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BACKGROUND: Tuberculosis has been associated with genetic variation in host immunity. We hypothesized that single-nucleotide polymorphisms (SNPs) in SIGIRR, a negative regulator of Toll-like receptor/IL-1R signaling, are associated with susceptibility to tuberculosis. METHODS: We used a case-population study design in Vietnam with cases that had either tuberculous meningitis or pulmonary tuberculosis. We genotyped 6 SNPs in the SIGIRR gene region (including the adjacent genes PKP3 and TMEM16J) in a discovery cohort of 352 patients with tuberculosis and 382 controls. Significant associations were genotyped in a validation cohort (339 patients with tuberculosis, 376 controls). RESULTS: Three SNPs (rs10902158, rs7105848, rs7111432) were associated with tuberculosis in discovery and validation cohorts. The polymorphisms were associated with both tuberculous meningitis and pulmonary tuberculosis and were strongest with a recessive genetic model (odds ratios, 1.5-1.6; P = .0006-.001). Coinheritance of these polymorphisms with previously identified risk alleles in Toll-like receptor 2 and TIRAP was associated with an additive risk of tuberculosis susceptibility. CONCLUSIONS: These results demonstrate a strong association of SNPs in the PKP3-SIGIRR-TMEM16J gene region and tuberculosis in discovery and validation cohorts. To our knowledge, these are the first associations of polymorphisms in this region with any disease.

Although respiratory infections cause significant morbidity and mortality throughout the world, the immunologic factors that mediate host susceptibility to these infections remain poorly understood. The lung contains a vast surface at the host-environment interface and acts as a crucial barrier to invading pathogens. The lung is equipped with specialized epithelial and hematopoietic cells, which express pattern recognition receptors that act as both sentinels and mediators of pulmonary innate immunity. Toll-like receptors (TLRs) mediate a particularly critical role in pathogen recognition and subsequent initiation of the host immune response. In this review, we will summarize current knowledge of TLRs and their bacterial ligands and explore their role in respiratory infections. Moreover, we will highlight recent advances in the role of TLRs in pulmonary infections from a human immunogenetics perspective.

Genetic epidemiological studies of complex diseases often rely on data from the International HapMap Consortium for identification of single nucleotide polymorphisms (SNPs), particularly those that tag haplotypes. However, little is known about the relevance of the African populations used to collect HapMap data for study populations conducted elsewhere in Africa. Toll-like receptor (TLR) genes play a key role in susceptibility to various infectious diseases, including tuberculosis. We conducted full-exon sequencing in samples obtained from Uganda (n = 48) and South Africa (n = 48), in four genes in the TLR pathway: TLR2, TLR4, TLR6, and TIRAP. We identified one novel TIRAP SNP (with minor allele frequency [MAF] 3.2%) and a novel TLR6 SNP (MAF 8%) in the Ugandan population, and a TLR6 SNP that is unique to the South African population (MAF 14%). These SNPs were also not present in the 1000 Genomes data. Genotype and haplotype frequencies and linkage disequilibrium patterns in Uganda and South Africa were similar to African populations in the HapMap datasets. Multidimensional scaling analysis of polymorphisms in all four genes suggested broad overlap of all of the examined African populations. Based on these data, we propose that there is enough similarity among African populations represented in the HapMap database to justify initial SNP selection for genetic epidemiological studies in Uganda and South Africa. We also discovered three novel polymorphisms that appear to be population-specific and would only be detected by sequencing efforts.

The development of effective immunoprophylaxis against tuberculosis (TB) remains a global priority, but is hampered by a partially protective Bacillus Calmette-Guérin (BCG) vaccine and an incomplete understanding of the mechanisms of immunity to Mycobacterium tuberculosis. Although host genetic factors may be a primary reason for BCG's variable and inadequate efficacy, this possibility has not been intensively examined. We hypothesized that Toll-like receptor (TLR) variation is associated with altered in vivo immune responses to BCG. We examined whether functionally defined TLR pathway polymorphisms were associated with T cell cytokine responses in whole blood stimulated ex vivo with BCG 10 weeks after newborn BCG vaccination of South African infants. In the primary analysis, polymorphism TLR6_C745T (P249S) was associated with increased BCG-induced IFN-γ in both discovery (n = 240) and validation (n = 240) cohorts. In secondary analyses of the combined cohort, TLR1_T1805G (I602S) and TLR6_G1083C (synonymous) were associated with increased IFN-γ, TLR6_G1083C and TLR6_C745T were associated with increased IL-2, and TLR1_A1188T was associated with increased IFN-γ and IL-2. For each of these polymorphisms, the hypo-responsive allele, as defined by innate immunity signaling assays, was associated with increased production of TH1-type T cell cytokines (IFN-γ or IL-2). After stimulation with TLR1/6 lipopeptide ligands, PBMCs from TLR1/6-deficient individuals (stratified by TLR1_T1805G and TLR6_C745T hyporesponsive genotypes) secreted lower amounts of IL-6 and IL-10 compared to those with responsive TLR1/6 genotypes. In contrast, no IL-12p70 was secreted by PBMCs or monocytes. These data support a mechanism where TLR1/6 polymorphisms modulate TH1 T-cell polarization through genetic regulation of monocyte IL-10 secretion in the absence of IL-12. These studies provide evidence that functionally defined innate immune gene variants are associated with the development of adaptive immune responses after in vivo vaccination against a bacterial pathogen in humans. These findings could potentially guide novel adjuvant vaccine strategies as well as have implications for IFN-γ-based diagnostic testing for TB., Author Summary Tuberculosis (TB) is one of the leading infectious causes of death worldwide. The current vaccine for TB, BCG, is widely used but it is not highly effective in preventing disease. We investigated the role of host genetics in the immune response to BCG vaccination. We found that variants of innate immunity genes (TLR1 and TLR6) were associated with BCG-induced immune responses after vaccination. These findings may guide new strategies for vaccine development as well as diagnosis of TB.

Toll-like receptors (TLRs) are critical mediators of the immune response to pathogens. The influence of human TLR6 polymorphisms on susceptibility to infection is only partially understood. Most microbes contain lipopeptides recognized by TLR2/1 or TLR2/6 heterodimers. Our aim was to determine whether single nucleotide polymorphisms in TLR6 are associated with altered immune responses to lipopeptides and whole mycobacteria. We sequenced the TLR6 coding region in 100 healthy South African adults to assess genetic variation and determined associations between polymorphisms and lipopeptide- and mycobacteria-induced interleukin (IL)-6 production in whole blood. We found two polymorphisms, C745T and G1083C, that were associated with altered IL-6 secretion. G1083C was associated with altered IL-6 levels in response to lipopeptides, Mycobacterium tuberculosis lysate (Mtb lysate, P=0.018) and Bacille Calmette-Guerin (BCG P=0.039). The 745T allele was also associated with lower NF-κB signaling in response to di-acylated lipopeptide, PAM2 (P=0.019) or Mtb (P=0.026) in an HEK293 cell line reconstitution assay, compared with the 745C allele. We conclude that TLR6 polymorphisms may be associated with altered lipopeptide-induced cytokine responses and recognition of Mtb. These studies provide new insight into the role of TLR6 variation and the innate immune response to human infection.

Establishment of Tuberculosis infection begins with the successful entry and survival of the pathogen within macrophages. We previously showed that macrophage CD43 is required for optimal uptake and growth inhibition of Mycobacterium tuberculosis both in vitro and in vivo. Here, we explore the mechanisms by which CD43 restricts mycobacterial growth in murine macrophages. We found that although M. tuberculosis grows more readily in resting CD43-/- macrophages, priming of cells with IFN-gamma returns the bacterial growth rate to that seen in CD43+/+ cells. To discern the mechanisms by which M. tuberculosis exhibits enhanced growth within resting CD43-/- macrophages, we assessed the induction of inflammatory mediators in response to infection. We found that absence of CD43 resulted in reduced production of TNF-alpha, IL-12 and IL-6 by M. tuberculosis-infected macrophages. We also found that infected resting, but not activated CD43-/- macrophages, showed decreased apoptosis and increased necrosis. Exogenous addition of the pro-inflammatory cytokine TNF-alpha restored control of M. tuberculosis growth and induction of apoptosis to CD43+/+ levels. We propose that CD43 is involved in the inflammatory response to M. tuberculosis and, through the induction of pro-inflammatory mediators, can regulate apoptosis to control intracellular growth of the bacterium.

We explored the role of macrophage (Mφ) CD43, a transmembrane glycoprotein, in the pathogenesis of Mycobacterium tuberculosis. Using gene-deleted mice (CD43−/−), we assessed the association of the bacterium with distinct populations of Mφ and found that CD43−/− Mφ bound less M. tuberculosis than CD43+/+ Mφ. Increased infective doses did not abrogate this difference. However, reduced association due to the absence of CD43 could be overcome by serum components. Mφ from heterozygote mice, which express 50% of wild-type CD43, bound more bacteria than CD43−/− but less than CD43+/+, proving that the gene dose of CD43 correlates with binding of M. tuberculosis. Furthermore, the reduced ability of CD43−/− Mφ to bind bacteria was restricted to mycobacterial species. We also found that the survival and replication of M. tuberculosis within Mφ was enhanced significantly in the absence of CD43, making this the first demonstration that the mechanism of mycobacterial entry influences its subsequent growth. Most importantly, we show here that the absence of CD43 in mice aerogenically infected with M. tuberculosis results in an increased bacterial load during both the acute and chronic stages of infection and more rapid development of granulomas, with greater lung involvement and distinctive cellularity.