p53 suppresses MHC class II presentation by intestinal epithelium to protect against radiation-induced gastrointestinal syndrome

Radiation-induced gastrointestinal syndrome is a major complication and limiting factor for radiotherapy. Tumor suppressor p53 has a protective role in radiation-induced gastrointestinal toxicity. However, its underlying mechanism remains unclear. Here we report that regulating the IL12-p40/MHC class II signaling pathway is a critical mechanism by which p53 protects against radiation-induced gastrointestinal syndrome. p53 inhibits the expression of inflammatory cytokine IL12-p40, which in turn suppresses the expression of MHC class II on intestinal epithelial cells to suppress T cell activation and inflammation post-irradiation that causes intestinal stem cell damage. Anti-IL12-p40 neutralizing antibody inhibits inflammation and rescues the defects in intestinal epithelial regeneration post-irradiation in p53-deficient mice and prolongs mouse survival. These results uncover that the IL12-p40/MHC class II signaling mediates the essential role of p53 in ensuring intestinal stem cell function and proper immune reaction in response to radiation to protect mucosal epithelium, and suggest a potential therapeutic strategy to protect against radiation-induced gastrointestinal syndrome.

Representative images from at least 3 independent mice showing IF staining of CD45 and Olfm4 in the SI of p53 +/+ and p53 -/-mice at different days post 12 Gy TBI.Representative images from at least 3 independent mice (left panels) and quantification (right panel) of IF staining for lysozyme, a Paneth cell marker, in the SI of p53 +/+ and p53 -/-mice at 3 days post-TBI.n = 30 crypts from at least 3 mice/group.Data are presented as mean ± SD from at least 3 independent experiments.ns: not significant, two-tailed Student's t-test.Source data are provided as a Source Data file.

Figure S6. p53 deficiency induces MHC-II expression on both villus and crypt cells in the SI of mice post-TBI.
Representative images from at least 3 independent mice showing IF staining of MHC-II in the SI of p53 +/+ and p53 -/-mice at 3 days post-TBI.Representative images from at least 3 independent mice (upper panels) and quantification (bottom panel) of TUNEL assays in the SI of p53 +/+ and p53 -/-mice with or without rLIF administration at 3 days post 12 Gy TBI.n = 20 fields from at least 3 mice/group.Data are presented as mean ± SD from at least 3 independent experiments.ns: not significant, two-tailed Student's t-test followed by Bonferroni correction.Source data are provided as a Source Data file.Representative images from at least 3 independent mice (left panels) and quantification (right panel) of IF staining of lysozyme in the SI of mice at 3 days post-TBI.n = 30 crypts from at least 3 mice/group.Data are presented as mean ± SD from at least 3 independent experiments.ns: not significant, two-tailed Student's t-test followed by Bonferroni correction.Source data are provided as a Source Data file.

Figure S2 .
Figure S2.p53 deficiency enhances the inflammatory immune response and ISC damage in the SI of mice post-TBI.

Figure S3 .
Figure S3.p53 deficiency enhances the damage of Paneth cells in the SI of mice post-TBI.

Figure S5 .
Figure S5.Gating strategies for different immune cell populations and IEC population.

Figure S7 .
Figure S7.p53 deficiency has no obvious effect on MHC-II expression on macrophages and DCs in the LP of mice post-TBI.

Figure
Figure S8.p53 deficiency promotes TNFα secretion from T cells in the LP of mice post-TBI.

Figure S9 .
Figure S9.The depletion of CD3 + T cells in mice upon the aCD3 antibody administration.Representative flow cytometric images from at least 3 independent mice showing the depletion of CD3 + T cells in the MLN and LP of p53 +/+ mice at 6 days upon IgG or the aCD3 antibody (200 μg/mouse, once) treatment.

Figure S10 .
Figure S10.Adoptive transfer of the BM derived from p53 -/-mice into p53 +/+ mice exacerbates the damage of Paneth cells post-TBI.a. CD45.1 + mice were adoptively transferred with BM cells isolated from CD45.2 + p53 +/+ and p53 - /-mice.The reconstitution of donor CD45.2 + CD45 + immune cells in the MLN and LP was examined at 28 days after adoptive transfer by flow cytometric assays.n = 3 mice/group.b.Representative images from at least 3 independent mice (left panels) and quantification (right panel) of IF staining of the lysozyme in the SI of mice at 5 days post 12 Gy TBI.n = 30 crypts from at least 3 mice/group.Data are presented as mean ± SD from at least 3 independent experiments.ns: not significant, two-tailed Student's t-test.Source data are provided as a Source Data file.

Figure
Figure S11.aIL12-p40 antibody reduces the damage of Paneth cells in the SI of mice post-TBI.a.The mRNA levels of BAX, PUMA and p21 in the SI of p53 +/+ mice at 6 hours post 12 Gy TBI along with or without RG7112 treatment (i.p., 50 μg/g body weight, twice, 8 hours before and 1 hour post-TBI) determined by qPCR assays.n = 3 mice/group.b.The number of CD69 + activated CD4 + (left panel) and CD8 + (right panel) T cells in the LP of mice at 3 days post-TBI determined by flow cytometric assays.n = 5 mice/group.c.Representative images from at least 3 independent mice (left panels) and quantification (right panel) of IF staining of lysozyme in the SI of mice at 3 days post-TBI.n = 30 crypts from at least 3 mice/group.Data are presented as mean ± SD from at least 3 independent experiments.ns: not significant, two-tailed Student's t-test followed by Bonferroni correction.Source data are provided as a Source Data file.

Figure S12 .
Figure S12.rLIF administration inhibits the IL12-p40 production in p53 -/-mice post-TBI.Left: representative images of cytokine levels in the serum from p53 +/+ and p53 -/-mice with or without rLIF administration at 3 days post 12 Gy TBI detected by using the Proteome Profiler Mouse XL Cytokine Array.Right: quantification of IL12-p40 levels in Cytokine Array.Each array represents a pool of 3 mice with the same treatment.Dots in yellow box represent IL12-p40.Source data are provided as a Source Data file.

Figure S13 .
Figure S13.p53 deficiency reduces LIF expression levels in intestinal organoids.The mRNA levels of LIF in intestinal organoids derived from p53 +/+ and p53 -/-mice determined by qPCR assays.n = 5/group.Data are presented as mean ± SD from at least 3 independent experiments.Two-tailed Student's t-test.Source data are provided as a Source Data file.