Tuberculosis (TB), a chronic infectious disease caused by the pathogen Mycobacterium tuberculosis, remains a major cause of morbidity and mortality worldwide. Until COVID-19 pandemic, TB was the most common cause of death from a single infectious agent, surpassing HIV/AIDS (World Health Organization (WHO), 2022). The impacts generated in 2020 by the pandemic included reduced access to diagnosis and treatment with the consequent increase in the number of deaths. The WHO estimated 1.4 million TB deaths among HIV-negative patients and an additional of 187.000 among HIV-positive people (World Health Organization (WHO), 2022). In Argentina, the last report indicated 10.896 new cases and 656 deaths in 2020 with a rate of mortality of 1.4/100.000 inhabitants (Ministerio De Salud Argentina, 2022). It is estimated that about a quarter of the world’s population has latent TB infection, and is therefore at risk of developing active disease during their lifetime (World Health Organization (WHO), 2022). However, only 5-10% of individuals infected with M. tuberculosis will progress to active TB, suggesting that both host genetic and environmental factors might influence the susceptibility to TB (Fortin et al., 2007). Moreover, it has been shown that M. tuberculosis sublineages evolved in different human populations showing significant differences in virulence and immunomodulatory functions (Saelens et al., 2019), demonstrating that the genetic variability of M. tuberculosis is also an important factor affecting the pathogenesis of the disease. Although most environmental mycobacteria are non-pathogenic species, M. tuberculosis has evolved from an opportunist to a professional pathogen capable of infecting and surviving within the host.

Cell-mediated immunity plays an essential role in eliciting a protective immune response against M. tuberculosis infection. The secretion of Th1 cytokines by antigen-specific T cells collaborates with protective granuloma formation and stimulates the antimicrobial activity of infected macrophages (Flynn and Chan, 2001). In particular, IFN-γ is a key Th1 cytokine produced primarily by natural killer cells and T cells. IFNG KO mice infected with M. tuberculosis fail to produce reactive nitrogen intermediates that restrict the growth of the bacilli (Cooper et al., 1993; Flynn et al., 1993). Genetic studies in families with Mendelian Susceptibility to Mycobacterial Disease (MSMD), a primary immune deficiency that results in partial or complete defects in IFN-γ secretion, production, binding, or signaling, highlighted the critical role of this cytokine in mycobacterial infections (Rosain et al., 2019).

Family-based genetic studies and population-based case-control association analyses have been used to identify candidate genes for susceptibility to tuberculosis. Besides, cytokine gene polymorphisms underlie the complexity of inter-individual differences in the susceptibility, severity and clinical outcomes of several infectious diseases (Cooke and Hill, 2001; Patarčić et al., 2015). In particular, the +874 A/T (rs2430561) single nucleotide polymorphism (SNP) is located at the 5’ end of the CA repeat region in the first intron of the IFN-γ gene (Pravica et al., 2000). Pravica et al. suggested that the functional role of the +874 A/T SNP is related to its location within a putative NF-κB binding site (Pravica et al., 2000). The T allele predisposes to the binding of the transcription factor NF-κB, whereas the A allele reduces the affinity and therefore the expression of the gene in response to stimulus (Pravica et al., 2000). Thus, AA genotype or A allele have been associated with low levels of IFN-γ production, otherwise TT genotype or T allele are associated with higher production of this cytokine, and AT genotype with intermediate levels (Pravica et al., 2000). A significant association between this SNP and TB has been described in several populations around the world, as in the Sicilian, Brazilian, Chinese, Egyptian children, Spanish, Warao indigenous, South Indian and Iranian population (Lio et al., 2002; Lopez-Maderuelo et al., 2003; Tso et al., 2005; Amim et al., 2008; Mosaad et al., 2010; Bhanothu et al., 2015; Beiranvand et al., 2016; Araujo et al., 2017). Moreover, +874 A/T IFNG polymorphism has been associated with TB and extrapulmonary TB susceptibility in meta-analysis studies (Pacheco et al., 2008; Wei et al., 2017; Mandal et al., 2019; Areeshi et al., 2021). However, this SNP has not been studied in TB patients from Argentina.

On the other hand, IFN-γ gene is subject to both genetic and epigenetic modifications. This could imply a critical link between epigenetics and transcription factors in the regulation of IFN-γ and, consequently, in T cells responses elicited against M. tuberculosis. Host, pathogen & environment are the triad, which influences any disease, and epigenetics can bridge the gaps between them. The association between different epigenetic modifications and disease progression in M. tuberculosis is becoming an area of growing interest (Fatima et al., 2021; Gauba et al., 2021). Epigenetic changes, for example, histone modifications, DNA methylation, and miRNA-mediated up/downregulation of immune genes, play an important role in immunomodulation of the host at the post-TB infective phase. The reversibility of these epigenetic modifications makes them ideal and innovative targets that can be exploited for drug development and control strategies.

In particular, methylation in the promoter region can lead to the failure of gene transcription initiation and gene silencing by hampering transcription factor binding to specific motifs. The proximal region of the IFNG promoter contains binding sites for several transcription factors, including NFAT, NF-κB, and CREB-ATF1. Interestingly, methylation at the -53 site of the IFNG promoter results in inhibition of CREB and ATF2/c-Jun binding to the proximal AP1 site and is sufficient to inhibit expression of this cytokine in a Th1 murine cell line (Jones and Chen, 2006). Moreover, hypermethylation in human Th2 cells results in chromatin condensation and exclusion of CREB proteins from the IFNG promoter (Yano et al., 2003). CREB upregulates IFN-γ production in human T cells that respond to M. tuberculosis (Samten et al., 2002; Samten et al., 2005). We have previously shown that costimulation through the Signal Lymphocyte Activation Molecule (SLAM) in M. tuberculosis-stimulated T cells induces CREB activation, leading to IFN-γ secretion (Pasquinelli et al., 2009). Thus, our hypothesis was that the methylation of the CpG -53 site of IFNG could lead to the negative regulation of this cytokine during the immune response against M. tuberculosis.

The aim of this study was to further characterize the genetic and epigenetic mechanisms that regulate IFN-γ responses during M. tuberculosis infection. To this end, we performed a study in patients with active TB and healthy donors (HD) to determine the influence of +874 A/T polymorphism on tuberculosis susceptibility. We additionally evaluated DNA methylation at the -53 CpG site of the promoter region of IFNG as a new mechanism of epigenetic regulation of IFN-γ in the immune response against M. tuberculosis.

Tuberculosis, an ancient disease, is still one of the biggest killers worldwide, with a high morbidity rate. It is known that only about 10% of the M. tuberculosis-infected population progresses to active tuberculosis which leads to the following question: Do patients with active TB have a genetic predisposition that makes them more susceptible to disease progression? Single nucleotide polymorphisms in several candidate genes, especially polymorphisms in cytokine genes are known to modulate cytokine levels, which may influence susceptibility to tuberculosis infection and disease. Given the essential role of IFN-γ to control mycobacterial replication and development of cellular immune response (Flesch and Kaufmann, 1987; Serbina et al., 2001; Salgame, 2005), our case-control study focused on determining whether rs2430561 SNP (+874 A/T) is associated with susceptibility to tuberculosis in Argentinean population.

In this study, we found a significant increase in the frequency of A allele (79%) and AA genotype (64%) in TB patients as compared with HD suggesting an association of the rs2430561 SNP A variant with susceptibility to tuberculosis disease in Argentina. Remarkably, more than 45% of individuals from the HD population (91 out of 199, QFT-GIT negative) were subjects exposed to M. tuberculosis (TB contacts or healthcare staff). Among them, 60.44% has a T allele, reinforcing that the presence of the T allele is indeed associated with resistance to M. tuberculosis infection. Despite Etokebe et al. did not find correlation between the +874 A/T polymorphism and tuberculosis disease in a Croatian population (Etokebe et al., 2006), our results are consistent with previous studies that have demonstrated an association of A allele and AA genotype with susceptibility to tuberculosis in Sicilian, Spanish, Hong Kong, Brazilians, South African and Egyptian populations (Lio et al., 2002; Lopez-Maderuelo et al., 2003; Rossouw et al., 2003; Tso et al., 2005; Amim et al., 2008; Mosaad et al., 2010).

A few reports have analyzed the association of rs2430561 SNP with the clinical manifestations of the disease (Lopez-Maderuelo et al., 2003; Etokebe et al., 2006; Ansari et al., 2011). Lopez-Maderuelo et al. showed an association of AA genotype with a far advanced form of radiographic extent of disease (Lopez-Maderuelo et al., 2003). Likewise, a study in a Pakistan population reported an overrepresentation of the T allele in less severe forms of pulmonary TB (Ansari et al., 2011). However, our results showed no association with clinical parameters demonstrating that IFN-γ +874 A/T polymorphism is associated with TB susceptibility but not with disease severity in our population of study.

We have previously demonstrated that low levels of IFN-γ are associated with disease severity (Pasquinelli et al., 2004; Jurado et al., 2012). Since it has been reported that +874 T to A polymorphism overlaps with a putative NF-κ B binding site which might have functional consequences for the transcription of the human IFN-γ gene (Pravica et al., 2000); we investigated the functional association of the different +874 genotypes with IFN-γ production. Our study demonstrates that M. tuberculosis-stimulated PBMCs from HD carrying the AA genotype produced strikingly significantly lower levels of IFN-γ than those carrying the AT and TT genotype. However, while a tendency was observed in TB patients, we did not find significant differences on the production of IFN-γ. On the other side, we did observe a significant difference in the production of IFN-γ between HD and TB patients carrying a T allele, which suggests that besides the presence of the T allele other mechanisms are regulating IFN-γ during active disease.

Indeed, genetic polymorphisms are not the only inheritable character of DNA that could influence susceptibility to disease. Epigenetic mechanisms such as DNA methylation and histone acetylation regulate the rate of transcription and/or specific tissue expression of certain genes without altering the DNA sequence.

Recent studies suggest that M. tuberculosis can alter the host epigenome to modulate the transcriptional machinery and plays a major role in modulating the immune response of the host (Kathirvel and Mahadevan, 2016). However, the mechanisms involved in epigenetic alterations during M. tuberculosis infection have not been fully understood yet. In addition to a potential causal role in pathogenic processes, epigenetic alterations – such as de novo methylation of DNA – can also occur as a direct or indirect consequence of disease and might serve as biomarkers of disease activity (Esterhuyse et al., 2012).

Owing to the major role of the epigenome in the phenotypic plasticity of the immune system and its ability to link environment and cellular phenotypes, host susceptibility to TB is expected to have an epigenetic predisposition component. In addition, epigenetic analyses will probably reveal changes at the epigenome resulting either directly or indirectly from infection. Growing evidence highlights the impact of pathogens genetic variation on susceptibility and severity of infections.

In Argentina, Monteserin et al. studied the genetic diversity of M. tuberculosis strains circulating in the Metropolitan Area of Buenos Aires (Monteserin et al., 2018). They showed that as in the rest of South America, genotypes of the Lineage 4 Euro-American (one of the seven globally described M. tuberculosis strains) predominate in our country and in contrast with the neighboring country Brazil, the RD^rio type (associated with high rate of transmission and drug resistance) does not play a major role in the TB epidemic (Monteserin et al., 2018). Moreover, the multidrug resistant M strain which belongs to the Lineage 4 Euro-American is highly prevalent in Argentina and has been shown to induce poor immune responses (Yokobori et al., 2022). However, the M. tuberculosis population structure in Argentina is still not fully comprehend.

It has been demonstrated that the IFNG promoter undergoes differential methylation during in vitro polarization of human peripheral blood T cells (Yano et al., 2003). This promoter is hypermethylated at the -53 CpG site during Th2 differentiation whereas it becomes hypomethylated in Th1 differentiated cells, existing a correlation between the degree of promoter methylation and the production of IFN-γ (Yano et al., 2003). Janson et al. also described different methylation levels at the -53 CpG site between human naive CD4 lymphocytes which were hypermethylated and Th1 lymphocytes which showed demethylation during differentiation (Janson et al., 2008). In other work, Dong et al. demonstrated that in human T cells early Th1-cell differentiation is accompanied by dynamic demethylation of CpGs at both the promoter and the CNS-1 regions of the IFNG locus (Dong et al., 2013). Moreover, epigenetic modifications in this locus were indispensable for the establishment of stable functional Th1 cytokine memory. The authors showed considerable levels of demethylation among different CpG of the promoter and the CNS1 region, including the -53 CpG site studied in our work (Dong et al., 2013).

In mice, naive T cells show a hypomethylated state of the IFNG promoter, which is maintained as they differentiate into Th1 cells (Winders et al., 2004). On the contrary, under Th2-polarizing conditions, CD4 cells display higher levels of methylation in the IFNG promoter, including the -53 CpG site (Winders et al., 2004). Besides, it has been shown that the methylation of the -53 CpG site impairs IFNG promoter activity and ATF2/c-Jun and CREB binding in vitro (Jones and Chen, 2006).

Despite the presence of some differences in the regulation of IFNG transcription between mice and human T lymphocytes, these data demonstrate the epigenetic involvement in immune regulation and evidence the -53 CpG site as an evolutionarily conserved position essential for IFN-γ production. When we evaluated the methylation status of the IFNG CpG -53 site in whole blood, our results showed increased methylation levels in patients with active TB compared to LTBI people. Surprisingly, we did not find differences in the methylation status between HD and TB patients, which could be attributed to a reactive epigenome. The lowest levels of methylation were observed for LTBI, who has the ability to control the infection. Considering that patients with active TB are not able to contain the infection at the time they were studied, a hypermethylation status could be expected.

Moreover, ROC and AUC analysis demonstrated that hypermethylation of this site could be a good biomarker of disease status; which reinforce the importance of this epigenetic modification as a key mechanism during active disease.

In accordance, some reports have shown changes in human methylome during TB or in response to BCG. Lyu et al. conclude that DNA methylation might be a promising TB diagnostic biomarker, as they identified TB-related targets differentially methylated by logistic regression and elastic net regression that were validated by bisulfite conversion and PCR (Lyu et al., 2022). DNA methylation has been also associated to anti-mycobacterial activity in PBMCs from BCG-vaccinated individuals (Verma et al., 2017). Recently, DiNardo et al. characterized DNA methylation of different genes that are critical to anti-mycobacterial immunity (DiNardo et al., 2020). They showed that immune cells from patients with active TB present a hypermethylated state, leading to nonspecific immune responsiveness (DiNardo et al., 2020).

These studies demonstrate the importance of evaluating the epigenome in the context of TB, however, none of them have evaluated the relevance of the -53 site in particular. Although the number of samples evaluated in the present study limits the extrapolation of our findings, since DNA methylome patterns are sensitive to environmental and seasonal factors, age, and ethnicity, it is attractive to focus on a well-defined study group as we did in our work. Another issue to highlight from our study is that we analyzed changes in the methylation of the -53 site in peripheral blood. Peripheral blood biomarkers, whether at the genomic, transcriptomic, epigenetic, or proteomic level, have received special attention in recent years. It is noteworthy that epigenetics can establish bridges between the host, M. tuberculosis and the environment. Therefore, epigenetic biomarkers in peripheral blood have great potential in the diagnosis and monitoring of progression to active TB. Epigenetic markers may provide a novel biosignature for discrimination between LTBI and active TB disease, for monitoring drug treatment outcome, and, in the long run, for predicting the risk of progression of LTBI to active TB disease (Esterhuyse et al., 2012).

To our knowledge, there are only three reports in South America population that study the association of the +874 A/T SNP with resistance/susceptibility to TB (Henao et al., 2006; Amim et al., 2008; Araujo et al., 2017). Recently, another IFN-γ SNP, the IFNG rs1861494, was associated with TB susceptibility in Argentinean population (Rolandelli et al., 2018). Our work, is the first to study association and functional relevance of rs1861494 SNP in Argentina. Even though we did not find correlation with the severity of the disease, we demonstrated that AA genotype and A allele are correlated with low IFN-γ production and increased susceptibility to develop active TB. Moreover, we also confirmed the methylation of -53 CpG of IFNG in TB patients. Many other polymorphic genes should be investigated in active and latent TB, but our results indicate that the AA genotype of the rs2430561 SNP could be a potential genetic biomarker for tuberculosis susceptibility in Argentina.

The original contributions presented in the study are included in the article/ Supplementary Materials , further inquiries can be directed to the corresponding author/s.

The studies involving human participants were reviewed and approved by Hospital Dr. F. J. Muñiz. The patients/participants provided their written informed consent to participate in this study.

GA, RH, AB, VG and VP contributed to the conception and design of the study. GA and RH performed the experiments and analyzed the data. AB, ME and JC contributed with some of the experiments and provided methodological support and insightful data discussion. RH, AB and VP wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version. DP and RM were in charge of patients’ diagnosis, obtained the blood samples and contributed with the analysis of the clinical data. All authors contributed to the article and approved the submitted version.