</given-names>
</name>
<name>
<surname>Olive</surname> <given-names>AJ</given-names>
</name>
<name>
<surname>Zhang</surname> <given-names>G</given-names>
</name>
<name>
<surname>Wang</surname> <given-names>W</given-names>
</name>
<name>
<surname>Eugenin</surname> <given-names>E</given-names>
</name>
<etal/>
</person-group>. <article-title>Nitric oxide prevents a pathogen-permissive granulocytic inflammation during tuberculosis</article-title>. <source>Nat Microbiol</source>. (<year>2017</year>) <volume>2</volume>:<fpage>1</fpage>&#x2013;<lpage>11</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1038/nmicrobiol.2017.72</pub-id>.</citation>
</ref>
<ref id="B19">
<label>19</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Hansen</surname> <given-names>SG</given-names>
</name>
<name>
<surname>Zak</surname> <given-names>DE</given-names>
</name>
<name>
<surname>Xu</surname> <given-names>G</given-names>
</name>
<name>
<surname>Ford</surname> <given-names>JC</given-names>
</name>
<name>
<surname>Marshall</surname> <given-names>EE</given-names>
</name>
<name>
<surname>Malouli</surname> <given-names>D</given-names>
</name>
<etal/>
</person-group>. <article-title>Prevention of tuberculosis in rhesus macaques by a cytomegalovirus-based vaccine</article-title>. <source>Nat Med</source>. (<year>2018</year>) <volume>24</volume>:<page-range>130&#x2013;43</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1038/nm.4473</pub-id>.</citation>
</ref>
<ref id="B20">
<label>20</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Yang</surname> <given-names>C-T</given-names>
</name>
<name>
<surname>Cambier</surname> <given-names>C</given-names>
</name>
<name>
<surname>Davis</surname> <given-names>JM</given-names>
</name>
<name>
<surname>Hall</surname> <given-names>CJ</given-names>
</name>
<name>
<surname>Crosier</surname> <given-names>PS</given-names>
</name>
<name>
<surname>Ramakrishnan</surname> <given-names>L</given-names>
</name>
</person-group>. <article-title>Neutrophils exert protection in the early tuberculous granuloma by oxidative killing of mycobacteria phagocytosed from infected macrophages</article-title>. <source>Cell Host Microbe</source>. (<year>2012</year>) <volume>12</volume>:<page-range>301&#x2013;12</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1016/j.chom.2012.07.009</pub-id>.</citation>
</ref>
<ref id="B21">
<label>21</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Filep</surname> <given-names>JG</given-names>
</name>
</person-group>. <article-title>Targeting neutrophils for promoting the resolution of inflammation</article-title>. <source>Front Immunol</source>. (<year>2022</year>) <volume>1008</volume>. doi:&#xa0;<pub-id pub-id-type="doi">10.3389/fimmu.2022.866747</pub-id>.</citation>
</ref>
<ref id="B22">
<label>22</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Talwar</surname> <given-names>S</given-names>
</name>
<name>
<surname>Pandey</surname> <given-names>M</given-names>
</name>
<name>
<surname>Sharma</surname> <given-names>C</given-names>
</name>
<name>
<surname>Kutum</surname> <given-names>R</given-names>
</name>
<name>
<surname>Lum</surname> <given-names>J</given-names>
</name>
<name>
<surname>Carbajo</surname> <given-names>D</given-names>
</name>
<etal/>
</person-group>. <article-title>Role of VapBC12 toxin-antitoxin locus in cholesterol-induced mycobacterial persistence</article-title>. <source>M Syst</source>. (<year>2020</year>) <volume>5</volume>:<page-range>e00855&#x2013;00820</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1128/mSystems.00855-20</pub-id>.</citation>
</ref>
<ref id="B23">
<label>23</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Pandey</surname> <given-names>M</given-names>
</name>
<name>
<surname>Talwar</surname> <given-names>S</given-names>
</name>
<name>
<surname>Bose</surname> <given-names>S</given-names>
</name>
<name>
<surname>Pandey</surname> <given-names>AK</given-names>
</name>
</person-group>. <article-title>Iron homeostasis in Mycobacterium tuberculosis is essential for persistence</article-title>. <source>Sci Rep</source>. (<year>2018</year>) <volume>8</volume>:<fpage>17359</fpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1038/s41598-018-35012-3</pub-id>.</citation>
</ref>
<ref id="B24">
<label>24</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Sadhu</surname> <given-names>S</given-names>
</name>
<name>
<surname>Rizvi</surname> <given-names>ZA</given-names>
</name>
<name>
<surname>Pandey</surname> <given-names>RP</given-names>
</name>
<name>
<surname>Dalal</surname> <given-names>R</given-names>
</name>
<name>
<surname>Rathore</surname> <given-names>DK</given-names>
</name>
<name>
<surname>Kumar</surname> <given-names>B</given-names>
</name>
<etal/>
</person-group>. <article-title>Gefitinib results in robust host-directed immunity against Salmonella infection through proteo-metabolomic reprogramming</article-title>. <source>Front Immunol</source>. (<year>2021</year>) <volume>12</volume>:<elocation-id>648710</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.3389/fimmu.2021.648710</pub-id>.</citation>
</ref>
<ref id="B25">
<label>25</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Sadhu</surname> <given-names>S</given-names>
</name>
<name>
<surname>Dalal</surname> <given-names>R</given-names>
</name>
<name>
<surname>Dandotiya</surname> <given-names>J</given-names>
</name>
<name>
<surname>Binayke</surname> <given-names>A</given-names>
</name>
<name>
<surname>Singh</surname> <given-names>V</given-names>
</name>
<name>
<surname>Tripathy</surname> <given-names>MR</given-names>
</name>
<etal/>
</person-group>. <article-title>IL-9 aggravates SARS-CoV-2 infection and exacerbates associated airway inflammation</article-title>. <source>Nat Commun</source>. (<year>2023</year>) <volume>14</volume>:<fpage>4060</fpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1038/s41467-023-39815-5</pub-id>.</citation>
</ref>
<ref id="B26">
<label>26</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Petryszak</surname> <given-names>R</given-names>
</name>
<name>
<surname>Burdett</surname> <given-names>T</given-names>
</name>
<name>
<surname>Fiorelli</surname> <given-names>B</given-names>
</name>
<name>
<surname>Fonseca</surname> <given-names>NA</given-names>
</name>
<name>
<surname>Gonzalez-Porta</surname> <given-names>M</given-names>
</name>
<name>
<surname>Hastings</surname> <given-names>E</given-names>
</name>
<etal/>
</person-group>. <article-title>Expression Atlas update&#x2014;a database of gene and transcript expression from microarray-and sequencing-based functional genomics experiments</article-title>. <source>Nucleic Acids Res</source>. (<year>2014</year>) <volume>42</volume>:<page-range>D926&#x2013;32</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1093/nar/gkt1270</pub-id>
</citation>
</ref>
<ref id="B27">
<label>27</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Storey</surname> <given-names>JD</given-names>
</name>
</person-group>. <article-title>A direct approach to false discovery rates</article-title>. <source>J R Stat Society: Ser B</source>. (<year>2002</year>) <volume>64</volume>:<page-range>479&#x2013;98</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1111/1467-9868.00346</pub-id>.</citation>
</ref>
<ref id="B28">
<label>28</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Malik</surname> <given-names>S</given-names>
</name>
<name>
<surname>Sadhu</surname> <given-names>S</given-names>
</name>
<name>
<surname>Elesela</surname> <given-names>S</given-names>
</name>
<name>
<surname>Pandey</surname> <given-names>RP</given-names>
</name>
<name>
<surname>Chawla</surname> <given-names>AS</given-names>
</name>
<name>
<surname>Sharma</surname> <given-names>D</given-names>
</name>
<etal/>
</person-group>. <article-title>Transcription factor Foxo1 is essential for IL-9 induction in T helper cells</article-title>. <source>Nat Commun</source>. (<year>2017</year>) <volume>8</volume>:<fpage>815</fpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1038/s41467-017-00674-6</pub-id>.</citation>
</ref>
<ref id="B29">
<label>29</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Du Plessis</surname> <given-names>N</given-names>
</name>
<name>
<surname>Jacobs</surname> <given-names>R</given-names>
</name>
<name>
<surname>Gutschmidt</surname> <given-names>A</given-names>
</name>
<name>
<surname>Fang</surname> <given-names>Z</given-names>
</name>
<name>
<surname>van Helden</surname> <given-names>PD</given-names>
</name>
<name>
<surname>Lutz</surname> <given-names>MB</given-names>
</name>
<etal/>
</person-group>. <article-title>Phenotypically resembling myeloid derived suppressor cells are increased in children with HIV and exposed/infected with Mycobacterium tuberculosis</article-title>. <source>Eur J Immunol</source>. (<year>2017</year>) <volume>47</volume>:<page-range>107&#x2013;18</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1002/eji.201646658</pub-id>.</citation>
</ref>
<ref id="B30">
<label>30</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Tsiganov</surname> <given-names>EN</given-names>
</name>
<name>
<surname>Verbina</surname> <given-names>EM</given-names>
</name>
<name>
<surname>Radaeva</surname> <given-names>TV</given-names>
</name>
<name>
<surname>Sosunov</surname> <given-names>VV</given-names>
</name>
<name>
<surname>Kosmiadi</surname> <given-names>GA</given-names>
</name>
<name>
<surname>Nikitina</surname> <given-names>IY</given-names>
</name>
<etal/>
</person-group>. <article-title>Gr-1dimCD11b+ immature myeloid-derived suppressor cells but not neutrophils are markers of lethal tuberculosis infection in mice</article-title>. <source>J Immunol</source>. (<year>2014</year>) <volume>192</volume>:<page-range>4718&#x2013;27</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.4049/jimmunol.1301365</pub-id>.</citation>
</ref>
<ref id="B31">
<label>31</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Kato</surname> <given-names>K</given-names>
</name>
<name>
<surname>Yamamoto</surname> <given-names>K</given-names>
</name>
</person-group>. <article-title>Suppression of BCG cell wall-induced delayed-type hypersensitivity by BCG pre-treatment. II. Induction of suppressor T cells by heat-killed BCG injection</article-title>. <source>Immunology</source>. (<year>1982</year>) <volume>45</volume>:<fpage>655</fpage>.</citation>
</ref>
<ref id="B32">
<label>32</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Klimpel</surname> <given-names>GR</given-names>
</name>
<name>
<surname>Okada</surname> <given-names>M</given-names>
</name>
<name>
<surname>Henney</surname> <given-names>CS</given-names>
</name>
</person-group>. <article-title>Inhibition of in <italic>vitro</italic> Cytotoxic Responses by BCG-Induced Macrophage-Like Suppressor Cells: II. Suppression Occurs at the Level of a &#x201c;Helper&#x201d; T Cell</article-title>. <source>J Immunol</source>. (<year>1979</year>) <volume>123</volume>:<page-range>350&#x2013;7</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.4049/jimmunol.123.1.350</pub-id>.</citation>
</ref>
<ref id="B33">
<label>33</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Nouailles</surname> <given-names>G</given-names>
</name>
<name>
<surname>Dorhoi</surname> <given-names>A</given-names>
</name>
<name>
<surname>Koch</surname> <given-names>M</given-names>
</name>
<name>
<surname>Zerrahn</surname> <given-names>J</given-names>
</name>
<name>
<surname>Weiner</surname> <given-names>J</given-names>
</name>
<name>
<surname>Fae</surname> <given-names>KC</given-names>
</name>
<etal/>
</person-group>. <article-title>CXCL5-secreting pulmonary epithelial cells drive destructive neutrophilic inflammation in tuberculosis</article-title>. <source>J Clin Invest</source>. (<year>2014</year>) <volume>124</volume>:<page-range>1268&#x2013;82</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1172/JCI72030</pub-id>.</citation>
</ref>
<ref id="B34">
<label>34</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Kimmey</surname> <given-names>JM</given-names>
</name>
<name>
<surname>Huynh</surname> <given-names>JP</given-names>
</name>
<name>
<surname>Weiss</surname> <given-names>LA</given-names>
</name>
<name>
<surname>Park</surname> <given-names>S</given-names>
</name>
<name>
<surname>Kambal</surname> <given-names>A</given-names>
</name>
<name>
<surname>Debnath</surname> <given-names>J</given-names>
</name>
<etal/>
</person-group>. <article-title>Unique role for ATG5 in neutrophil-mediated immunopathology during M. tuberculosis infection</article-title>. <source>Nature</source>. (<year>2015</year>) <volume>528</volume>:<page-range>565&#x2013;9</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1038/nature16451</pub-id>.</citation>
</ref>
<ref id="B35">
<label>35</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Montoya</surname> <given-names>CJ</given-names>
</name>
<name>
<surname>Catano</surname> <given-names>JC</given-names>
</name>
<name>
<surname>Ramirez</surname> <given-names>Z</given-names>
</name>
<name>
<surname>Rugeles</surname> <given-names>MT</given-names>
</name>
<name>
<surname>Wilson</surname> <given-names>SB</given-names>
</name>
<name>
<surname>Landay</surname> <given-names>AL</given-names>
</name>
</person-group>. <article-title>Invariant NKT cells from HIV-1 or Mycobacterium tuberculosis-infected patients express an activated phenotype</article-title>. <source>Clin Immunol</source>. (<year>2008</year>) <volume>127</volume>:<fpage>1</fpage>&#x2013;<lpage>6</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1016/j.clim.2007.12.006</pub-id>.</citation>
</ref>
<ref id="B36">
<label>36</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Snyder-Cappione</surname> <given-names>JE</given-names>
</name>
<name>
<surname>Nixon</surname> <given-names>DF</given-names>
</name>
<name>
<surname>Loo</surname> <given-names>CP</given-names>
</name>
<name>
<surname>Chapman</surname> <given-names>JM</given-names>
</name>
<name>
<surname>Meiklejohn</surname> <given-names>DA</given-names>
</name>
<name>
<surname>Melo</surname> <given-names>FF</given-names>
</name>
<etal/>
</person-group>. <article-title>Individuals with pulmonary tuberculosis have lower levels of circulating CD1d-restricted NKT cells</article-title>. <source>J Infect Dis</source>. (<year>2007</year>) <volume>195</volume>:<page-range>1361&#x2013;4</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1086/513567</pub-id>
</citation>
</ref>
<ref id="B37">
<label>37</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Muraille</surname> <given-names>E</given-names>
</name>
<name>
<surname>Leo</surname> <given-names>O</given-names>
</name>
<name>
<surname>Moser</surname> <given-names>M</given-names>
</name>
</person-group>. <article-title>TH1/TH2 paradigm extended: macrophage polarization as an unappreciated pathogen-driven escape mechanism</article-title>? <source>Front Immunol</source>. (<year>2014</year>) <volume>5</volume>:<elocation-id>603</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.3389/fimmu.2014.00603</pub-id>.</citation>
</ref>
<ref id="B38">
<label>38</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Huang</surname> <given-names>D</given-names>
</name>
<name>
<surname>Shen</surname> <given-names>Y</given-names>
</name>
<name>
<surname>Qiu</surname> <given-names>L</given-names>
</name>
<name>
<surname>Chen</surname> <given-names>CY</given-names>
</name>
<name>
<surname>Shen</surname> <given-names>L</given-names>
</name>
<name>
<surname>Estep</surname> <given-names>J</given-names>
</name>
<etal/>
</person-group>. <article-title>Immune distribution and localization of phosphoantigen-specific V&#x3b3;2V&#x3b4;2 T cells in lymphoid and nonlymphoid tissues in Mycobacterium tuberculosis infection</article-title>. <source>Infection Immun</source>. (<year>2008</year>) <volume>76</volume>:<page-range>426&#x2013;36</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1128/IAI.01008-07</pub-id>.</citation>
</ref>
<ref id="B39">
<label>39</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Gopal</surname> <given-names>R</given-names>
</name>
<name>
<surname>Monin</surname> <given-names>L</given-names>
</name>
<name>
<surname>Torres</surname> <given-names>D</given-names>
</name>
<name>
<surname>Slight</surname> <given-names>S</given-names>
</name>
<name>
<surname>Mehra</surname> <given-names>S</given-names>
</name>
<name>
<surname>McKenna</surname> <given-names>KC</given-names>
</name>
<etal/>
</person-group>. <article-title>S100A8/A9 proteins mediate neutrophilic inflammation and lung pathology during tuberculosis</article-title>. <source>Am J Respir Crit Care Med</source>. (<year>2013</year>) <volume>188</volume>:<page-range>1137&#x2013;46</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1164/rccm.201304-0803OC</pub-id>.</citation>
</ref>
<ref id="B40">
<label>40</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Niazi</surname> <given-names>MK</given-names>
</name>
<name>
<surname>Dhulekar</surname> <given-names>N</given-names>
</name>
<name>
<surname>Schmidt</surname> <given-names>D</given-names>
</name>
<name>
<surname>Major</surname> <given-names>S</given-names>
</name>
<name>
<surname>Cooper</surname> <given-names>R</given-names>
</name>
<name>
<surname>Abeijon</surname> <given-names>C</given-names>
</name>
<etal/>
</person-group>. <article-title>Lung necrosis and neutrophils reflect common pathways of susceptibility to Mycobacterium tuberculosis in genetically diverse, immune-competent mice</article-title>. <source>Dis Models Mech</source>. (<year>2015</year>) <volume>8</volume>:<page-range>1141&#x2013;53</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1242/dmm.020867</pub-id>.</citation>
</ref>
<ref id="B41">
<label>41</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Silverio</surname> <given-names>D</given-names>
</name>
<name>
<surname>Goncalves</surname> <given-names>R</given-names>
</name>
<name>
<surname>Appelberg</surname> <given-names>R</given-names>
</name>
<name>
<surname>Saraiva</surname> <given-names>M</given-names>
</name>
</person-group>. <article-title>Advances on the role and applications of interleukin-1 in tuberculosis</article-title>. <source>Mbio</source>. (<year>2021</year>) <volume>12</volume>:<page-range>e03134&#x2013;03121</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1128/mBio.03134-21</pub-id>.</citation>
</ref>
<ref id="B42">
<label>42</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Anes</surname> <given-names>E</given-names>
</name>
<name>
<surname>Azevedo-Pereira</surname> <given-names>JM</given-names>
</name>
<name>
<surname>Pires</surname> <given-names>D</given-names>
</name>
</person-group>. <article-title>Cathepsins and their endogenous inhibitors in host defense during mycobacterium tuberculosis and HIV infection</article-title>. <source>Front Immunol</source>. (<year>2021</year>) <volume>12</volume>:<elocation-id>726984</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.3389/fimmu.2021.726984</pub-id>.</citation>
</ref>
<ref id="B43">
<label>43</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Gaffney</surname> <given-names>E</given-names>
</name>
<name>
<surname>Murphy</surname> <given-names>D</given-names>
</name>
<name>
<surname>Walsh</surname> <given-names>A</given-names>
</name>
<name>
<surname>Connolly</surname> <given-names>S</given-names>
</name>
<name>
<surname>Basdeo</surname> <given-names>S</given-names>
</name>
<name>
<surname>Keane</surname> <given-names>J</given-names>
</name>
<etal/>
</person-group>. <article-title>Defining the role of neutrophils in the lung during infection: Implications for tuberculosis disease</article-title>. <source>Front Immunol</source>. (<year>2022</year>) <volume>13</volume>:<elocation-id>984293</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.3389/fimmu.2022.984293</pub-id>.</citation>
</ref>
<ref id="B44">
<label>44</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Witko-Sarsat</surname> <given-names>V</given-names>
</name>
<name>
<surname>Rieu</surname> <given-names>P</given-names>
</name>
<name>
<surname>Descamps-Latscha</surname> <given-names>B</given-names>
</name>
<name>
<surname>Lesavre</surname> <given-names>P</given-names>
</name>
<name>
<surname>Halbwachs-Mecarelli</surname> <given-names>L</given-names>
</name>
</person-group>. <article-title>Neutrophils: molecules, functions and pathophysiological aspects</article-title>. <source>Lab Invest</source>. (<year>2000</year>) <volume>80</volume>:<page-range>617&#x2013;53</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1038/labinvest.3780067</pub-id>.</citation>
</ref>
<ref id="B45">
<label>45</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Olofsen</surname> <given-names>PA</given-names>
</name>
<name>
<surname>Stip</surname> <given-names>MC</given-names>
</name>
<name>
<surname>Jansen</surname> <given-names>JM</given-names>
</name>
<name>
<surname>Chan</surname> <given-names>C</given-names>
</name>
<name>
<surname>Nederend</surname> <given-names>M</given-names>
</name>
<name>
<surname>Tieland</surname> <given-names>RG</given-names>
</name>
<etal/>
</person-group>. <article-title>Effective, long-term, neutrophil depletion using a murinized anti-ly-6G 1A8 antibody</article-title>. <source>Cells</source>. (<year>2022</year>) <volume>11</volume>:<fpage>3406</fpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.3390/cells11213406</pub-id>.</citation>
</ref>
<ref id="B46">
<label>46</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Vogl</surname> <given-names>T</given-names>
</name>
<name>
<surname>Tenbrock</surname> <given-names>K</given-names>
</name>
<name>
<surname>Ludwig</surname> <given-names>S</given-names>
</name>
<name>
<surname>Leukert</surname> <given-names>N</given-names>
</name>
<name>
<surname>Ehrhardt</surname> <given-names>C</given-names>
</name>
<name>
<surname>Van Zoelen</surname> <given-names>MA</given-names>
</name>
<etal/>
</person-group>. <article-title>Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxin-induced shock</article-title>. <source>Nat Med</source>. (<year>2007</year>) <volume>13</volume>:<page-range>1042&#x2013;9</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1038/nm1638</pub-id>.</citation>
</ref>
<ref id="B47">
<label>47</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>El Kasmi</surname> <given-names>KC</given-names>
</name>
<name>
<surname>Qualls</surname> <given-names>JE</given-names>
</name>
<name>
<surname>Pesce</surname> <given-names>JT</given-names>
</name>
<name>
<surname>Smith</surname> <given-names>AM</given-names>
</name>
<name>
<surname>Thompson</surname> <given-names>RW</given-names>
</name>
<name>
<surname>Henao-Tamayo</surname> <given-names>M</given-names>
</name>
<etal/>
</person-group>. <article-title>Toll-like receptorinduced arginase 1 in macrophages thwarts effective immunity against intracellular pathogens</article-title>. <source>Nat Immunol</source>. (<year>2008</year>) <volume>9</volume>:<page-range>1399&#x2013;406</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1038/ni.1671</pub-id>.</citation>
</ref>
<ref id="B48">
<label>48</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Pessanha</surname> <given-names>AP</given-names>
</name>
<name>
<surname>Martins</surname> <given-names>RA</given-names>
</name>
<name>
<surname>Mattos-Guaraldi</surname> <given-names>AL</given-names>
</name>
<name>
<surname>Vianna</surname> <given-names>A</given-names>
</name>
<name>
<surname>Moreira</surname> <given-names>LO</given-names>
</name>
</person-group>. <article-title>Arginase-1 expression in granulomas of tuberculosis patients</article-title>. <source>FEMS Immunol Med Microbiol</source>. (<year>2012</year>) <volume>66</volume>:<page-range>265&#x2013;8</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1111/j.1574-695X.2012.01012.x</pub-id>.</citation>
</ref>
<ref id="B49">
<label>49</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Abebe</surname> <given-names>T</given-names>
</name>
<name>
<surname>Hailu</surname> <given-names>A</given-names>
</name>
<name>
<surname>Woldeyes</surname> <given-names>M</given-names>
</name>
<name>
<surname>Mekonen</surname> <given-names>W</given-names>
</name>
<name>
<surname>Bilcha</surname> <given-names>K</given-names>
</name>
<name>
<surname>Cloke</surname> <given-names>T</given-names>
</name>
<etal/>
</person-group>. <article-title>Local increase of arginase activity in lesions of patients with cutaneous leishmaniasis in Ethiopia</article-title>. <source>PloS Negl Trop Dis</source>. (<year>2012</year>) <volume>6</volume>:<fpage>e1684</fpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1371/journal.pntd.0001684</pub-id>.</citation>
</ref>
<ref id="B50">
<label>50</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Mattila</surname> <given-names>JT</given-names>
</name>
<name>
<surname>Ojo</surname> <given-names>OO</given-names>
</name>
<name>
<surname>Kepka-Lenhart</surname> <given-names>D</given-names>
</name>
<name>
<surname>Marino</surname> <given-names>S</given-names>
</name>
<name>
<surname>Kim</surname> <given-names>JH</given-names>
</name>
<name>
<surname>Eum</surname> <given-names>SY</given-names>
</name>
<etal/>
</person-group>. <article-title>via LE Kirschner DE: Microenvironments in tuberculous granulomas are delineated by distinct populations of macrophage subsets and expression of nitric oxide synthase and arginase isoforms</article-title>. <source>J Immunol</source>. (<year>2013</year>) <volume>191</volume>:<page-range>773&#x2013;84</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.4049/jimmunol.1300113</pub-id>.</citation>
</ref>
<ref id="B51">
<label>51</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Abdullah</surname> <given-names>Z</given-names>
</name>
<name>
<surname>Geiger</surname> <given-names>S</given-names>
</name>
<name>
<surname>Nino-Castro</surname> <given-names>A</given-names>
</name>
<name>
<surname>Bottcher</surname> <given-names>JP</given-names>
</name>
<name>
<surname>Muraliv</surname> <given-names>E</given-names>
</name>
<name>
<surname>Gaidt</surname> <given-names>M</given-names>
</name>
<etal/>
</person-group>. <article-title>Lack of PPAR&#x3b3; in myeloid cells confers resistance to Listeria monocytogenes infection</article-title>. <source>PloS One</source>. (<year>2012</year>) <volume>7</volume>:<elocation-id>e37349</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.1371/journal.pone.0037349</pub-id>.</citation>
</ref>
<ref id="B52">
<label>52</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Shirey</surname> <given-names>KA</given-names>
</name>
<name>
<surname>Cole</surname> <given-names>LE</given-names>
</name>
<name>
<surname>Keegan</surname> <given-names>AD</given-names>
</name>
<name>
<surname>Vogel</surname> <given-names>SN</given-names>
</name>
</person-group>. <article-title>Francisella tularensis live vaccine strain induces macrophage alternative activation as a survival mechanism</article-title>. <source>J Immunol</source>. (<year>2008</year>) <volume>181</volume>:<page-range>4159&#x2013;67</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.4049/jimmunol.181.6.4159</pub-id>.</citation>
</ref>
<ref id="B53">
<label>53</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Xavier</surname> <given-names>MN</given-names>
</name>
<name>
<surname>Winter</surname> <given-names>MG</given-names>
</name>
<name>
<surname>Spees</surname> <given-names>AM</given-names>
</name>
<name>
<surname>den Hartigh</surname> <given-names>AB</given-names>
</name>
<name>
<surname>Nguyen</surname> <given-names>K</given-names>
</name>
<name>
<surname>Roux</surname> <given-names>CM</given-names>
</name>
<etal/>
</person-group>. <article-title>PPAR&#x3b3;-mediated increase in glucose availability sustains chronic Brucella abortus infection in alternatively activated macrophages</article-title>. <source>Cell Host Microbe</source>. (<year>2013</year>) <volume>14</volume>:<page-range>159&#x2013;70</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1016/j.chom.2013.07.009</pub-id>.</citation>
</ref>
<ref id="B54">
<label>54</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Almeida</surname> <given-names>PE</given-names>
</name>
<name>
<surname>Carneiro</surname> <given-names>AB</given-names>
</name>
<name>
<surname>Silva</surname> <given-names>AR</given-names>
</name>
<name>
<surname>Bozza</surname> <given-names>PT</given-names>
</name>
</person-group>. <article-title>PPAR&#x3b3; expression and function in mycobacterial infection: roles in lipid metabolism, immunity, and bacterial killing</article-title>. <source>PPAR Res</source>. (<year>2012</year>). doi:&#xa0;<pub-id pub-id-type="doi">10.1155/2012/383829</pub-id>
</citation>
</ref>
<ref id="B55">
<label>55</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Eisele</surname> <given-names>NA</given-names>
</name>
<name>
<surname>Ruby</surname> <given-names>T</given-names>
</name>
<name>
<surname>Jacobson</surname> <given-names>A</given-names>
</name>
<name>
<surname>Manzanillo</surname> <given-names>PS</given-names>
</name>
<name>
<surname>Cox</surname> <given-names>JS</given-names>
</name>
<name>
<surname>Lam</surname> <given-names>L</given-names>
</name>
<etal/>
</person-group>. <article-title>Salmonella require the fatty acid regulator PPAR&#x3b4; for the establishment of a metabolic environment essential for long-term persistence</article-title>. <source>Cell Host Microbe</source>. (<year>2013</year>) <volume>14</volume>:<page-range>171&#x2013;82</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1016/j.chom.2013.07.010</pub-id>.</citation>
</ref>
<ref id="B56">
<label>56</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Condos</surname> <given-names>R</given-names>
</name>
<name>
<surname>Rom</surname> <given-names>WN</given-names>
</name>
<name>
<surname>Liu</surname> <given-names>YM</given-names>
</name>
<name>
<surname>Schluger</surname> <given-names>NW</given-names>
</name>
</person-group>. <article-title>Local immune responses correlate with presentation and outcome in tuberculosis</article-title>. <source>Am J Respir Crit Care Med</source>. (<year>1998</year>) <volume>157</volume>:<page-range>729&#x2013;35</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1164/ajrccm.157.3.9705044</pub-id>.</citation>
</ref>
<ref id="B57">
<label>57</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Keller</surname> <given-names>C</given-names>
</name>
<name>
<surname>Hoffmann</surname> <given-names>R</given-names>
</name>
<name>
<surname>Lang</surname> <given-names>R</given-names>
</name>
<name>
<surname>Brandau</surname> <given-names>S</given-names>
</name>
<name>
<surname>Hermann</surname> <given-names>C</given-names>
</name>
<name>
<surname>Ehlers</surname> <given-names>S</given-names>
</name>
</person-group>. <article-title>Genetically determined susceptibility to tuberculosis in mice causally involves accelerated and enhanced recruitment of granulocytes</article-title>. <source>Infection Immun</source>. (<year>2006</year>) <volume>74</volume>:<page-range>4295&#x2013;309</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1128/IAI.00057-06</pub-id>.</citation>
</ref>
<ref id="B58">
<label>58</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Nandi</surname> <given-names>B</given-names>
</name>
<name>
<surname>Behar</surname> <given-names>SM</given-names>
</name>
</person-group>. <article-title>Regulation of neutrophils by interferon-&#x3b3; limits lung inflammation during tuberculosis infection</article-title>. <source>J Exp Med</source>. (<year>2011</year>) <volume>208</volume>:<page-range>2251&#x2013;62</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1084/jem.20110919</pub-id>.</citation>
</ref>
<ref id="B59">
<label>59</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Lowe</surname> <given-names>DM</given-names>
</name>
<name>
<surname>Redford</surname> <given-names>PS</given-names>
</name>
<name>
<surname>Wilkinson</surname> <given-names>RJ</given-names>
</name>
<name>
<surname>OGarra</surname> <given-names>A</given-names>
</name>
<name>
<surname>Martineau</surname> <given-names>AR</given-names>
</name>
</person-group>. <article-title>Neutrophils in tuberculosis: friend or foe</article-title>? <source>Trends Immunol</source>. (<year>2012</year>) <volume>33</volume>:<fpage>14</fpage>&#x2013;<lpage>25</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1016/j.it.2011.10.003</pub-id>.</citation>
</ref>
<ref id="B60">
<label>60</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Szulc-Dabrowska</surname> <given-names>L</given-names>
</name>
<name>
<surname>Bossowska-Nowicka</surname> <given-names>M</given-names>
</name>
<name>
<surname>Struzik</surname> <given-names>J</given-names>
</name>
<name>
<surname>Toka</surname> <given-names>FN</given-names>
</name>
</person-group>. <article-title>Cathepsins in bacteria-macrophage interaction: defenders or victims of circumstance</article-title>? <source>Front Cell infection Microbiol</source>. (<year>2020</year>) <volume>10</volume>:<elocation-id>601072</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.3389/fcimb.2020.601072</pub-id>.</citation>
</ref>
<ref id="B61">
<label>61</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Sharma</surname> <given-names>T</given-names>
</name>
<name>
<surname>Grover</surname> <given-names>S</given-names>
</name>
<name>
<surname>Arora</surname> <given-names>N</given-names>
</name>
<name>
<surname>Ehtesham</surname> <given-names>NZ</given-names>
</name>
<name>
<surname>Hasnain</surname> <given-names>SE</given-names>
</name>
</person-group>. <article-title>PGRS domain of Rv0297 of Mycobacterium tuberculosis is involved in modulation of macrophage functions to favor bacterial persistence</article-title>. <source>Front Cell Infection Microbiol</source>. (<year>2020</year>) <volume>10</volume>:<elocation-id>451</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.3389/fcimb.2020.00451</pub-id>.</citation>
</ref>
<ref id="B62">
<label>62</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Gutierrez</surname> <given-names>MG</given-names>
</name>
<name>
<surname>Mishra</surname> <given-names>BB</given-names>
</name>
<name>
<surname>Jordao</surname> <given-names>L</given-names>
</name>
<name>
<surname>Elliott</surname> <given-names>E</given-names>
</name>
<name>
<surname>Anes</surname> <given-names>E</given-names>
</name>
<name>
<surname>Griffiths</surname> <given-names>G</given-names>
</name>
</person-group>. <article-title>NF-&#x3ba;B activation controls phagolysosome fusion-mediated killing of mycobacteria by macrophages</article-title>. <source>J Immunol</source>. (<year>2008</year>) <volume>181</volume>:<page-range>2651&#x2013;63</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.4049/jimmunol.181.4.2651</pub-id>.</citation>
</ref>
<ref id="B63">
<label>63</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Amaral</surname> <given-names>EP</given-names>
</name>
<name>
<surname>Riteau</surname> <given-names>N</given-names>
</name>
<name>
<surname>Moayeri</surname> <given-names>M</given-names>
</name>
<name>
<surname>Maier</surname> <given-names>N</given-names>
</name>
<name>
<surname>Mayer-Barber</surname> <given-names>KD</given-names>
</name>
<name>
<surname>Pereira</surname> <given-names>RM</given-names>
</name>
<etal/>
</person-group>. <article-title>Lysosomal cathepsin release is required for NLRP3-inflammasome activation by Mycobacterium tuberculosis in infected macrophages</article-title>. <source>Front Immunol</source>. (<year>2018</year>) <volume>9</volume>:<elocation-id>1427</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.3389/fimmu.2018.01427</pub-id>.</citation>
</ref>
<ref id="B64">
<label>64</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Pires</surname> <given-names>D</given-names>
</name>
<name>
<surname>Marques</surname> <given-names>J</given-names>
</name>
<name>
<surname>Pombo</surname> <given-names>JP</given-names>
</name>
<name>
<surname>Carmo</surname> <given-names>N</given-names>
</name>
<name>
<surname>Bettencourt</surname> <given-names>P</given-names>
</name>
<name>
<surname>Neyrolles</surname> <given-names>O</given-names>
</name>
<etal/>
</person-group>. <article-title>Role of cathepsins in Mycobacterium tuberculosis survival in human macrophages</article-title>. <source>Sci Rep</source>. (<year>2016</year>) <volume>6</volume>:<fpage>32247</fpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1038/srep32247</pub-id>.</citation>
</ref>
<ref id="B65">
<label>65</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Knaul</surname> <given-names>JK</given-names>
</name>
<name>
<surname>Jorg</surname> <given-names>S</given-names>
</name>
<name>
<surname>Oberbeck-Mueller</surname> <given-names>D</given-names>
</name>
<name>
<surname>Heinemann</surname> <given-names>E</given-names>
</name>
<name>
<surname>Scheuermann</surname> <given-names>L</given-names>
</name>
<name>
<surname>Brinkmann</surname> <given-names>V</given-names>
</name>
<etal/>
</person-group>. <article-title>Lung-residing myeloid-derived suppressors display dual functionality in murine pulmonary tuberculosis</article-title>. <source>Am J Respir Crit Care Med</source>. (<year>2014</year>) <volume>190</volume>:<page-range>1053&#x2013;66</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1164/rccm.201405-0828OC</pub-id>.</citation>
</ref>
<ref id="B66">
<label>66</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Agrawal</surname> <given-names>N</given-names>
</name>
<name>
<surname>Streata</surname> <given-names>I</given-names>
</name>
<name>
<surname>Pei</surname> <given-names>G</given-names>
</name>
<name>
<surname>Weiner</surname> <given-names>J</given-names>
</name>
<name>
<surname>Kotze</surname> <given-names>L</given-names>
</name>
<name>
<surname>Bandermann</surname> <given-names>S</given-names>
</name>
<etal/>
</person-group>. <article-title>Human monocytic suppressive cells promote replication of Mycobacterium tuberculosis and alter stability of in <italic>vitro</italic> generated granulomas</article-title>. <source>Front Immunol</source>. (<year>2018</year>) <volume>9</volume>:<elocation-id>2417</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.3389/fimmu.2018.02417</pub-id>.</citation>
</ref>
<ref id="B67">
<label>67</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Magcwebeba</surname> <given-names>T</given-names>
</name>
<name>
<surname>Dorhoi</surname> <given-names>A</given-names>
</name>
<name>
<surname>du Plessis</surname> <given-names>N</given-names>
</name>
</person-group>. <article-title>The emerging role of myeloid-derived suppressor cells in tuberculosis</article-title>. <source>Front Immunol</source>. (<year>2019</year>) <volume>10</volume>:<elocation-id>917</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.3389/fimmu.2019.00917</pub-id>.</citation>
</ref>
<ref id="B68">
<label>68</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Parveen</surname> <given-names>S</given-names>
</name>
<name>
<surname>Lun</surname> <given-names>S</given-names>
</name>
<name>
<surname>Urbanowski</surname> <given-names>ME</given-names>
</name>
<name>
<surname>Cardin</surname> <given-names>M</given-names>
</name>
<name>
<surname>Shen</surname> <given-names>J</given-names>
</name>
<name>
<surname>Murphy</surname> <given-names>JR</given-names>
</name>
<etal/>
</person-group>. <article-title>Effective host-directed therapy for tuberculosis by depletion of myeloid-derived suppressor cells and related cells using a diphtheria toxin fusion protein</article-title>. <source>J Infect Dis</source>. (<year>2021</year>) <volume>224</volume>:<page-range>1962&#x2013;72</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1093/infdis/jiab235</pub-id>.</citation>
</ref>
<ref id="B69">
<label>69</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Scott</surname> <given-names>NR</given-names>
</name>
<name>
<surname>Swanson</surname> <given-names>RV</given-names>
</name>
<name>
<surname>Al-Hammadi</surname> <given-names>N</given-names>
</name>
<name>
<surname>Domingo-Gonzalez</surname> <given-names>R</given-names>
</name>
<name>
<surname>Rangel-Moreno</surname> <given-names>J</given-names>
</name>
<name>
<surname>Kriel</surname> <given-names>BA</given-names>
</name>
<etal/>
</person-group>. <article-title>S100A8/A9 regulates CD11b expression and neutrophil recruitment during chronic tuberculosis</article-title>. <source>J Clin Invest</source>. (<year>2020</year>) <volume>130</volume>:<page-range>3098&#x2013;112</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1172/JCI130546</pub-id>.</citation>
</ref>
<ref id="B70">
<label>70</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Guo</surname> <given-names>Q</given-names>
</name>
<name>
<surname>Zhao</surname> <given-names>Y</given-names>
</name>
<name>
<surname>Li</surname> <given-names>J</given-names>
</name>
<name>
<surname>Liu</surname> <given-names>J</given-names>
</name>
<name>
<surname>Yang</surname> <given-names>X</given-names>
</name>
<name>
<surname>Guo</surname> <given-names>X</given-names>
</name>
<etal/>
</person-group>. <article-title>Induction of alarmin S100A8/A9 mediates activation of aberrant neutrophils in the pathogenesis of COVID-19</article-title>. <source>Cell Host Microbe</source>. (<year>2021</year>) <volume>29</volume>:<page-range>222&#x2013;35</page-range>:<elocation-id>e224</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.1016/j.chom.2020.12.016</pub-id>.</citation>
</ref>
<ref id="B71">
<label>71</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Narumi</surname> <given-names>K</given-names>
</name>
<name>
<surname>Miyakawa</surname> <given-names>R</given-names>
</name>
<name>
<surname>Ueda</surname> <given-names>R</given-names>
</name>
<name>
<surname>Hashimoto</surname> <given-names>H</given-names>
</name>
<name>
<surname>Yamamoto</surname> <given-names>Y</given-names>
</name>
<name>
<surname>Yoshida</surname> <given-names>T</given-names>
</name>
<etal/>
</person-group>. <article-title>Proinflammatory proteins S100A8/S100A9 activate NK cells via interaction with RAGE</article-title>. <source>J Immunol</source>. (<year>2015</year>) <volume>194</volume>:<page-range>5539&#x2013;48</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.4049/jimmunol.1402301</pub-id>.</citation>
</ref>
<ref id="B72">
<label>72</label>
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Goyette</surname> <given-names>J</given-names>
</name>
<name>
<surname>Geczy</surname> <given-names>CL</given-names>
</name>
</person-group>. <article-title>Inflammation-associated S100 proteins: new mechanisms that regulate function</article-title>. <source>Amino Acids</source>. (<year>2011</year>) <volume>41</volume>:<page-range>821&#x2013;42</page-range>. doi:&#xa0;<pub-id pub-id-type="doi">10.1007/s00726-010-0528-0</pub-id>.</citation>
</ref>
</ref-list>
</back>
</article>