Persistent salmonellosis causes pancreatitis in a murine model of infection

Abstract

Pancreatitis, a known risk factor for the development of pancreatic ductal adenocarcinoma, is a serious, widespread medical condition usually caused by alcohol abuse or gallstone-mediated ductal obstruction. However, many cases of pancreatitis are of an unknown etiology. Pancreatitis has been linked to bacterial infection, but causality has yet to be established. Here, we found that persistent infection of mice with the bacterial pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) was sufficient to induce pancreatitis reminiscent of the human disease. Specifically, we found that pancreatitis induced by persistent S. Typhimurium infection was characterized by a loss of pancreatic acinar cells, acinar-to-ductal metaplasia, fibrosis and accumulation of inflammatory cells, including CD11b+ F4/80+, CD11b+ Ly6Cint Ly6G+ and CD11b+ Ly6Chi Ly6G- cells. Furthermore, we found that S. Typhimurium colonized and persisted in the pancreas, associated with pancreatic acinar cells in vivo, and could invade cultured pancreatic acinar cells in vitro. Thus, persistent infection of mice with S. Typhimurium may serve as a useful model for the study of pancreatitis as it relates to bacterial infection. Increased knowledge of how pathogenic bacteria can cause pancreatitis will provide a more integrated picture of the etiology of the disease and could lead to the development of new therapeutic approaches for treatment and prevention of pancreatitis and pancreatic ductal adenocarcinoma.

Figure 1. S. Typhimurium LPS induces pancreatic inflammation.

(A) Histological analysis of pancreatic tissue sections from mock-treated or LPS-treated C57BL/6J mice (n = 4 per group). Tissue sections were stained using H&E or subjected to IHC using antibodies specific for F4/80. Scale bars for H&E = 200 μm and for IHC = 100 μm. (B) Quantitation of IHC data shown in (A). (C and D) Expression of surface F4/80 and CD11b by cells harvested from pancreata of mock-treated or LPS-treated C57BL/6J mice (n = 4 per group) as measured using flow cytometry. Numbers in (C) refer to CD11b⁺ F4/80⁺ cells as percentages of the total numbers of cells. (E and F) Expression of surface Ly6C and Ly6G by CD11b⁺ cells present in pancreata of mock-treated or LPS-treated C57BL/6J mice (n = 4 per group) as measured using flow cytometry. Numbers in (E) refer to CD11b⁺ Ly6C^hi Ly6G⁻ and CD11b⁺ Ly6C^int Ly6G⁺ cells as percentages of the total numbers of CD11b⁺ cells. Data are representative of (A, C, and E), or show mean with SEM from (B, D and F), two independent experiments. Data were analyzed using a two-tailed, paired Student’s t-test; p values<0.05 were considered to be statistically significant. Asterisks indicate statistically significant differences (***p<0.001, *p<0.05).

Figure 2. S. Typhimurium infection induces pancreatitis.

(A and B) Histological analysis of pancreatic tissue sections from C57BL/6J Nramp1^G169 mice (n = 3–4 per group) left uninfected or infected for 10 days with S. Typhimurium (STm). Tissue sections were stained using H&E, cytokeratin 19, or Picrosirius Red Stain Kit. In addition, tissue sections were subjected to IHC using antibodies specific for collagen I (A) or F4/80 or Ly6B.2 (B). Scale bars for H&E = 200 μm and for IHC = 100 μm. (C) Quantitation of IHC data shown in (B). (D and E) Expression of surface F4/80 and CD11b by cells harvested from pancreata of C57BL/6J Nramp1^G169 mice (n = 3–4 per group) left uninfected or infected for 10 days with STm as measured using flow cytometry. Numbers in (D) refer to CD11b⁺ F4/80⁺ cells as percentages of the total numbers of cells. (F and G) Expression of surface Ly6C and Ly6G by CD11b⁺ cells present in pancreata of C57BL/6J Nramp1^G169 mice (n = 3–4 per group) left uninfected or infected for 10 days with STm as measured using flow cytometry. Numbers in (F) refer to CD11b⁺ Ly6C^hi Ly6G⁻ and CD11b⁺ Ly6C^int Ly6G⁺ cells as percentages of the total numbers of CD11b⁺ cells. Data are representative of (A, B, D and F), or show mean with SEM from (C, E and G), two independent experiments. Data were analyzed using a two-tailed, paired Student’s t-test; p values<0.05 were considered to be statistically significant. Asterisks indicate statistically significant differences (***p<0.001, *p<0.05).

Figure 3. S. Typhimurium colonize and persist in the pancreas, associate with pancreatic acinar cells in vivo, and can invade pancreatic acinar cells in vitro.

(A–C) Bacterial loads per gram of pancreas (A), liver (B) and spleen (C) tissue harvested from C57BL/6J Nramp1^G169 mice (n = 3 per group) at indicated times after infection with S. Typhimurium. (D) Representative confocal images of pancreatic tissue sections harvested from C57BL/6J Nramp1^G169 mice (n = 3 per group) infected with S. Typhimurium expressing GFP. Tissue sections were stained with Alexa Fluor 594 phalloidin (red) and DAPI (blue). Arrows point to GFP-expressing S. Typhimurium. (E) Invasion of cultured pancreatic acinar cells (line 266-6) by wild-type or invA-deficient S. Typhimurium as measured by gentamicin protection assay. (F and G) Detection of GFP associated with cultured pancreatic acinar cells (line 266-6) infected with wild-type or invA-deficient S. Typhimurium expressing GFP. Data shown in (A–D) show mean with spread from (A–C), or are representative of (D), two independent experiments. Data shown in (E–G) show mean with SEM from (E and G), or are representative of (F), four independent experiments. Data in (E and G) were analyzed using a two-tailed, paired Student’s t-test; p values<0.05 were considered to be statistically significant. Asterisks indicate statistically significant differences (***p<0.001, **p<0.01).

Figure 4. Pancreatitis progresses with persistent S. Typhimurium infection.

(A and C) Histological analysis of pancreatic tissue sections from C57BL/6J Nramp1^G169 mice (n = 3–4 per group) left uninfected or infected for 60 days with S. Typhimurium (STm). Tissue sections were stained using H&E, cytokeratin 19, or Picrosirius Red Stain Kit. In addition, tissue sections were subjected to IHC using antibodies specific for collagen I (A) or F4/80 or Ly6B.2 (C). Scale bars for H&E = 200 μm and for IHC = 100 μm. (B and D) Quantitation of IHC data shown in (A and C). (E and F) Expression of surface F4/80 and CD11b by cells harvested from pancreata of C57BL/6J Nramp1^G169 mice (n = 3–4 per group) left uninfected or infected for 60 days with STm as measured using flow cytometry. Numbers in (E) refer to CD11b⁺ F4/80⁺ cells as percentages of the total numbers of cells. (G and H) Expression of surface Ly6C and Ly6G by CD11b⁺ cells present in pancreata of C57BL/6J Nramp1^G169 mice (n = 3–4 per group) left uninfected or infected for 60 days with STm as measured using flow cytometry. Numbers in (G) refer to CD11b⁺ Ly6C^hi Ly6G⁻ and CD11b⁺ Ly6C^int Ly6G⁺ cells as percentages of the total numbers of CD11b⁺ cells. Data are representative of (A, C, E and G), or show mean with SEM from (B, D, F and H), two independent experiments. Data were analyzed using a two-tailed, paired Student’s t-test (B and D) or a one-way ANOVA (F and H); p values<0.05 were considered to be statistically significant. Asterisks indicate statistically significant differences (***p<0.001).