Search

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.

Developers of severe acute respiratory syndrome coronavirus 2 vaccines should consider some of the lessons from a “new” vaccine introduced in 1921, namely bacille Calmette-Guérin.

Tuberculosis (TB) has surpassed HIV to become the leading infectious killer of adults globally, causing almost 2 million deaths annually. Although this airborne disease has been treatable since 1948, global rates of TB have dropped less than two percent per year; an estimated 10 million incident cases continue to occur annually, including one million in children. While transmission of active disease is an important driver of the epidemic, the seedbed that feeds the epidemic is the more than two billion people estimated to have TB infection, five to ten percent of whom will progress to active disease during their lifetime. While any successful strategy aimed at TB elimination needs to address this reservoir of TB infection worldwide, much remains to be understood about host and pathogen factors that can be used to identify increased risk for progression to disease, and intervened upon to prevent progression from occurring. The Division of AIDS of the National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA, and the Harvard Medical School Center for Global Health Delivery-Dubai convened a group of scientists and stakeholders on September 28 and 29, 2017, to address knowledge gaps that affect our ability to rapidly find and treat individuals infected with Mycobacterium tuberculosis who are most likely to progress to active disease. The meeting identified a number of efforts underway to address this important gap in the collective ability to stop the global TB epidemic. Here, we review and outline the priority areas for research, diagnosis and treatment of TB infection that emerged from the meeting (Table 1), building on recent reviews in this area.

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.

N/A

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.

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

Complex traits are known to be influenced by a combination of environmental factors and rare and common genetic variants. However, detection of such multivariate associations can be compromised by low statistical power and confounding by population structure. Linear mixed effects models (LMM) can account for correlations due to relatedness but have not been applicable in high-dimensional (HD) settings where the number of fixed effect predictors greatly exceeds the number of samples. False positives or false negatives can result from two-stage approaches, where the residuals estimated from a null model adjusted for the subjects’ relationship structure are subsequently used as the response in a standard penalized regression model. To overcome these challenges, we develop a general penalized LMM with a single random effect called ggmix for simultaneous SNP selection and adjustment for population structure in high dimensional prediction models. We develop a blockwise coordinate descent algorithm with automatic tuning parameter selection which is highly scalable, computationally efficient and has theoretical guarantees of convergence. Through simulations and three real data examples, we show that ggmix leads to more parsimonious models compared to the two-stage approach or principal component adjustment with better prediction accuracy. Our method performs well even in the presence of highly correlated markers, and when the causal SNPs are included in the kinship matrix. ggmix can be used to construct polygenic risk scores and select instrumental variables in Mendelian randomization studies. Our algorithms are available in an R package available on CRAN (https://cran.r-project.org/package=ggmix)., Author summary This work addresses a recurring challenge in the analysis and interpretation of genetic association studies: which genetic variants can best predict and are independently associated with a given phenotype in the presence of population structure? Not controlling confounding due to geographic population structure, family and/or cryptic relatedness can lead to spurious associations. Much of the existing research has therefore focused on modeling the association between a phenotype and a single genetic variant in a linear mixed model with a random effect. However, this univariate approach may miss true associations due to the stringent significance thresholds required to reduce the number of false positives and also ignores the correlations between markers. We propose an alternative method for fitting high-dimensional multivariable models, which selects SNPs that are independently associated with the phenotype while also accounting for population structure. We provide an efficient implementation of our algorithm and show through simulation studies and real data examples that our method outperforms existing methods in terms of prediction accuracy and controlling the false discovery rate.

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.

Increased prevalence of latent tuberculosis infection (LTBI) has been observed among high-risk populations such as healthcare workers (HCWs). The results may depend on the method of LTBI assessment, interferon-gamma release assay (IGRA) and/or tuberculin skin test (TST). Here, we investigated the prevalence and risk factors for LTBI assessed by both IGRAs and TST in HCWs living in Morocco, a country with intermediate tuberculosis (TB) endemicity and high BCG vaccination coverage. HCWs were recruited in two Moroccan hospitals, Rabat and Meknes. All the participants underwent testing for LTBI by both IGRA (QuantiFERON-TB Gold In-Tube, QFT-GIT) and TST. Different combinations of IGRA and TST results defined the LTBI status. Risk factors associated with LTBI were investigated using a mixed-effect logistic regression model. The prevalence of LTBI among 631 HCWs (age range 18-60 years) varied from 40.7% (95%CI 36.9-44.5%) with QFT-GIT to 52% (95%CI 48.2-56.0%) with TST using a 10 mm cut-off. The highest agreement between QFT-GIT and TST (κ = 0.50; 95%CI 0.43-0.56) was observed with the 10 mm cut-off for a positive TST. For a definition of LTBI status using a double positive result for both QFT-GIT and TST, significant associations were found with the following risk factors: being male (OR = 2.21; 95%CI 1.40-3.49; p = 0.0007), belonging to age groups 35-44 years (OR = 2.43; 95%CI 1.45-4.06; p = 0.0007) and even more 45-60 years (OR = 4.81; 95%CI 2.72-8.52; p = 7.10-8), having a family history of TB (OR = 6.62; 95%CI 2.59-16.94; p = 8.10-5), and working at a pulmonology unit (OR = 3.64; 95%CI 1.44-9.23; p = 0.006). Smoking was associated with LTBI status when defined by a positive QFT-GIT result (OR = 1.89; 95%CI 1.12-3.21; p = 0.02). A high prevalence of LTBI was observed among HCWs in two Moroccan hospitals. Male gender, increased age, family history of TB, and working at a pulmonology unit were consistent risk factors associated with LTBI.

Tuberculosis claims more human lives than any other infectious disease. This alarming epidemic has fuelled the development of novel antimicrobials and diagnostics. However, public health interventions that interrupt transmission have been slow to emerge, particularly in HIV-endemic settings. Transmission of tuberculosis is complex, involving various environmental, bacteriological, and host factors, among which concomitant HIV infection is important. Preventing person-to-person spread is central to halting the epidemic and, consequently, tuberculosis transmission is now being studied with renewed interest. In this Series paper, we review recent advances in the understanding of tuberculosis transmission, from the view of source-case infectiousness, inherent susceptibility of exposed individuals, appending tools for predicting risk of disease progression, the biophysical nature of the contagion, and the environments in which transmission occurs and is sustained in populations. We focus specifically on how HIV infection affects these features with a view to describing novel transmission blocking strategies in HIV-endemic settings.

Background. Interferon γ (IFN-γ) release assays (IGRAs) provide an in vitro measurement of antimycobacterial immunity that is widely used as a test for Mycobacterium tuberculosis infection. IGRA outcomes are highly heritable in various populations, but the nature of the involved genetic factors remains unknown. Methods. We conducted a genome-wide linkage analysis of IGRA phenotypes in families from a tuberculosis household contact study in France and a replication study in families from South Africa to confirm the loci identified. Results. We identified a major locus on chromosome 8q controlling IFN-γ production in response to stimulation with live bacillus Calmette-Guerin (BCG; LOD score, 3.81; P = 1.40 × 10−5). We also detected a second locus, on chromosome 3q, that controlled IFN-γ levels in response to stimulation with 6-kDa early secretory antigen target, when accounting for the IFN-γ production shared with that induced by BCG (LOD score, 3.72; P = 1.8 × 10−5). Both loci were replicated in South African families, where tuberculosis is hyperendemic. These loci differ from those previously identified as controlling the response to the tuberculin skin test (TST1 and TST2) and the production of TNF-α (TNF1). Conclusions. The identification of 2 new linkage signals in populations of various ethnic origins living in different M. tuberculosis exposure settings provides new clues about the genetic control of human antimycobacterial immunity.

Setting Tuberculosis spondylodiscitis (TS), or Pott's disease, an extra-pulmonary form of tuberculosis (TB), is rare and difficult to diagnose in children. Some cases of severe TB in children were recently explained by inborn errors of immunity affecting the interleukin-12/interferon-gamma (IL-12/IFN-γ) axis. Objective To analyse clinical data on Moroccan children with TS, and to perform immunological and genetic explorations of the IL-12/IFN-γ axis. Design We studied nine children with TS diagnosed between 2012 and 2014. We investigated the IL-12/IFN-γ circuit by both whole-blood assays and sequencing of the coding regions of 14 core genes of this pathway. Results A diagnosis of TS was based on a combination of clinical, biological, histological and radiological data. QuantiFERON(®)-TB Gold In-Tube results were positive in 75% of patients. Whole-blood assays showed normal IL-12 and IFN-γ production in all but one patient, who displayed impaired decreased response to IL-12. No candidate disease-causing mutations were detected in the exonic regions of the 14 genes. Conclusions TS diagnosis in children remains challenging, and is based largely on imaging. Further investigations of TS in children are required to determine the role of genetic defects in pathways that may or may not be related to the IL-12/IFN-γ axis.

Mycobacterium tuberculosis is a leading cause of mortality worldwide and establishes a long-lived latent infection in a substantial proportion of the human population. Multiple lines of evidence suggest that some individuals are resistant to latent M. tuberculosis infection despite long-term and intense exposure, and we term these individuals ‘resisters’. In this Review, we discuss the epidemiological and genetic data that support the existence of resisters and propose criteria to optimally define and characterize the resister phenotype. We review recent insights into the immune mechanisms of M. tuberculosis clearance, including responses mediated by macrophages, T cells and B cells. Understanding the cellular mechanisms that underlie resistance to M. tuberculosis infection may reveal immune correlates of protection that could be utilized for improved diagnostics, vaccine development and novel host-directed therapeutic strategies. Resisters are individuals who show resistance to infection despite long-term, high exposure to Mycobacterium tuberculosis. In this Review, Simmons and colleagues discuss potential mechanisms underlying this resistance, such as those mediated by macrophages, T cells and B cells, and how an understanding of these mechanisms might aid in the development of therapies for tuberculosis.

A substantial proportion of subjects exposed to a contagious tuberculosis case display lack of tuberculin skin test (TST) reactivity. We previously mapped a major locus (TST1) controlling lack of TST reactivity in families from an area in South Africa where tuberculosis is hyperendemic. Here, we conducted a household tuberculosis contact study in a French area where the endemicity of tuberculosis is low. A genome-wide analysis of TST negativity identified a significant linkage signal (P3 × 10(-5)) in close vicinity of TST1. Combined analysis of the 2 samples increased evidence of linkage (P = 2.4 × 10(-6)), further implicating genetic factors located on 11p14-15. This region overlaps the TNF1 locus controlling mycobacteria-driven tumor necrosis factor α production.

Background. Tumor necrosis factor (TNF) is a key immune regulator of tuberculosis resistance, as exemplified by the highly increased risk of tuberculosis disease among individuals receiving TNF-blocker therapy. Methods. We determined the extent of TNF production after stimulation with BCG or BCG plus interferon gamma (IFN-γ) using a whole blood assay in 392 children belonging to 135 nuclear families from an area hyperendemic for tuberculosis in South Africa. We conducted classical univariate and bivariate genome-wide linkage analysis of TNF production using the data from both stimulation protocols by means of an extension of the maximum-likelihoodbinomial method for quantitative trait loci to multivariate analysis. Results. Stimulation of whole blood by either BCG or BCG plus IFN-γ resulted in a range of TNF release across subjects. Extent of TNF production following both stimulation protocols was highly correlated (r =0 .81). We failed to identify genetic linkage of TNF release when considering each stimulus separately. However, using a multivariate approach, we detected a major pleiotropic locus (P

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a major public health problem worldwide, resulting in 8.7 million new cases and 1.4 million deaths each year. One third of the world's population is exposed to M. tuberculosis and, after exposure, most, but not all, individuals become infected. Among infected subjects, only a minority (∼10%) will eventually develop clinical disease, which is typically either a primary, often extra-pulmonary, TB in children, or a reactivation, pulmonary TB in adults. Considerable genetic epidemiological evidence has accumulated to support a major role for human genetic factors in the development of TB. Numerous association studies with various candidate genes have been conducted in pulmonary TB, with very few consistent results. Recent genome-wide association studies revealed only a modest role for two inter-genic polymorphisms. However, a first major locus for pulmonary TB was mapped to chromosome 8q12-q13 in a Moroccan population after a genome-wide linkage screen. Using a similar strategy, two other major loci controlling TB infection were recently identified. While the precise identification of these major genes is ongoing, the other fascinating observation of these last years was the demonstration that TB can also reflect a Mendelian predisposition. Following the findings obtained in the syndrome of Mendelian susceptibility to mycobacterial diseases, several children with complete IL-12Rβ1 deficiency, were found to have severe TB as their sole phenotype. Overall, these recent findings provide the proof of concept that the human genetics of TB involves a continuous spectrum from Mendelian to complex predisposition with intermediate major gene involvement. The understanding of the molecular genetic basis of TB will have fundamental immunological and medical implications, in particular for the development of new vaccines and treatments.

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.

The tuberculin skin test (TST) measures the intensity of antimycobacterial acquired immunity and is used to diagnose latent infection with Mycobacterium tuberculosis. We report evidence for a codominant gene explaining ∼65% of the TST variability. Disregarding the host genetic background may lead to misclassifications of TST-based diagnosis of latent M. tuberculosis infection.

Mycobacteria, the pathogens that cause tuberculosis and leprosy, establish long-term infections in host macrophages. Recent studies, including two genetic screens reported in this issue of Cell (Kumar et al., 2010; Tobin et al., 2010), reveal that virulent mycobacteria evade the host immune system by stimulating production of anti-inflammatory molecules and inhibiting autophagy.

Susceptibility to Mycobacterium bovis Bacille Calmette-Guérin (BCG) is genetically controlled by Nramp1 (Slc11a1). Inbred mouse strains harbor either the resistance (Nramp1(G169)) or the susceptibility (Nramp1(D169)) allele at Nramp1. Mus spretus (Nramp1(G169); SPRET/EiJ) is shown to display an intermediate level of BCG replication in the spleen (log(10) colony-forming units (CFU) approximately 5), compared to resistant A/J (log(10)CFU approximately 4.0) and susceptible C57BL/6J (log(10)CFU approximately 6.0) mice. The presence of genetic modifiers of Nramp1-dependent susceptibility to M. bovis (BCG) infection in Mus spretus was analyzed by whole-genome scanning in 175 mice of an informative (C57BL/6J x SPRET/EiJ) x C57BL/6J backcross. Nramp1 showed a major effect (D1Mcg4, P1e(-4)), but additional single marker effects were identified on chromosomes 4 (D4Mit150) and x (DXMit249) in male mice, and on chromosome 9 (D9Mit77) and 17 (D17Mit81) in female mice. A strong interaction between Nramp1 and the major histocompatibility locus was also noted in female mice. The mapped loci may act as modifiers of Nramp1 action, and constitute novel entry points for the parallel search of loci regulating susceptibility to mycobacterial infections in humans.

Efforts to explore a mycobacterial origin for Crohn's disease typically have involved an epidemiological approach, searching for Mycobacterium avium subsp. paratuberculosis in patient tissue. An alternative approach involves consideration of genetic and experimental data regarding host resistance to mycobacteria. From human and mycobacterial genetics, it is known that mycobacterial diseases depend on both pathogen and host factors and that tuberculosis and leprosy are effectively genetic diseases. The discovery of a number of Crohn's susceptibility genes, including NOD2/CARD15, demonstrates that Crohn's also is a complex genetic disease. Mutations in NOD2/CARD15 do not necessarily lead to Crohn's disease, so other mitigating factors, genetic and/or environmental, probably are required to produce illness. Recent work has shown that NOD2/CARD15 serves a role in bacterial sensing and activation of innate immune responses, providing a link between Crohn's genetics and an environmental factor, potentially a bacterial trigger. In this review, we discuss the current understanding of mycobacterial and Crohn's genetic susceptibility and review the evidence that NOD2/CARD15 may mediate host resistance to mycobacterial infection.

Tuberculosis, mainly caused by the pathogenic bacterium Mycobacterium tuberculosis, remains an inestimable public health problem, despite the established use of the Bacillus Calmette-Guerin (BCG) vaccine, multidrug therapy and the existence of global tuberculosis control programmes. Statistics show that nearly 2 billion people (approximately one-third of the world's population) are infected with M. tuberculosis. For unknown reasons, only about 10 per cent of those infected by M. tuberculosis will develop tuberculosis, resulting in 9 million new cases yearly and 2 million deaths. A better understanding of the host--mycobacterial--environmental interplay is central to developing better antituberculosis vaccines and treatments. This review will discuss how a clearer idea of this interplay is emerging with new genomic strategies in mouse models.

Five disease-causing genes, including the IL12RB1 gene that encodes the beta 1 chain of the receptor for interleukin (IL)-12 (IL-12R beta 1), are known to be associated with the syndrome of Mendelian susceptibility to mycobacterial diseases. Some IL-12R beta 1-deficient patients present with tuberculosis as the only clinical phenotype. A comprehensive genetic study of IL12RB1 was conducted among 101 Moroccan families, including 157 offspring (age, >15 years) who had culture-positive pulmonary tuberculosis (PTB). The promoter, exons, and flanking intron regions of IL12RB1 in 40 randomly selected patients with PTB were entirely sequenced, leading to the detection of 19 variants (including 10 novel mutations). Blood cells obtained from individuals who were homozygous for any of the 13 most common variants responded to IL-12, indicating that these polymorphisms were not loss-of-function mutations. By use of a family-based study, 2 promoter polymorphisms that were in strong linkage disequilibrium were found to be associated with PTB, especially -2C-->T (odds ratio for CT or TT vs. CC, 2.69 [95% confidence interval, 1.19-6.09]). This result suggests that IL12RB1 polymorphisms might influence the risk of development of PTB in adults.

This chapter presents an overview of the specific mouse loci identified as playing a key role in susceptibility to infection with human bacterial pathogens. The role of natural resistance-associated macrophage protein 1 (Nramp1) in host defenses against clinically relevant strains of Mycobacterium tuberculosis and Mycobacterium leprae was addressed in genetic studies in humans from areas of endemic tuberculosis (The Gambia) and leprosy (South Vietnam) infection, using informative markers derived from the NRAMP1 gene region. Mice bearing naturally occurring (Nramp1D169) or experimentally induced (Nramp1-/-) mutations at Nramp1 are extremely susceptible to intravenous or subcutaneous infection with low doses of S. enterica serovar Typhimurium (103 CFU), with uncontrolled bacterial replication in the spleen and liver of susceptible mice leading to uniform death within 4 to 5 days of infection. Legionella pneumophila induces apoptosis during infection of permissive human macrophages and in alveolar epithelial cell lines. Avirulent L. pneumophila mutants cannot induce either apoptosis or caspase 3 activation, and specific inhibition of caspase 3 activity can block both L. pneumophila-induced apoptosis and cytopathogenicity. Hence, it seems likely that the number of described susceptibility and resistance loci for divergent bacterial infections in the mouse will increase dramatically over the coming years.

Background. Only a minority of individuals infected with Mycobacterium tuberculosis develop clinical tuberculosis. Genetic epidemiological evidence suggests that pulmonary tuberculosis has a strong human genetic component. Previous genetic findings in Mendelian predisposition to more severe mycobacterial infections, including by M. tuberculosis, underlined the importance of the interleukin 12 (IL-12)/interferon γ (IFN-γ) circuit in antimycobacterial immunity. Methods. We conducted an association study in Morocco between pulmonary tuberculosis and a panel of single-nucleotide polymorphisms (SNPs) covering 14 core IL-12/IFN-γ circuit genes. The analyses were performed in a discovery family-based sample followed by replication in a case-control population. Results. Out of 228 SNPs tested in the family-based sample, 6 STAT4 SNPs were associated with pulmonary tuberculosis (P = .0013–.01). We replicated the same direction of association for 1 cluster of 3 SNPs encompassing the promoter region of STAT4. In the combined sample, the association was stronger among younger subjects (pulmonary tuberculosis onset

The scope of the tuberculosis (TB) epidemic in the world today is enormous, with about 30 million active cases. Current research into preventing the spread of TB is focused on development of new drugs to inactivate Mycobacterium tuberculosis, the causative agent of TB, as well as on identifying the critical steps of host defense to infection with Mycobacteria, which might also yield therapeutic targets. Our infection genomics approach toward the latter strategy has been to isolate and characterize a mouse gene, Bcg (Nramp1), which controls natural susceptibility to infection with Mycobacteria, as well as Salmonella and Leishmania. Through comparative genomics, we have identified the homologous human NRAMP1 gene, alleles of which are now being used for tests of linkage with TB and leprosy.

The host response to mycobacterial infection including tuberculosis depends on genetically controlled host and bacterial factors and their interaction. A largely unknown aspect of this interaction is whether disease results from an additive and independent effect of host and pathogen or from specific host-pathogen combinations. The preferential association of specific mycobacterial strains with specific ethnic groups provided tentative evidence in favor of host-pathogen specificity in tuberculosis and is consistent with the hypothesis of host-mycobacterial co-adaptation. Substantial evidence for specificity has now been provided by animal models and human case-control association studies. These studies indicate that differences in the host response to infection are at least in part due to specific combinations of host genetic factors and genetic and phenotypic characteristics of the infecting mycobacterial strain.

International audience; Only a small fraction of individuals infected with Mycobacterium tuberculosis develop clinical tuberculosis (TB) in their lifetime. Genetic epidemiological evidence suggests a genetic determinism of pulmonary TB (PTB), but the molecular basis of genetic predisposition to PTB remains largely unknown. We used a positional-cloning approach to carry out ultrafine linkage-disequilibrium mapping of a previously identified susceptibility locus in chromosomal region 8q12-13 by genotyping 3,216 SNPs in a family-based Moroccan sample including 286 offspring with PTB. We observed 44 PTB-associated SNPs (p < 0.01), which were genotyped in an independent set of 317 cases and 650 controls from Morocco. A single signal, consisting of two correlated SNPs close to TOX, rs1568952 and rs2726600 (combined p = 1.1 × 10(-5) and 9.2 × 10(-5), respectively), was replicated. Stronger evidence of association was found in individuals who developed PTB before the age of 25 years (combined p for rs1568952 = 4.4 × 10(-8); odds ratio of PTB for AA versus AG/GG = 3.09 [1.99-4.78]). The association with rs2726600 (p = 0.04) was subsequently replicated in PTB-affected subjects under 25 years in a study of 243 nuclear families from Madagascar. Stronger evidence of replication in Madagascar was obtained for additional SNPs in strong linkage disequilibrium with the two initial SNPs (p = 0.003 for rs2726597), further confirming the signal. We thus identified around rs1568952 and rs2726600 a cluster of SNPs strongly associated with early-onset PTB in Morocco and Madagascar. SNP rs2726600 is located in a transcription-factor binding site in the 3' region of TOX, and further functional explorations will focus on CD4 T lymphocytes.

The host response to mycobacterial infection including tuberculosis depends on genetically controlled host and bacterial factors and their interaction. A largely unknown aspect of this interaction is whether disease results from an additive and independent effect of host and pathogen or from specific host-pathogen combinations. The preferential association of specific mycobacterial strains with specific ethnic groups provided tentative evidence in favor of host-pathogen specificity in tuberculosis and is consistent with the hypothesis of host-mycobacterial co-adaptation. Substantial evidence for specificity has now been provided by animal models and human case-control association studies. These studies indicate that differences in the host response to infection are at least in part due to specific combinations of host genetic factors and genetic and phenotypic characteristics of the infecting mycobacterial strain.

In an accident later known as the Lübeck disaster, 251 neonates were orally given three doses of the new Bacille Calmette–Guérin (BCG) antituberculosis (TB) vaccine contaminated with Mycobacterium tuberculosis. A total of 173 infants developed clinical or radiological signs of TB but survived the infection, while 72 died from TB. While some blamed the accident on BCG itself by postulating reversion to full virulence, such a possibility was conclusively disproven. Rather, by combining clinical, microbiological, and epidemiological data, the chief public health investigator Dr. A. Moegling concluded that the BCG vaccine had been contaminated with variable amounts of fully virulent M. tuberculosis. Here, we summarize the conclusions drawn by Moegling and point out three lessons that can be learned. First, while mortality was high (approximately 29%), the majority of neonates inoculated with M. tuberculosis eventually overcame TB disease. This shows the high constitutional resistance of humans to the bacillus. Second, four semiquantitative levels of contamination were deduced by Moegling from the available data. While at low levels of M. tuberculosis there was a large spread of clinical phenotypes reflecting a good degree of innate resistance to TB, at the highest dose, the majority of neonates were highly susceptible to TB. This shows the dominating role of dose for innate resistance to TB. Third, two infants inoculated with the lowest dose nevertheless died of TB, and their median time from inoculation to death was substantially shorter than for those who died after inoculation with higher doses. This suggests that infants who developed disease after low dose inoculation are those who are most susceptible to the disease. We discuss some implications of these lessons for current study of genetic susceptibility to TB., Author Summary The Lübeck disaster is a unique event in the history of tuberculosis when 251 newborns were accidentally infected with a virulent strain of Mycobacterium tuberculosis. The disaster happened while BCG was introduced as an anti-TB vaccine. In an exemplary multidisciplinary investigation, the disaster was shown to be due to the accidental contamination of BCG vaccine preparations with virulent M. tuberculosis and not BCG itself. A meticulous description of the accident, including epidemiological, histopathological, and bacteriological observations, was published; however, this report is only available in German, thus limiting access to the data. Here, we present a summary of the accident and its clinical consequences. Based on the results of the initial report, we draw three important conclusions: (i) overall, the newborns demonstrated remarkable resistance to virulent M. tuberculosis; (ii) the extent of contamination had a strong impact on the observed TB clinical spectrum, demonstrating the importance of the infectious dose for innate resistance to TB; and (iii) the possible effect of host genetics on TB susceptibility appeared most prominent in the context of a low dose of M. tuberculosis. The latter observation suggests that low exposure settings may be best suited for host genetics studies of susceptibility to TB.

The present study examined the differential contribution of host genetic background and mycobacterial pathogen variability to biological and mechanistic phenotypes of infection. For this purpose, A/J and C57BL/6J mice were infected intravenously with a low dose of Mycobacterium tuberculosis H37Rv or the Russia, Japan, and Pasteur substrains of Mycobacterium bovis bacille Calmette-Guérin (BCG). The pulmonary bacterial counts (number of CFU) and transcript levels of select cytokines (e.g., Ifng , Il12b , and Il4 ) at 1, 3, and 6 weeks postinfection were measured as biological and mechanistic phenotypes, respectively. The individual and combined impact of the host and mycobacteria on these phenotypes was assessed using three-way analysis of variance (ANOVA), which partitions phenotypic variation into host, pathogen, time, and interaction effects. All phenotypes, except pulmonary Il4 transcript levels, displayed evidence for host-mycobacterium specificity by means of significant interaction terms. Pulmonary expression profiles of 34 chemokines and chemokine-related genes were compared across the hosts and mycobacteria. The differences in induction of these immune messenger genes between A/J and C57BL/6J mice were modest and generally failed to reach significance. In contrast, the mycobacteria induced significant variance in a subset of the immune messenger genes, which was more evident in A/J mice relative to that in C57BL/6J mice. Overall, the results demonstrated the importance of considering the joint effects of the mycobacterial and host genetic backgrounds on susceptibility to mycobacterial infections.

Assessment of the contribution of host genetics to human tuberculosis is a long-standing research challenge. Evidence of genetic factors has come primarily from twin studies and risks to first-degree relatives of cases. In addition, inferences of strong genetic influences have come from anecdotal accounts of socially prominent families, population variation in TB incidence and susceptibility to infection, and secular changes in TB severity, incidence and mortality inferred from historical information of contact between different populations, as well as accidental inoculation of vaccinees with M. tuberculosis. Recently, a major tuberculosis susceptibility locus has been mapped to the long arm of human chromosome. A number of host genetic factors have been directly implicated in tuberculosis susceptibility but strong genetic effects on tuberculosis risk have been difficult to detect both by candidate gene and genome-wide association studies. The reason for our current inability to trace strong genetic effects is unknown. However, a number of possible explanations are supported by direct experimental data. For example, it has been shown that host genetic control of susceptibility is limited to specific host M. tuberculosis strain combinations. In addition, it is known that proper inclusion of gene environment interactions is of critical importance for the detection of strong host genetic effects on tuberculosis susceptibility. By contrast, few genetic studies stratify on M. tuberculosis or try to model gene-environment interactions. Until now, most of the human genetics studies in tuberculosis have focused on the identification of genetic variants that impact on progression from infection to disease. There are few studies that aim at the identification of genes that impact on resistance to infection with M. tuberculosis or genes that control the extent of antimycobacterial immunity. Yet, estimates of heritability for these quantitative traits provide clear evidence for an important role of host genetics in anti-mycobacterial immunity. Recent work involving scientists from South Africa, France and Canada has focused on the study of innate resistance to infection with M. tuberculosis. Employing the tuberculin skin test as a tool to evaluate resistance to infection, a major locus (TST1) on chromosomal region 11p14 was identified that T-cell independent resistance to M. tuberculosis. In addition, a second major locus (TST2), on chromosomal region 5p15 was identified that controls the intensity of T-cell mediated delayed type hypersensitivity (DTH) to tuberculin. These results pave the way for the understanding of the molecular mechanisms involved in resistance to M. tuberculosis infection in endemic areas (TST1), and for the identification of critical regulators of T-cell dependent DTH to tuberculin (TST2). The finding of a strong host genetic control of anti-mycobacterial immunity raises the questions to what extent host genetics will be a barrier to the development of a universally efficacious tuberculosis vaccine. In fact, epidemiological studies in highly endemic areas and experiments in animal models suggest a strong contribution of host genetic factors to vaccine efficacy making the identification of the corresponding genes one of the new frontiers of mycobacterial research.

Although many studies have compared in vitro TB diagnostic tests with the venerable tuberculin skin test (TST), there is little understanding of the quantitative relationship between critical measures of antimycobacterial immunity used to detect TB infection. We, therefore, decided to determine the degree of redundancy between quantitative read-outs of in vivo and in vitro assays of antimycobacterial immunity.We enrolled 475 healthy HIV-negative children and young adults living in a hyperendemic area of TB. We measured in vivo TST responses, and a 1:10 diluted 3- or 7-day whole-blood assay was used to determine the in vitro antigen-specific interferon (IFN)-gamma cytokine release. The frequency of antigen-specific IFN-gamma(+)CD4(+) and IFN-gamma(+)CD8(+) cells was tested using intracellular cytokine staining after 1 day incubation.In vivo TST responses segregated into two well-separated groups with either no measurable response (TST induration5 mm; n = 164) or a normally distributed group with TST indurationsor = 5 mm with peak at 15 mm (n = 260). In vitro assays provided a less pronounced separation of responders and nonresponders. Correlation analysis of responses among persons with TSTor = 5 mm demonstrated that extent of TST response was poorly correlated with IFN-gamma release (coefficients of correlation rho = 0.17-0.22) and frequency of IFN-gamma(+)CD4(+)/CD8(+) cells (rho = 0.05-0.17) across three stimulating antigens (Mycobacterium bovis bacillus Calmette-Guérin, purified protein derivative, early-secreted antigenic target-6).We conclude that in vivo and in vitro assays are nonredundant, complementary measures of antimycobacterial immunity. Both TST and in vitro assays provided valuable information about antimycobacterial immunity and by inference latent TB in the studied high-incidence TB settings.

Human antimycobacterial immunity is a critical component of tuberculosis (TB) pathogenesis that is often used to infer the presence of TB infection. We report high heritability (>50%) for in vitro secretion of tumor necrosis factor alpha and interferon gamma (IFN-gamma), and the frequency of antigen-specific IFN-gamma(+)CD4(+) and IFN-gamma(+)CD8(+) cells in the response of whole blood to mycobacterial challenge. In principal component analysis, the first 3 components explain 78% of the overall variance consistent with the effect of pleiotropic regulatory genes of human antimycobacterial immunity. These results directly demonstrate the pivotal role played by host genetics in quantitative measures of antimycobacterial immunity underlying immune diagnosis of TB infection.

Approximately 20% of persons living in areas hyperendemic for tuberculosis (TB) display persistent lack of tuberculin skin test (TST) reactivity and appear to be naturally resistant to infection by Mycobacterium tuberculosis. Among those with a positive response, the intensity of TST reactivity varies greatly. The genetic basis of TST reactivity is not known. We report on a genome-wide linkage search for loci that have an impact on TST reactivity, which is defined either as zero versus nonzero (TST-BINa) or as extent of TST in millimeters (TST–quantitative trait locus [QTL]) in a panel of 128 families, including 350 siblings, from an area of South Africa hyperendemic for TB. We detected a major locus (TST1) on chromosomal region 11p14 (P = 1.4 × 10−5), which controls TST-BINa, with a lack of responsiveness indicating T cell–independent resistance to M. tuberculosis. We also detected a second major locus (TST2) on chromosomal region 5p15 (P < 10−5), which controls TST-QTL or the intensity of T cell–mediated delayed type hypersensitivity (DTH) to tuberculin. Fine mapping of this region identified SLC6A3, encoding the dopamine transporter DAT1, as a promising gene for further studies. Our results pave the way for the understanding of the molecular mechanisms involved in resistance to M. tuberculosis infection in endemic areas (TST1) and for the identification of critical regulators of T cell–dependent DTH to tuberculin (TST2).

The Mendelian segregation of resistance to infection in different strains of mice infected with mycobacteria, Salmonella and Leishmania spp, all of which live in macrophages, is currently under close scrutiny. Here, Erwin Schurr and colleagues review the nature and function of the Bcg gene in controlling innate resistance to mycobacterial infection in mice and speculate on the occurrence of a possible human equivalent.

Polymorphic variants within the human natural resistance-associated macrophage protein-1 (NRAMP1, also known as SLC11A1) gene have been shown to impact on susceptibility to tuberculosis in different human populations. In the mouse, Nramp1 is expressed at the macrophage phagosomal membrane and its activity can be assayed by the relative acquisition of mannose 6-phosphate receptor (M6PR) in Salmonella-containing vacuoles. Based on this M6PR recruitment assay, we have now developed an assay in primary human macrophages to test the function of human NRAMP1 gene variants. First, we established that M6PR acquisition was significantly higher (P = 0.002) in human U-937 monocytic cell lines transfected with NRAMP1 as compared to untransfected U-937 cells. Second, the M6PR assay was shown to be highly reproducible for NRAMP1 activity in monocyte-derived macrophages (MDM) from healthy volunteers. Finally, the assay was investigated in MDM from pediatric tuberculosis patients and significantly lower NRAMP1 activity was detected in MDM from individuals homozygous for the NRAMP1-274 high-risk allele (CC genotype) in comparison to heterozygous individuals (CT genotype; P=0.013). The present study describes both an assay for human NRAMP1 functional activity and concomitant evidence for reduced NRAMP1 function in the common genetic variant shown to be associated with tuberculosis susceptibility in pediatric patients.

Interstitial lung macrophages from tuberculosis-susceptible I/St and tuberculosis-resistant A/Sn mice demonstrated significant constitutive differences in gene expression levels, whereas in vitro infection of these cells withMycobacterium tuberculosishad only a modulatory impact on gene expression. We conclude that intrinsic gene expression profiles are an important determinant of tuberculosis pathogenesis in mice.

Lungs are the central organ affected and targeted by Mycobacterium tuberculosis and immune processes in the lung are of critical importance in the pathogenesis of tuberculosis. A major lung defense against invading pathogens is provided by surfactant protein A, a multi-chain protein encoded by the SFTPA1 and SFTPA2 genes. Here, we investigated polymorphisms in the SFTPA1 and SFTPA2 genes for association with tuberculosis in 181 Ethiopian families comprising 226 tuberculosis cases. Four polymorphisms, SFTPA1 307A, SFTPA1 776T, SFTPA2 355C, and SFTPA2 751C, were associated with tuberculosis (P=0.00008; P=0.019, P=0.029 and P=0.042, respectively). Additional subgroup analysis in male, female and more severely affected patients provided evidence for SFTPA1/2-covariate interaction. Finally, out of five intragenic haplotypes identified in the SFTPA1 gene and nine identified in the SFTPA2 gene, 1A 3 was most significantly associated with tuberculosis susceptibility (P=0.026). These findings suggest that SFTPA1 and SFTPA2 modify the risk of tuberculosis susceptibility and that this risk is influenced by additional covariates.