Stromal IFN-γR-signaling modulates goblet cell function during Salmonella Typhimurium infection - PubMed (original) (raw)

Stromal IFN-γR-signaling modulates goblet cell function during Salmonella Typhimurium infection

Pascal Songhet et al. PLoS One. 2011.

Abstract

Enteropathogenic bacteria are a frequent cause of diarrhea worldwide. The mucosal defenses against infection are not completely understood. We have used the streptomycin mouse model for Salmonella Typhimurium diarrhea to analyze the role of interferon gamma receptor (IFN-γR)-signaling in mucosal defense. IFN-γ is known to contribute to acute S. Typhimurium diarrhea. We have compared the acute mucosal inflammation in IFN-γR(-/-) mice and wild type animals. IFN-γR(-/-) mice harbored increased pathogen loads in the mucosal epithelium and the lamina propria. Surprisingly, the epithelium of the IFN-γR(-/-) mice did not show the dramatic "loss" of mucus-filled goblet cell vacuoles, a hallmark of the wild type mucosal infection. Using bone marrow chimeric mice we established that IFN-γR-signaling in stromal cells (e.g. goblet cells, enterocytes) controlled mucus excretion/vacuole loss by goblet cells. In contrast, IFN-γR-signaling in bone marrow-derived cells (e.g. macrophages, DCs, PMNs) was required for restricting pathogen growth in the gut tissue. Thus IFN-γR-signaling influences different mucosal responses to infection, including not only pathogen restriction in the lamina propria, but, as shown here, also goblet cell function.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Mucosal S. Typhimurium infection in IFN-γR-/- and isogenic control mice.

IFN-γR-/- mice (C57BL/6 background; open symbols), C57BL/6 controls (closed black symbols) and IFN-γR+/− control mice (closed grey symbols) were pre-treated with streptomycin and infected for 1 day (1 dpi) or 2 days (2 dpi) with S. Typhimurium (SL1344 pM973; Material and Methods). We analyzed colonization levels in the gut lumen (A), the MLNs (B), the livers (C), the spleens (D) and the mucosal tissues (E; red = lamina propria; blue = epithelial cells). Data point color code: closed symbols (red, dark blue) = C57BL/6 mice; closed symbols (orange, light blue) = IFN-γR+/− control littermates; open symbols (red, dark blue) = IFN-γR+/− mice. *: p<0.05.

Figure 2

Figure 2. Mucosal pathology of S. Typhimurium infected IFN-γR-/- mice.

Infected animals from Fig. 1 (IFN-γR-/- mice, open symbols; C57BL/6 control, closed black symbols; and IFN-γR+/− control mice, closed grey symbols) as well as streptomycin pre-treated non-infected animals (0 dpi) were analyzed (A–F). (A,B) HE-stained tissue sections from wt C57BL/6 controls and IFN-γR-/- mice at day 2 p.i.. (C) Inflammation of the cecal mucosa. (D) The number of goblet cells harboring mucus-filled vacuoles was analyzed using HE-stained tissue sections. (E) WGA-AlexaFluor647 staining for mucin (incl. mucus filled vacuoles, secreted mucins). (F) Quantitative analysis of the number of goblet cells which harbor mucus-filled vacuoles as determined by WGA-AlexaFluor647 fluorescence microscopy (see E). GHE: mucus-filled goblet cell vacuoles detected in HE-stained tissue sections. GWGA: mucin-filled goblet cell vacuoles detected in tissue sections stained with the lectin WGA-AlexaFluor647. (G, H). Quantitative real time PCR analysis of cxcl2 and muc2 expression. RNA samples from IFN-γR-/- mice (open symbols), C57BL/6 control, (closed black symbols) and IFN-γR+/− control mice (closed grey symbols) were selected from the experiment shown above. mRNA expression levels were normalized to Gapdh (Materials and Methods). *: p<0.05; ns: not statistically significant. Black line: median. Errors bars (F): standard error of mean; bar = 100 µm.

Figure 3

Figure 3. Mucosal S. Typhimurium infection in IFN-γR-/- bone marrow chimeras and IFN-γR+/+ controls.

We generated bone marrow chimeras (BM chimeras) and challenged the animals with S. Typhimurium (SL1344; harboring pM973) in the streptomycin mouse model for 2 days (A-E). Types of BM chimeric animals generated: "B6/IFN-γR" [C57BL/6 mice (Ly5.1 marker) reconstituted with IFN-γR-/- (Ly5.2 marker) bone marrow; circles], "IFN-γR/B6" [IFN-γR-/- mice (Ly5.2 marker) reconstituted with C57BL/6 (Ly5.1 marker) bone marrow; squares] and "B6/B6" [C57BL/6 mice (Ly5.2 marker) reconstituted with C57BL/6 (Ly5.1 marker) bone marrow; triangles] . We analyzed colonization levels in the gut lumen (A), the MLNs (B), the livers (C), the spleens (D) and the mucosal tissues (E; red = lamina propria; blue = epithelial cells). *: p<0.05. Black line: median. n.s.: not significant (p≥0.05). Stippled line: minimal detectable value.

Figure 4

Figure 4. Mucosal pathology and mucus-filled vacuoles in the mucosa of S. Typhimurium-infected IFN-γR-/--bone marrow chimeras.

We analyzed the cecum mucosa of S. Typhimurium-infected BM chimeras shown in Fig. 3. (A), HE-stained gut tissue sections. (B) Mucosal tissue pathology. (C) Quantitative analysis of the number of mucus vacuoles in the cecal mucosa of infected BM chimeras as determined from HE-stained tissue sections. (D) UEA-1-rhodamine or WGA-AlexaFluor647 staining of glycoconjugates /mucins. (E) Staining of the cell-proliferation marker Ki67 and the lamina propria marker CD54 (E). *: p<0.05; Black line: median. n.s.: not significant (p≥0.05). Bar = 100 µm.

Figure 5

Figure 5. Mucus-filled goblet cell vacuoles in the cecal mucosa of S. Typhimurium infected BM chimeras, C57BL/6 controls, IFN-γR+/− mice and IFN-γR-/- animals.

Cecum tissues of BM chimeras from Fig. 3 and 4 (2 dpi), as well as cecum tissue from C57BL/6 controls, IFN-γR+/− littermates and IFN-γR-/- mice from Fig. 2 (0 dpi and 1 dpi) were analyzed. (A–C) Immunofluorescence microscopy images of cecal tissue sections from non-infected controls (A) and mice at days 1 p.i. (B) and day 2 p.i. (C). Green, anti-Muc2-antibody staining; The Muc2-specific antibody recognizes an epitope of immature mucin, located within goblet cells, but absent from secreted Muc2. red: actin (phalloidin staining); blue: DNA (DAPI staining). (D) Quantitative analysis of the number of goblet cells which harbor mucus-filled vacuoles as determined by Muc2 fluorescence microscopy. No significant differences were observed when compared to non-infected C57BL/6 mice (p≥0.05). Errors bars (D): standard error of mean. Bar = 100 µm.

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