James J. Gilchrist

Dysregulated humoral immunity to nontyphoidal Salmonella in HIV-infected African adults.

HIV and Salmonella HIV-positive individuals who are infected with nontyphoidal strains of Salmonella enterica often succumb to high morbidity and mortality. Why this is the case is unknown. MacLennan et al. (p. 508; see the Perspective by Moir and Fauci) have uncovered a dysregulated antibody response to Salmonella that is the likely culprit. Sera from HIV-infected individuals do a poor job of killing S. Typhimurium, despite surprisingly elevated antibody titers. Experiments showed that HIV-infected serum inhibited the power of normal serum to kill Salmonella. Inhibition was specific to antibodies against lipopolysaccharide (LPS), a component of the cell wall of Salmonella. Hence, HIV-infected sera was able to kill Salmonella strains lacking LPS, and removing LPS immunoglobulin G from infected sera permitted Salmonella killing. Thus, not only does HIV cause defects in cell-mediated immunity but it also seems to impair humoral immunity, with severe consequences for multiple infections. Abnormal antibody responses produced in HIV-infected individuals are ineffective at clearing food-poisoning bacteria. Nontyphoidal Salmonellae are a major cause of life-threatening bacteremia among HIV-infected individuals. Although cell-mediated immunity controls intracellular infection, antibodies protect against Salmonella bacteremia. We report that high-titer antibodies specific for Salmonella lipopolysaccharide (LPS) are associated with a lack of Salmonella-killing in HIV-infected African adults. Killing was restored by genetically shortening LPS from the target Salmonella or removing LPS-specific antibodies from serum. Complement-mediated killing of Salmonella by healthy serum is shown to be induced specifically by antibodies against outer membrane proteins. This killing is lost when excess antibody against Salmonella LPS is added. Thus, our study indicates that impaired immunity against nontyphoidal Salmonella bacteremia in HIV infection results from excess inhibitory antibodies against Salmonella LPS, whereas serum killing of Salmonella is induced by antibodies against outer membrane proteins.

Genetic susceptibility to invasive Salmonella disease

Invasive Salmonella disease, in the form of enteric fever and invasive non-typhoidal Salmonella (iNTS) disease, causes substantial morbidity and mortality in children and adults in the developing world. The study of genetic variations in humans and mice that influence susceptibility of the host to Salmonella infection provides important insights into immunity to Salmonella. In this Review, we discuss data that have helped to elucidate the host genetic determinants of human enteric fever and iNTS disease, alongside data from the mouse model of Salmonella infection. Considered together, these studies provide a detailed picture of the immunobiology of human invasive Salmonella disease.

Sequential Acquisition of T Cells and Antibodies to Nontyphoidal Salmonella in Malawian Children

Background. Salmonella Typhimurium (STm) remain a prominent cause of bacteremia in sub-Saharan Africa. Complement-fixing antibodies to STm develop by 2 years of age. We hypothesized that STm-specific CD4+ T cells develop alongside this process. Methods. Eighty healthy Malawian children aged 0–60 months were recruited. STm-specific CD4+ T cells producing interferon γ, tumor necrosis factor α, and interleukin 2 were quantified using intracellular cytokine staining. Antibodies to STm were measured by serum bactericidal activity (SBA) assay, and anti-STm immunoglobulin G antibodies by enzyme-linked immunosorbent assay. Results. Between 2006 and 2011, STm bacteremias were detected in 449 children <5 years old. STm-specific CD4+ T cells were acquired in infancy, peaked at 14 months, and then declined. STm-specific SBA was detectable in newborns, declined in the first 8 months, and then increased to a peak at age 35 months. Acquisition of SBA correlated with acquisition of anti–STm–lipopolysaccharide (LPS) immunoglobulin G (r = 0.329 [95% confidence interval, .552–.062]; P = .01) but not anti–STm–outer membrane protein or anti–STm-flagellar protein (FliC). Conclusions. Acquisition of STm-specific CD4+ T cells in early childhood is consistent with early exposure to STm or cross-reactive protein antigens priming this T-cell development. STm-specific CD4+ T cells seem insufficient to protect against invasive nontyphoidal Salmonella disease, but sequential acquisition of SBA to STm LPS is associated with a decline in its incidence.

Human genetic and metabolite variation reveals that methylthioadenosine is a prognostic biomarker and an inflammatory regulator in sepsis.

The substrate of the methionine salvage pathway, methylthioadenosine, is a predictor of death in sepsis patients. Sepsis is a deleterious inflammatory response to infection with high mortality. Reliable sepsis biomarkers could improve diagnosis, prognosis, and treatment. Integration of human genetics, patient metabolite and cytokine measurements, and testing in a mouse model demonstrate that the methionine salvage pathway is a regulator of sepsis that can accurately predict prognosis in patients. Pathway-based genome-wide association analysis of nontyphoidal Salmonella bacteremia showed a strong enrichment for single-nucleotide polymorphisms near the components of the methionine salvage pathway. Measurement of the pathway’s substrate, methylthioadenosine (MTA), in two cohorts of sepsis patients demonstrated increased plasma MTA in nonsurvivors. Plasma MTA was correlated with levels of inflammatory cytokines, indicating that elevated MTA marks a subset of patients with excessive inflammation. A machine-learning model combining MTA and other variables yielded approximately 80% accuracy (area under the curve) in predicting death. Furthermore, mice infected with Salmonella had prolonged survival when MTA was administered before infection, suggesting that manipulating MTA levels could regulate the severity of the inflammatory response. Our results demonstrate how combining genetic data, biomolecule measurements, and animal models can shape our understanding of disease and lead to new biomarkers for patient stratification and potential therapeutic targeting.

Lymphocyte Perturbations in Malawian Children with Severe and Uncomplicated Malaria

ABSTRACT Lymphocytes are implicated in immunity and pathogenesis of severe malaria. Since lymphocyte subsets vary with age, assessment of their contribution to different etiologies can be difficult. We immunophenotyped peripheral blood from Malawian children presenting with cerebral malaria, severe malarial anemia, and uncomplicated malaria (n = 113) and healthy aparasitemic children (n = 42) in Blantyre, Malawi, and investigated lymphocyte subset counts, activation, and memory status. Children with cerebral malaria were older than those with severe malarial anemia. We found panlymphopenia in children presenting with cerebral malaria (median lymphocyte count, 2,100/μl) and uncomplicated malaria (3,700/μl), which was corrected in convalescence and was absent in severe malarial anemia (5,950/μl). Median percentages of activated CD69+ NK (73%) and γδ T (60%) cells were higher in cerebral malaria than in other malaria types. Median ratios of memory to naive CD4+ lymphocytes were higher in cerebral malaria than in uncomplicated malaria and low in severe malarial anemia. The polarized lymphocyte subset profiles of different forms of severe malaria are independent of age. In conclusion, among Malawian children cerebral malaria is characterized by lymphocyte activation and increased memory cells, consistent with immune priming. In contrast, there are reduced memory cells and less activation in severe malaria anemia. Further studies are required to understand whether these immunological profiles indicate predisposition of some children to one or another form of severe malaria.

Polymorphism in a lincRNA Associates with a Doubled Risk of Pneumococcal Bacteremia in Kenyan Children

Bacteremia (bacterial bloodstream infection) is a major cause of illness and death in sub-Saharan Africa but little is known about the role of human genetics in susceptibility. We conducted a genome-wide association study of bacteremia susceptibility in more than 5,000 Kenyan children as part of the Wellcome Trust Case Control Consortium 2 (WTCCC2). Both the blood-culture-proven bacteremia case subjects and healthy infants as controls were recruited from Kilifi, on the east coast of Kenya. Streptococcus pneumoniae is the most common cause of bacteremia in Kilifi and was thus the focus of this study. We identified an association between polymorphisms in a long intergenic non-coding RNA (lincRNA) gene (AC011288.2) and pneumococcal bacteremia and replicated the results in the same population (p combined = 1.69 × 10−9; OR = 2.47, 95% CI = 1.84–3.31). The susceptibility allele is African specific, derived rather than ancestral, and occurs at low frequency (2.7% in control subjects and 6.4% in case subjects). Our further studies showed AC011288.2 expression only in neutrophils, a cell type that is known to play a major role in pneumococcal clearance. Identification of this novel association will further focus research on the role of lincRNAs in human infectious disease.

Genetic variants associated with non-typhoidal Salmonella bacteraemia in African children.

BACKGROUND Non-typhoidal Salmonella (NTS) causes invasive and frequently fatal disease in African children. Existing strategies to prevent, diagnose, and treat NTS disease are inadequate. An improved understanding of the biology of invasive Salmonella infection will facilitate the development of novel NTS control measures. Despite evidence in mice and man showing a clear role for host genetics in NTS susceptibility, there are no published studies investigating host genetic susceptibility to NTS in African populations. METHODS We conducted a genome-wide association study (SNP Array 6.0, Affymetrix, CA, USA) of NTS bacteraemia in Kenyan children, with replication in Malawian children. We assessed the function of NTS-associated variants in an expression quantitative trait locus (eQTL) dataset of interferon γ (IFNγ) and lipopolysaccharide-stimulated monocytes from 432 healthy European adults. Serum IFNγ (Bio-Plex immunoassay, Bio-Rad Laboratories, CA, USA) in Malawian NTS cases (n=106) during acute disease was correlated with genotype by linear regression. FINDINGS After whole-genome imputation and quality control, 180 Kenyan cases and 2677 controls were included in an association analysis at 7 951 614 (additive model) and 4 669 537 (genotypic model) loci. After quality control, 143 Malawian cases and 336 controls were included in the replication analysis. An intronic variant in STAT4 was associated (recessive model) with NTS in both Kenyan and Malawian children (Kenya p=5·6 × 10(-9), Malawi p=0·02, combined p=1·4 × 10(-9); odds ratio 7·2, 95% CI 3·8-13·5). The NTS-associated variant was an eQTL for STAT4 expression in IFNγ-stimulated monocytes (p=9·59 × 10(-6)), the NTS risk allele being associated with lower STAT4 expression. In Malawian children with NTS bacteraemia, the same NTS risk allele was associated with lower serum concentrations of IFNγ (p=0·02) at presentation. INTERPRETATION STAT4 is highly plausible as a susceptibility locus for invasive NTS disease. STAT4 mediates IFNγ release in T cells and natural killer cells in response to interleukin 12 (IL12). Individuals with rare mutations elsewhere in the IL12-IFNγ axis are at risk of disseminated NTS infection. We provide the first evidence, to our knowledge, of a host genetic determinant of NTS disease in African children, and of a STAT4 variant conferring susceptibility to an infectious disease in man. FUNDING Wellcome Trust.

Context-specific regulation of monocyte surface IL7R expression and soluble receptor secretion by a common autoimmune risk allele

IL-7 is a key factor in T-cell immunity and IL7R polymorphisms are implicated in autoimmune pathogenesis. We previously reported an expression quantitative trait locus (eQTL) at rs931555, 59 to IL7R, associated with stimulated monocyte IL7R mRNA expression. Unlike in T-cells, a role for IL7R in monocyte biology is poorly described. Here we detail replication and characterization of this eQTL at protein level across cell subsets and conditions in a separate cohort. We find rs6897932, a non-synonymous IL7R polymorphism associated with susceptibility to Multiple Sclerosis, Ankylosing Spondylitis and Primary Biliary Cirrhosis, is the key determinant of monocyte IL7R surface expression and soluble IL7R (sIL7R) and functions in a context-specific manner. Monocyte surface IL7R is markedly induced by LPS and TNF stimulation under the genotypic regulation of rs6897932, whereas no effect of this allele was observed on CD4+, CD8+ or CD56+ cell surface IL7R or in unstimulated monocytes. LPS-induced monocyte release of sIL7R was strongly associated with both rs6897932 genotype and expression of the splicing factor gene DDX39A. After induction of IL7R expression, human monocytes display a robust and pleiotropic transcriptional response to exogenous IL-7. Monocytes from the synovial fluid of patients with Spondyloarthritis were similarly found to express high levels of surface IL7R. These data demonstrate that disease-associated genetic variants in the IL7R gene critically impact monocyte IL7R and sIL7R expression following innate immune stimulation, suggesting a previously unappreciated key role for monocytes in IL-7 pathway biology and IL7R-associated diseases.IL-7 is a key factor in T-cell immunity and IL7R polymorphisms are implicated in autoimmune pathogenesis. IL7R mRNA is induced in stimulated monocytes in a genetically determined manner, yet a role for IL7R in monocyte biology remains unexplored. Here we characterize genetic regulation of IL7R at the protein level across multiple cell subsets and conditions in healthy individuals. We find monocyte surface and soluble IL7R (sIL7R) protein are markedly expressed in response to lipopolysaccharide (LPS). We further demonstrate alleles of rs6897932, a non-synonymous IL7R polymorphism associated with susceptibility to Multiple Sclerosis, Ankylosing Spondylitis and Primary Biliary Cirrhosis, form the key determinant of both surface IL7R and sIL7R in the context of inflammation. No effect of this allele was observed in unstimulated monocytes or across lymphoid subsets. Production of sIL7R by monocytes greatly exceeded that of CD4+ T-cells, and was strongly associated with both rs6897932 genotype and expression of the splicing factor gene DDX39A. Stimulated monocytes were sensitive to exogenous IL-7, which elicits a defined transcriptional signature. Flow cytometry and single-cell sequencing of synovial fluid derived monocytes from patients with spondyloarthritis showed an enlarged subset of IL7R+ monocytes with a unique transcriptional profile that markedly overlaps that induced by IL-7 in-vitro and shows similarity to the previously described ‘Mono4’ subset. These data demonstrate disease-associated genetic variants at IL7R specifically impact monocyte surface IL7R and sIL7R following innate immune stimulation, suggesting a previously unappreciated key role for monocytes in IL-7 pathway biology and IL7R-associated diseases.

Genetic variation in VAC14 is associated with bacteremia secondary to diverse pathogens in African children

Performing a genome-wide association study of Salmonella enterica serovar Typhi ( S. Typhi) invasion, Alvarez et al. (1) identify a trait-associated SNP, rs8060947, in VAC14 . rs8060947 is an expression quantitative trait locus for VAC14 RNA expression, and carriage of the A allele is associated with reduced VAC14 RNA and protein expression, and increased invasion of S . Typhi. VAC14-associated inhibition of S . Typhi invasion is mediated by a reduction in host cell membrane cholesterol. Carriage of the A allele at rs8060947 is associated with typhoid fever in Vietnamese individuals [cases = 496, controls = 500; P = 0.01, additive odds ratio (OR) = 1.38]. The authors further identify a SNP in high linkage disequilibrium with rs8060947 and rs8044133, located in a transcription factor binding site, which merits further investigation as the causative SNP. Alvarez et al. (1) note that cholesterol has been implicated in the invasion and pathogenesis of a wide range of pathogens (2⇓⇓–5). We therefore used previously published genome-wide association study data, describing susceptibility to bacteraemia secondary to diverse pathogens in Kenyan children (6), to explore whether genetic variation at the VAC14 locus is associated with susceptibility to invasive bacterial infections other than typhoid fever. In Kenyan children (cases = 1,536, controls = 2,677), rs8060947 (Fig. 1 A ) is significantly associated with all-cause bacteremia: P additive = 0.02, OR = 1.11 [95% confidence interval (CI) 1.02–1.22]. In keeping with the effect observed in typhoid, carriage of the A allele at rs8060947 increases risk of bacteremia. Fig. 1. rs8060947 and major causes of bacteremia … [↵][1]1To whom correspondence should be addressed. Email: james.gilchrist{at}well.ox.ac.uk. [1]: #xref-corresp-1-1

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

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.Nontyphoidal Salmonella (NTS) is a major cause of bacteraemia in children and HIV-infected adults in Africa. Here, Gilchrist et al. conduct a genome-wide association study and show that genetic variation in STAT4 is a determinant of NTS in Kenyan and Malawian children.