Helicobacter pylori infection in a pig model is dominated by Th1 and cytotoxic CD8+ T cell responses

Abstract

Helicobacter pylori infection is the leading cause for peptic ulcer disease and gastric adenocarcinoma. Mucosal T cell responses play an important role in mediating H. pylori-related gastric immunopathology. While induced regulatory T (iTreg) cells are required for chronic colonization without disease, T helper 1 (Th1) effector responses are associated with lower bacterial loads at the expense of gastric pathology. Pigs were inoculated with either H. pylori strain SS1 or J99. Phenotypic and functional changes in peripheral blood mononuclear cell (PBMC) populations were monitored weekly, and mucosal immune responses and bacterial loads were assessed up to 2 months postinfection. Both H. pylori strains elicited a Th1 response characterized by increased percentages of CD4(+)Tbet(+) cells and elevated gamma interferon (IFN-γ) mRNA in PBMCs. A subset of CD8(+) T cells expressing Tbet and CD16 increased following infection. Moreover, a significant increase in perforin and granzyme mRNA expression was observed in PBMCs of infected pigs, indicating a predominant cytotoxic immune response. Infiltration of B cells, myeloid cells, T cells expressing Treg- and Th17-associated transcription factors, and cytotoxic T cells was found in the gastric lamina propria of both infected groups. Interestingly, based on bacterial reisolation data, strain SS1 showed greater capacity to colonize and/or persist in the gastric mucosa than did strain J99. This novel pig model of infection closely mimics human gastric pathology and presents a suitable avenue for studying effector and regulatory responses toward H. pylori described in humans.

Fig 1

Increase in circulating Th1 cells and IFN-γ expression upon H. pylori infection. CD4⁺ T cells expressing the T cell lineage-specific transcription factors Tbet (Th1) (A), RORγt (TH17) (C), and FOXP3 (iTreg, nTreg) (D) were detected by flow cytometry. IFN-γ mRNA levels in PBMC were measured by qRT-PCR (B). Data were derived from study 2. Symbols indicate statistical differences between either the H. pylori J99 (#)- or the SS1 (*)-infected group and the control group and between the two infected groups (&); n = 8 or 9; data are means ± standard errors of the means (SEM); P ≤ 0.05.

Fig 2

T cells and B cells upregulate Tbet expression upon infection with H. pylori. Expression of Tbet was determined in peripheral T cells (CD3⁺), T helper cells (CD4⁺), B cells (CD21⁺), and γδ T cells (TCRγδ⁺). Representative flow cytometry dot blots for uninfected and H. pylori J99- and SS1-infected pigs at day 42 postinfection of study 1 are presented. Numbers indicate the percentages of positive cells within the single cell population.

Fig 3

Increased circulating and functional cytotoxic cells upon H. pylori infection. Expression of Tbet was assessed in peripheral CTL (CD8β⁺) on day 42 postinfection (A) and over time (B). Representative flow cytometry dot blots for noninfected and H. pylori J99- and SS1-infected pigs are presented. Numbers indicate percentages of positive cells within the single cell population (A). CTL (CD8β⁺) (C) and NK cells (CD3⁻CD8α⁺) (E) were enumerated in PBMC over time. Numbers of cells per ml of blood were calculated by applying the percentage of immune cells obtained by flow cytometry to the concentration of cells in whole blood. The percentage of CD16-expressing CTL was assessed throughout the study (D). Gene expression levels of perforin (F), granzyme A (G), and granzyme B (H) in PBMC were analyzed over time. Data were derived from study 2. Symbols indicate statistical differences between either the H. pylori J99 (#)- or the SS1 (*)-infected group and the control group and between the two infected groups (&); n = 8 or 9; data are means ± SEM; P ≤ 0.05.

Fig 4

Infiltration of immune cells into the gastric lamina propria upon H. pylori infection. The phenotypes of lamina propria leukocytes within the pyloric, cardiac, and fundus regions of the pig stomach were determined by flow cytometry. The percentages of B cells (CD21⁺) (A), myeloid cells (SWC3⁺) (B), T cells (CD3⁺) (C), CD4⁺ T cells expressing FOXP3 (D) or RORγt (E), and CD8β⁺Tbet⁺ T cells (F) were assessed in noninfected and H. pylori J99- and SS1-infected pigs. Data were derived from study 2, and statistically significant differences are represented by an asterisk (*); n = 8 or 9; data are means ± SEM; P ≤ 0.05.

Fig 5

Increased Tbet expression in spleen and induction of cytokine secretion in cells from GLN and spleen upon reexposure to antigen. The percentages of CD8β⁺ Tbet⁺ (A) and CD4⁺ FOXP3⁺ and CD4⁺ RORγt⁺ T cells (B) were assessed in spleen of noninfected and H. pylori J99- and SS1-infected pigs. The number of splenocytes secreting IFN-γ (C) and IL-17 (D) and the number of cells from GLN secreting IL-17 (E) were analyzed by ELISpot. Cells from noninfected and infected pigs were stimulated with whole-cell sonicated (WCS) bacterial antigen in vitro for 36 h. Cells treated with cRPMI alone served as negative control. Data were expressed as fold of induction (FOI) to the negative control. Data were derived from study 2, and statistically significant differences are represented by an asterisk (*); n = 8 or 9; data are means ± SEM; P ≤ 0.05.

Fig 6

Lesion development upon H. pylori infection and lack of bacterial clearance upon long-term infection with strain SS1. Representative images were taken from hematoxylin- and eosin-stained specimens collected from the stomach regions cardiac B (CB) and pyloric A (PA) of noninfected and infected pigs at a magnification of ×10 (A). H. pylori J99 and SS1 were reisolated from 6 stomach locations (see Fig. S1 in the supplemental material) at 2 months postinfection. Reisolation data from studies 1 and 2 were combined and expressed as percentages (B).