Neutrophil prime unique transcriptional responses in intestinal organoids during infection with nontyphoidal Salmonella enterica serovars

Abstract

Nontyphoidal strains of Salmonella enterica are a major cause of foodborne illnesses, and infection with these bacteria results in inflammatory gastroenteritis. Polymorphonuclear leukocytes (PMNs), also known as neutrophils, are a dominant immune cell type found at the site of infection in Salmonella-infected individuals, but how they regulate infection outcome is not well understood. Here, we used a co-culture model of primary human PMNs and human intestinal organoids to probe the role of PMNs during infection with two of the most prevalent Salmonella serovars: Salmonella enterica serovar Enteritidis and Typhimurium. Using a transcriptomics approach, we identified a dominant role for PMNs in mounting differential immune responses including production of pro-inflammatory cytokines, chemokines, and antimicrobial peptides. We also identified specific gene sets that were induced by PMNs in response to Enteritidis or Typhimurium infection. By comparing host responses to these serovars, we uncovered differential regulation of host metabolic pathways particularly induction of cholesterol biosynthetic pathways during Typhimurium infection and suppression of RNA metabolism during Enteritidis infection. Together, these findings provide insight into the role of human PMNs in modulating different host responses to pathogens that cause similar disease in humans.IMPORTANCENontyphoidal serovars of Salmonella enterica are known to induce robust recruitment of polymorphonuclear leukocytes (PMNs) in the gut during early stages of infection, but the specific role of PMNs in regulating infection outcome of different serovars is poorly understood. Due to differences in human infection progression compared to small animal models, characterizing the role of PMNs during infection has been challenging. Here, we used a co-culture model of human intestinal organoids with human primary PMNs to study the role of PMNs during infection of human intestinal epithelium. Using a transcriptomics approach, we define PMN-dependent reprogramming of the host response to Salmonella, establishing a clear role in amplifying pro-inflammatory gene expression. Additionally, the host response driven by PMNs differed between two similar nontyphoidal Salmonella serovars. These findings highlight the importance of building more physiological infection models to replicate human infection conditions to study host responses specific to individual pathogens.

Keywords: Salmonella; enteric pathogens; epithelial cells; innate immunity; neutrophils.

Fig 1

PMNs enhance HIO immune activation and other transcriptional responses during Salmonella infection. (A) PCA plot of HIOs and PMN-HIOs infected with STM or SE with PBS as the mock control for 8 h. (B) Loadings data from PC1 were extracted from (A) to identify the top 50 genes driving separation of samples via PC1. Genes were then assessed for pathway enrichment using the Reactome database. Pie chart shows the percent of the top 50 pathways belonging to each of the major Reactome pathway categories. (C) Dot plot assessing pathway enrichment of the top 50 identified pathways in (B) using all differentially expressed genes (P-adjusted value < 0.05 compared to PBS-injected HIOs). Gene ratio is shown on the x-axis and the dot size corresponds to the −log10(P-value). HIO samples are outlined in black while PMN-HIOs are outlined in gray. PBS injection (red), SE injection (blue), and STM injection (green). (D) Number of diarrhea-associated genes with P-adjusted < 0.05 and log2(fold change) > 1.5 in each condition. Gene list for this analysis was used from reference (22). (E) Venn diagram comparing differentially regulated genes with P-adjusted value < 0.05 relative to PBS-injected HIOs for SE-injected HIOs, PBS-injected PMN-HIOs, and SE-injected PMN-HIOs (i) and for STM-injected HIOs, PBS-injected PMN-HIOs, and STM-injected PMN-HIOs (ii).

Fig 2

PMN-HIO co-culture amplifies production of cytokines and chemokines in infected HIOs. (A-B) Gene expression data presented as log₂(fold change) relative to PBS-injected HIOs for (A) cytokines and (B) chemokines. Genes that were significantly changed from PBS-injected HIOs in at least one condition with P-adjusted < 0.05 are included. (C) ELISA data from culture media sampled at 8 hpi with five HIOs per well with n = 4 replicates. Significance was determined by two-way analysis of variance (ANOVA) where *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig 3

Presence of PMNs strengthens the antimicrobial environment. (A) Gene expression data presented as log₂(fold change) relative to PBS-injected HIOs for antimicrobial genes. Heatmap is divided into functional categories: Microbicidal, Nutritional Immunity, and Opsonins. Genes that were significantly changed from PBS-injected HIOs in at least one condition with P-adjusted < 0.05 are included. (B) ELISA data from culture media sampled at 8 hpi with five HIOs per well with n = 4 replicates. Significance was determined by two-way ANOVA where *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig 4

PMN-HIOs distinguish between SE and STM infection. (A) Venn diagram comparing differentially regulated genes with P-adjusted value < 0.05 relative to PBS-injected HIOs for STM-, SE-, or PBS-injected PMN-HIOs. (B) Heatmap of unique differentially expressed genes. Top 25 genes based on adjusted P-value that were significant in only SE-injected PMN-HIOs (i) or in STM-injected PMN-HIOs (ii). Data are presented as log₂(fold change) relative to PBS-injected HIOs. *Cholesterol biosynthetic genes; ^#RNA modification genes. (C) Pathway enrichment analysis of 1,012 SE-induced genes (i) or 2,098 STM-induced genes identified in (A) from the Reactome database. Pie chart shows the number of each major Reactome pathway category represented by the pathway enrichment results out of two significant pathways in SE-infected PMN-HIOs or 143 significant pathways in STM-infected PMN-HIOs. (D) Dot plot assessing pathway enrichment in PMN-HIOs of all differentially expressed genes (P-adjusted value < 0.05 compared to PBS-injected HIOs). Top pathways were selected by the biggest difference in gene ratio between SE- and STM-injected PMN-HIOs. Gene ratio is shown on the x-axis, and the dot size corresponds to the −log₁₀(P-value). PBS injection (red), SE injection (blue), and STM injection (green).