Fig 2: IL22 effects on enteroid size and number are resistant to rescue by exogenous wnt. (A) Jejunal enteroids were cultured in WRN without or with IL22 (1 or 5 ng/mL) for 4 days. Representative images are shown. Bar, 500 μm and 200 μm (insets). (B and C) Quantification of cross-sectional area (n = 20–30) and number (n = 3) of jejunal enteroids cultured in WRN with indicated concentrations of IL22. (D) Cldn2 mRNA expression in enteroids (spheroids) cultured in WRN without (blue symbols) or with IL22 (black symbols) (5 ng/mL, PeproTech). *P < .05. (E and F) Ki67 and Pcna expression was markedly reduced in IL22-treated spheroids. Data are representative of at least 3 independent experiments. *P < .05, **P < .01. (G) Jejunal enteroids were cultured in WNR without or with IL22 (5 ng/mL, PeproTech) for 3 days and then pulsed with EdU (red) for 1 hour before fixation. Bar, 25 μm.

Fig 3: IL22 promotes cell death. (A) Merged bright-field and fluorescent images and flow cytometric analysis of enteroids cultured in ENR without or with IL22 (5 ng/mL, PeproTech) for 3 days and stained with propidium iodide (red in images). Histograms of flow cytometry data from enteroids cultured without (blue) or with (black) IL22. Fractions of cells within the window are indicated. Data are representative of 3 separate analyses. Bar, 100 μm. **P < .01. (B) Merged bright-field and fluorescent images and flow cytometric analysis of enteroids cultured in ENR without or with IL22 (5 ng/mL, PeproTech) for 3 days and labeled for caspase 3/7 activation (green in images). Histograms of flow cytometry data from enteroids cultured without (blue) or with (black) IL22. Fractions of cells within the window are indicated. Data are representative of 3 separate analyses. Bar, 100 μm.*P < .05.

Fig 4: IL22 reduces Lgr5+stem cell numbers in vivo. (A) Mice were treated with IL22 (1 μg/day for 7 consecutive days). Tissues were collected on day 8. Isolated jejunal crypts were grown as enteroids in ENR medium without added IL22. Bright-field images after 2 days of culture are shown. Images are representative of 3 independent experiments. Bar, 100 μm. (B and C) Enteroid cross-sectional area (n = 8) and number (n = 8) were quantified. *P < .05, **P < .01. (D) Quantitative RT-PCR of relative mRNA expression in isolated jejunal epithelium. n = 3–7 in the representative experiment shown. *P < .05. (E and F) Lgr5-EGFP (detected using anti-GFP) and Olfm4 immunostains of jejunal tissue sections from IL22- or saline-treated mice. Numbers of positive cells per well-oriented crypt are shown. Each point (n = 12) represents a separate field from a total of 5–7 mice per condition. Bar, 100 μm. Bar, 20 μm (insets). **P < .01. (G and H) Lgr5-EGFP and Olfm4 immunostains of ileal tissue sections from IL22- or saline-treated mice. Numbers of positive cells per well-oriented crypt are shown. Each point (n = 12) represents a separate field from a total of 5–7 mice per condition. Bar, 100 μm. Bar, 20 μm (insets). **P < .01.

Fig 5: IL22 increases epithelial proliferation in vitro and in vivo. (A) Jejunal enteroids were cultured in ENR without or with IL22 (5 ng/mL, PeproTech) for 3 days and then pulsed with EdU (red) for 1 hour before fixation. Bar, 100 μm. Histograms of flow cytometry data from enteroids cultured without (blue) or with (black) IL22 are shown, along with the fraction of cells within the indicated window. Data are representative of 3 separate analyses. **P < .01. (B) Relative mRNA expression of Ki67 (proliferative marker), Prom1 (stem- and transit-amplifying cell marker), Zfp652 (transit-amplifying cell marker), and Prss32 (immature enterocyte marker) in jejunal enteroids cultured without (blue symbols) or with IL22 (black symbols) (5 ng/mL, PeproTech; in ENR.) n = 5–6 in the representative experiment shown. *P < .05, **P < .01. (C and D) Jejunal (C) and ileal (D) sections from mice treated with saline or IL22 (as in Figure 5) were stained by hematoxylin-eosin or immunostained for Ki67. Representative complete cross sections (H&E) are shown along with higher magnification images and depth of well-oriented crypts (n = 12). Numbers of positive cells per crypt were assessed in Ki67 immunostains. Bars, 500 μm (upper images), 100 μm (higher magnification H&E and immunostain images). For all graphs, each point (n = 12) represents a separate field of view from a total of 5–7 mice per condition. Representative data are shown. **P < .01. (E) Snap-frozen jejunum from IL22- or saline-treated mice was immunostained for CD3, F4/80, or myeloperoxidase (MPO) to assess infiltrates of lymphocytes, macrophages, and granulocytes, respectively. Relative mRNA expression of Cd3, F4/80, and MPO in jejunum isolated from saline- and IL22-treated mice is shown. n = 5–7. IL22 did not induce influx of any immune cell population. Bar, 50 μm.