History of tuberculosis disease is associated with genetic regulatory variation in Peruvians.

A quarter of humanity is estimated to be latently infected with Mycobacterium tuberculosis (Mtb) with a 5-10% risk of developing tuberculosis (TB) disease. Variability in responses to Mtb infection could be due to host or pathogen heterogeneity. Here, we focused on host genetic variation in a Peruvian population and its associations with gene regulation in monocyte-derived macrophages and dendritic cells (DCs). We recruited former household contacts of TB patients who previously progressed to TB (cases, n=63) or did not progress to TB (controls, n=63). Transcriptomic profiling of monocyte-derived dendritic cells (DCs) and macrophages measured the impact of genetic variants on gene expression by identifying expression quantitative trait loci (eQTL). We identified 330 and 257 eQTL genes in DCs and macrophages (False Discovery Rate (FDR) < 0.05), respectively. Five genes in DCs showed interaction between eQTL variants and TB progression status. The top eQTL interaction for a protein-coding gene was with FAH, the gene encoding fumarylacetoacetate hydrolase, which mediates the last step in mammalian tyrosine catabolism. FAH expression was associated with genetic regulatory variation in cases but not controls. Using public transcriptomic and epigenomic data of Mtb-infected monocyte-derived dendritic cells, we found that Mtb infection results in FAH downregulation and DNA methylation changes in the locus. Overall, this study demonstrates effects of genetic variation on gene expression levels that are dependent on history of infectious disease and highlights a candidate pathogenic mechanism through pathogen-response genes. Furthermore, our results point to tyrosine metabolism and related candidate TB progression pathways for further investigation.

Multimodally profiling memory T cells from a tuberculosis cohort identifies cell state associations with demographics, environment and disease.

Multimodal T cell profiling can enable more precise characterization of elusive cell states underlying disease. Here, we integrated single-cell RNA and surface protein data from 500,089 memory T cells to define 31 cell states from 259 individuals in a Peruvian tuberculosis (TB) progression cohort. At immune steady state >4 years after infection and disease resolution, we found that, after accounting for significant effects of age, sex, season and genetic ancestry on T cell composition, a polyfunctional type 17 helper T (TH17) cell-like effector state was reduced in abundance and function in individuals who previously progressed from Mycobacterium tuberculosis (M.tb) infection to active TB disease. These cells are capable of responding to M.tb peptides. Deconvoluting this state-uniquely identifiable with multimodal analysis-from public data demonstrated that its depletion may precede and persist beyond active disease. Our study demonstrates the power of integrative multimodal single-cell profiling to define cell states relevant to disease and other traits.

Dysglycemia is associated with Mycobacterium tuberculosis lineages in tuberculosis patients of North Lima-Peru.

This study was performed to investigate the role of dysglycemia on the genetic diversity of Mycobacterium tuberculosis (MTB) among pulmonary tuberculosis (TB) patients to build scientific evidence about the possible mechanisms of TB transmission. MTB isolates obtained of patients affected by pulmonary tuberculosis from health care facilities of North Lima-Peru, were analyzed using whole genome sequencing and 24-locus mycobacterial interspersed repetitive-unit -variable-number tandem repeats (MIRU-VNTR). Subsequently, clinical and epidemiological characteristics were associated with clustering, lineages and comorbid conditions. The analysis carried out 112 pulmonary TB patients from various health centers in North Lima, 17 (15%) had diabetes mellitus (DM) and 33 (29%) had pre-diabetes (PDM). Latin American-Mediterranean, Haarlem and Beijing were the most frequent MTB lineages found in those patients. Previous TB (adjusted odds ratio [aOR] = 3.65; 95%CI: 1.32-17.81), age (aOR = 1.12; 95%CI: 1.03-1.45) and Beijing lineage (aOR = 3.53; 95%CI: 1.08-13.2) were associated with TB-DM comorbidity. Alcoholism (aOR = 2.92; 95%CI: 1.10-8.28), age (aOR = 1.05; 95%CI: 1.03-1.12) and Haarlem lineage (aOR = 2.54; 95%CI: 1.04-6.51) were associated with TB-PDM comorbidity. Beijing and Haarlem lineages were independently associated with TB-DM and TB-PDM comorbidities, respectively. Although these findings may be surprising, we must be cautious to suggest that dysglycemia could be associated with a highly clustering and predisposition of MTB lineages related to a serious impact on the severity of TB disease, which requires further research.

Synthetic mycobacterial diacyl trehaloses reveal differential recognition by human T cell receptors and the C-type lectin Mincle.

The cell wall of Mycobacterium tuberculosis is composed of diverse glycolipids which potentially interact with the human immune system. To overcome difficulties in obtaining pure compounds from bacterial extracts, we recently synthesized three forms of mycobacterial diacyltrehalose (DAT) that differ in their fatty acid composition, DAT1, DAT2, and DAT3. To study the potential recognition of DATs by human T cells, we treated the lipid-binding antigen presenting molecule CD1b with synthetic DATs and looked for T cells that bound the complex. DAT1- and DAT2-treated CD1b tetramers were recognized by T cells, but DAT3-treated CD1b tetramers were not. A T cell line derived using CD1b-DAT2 tetramers showed that there is no cross-reactivity between DATs in an IFN-γ release assay, suggesting that the chemical structure of the fatty acid at the 3-position determines recognition by T cells. In contrast with the lack of recognition of DAT3 by human T cells, DAT3, but not DAT1 or DAT2, activates Mincle. Thus, we show that the mycobacterial lipid DAT can be both an antigen for T cells and an agonist for the innate Mincle receptor, and that small chemical differences determine recognition by different parts of the immune system.

CD1b Tetramers Broadly Detect T Cells That Correlate With Mycobacterial Exposure but Not Tuberculosis Disease State.

The non-polymorphic nature of CD1 proteins creates a situation in which T cells with invariant T cell receptors (TCRs), like CD1d-specific NKT cells, are present in all humans. CD1b is an abundant protein on human dendritic cells that presents M. tuberculosis (Mtb) lipid antigens to T cells. Analysis of T cell clones suggested that semi-invariant TCRs exist in the CD1b system, but their prevalence in humans is not known. Here we used CD1b tetramers loaded with mycolic acid or glucose monomycolate to study polyclonal T cells from 150 Peruvian subjects. We found that CD1b tetramers loaded with mycolic acid or glucose monomycolate antigens stained TRAV1-2+ GEM T cells or TRBV4-1+ LDN5-like T cells in the majority of subjects tested, at rates ~10-fold lower than NKT cells. Thus, GEM T cells and LDN5-like T cells are a normal part of the human immune system. Unlike prior studies measuring MHC- or CD1b-mediated activation, this large-scale tetramer study found no significant differences in rates of CD1b tetramer-mycobacterial lipid staining of T cells among subjects with Mtb exposure, latent Mtb infection or active tuberculosis (TB) disease. In all subjects, including "uninfected" subjects, CD1b tetramer+ T cells expressed memory markers at high levels. However, among controls with lower mycobacterial antigen exposure in Boston, we found significantly lower frequencies of T cells staining with CD1b tetramers loaded with mycobacterial lipids. These data link CD1b-specific T cell detection to mycobacterial exposure, but not TB disease status, which potentially explains differences in outcomes among CD1-based clinical studies, which used control subjects with low Mtb exposure.

Severe pulmonary radiological manifestations are associated with a distinct biochemical profile in blood of tuberculosis patients with dysglycemia.

BACKGROUND: Diabetes mellitus (DM) is thought to affect tuberculosis (TB) clinical presentation and treatment response. Whether DM impacts radiological manifestations of pulmonary TB is still not clear. This study investigated the impact of glycemic status on radiological manifestations of pulmonary TB cases and its relationship with concentration of biochemical parameters in peripheral blood. METHODS: A retrospective cross-sectional study used data from 132 microbiologically confirmed pulmonary TB patients from Lima, Peru, evaluated in a previous investigation performed between February and December 2017. Chest radiographs were analyzed by a radiologist and a pulmonologist. Radiographic lesions were identified as cavities, alveolar infiltrates and fibrous tracts. Hyperglycemia in TB patients was identified by use of fasting plasma glucose, HbA1c and oral glucose tolerance test. Clinical, biochemical and hematological parameters were also analyzed. RESULTS: TB patients with hyperglycemia presented more frequently with cavities, alveolar infiltrates and fibrous tracts than those with normoglycemia. Hierarchical clustering analysis indicated that patients with more diverse and higher number of lung lesions exhibited a distinct laboratorial profile characterized by heightened white blood cell counts and circulating levels of total cholesterol, triglycerides and transaminases and simultaneously low levels of albumin and hemoglobin. Multivariable regression analyses adjusted for age, sex, prior TB, hemoglobin levels and acid-fast bacilli ≥2+ in sputum smears, demonstrated that presence of prediabetes or diabetes in TB patients was associated with increased odds of having 3 pulmonary lesion types (p = 0.003 and p < 0.01 respectively) or ≥ 4 lesions (p = 0.001 and p = 0.01 respectively). CONCLUSION: Hyperglycemia (both DM and prediabetes) significantly affected the presentation of radiographic manifestations and the number of lesions in pulmonary TB patients as well as the biochemical profile in peripheral blood.

A TCR ß-Chain Motif Biases toward Recognition of Human CD1 Proteins.

High-throughput TCR sequencing allows interrogation of the human TCR repertoire, potentially connecting TCR sequences to antigenic targets. Unlike the highly polymorphic MHC proteins, monomorphic Ag-presenting molecules such as MR1, CD1d, and CD1b present Ags to T cells with species-wide TCR motifs. CD1b tetramer studies and a survey of the 27 published CD1b-restricted TCRs demonstrated a TCR motif in humans defined by the TCR ß-chain variable gene 4-1 (TRBV4-1) region. Unexpectedly, TRBV4-1 was involved in recognition of CD1b regardless of the chemical class of the carried lipid. Crystal structures of two CD1b-specific TRBV4-1+ TCRs show that germline-encoded residues in CDR1 and CDR3 regions of TRBV4-1-encoded sequences interact with each other and consolidate the surface of the TCR. Mutational studies identified a key positively charged residue in TRBV4-1 and a key negatively charged residue in CD1b that is shared with CD1c, which is also recognized by TRBV4-1 TCRs. These data show that one TCR V region can mediate a mechanism of recognition of two related monomorphic Ag-presenting molecules that does not rely on a defined lipid Ag.

High prevalence and heterogeneity of Dysglycemia in patients with tuberculosis from Peru: a prospective cohort study.

BACKGROUND: The accuracy of different laboratory tests for diagnosis of diabetes mellitus (DM) and prediabetes (preDM) in populations exposed to tuberculosis (TB) remains poorly understood. Here, we examined the prevalence of DM and preDM in TB affected people in Lima, Peru. METHODS: A prospective cohort study of patients affected TB and their household contacts (HHC), was conducted between February and November 2017 in Lima, Peru. Fasting plasma glucose (FPG), HbA1c and oral glucose tolerance test (OGTT) were used to detect DM and preDM in a prospective cohort of TB patients (n = 136) and household contacts (n = 138). Diagnostic performance of the laboratory tests was analyzed. Potential effects of sociodemographic and clinical factors on detection of dysglycemia were analyzed. RESULTS: In TB patients, prevalence of DM and preDM was 13.97 and 30.88% respectively. Lower prevalence of both DM (6.52%) and preDM (28.99%) were observed in contacts. FPG, HbA1c and OGTT had poor agreement in detection of preDM in either TB cases or contacts. TB-DM patients had substantially lower hemoglobin levels, which resulted in low accuracy of HbA1c-based diagnosis. Classic sociodemographic and clinical characteristics were not different between TB patients with or without dysglycemia. CONCLUSION: High prevalence of DM and preDM was found in both TB patients and contacts in Lima. Anemia was strongly associated with TB-DM, which directly affected the diagnostic performance of HbA1c in such population.