Early and innate host reponses to enteric fever
Keywords: enteric fever
transcriptomics
paratyphoid
human challenge models
typhoid
genetic

Salmonella enterica serovar Typhi (S. Typhi) and S. Paratyphi A are human-restricted pathogens estimated to cause 20 million cases of enteric fever each year. Due to lack of an animal model which fully recapitulates human disease, and difficulty studying infection prior to clinical presentation in field settings, little is known about host-pathogen interactions during the incubation period. It is likely during this period of time that both vaccine-mediated and natural elimination of infection occurs in those who remain healthy despite exposure. Stool shedding, early plasma cytokine responses and S. Typhi DNAaemia have previously been detected in both volunteers who did and did not develop acute enteric fever following ingestion of S. Typhi Quailes strain. Here we have applied functional genomics and multiplex cytokine assays to further investigate the nature of the early host response and identify genetic risk factors for development of disease in human volunteers challenged with S. Typhi and S. Paratyphi A.
Perturbation of blood plasma cytokines was greatest 12 hours post-challenge and during acute disease. Early after challenge there was a significant rise in BDNF and EGF (p < 0.0001 compared with baseline, linear modelling), validated by independent assays and significant relative to unchallenged controls (p < 0.005, Mann–Whitney U test). In a multiplex cytokine assay produced by a different manufacturer, CD40L and GRO-α were also significantly raised after challenge (p < 0.01, Mann–Whitney U test). BDNF, EGF and GRO-α were increased in the supernatants of leukocytes stimulated with S. Typhi (p < 0.1, t-test). While GRO-α was secreted by S. Typhi infected granulocytes, monocytes, lymphocytes and intestinal organoids, EGF and CD40L were secreted by granulocytes only. IL1RA, MIG and IP10 were identified as correlates of symptomology and inflammation during acute disease (p < 0.001 for overall correlations with symptom score and C-reactive protein, Pearson correlation coefficient), and validated using data from two independent cohorts. In vitro, a greater amount of IL1RA was produced in response to S. Paratyphi A and S. Typhimurium than S. Typhi Quailes strain (p = 0.03 and 0.01 respectively, t-test), and appeared to be specifically secreted by granulocytes. No marked differences in plasma cytokine profile were seen between participants challenged with wild-type S. Typhi Quailes strain and an isogenic typhoid toxin knockout.
316 genes were significantly differentially expressed (FDR < 0.05, linear modelling) 12 hours post-challenge, normalised to gene expression changes in an unchallenged cohort. These changes were validated in two independent challenge studies, and correlated with gene expression changes following in vitro infection of cell lines (ρ = 0.7 and p = 1 x 10-48 for Hep2 cells, Pearson correlation coefficient) and inflammatory bowel disease (ρ = 0.3 and p = 3 x 10-5 with Crohn’s disease, Pearson correlation coefficient). Gene sets relating to innate immunity were over-represented in the early host response, with deconvolution suggesting an early peak in neutrophils (p = 1.6 x 10-6 for the discovery cohort, Wilcoxon signed-rank test). In vitro, S. Typhi infection induced neutrophil activation, bacterial uptake and a rise in reactive oxygen species. Despite activation, bacterial survival was no different between granulocytes and monocytes four hours after in vitro infection (p = 0.3, t-test). No significant differences were observed between those who remained healthy and those who developed acute disease, although the magnitude of the early response was on average higher in those remaining healthy (p = 3.7 x 10-12, Mann–Whitney–Wilcoxon test).
At the genome-wide level, no polymorphisms were significantly associated with susceptibility or resistance to enteric fever. C1QC mRNA was significantly higher at baseline in those exposed to S. Paratyphi A who remained healthy after challenge (p = 0.0004, linear modelling), but no differences in C1q protein were seen between outcome groups (p = 0.9, Mann-Whitney-Wilcoxon test). The prevalence of HLA type HLA-B*27:05 was higher in those who developed acute disease (p = 0.006, logistic regression), and in vitro resulted in greater bacterial burden 24 hours after S. Typhi infection (p = 0.05, t-test).
Overall, there appears to be a marked and reproducible early host response in both those who do and do not go on to develop enteric fever following exposure. Although underpowered to detect baseline differences between outcome groups, we identify several promising candidate factors such as HLA type and early transcriptional response.