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

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

BackgroundA 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).ObjectiveUsing 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 methodsMonocytes 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.ResultsWe identified 81 differentially expressed transcripts (DETs) in 70 genes (FDR Conclusion and limitationsTranscript 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

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

IntroductionThe 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.MethodsWe 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.Resultscis-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.DiscussionThese 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

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

RationaleThe 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.ObjectivesTo identify functional TOLLIP variants and evaluate the role of TOLLIP variation on innate and adaptive immune responses to mycobacteria and susceptibility to tuberculosis.MethodsWe 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 resultsWe 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.ConclusionsTOLLIP 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

<i>CCL17</i> and <i>CCL18</i> by Ridley Jopling classification.

<p>Relative tissue mRNA levels of <i>CCL17</i> (A) and <i>CCL18</i> (B) normalized to GAPDH were arranged in order of specific leprosy classification and analyzed for significance by non-parametric trend test with variables TT, BT, BB, BL, and LL treated as increasing categorical variables, p values displayed. Lines are medians. A value of 40 was given to undetectable Ct Values in order to graphically represent on log scale.</p

CCL17 and CCL18 mRNA are not associated with reactive state.

<p>CCL17 and CCL18 mRNA levels (normalized by GAPDH expression) were compared in LL+BL versus TT+BT patients with T1R (L pole (n = 16), T pole (n = 20)) versus those without T1R (L pole (n = 17), T pole (n = 22)). Lines are medians. A value of 40 was given to undetectable Ct Values in order to graphically represent on log scale. P values were **<0.01, *<0.05, based on Mann-Whitney analysis.</p

CCL17 and CCL18 serum levels.

<p>Serum levels of CCL17 and CCL18 protein were measured in leprosy patients with lesions defined by RJ classification (n = 20) compared to EC (n = 6). P values by non-parametric trend test are depicted. EC (patients without leprosy) were given a categorical value of 0, while those with leprosy were given increasing categorical values as progressing to LL pole. Lines depict median values.</p

Cell marker expression in dermal leprosy lesions.

1<p>Tuberculoid polar leprosy,</p>2<p>lepromatous polar leprosy. Values of 0.000 are Ct values less than 1/2⁴⁰.</p>3<p>P values were calculated by Mann-Whitney tests. Both median and 75^th percentile (75(%)) are depicted.</p>4<p>Adjusted p values are the original p values corrected for multiple comparisons by traditional Bonferroni correction.</p><p>In bold are cytokines that were significant following Bonferroni correction.</p><p>*represents cytokines that were significant by Mann-Whitney, but failed multiple comparisons corrections.</p><p>Cell marker expression in dermal leprosy lesions.</p