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. 2009 Jan;77(1):387-98.
doi: 10.1128/IAI.00933-08. Epub 2008 Oct 27.

Interleukin-23 orchestrates mucosal responses to Salmonella enterica serotype Typhimurium in the intestine

Ivan Godinez  1 , Manuela Raffatellu, Hiutung Chu, Tatiane A Paixão, Takeshi Haneda, Renato L Santos, Charles L Bevins, Renée M Tsolis, Andreas J Bäumler
Affiliations

Interleukin-23 orchestrates mucosal responses to Salmonella enterica serotype Typhimurium in the intestine

Ivan Godinez et al. Infect Immun. 2009 Jan.

Abstract

Salmonella enterica serotype Typhimurium causes an acute inflammatory reaction in the ceca of streptomycin-pretreated mice that involves T-cell-dependent induction of gamma interferon (IFN-gamma), interleukin-22 (IL-22), and IL-17 expression (genes Ifn-gamma, Il-22, and Il-17, respectively). We investigated here the role of IL-23 in initiating these inflammatory responses using the streptomycin-pretreated mouse model. Compared to wild-type mice, the expression of IL-17 was abrogated, IL-22 expression was markedly reduced, but IFN-gamma expression was normal in the ceca of IL-23p19-deficient mice during serotype Typhimurium infection. IL-23p19-deficient mice also exhibited a markedly reduced expression of regenerating islet-derived 3 gamma, keratinocyte-derived cytokine, and reduced neutrophil recruitment into the cecal mucosa during infection. Analysis of CD3(+) lymphocytes in the intestinal mucosa by flow cytometry revealed that alphabeta T cells were the predominant cell type expressing the IL-23 receptor in naive mice. However, a marked increase in the number of IL-23 receptor-expressing gammadelta T cells was observed in the lamina propria during serotype Typhimurium infection. Compared to wild-type mice, gammadelta T-cell-receptor-deficient mice exhibited blunted expression of IL-17 during serotype Typhimurium infection, while IFN-gamma expression was normal. These data suggested that gammadelta T cells are a significant source, but not the sole source, of IL-17 in the acutely inflamed cecal mucosa of mice. Collectively, our results point to IL-23 as an important player in initiating a T-cell-dependent amplification of inflammatory responses in the intestinal mucosa during serotype Typhimurium infection.

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Figures

FIG. 1.
FIG. 1.
Cytokine expression elicited by serotype Typhimurium in streptomycin pretreated wild-type mice (▪) and streptomycin-pretreated IL-23-deficient mice (░⃞) 48 h after infection measured by quantitative real-time PCR. (A to D) Bars represent fold changes in mRNA levels of Il-23 (A), Il-17 (B), Il-22 (C), and Ifn-γ (D) compared to mRNA levels detected in a group of mock-infected wild-type mice (n = 8). The data are shown as geometric means of fold changes ± the standard error determined for RNA from individual mice. (E) Average bacterial numbers (in CFU) recovered 48 h after serotype Typhimurium infection from colon contents or Peyer's patch tissues of wild-type mice (▪) or IL-23-deficient mice (░⃞). The statistical significance of differences is indicated by P values above brackets. NS, not significant.
FIG. 2.
FIG. 2.
Absolute transcript levels of Il-17 (A) and Reg3g (B) in IL-23p19-deficient mice (IL-23p19−/−, ░⃞) or wild-type littermates (▪) determined by quantitative real-time PCR 48 h after mock infection or infection with serotype Typhimurium. The data represent mean mRNA copy numbers per 20 ng of RNA ± the standard error. Statistically significant differences are indicated by P values.
FIG. 3.
FIG. 3.
Neutrophil recruitment into the cecal mucosa. (A) Expression of the neutrophil chemoattractant Kc gene elicited by serotype Typhimurium in streptomycin-pretreated wild-type mice (▪) and streptomycin-pretreated IL-23-deficient mice (░⃞) 48 h after infection measured by quantitative real-time PCR. Bars represent fold changes in mRNA levels compared to mRNA levels detected in a group of mock-infected wild-type mice (n = 8). The data are shown as geometric means of fold changes ± the standard error determined for RNA from individual mice. (B) The numbers of neutrophils per microscopic field were determined by a veterinary pathologist during a blinded examination of slides from the cecal mucosa. The data represent means ± the standard error. Statistical significance of differences is indicated by P values. (C to F) Histopathological appearance of the murine ceca of serotype Typhimurium-infected IL-23-deficient mice (C), serotype Typhimurium-infected wild-type mice (D), mock-infected IL-23-deficient mice (E), or mock-infected wild-type mice (F). All images were taken from hematoxylin-and-eosin-stained cecal sections at the same magnification (×100).
FIG. 4.
FIG. 4.
Isolation of T cells from the intestinal epithelium and lamina propria. The total numbers of live CD3+ cells present in preparations of IEL (A) and LPL (B) from naive mice (n = 6, ░⃞) or serotype Typhimurium-infected mice (n = 6, ▪) were determined. (C) Representative example of the gating strategy used to define γδ+ and γδ− T-cell populations among live IEL. (D) Representative example of the gating strategy used to separate intraepithelial γδ− T cells into different subsets. (E) Representative example of the gating strategy used to define γδ+ and γδ− T-cell populations among live LPL. (F) Representative example of the gating strategy used to separate lamina propria γδ− T cells into different subsets. In panels C to F, the axis represents the fluorescence intensity produced by fluorescent antibody conjugates recognizing the γδ T-cell receptor (γδ TCR), CD3, CD4, or CD8.
FIG. 5.
FIG. 5.
Characterization of T-cell subsets in the intestines of naive mice (n = 6, ░⃞) or serotype Typhimurium-infected mice (n = 6, ▪). (A) T-cell subsets in the IEL population are shown as a percentage of the total number of intraepithelial T cells (CD3+ IEL). (B) T-cell subsets in the LPL population are shown as a percentage of the total number of lamina propria T cells (CD3+ LPL). The data are shown as means ± the standard error.
FIG. 6.
FIG. 6.
Expression of IL-23 receptor by intraepithelial T cells (A) and lamina propria T cells (B) in the intestines of naive mice (n = 6, ░⃞) or serotype Typhimurium-infected mice (n = 6, ▪). (A) IL-23 receptor-expressing cells expressing the indicated markers (CD4, CD8, and/or γδ T-cell receptor [TCR]) are shown as a percentage of the total number of intraepithelial T cells (CD3+ IEL). (B) IL-23 receptor-expressing cells expressing the indicated markers (CD4, CD8 and/or γδ TCR) are shown as a percentage of the total number of lamina propria T cells (CD3+ LPL). The data are shown as means ± the standard error. Statistical significance of differences is indicated by P values. (C) Representative example of IL-23 receptor expression by CD8− γδ+ lamina propria T cells pooled from the intestine of two naive mice (left panel) or two serotype Typhimurium-infected mice (right panel). IL-23R, IL-23 receptor.
FIG. 7.
FIG. 7.
Cytokine expression elicited by serotype Typhimurium in streptomycin-pretreated wild-type mice (C57BL/6, ▪) or streptomycin-pretreated T-cell-receptor δ-chain-deficient mice (Trd−/−, ░⃞) 48 h after infection measured by quantitative real-time PCR. Bars represent fold changes in the mRNA levels of Il-23 (A), Il-17 (B), Ifn-γ (C), and Il-22 (D) compared to mRNA levels detected in a group of mock-infected wild-type mice (n = 8). The data are shown as geometric means of fold changes ± the standard error determined for RNA from individual mice. (E) Average bacterial numbers (in CFU) recovered 48 h after serotype Typhimurium infection from the colon contents or Peyer's patch tissues of wild-type mice (▪) or γδ T-cell-receptor-deficient mice (░⃞). Statistical significance of differences is indicated by P values above brackets. NS, not significant.
FIG. 8.
FIG. 8.
Absolute transcript levels of Il-17 (A) and Reg3g (B) in γδ T-cell-receptor-deficient mice (Trd−/−, ░⃞) or wild-type mice (C57BL/6, ▪) determined by quantitative real-time PCR 48 h after mock infection or infection with serotype Typhimurium. The data represent mean mRNA copy numbers per 20 ng of RNA ± the standard error. (C) Numbers of neutrophils per microscopic field were determined by a veterinary pathologist during a blinded examination of slides from the cecal mucosa. The data represent means ± the standard error. Statistically significant differences are indicated by P values above brackets. NS, not significant.
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