Dynamics of pulmonary mucosal cytotoxic CD8 T-cells in people living with HIV under suppressive antiretroviral therapy.

BACKGROUND: Despite the success of antiretroviral therapy (ART), people living with HIV (PLWH) suffer from a high burden of pulmonary diseases, even after accounting for their smoking status. Cytotoxic CD8 T-cells are likely implicated in this phenomenon and may act as a double-edged sword. While being essential in viral infection control, their hyperactivation can also contribute to lung mucosal tissue damage. The effects of HIV and smoking on pulmonary mucosal CD8 T-cell dynamics has been a neglected area of research, which we address herein. METHODS: Bronchoalveolar lavage (BAL) fluid were obtained from ART-treated PLWH (median duration of supressed viral load: 9 years; smokers: n = 14; non-smokers: n = 21) and HIV-uninfected controls (smokers: n = 11; non-smokers: n = 20) without any respiratory symptoms or active infection. Lymphocytes were isolated and CD8 T-cell subsets and homing markers were characterized by multiparametric flow cytometry. RESULTS: Both smoking and HIV infection were independently associated with a significant increase in frequencies of total pulmonary mucosal CD8 T-cell. BAL CD8 T-cells were primarily CD69 + expressing CD103 and/or CD49a, at least one of the two granzymes (GzmA/GzmB), and little Perforin. Higher expression levels of CD103, CD69, and GzmB were observed in smokers versus non-smokers. The ex vivo phenotype of GzmA + and GzmB + cells revealed increased expression of CD103 and CXCR6 in smokers, while PLWH displayed elevated levels of CX3CR1 compared to controls. CONCLUSION: Smoking and HIV could promote cytotoxic CD8 T-cell retention in small airways through different mechanisms. Smoking likely increases recruitment and retention of GzmB + CD8 Trm via CXCR6 and CD103. Heightened CX3CR1 expression could be associated with CD8 non-Trm recruitment from the periphery in PLWH.

Mycobacterium tuberculosis resisters despite HIV exhibit activated T cells and macrophages in their pulmonary alveoli.

To understand natural resistance to Mycobacterium tuberculosis ( Mtb ) infection, we studied people living with HIV (PLWH) in an area of high Mtb transmission. Given that alveolar leukocytes may contribute to this resistance, we performed single cell RNA-sequencing of bronchoalveolar lavage cells, unstimulated or ex vivo stimulated with Mtb . We obtained high quality cells for 7 participants who were TST & IGRA positive (called LTBI) and 6 who were persistently TST & IGRA negative (called resisters). Alveolar macrophages (AM) from resisters displayed more of an M1 phenotype relative to LTBI AM at baseline. Alveolar lymphocytosis (10%-60%) was exhibited by 5/6 resisters, resulting in higher numbers of CD4 + and CD8 + IFNG -expressing cells at baseline and upon Mtb challenge than LTBI samples. Mycobactericidal granulysin was expressed almost exclusively by a cluster of CD8 + T cells that co-expressed granzyme B, perforin and NK cell receptors. For resisters, these poly-cytotoxic T cells over-represented activating NK cell receptors and were present at 15-fold higher numbers in alveoli compared to LTBI. Altogether, our results showed that alveolar lymphocytosis, with increased numbers of alveolar IFNG -expressing cells and CD8 + poly-cytotoxic T cells, as well as activated AM were strongly associated with protection from persistent Mtb infection in PLWH.

Neutrophil extracellular trap formation and gene programs distinguish TST/IGRA sensitization outcomes among Mycobacterium tuberculosis exposed persons living with HIV.

Persons living with HIV (PLWH) have an increased risk for tuberculosis (TB). After prolonged and repeated exposure, some PLWH never develop TB and show no evidence of immune sensitization to Mycobacterium tuberculosis (Mtb) as defined by persistently negative tuberculin skin tests (TST) and interferon gamma release assays (IGRA). This group has been identified and defined as HIV+ persistently TB, tuberculin and IGRA negative (HITTIN). To investigate potential innate mechanisms unique to individuals with the HITTIN phenotype we compared their neutrophil Mtb infection response to that of PLWH, with no TB history, but who test persistently IGRA positive, and tuberculin positive (HIT). Neutrophil samples from 17 HITTIN (PMNHITTIN) and 11 HIT (PMNHIT) were isolated and infected with Mtb H37Rv for 1h and 6h. RNA was extracted and used for RNAseq analysis. Since there was no significant differential transcriptional response at 1h between infected PMNHITTIN and PMNHIT, we focused on the 6h timepoint. When compared to uninfected PMN, PMNHITTIN displayed 3106 significantly upregulated and 3548 significantly downregulated differentially expressed genes (DEGs) (absolute cutoff of a log2FC of 0.2, FDR < 0.05) whereas PMNHIT demonstrated 3816 significantly upregulated and 3794 significantly downregulated DEGs following 6h Mtb infection. Contrasting the log2FC 6h infection response to Mtb from PMNHITTIN against PMNHIT, 2285 genes showed significant differential response between the two groups. Overall PMNHITTIN had a lower fold change response to Mtb infection compared to PMNHIT. According to pathway enrichment, Apoptosis and NETosis were differentially regulated between HITTIN and HIT PMN responses after 6h Mtb infection. To corroborate the blunted NETosis transcriptional response measured among HITTIN, fluorescence microscopy revealed relatively lower neutrophil extracellular trap formation and cell loss in PMNHITTIN compared to PMNHIT, showing that PMNHITTIN have a distinct response to Mtb.

A systems biology approach identifies candidate drugs to reduce mortality in severely ill patients with COVID-19.

Despite the availability of highly efficacious vaccines, coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lacks effective drug treatment, which results in a high rate of mortality. To address this therapeutic shortcoming, we applied a systems biology approach to the study of patients hospitalized with severe COVID. We show that, at the time of hospital admission, patients who were equivalent on the clinical ordinal scale displayed significant differential monocyte epigenetic and transcriptomic attributes between those who would survive and those who would succumb to COVID-19. We identified messenger RNA metabolism, RNA splicing, and interferon signaling pathways as key host responses overactivated by patients who would not survive. Those pathways are prime drug targets to reduce mortality of critically ill patients with COVID-19, leading us to identify tacrolimus, zotatifin, and nintedanib as three strong candidates for treatment of severely ill patients at the time of hospital admission.

Human alveolar macrophage metabolism is compromised during Mycobacterium tuberculosis infection.

Pulmonary macrophages have two distinct ontogenies: long-lived embryonically-seeded alveolar macrophages (AM) and bone marrow-derived macrophages (BMDM). Here, we show that after infection with a virulent strain of Mycobacterium tuberculosis (H37Rv), primary murine AM exhibit a unique transcriptomic signature characterized by metabolic reprogramming distinct from conventional BMDM. In contrast to BMDM, AM failed to shift from oxidative phosphorylation (OXPHOS) to glycolysis and consequently were unable to control infection with an avirulent strain (H37Ra). Importantly, healthy human AM infected with H37Ra equally demonstrated diminished energetics, recapitulating our observation in the murine model system. However, the results from seahorse showed that the shift towards glycolysis in both AM and BMDM was inhibited by H37Rv. We further demonstrated that pharmacological (e.g. metformin or the iron chelator desferrioxamine) reprogramming of AM towards glycolysis reduced necrosis and enhanced AM capacity to control H37Rv growth. Together, our results indicate that the unique bioenergetics of AM renders these cells a perfect target for Mtb survival and that metabolic reprogramming may be a viable host targeted therapy against TB.

Deep resequencing identifies candidate functional genes in leprosy GWAS loci.

Leprosy is the second most prevalent mycobacterial disease globally. Despite the existence of an effective therapy, leprosy incidence has consistently remained above 200,000 cases per year since 2010. Numerous host genetic factors have been identified for leprosy that contribute to the persistently high case numbers. In the past decade, genetic epidemiology approaches, including genome-wide association studies (GWAS), identified more than 30 loci contributing to leprosy susceptibility. However, GWAS loci commonly encompass multiple genes, which poses a challenge to define causal candidates for each locus. To address this problem, we hypothesized that genes contributing to leprosy susceptibility differ in their frequencies of rare protein-altering variants between cases and controls. Using deep resequencing we assessed protein-coding variants for 34 genes located in GWAS or linkage loci in 555 Vietnamese leprosy cases and 500 healthy controls. We observed 234 nonsynonymous mutations in the targeted genes. A significant depletion of protein-altering variants was detected for the IL18R1 and BCL10 genes in leprosy cases. The IL18R1 gene is clustered with IL18RAP and IL1RL1 in the leprosy GWAS locus on chromosome 2q12.1. Moreover, in a recent GWAS we identified an HLA-independent signal of association with leprosy on chromosome 6p21. Here, we report amino acid changes in the CDSN and PSORS1C2 genes depleted in leprosy cases, indicating them as candidate genes in the chromosome 6p21 locus. Our results show that deep resequencing can identify leprosy candidate susceptibility genes that had been missed by classic linkage and association approaches.

Alveolar macrophages from persons living with HIV show impaired epigenetic response to Mycobacterium tuberculosis.

Persons living with HIV (PLWH) are at increased risk of tuberculosis (TB). HIV-associated TB is often the result of recent infection with Mycobacterium tuberculosis (M. tuberculosis) followed by rapid progression to disease. Alveolar macrophages (AMs) are the first cells of the innate immune system that engage M. tuberculosis, but how HIV and antiretroviral therapy (ART) affect the anti-mycobacterial response of AMs is not known. To investigate the impact of HIV and ART on the transcriptomic and epigenetic response of AMs to M. tuberculosis, we obtained AMs by bronchoalveolar lavage from 20 PLWH receiving ART, 16 control subjects who were HIV-free (HC), and 14 subjects who received ART as preexposure prophylaxis (PrEP) to prevent HIV infection. Following in vitro challenge with M. tuberculosis, AMs from each group displayed overlapping but distinct profiles of significantly up- and downregulated genes in response to M. tuberculosis. Comparatively, AMs isolated from both PLWH and PrEP subjects presented a substantially weaker transcriptional response. In addition, AMs from HC subjects challenged with M. tuberculosis responded with pronounced chromatin accessibility changes while AMs obtained from PLWH and PrEP subjects displayed no significant changes in their chromatin state. Collectively, these results revealed a stronger adverse effect of ART than HIV on the epigenetic landscape and transcriptional responsiveness of AMs.

Genome-wide association study of resistance to Mycobacterium tuberculosis infection identifies a locus at 10q26.2 in three distinct populations.

The natural history of tuberculosis (TB) is characterized by a large inter-individual outcome variability after exposure to Mycobacterium tuberculosis. Specifically, some highly exposed individuals remain resistant to M. tuberculosis infection, as inferred by tuberculin skin test (TST) or interferon-gamma release assays (IGRAs). We performed a genome-wide association study of resistance to M. tuberculosis infection in an endemic region of Southern Vietnam. We enrolled household contacts (HHC) of pulmonary TB cases and compared subjects who were negative for both TST and IGRA (n = 185) with infected individuals (n = 353) who were either positive for both TST and IGRA or had a diagnosis of TB. We found a genome-wide significant locus on chromosome 10q26.2 with a cluster of variants associated with strong protection against M. tuberculosis infection (OR = 0.42, 95%CI 0.35-0.49, P = 3.71×10-8, for the genotyped variant rs17155120). The locus was replicated in a French multi-ethnic HHC cohort and a familial admixed cohort from a hyper-endemic area of South Africa, with an overall OR for rs17155120 estimated at 0.50 (95%CI 0.45-0.55, P = 1.26×10-9). The variants are located in intronic regions and upstream of C10orf90, a tumor suppressor gene which encodes an ubiquitin ligase activating the transcription factor p53. In silico analysis showed that the protective alleles were associated with a decreased expression in monocytes of the nearby gene ADAM12 which could lead to an enhanced response of Th17 lymphocytes. Our results reveal a novel locus controlling resistance to M. tuberculosis infection across different populations.

Peculiar Phenotypic and Cytotoxic Features of Pulmonary Mucosal CD8 T Cells in People Living with HIV Receiving Long-Term Antiretroviral Therapy.

People living with HIV have high burdens of chronic lung disease, lung cancers, and pulmonary infections despite antiretroviral therapy (ART). The rates of tobacco smoking by people living with HIV vastly exceed that of the general population. Furthermore, we showed that HIV can persist within the lung mucosa despite long-term ART. As CD8 T cell cytotoxicity is pivotal for controlling viral infections and eliminating defective cells, we explored the phenotypic and functional features of pulmonary versus peripheral blood CD8 T cells in ART-treated HIV+ and uninfected controls. Bronchoalveolar lavage fluid and matched blood were obtained from asymptomatic ART-treated HIV+ smokers (n = 11) and nonsmokers (n = 15) and uninfected smokers (n = 7) and nonsmokers (n = 10). CD8 T cell subsets and phenotypes were assessed by flow cytometry. Perforin/granzyme B content, degranulation (CD107a expression), and cytotoxicity against autologous Gag peptide-pulsed CD4 T cells (Annexin V+) following in vitro stimulation were assessed. In all groups, pulmonary CD8 T cells were enriched in effector memory subsets compared with blood and displayed higher levels of activation (HLA-DR+) and exhaustion (PD1+) markers. Significant reductions in proportions of senescent pulmonary CD28-CD57+ CD8 T cells were observed only in HIV+ smokers. Pulmonary CD8 T cells showed lower perforin expression ex vivo compared with blood CD8 T cells, with reduced granzyme B expression only in HIV+ nonsmokers. Bronchoalveolar lavage CD8 T cells showed significantly less in vitro degranulation and CD4 killing capacity than blood CD8 T cells. Therefore, pulmonary mucosal CD8 T cells are more differentiated, activated, and exhausted, with reduced killing capacity in vitro than blood CD8 T cells, potentially contributing to a suboptimal anti-HIV immune response within the lungs.

An observational study identifying highly tuberculosis-exposed, HIV-1-positive but persistently TB, tuberculin and IGRA negative persons with M. tuberculosis specific antibodies in Cape Town, South Africa.

BACKGROUND: Mycobacterium tuberculosis (Mtb) infection is inferred from positive results of T-cell immune conversion assays measuring Mtb-specific interferon gamma production or tuberculin skin test (TST) reactivity. Certain exposed individuals do not display T-cell immune conversion in these assays and do not develop TB. Here we report a hitherto unknown form of this phenotype: HIV-1-positive persistently TB, tuberculin and IGRA negative (HITTIN). METHODS: A community-based case-control design was used to systematically screen and identify adults living with HIV (HIV+), aged 35-60 years, who met stringent study criteria, and then longitudinally followed up for repeat IGRA and TST testing. Participants had no history of TB despite living in TB hyper-endemic environments in Cape Town, South Africa with a provincial incidence of 681/100,000. Mtb-specific antibodies were measured using ELISA and Luminex. FINDINGS: We identified 48/286 (17%) individuals who tested persistently negative for Mtb-specific T-cell immunoreactivity (three negative Quantiferon results and one TST = 0mm) over 206±154 days on average. Of these, 97·2% had documented CD4 counts<200 prior to antiretroviral therapy (ART). They had received ART for 7·0±3·0 years with a latest CD4 count of 505·8±191·4 cells/mm3. All HITTIN sent for further antibody testing (n=38) displayed  Mtb-specific antibody titres. INTERPRETATION: Immune reconstituted HIV+ persons can be persistently non-immunoreactive to TST and interferon-γ T-cell responses to Mtb, yet develop species-specific antibody responses. Exposure is evidenced by Mtb-specific antibody titres. Our identification of HIV+ individuals displaying a persisting lack of response to TST and IGRA T-cell immune conversion paves the way for future studies to investigate this phenotype in the context of HIV-infection that so far have received only scant attention.

HIV Infection and Persistence in Pulmonary Mucosal Double Negative T Cells In Vivo.

The lungs are relatively unexplored anatomical human immunodeficiency virus (HIV) reservoirs in the antiretroviral therapy (ART) era. Double negative (DN) T cells are a subset of T cells that lack expression of CD4 and CD8 (CD4- CD8-) and may have both regulatory and effector functions during HIV infection. Notably, circulating DN T cells were previously described as cellular HIV reservoirs. Here, we undertook a thorough analysis of pulmonary versus blood DN T cells of people living with HIV (PLWH) under ART. Bronchoalveolar lavage (BAL) fluid and matched peripheral blood were collected from 35 PLWH on ART and 16 uninfected volunteers without respiratory symptoms. Both PLWH and HIV-negative (HIV-) adults displayed higher frequencies of DN T cells in BAL versus blood, and these cells mostly exhibited an effector memory phenotype. In PLWH, pulmonary mucosal DN T cells expressed higher levels of HLA-DR and several cellular markers associated with HIV persistence (CCR6, CXCR3, and PD-1) than blood. We also observed that DN T cells were less senescent (CD28- CD57+) and expressed less immunosuppressive ectonucleotidase (CD73/CD39), granzyme B, and perforin in the BAL fluid than in the blood of PLWH. Importantly, fluorescence-activated cell sorter (FACS)-sorted DN T cells from the BAL fluid of PLWH under suppressive ART harbored HIV DNA. Using the humanized bone marrow-liver-thymus (hu-BLT) mouse model of HIV infection, we observed higher infection frequencies of lung DN T cells than those of the blood and spleen in both early and late HIV infection. Overall, our findings show that HIV is seeded in pulmonary mucosal DN T cells early following infection and persists in these potential cellular HIV reservoirs even during long-term ART.IMPORTANCE Reservoirs of HIV during ART are the primary reasons why HIV/AIDS remains an incurable disease. Indeed, HIV remains latent and unreachable by antiretrovirals in cellular and anatomical sanctuaries, preventing its eradication. The lungs have received very little attention compared to other anatomical reservoirs despite being immunological effector sites exhibiting characteristics ideal for HIV persistence. Furthermore, PLWH suffer from a high burden of pulmonary non-opportunistic infections, suggesting impaired pulmonary immunity despite ART. Meanwhile, various immune cell populations have been proposed to be cellular reservoirs in blood, including CD4- CD8- DN T cells, a subset that may originate from CD4 downregulation by HIV proteins. The present study aims to describe DN T cells in human and humanized mice lungs in relation to intrapulmonary HIV burden. The characterization of DN T cells as cellular HIV reservoirs and the lungs as an anatomical HIV reservoir will contribute to the development of targeted HIV eradication strategies.

The complex pattern of genetic associations of leprosy with HLA class I and class II alleles can be reduced to four amino acid positions.

Leprosy is a chronic disease caused by Mycobacterium leprae. Worldwide, more than 200,000 new patients are affected by leprosy annually, making it the second most common mycobacterial disease after tuberculosis. The MHC/HLA region has been consistently identified as carrying major leprosy susceptibility variants in different populations at times with inconsistent results. To establish the unambiguous molecular identity of classical HLA class I and class II leprosy susceptibility factors, we applied next-generation sequencing to genotype with high-resolution 11 HLA class I and class II genes in 1,155 individuals from a Vietnamese leprosy case-control sample. HLA alleles belonging to an extended haplotype from HLA-A to HLA-DPB1 were associated with risk to leprosy. This susceptibility signal could be reduced to the HLA-DRB1*10:01~ HLA-DQA1*01:05 alleles which were in complete linkage disequilibrium (LD). In addition, haplotypes containing HLA-DRB3~ HLA-DRB1*12:02 and HLA-C*07:06~ HLA-B*44:03~ HLA-DRB1*07:01 alleles were found as two independent protective factors for leprosy. Moreover, we replicated the previously associated HLA-DRB1*15:01 as leprosy risk factor and HLA-DRB1*04:05~HLA-DQA1*03:03 as protective alleles. When we narrowed the analysis to the single amino acid level, we found that the associations of the HLA alleles were largely captured by four independent amino acids at HLA-DRß1 positions 57 (D) and 13 (F), HLA-B position 63 (E) and HLA-A position 19 (K). Hence, analyses at the amino acid level circumvented the ambiguity caused by strong LD of leprosy susceptibility HLA alleles and identified four distinct leprosy susceptibility factors.

Family-based genome-wide association study of leprosy in Vietnam.

Leprosy is a chronic infectious disease of the skin and peripheral nerves with a strong genetic predisposition. Recent genome-wide approaches have identified numerous common variants associated with leprosy, almost all in the Chinese population. We conducted the first family-based genome-wide association study of leprosy in 622 affected offspring from Vietnam, followed by replication in an independent sample of 1181 leprosy cases and 668 controls of the same ethnic origin. The most significant results were observed within the HLA region, in which six SNPs displayed genome-wide significant associations, all of which were replicated in the independent case/control sample. We investigated the signal in the HLA region in more detail, by conducting a multivariate analysis on the case/control sample of 319 GWAS-suggestive HLA hits for which evidence for replication was obtained. We identified three independently associated SNPs, two located in the HLA class I region (rs1265048: OR = 0.69 [0.58-0.80], combined p-value = 5.53x10-11; and rs114598080: OR = 1.47 [1.46-1.48], combined p-value = 8.77x10-13), and one located in the HLA class II region (rs3187964 (OR = 1.67 [1.55-1.80], combined p-value = 8.35x10-16). We also validated two previously identified risk factors for leprosy: the missense variant rs3764147 in the LACC1 gene (OR = 1.52 [1.41-1.63], combined p-value = 5.06x10-14), and the intergenic variant rs6871626 located close to the IL12B gene (OR = 0.73 [0.61-0.84], combined p-value = 6.44x10-8). These results shed new light on the genetic control of leprosy, by dissecting the influence of HLA SNPs, and validating the independent role of two additional variants in a large Vietnamese sample.

Pleiotropic effects for Parkin and LRRK2 in leprosy type-1 reactions and Parkinson's disease.

Type-1 reactions (T1R) are pathological inflammatory episodes and main contributors to nerve damage in leprosy. Here, we evaluate the genewise enrichment of rare protein-altering variants in 7 genes where common variants were previously associated with T1R. We selected 474 Vietnamese leprosy patients of which 237 were T1R-affected and 237 were T1R-free matched controls. Genewise enrichment of nonsynonymous variants was tested with both kernel-based (sequence kernel association test [SKAT]) and burden methods. Of the 7 genes tested 2 showed statistical evidence of association with T1R. For the LRRK2 gene an enrichment of nonsynonymous variants was observed in T1R-free controls (PSKAT-O = 1.6 × 10-4). This genewise association was driven almost entirely by the gain-of-function variant R1628P (P = 0.004; odds ratio = 0.29). The second genewise association was found for the Parkin coding gene PRKN (formerly PARK2) where 7 rare variants were enriched in T1R-affected cases (PSKAT-O = 7.4 × 10-5). Mutations in both PRKN and LRRK2 are known causes of Parkinson's disease (PD). Hence, we evaluated to what extent such rare amino acid changes observed in T1R are shared with PD. We observed that amino acids in Parkin targeted by nonsynonymous T1R-risk mutations were also enriched for mutations implicated in PD (P = 1.5 × 10-4). Hence, neuroinflammation in PD and peripheral nerve damage due to inflammation in T1R share overlapping genetic control of pathogenicity.

Neutrophils: Innate Effectors of TB Resistance?

Certain individuals are able to resist Mycobacterium tuberculosis infection despite persistent and intense exposure. These persons do not exhibit adaptive immune priming as measured by tuberculin skin test (TST) and interferon-γ (IFN-γ) release assay (IGRA) responses, nor do they develop active tuberculosis (TB). Genetic investigation of individuals who are able to resist M. tuberculosis infection shows there are likely a combination of genetic variants that contribute to the phenotype. The contribution of the innate immune system and the exact cells involved in this phenotype remain incompletely elucidated. Neutrophils are prominent candidates for possible involvement as primers for microbial clearance. Significant variability is observed in neutrophil gene expression and DNA methylation. Furthermore, inter-individual variability is seen between the mycobactericidal capacities of donor neutrophils. Clearance of M. tuberculosis infection is favored by the mycobactericidal activity of neutrophils, apoptosis, effective clearance of cells by macrophages, and resolution of inflammation. In this review we will discuss the different mechanisms neutrophils utilize to clear M. tuberculosis infection. We discuss the duality between neutrophils' ability to clear infection and how increasing numbers of neutrophils contribute to active TB severity and mortality. Further investigation into the potential role of neutrophils in innate immune-mediated M. tuberculosis infection resistance is warranted since it may reveal clinically important activities for prevention as well as vaccine and treatment development.

Genetic Resistance to Mycobacterium tuberculosis Infection and Disease.

Natural history studies of tuberculosis (TB) have revealed a spectrum of clinical outcomes after exposure to Mycobacterium tuberculosis, the cause of TB. Not all individuals exposed to the bacterium will become diseased and depending on the infection pressure, many will remain infection-free. Intriguingly, complete resistance to infection is observed in some individuals (termed resisters) after intense, continuing M. tuberculosis exposure. After successful infection, the majority of individuals will develop latent TB infection (LTBI). This infection state is currently (and perhaps imperfectly) defined by the presence of a positive tuberculin skin test (TST) and/or interferon gamma release assay (IGRA), but no detectable clinical disease symptoms. The majority of healthy individuals with LTBI are resistant to clinical TB, indicating that infection is remarkably well-contained in these non-progressors. The remaining 5-15% of LTBI positive individuals will progress to active TB. Epidemiological investigations have indicated that the host genetic component contributes to these infection and disease phenotypes, influencing both susceptibility and resistance. Elucidating these genetic correlates is therefore a priority as it may translate to new interventions to prevent, diagnose or treat TB. The most successful approaches in resistance/susceptibility investigation have focused on specific infection and disease phenotypes and the resister phenotype may hold the key to the discovery of actionable genetic variants in TB infection and disease. This review will not only discuss lessons from epidemiological studies, but will also focus on the contribution of epidemiology and functional genetics to human genetic resistance to M. tuberculosis infection and disease.

HIV persistence in mucosal CD4+ T cells within the lungs of adults receiving long-term suppressive antiretroviral therapy.

BACKGROUND: The lungs were historically identified as one of the major anatomic sites for HIV replication in the pre-antiretroviral therapy (ART) era. However, their contribution to HIV persistence in individuals under suppressive ART remains understudied. DESIGN: We assessed HIV persistence and comprehensively characterized pulmonary mucosal CD4 T cells in HIV-infected (HIV) individuals receiving long-term suppressive ART versus uninfected participants. METHODS: Bronchoalveolar lavage (BAL), bronchial biopsies, and matched peripheral blood were obtained from n = 24 HIV-infected adults receiving long-term suppressive ART (median: 9 years) and n = 8 healthy volunteers without respiratory symptoms. HIV-DNA and cell-associated HIV-RNA were quantified by ultra-sensitive PCR, and lung mucosal CD4 T-cell subsets were characterized by multiparameter flow cytometry. RESULTS: The levels of HIV-DNA were 13-fold higher in total BAL cells compared to blood. Importantly, FACS-sorted CD4 T cells from BAL contained greater levels of HIV-DNA compared to peripheral CD4 T cells. BAL CD4 T cells in HIV individuals were characterized mostly by an effector memory phenotype, whereas naive and terminally differentiated cells were underrepresented compared to blood. Furthermore, BAL CD4 T cells expressed higher levels of immune activation (HLA-DR/CD38) and senescence (CD57) markers. Importantly, BAL was enriched in T-cell subsets proposed to be preferential cellular HIV reservoirs, including memory CD4CCR6, Th1Th17 (CD4CCR6CCR4CXCR3), CD4CCR6CXCR3CCR4, and CD4CD32a T cells. CONCLUSION: The pulmonary mucosa represents an important immunological effector site highly enriched in activated and preferential CD4 T-cell subsets for HIV persistence during long-term ART in individuals without respiratory symptoms. Our findings raise new challenges for the design of novel HIV eradication strategies in mucosal tissues.

Mitochondrial Voltage-Dependent Anion Channel Protein Por1 Positively Regulates the Nuclear Localization of Saccharomyces cerevisiae AMP-Activated Protein Kinase.

Snf1 protein kinase of the yeast Saccharomyces cerevisiae is a member of the highly conserved eukaryotic AMP-activated protein kinase (AMPK) family, which is involved in regulating responses to energy limitation. Under conditions of carbon/energy stress, such as during glucose depletion, Snf1 is catalytically activated and enriched in the nucleus to regulate transcription. Snf1 catalytic activation requires phosphorylation of its conserved activation loop threonine (Thr210) by upstream kinases. Catalytic activation is also a prerequisite for Snf1's subsequent nuclear enrichment, a process that is mediated by Gal83, one of three alternate ß-subunits of the Snf1 kinase complex. We previously reported that the mitochondrial voltage-dependent anion channel (VDAC) proteins Por1 and Por2 play redundant roles in promoting Snf1 catalytic activation by Thr210 phosphorylation. Here, we show that the por1Δ mutation alone, which by itself does not affect Snf1 Thr210 phosphorylation, causes defects in Snf1 and Gal83 nuclear enrichment and Snf1's ability to stimulate transcription. We present evidence that Por1 promotes Snf1 nuclear enrichment by promoting the nuclear enrichment of Gal83. Overexpression of Por2, which is not believed to have channel activity, can suppress the localization and transcription activation defects of the por1Δ mutant, suggesting that the regulatory role played by Por1 is separable from its channel function. Thus, our findings expand the positive roles of the yeast VDACs in carbon/energy stress signaling upstream of Snf1. Since AMPK/Snf1 and VDAC proteins are conserved in evolution, our findings in yeast may have implications for AMPK regulation in other eukaryotes, including humans. IMPORTANCE AMP-activated protein kinases (AMPKs) sense energy limitation and regulate transcription and metabolism in eukaryotes from yeast to humans. In mammals, AMPK responds to increased AMP-to-ATP or ADP-to-ATP ratios and is implicated in diabetes, heart disease, and cancer. Mitochondria produce ATP and are generally thought to downregulate AMPK. Indeed, some antidiabetic drugs activate AMPK by affecting mitochondrial respiration. ATP release from mitochondria is mediated by evolutionarily conserved proteins known as voltage-dependent anion channels (VDACs). One would therefore expect VDACs to serve as negative regulators of AMPK. However, our experiments in yeast reveal the existence of an opposite relationship. We previously showed that Saccharomyces cerevisiae VDACs Por1 and Por2 positively regulate AMPK/Snf1 catalytic activation. Here, we show that Por1 also plays an important role in promoting AMPK/Snf1 nuclear localization. Our counterintuitive findings could inform research in areas ranging from diabetes to cancer to fungal pathogenesis.

Human Genomics of Mycobacterium tuberculosis Infection and Disease.

PURPOSE OF REVIEW: The study of the genetic basis of tuberculosis pathogenesis has benefited from powerful technological innovations, a more structured definition of latent and clinical manifestations of the disease, and the application of functional genomics approaches. This short review aims to summarize recent advances and to provide a link with results of previous human genetic studies of tuberculosis susceptibility. RECENT FINDINGS: Transcriptomics has been shown to be a useful tool to predict progression from latency to clinical disease while functional genomics has traced the molecular events that link pathogen-triggered gene expression and host genetics. Resistance to infection with Mycobacterium tuberculosis has been revealed to be strongly impacted by host genetics. Host genomics of clinical disease has been shown to be most powerful when focusing on carefully selected clinical entities and possibly by considering host pathogen combinations. SUMMARY: Future studies need to build on the latest molecular findings to define disease subtypes to successfully elucidate the human genetic component in tuberculosis pathogenesis.

An eQTL variant of ZXDC is associated with IFN-γ production following Mycobacterium tuberculosis antigen-specific stimulation.

There is a large inter-individual variability in the response to Mycobacterium tuberculosis infection. In previous linkage analyses, we identified a major locus on chromosome region 8q controlling IFN-γ production after stimulation with live BCG (Bacillus Calmette-Guérin), and a second locus on chromosome region 3q affecting IFN-γ production triggered by the 6-kDa early secretory antigen target (ESAT-6), taking into account the IFN-γ production induced by BCG (IFNγ-ESAT6BCG). High-density genotyping and imputation identified ~100,000 variants within each linkage region, which we tested for association with the corresponding IFN-γ phenotype in families from a tuberculosis household contact study in France. Significant associations were replicated in a South African familial sample. The most convincing association observed was that between the IFNγ-ESAT6BCG phenotype and rs9828868 on chromosome 3q (p = 9.8 × 10-6 in the French sample). This variant made a significant contribution to the linkage signal (p < 0.001), and a trend towards the same association was observed in the South African sample. This variant was reported to be an eQTL of the ZXDC gene, biologically linked to monocyte IL-12 production through CCL2/MCP1. The identification of rs9828868 as a genetic driver of IFNγ production in response to mycobacterial antigens provides new insights into human anti-tuberculosis immunity.