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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.

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.

Despite the availability of highly efficacious vaccines, Coronavirus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome-related 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 system 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 mRNA 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 COVID-19 patients leading us to identify Tacrolimus, Zotatifin, and Nintedanib as three strong candidates for treatment of severely ill patients at the time of hospital admission.TeaserEpigenetics distinguishes COVID-19 survivors already at hospital admission: lessons for drug repurposing.

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 (Mtb) followed by rapid progression to disease. Alveolar macrophages (AM) are the first cells of the innate immune system that engage Mtb, but how HIV and antiretroviral therapy (ART) impact on the anti-mycobacterial response of AM is not known. To investigate the impact of HIV and ART on the transcriptomic and epigenetic response of AM to Mtb, we obtained AM by bronchoalveolar lavage from 20 PLWH receiving ART, 16 control subjects who were HIV-free (HC), and 14 subjects who received ART as pre-exposure prophylaxis (PrEP) to prevent HIV infection.Following in-vitro challenge with Mtb, AM from each group displayed overlapping but distinct profiles of significantly up- and down-regulated genes in response to Mtb. Compared to HC subjects, AM isolated from PLWH and PrEP subjects presented a substantially weaker transcriptional response. Further investigation of chromatin structure revealed that AM from control subjects challenged with Mtb responded with pronounced accessibility changes in over ten thousand regions. In stark contrast, AM obtained from PLWH and PrEP subjects displayed no significant changes in their chromatin state in response to Mtb. Collectively, these results revealed a previously unknown adverse effect of ART on the epigenetic landscape and transcriptional responsiveness of AM.

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., Author summary There is strong epidemiological evidence that a proportion of highly exposed individuals remain resistant to M. tuberculosis infection, as shown by a negative result for Tuberculin Skin Test (TST) or IFN-γ Release Assays (IGRAs). We performed a genome-wide association study between resistant and infected individuals, which were carefully selected employing a household contact design to maximize exposure by infectious index patients. We employed stringently defined concordant results for both TST and IGRA assays to avoid misclassifications. We discovered a locus at 10q26.2 associated with resistance to M. tuberculosis infection in a Vietnamese discovery cohort. This locus could be replicated in two independent cohorts from different epidemiological settings and of diverse ancestries enrolled in France and South Africa.

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 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.

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.

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-DRβ1 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., Author summary Despite global efforts to eliminate leprosy over the past 25 years, more than 200,000 new cases are reported annually, and leprosy still represents a major public health problem in endemic regions. Leprosy presents a strong link with the host genetic background. The most significant susceptibility factors are located in the MHC region and likely involve classical HLA genes. However, the molecular identity of the HLA class I/II-leprosy risk factor(s) has been a matter of longstanding scientific dispute. By conducting a comprehensive sequenced-based analysis of HLA class I and class II genes, we are able to provide a unifying view of the complex relationship of leprosy susceptibility and HLA alleles. In addition, we show that four amino acid polymorphisms in HLA-DRβ1, HLA-B and HLA-A are sufficient to explain the majority of leprosy-HLA associations which opens the way for select protein-HLA peptide binding studies.

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.

BackgroundMycobacterium 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).MethodsA 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.FindingsWe 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 counts3. All HITTIN sent for further antibody testing (n=38) displayed Mtb-specific antibody titres.InterpretationImmune reconstituted HIV+ persons can be persistently non-immunoreactive 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.FundingFunding provided by National Institutes of Health [1R01AI124349-01].Research in Context sectionEvidence before this studyThe majority of individuals who are exposed to Mycobacterium tuberculosis (Mtb) appear to have natural immunity against developing tuberculosis (TB). A subset of these individuals may clear or control Mtb without developing a classical T-cell immune response as measured by a tuberculin skin test (TST) or IGRA. While the gold standard for TB diagnosis is culturing Mtb from a specimen, there is no direct test to prove current Mtb infection. Hence, infection needs to be inferred from tests that measure Mtb T-cell immunoreactivity. Once Mtb is inhaled, pulmonary innate immune cells, so-called alveolar macrophages, are the first to make contact with the bacilli. Mtb-infected alveolar macrophages traverse from the alveoli to the lung interstitium where Mtb is transferred to inflammatory macrophages that present the bacilli to T-cells and initiate the adaptive immune response, including the generation of memory T-cells. The current Mtb T-cell immunoreactivity tests indicate infection if positive. A major caveat with these tests is an inability to distinguish between a current infection or a persistent immune response after a previous infection that was cleared. In addition it is important to clarify that persisting negative immunoreactivity does not simply imply that individuals are not infected. They could have had previous immunoreactivity which has reverted, could not have been exposed to Mtb or they were exposed but cleared Mtb without a classical IFN-γ T-cell immunoreactive response. The latter is a novel concept and previous studies in HIV-negative subjects have focused on this resistance to ‘infection’- or to be more precise failure of IFN-γ T-cell and TST immune conversion to Mtb. However, these studies found Mtb-specific antibodies in their HIV-negative ‘resister’ subjects. The presence of especially IgG antibodies confirmed and indicated long term exposure to Mtb and pointed to current or cleared infection. T-cell responses are required for antigen-specific B-cells to release class-switched IgG. Since these individuals test persistently negative for TST and IFN-γ T-cell immunoreactivity, innate and alternative adaptive IFN-γ independent T-cell responses should be investigated. This is a developing field and as research emerges, it is increasingly important to clearly characterize and define the TB resistance phenotype.Added value of this studyHIV-positive (HIV+) persons are at increased risk of infection with Mtb and rapid progression to tuberculosis making the phenotype we describe here especially important in this population segment. However, there is a major lack of data in HIV+ persons describing persisting lack of response to TST and Mtb IFN-γ T-cell immune conversion. Here we show that this phenotype can be identified in immune-reconstituted HIV+ persons. We describe the recruitment and define this conversion resistance phenotype as HIV-1-positive persistently TB, tuberculin and IGRA negative (HITTIN). The presence of specific antibodies confirms exposure to Mtb. Our study suggests possible innate mechanisms and non-classical adaptive mechanisms that subvert early stages of tuberculosis pathogenesis in a substantial proportion of HIV-infected patients. Harnessing such TST and IFN-γ T- cell independent mechanisms of resistance is of particular interest for prevention of tuberculosis in the HIV+ population.Implications of all the available evidenceUnderstanding the mechanisms of resistance will enable us to develop TB prevention and treatment modalities.

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

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 CD4+CCR6+, Th1Th17 (CD4+CCR6+CCR4−CXCR3+), CD4+CCR6+CXCR3−CCR4−, and CD4+CD32a+ 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.

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 genomic approaches. This short review aims to summarize recent advances and to provide a link with results of previous human genetic studies of tuberculosis susceptibility. 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. Future studies need to build on the latest molecular findings to define disease subtypes to successfully elucidate the human genetic component in tuberculosis pathogenesis.

Significance Type-1 reactions (T1R) are pathological immune responses in leprosy and a frequent cause of peripheral nerve damage. Employing a candidate gene approach combined with deep resequencing, we identified amino acid mutations in the E3 ligase Parkin and the polyfunctional kinase LRRK2 that were associated with T1R. This finding directly linked both proteins with the extent of the immune response in an infectious disease. Moreover, amino acids associated with T1R mutations were significantly enriched for mutations found in patients suffering from Parkinson’s disease (PD). These findings confirm Parkin and LRRK2 as 2 key inflammatory regulators and suggest that T1R and PD share overlapping pathways of pathogenesis., 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.

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., Author summary Due to its extreme reductive evolution Mycobacterium leprae is a rare example of an essentially clonal bacterial pathogen that affects humans. However, exposed individuals display a wide diversity of symptoms reflecting the interactions between the host immune response and the bacterium. There is now accumulating evidence, in particular from genome-wide association study (GWAS), that common genetic variants play a role explaining this variability. Previous GWAS were based on case control design and almost all of them were conducted in the Chinese population. We conducted the first family-based GWAS of leprosy in the Vietnamese population and identified several genome-wide significant association signals within the HLA region. By performing a multivariate analysis in an independent case-control sample of the same ethnic origin we were able to decipher the HLA association signal and to reduce it to three independent SNPs, two in the class I and one in the class II region. We also validated two previously identified risk factors for leprosy, a missense variant in the LACC1 gene, and an intergenic variant located close to the IL12B gene. These results shed new light on the genetic control of leprosy and, in particular, on the influence of HLA SNPs in a large Vietnamese sample.

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.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 withFuture studies need to build on the latest molecular findings to define disease subtypes to successfully elucidate the human genetic component in tuberculosis pathogenesis.

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

Leprosy is a human infectious disease caused by Mycobacterium leprae. A strong host genetic contribution to leprosy susceptibility is well established. However, the modulation of the transcriptional response to infection and the mechanism(s) of disease control are poorly understood. To address this gap in knowledge of leprosy pathogenicity, we conducted a genome-wide search for expression quantitative trait loci (eQTL) that are associated with transcript variation before and after stimulation with M. leprae sonicate in whole blood cells. We show that M. leprae antigen stimulation mainly triggered the upregulation of immune related genes and that a substantial proportion of the differential gene expression is genetically controlled. Indeed, using stringent criteria, we identified 318 genes displaying cis-eQTL at an FDR of 0.01, including 66 genes displaying response-eQTL (reQTL), i.e. cis-eQTL that showed significant evidence for interaction with the M. leprae stimulus. Such reQTL correspond to regulatory variations that affect the interaction between human whole blood cells and M. leprae sonicate and, thus, likely between the human host and M. leprae bacilli. We found that reQTL were significantly enriched among binding sites of transcription factors that are activated in response to infection, and that they were enriched among single nucleotide polymorphisms (SNPs) associated with susceptibility to leprosy per se and Type-I Reaction, and seven of them have been targeted by recent positive selection. Our study suggested that natural selection shaped our genomic diversity to face pathogen exposure including M. leprae infection., Author summary Each year, 200,000 new leprosy cases are reported worldwide. While there is unambiguous evidence for a role of host genetics in leprosy pathogenesis, the mechanisms by which the human host fights the infection are poorly understood. Here, we highlight the search for naturally occurring genetic variations that modulate gene expression levels following exposure to sonicate of Mycobacterium leprae, the bacterium causing the disease. Because M. leprae is not cultivable and the genuine immune cells involved in the host response during infection are still unknown, we performed a genome-wide search for such genetic variations after stimulation of whole-blood from leprosy patients with M. leprae sonicate. This design allowed to provide a general framework for the genetic control of host responses to M. leprae and outlined the contribution of host genetics to leprosy pathogenesis. Among the M. leprae-dependent genetic regulators of gene expression levels there was an enrichment of variants (i) associated with leprosy, (ii) located in transcription factor binding sites and (iii) targeted by recent positive selection.

Leprosy Type-1 Reactions (T1Rs) are pathological inflammatory responses that afflict a sub-group of leprosy patients and result in peripheral nerve damage. Here, we employed a family-based GWAS in 221 families with 229 T1R-affect offspring with stepwise replication to identify risk factors for T1R. We discovered, replicated and validated T1R-specific associations with SNPs located in chromosome region 10p21.2. Combined analysis across the three independent samples resulted in strong evidence of association of rs1875147 with T1R (p = 4.5x10-8; OR = 1.54, 95% CI = 1.32–1.80). The T1R-risk locus was restricted to a lncRNA-encoding genomic interval with rs1875147 being an eQTL for the lncRNA. Since a genetic overlap between leprosy and inflammatory bowel disease (IBD) has been detected, we evaluated if the shared genetic control could be traced to the T1R endophenotype. Employing the results of a recent IBD GWAS meta-analysis we found that 10.6% of IBD SNPs available in our dataset shared a common risk-allele with T1R (p = 2.4x10-4). This finding points to a substantial overlap in the genetic control of clinically diverse inflammatory disorders., Author summary Leprosy still affects approximately 200,000 new victims each year. A major challenge of leprosy control is the prevention of permanent disability due to nerve damage. Nerve damage occurs if leprosy remains undiagnosed for extended periods or when patients undergo pathological inflammatory responses termed Type-1 Reactions (T1R). T1R is a rare example where beneficial inflammatory responses are temporal separated from host pathological responses. There is strong experimental evidence that supports a role of host genetic factors in T1R susceptibility. Here, we employed a genome-wide association study (GWAS) to investigate susceptibility factors for T1R in Vietnamese families. We followed up the initial GWAS findings in independent population samples from Vietnam and Brazil and identified a set of cis-eQTL genetic variants for the ENSG00000235140 lncRNA as global risk factors for T1R. To test our proposal that T1R is a strong model for pathological inflammatory responses we evaluated if inflammatory bowel disease (IBD) genetic risk-factors were enriched among T1R risk factors. We observed that more than 10% of IBD-risk loci were nominally associated with risk for T1R suggesting a shared mechanism of excessive inflammatory response in the both disease etiologies.

Background. Type 1 reactions (T1R) affect a considerable proportion of patients with leprosy. In those with T1R, the host immune response pathologically overcompensates for the actual infectious threat, resulting in nerve damage and permanent disability. Based on the results of a genome-wide association study of leprosy per se, we investigated the TNFSF15 chromosomal region for a possible contribution to susceptibility to T1R. Methods. We performed a high-resolution association scan of the TNFSF15 locus to evaluate the association with T1R in 2 geographically and ethnically distinct populations: a family-based sample from Vietnam and a case-control sample from Brazil, comprising a total of 1768 subjects. Results. In the Vietnamese sample, 47 single-nucleotide polymorphisms (SNPs) overlapping TNFSF15 and the adjacent TNFSF8 gene were associated with T1R but not with leprosy. Of the 47 SNPs, 39 were cis-expression quantitative trait loci (cis-eQTL) for TNFSF8 including SNPs located within the TNFSF15 gene. In the Brazilian sample, 18 of these cis-eQTL SNPs overlapping the TNFSF8 gene were validated for association with T1R. Conclusions. Taken together, these results indicate TNFSF8 and not TNFSF15 as an important T1R susceptibility gene. Our data support the need for infection genetics to go beyond genes for pathogen control to explore genes involved in a commensurate host response.

Multiphoton micro-spectroscopy, employing diffraction optics and electron-multiplying CCD (EMCCD) cameras, is a suitable method for determining protein complex stoichiometry, quaternary structure, and spatial distribution in living cells using Förster resonance energy transfer (FRET) imaging. The method provides highly resolved spectra of molecules or molecular complexes at each image pixel, and it does so on a timescale shorter than that of molecular diffusion, which scrambles the spectral information. Acquisition of an entire spectrally resolved image, however, is slower than that of broad-bandwidth microscopes because it takes longer times to collect the same number of photons at each emission wavelength as in a broad bandwidth. Here, we demonstrate an optical micro-spectroscopic scheme that employs a laser beam shaped into a line to excite in parallel multiple sample voxels. The method presents dramatically increased sensitivity and/or acquisition speed and, at the same time, has excellent spatial and spectral resolution, similar to point-scan configurations. When applied to FRET imaging using an oligomeric FRET construct expressed in living cells and consisting of a FRET acceptor linked to three donors, the technique based on line-shaped excitation provides higher accuracy compared to the point-scan approach, and it reduces artifacts caused by photobleaching and other undesired photophysical effects.

Tuberculosis (TB), caused by the human pathogenic bacterium Mycobacterium tuberculosis, poses a major global health problem. The tubercle bacillus is transmitted from person to person by aerosol, but only a proportion of those in contact with infectious aerosol particles will become infected. If infection occurs, less than 10% of those infected will develop clinical signs of TB, while the majority will develop latent TB infection (LTBI). The identification and treatment of LTBI persons is a major aspect of TB control, especially in low-incidence, highly developed nations. In the absence of a gold standard test for latent TB, infection is inferred with the help of either the in vivo tuberculin skin test or in vitro interferon gamma release assays of anti-mycobacterial immunity. Recent work has observed high heritability of these immune assays indicating the critical role of the host genetic background on the establishment of infection and latency. Additional genetic studies have identified the host genetic background as an important covariate for the proper interpretation of the results obtained from LTBI assays. Taken together, these data suggest TB surveillance and control can likely be improved by including host genetic information into the interpretation of these widely used assays.

Saccharomyces cerevisiae Snf1 is a member of the conserved Snf1/AMP-activated protein kinase (Snf1/AMPK) family involved in regulating responses to energy limitation, which is detected by mechanisms that include sensing adenine nucleotides. Mitochondrial voltage-dependent anion channel (VDAC) proteins, also known as mitochondrial porins, are conserved in eukaryotes from yeast to humans and play key roles in mediating mitochondrial outer membrane permeability to small metabolites, including ATP, ADP, and AMP. We previously recovered the yeast mitochondrial porin Por1 (yVDAC1) from a two-hybrid screen for Snf1-interacting proteins. Here, we present evidence that Snf1 interacts with Por1 and its homolog Por2 (yVDAC2). Cells lacking Por1 and Por2, but not respiratory-deficient rho 0 cells lacking the mitochondrial genome, exhibit reduced Snf1 activation loop phosphorylation in response to glucose limitation. Thus, Por1 and Por2 contribute to the positive control of Snf1 protein kinase. Physical proximity to the VDAC proteins and mitochondrial surface could facilitate Snf1's ability to sense energy limitation.

Type-1 (T1R) and Type-2 (T2R) leprosy reactions (LR), which affect up to 50% of leprosy patients, are aggressive inflammatory episodes of sudden onset and highly variable incidence across populations. LR are often diagnosed concurrently with leprosy, but more frequently occur several months after treatment onset. It is not uncommon for leprosy patients to develop recurring reactional episodes; however, they rarely undergo both types of LR. Today, LR are the main cause of permanent disabilities associated with leprosy and represent a major challenge in the clinical management of leprosy patients. Although progress has been made in understanding the immunopathology of LR, the factors that cause a leprosy patient to suffer from LR are largely unknown. Given the impact that ethnic background has on the risk of developing LR, host genetic factors have long been suspected of contributing to LR. Indeed, polymorphisms in seven genes [Toll-like receptors (TLR)1, TLR2, nucleotide-binding oligomerisation domain containing 2, vitamin D receptor, natural resistance-associated macrophage protein 1, C4B and interleukin-6] have been found to be associated with one or more LR outcomes. The identification of host genetic markers with predictive value for LR would have a major impact on nerve damage control in leprosy. In this review, we present the recent advances achieved through genetic studies of LR.

After sustained exposure to Mycobacterium leprae, only a subset of exposed individuals develops clinical leprosy. Moreover, leprosy patients show a wide spectrum of clinical manifestations that extend from the paucibacillary (PB) to the multibacillary (MB) form of the disease. This “polarization” of leprosy has long been a major focus of investigation for immunologists because of the different immune response in these two forms. But while leprosy per se has been shown to be under tight human genetic control, few epidemiological or genetic studies have focused on leprosy subtypes. Using PubMed, we collected available data in English on the epidemiology of leprosy polarization and the possible role of human genetics in its pathophysiology until September 2015. At the genetic level, we assembled a list of 28 genes from the literature that are associated with leprosy subtypes or implicated in the polarization process. Our bibliographical search revealed that improved study designs are needed to identify genes associated with leprosy polarization. Future investigations should not be restricted to a subanalysis of leprosy per se studies but should instead contrast MB to PB individuals. We show the latter approach to be the most powerful design for the identification of genetic polarization determinants. Finally, we bring to light the important resource represented by the nine-banded armadillo model, a unique animal model for leprosy.

Although epidemiological evidence suggests a human genetic basis of pulmonary tuberculosis (PTB) susceptibility, the identification of specific genes and alleles influencing PTB risk has proven to be difficult. Previous genome-wide association (GWA) studies have identified only three novel loci with modest effect sizes in sub-Saharan African and Russian populations. We performed a GWA study of 550,352 autosomal SNPs in a family-based discovery Moroccan sample (on the full population and on the subset with PTB diagnosis at

One of the persistent challenges of genetic association studies is the replication of genetic marker-disease associations across ethnic groups. Here, we conducted high-density association mapping of PARK2/PACRG SNPs with leprosy and identified 69 SNPs significantly associated with leprosy in 198 single-case Vietnamese leprosy families. A total of 56 associated SNPs localized to the overlapping promoter regions of PARK2/PACRG. For this region, multivariate analysis identified four SNPs belonging to two major SNP bins (rs1333955, rs7744433) and two single SNP bins (rs2023004, rs6936895) that capture the combined statistical evidence (P = 1.1 × 10−5) for association among Vietnamese patients. Next, we enrolled a case–control sample of 364 leprosy cases and 370 controls from Northern India. We genotyped all subjects for 149 SNPs that capture >80 % of the genetic variation in the Vietnamese sample and found 24 SNPs significantly associated with leprosy. Multivariate analysis identified three SNPs (rs1333955, rs9356058 and rs2023004) that capture the association with leprosy (P

Snf1 protein kinase regulates responses to glucose limitation and other stresses. Snf1 activation requires phosphorylation of its T-loop threonine by partially redundant upstream kinases (Sak1, Tos3, and Elm1). Under favorable conditions, Snf1 is turned off by Reg1-Glc7 protein phosphatase. The reg1 mutation causes increased Snf1 activation and slow growth. To identify new components of the Snf1 pathway, we searched for mutations that, like snf1 , suppress reg1 for the slow-growth phenotype. In addition to mutations in genes encoding known pathway components ( SNF1 , SNF4 , and SAK1 ), we recovered “fast” mutations, designated fst1 and fst2 . Unusual morphology of the mutants in the Σ1278b strains employed here helped us identify fst1 and fst2 as mutations in the RasGAP genes IRA1 and IRA2 . Cells lacking Ira1, Ira2, or Bcy1, the negative regulatory subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA), exhibited reduced Snf1 pathway activation. Conversely, Snf1 activation was elevated in cells lacking the Gpr1 sugar receptor, which contributes to PKA signaling. We show that the Snf1-activating kinase Sak1 is phosphorylated in vivo on a conserved serine (Ser1074) within an ideal PKA motif. However, this phosphorylation alone appears to play only a modest role in regulation, and Sak1 is not the only relevant target of the PKA pathway. Collectively, our results suggest that PKA, which integrates multiple regulatory inputs, could contribute to Snf1 regulation under various conditions via a complex mechanism. Our results also support the view that, like its mammalian counterpart, AMP-activated protein kinase (AMPK), yeast Snf1 participates in metabolic checkpoint control that coordinates growth with nutrient availability.