Figure 1.
IL23R and IL12RB2 are not transcribed within the same lymphocyte populations.
The relative mRNA transcription level of IL23R, IL12RB1 and IL12RB2 in (A) different sorted PBMC populations of healthy donors and in (B) murine sorted spleen cell populations is shown. (C) The relative mRNA transcription level of Il23r, Il12rb1 and Il12rb2 in sorted CD27+/− γδ T cell populations from mouse spleens is shown. For all panels, the horizontal bar represents the mean of each group and each symbol represents one sample.*, p<0.05, **p<0.01.
Figure 2.
Il23r expressing cells exhibit a mixed Th1/Th17 signature profile.
(A) The relative mRNA transcription level of Rorc, Il17a, Il22, Tbx21, and Ifng in sorted murine spleen cells is shown. (B) The relative mRNA transcription level of Il23r, Il12rb1, Il12rb2, Rorc, Il17a, Tbx21 and Ifng in γδ T cells from RORγt−/− mice is shown. (C) The relative mRNA transcription level of Il23r, Il12rb1, Il12rb2, Rorc, Il17a, Il22, Tbx21 and Ifng in IL23R-eGFP sorted spleen cells and NK cells is shown. For all panels, the horizontal bar represents the mean of each group and each symbol represents one sample.*, p<0.05 compared to each group.
Figure 3.
IL-23R is not limited to T cells.
(A) Extracellular staining of spleen cells in IL23R-eGFP+/− mice, gated on eGFP positive cells. (B) Extracellular staining of spleen in IL23R+/−.Rag1−/− mice, gated on eGFP positive cells. (C) Pie chart representation of subpopulations within the IL23R-eGFP positive cells, in the IL-23R-eGFP+/− mice and IL-23R-eGFP+/−.Rag1−/− mice. n = 3.
Figure 4.
IL-23R+ cells in the intestine are mostly innate lymphoid cells.
(A) The percentage and the absolute number of IL-23R-eGFP+ cells in lymphoid organs is depicted. The horizontal bar represents the mean of each group. Each symbol represents data from one mouse. (B) The extracellular staining strategy for the identification of specific immune cellular subsets from total cells in the lamina propria of the small intestine and colon is shown. Electronic gating of viable hematopoietic cells was achieved by using the viable exclusion dye and CD45 expression. γδ T cells (CD3ε+ TCR γδ+), NK cells (CD49b+), B cells (CD19+) and innate lymphoid cells (CD117+CD90+). (C) Identification of IL-23R-eGFP+ immune cell subsets from IL-23R-eGFP+/− mice based on the strategy defined in B. (D) Flow cytometry profiles of IL-23R-eGFP expression in CD11c and CD11b expressing cells. n = 2.
Figure 5.
Anti-CD40 injection in Rag-deficient mice leads to multi-organ inflammatory cellular infiltration.
Representative H&E microphotographs of liver, lung, heart and colon from C57BL6.Rag1−/− mice are shown. At day 0, mice received 200 µl of saline solution (control, top panels) or 200 µg of anti-CD40 (bottom panels) by intraperitoneal route. Neither inflammatory nor structural changes are observed in the tissues of mice that received the saline solution. The liver, lung and heart of mice treated with anti-CD40 show inflammatory cellular infiltrations (asterisk) near blood vessels (arrows). The colon of anti-CD40 treated mice presents with a mild inflammatory response at the mucosa, a decrease in goblet cells as well as gland arborization.
Figure 6.
IL-12Rβ2+ and IL-23R+ innate immune cells both contribute to the systemic inflammatory response.
C57BL/6.Rag1−/−, IL-23R−/−.Rag1−/−, and IL-12Rβ2−/−.Rag1−/− mice were injected with anti-CD40 antibody or PBS (6 to 11 mice per group). (A) Mouse weight relative to the initial weight is shown. a, b p<0.05. (B) Day 7 serum concentrations of IFN-γ, IL-1β, IL-6, IL-12p70, IL-18, IL-23p19, TNF-α, CCL-2 and CXCL1 were quantified. Each symbol represents data for one mouse. *p<0.05, **p<0.01.
Figure 7.
IL-23R+ innate lymphocytes play a dominant role in the intestinal inflammatory response.
(A) Histological examination of the colon. Increase in the thickness of the mucosa, inflammatory infiltration (indicated by arrows), goblet cell depletion, discrete glandular hyperplasia and edema of the submucosa were found in C57BL/6.Rag1−/− and IL-12Rβ2−/−.Rag1−/− mice which received anti-CD40 antibody. Original magnification 100X. Scale = 100 µm. (B) The relative mRNA transcription levels of cytokines and chemokines in the proximal colon are shown. Each symbol represents data for one mouse. *p<0.05. **p<0.01. ***p<0.001.
Figure 8.
The anti-CD40-induced inflammatory response does not alter the distribution of immune cells in the gut-associated lymphoid tissue.
IL-23R-eGFP−/−.Rag1−/− mice were treated with anti-CD40 and the intestines were processed at day 2. (A) The extracellular staining strategy for the identification of innate lymphoid cells (CD117+ CD90+) and NK cells (CD49b) within total cells in the lamina propria of the small intestine and colon. Electronic gating of viable hematopoietic cells was achieved by using the viable exclusion dye and CD45 expression. (B) Identification of IL-23R-eGFP+ immune cell subsets from IL-23R-eGFP−/−.Rag1−/− anti-CD40-treated mice based on the strategy defined in A. (C) Flow cytometry profiles of IL-23R-eGFP expression in CD11c and CD11b expressing cells. n = 2.