Risk of nontyphoidal Salmonella bacteraemia in African children is modified by STAT4

Abstract

Nontyphoidal Salmonella (NTS) is a major cause of bacteraemia in Africa. The disease typically affects HIV-infected individuals and young children, causing substantial morbidity and mortality. Here we present a genome-wide association study (180 cases, 2677 controls) and replication analysis of NTS bacteraemia in Kenyan and Malawian children. We identify a locus in STAT4, rs13390936, associated with NTS bacteraemia. rs13390936 is a context-specific expression quantitative trait locus for STAT4 RNA expression, and individuals carrying the NTS-risk genotype demonstrate decreased interferon-γ (IFNγ) production in stimulated natural killer cells, and decreased circulating IFNγ concentrations during acute NTS bacteraemia. The NTS-risk allele at rs13390936 is associated with protection against a range of autoimmune diseases. These data implicate interleukin-12-dependent IFNγ-mediated immunity as a determinant of invasive NTS disease in African children, and highlight the shared genetic architecture of infectious and autoimmune disease.

Fig. 1

Association plot of NTS bacteraemia susceptibility at the STAT4 region in Kenyan children. NTS association at the STAT4 region under the recessive model in Kenyan discovery samples (n = 180 cases, 2677 controls). rs13390936 is highlighted in blue. SNPs are coloured according to the strength of linkage disequilibrium (r²) to rs13390936. SNPs in a credible SNP set, which includes the causal variant with >95% probability, are ringed with black circles

Fig. 2

RNA and protein phenotypes of rs13390936 genotype. Genotype–phenotype correlations are analysed by regression and analysis of variance. P values are calculated by F-tests with one degree of freedom. a rs13390936 genotype is not significantly associated with STAT4 RNA expression in unstimulated B cells, monocytes, neutrophils or NK cells. b Healthy European adults carrying the NTS-susceptibility (T) allele have reduced STAT4 expression following LPS (2 and 24 h) and IFNγ (24 h) stimulation in monocytes. c Gating strategy for IFNγ ⁺ NK cells. Representative IFNγ expression in naïve, and IL-12-simulated or NTS-simulated whole blood is shown for healthy European adult donors carrying the NTS-protective AA genotype (blue) and the NTS-susceptible TT genotype (red). d Healthy European adults with the NTS-susceptible (TT) genotype have significantly fewer IFNγ⁺ NK cells at 6 h following IL-12 (mean_AA = 1.54%, mean_TT = 0.91%) or NTS (mean_AA = 17.09%, mean_TT = 10.76%) stimulation. rs13390936 genotype does not significantly perturb IFNγ⁺ NK cell numbers in unstimulated whole blood (mean_AA = 0.25%, mean_TT = 0.19%). e Malawian children (total n = 106, 97 with complete covariate data included in final model; rs13390936: AA, n = 69; TA, n = 24; TT, n = 4) with the NTS-susceptibility (T) allele have reduced circulating levels of IFNγ during acute NTS bacteraemia (mean_AA = 613.9 pg/ml, mean_TT = 281.4 pg/ml) in a linear model adjusted for age, sex and NTS-associated co-morbidity (HIV infection, malnutrition and malaria). Box and whisker plots; boxes depict the upper and lower quartiles of the data, and whiskers depict the range of the data excluding outliers (outliers are defined as data-points >1.5× the inter-quartile range from the upper or lower quartiles)

Fig. 3

a STAT4 association with major bacterial pathogens in Kenyan children. Log-transformed odds ratios and 95% confidence intervals of rs13390936 association (recessive model) in Kenyan discovery samples. b Posterior probabilities of models of association at rs13390936: NULL, no association with any bacterial pathogen; SAME, the same effect across all bacterial pathogens; REL, related effects across all bacterial pathogens; NTS, a non-zero effect in NTS alone. In a comparison of all possible combinations of rs13390936 association with these pathogens, the model in which NTS alone (highlighted in red) is associated with rs13390936 is the most probable (Bayes factor cf. NULL = 1327). c STAT4 association with autoimmune disease in populations of European ancestry. Log-transformed odds ratios and 95% confidence intervals of rs16833239 (in perfect linkage disequilibrium with rs13390936 in European populations; r² = 1, D′ = 1) association (additive model) with autoimmune disease in European populations

Fig. 4

The role of STAT4 in the control of intracellular Salmonella infection. Internalization of NTS by a phagocyte, in a Salmonella-containing vacuole (SCV), results in interleukin-12 (IL-12) release. IL-12 signals via the IL-12 receptor complex on NK cells and T cells leading to the phosphorylation and activation of STAT4 (blue). Activated STAT4 drives IFNγ production from both T cells and NK cells, and T_H1 polarization. Released IFNγ activates infected phagocytes via the IFNγ receptor, resulting in the phosphorylation and homodimerization of STAT1 to form γ-activating factor (GAF), which upregulates anti-bacterial effector mechanisms. Genes causing Mendelian susceptibility to mycobacterial disease (MSMD) in which susceptibility to invasive NTS infection has been described are highlighted in pink. (Figure modified from reference [29], first published in Nature Reviews Immunology, volume 15, pages 452–463, 2015, by Nature Publishing Group)