Epigenetic programming of host lipid metabolism associated with resistance to TST/IGRA conversion after exposure to Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) exposure leads to a range of outcomes including clearance, latent TB infection (LTBI), and pulmonary tuberculosis (TB). Some heavily exposed individuals resist tuberculin skin test (TST) and interferon-gamma (IFNγ) release assay (IGRA) conversion (RSTR), which suggests that they employ IFNγ-independent mechanisms of Mtb control. Here, we compare monocyte epigenetic profiles of RSTR and LTBI from a Ugandan household contact cohort. Chromatin accessibility did not differ between uninfected RSTR and LTBI monocytes. By contrast, methylation significantly differed at 174 CpG sites and across 63 genomic regions. Consistent with previous transcriptional findings in this cohort, differential methylation was enriched in lipid- and cholesterol-associated pathways including the genes APOC3, KCNQ1, and PLA2G3. In addition, methylation was enriched in Hippo signaling, which is associated with cholesterol homeostasis and includes CIT and SHANK2. Lipid export and Hippo signaling pathways were also associated with gene expression in response to Mtb in RSTR as well as IFN stimulation in monocyte-derived macrophages (MDMs) from an independent healthy donor cohort. Moreover, serum-derived high-density lipoprotein from RSTR had elevated ABCA1-mediated cholesterol efflux capacity (CEC) compared to LTBI. Our findings suggest that resistance to TST/IGRA conversion is linked to regulation of lipid accumulation in monocytes, which could facilitate early Mtb clearance among RSTR subjects through IFNγ-independent mechanisms.IMPORTANCETuberculosis (TB) remains an enduring global health challenge with millions of deaths and new cases each year. Despite recent advances in TB treatment, we lack an effective vaccine or a durable cure. While heavy exposure to Mycobacterium tuberculosis often results in latent TB latent infection (LTBI), subpopulations exist that are either resistant to infection or contain Mtb with interferon-gamma (IFNγ)-independent mechanisms not indicative of LTBI. These resisters provide an opportunity to investigate the mechanisms of TB disease and discover novel therapeutic targets. Here, we compare monocyte epigenetic profiles of RSTR and LTBI from a Ugandan household contact cohort. We identify methylation signatures in host lipid and cholesterol pathways with potential relevance to early TB clearance before the sustained IFN responses indicative of LTBI. This adds to a growing body of literature linking TB disease outcomes to host lipids.

Mycobacterium tuberculosis cough aerosol culture status associates with host characteristics and inflammatory profiles.

Interrupting transmission events is critical to tuberculosis control. Cough-generated aerosol cultures predict tuberculosis transmission better than microbiological or clinical markers. We hypothesize that highly infectious individuals with pulmonary tuberculosis (positive for cough aerosol cultures) have elevated inflammatory markers and unique transcriptional profiles compared to less infectious individuals. We performed a prospective, longitudinal study using cough aerosol sampling system. We enrolled 142 participants with treatment-naïve pulmonary tuberculosis in Kenya and assessed the association of clinical, microbiologic, and immunologic characteristics with Mycobacterium tuberculosis aerosolization and transmission in 129 household members. Contacts of the forty-three aerosol culture-positive participants (30%) are more likely to have a positive interferon-gamma release assay (85% vs 53%, P = 0.006) and higher median IFNγ level (P < 0.001, 4.28 IU/ml (1.77-5.91) vs. 0.71 (0.01-3.56)) compared to aerosol culture-negative individuals. We find that higher bacillary burden, younger age, larger mean upper arm circumference, and host inflammatory profiles, including elevated serum C-reactive protein and lower plasma TNF levels, associate with positive cough aerosol cultures. Notably, we find pre-treatment whole blood transcriptional profiles associate with aerosol culture status, independent of bacillary load. These findings suggest that tuberculosis infectiousness is associated with epidemiologic characteristics and inflammatory signatures and that these features may identify highly infectious persons.

Human Alveolar and Monocyte-Derived Human Macrophage Responses to Mycobacterium tuberculosis.

Alveolar macrophages (AMs) and recruited monocyte-derived macrophages (MDMs) mediate early lung immune responses to Mycobacterium tuberculosis. Differences in the response of these distinct cell types are poorly understood and may provide insight into mechanisms of tuberculosis pathogenesis. The objective of this study was to determine whether M. tuberculosis induces unique and essential antimicrobial pathways in human AMs compared with MDMs. Using paired human AMs and 5-d MCSF-derived MDMs from six healthy volunteers, we infected cells with M. tuberculosis H37Rv for 6 h, isolated RNA, and analyzed transcriptomic profiles with RNA sequencing. We found 681 genes that were M. tuberculosis dependent in AMs compared with MDMs and 4538 that were M. tuberculosis dependent in MDMs, but not AMs (false discovery rate [FDR] < 0.05). Using hypergeometric enrichment of DEGs in Broad Hallmark gene sets, we found that type I and II IFN Response were the only gene sets selectively induced in M. tuberculosis-infected AM (FDR < 0.05). In contrast, MYC targets, unfolded protein response and MTORC1 signaling, were selectively enriched in MDMs (FDR < 0.05). IFNA1, IFNA8, IFNE, and IFNL1 were specifically and highly upregulated in AMs compared with MDMs at baseline and/or after M. tuberculosis infection. IFNA8 modulated M. tuberculosis-induced proinflammatory cytokines and, compared with other IFNs, stimulated unique transcriptomes. Several DNA sensors and IFN regulatory factors had higher expression at baseline and/or after M. tuberculosis infection in AMs compared with MDMs. These findings demonstrate that M. tuberculosis infection induced unique transcriptional responses in human AMs compared with MDMs, including upregulation of the IFN response pathway and specific DNA sensors.

Mycobacterium tuberculosis-dependent monocyte expression quantitative trait loci, cytokine production, and TB pathogenesis.

Introduction: The heterogeneity of outcomes after Mycobacterium tuberculosis (Mtb) exposure is a conundrum associated with millennia of host-pathogen co-evolution. We hypothesized that human myeloid cells contain genetically encoded, Mtb-specific responses that regulate critical steps in tuberculosis (TB) pathogenesis. Methods: We mapped genome-wide expression quantitative trait loci (eQTLs) in Mtb-infected monocytes with RNAseq from 80 Ugandan household contacts of pulmonary TB cases to identify monocyte-specific, Mtb-dependent eQTLs and their association with cytokine expression and clinical resistance to tuberculin skin test (TST) and interferon-γ release assay (IGRA) conversion. Results: cis-eQTLs (n=1,567) were identified in Mtb-infected monocytes (FDR<0.01), including 29 eQTLs in 16 genes which were Mtb-dependent (significant for Mtb:genotype interaction [FDR<0.1], but not classified as eQTL in uninfected condition [FDR≥0.01]). A subset of eQTLs were associated with Mtb-induced cytokine expression (n=8) and/or clinical resistance to TST/IGRA conversion (n=1). Expression of BMP6, an Mtb-dependent eQTL gene, was associated with IFNB1 induction in Mtb-infected and DNA ligand-induced cells. Network and enrichment analyses identified fatty acid metabolism as a pathway associated with eQTL genes. Discussion: These findings suggest that monocyte genes contain Mtb-dependent eQTLs, including a subset associated with cytokine expression and/or clinical resistance to TST/IGRA conversion, providing insight into immunogenetic pathways regulating susceptibility to Mtb infection and TB pathogenesis.

Epigenetic programming of host lipid metabolism associates with resistance to TST/IGRA conversion after exposure to Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) exposure leads to a range of outcomes including clearance, latent TB infection (LTBI), and pulmonary tuberculosis (TB). Some heavily exposed individuals resist tuberculin skin test (TST) and interferon gamma release assay (IGRA) conversion (RSTR), which suggests that they employ IFNγ-independent mechanisms of Mtb control. Here, we compare monocyte epigenetic profiles of RSTR and LTBI from a Ugandan household contact cohort. Chromatin accessibility did not differ between uninfected RSTR and LTBI monocytes. In contrast, methylation significantly differed at 174 CpG sites and across 63 genomic regions. Consistent with previous transcriptional findings in this cohort, differential methylation was enriched in lipid and cholesterol associated pathways including in the genes APOC3, KCNQ1, and PLA2G3. In addition, methylation was enriched in Hippo signaling, which is associated with cholesterol homeostasis and includes CIT and SHANK2. Lipid export and Hippo signaling pathways were also associated with gene expression in response to Mtb in RSTR as well as IFN stimulation in monocyte-derived macrophages (MDMs) from an independent healthy donor cohort. Moreover, serum-derived HDL from RSTR had elevated ABCA1-mediated cholesterol efflux capacity (CEC) compared to LTBI. Our findings suggest that resistance to TST/IGRA conversion is linked to regulation of lipid accumulation in monocytes, which could facilitate early Mtb clearance among RSTR subjects through IFNγ-independent mechanisms.

Tuberculosis Infectiousness is Associated with Distinct Clinical and Inflammatory Profiles.

Interrupting transmission events to prevent new acquisition of infection and disease is a critical part of tuberculosis (TB) control efforts. However, knowledge gaps in understanding the biology and determinants of TB transmission, including poor estimates of individual infectiousness and the lack of accurate and convenient biomarkers, undermine efforts to develop interventions. Cough-generated aerosol cultures have been found to predict TB transmission better than any microbiological or clinical markers in cohorts from Uganda and Brazil. We hypothesized that highly infectious individuals with pulmonary TB (defined as positive for cough aerosol cultures) have elevated inflammatory markers and unique transcriptional profiles compared to less infectious individuals (negative for cough aerosol cultures). We performed a prospective, longitudinal study using a cough aerosol sampling system as in other studies. We enrolled 142 participants with treatment-naïve pulmonary TB in Nairobi, Kenya, and assessed the association of clinical, microbiologic, and immunologic characteristics with Mtb aerosolization and transmission in 143 household members. Contacts of the forty-three aerosol culture-positive participants (30%) were more likely to have a positive IGRA (85% vs 53%, P = 0.005) and a higher median IGRA IFNγ level (P < 0.001, median 4.25 IU/ml (0.90-5.91) vs. 0.71 (0.01-3.56)) compared to aerosol culture-negative individuals. We found that higher bacillary burden, younger age, and larger mean upper arm circumference were associated with positive aerosol cultures. In addition, novel host inflammatory profiles, including elevated serum C-reactive protein and sputum cytokines, were associated with aerosol culture status. Notably, we found pre-treatment whole blood transcriptional profiles associated with aerosol culture status, independent of bacillary load. Together, these findings suggest that TB infectiousness is associated with epidemiologic characteristics and inflammatory signatures and that these features may be used to identify highly infectious persons. These results provide new public health tools and insights into TB pathogenesis.

Mycobacterium tuberculosis -induced monocyte transcriptional responses associated with resistance to tuberculin skin test/interferon-γ release assay conversion in people with HIV.

OBJECTIVE: To determine whether Mycobacterium tuberculosis (Mtb)-induced monocyte transcriptional responses differ in people with HIV (PWH) who do (RSTR) or do not (LTBI) resist tuberculin skin test/interferon-γ (IFN-γ) release assay (TST/IGRA) conversion after exposure. DESIGN: We compared ex-vivo Mtb-induced monocyte transcriptional responses in a Ugandan tuberculosis (TB) household contact study of RSTR and LTBI individuals among PWH. METHODS: Monocytes were isolated from peripheral blood mononuclear cells from 19 household contacts of pulmonary TB patients, and their transcriptional profiles were measured with RNA-Seq after a 6 h infection with Mtb (H37Rv) or media. Differentially expressed genes (DEGs) were identified by a linear mixed effects model and pathways by gene set enrichment analysis that compared RSTR and LTBI phenotypes with and without Mtb stimulation. RESULTS: Among PWH, we identified 8341 DEGs that were dependent on Mtb stimulation [false discovery rate (FDR) <0.01]. Of these, 350 were not significant (FDR >0.2) in individuals without HIV. Additionally, we found 26 genes that were differentially expressed between RSTR and LTBI monocytes in PWH, including 20 which were Mtb-dependent (FDR <0.2). In unstimulated monocytes, several gene sets [TGF-ß signaling, TNF-α signaling via NF-κB, NOTCH signaling, coagulation, and epithelial mesenchymal transition (EMT)] were enriched in RSTR relative to LTBI monocytes (FDR <0.1). These patterns were not observed in individuals without HIV. CONCLUSION: RSTR monocytes in PWH show different gene expressions in response to Mtb infection when compared with those with LTBI and RSTR without HIV. These differential expression patterns are enriched in inflammatory pathways.

Mycobacterium tuberculosis-dependent Monocyte Expression Quantitative Trait Loci and Tuberculosis Pathogenesis.

The heterogeneity of outcomes after Mycobacterium tuberculosis (Mtb) exposure is a conundrum associated with millennia of host-pathogen co-evolution. We hypothesized that human myeloid cells contain genetically encoded, Mtb-specific responses that regulate critical steps in tuberculosis (TB) pathogenesis. We mapped genome-wide expression quantitative trait loci (eQTLs) in Mtb-infected monocytes with RNAseq from 80 Ugandan household contacts of pulmonary TB cases to identify monocyte-specific, Mtb-dependent eQTLs and their association with cytokine expression and clinical resistance to tuberculin skin test (TST) and interferon-γ release assay (IGRA) conversion. cis-eQTLs (n=1,567) were identified in Mtb-infected monocytes (FDR<0.01), including 29 eQTLs in 16 genes which were Mtb-dependent (significant for Mtb:genotype interaction [FDR<0.1], but not classified as eQTL in media condition [FDR≥0.01]). A subset of eQTLs were associated with Mtb-induced cytokine expression (n=8) and/or clinical resistance to TST/IGRA conversion (n=1). Expression of BMP6, an Mtb-dependent eQTL gene, was associated with IFNB1 induction in Mtb-infected and DNA ligand-induced cells. Network and enrichment analyses identified fatty acid metabolism as a pathway associated with eQTL genes. These findings suggest that monocyte genes contain Mtb-dependent eQTLs, including a subset associated with cytokine expression and/or clinical resistance to TST/IGRA conversion, providing insight into immunogenetic pathways regulating susceptibility to Mtb infection and TB pathogenesis.

Differentially expressed transcript isoforms associate with resistance to tuberculin skin test and interferon gamma release assay conversion.

BACKGROUND: A mechanistic understanding of uncommon immune outcomes such as resistance to infection has led to the development of novel therapies. Using gene level analytic methods, we previously found distinct monocyte transcriptional responses associated with resistance to Mycobacterium tuberculosis (Mtb) infection defined as persistently negative tuberculin skin test (TST) and interferon gamma release assay (IGRA) reactivity among highly exposed contacts (RSTR phenotype). OBJECTIVE: Using transcript isoform analyses, we aimed to identify novel RSTR-associated genes hypothesizing that previous gene-level differential expression analysis obscures isoform-specific differences that contribute to phenotype. MATERIALS AND METHODS: Monocytes from 49 RSTR versus 52 subjects with latent Mtb infection (LTBI) were infected with M. tuberculosis (H37Rv) or left unstimulated (media) prior to RNA isolation and sequencing. RSTR-associated gene expression was then identified using differential transcript isoform analysis. RESULTS: We identified 81 differentially expressed transcripts (DETs) in 70 genes (FDR <0.05) comparing RSTR and LTBI phenotypes with the majority (n = 79 DETs) identified under Mtb-stimulated conditions. Seventeen of these genes were previously identified with gene-level bulk RNAseq analyses including genes in the IFNγ response that had increased expression among LTBI subjects, findings consistent with a clinical phenotype based on IGRA reactivity. Among the subset of 23 genes with positive differential expression among Mtb-infected RSTR monocytes, 13 were not previously identified. These novel DET genes included PDE4A and ZEB2, which each had multiple DETs with higher expression among RSTR subjects, and ACSL4 and GAPDH that each had a single transcript isoform associated with RSTR. CONCLUSION AND LIMITATIONS: Transcript isoform-specific analyses identify transcriptional associations, such as those associated with resistance to TST/IGRA conversion, that are obscured when using gene-level approaches. These findings should be validated with additional RSTR cohorts and whether the newly identified candidate resistance genes directly influence the monocyte Mtb response requires functional study.

T-cell deficiency and hyperinflammatory monocyte responses associate with Mycobacterium avium complex lung disease.

Immunological mechanisms of susceptibility to nontuberculous mycobacterial (NTM) disease are poorly understood. To understand NTM pathogenesis, we evaluated innate and antigen-specific adaptive immune responses to Mycobacterium avium complex (MAC) in asymptomatic individuals with a previous history of MAC lung disease (MACDZ). We hypothesized that Mav-specific immune responses are associated with susceptibility to MAC lung disease. We measured MAC-, NTM-, or MAC/Mtb-specific T-cell responses by cytokine production, expression of surface markers, and analysis of global gene expression in 27 MACDZ individuals and 32 healthy controls. We also analyzed global gene expression in Mycobacterium avium-infected and uninfected peripheral blood monocytes from 17 MACDZ and 17 healthy controls. We were unable to detect increased T-cell responses against MAC-specific reagents in MACDZ compared to controls, while the responses to non-mycobacteria derived antigens were preserved. MACDZ individuals had a lower frequency of Th1 and Th1* T-cell populations. In addition, MACDZ subjects had lower transcriptional responses in PBMCs stimulated with a mycobacterial peptide pool (MTB300). By contrast, global gene expression analysis demonstrated upregulation of proinflammatory pathways in uninfected and M. avium-infected monocytes, i.e. a hyperinflammatory in vitro response, derived from MACDZ subjects compared to controls. Together, these data suggest a novel immunologic defect which underlies MAC pathogenesis and includes concurrent innate and adaptive dysregulation which persists years after completion of treatment.

Monocyte Transcriptional Responses to Mycobacterium tuberculosis Associate with Resistance to Tuberculin Skin Test and Interferon Gamma Release Assay Conversion.

Heavy exposure to Mycobacterium tuberculosis, the etiologic agent of tuberculosis (TB) and among the top infectious killers worldwide, results in infection that is cleared, contained, or progresses to disease. Some heavily exposed tuberculosis contacts show no evidence of infection using the tuberculin skin test (TST) and interferon gamma release assay (IGRA); yet the mechanisms underlying this "resister" (RSTR) phenotype are unclear. To identify transcriptional responses that distinguish RSTR monocytes, we performed transcriptome sequencing (RNA-seq) on monocytes isolated from heavily exposed household contacts in Uganda and gold miners in South Africa after ex vivo M. tuberculosis infection. Gene set enrichment analysis (GSEA) revealed several gene pathways that were consistently enriched in response to M. tuberculosis among RSTR subjects compared to controls with positive TST/IGRA testing (latent TB infection [LTBI]) across Uganda and South Africa. The most significantly enriched gene set in which expression was increased in RSTR relative to LTBI M. tuberculosis-infected monocytes was the tumor necrosis factor alpha (TNF-α) signaling pathway whose core enrichment (leading edge) substantially overlapped across RSTR populations. These leading-edge genes included candidate resistance genes (ABCA1 and DUSP2) with significantly increased expression among Uganda RSTRs (false-discovery rate [FDR], <0.1). The distinct monocyte transcriptional response to M. tuberculosis among RSTR subjects, including increased expression of the TNF signaling pathway, highlights genes and inflammatory pathways that may mediate resistance to TST/IGRA conversion and provides therapeutic targets to enhance host restriction of M. tuberculosis intracellular infection. IMPORTANCE After heavy M. tuberculosis exposure, the events that determine why some individuals resist TST/IGRA conversion are poorly defined. Enrichment of the TNF signaling gene set among RSTR monocytes from multiple distinct cohorts suggests an important role for the monocyte TNF response in determining this alternative immune outcome. These TNF responses to M. tuberculosis among RSTRs may contribute to antimicrobial programs that result in early clearance or the priming of alternative (gamma interferon-independent) cellular responses.

Monocyte metabolic transcriptional programs associate with resistance to tuberculin skin test/interferon-γ release assay conversion.

After extensive exposure to Mycobacterium tuberculosis (Mtb), most individuals acquire latent Mtb infection (LTBI) defined by a positive tuberculin skin test (TST) or interferon-γ release assay (IGRA). To identify mechanisms of resistance to Mtb infection, we compared transcriptional profiles from highly exposed contacts who resist TST/IGRA conversion (resisters, RSTRs) and controls with LTBI using RNAseq. Gene sets related to carbon metabolism and free fatty acid (FFA) transcriptional responses enriched across 2 independent cohorts suggesting RSTR and LTBI monocytes have distinct activation states. We compared intracellular Mtb replication in macrophages treated with FFAs and found that palmitic acid (PA), but not oleic acid (OA), enhanced Mtb intracellular growth. This PA activity correlated with its inhibition of proinflammatory cytokines in Mtb-infected cells. Mtb growth restriction in PA-treated macrophages was restored by activation of AMP kinase (AMPK), a central host metabolic regulator known to be inhibited by PA. Finally, we genotyped AMPK variants and found 7 SNPs in PRKAG2, which encodes the AMPK-γ subunit, that strongly associated with RSTR status. Taken together, RSTR and LTBI phenotypes are distinguished by FFA transcriptional programs and by genetic variation in a central metabolic regulator, which suggests immunometabolic pathways regulate TST/IGRA conversion.

HDAC3 inhibitor RGFP966 controls bacterial growth and modulates macrophage signaling during Mycobacterium tuberculosis infection.

RATIONALE: Host-directed therapeutics for Mycobacterium tuberculosis (Mtb) offer potential strategies for combatting antibiotic resistance and for killing non-replicating bacilli. Phenylbutyrate, a partially selective histone-deacetylase (HDAC) inhibitor, was previously shown to control Mtb growth and alter macrophage inflammatory pathways at 2-4 mM concentrations. OBJECTIVE: To identify a more potent and selective HDAC inhibitor that modulates macrophage responses to mycobacteria and has direct antibacterial effects against Mtb. METHODS: We used cellular approaches to characterize the role of pharmacologic inhibition of HDAC3 on Mtb growth and Mtb-induced peripheral and alveolar macrophage immune functions. MEASUREMENTS AND MAIN RESULTS: RGFP966, an HDAC3 inhibitor, controlled Mtb, BCG and M. avium growth directly in broth culture and in human peripheral blood monocyte-derived and alveolar macrophages with an MIC50 of approximately 5-10 µM. In contrast, RGFP966 did not inhibit growth of several other intracellular and extracellular bacteria. We also found that RGFP966 modulated macrophage pro-inflammatory cytokine secretion in response to Mtb infection with decreased IL6 and TNF secretion. CONCLUSIONS: We identified a potent and selective small molecule inhibitor of HDAC3 with direct antimicrobial activity against Mtb and modulation of macrophage signaling pathways.

Nicotinamide Limits Replication of Mycobacterium tuberculosis and Bacille Calmette-Guérin Within Macrophages.

Novel antimicrobials for treatment of Mycobacterium tuberculosis are needed. We hypothesized that nicotinamide (NAM) and nicotinic acid (NA) modulate macrophage function to restrict M. tuberculosis replication in addition to their direct antimicrobial properties. Both compounds had modest activity in 7H9 broth, but only NAM inhibited replication in macrophages. Surprisingly, in macrophages NAM and the related compound pyrazinamide restricted growth of bacille Calmette-Guérin but not wild-type Mycobacterium bovis, which both lack a functional nicotinamidase/pyrazinamidase (PncA) rendering each strain resistant to these drugs in broth culture. Interestingly, NAM was not active in macrophages infected with a virulent M. tuberculosis mutant encoding a deletion in pncA. We conclude that the differential activity of NAM and nicotinic acid on infected macrophages suggests host-specific NAM targets rather than PncA-dependent direct antimicrobial properties. These activities are sufficient to restrict attenuated BCG, but not virulent wild-type M. bovis or M. tuberculosis.

A Functional Toll-Interacting Protein Variant Is Associated with Bacillus Calmette-Guérin-Specific Immune Responses and Tuberculosis.

RATIONALE: The molecular mechanisms that regulate tuberculosis susceptibility and bacillus Calmette-Guérin (BCG)-induced immunity are mostly unknown. However, induction of the adaptive immune response is a critical step in host control of Mycobacterium tuberculosis. Toll-interacting protein (TOLLIP) is a ubiquitin-binding protein that regulates innate immune responses, including Toll-like receptor signaling, which initiate adaptive immunity. TOLLIP variation is associated with susceptibility to tuberculosis, but the mechanism by which it regulates tuberculosis immunity is poorly understood. OBJECTIVES: To identify functional TOLLIP variants and evaluate the role of TOLLIP variation on innate and adaptive immune responses to mycobacteria and susceptibility to tuberculosis. METHODS: We used human cellular immunology approaches to characterize the role of a functional TOLLIP variant on monocyte mRNA expression and M. tuberculosis-induced monocyte immune functions. We also examined the association of TOLLIP variation with BCG-induced T-cell responses and susceptibility to latent tuberculosis infection. MEASUREMENTS AND MAIN RESULTS: We identified a functional TOLLIP promoter region single-nucleotide polymorphism, rs5743854, which was associated with decreased TOLLIP mRNA expression in infant monocytes. After M. tuberculosis infection, TOLLIP-deficient monocytes demonstrated increased IL-6, increased nitrite, and decreased bacterial replication. The TOLLIP-deficiency G/G genotype was associated with decreased BCG-specific IL-2+ CD4+ T-cell frequency and proliferation. This genotype was also associated with increased susceptibility to latent tuberculosis infection. CONCLUSIONS: TOLLIP deficiency is associated with decreased BCG-specific T-cell responses and increased susceptibility to tuberculosis. We hypothesize that the heightened antibacterial monocyte responses after vaccination of TOLLIP-deficient infants are responsible for decreased BCG-specific T-cell responses. Activating TOLLIP may provide a novel adjuvant strategy for BCG vaccination.

Transcriptional networks are associated with resistance to Mycobacterium tuberculosis infection.

RATIONALE: Understanding mechanisms of resistance to M. tuberculosis (M.tb) infection in humans could identify novel therapeutic strategies as it has for other infectious diseases, such as HIV. OBJECTIVES: To compare the early transcriptional response of M.tb-infected monocytes between Ugandan household contacts of tuberculosis patients who demonstrate clinical resistance to M.tb infection (cases) and matched controls with latent tuberculosis infection. METHODS: Cases (n = 10) and controls (n = 18) were selected from a long-term household contact study in which cases did not convert their tuberculin skin test (TST) or develop tuberculosis over two years of follow up. We obtained genome-wide transcriptional profiles of M.tb-infected peripheral blood monocytes and used Gene Set Enrichment Analysis and interaction networks to identify cellular processes associated with resistance to clinical M.tb infection. MEASUREMENTS AND MAIN RESULTS: We discovered gene sets associated with histone deacetylases that were differentially expressed when comparing resistant and susceptible subjects. We used small molecule inhibitors to demonstrate that histone deacetylase function is important for the pro-inflammatory response to in-vitro M.tb infection in human monocytes. CONCLUSIONS: Monocytes from individuals who appear to resist clinical M.tb infection differentially activate pathways controlled by histone deacetylase in response to in-vitro M.tb infection when compared to those who are susceptible and develop latent tuberculosis. These data identify a potential cellular mechanism underlying the clinical phenomenon of resistance to M.tb infection despite known exposure to an infectious contact.

The SIGLEC14 null allele is associated with Mycobacterium tuberculosis- and BCG-induced clinical and immunologic outcomes.

Humans exposed to Mycobacterium tuberculosis (Mtb) have variable susceptibility to tuberculosis (TB) and its outcomes. Siglec-5 and Siglec-14 are members of the sialic-acid binding lectin family that regulate immune responses to pathogens through inhibitory (Siglec-5) and activating (Siglec-14) domains. The SIGLEC14 coding sequence is deleted in a high proportion of individuals, placing a SIGLEC5-like gene under the expression of the SIGLEC14 promoter (the SIGLEC14 null allele) and causing expression of a Siglec-5 like protein in monocytes and macrophages. We hypothesized that the SIGLEC14 null allele was associated with Mtb replication in monocytes, T-cell responses to the BCG vaccine, and clinical susceptibility to TB. The SIGLEC14 null allele was associated with protection from TB meningitis in Vietnamese adults but not with pediatric TB in South Africa. The null allele was associated with increased IL-2 and IL-17 production following ex-vivo BCG stimulation of blood from 10 week-old South African infants vaccinated with BCG at birth. Mtb replication was increased in THP-1 cells overexpressing either Siglec-5 or Siglec-14 relative to controls. To our knowledge, this is the first study to demonstrate an association between SIGLEC expression and clinical TB, Mtb replication, or BCG-specific T-cell cytokines.

Human ULK1 Variation and Susceptibility to Mycobacterium tuberculosis Infection.

BACKGROUND: Unlike tuberculosis, few studies have evaluated a host genetic basis for variability in susceptibility to latent Mycobacterium tuberculosis infection (LTBI). We performed a candidate gene association study of autophagy-related genes and LTBI. METHODS: We enrolled close contacts of individuals with pulmonary tuberculosis, assessed LTBI status, and determined clinical and sociodemographic risk factors for LTBI. In participants who self-identified as Asian or black, we compared haplotype-tagging single-nucleotide polymorphisms (SNPs) in ULK1 and GABARAP between cases (n = 143) and controls (n = 106). Using CRISPR/Cas9 in U937 monocytes, we investigated the effect of ULK1 deficiency on cytokine expression, autophagy, and M. tuberculosis replication. RESULTS: In Asian participants, we identified 2 ULK1 SNPs (rs12297124 and rs7300908) associated with LTBI. After adjustment for population admixture and clinical risk for LTBI, each rs12297124 minor allele conferred 80% reduction in LTBI risk (odds ratio, 0.18; 95% confidence interval, .07-.46). Compared with controls, ULK1-deficient cells exhibited decreased tumor necrosis factor secretion after stimulation with Toll-like receptor ligands and M. tuberculosis whole-cell lysate, increased M. tuberculosis replication, and decreased selective autophagy. CONCLUSIONS: These results demonstrate a strong association of rs12297124, a noncoding ULK1 SNP, with LTBI and a role for ULK1 regulation of TNF secretion, nonspecific and M. tuberculosis-induced autophagy, and M. tuberculosis replication in monocytes.

Genetic Variation in Toll-Interacting Protein Is Associated With Leprosy Susceptibility and Cutaneous Expression of Interleukin 1 Receptor Antagonist.

Leprosy is a chronic disease characterized by skin and peripheral nerve pathology and immune responses that fail to control Mycobacterium leprae. Toll-interacting protein (TOLLIP) regulates Toll-like receptor (TLR) and interleukin 1 receptor (IL-1R) signaling against mycobacteria. We analyzed messenger RNA (mRNA) expression of candidate immune genes in skin biopsy specimens from 85 individuals with leprosy. TOLLIP mRNA was highly and specifically correlated with IL-1R antagonist (IL-1Ra). In a case-control gene-association study with 477 cases and 1021 controls in Nepal, TOLLIP single-nucleotide polymorphism rs3793964 TT genotype was associated with increased susceptibility to leprosy (recessive, P = 1.4 × 10(-3)) and with increased skin expression of TOLLIP and IL-1Ra. Stimulation of TOLLIP-deficient monocytes with M. leprae produced significantly less IL-1Ra (P < .001), compared with control. These data suggest that M. leprae upregulates IL-1Ra by a TOLLIP-dependent mechanism. Inhibition of TOLLIP may decrease an individual's susceptibility to leprosy and offer a novel therapeutic target for IL-1-dependent diseases.

T Cell Responses against Mycobacterial Lipids and Proteins Are Poorly Correlated in South African Adolescents.

Human T cells are activated by both peptide and nonpeptide Ags produced by Mycobacterium tuberculosis. T cells recognize cell wall lipids bound to CD1 molecules, but effector functions of CD1-reactive T cells have not been systematically assessed in M. tuberculosis-infected humans. It is also not known how these features correlate with T cell responses to secreted protein Ags. We developed a flow cytometric assay to profile CD1-restricted T cells ex vivo and assessed T cell responses to five cell wall lipid Ags in a cross-sectional study of 19 M. tuberculosis-infected and 22 M. tuberculosis-uninfected South African adolescents. We analyzed six T cell functions using a recently developed computational approach for flow cytometry data in high dimensions. We compared these data with T cell responses to five protein Ags in the same cohort. We show that CD1b-restricted T cells producing antimycobacterial cytokines IFN-γ and TNF-α are detectable ex vivo in CD4(+), CD8(+), and CD4(-)CD8(-) T cell subsets. Glucose monomycolate was immunodominant among lipid Ags tested, and polyfunctional CD4 T cells specific for this lipid simultaneously expressed CD40L, IFN-γ, IL-2, and TNF-α. Lipid-reactive CD4(+) T cells were detectable at frequencies of 0.001-0.01%, and this did not differ by M. tuberculosis infection status. Finally, CD4 T cell responses to lipids were poorly correlated with CD4 T cell responses to proteins (Spearman rank correlation -0.01; p = 0.95). These results highlight the functional diversity of CD1-restricted T cells circulating in peripheral blood as well as the complementary nature of T cell responses to mycobacterial lipids and proteins. Our approach enables further population-based studies of lipid-specific T cell responses during natural infection and vaccination.