Abstract
Introduction:
Type I interferon (IFN-I) responses correlate with the severity of Tuberculosis. ISG15, a ubiquitin-like protein, regulates immune responses intracellularly by protein posttranslational modification (ISGylation) or extracellularly, in a cytokine-like manner. The ubiquitin specific protease USP18 deconjugates ISG15 from target proteins and is a major negative regulator of IFN-I signaling.
Methods:
Here we show that M. tuberculosis infection induces ISGylation associated transcripts in murine bone marrow-derived macrophages (BMM). The expression of these transcripts was dependent on IFN-I signaling and was further enhanced by supplementation of IFN-β, but not with IFN-γ or IL-1β. While stimulation with IFN-β impaired the intracellular control of M. tuberculosis in BMM, the bacterial growth was not altered by addition of extracellular ISG15. Treatment with two USP18 inhibitors, increased the ISGylation in uninfected and M. tuberculosis-infected BMM.
Results:
The inhibition of USP18 enhanced both IFN-I responses and antimicrobial responses in BMM and increased NF-κB-activation and IL-1β expression. Moreover, the USP18 inhibitors improved the intracellular macrophage control of M. tuberculosis in an IFN-I independent manner. Instead, the addition of IFN-β to the BMM cultures at least partially reversed the bacterial control conferred by the USP18 inhibition.
Discussion:
Our results suggest that the pharmacological inhibition of USP18 can be further explored as a therapeutic tool in the control of tuberculosis.
1 Introduction
Tuberculosis (TB), caused by the infection with Mycobacterium tuberculosis (M. tuberculosis), remains a leading public health problem worldwide. The worldwide incidence of TB is approximately 10 million new cases and 1.4 million deaths annually (https://www.who.int/news-room/fact-sheets/detail/tuberculosis). The rate of progression from infection to disease is highly variable. While approximately 90% of infected individuals never develop clinical disease, in 5-10% of cases M. tuberculosis causes an extensive lung damage and efficient airborne transmission of bacteria (1).
Macrophages are both the major host cell niche where M. tuberculosis survives and proliferates, and the cell where the bacilli can be controlled or eliminated. A host counters mycobacterial infections primarily via IFN-γ-mediated macrophage activation (2, 3). In contrast, the type I IFNs family (IFN-I) exacerbates clinical and experimental TB pathogenesis (4–6). IFN-I inhibits the production of the protective cytokine IL-1β by hampering inflammasome activation, and by inducing IL-10, which further reduced IL-1 and IFN-γ gene transcription (7), as well as responsiveness to IFNγ, leading to an initial loss of bacterial control. This action is amplified by neutrophil and plasmacytoid dendritic cells (PDC) activation (5). Although high levels of IFN-I have negative effects in TB, tonic IFN-I signaling or low IFN-I levels may exert beneficial effects by priming host-protective responses (8).
IFN-I mediates the production of numerous innate immune genes, including ISG15, a ubiquitin-like intracellular protein modifier and other molecules involved in the control of ISG15 production. ISG15 expression is upregulated in response to cellular stress caused by bacterial and viral infections (9).
The covalent modification of proteins by ISG15, termed ISGylation, occurs in more than 100 intracellular proteins. ISG15 is synthesized as an inactive precursor the C-terminal peptide cap needs to be removed, to expose the C-terminal glycine required for target binding (10). ISG15 is covalently conjugated onto target proteins via an enzymatic cascade, although the fate of these modified proteins is still largely unknown. However, protein ISGylation has been shown to disrupt the replication of several viruses (9, 11). Unlike ubiquitylation, ISGylation does not directly label the target proteins for proteosomal degradation. In addition to its intracellular conjugation, extracellular ISG15 has been proposed to function as a cytokine with several immunomodulatory activities (12).
The ISG15-binding motif is recognized by the ubiquitin-specific protease 18 (USP18), which removes ISG15 from the conjugated proteins (13). In contrast to other members of the USP family, USP18 shows no reactivity toward ubiquitin but specifically deconjugates ISG15 (14, 15).
All type I IFNs bind to a common cell-surface receptor, known as the type I IFN receptor, which is composed of two subunits, IFNAR1 and IFNAR2. These subunits are associated with the Janus activated kinases (JAKs) tyrosine kinase 2 and JAK1, respectively. Activation of the JAKs that are associated with the type I IFN receptor results in tyrosine phosphorylation of STAT2 and STAT1, leading to the formation of STAT1–STAT2–IRF9 trimers that translocate to the nucleus and binding to interferon-stimulated response elements (ISREs) in the DNA to initiate gene transcription (16). Mutational studies have shown that USP18 binds to the intracellular region of IFN-I receptor subunit IFNAR2 outcompeting the downstream kinase JAK1 and thereby abrogating IFN-I-signaling independent of the peptidase activity (17). Patients with USP18 deficiency showed a type I interferonopathy and predisposition to mycobacterial disease (18, 19). ISG15 interacts with USP18, protecting it from ubiquitylation and subsequent proteasomal degradation. In ISG15-deficient patients USP18 is thus degraded, resulting in a prolonged IFN-I signaling causing a type I interferonopathy and enhanced susceptibility to mycobacterial infection (20–22).
Here, we analyzed the immunological features of ISG15 and USP18 as intracellular regulators of the outcome of M. tuberculosis infection in macrophages. Our studies confirm that USP18 modulates IFN-I responses by reducing ISGylation. Moreover, we show that USP18 impairs the intracellular control of M. tuberculosis intracellular growth in macrophages through a mechanism that is independent of IFN-I signaling.
2 Materials and methods
2.1 Ethics
Mice were housed in accordance with the directives and guidelines of the Swedish Board of Agriculture, the Swedish Animal Protection Agency, and the Karolinska Institutet (djurskyddslagen 1988:534; djurskyddsförordningen 1988:539; djurskyddsmyndigheten DFS 2004:4). The study was performed under approval of the Stockholm North Ethical Committee on Animal Experiments permit number 1374–2020 and 2476-2025.
2.2 Generation of bone marrow-derived macrophages
Bone marrow cells were flushed from tibia and femurs with PBS, filtered through a 70μm cell strainer, resuspended in DMEM supplemented with 10% FCS and 30% L929 cell-conditioned medium (as a source of macrophage-colony stimulating factor) and incubated for 6 days at 37°C, 5% CO2. Bone marrow-derived macrophage (BMM) cultures were then washed with PBS, detached with 1%trypsin, and 5x105 cells were seeded per well in 24-well plates. BMM were further incubated for 24 h at 37°C before infections or treatment with diverse compounds. BMM were F4/80+, CD11b+, CD11c- and SiglecF- (23).
2.3 Infection of BMM with M. tuberculosis
M. tuberculosis Harlingen or H37Rv carrying the green fluorescent protein (GFP)-encoding pFPV2 plasmid were grown in Middlebrook 7H9 (Difco, Detroit, MI) supplemented with albumin, dextrose and catalase and quantified by densitometry. BMM were infected with sonicated bacteria at a multiplicity of infection (MOI) of 2 unless otherwise indicated. After 4 h, cells were washed twice with PBS to remove extracellular bacteria and further incubated for 1 to 5 days. The infected BMM were then detached using trypsin-EDTA at different times after infection, incubated with live/dead stain (LIVE/DEAD™ Fixable Yellow Dead Cell Stain, Invitrogen), washed with FACS buffer (PBS containing 0.5% FCS and 0.5 mM EDTA] and fixed with 4% formaldehyde (Sigma-Aldrich) at room temperature for 10 min. Data were acquired on a Sony ID7000 spectral cytometer and analyzed with FlowJo software (Tree star Inc., Ashland, OR). Cells within the mononuclear gate were ≥ 95% viable, and ≥ 95% of live cells are CD11b+ F4/80+. This procedure allowed the exclusion of Mtb associated with dead cells and enable determination of the frequency of infected cells and the relative levels of intracellular bacteria (23).
2.4 Small interfering RNA gene silencing
siRNA transfections were performed as previously described (24), using sequences listed in the Supplementary Table 2. BMM were plated at 1 × 106 cells per ml in 24-well plates overnight. On the day of transfection, the media was replaced with 450 μl DMEM without penicillin/streptomycin or L-929 cell supernatants. For each target gene, two tubes were prepared. Optimem (25 μl per well) was added to each tube. Lipofectamine RNAimax (2.5 μl per well; Invitrogen) was added to one tube and siRNA (50 nM per well, unless otherwise indicated) was added to the second tube. The siRNA was then combined with the vial with RNAimax, mixed well by pipetting and incubated for 15 min. Then, 50 μl of the mix was incubated with the BMM cell culture. Twenty-four hours after transfection, cells were infected or incubated with different molecules.
2.5 Real time PCR
Total RNA was extracted from lung samples or culture cells using Trizol (Sigma Aldrich) and cDNA was obtained by reverse transcription. Transcripts were quantified by real time PCR as described (25). Transcripts were quantified using hprt as a control house-keeping gene to calculate the ΔCt values for individual samples. The relative number of transcripts was calculated using the 2-(ΔΔCt) method. The primer sequences used are listed in Supplementary Table 1. These values were then used to calculate the relative expression of mRNA in the different conditions (infection and/or treatment) used in tissues and cells.
2.6 IL-1β measurement in culture supernatants
The concentration of IL-1β in BMM supernatants were quantified by ELISA, according to the manufacturer’s instructions (BioLegend ELISA MAX™ Standard Set Mouse IL-1β).
2.7 RNA sequencing
RNA was extracted from BMM, infected or not with M. tuberculosis and treated with the USP18 inhibitor 4 h before infection. RNA was isolated using the miRNeasy micro kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions and processed for sequencing at bioinformatics and expression analysis at Novogene Biotech Co (Cambridge, UK). The RNA quality was assessed on a 2200 TapeStation Instrument (Agilent, Santa Clara, CA). PolyA RNA selection was performed using the Illumina TruSeq RNA Sample Preparation Kit according to the manufacturer’s protocol. RNA-seq libraries were prepared and sequenced on the Illumina HiSeq 2000 platform. All the FASTQ files that passed QC were quantified by Salmon (26) using GRCm39 as the reference transcriptome. Differential gene expression analysis was performed with R (version 4.3.3) using the Limma package (27). Genes with adjusted p-value less than 0.05 were and fold change larger than 2 were identified as differentially expressed. All the FASTQ files that passed QC were quantified by Salmon using GRCm39 as the reference transcriptome (26). Differential gene expression analysis was performed with R (version 4.3.3) using the Limma package (27). Genes with adjusted p-value less than 0.05 were and fold change larger than 2 were identified as differentially expressed genes (DEGs). Gene Ontology enrichment analysis (Biological Process, Molecular Functions) or KEGG pathway enrichment was performed with WebGestalt (http://www.webgestalt.org) using default parameters. The raw and processed RNA sequencing data can be found at Gene Expression Omnibus (GEO) repository.
2.8 Small molecule inhibitors of USP18
The selection and development of the small molecular inhibitors of USP18 used 2K04 and BB7 have been recently described (28). Briefly, 2K04 was selected from a chemical library of candidate DUBs screened for the inhibition of the USP18 biochemical activity. BB7 is an optimized inhibitor derived by medicinal chemistry and NC is a stereoisomer of BB7 that shows no USP18 inhibition. The inhibitors formed covalent interactions with USP18, and were cell penetrating. The compounds demonstrated an exceptional specificity for murine USP18 across 41 deubiquitinases including 22 USPs (Ubiquitin Specific Proteases) from the related USP family of DUBs (28). Both compounds inhibited mUSP18 enzymatic activity with IC50 at the nanomolar range and inhibited the engagement of cellular USP18 with ISG15 at 1 μM. No BMM toxicity was observed at the concentrations used in our study.
2.9 Western blot
BMMs were lysed in RIPA buffer (25mM Tris-Cl pH 7.5, 50 mM NaCl, 0.5% NP40, 0.1% SDS, 0.5% DOC, 1mM DTT, 20mM NEM supplied with fresh Protease and Phosphatase inhibitors). A syringe with 29G needle was used to mechanically disrupt genomic DNA. Lysates were clarified at 10000 g for 15 min at 4°C. Protein concentration (DC Protein Assay Kit, Bio-Rad) was determined on the clear supernatants. Desired concentrations of cell lysates were denatured for 10 min at 95°C in loading buffer (NuPage 4X, Reducing Agent 10X, Invitrogen, Carlsbad, CA) and loaded in acrylamide Bis-Tris 4-12% gradient gels (Invitrogen, Carlsbad, CA). After transfer onto PVDF membranes (Merck Millipore, Bedford, MA) for 60 min at 0.34 A, the filters were blocked in TBS-T solution (50 mM Tris-Cl, 150 mM NaCl, 0.1% Tween-20 and 5% non-fat milk, pH 7,6), and incubated with specific primary antibodies (polyclonal rabbit anti-ISG15, monoclonal rabbit anti-phospho-IκBα pSer32, monoclonal rabbit anti-total IκBα), overnight at 4°C, following an incubation with the appropriate horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Proteins were visualized by chemiluminescence (ECL, GE Healthcare, Uppsala, Sweden), detected by the ChemiDoc MP Imaging System (Bio-Rad Laboratory, CA), and chemiluminescence intensity was analyzed only for non-saturated bands using the corresponding imaging analysis software, ImageJ version 5.0.
2.10 Recombinant mature and capped ISG15 production
The coding sequences for the full-length and the cap free (removing the C-terminal hexapeptide cap) mouse ISG15 protein (Uniprot Acc. Nr Q64339) were obtained as synthetic genes (Thermo Fisher, GeneArt). The coding sequences were cloned in the pNIC28Bsa4 expression vector (GenBank Acc. Nr. EF198106). The expression constructs contain an N-terminal hexahistidine tag and a tobacco etch virus (TEV) protease recognition site for affinity tag removal; the resulting products carry one extra N-terminal serine residue. The His6-tagged proteins were expressed in E. coli BL21(DE3). The cells were harvested by centrifugation, resuspended in 25 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole (pH 8.0), and lysed by 40 µg/mL lysozyme, 6 µg/mL DNase-I, and sonication. The clarified lysates were loaded onto a 0.5 mL Ni-NTA column (Thermo Scientific). The His6-tagged proteins were eluted by an increasing imidazole gradient.
The imidazole was removed by a PD10 desalting column (Cytiva). The protein preparations were treated with TEV protease for affinity tag removal. The His6-tagged TEV protease and un-cleaved proteins were removed using a Ni-NTA column. The tag-free proteins were concentrated using the Amicon concentrator device (Merck) with 3 kDa molecular weight cut-off and loaded on size exclusion chromatography column (Superdex-75, GE Healthcare). The mouse ISG15-capped (immature) was made available at 545 µM concentration, and that ISG15-uncapped (mature) at 1453 µM. The protein preps were aliquoted and stored at –80°C until use. The recombinant mouse ISG15 protein preparations were analyzed in SDS-PAGE and the by mass-spectrometry to control purity and identity (Supplementary Figure 4). The sequence coverage of the tryptic peptides from the preparation was 98% for the capped construct and 94,8% for the uncapped product, strongly indicating proper biological functions.
2.11 Statistical analysis
All in vitro assays were performed at least twice, each time using biological triplicates in each assay. Differences in transcript and protein levels and % infected cells were analyzed by unpaired Student’s t-test considering unequal variances (Welch’s test) or by one-way ANOVA using the Welch’s adjustment when comparing three or more groups. When more than two parameters (i.e infection and treatments) were analyzed a two-way ANOVA was applied. Statistical analyses were performed in Prism (GraphPad, La Jolla, CA).
3 Results
3.1 IFN-βincreases the expression of inflammatory and ISGylation-related genes in M. tuberculosis-infected BMM
Type I IFN responses, including several regulators of ISGylation, are increased in TB patients as well as in individuals who progress to active disease (4). In addition to being strongly induced by IFN-I, ISG15 has also been shown to be upregulated by viral and bacterial infections, LPS and retinoic acid (29–31). Therefore, we asked whether M. tuberculosis infection influences ISGylation in bone marrow-derived macrophages (BMM). Notably, upon infection, BMM showed a clear upregulation of the expression genes involved in the ISGylation pathway including isg15, usp18 and ube2l6, (Figures 1A–C). Similarly, analysis of previously published transcriptomic datasets revealed increased expression of ISGylation-related genes in M. tuberculosis-infected human monocyte derived macrophages, and alveolar macrophages, as well as bone marrow-derived macrophages (Supplementary Figure 1).
Figure 1
The effect of IFN-I supplementation on ISGylation responses of M. tuberculosis-infected BMM was evaluated. Stimulation with IFN-β increased the expression of the ISGylation-related transcripts in uninfected BMM and further increased their levels in M. tuberculosis-infected cells. Transcripts encoding for il1b, inos and tnf, immune mediators that contribute to intracellular M. tuberculosis control, were increased in infected BMM (Figures 1D–F). The addition of IFN-β further enhanced the levels of these transcripts in infected, but not in uninfected, BMM.
In contrast to the stimulatory effects of IFN-β on ISGylation-related transcripts, stimulation of infected BMM with IFN-γ or IL-1β cytokines that improve control of M. tuberculosis infection (2, 23, 32, 33), did not further increase isg15, usp18 and ube2l6 mRNA levels beyond those induced by the infection alone (Figures 1G–J, L-N). Both IFN-γ and IL-1β increased levels of inos and il1b mRNA expression (Figures 1K, O, P).
3.2 Pharmacological inhibition of USP18 improves the intracellular control of infection with M. tuberculosis
Stimulation of BMM with various concentrations of IFN-β resulted in higher intracellular levels of M. tuberculosis, as measured by an increased frequency of infected BMM and a greater bacteria density per infected BMM (Figures 2A, B).
Figure 2
ISG15 is synthesized as 161 aminoacid-long precursor that is rapidly processed into its mature 155 aminoacid form by proteolytic cleavage, thereby exposing the conjugation site at its C-terminus (34). The immature (capped) ISG15 its not conjugated to substrates but may upon secretion, act extracellularly in a cytokine-like manner (12, 21). We produced both mature and capped ISG15 and tested whether these could influence M. tuberculosis control in BMM. The intracellular bacterial levels in BMM treated with different concentrations of mature or immature ISG15 were similar (Figures 2C, D).
Next, we investigated whether USP18 modulates macrophage responses to M. tuberculosis infection. For this purpose, two USP18 inhibitors, BB7 and 2K04, were used. These compounds were selected from a small-molecule compound library through a deISGylase assay-based high-throughput screening (2K04) and further optimized through medicinal chemistry (BB7) (28). BMM were treated with BB7 or 2K04 along with a structurally related inactive control compound (NC), before and during M. tuberculosis infection. Treatment of infected BMM with either 1 or 10 μM of BB7 or 2K04 but not with NC resulted in increased ISGylation (Figures 2E, F). Both inhibitors increased ISGylation in uninfected BMM (Figures 2G, H). M. tuberculosis infection resulted in increased ISGylation in BMM relative to uninfected controls (Figures 2F, H). In some experiments incubation with NC showed slightly increased ISGylation levels, although to a lesser extent than BB7 or 2K04 (Figures 2F, H). Incubation with the inhibitors did not reduce BMM viability (Supplementary Figure 2A), consistent with previous observations in cell lines at higher concentrations (28).
We then tested whether USP18 inhibition by BB7 and 2K04 affected the control of M. tuberculosis infection in BMM. Cells treated with either BB7 or 2K04 showed improved control of intracellular M. tuberculosis when compared to BMM NC- treated controls, as indicated by the reduced frequency of infected BMM and the lower bacterial density per cell (Figures 3A, B). Coincubation of M. tuberculosis in axenic cultures with 10 μM USP18 inhibitors did not reduce the bacterial growth (Supplementary Figure 2B). Treatment of M. tuberculosis-infected BMM with either BB7 or 2K04 increased the levels of the IFN-I regulated usp18, isg15 and cxcl10 mRNA (Figures 3C–E), as well as il1b and inos (Figures 3F, G), which are transcriptionally regulated by NF-κB. The transcriptional activity of NF-κB is tightly repressed by binding to the IκBα inhibitor. Upon stimulation, IκBα is phosphorylated, leading to its the degradation and the release of NF-κB to activate gene transcription (35). Infection of BMM with M. tuberculosis increased IκBα phosphorylation, and treatment with the USP18 inhibitors further enhanced phospho-IκBα levels in infected BMM, demonstrating strong NF-κB activation by these inhibitors (Figures 3H, I).
Figure 3
3.3 Inflammasome activation and innate immune pathways are enriched in the transcriptome of M. tuberculosis infected BMM treated with USP18 inhibitor
We next compared the transcriptomic profiles of M. tuberculosis-infected BMM treated with BB7 or NC. A differential clustering among uninfected, M. tuberculosis (Mtb) and M. tuberculosis-BB7-treated BMM was revealed by PCA analysis and Pearson’s correlation, highlighting the variation in gene expression between the diverse groups (Figure 4A, Supplementary Figure 3A). We identified 710 differentially expressed genes (DEGs) between uninfected and Mtb BMM and 243 DEGs comparing Mtb-BB7 vs Mtb BMM, 129 of these DEGs were upregulated (Figures 4B, C, Supplementary Figure 3B).
Figure 4
Gene ontology (GO) analysis revealed enrichment of pathways related to antiviral responses, innate immune activation and inflammasome signaling when comparing Mtb-BB7 vs Mtb BMM (Figure 4D). Conversely, genes associated with stress responses, hypoxia and antioxidant activity were downregulated in Mtb-BB7 (Figure 4E). A detailed analysis of individual transcripts within these pathways is shown in Figures 4F–I and Supplementary Figures 3C, D. ISGylation-related genes usp18, ube2l6 and isg15 were detected among transcripts of GO “responses to virus” that were upregulated by BB7 treatment. Additionally, the genes inos, igtp, gbp2 and gbp5 which have been shown to contribute to the intracellular control of M. tuberculosis in macrophages (2, 36–38) were also increased in BB7-treated BMM (Supplementary Figure 3C). In contrast, expression of arg1, socs3, and nr4a1 mRNA, which might impair anti-M. tuberculosis responses by macrophages was downregulated following BB7 treatment (Supplementary Figure 3D).
Because the inflammasome pathway was upregulated in BB7-treated, M. tuberculosis-infected BMM, mature IL-1β levels in culture supernatants were measured as a readout of inflammasome activation. IL-1β levels were stimulated by the M. tuberculosis infection but not by BB7 treatment alone. However, the addition of BB7 to M. tuberculosis-infected BMM increased IL-1β levels as compared to untreated controls, indicating that the USP18 inhibitor enhances the inflammasome pathway during infection (Figure 4J).
3.4 USP18 inhibition improves the control of M. tuberculosis by BMM in a IFN-I independent manner
Silencing of USP18 by siRNA transfection, reduced the accumulation of usp18 mRNA in M. tuberculosis-infected BMM (Figure 5A). Conversely, levels of the ISGylation related transcript isg15 and ube2l6, as well as the IFN-I regulated genes ifit1 and cxcl10, were increased following usp18 silencing (Figures 5B–E). Similarly, usp18-silenced infected BMM displayed increased il1b and inos mRNA levels (Figures 5F, G), recapitulating the effect of pharmacological USP18 inhibition.
Figure 5
Then, we investigated whether IFN-I signaling contributes to the role of USP18 in M. tuberculosis infection of BMM. Silencing ifnar1, which encodes the IFN-I receptor α chain, reduced the accumulation of ifnar1 mRNA (Figure 5H) and decreased the expression of IFN-I regulated genes usp18, isg15, ube2l6 and ifit1 as compared with control siRNA-transfected BMM (Figures 5I–L), indicating that the IFN-I signal was hampered by the ifnar1 siRNA. Levels of il1b and inos mRNA were increased in ifnar1 siRNA transfected BMM as compared to those in BMM transfected with control siRNA (Figures 5M, N).
Next, we examinate whether ifn1ar silencing hindered the ability of USP18 inhibitors to control M. tuberculosis. Treatment with 2K04 reduced both the proportion of infected BMM and bacterial density per infected cell compared with controls (Figures 5O, P). Blocking IFN-I signaling through ifna1r siRNAs similarly decreased the intracellular bacterial load, and combining 2K04 treatment with ifna1r silencing did not alter the enhanced M. tuberculosis control mediated USP18 inhibition (Figures 5O, P). These results indicated that the improved control of the intracellular bacteria after USP18 inhibition is independent of IFN-I signaling.
The effect of IFN-β supplementation in combination with USP18 inhibition in the responses of M. tuberculosis-infected BMM was then evaluated. The levels of isg15 and usp18 mRNA in M. tuberculosis-infected BMM increased after the stimulation with IFN-β. These effects were further amplified upon IFN-β and 2K04 co-treatment (Figures 6A, B). Levels of il1b and inos transcripts were also higher in IFN-β-treated M. tuberculosis-infected BMM exposed to 2K04 than in NC-treated controls (Figures 6C, D). However, IFN-β stimulation partially impaired the improved bacterial control conferred by the USP18 inhibitor (Figures 6E, F). Thus, high levels of IFN-I combined increased the susceptibility to M. tuberculosis in BMM with impaired USP18 regulation.
Figure 6
Finally, we assessed whether stimulation with the TLR3 agonist polyIC, a potent inducer of IFN-I expression, would mimic the effects of IFN-β addition on USP18 inhibitor-treated BMM. PolyIC induced the accumulation isg15, usp18, ifit1 and cxcl10 transcripts in both uninfected and M. tuberculosis-infected BMM (Figures 6G–K). PolyIC also increased the levels of il1b and inos mRNA in both uninfected and infected BMM (Figures 6K, L), while IFN-β-induced the accumulation of i1b and inos mRNA in infected but not in uninfected BMM (Figures 1D–F). Moreover, the addition of polyIC reduced the intracellular density of M. tuberculosis as compared to untreated controls (Figures 6M, N). Combined treatment with both polyIC and 2K04 further decreased the intracellular bacterial levels in BMM relative to either treatment alone (Figures 6M, N).
4 Discussion
Here we shown that infection of BMM with M. tuberculosis stimulates the expression of genes involved in the ISGylation pathway. ISGylation-related transcripts were also increased in human and murine inflammatory and alveolar macrophages. Studies indicating that transcript and protein levels of ISGylation components in blood may serve as biomarkers for TB diagnosis further support our findings (39–41).
The addition of IFN-β stimulated the expression of ISGylation-related transcripts in uninfected BMM and enhanced their expression in M. tuberculosis-infected BMM. Conversely, ISGylation transcript levels in M. tuberculosis-infected BMM with defective IFN-I-signaling were reduced. The stimulation of BMM with either IFN-γ or IL-1β did not modulate the concentration of ISGylation-related transcripts. Interestingly, the addition of IFN-I increased the of il1b and inos mRNA in M. tuberculosis-infected-, but not in uninfected BMM. Paradoxically, silencing of IFN-I signaling also resulted in increased il1b and inos expression in BMM.
In addition to functioning as a protein modifier, ISG15 has also been shown to act extracellularly by stimulating IFN-γ secretion through binding to the LFA-1 surface receptor (12, 21). We found that the addition of ISG15 to the BMM cultures did not affect the intracellular M. tuberculosis growth. However, the possibility that ISG15 may act indirectly, through interactions between different immune cell populations, to influence the intracellular replication of M. tuberculosis in macrophages remains to be explored.
USP18 dampens IFN-I signaling preventing IFN-I-mediated inflammation that is observed in patients with USP18 deficiency (18, 19, 42). The inhibitory effect of USP18 on IFN-I signaling is independent of its isopeptidase activity, since control of IFN-I responses is preserved in mice with catalytically inactive USP18 (17, 43). This function is also independent of ISG15, as demonstrated in usp18-/-/isg15-/- mice (44). Mechanistically, USP18 interacts with STAT2, facilitating its recruitment to the IFNAR2 subunit of the IFN-I receptor and inhibiting receptor dimerization (45, 46). Distinct regions of USP18 mediate deISGylation and inhibition of IFN-I signaling (17). The inhibitors BB7 and 2K04 used in this study were selected based on their ability to covalently bind USP18 and impair its enzymatic activity to cleave ISG15-protein substrates (28). Here, we demonstrate that these inhibitors also hamper the negative regulatory function of USP18 on the IFN-I signaling.
Although the role of ISG15 in our model has not been elucidated, ISG15 is required for rescuing USP18 from S‐phase kinase‐associated protein 2‐mediated proteasomal degradation, a mechanism that explains the interferonopathies and increased susceptibility to environmental mycobacterial infections observed in patients with ISG15 deficiencies (22, 47). In contrast, isg15-/- or ube1l−/− mice did not show higher susceptibility to M. tuberculosis infection (48, 49), likely because the stabilizing effect of ISG15 on USP18 is not conserved in mice (20).
Patients with active tuberculosis have a characteristic IFN-I-inducible gene signature (4). While inhibition of USP18 increases the IFN-I responses in BMM, we here showed that the USP18 inhibition impairs the intracellular growth of M. tuberculosis in BMM in an IFN-I-independent manner.
Usp18-/- and usp18Ity9 mice, the latter with a mutation on USP18 that disrupts the IFN-I inhibitory function but not its enzymatic activity, are more susceptible to Salmonella infection (48, 50, 51). Similarly, usp18Ity9 also showed increased M. tuberculosis susceptibility. In contrast, usp18 and ifnar2-deficient mice showed improved control of L. monocytogenes (52).
Previous studies have provided evidence that USP18 can inhibit NF-κB activation by promoting deubiquitination of the TAK1–TAB1 complex or NEMO (53, 54). However, USP18 exhibits no reactivity toward ubiquitin in vitro, suggesting that the inhibition of the NF-κB pathway is indirect (17, 43). USP18-mediated NF-κB inhibition also protected mice from LPS-induced sepsis (55). Consistent with this, we showed that the inhibition of USP18 increased the expression of both IFN-I mediated but also of NF-κB-controlled inflammatory genes that have been shown to mediate the control of M. tuberculosis growth in macrophages. In agreement with the observed enrichment of inflammasome activation pathways, mature IL-1β was also secreted to the supernatants of M. tuberculosis infected BMM treated with the USP18 inhibitors. Similarly with our results, IL-1β is increased at both the transcript and protein levels in usp18-/- mice infected with Salmonella (48).
While being a potent IFN-I activator, polyIC has also been shown to also stimulate the JNK, p38 kinases and NF-kB mediated signaling pathways (56, 57). Accordingly, we found that stimulation with polyIC induced both IFN-I and inflammatory responses, reduced the M. tuberculosis levels in BMM and that co-incubation with both polyIC and a USP18 inhibitor further improved M. tuberculosis intracellular control.
Thus, while IFN-I responses are increased in USP18 deficient macrophages, an IFN-I independent inhibition of M. tuberculosis growth associated to the activation of NF-κB dependent immune molecules prevailed. Supporting this conclusion, C57BL/6 mice, used in this study, mount relatively weak IFN-I responses to M. tuberculosis, and deletion of ifnar1 in this background does not consistently alter pulmonary bacterial burden (58–60). It is also possible that additional IFN-I producing cells, such as plasmacytoid DCs are required to potentiate the detrimental role of IFN-I during in vivo M. tuberculosis infections (5). Although our findings may appear to contrast with previous reports showing that IFN-I limits pro–IL-1β availability and IL-1β maturation (7) and that elevated IFN-I inhibits M. tuberculosis-induced IL-1β mRNA expression in macrophages (61), the addition of IFN-β to M. tuberculosis inhibited the protective effect of the USP18 inhibitors on the control of the bacterial growth in macrophages.
Overall, our findings using small-molecule inhibitors that disrupt both the deISGylating and scaffold functions of USP18, reveal that USP18 limits the full potential of BMM to control the M. tuberculosis infection in an IFN-I independent manner (Figure 7).
Figure 7
Statements
Data availability statement
The data presented in the study are deposited in the GEO repository, accession number GSE320445.
Ethics statement
Our study used 2 different datasets of human macrophages infected with M. tuberculosis, GSE164287 linked to Front Immunol. 2021;12:685237 approved by the Johns Hopkins University Institutional Review Board and GSE189996 linked to Commun Biol. 2025;8(1):950, approved by Ohio State University Wexner Medical Center ethical board. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required from the participants or the participants’ legal guardians/next of kin in accordance with the national legislation and institutional requirements. The animal study was approved by Stockholm North Ethical Committee on Animal Experiments permit number 1374-2020 and 2476-2025. The study was conducted in accordance with the local legislation and institutional requirements.
Author contributions
QZ: Formal analysis, Investigation, Writing – review & editing. ZG: Formal analysis, Investigation, Writing – review & editing, Supervision. RS: Resources, Formal analysis, Conceptualization, Writing – review & editing, Investigation. YZ: Writing – review & editing, Formal analysis. PG: Writing – review & editing, Resources. VL: Resources, Writing – review & editing. JX: Project administration, Funding acquisition, Conceptualization, Writing – review & editing. SG: Conceptualization, Investigation, Resources, Writing – review & editing, Data curation, Formal analysis. MR: Funding acquisition, Writing – original draft, Formal analysis, Project administration, Supervision, Conceptualization, Writing – review & editing, Data curation.
Funding
The author(s) declared that financial support was received for this work and/or its publication. This study was supported by the Swedish Heart and Lung foundation 2021-23/20200697, the Swedish Research Council 2019–01691 and 2019-04725, the Swedish Institute for Internationalization of Research and Higher Education (STINT) IMG 2021-9019, the Chinese Scholarship Council and the Karolinska Institutet to MR. We also acknowledge support by the ERC Consolidator Grant 3DMASH (101170408), the Swedish Research Council (2021-02801, 2023–03015 and 2024-03401) and the Robert Bosch Foundation, Stuttgart, Germany to VL.
Acknowledgments
We thank the expert help of the staff of Dept of Comparative Medicine, Karolinska Institutet, and to Dr Maria Johansson at the FACS facility Karolinska Institutet. and the comments from Dr Ruining Liu and Hanxiong Li on the manuscript and experimental details and set up. The M. tuberculosis-gfp bacteria were provided by Dr Susanna Brighenti, Department of Medicine Huddinge.
Conflict of interest
VL is CEO and shareholder of HepaPredict AB, as well as co-founder and shareholder of Shanghai Hepo Biotechnology Ltd.
The remaining author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The author MR 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.
Generative AI statement
The author(s) declared that generative AI was not used in the creation of this manuscript.
Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.
Publisher’s note
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Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2026.1739628/full#supplementary-material
References
1
SossenBRichardsASHeinsohnTFrascellaBBalzariniFOradini-AlacreuAet al. The natural history of untreated pulmonary tuberculosis in adults: a systematic review and meta-analysis. Lancet Respir Med. (2023) 11:367–79. doi: 10.1016/S2213-2600(23)00097-8
2
FlynnJLChanJTrieboldKJDaltonDKStewartTABloom.BR. An essential role for interferon gamma in resistance to Mycobacterium tuberculosis infection. J Exp Med. (1993) 178:2249–54. doi: 10.1084/jem.178.6.2249
3
CooperAMDaltonDKStewartTAGriffinJPRussellDGOrme.IM. Disseminated tuberculosis in interferon gamma gene-disrupted mice. J Exp Med. (1993) 178:2243–7. doi: 10.1084/jem.178.6.2243
4
BerryMPGrahamCMMcNabFWXuZBlochSAOniTet al. An interferon-inducible neutrophil-driven blood transcriptional signature in human tuberculosis. Nature. (2010) 466:973–7. doi: 10.1038/nature09247
5
KotovDILeeOVFattingerSALangnerCAGuillenJVPetersJMet al. Early cellular mechanisms of type I interferon-driven susceptibility to tuberculosis. Cell. (2023) 186:5536–53.e5522. doi: 10.1016/j.cell.2023.11.002
6
VanceRE. Tuberculosis as an unconventional interferonopathy. Curr Opin Immunol. (2025) 92:102508. doi: 10.1016/j.coi.2024.102508
7
GuardaGBraunMStaehliFTardivelAMattmannCForsterIet al. Type I interferon inhibits interleukin-1 production and inflammasome activation. Immunity. (2011) 34:213–23. doi: 10.1016/j.immuni.2011.02.006
8
Moreira-TeixeiraLMayer-BarberKSherAO’Garra.A. Type I interferons in tuberculosis: Foe and occasionally friend. J Exp Med. (2018) 215:1273–85. doi: 10.1084/jem.20180325
9
PerngYCLenschowDJ. ISG15 in antiviral immunity and beyond. Nat Rev Microbiol. (2018) 16:423–39. doi: 10.1038/s41579-018-0020-5
10
ZhaoCCollinsMNHsiangTYKrug.RM. Interferon-induced ISG15 pathway: an ongoing virus-host battle. Trends Microbiol. (2013) 21:181–6. doi: 10.1016/j.tim.2013.01.005
11
ZhangMLiJYanHHuangJWangFLiuTet al. ISGylation in innate antiviral immunity and pathogen defense responses: A review. Front Cell Dev Biol. (2021) 9:788410. doi: 10.3389/fcell.2021.788410
12
SwaimCDScottAFCanadeoLAHuibregtse.JM. Extracellular ISG15 signals cytokine secretion through the LFA-1 integrin receptor. Mol Cell. (2017) 68:581–90.e585. doi: 10.1016/j.molcel.2017.10.003
13
MalakhovMPMalakhovaOAKimKIRitchieKJZhang.DE. UBP43 (USP18) specifically removes ISG15 from conjugated proteins. J Biol Chem. (2002) 277:9976–81. doi: 10.1074/jbc.M109078200
14
BastersAGeurinkPPRockerAWittingKFTadayonRHessSet al. Structural basis of the specificity of USP18 toward ISG15. Nat Struct Mol Biol. (2017) 24:270–8. doi: 10.1038/nsmb.3371
15
BastersAGeurinkPPEl OualidFKetscherLCasuttMSKrauseEet al. Molecular characterization of ubiquitin-specific protease 18 reveals substrate specificity for interferon-stimulated gene 15. FEBS J. (2014) 281:1918–28. doi: 10.1111/febs.12754
16
PlataniasLC. Mechanisms of type-I- and type-II-interferon-mediated signalling. Nat Rev Immunol. (2005) 5:375–86. doi: 10.1038/nri1604
17
MalakhovaOAKimKILuoJKZouWKumarKGFuchsSYet al. UBP43 is a novel regulator of interferon signaling independent of its ISG15 isopeptidase activity. EMBO J. (2006) 25:2358–67. doi: 10.1038/sj.emboj.7601149
18
MeuwissenMESchotRButaSOudesluijsGTinschertSSpeerSDet al. Human USP18 deficiency underlies type 1 interferonopathy leading to severe pseudo-TORCH syndrome. J Exp Med. (2016) 213:1163–74. doi: 10.1084/jem.20151529
19
Martin-FernandezMButaSLe VoyerTLiZDynesenLTVuillierFet al. A partial form of inherited human USP18 deficiency underlies infection and inflammation. J Exp Med. (2022) 219. doi: 10.1084/jem.20211273
20
SpeerSDLiZButaSPayelle-BrogardBQianLVigantFet al. ISG15 deficiency and increased viral resistance in humans but not mice. Nat Commun. (2016) 7:11496. doi: 10.1038/ncomms11496
21
BogunovicDByunMDurfeeLAAbhyankarASanalOMansouriDet al. Mycobacterial disease and impaired IFN-gamma immunity in humans with inherited ISG15 deficiency. Science. (2012) 337:1684–8. doi: 10.1126/science.1224026
22
ZhangXBogunovicDPayelle-BrogardBFrancois-NewtonVSpeerSDYuanCet al. Human intracellular ISG15 prevents interferon-alpha/beta over-amplification and auto-inflammation. Nature. (2015) 517:89–93. doi: 10.1038/nature13801
23
LiHLiuRKathamuthuGRGenchevaRGongZScholzATet al. The inhibition of TXNRD1 by methylglyoxal impairs the intracellular control of Mycobacterium tuberculosis. Redox Biol. (2025) 85:103741. doi: 10.1016/j.redox.2025.103741
24
MillsELRyanDGPragHADikovskayaDMenonDZaslonaZet al. Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1. Nature. (2018) 556:113–7. doi: 10.1038/nature25986
25
CarowBYeXGavier-WidenDBhujuSOehlmannWSinghMet al. Silencing suppressor of cytokine signaling-1 (SOCS1) in macrophages improves Mycobacterium tuberculosis control in an interferon-gamma (IFN-gamma)-dependent manner. J Biol Chem. (2011) 286:26873–87. doi: 10.1074/jbc.M111.238287
26
PatroRDuggalGLoveMIIrizarryRAKingsford.C. Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods. (2017) 14:417–9. doi: 10.1038/nmeth.4197
27
RitchieMEPhipsonBWuDHuYLawCWShiWet al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. (2015) 43:e47. doi: 10.1093/nar/gkv007
28
KooijRPolVGanJvan DoodewaerdBRSapmazACampos AlonsoMet al. High-throughput synthesis and screening of a cyanimide library identifies selective inhibitors of ISG15-specific protease mUSP18. Angew Chem Int Ed Engl. (2025), e202510941. doi: 10.1002/anie.202510941
29
YuanWKrugRM. Influenza B virus NS1 protein inhibits conjugation of the interferon (IFN)-induced ubiquitin-like ISG15 protein. EMBO J. (2001) 20:362–71. doi: 10.1093/emboj/20.3.362
30
RadoshevichLImpensFRibetDQueredaJJNam ThamTNahoriMAet al. ISG15 counteracts Listeria monocytogenes infection. Elife. (2015) 4. doi: 10.7554/eLife.06848
31
MalakhovaOMalakhovMHetheringtonCZhang.DE. Lipopolysaccharide activates the expression of ISG15-specific protease UBP43 via interferon regulatory factor 3. J Biol Chem. (2002) 277:14703–11. doi: 10.1074/jbc.M111527200
32
FremondCMTogbeDDozERoseSVasseurVMailletIet al. IL-1 receptor-mediated signal is an essential component of MyD88-dependent innate response to Mycobacterium tuberculosis infection. J Immunol. (2007) 179:1178–89. doi: 10.4049/jimmunol.179.2.1178
33
Mayer-BarberKDAndradeBBOlandSDAmaralEPBarberDLGonzalesJ3rdSherAet al. Host-directed therapy of tuberculosis based on interleukin-1 and type I interferon crosstalk. Nature. (2014) 511:99–103. doi: 10.1038/nature13489
34
PotterJLNarasimhanJMende-MuellerLHaas.AL. Precursor processing of pro-ISG15/UCRP, an interferon-beta-induced ubiquitin-like protein. J Biol Chem. (1999) 274:25061–8. doi: 10.1074/jbc.274.35.25061
35
MathesEO’DeaELHoffmannAGhosh.G. NF-kappaB dictates the degradation pathway of IkappaBalpha. EMBO J. (2008) 27:1357–67. doi: 10.1038/emboj.2008.73
36
KimBHShenoyARKumarPDasRTiwariSMacMicking.JD. A family of IFN-gamma-inducible 65-kD GTPases protects against bacterial infection. Science. (2011) 332:717–21. doi: 10.1126/science.1201711
37
TaylorGACollazoCMYapGSNguyenKGregorioTATaylorLSet al. Pathogen-specific loss of host resistance in mice lacking the IFN-gamma-inducible gene IGTP. Proc Natl Acad Sci U.S.A. (2000) 97:751–5. doi: 10.1073/pnas.97.2.751
38
MacMickingJDNorthRJLaCourseRMudgettJSShahSKNathan.CF. Identification of nitric oxide synthase as a protective locus against tuberculosis. Proc Natl Acad Sci U.S.A. (1997) 94:5243–8. doi: 10.1073/pnas.94.10.5243
39
GaoJLiCLiWChenHFuYYi.Z. Increased UBE2L6 regulated by type 1 interferon as potential marker in TB. J Cell Mol Med. (2021) 25:11232–43. doi: 10.1111/jcmm.17046
40
GongZGuYXiongKNiuJZhengRSuBet al. The evaluation and validation of blood-derived novel biomarkers for precise and rapid diagnosis of tuberculosis in areas with high-TB burden. Front Microbiol. (2021) 12:650567. doi: 10.3389/fmicb.2021.650567
41
OttenhoffTHDassRHYangNZhangMMWongHESahiratmadjaEet al. Genome-wide expression profiling identifies type 1 interferon response pathways in active tuberculosis. PLoS One. (2012) 7:e45839. doi: 10.1371/journal.pone.0045839
42
GruberCMartin-FernandezMAilalFQiuXTaftJAltmanJet al. Homozygous STAT2 gain-of-function mutation by loss of USP18 activity in a patient with type I interferonopathy. J Exp Med. (2020) 217. doi: 10.1084/jem.20192319
43
KetscherLHannssRMoralesDJBastersAGuerraSGoldmannTet al. Selective inactivation of USP18 isopeptidase activity in vivo enhances ISG15 conjugation and viral resistance. Proc Natl Acad Sci U.S.A. (2015) 112:1577–82. doi: 10.1073/pnas.1412881112
44
KnobelochKPUtermohlenOKisserAPrinzMHorak.I. Reexamination of the role of ubiquitin-like modifier ISG15 in the phenotype of UBP43-deficient mice. Mol Cell Biol. (2005) 25:11030–4. doi: 10.1128/MCB.25.24.11030-11034.2005
45
ArimotoKILochteSStonerSABurkartCZhangYMiyauchiSet al. STAT2 is an essential adaptor in USP18-mediated suppression of type I interferon signaling. Nat Struct Mol Biol. (2017) 24:279–89. doi: 10.1038/nsmb.3378
46
WilmesSBeutelOLiZFrancois-NewtonVRichterCPJanningDet al. Receptor dimerization dynamics as a regulatory valve for plasticity of type I interferon signaling. J Cell Biol. (2015) 209:579–93. doi: 10.1083/jcb.201412049
47
VuillierFLiZCommerePHDynesenLTPellegrini.S. USP18 and ISG15 coordinately impact on SKP2 and cell cycle progression. Sci Rep. (2019) 9:4066. doi: 10.1038/s41598-019-39343-7
48
DauphineeSMRicherEEvaMMMcIntoshFPaquetMDangoorDet al. Contribution of increased ISG15, ISGylation and deregulated type I IFN signaling in Usp18 mutant mice during the course of bacterial infections. Genes Immun. (2014) 15:282–92. doi: 10.1038/gene.2014.17
49
KimmeyJMCampbellJAWeissLAMonteKJLenschowDJStallings.CL. The impact of ISGylation during Mycobacterium tuberculosis infection in mice. Microbes Infect. (2017) 19:249–58. doi: 10.1016/j.micinf.2016.12.006
50
RicherEPrendergastCZhangDEQureshiSTVidalSMMalo.D. N-ethyl-N-nitrosourea-induced mutation in ubiquitin-specific peptidase 18 causes hyperactivation of IFN-alphass signaling and suppresses STAT4-induced IFN-gamma production, resulting in increased susceptibility to Salmonella typhimurium. J Immunol. (2010) 185:3593–601. doi: 10.4049/jimmunol.1000890
51
KimKIMalakhovaOAHoebeKYanMBeutlerBZhang.DE. Enhanced antibacterial potential in UBP43-deficient mice against Salmonella typhimurium infection by up-regulating type I IFN signaling. J Immunol. (2005) 175:847–54. doi: 10.4049/jimmunol.175.2.847
52
ShaabaniNHonkeNNguyenNHuangZArimotoKILazarDet al. The probacterial effect of type I interferon signaling requires its own negative regulator USP18. Sci Immunol. (2018) 3. doi: 10.1126/sciimmunol.aau2125
53
LiuXLiHZhongBBlonskaMGorjestaniSYanMet al. USP18 inhibits NF-kappaB and NFAT activation during Th17 differentiation by deubiquitinating the TAK1-TAB1 complex. J Exp Med. (2013) 210:1575–90. doi: 10.1084/jem.20122327
54
YangZXianHHuJTianSQinYWangRFet al. USP18 negatively regulates NF-kappaB signaling by targeting TAK1 and NEMO for deubiquitination through distinct mechanisms. Sci Rep. (2015) 5:12738. doi: 10.1038/srep12738
55
HuBGeCZhu.C. Ubiquitin-specific peptidase 18 negatively regulates and inhibits lipopolysaccharide-induced sepsis by targeting transforming growth factor-beta-activated kinase 1 activity. Int Immunol. (2021) 33:461–8. doi: 10.1093/intimm/dxab029
56
JiangZMakTWSenGLi.X. Toll-like receptor 3-mediated activation of NF-kappaB and IRF3 diverges at Toll-IL-1 receptor domain-containing adapter inducing IFN-beta. Proc Natl Acad Sci U.S.A. (2004) 101:3533–8. doi: 10.1073/pnas.0308496101
57
MeylanEBurnsKHofmannKBlancheteauVMartinonFKelliherMet al. RIP1 is an essential mediator of Toll-like receptor 3-induced NF-kappa B activation. Nat Immunol. (2004) 5:503–7. doi: 10.1038/ni1061
58
McNabFWEwbankJRajsbaumRStavropoulosEMartirosyanARedfordPSet al. TPL-2-ERK1/2 signaling promotes host resistance against intracellular bacterial infection by negative regulation of type I IFN production. J Immunol. (2013) 191:1732–43. doi: 10.4049/jimmunol.1300146
59
StanleySAJohndrowJEManzanilloPCox.JS. The Type I IFN response to infection with Mycobacterium tuberculosis requires ESX-1-mediated secretion and contributes to pathogenesis. J Immunol. (2007) 178:3143–52. doi: 10.4049/jimmunol.178.5.3143
60
Moreira-TeixeiraLRedfordPSStavropoulosEGhilardiNMaynardCLWeaverCTet al. T cell-derived IL-10 impairs host resistance to mycobacterium tuberculosis infection. J Immunol. (2017) 199:613–23. doi: 10.4049/jimmunol.1601340
61
NovikovACardoneMThompsonRShenderovKKirschmanKDMayer-BarberKDet al. Mycobacterium tuberculosis triggers host type I IFN signaling to regulate IL-1beta production in human macrophages. J Immunol. (2011) 187:2540–7. doi: 10.4049/jimmunol.1100926
Summary
Keywords
ISG15, ISGylation, macrophage, Mycobacterium tuberculosis, type I interferon, USP18
Citation
Zhang Q, Gong Z, Schnell R, Zhong Y, Geurink PP, Lauschke VM, Xie J, Gastaldello S and Rottenberg ME (2026) Pharmacological inhibition of USP18 improves antibacterial responses and the intracellular control of Mycobacterium tuberculosis in macrophages. Front. Immunol. 17:1739628. doi: 10.3389/fimmu.2026.1739628
Received
04 November 2025
Revised
05 February 2026
Accepted
17 February 2026
Published
04 March 2026
Volume
17 - 2026
Edited by
Mohd Shariq, Gandhi Institute of Technology and Management (GITAM), India
Reviewed by
Qishun Feng, Affiliated Hospital of Guizhou Medical University, China
Meetu Agarwal, Jamia Hamdard University, India
Updates
Copyright
© 2026 Zhang, Gong, Schnell, Zhong, Geurink, Lauschke, Xie, Gastaldello and Rottenberg.
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.
*Correspondence: Martin E. Rottenberg, martin.rottenberg@ki.se
†These authors share senior authorship
Disclaimer
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.