Tuberculosis (TB) is an airborne communicable infection primarily caused by Mycobacterium tuberculosis (MTB) (Pai et al., 2016). Despite the advent of antibiotics and the Mycobacterium Bovis bacillus Calmette-Guérin (BCG) vaccine, millions of individuals are infected with TB each year (O'Garra et al., 2013). TB incidence is growing in part due to multidrug-resistant strains and to high rates of human immunodeficiency virus (HIV) co-infection (Sayes et al., 2012). In addition, the BCG vaccine is thought to be less effective as a means of maintaining long-term immunity in adults as compared to children (Yang et al., 2016), underscoring the importance of developing novel immunological approaches to preventing or treating this disease.

PE/PPE proteins containing highly conserved N-terminal Pro-Glu (PE) or Pro-Pro-Glu (PPE) motifs comprise approximately 10% of the MTB genome (Cole et al., 1998), and are key targets for vaccine development. The 99 and 69 characterized pe and ppe genes, respectively, contain extensive repetitive domains and a high GC content (Brennan, 2017). Comparative genomic analyses of mycobacterial species have shown that these PE/PPE proteins are primarily present in slower-growing mycobacterial pathogens (Cole, 2002), with the majority being proteins expressed on the cell surface or released into the extracellular space (Sampson, 2011; Mitra et al., 2017). These proteins are thought to be related to mycobacterial virulence, growth, and modulation or evasion of host immune responses (Vordermeier et al., 2012; Saini et al., 2016). PPE57 (Rv3425) is a PPE family protein encoded within the open reading frame in RD1 of M. tuberculosis H37Rv (Behr et al., 1999; Xu et al., 2015). Previous studies have indicated that Rv3425 was able to increase the production of IFN-γ in TB patients than in the healthy controls (Zhang et al., 2007; Chen et al., 2009). PPE57 can interact with Toll-like receptor 2 (TLR2) on macrophages to induce their activation and can promote type 1 helper T cell (Th1) polarization, while recombinant BCG-PPE57 can enhance protective immunity against MTB infection (Xu et al., 2015). Owing to its well-documented effects on human immune cells, PPE57 may thus alter the transcriptional profiles of exposed human immune cells.

Peripheral blood mononuclear cells (PBMCs) collected via venipuncture contain all of the key immune cells necessary for the coordination of MTB defenses. PBMCs can serve as sensitive biomarkers capable of differentiation between active TB infections and healthy individuals (Qian et al., 2016; Roe et al., 2016). RNA sequencing (RNA-seq) is a high-throughput technique to assess the full transcriptome of samples to identify gene expression in a manner more comprehensive than traditional microarray analyses (Stark et al., 2019). Blood transcriptomic profiling is frequently conducted to evaluate host immune responses to particular stimulants or pathogens (Wang et al., 2016; Fang et al., 2020). Herein, we employed an RNA-seq approach to identify genes that were differentially expressed in PBMCs upon stimulation with PPE57 to better understand global host-specific mechanisms related to the immune response in the context of TB infection.

PE/PPE family proteins are thought to serve as key mediators of MTB pathogenicity and immunogenicity (Sampson, 2011), yet the mechanisms whereby they affect host cells remain incompletely understood. A comprehensive analysis is thus essential in order to fully elucidate the complex responses of the host immune system to these mycobacterial antigens. As such, transcriptomic and bioinformatic analyses are of value as they offer a thorough understanding of the host defense mechanisms and immune evasion strategies associated with particular infections (Zhao et al., 2017; Wang et al., 2019; Fang et al., 2020). Several studies have explored the immune effect of PPE57 exposure (Zhang et al., 2007; Wang et al., 2008; Xu et al., 2015), but further work is needed to clarify host-pathogen interactions in this context. Relative to studies of ESAT-6/CFP-10, little is known regarding the biological impact of PPE57 exposure in PBMCs. Therefore, RNA-seq was performed in this study to understand the mechanisms underlying such interactions in host PBMCs.

A total of 1367 DEGs were identified in PBMCs following PPE57 stimulation. These DEGs were further subjected to bioinformatic analyses that seek to explore the pathways/mechanisms whereby they may regulate responses to this mycobacterial protein. Overall, we discovered that changes in the DEGs expression enriched in immune-related pathways including the type I IFN signaling pathway. Indeed, this signaling pathway was the most significantly enriched in our RNA-seq dataset, with 14 DEGs identified therein including ISG15, MX2, IRF9, IFIT3, IFIT2, OAS3, IFIT1, IFI6, OAS2, OASL, RSAD2, OAS1, IRF7, and MX1.

Type I IFN production can drive the upregulation and activation of a range of well-characterized antiviral genes, which stimulate or suppress immune function in a manner that can be either protective or detrimental, depending on the context in which it occurs. Type I IFN signaling is also against some bacteria, such as Escherichia coli, pneumococci, and group B streptococci (Mancuso et al., 2007). In this study, GO terms related to IFNs were significantly enriched in PBMCs following PPE57 stimulation, including the type I IFN signaling and response to IFNγ pathways. Type I IFN signaling was considered to be of particular importance, given that recent transcriptional analyses have highlighted a potentially deleterious role for type I IFNs in TB (Moreira-Teixeira et al., 2018). In a recent study conducted by Zhang et al. (2021), type I IFN signaling was identified as a critical mediator of Mtb-induced macrophage cell death, and it is one of the first specific mechanisms of Mtb-infected macrophage death to have been identified (Zhang et al., 2021). Yoshida et al. (1988) reported that type I IFNs were able to disrupt macrophage activation through type II IFN (IFN-γ), which is required for the control of TB in humans (Yoshida et al., 1988; Jouanguy et al., 1997). In vitro studies of the MTB infection of bone marrow-derived macrophages have revealed that type I IFN signaling is essential for the early production of inducible nitric oxide synthase (Shi et al., 2005), which is critical for defending against MTB. In clinical trials, type I IFNs have also been shown to be beneficial against pulmonary TB (Giosué et al., 1998). Therefore, it is difficult to define a clear role for this cytokine in the context of MTB infection despite its importance in many other immunological settings. A previous study suggested that PPE57 may be able to enhance protective responses against MTB by including macrophage activation through the mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) signaling pathways (Xu et al., 2015). In our study, PPE57 was able to stimulate type I IFN signaling, which may, in turn, be linked to MTB virulence and increased host susceptibility. A detailed examination of the type I IFN signaling pathway as it pertains to PPE57 may thus contribute to the design of host-directed therapies against MTB infection, although further validation will be required to confirm these results.

The type I IFN pathway is activated by the binding of this cytokine to the type I IFN receptor (IFNAR1 and IFNAR2) and the subsequent activation of the JAK/STAT signaling pathway, leading to the induction of about 300 interferon-stimulated genes (ISGs). Several of these ISGs have been found to be upregulated during MTB infection (Remoli et al., 2002; Chaussabel et al., 2003).

The expression of interferon-alpha inducible protein 6 (IFI6) has not been reported in the context of TB. In our study, IFI6 was found to be upregulated during bacterial infection. IFI6 encodes a mitochondrial ISG with immunomodulatory and anti-apoptotic activity (Porter et al., 1988). IFI6 is mainly localized inside the nucleus and restricts HBV gene expression and replication in vitro and in vivo (Sajid et al., 2021). The anti-apoptotic activity of IFI6 has been observed in breast cancer cells, gastric cancer cells, vascular endothelial cells, and human myeloma cells (Tahara et al., 2005; Cheriyath et al., 2007; Cheriyath et al., 2012; Qi et al., 2015). However, such pro-survival activity is also critical for MTB resistance in macrophages. As such, we hypothesized that PPE57 was able to induce the upregulation of IFI6 whereupon it may play an anti-apoptotic role in the context of TB via regulating JAK/STAT signaling or related pathways, although further validation will be necessary to test this hypothesis.

During MTB infection, we found that ISG15 was one of the most highly induced ISGs. Previous studies have shown that humans with an ISG15 deficiency are susceptible to mycobacterial disease, and secreted ISG15 can promote IFN-γ secretion (Bogunovic et al., 2012). ISG15 mRNA levels have also been identified as a potentially valuable biomarker in the context of active human TB (Dos Santos et al., 2018), as ISG15 can promote early MTB replication although it plays protective roles during the later stages of infection (Kimmey et al., 2017). Other ISGs with known antimicrobial roles (RSAD2, IFIT1, IFIT2, IFIT3, MX1, and MX2) were also highly upregulated in this study (Diamond, 2014; Helbig and Beard, 2014; Haller et al., 2015). To the best of our knowledge, the precise mechanisms by which many of these genes are upregulated during TB disease progression remain to be defined (Andreu et al., 2017). However, we have herein shown that PPE57 can promote the upregulation of these genes, and further research regarding their ability to shape host defense responses against MTB is warranted.

The 2’-5’-oligoadenylate synthetases3 (OAS3) exhibited the highest fold-change value of the 14 IFN-related DEGs in this study. The IFN-inducible gene profile includes both IFN-αβ and IFN-γ, as well OAS1-3, and 2’-5’-oligoadenylate synthetases-like (OASL), all of which can be regulated by both type I and type II IFNs (Zhou et al., 1997). As key ISGs, OAS1, OAS2, and OAS3 are evolutionarily conserved and are associated with the early inflammatory response during infection (Justesen et al., 2000; Yao et al., 2019). Transcriptomic analyses of blood from patients with active TB indicate that the upregulation of OAS1, OAS2, and OAS3 can differentiate between active and latent TB infection status (Berry et al., 2010; Maertzdorf et al., 2012; Ottenhoff et al., 2012). OAS1, OAS2, OAS3, and OASL expression levels are also directly linked to mycobacterial pathogenicity during the early post-infection period, as they can restrict MTB intracellular replication in macrophages by regulating cytokine secretion (Leisching et al., 2019; Leisching et al., 2020). As such, these ISGs may be involved in both promoting cellular survival and directly suppressing mycobacterial growth. Herein, we found that PPE57 exposure was sufficient to induce the upregulation of OAS1-3 and OASL in PBMCs, potentially indicating that PPE57 may indirectly induce anti-MTB effects related to the cell wall and the entrance of these bacteria into macrophages (Xu et al., 2015).

PPE proteins are able to readily induce host immune responses and are effective immunogens (Brennan, 2017). PPE18, for example, has already been used in completed phase IIb clinical trials of the M72/AS01 vaccine candidate (Day et al., 2013; Tait et al., 2019), and PPE42 has been used in a phase Ia trial of the ID93/GLA-SE, a subunit vaccine candidate that has exhibited good protection in animal models and has been shown reliable safety and robust immunogenicity (Coler et al., 2018; Khoshnood et al., 2018; Kwon et al., 2019). Unlike these two PPE proteins, the understanding pertaining to PPE57 is relatively limited, prompting the present study. Rv3425 (PPE57), which is also a promising protein for the design of mycobacterial vaccines, is herein reported as an MTB antigen associated with ISG induction for the first time. Prior studies have shown that other PPE proteins such as PPE26 and PPE39 can also affect the IFN signaling pathways (Su et al., 2015; Choi et al., 2019). Specifically, PPE39 is capable of eliciting dendritic cell (DC) activation in a TLR4-dependent manner through downstream MAPK and NF-κB signaling pathways, promoting Th1-cell polarization and increased IFN-γ production (Choi et al., 2019). PPE57 can activate the MAPK and NF-κB signaling pathways via TLR2 signaling (Xu et al., 2015). In our study, we found that PPE57 was able to affect the MAPK, JAK/STAT, and NF-κB pathways, potentially following type I IFN pathway activation. Therefore, we hypothesized that the type I IFN pathway may be activated by the binding of PPE57 to the type I IFN receptor (IFNAR1 and IFNAR2), subsequently regulating the activation of ISGs and other signaling pathways. PPE57 was also able to influence RLR pathway signaling, which controls type I IFN signaling pathway activation (Hou et al., 2021). RLRs include RIG-I, MDA5, and LGP2, all of which play important roles in the recognition of viral RNA in the cytoplasm (Bruns and Horvath, 2014). MTB is an intracellular parasite, and it is thus possible that PPE57 may bind to RLRs to regulate type I IFN signaling. Future studies testing these possibilities through antibody blocking experiments or related approaches are warranted.

PPE26 also induces TLR2-dependent macrophage activation and IFN-γ secretion (Su et al., 2015). Host proteomic changes in response to PPE26 stimulation have previously been analyzed via iTRAQ subcellular quantitative proteomics (Su et al., 2015), revealing a significant degree of overlap with the differentially expressed genes identified in the present study. For example, NFKB1, STAT1, STAT2, and TNF were upregulated in both studies, yet CD14 was downregulated in our study and upregulated in this prior study. CD14 is a surface receptor preferentially expressed on monocytes/macrophages that is critical for the induction of appropriate immune responses to aerogenic MTB infections (Cubillos-Angulo et al., 2021). PPE26 can upregulate CD14, thereby potentially promoting a more adequate innate immune response. Interestingly, targeting CD14 in the treatment of SARS-CoV-2-infected patients provides an opportunity to potentially inhibit multiple inflammatory responses (Martin et al., 2020). The pathogenic mechanisms of the intracellular MTB parasite may be similar to this virus in some ways such that PPE57 can downregulate the expression of CD14. PPE57 may thus indirectly lessen allergic reactions to MTB, although this requires further study. Overall, MTB PPE family proteins are known to be generally immunogenic, but they differ from one other with respect to their physiological effects. Exploring the pathogenicity and immunogenicity of each of these proteins is vital, thus prompting the present study of PPE57. Further research regarding other members of this protein family may similarly highlight novel clinical insights.

In this study, we employed PPE57-stimulated PBMCs to effectively study MTB-specific host immune responses while simultaneously minimizing unrelated background noise. Relative to other studies employing unstimulated whole blood samples, PPE57 stimulation was able to provoke more varied host immune responses, potentially improving our ability to resolve key transcriptional gene expression profiles of interest. However, as the results from the present study were obtained through a series of bioinformatics analyses, additional preclinical and clinical research will be essential to confirm and build upon these findings.

In conclusion, we comprehensively analyzed transcription profiles associated with PPE57 stimulation in PBMCs of healthy hosts and identified 14 genes (ISG15, MX2, IRF9, IFIT3, IFIT2, OAS3, IFIT1, IFI6, OAS2, OASL, RSAD2, OAS1, IRF7, and MX1) associated with type I IFN signaling that were enriched following such stimulation. These findings offer new insights into host global immune mechanisms underlying MTB infection and PPE57 exposure. However, additional functional studies are required to determine the specific roles of the identified genes in the context of TB pathogenesis. Overall, our data highlight the importance of examining whether type I IFN responses to PPE57 exposure induce protective or harmful effects for the host.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

The study was approved by the Ethics Committee of West China Hospital, Sichuan University (reference: 2021-756). The participants provided their written informed consent for the publication of any potentially identifiable data included in this article.

YX, FY, and YM conceived and designed the study. FY conducted the experiments of the study. JH, XZ, YW, QY, JD, and XH contributed reagents/materials. FY analyzed the data and wrote the paper. YX and YM critically revised the manuscript. All authors contributed to the article and approved the submitted version.

Project financial support was provided by the Science and Technology Department Project of Sichuan, China (2020YFS0555).

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.

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