Survival signal REG3α prevents crypt apoptosis to control acute gastrointestinal graft-versus-host disease

Abstract

Graft-versus-host disease (GVHD) in the gastrointestinal (GI) tract remains the major cause of morbidity and nonrelapse mortality after BM transplantation (BMT). The Paneth cell protein regenerating islet-derived 3α (REG3α) is a biomarker specific for GI GVHD. REG3α serum levels rose in the systematic circulation as GVHD progressively destroyed Paneth cells and reduced GI epithelial barrier function. Paradoxically, GVHD suppressed intestinal REG3γ (the mouse homolog of human REG3α), and the absence of REG3γ in BMT recipients intensified GVHD but did not change the composition of the microbiome. IL-22 administration restored REG3γ production and prevented apoptosis of both intestinal stem cells (ISCs) and Paneth cells, but this protection was completely abrogated in Reg3g-/- mice. In vitro, addition of REG3α reduced the apoptosis of colonic cell lines. Strategies that increase intestinal REG3α/γ to promote crypt regeneration may offer a novel, nonimmunosuppressive approach for GVHD and perhaps for other diseases involving the ISC niche, such as inflammatory bowel disease.

Keywords: Adaptive immunity; Bone marrow transplantation; Immunology; Innate immunity; Transplantation.

Figure 1. REG3α/γ levels in the plasma/serum and intestinal mucosa during GVHD.

(A–C) Samples were collected from 28 allogeneic BMT patients without GVHD (white circles, n = 13) and with GVHD (black circles, n = 15). (A) Plasma concentrations of REG3α measured by ELISA. (B) Average Paneth cell numbers per high-power field (HPF) in the same biopsies. (C) Semiquantitative REG3α expression in the duodenum. (D–F) B6 mice underwent BMT from syngeneic B6-Ly5.1 donors (GVHD –, white circles, n = 7) or allogeneic C3H.SW donors (GVHD +, black circles, n = 7), and samples were analyzed on day +7 after BMT. (D) Serum REG3γ levels measured by ELISA. (E) Average Paneth cell numbers per HPF in ileal tissue from the same mice. (F) Ileal tissue Reg3g mRNA expression measured by quantitative PCR (qPCR). (G–I) B6D2F1 mice underwent BMT from syngeneic B6D2F1 donors (GVHD –, white circles, n = 6) or allogeneic B6 donors (GVHD +, black circles, n = 6), and samples were analyzed as before on day +7 after BMT. (G) Serum REG3γ levels. (H) Average Paneth cell numbers per HPF in ileal tissue. (I) Ileal tissue Reg3g mRNA expression. **P < 0.01, unpaired 2-tailed t test. Data are expressed as mean ± SEM.

Figure 2. Time course of REG3γ and IL-22 expression during GVHD.

(A–C) B6D2F1 mice underwent BMT from syngeneic B6D2F1 donors (no GVHD, white circles) or allogeneic B6 donors (GVHD, black or blue circles), and samples were analyzed on days +3, +7, and +14 after BMT (n = 5 for each group on each day). (A) Serial REG3γ levels in the serum as measured by ELISA. (B) Serial average Paneth cell numbers per HPF in ileal tissue. (C) Serial Reg3g and IL-22 mRNA expression in the ileum was measured by qPCR. (D–G) B6D2F1 mice underwent BMT, without irradiation conditioning, from syngeneic B6D2F1 donors (no GVHD, white triangles) or allogeneic B6 donors (GVHD, black triangles). (D–F) Serial body weight measurement (D), clinical GVHD score (E), and survival (F) of mice after BMT. (G) Serial Reg3g mRNA expression in the ileum on days +3, +7, and +14 after BMT (n = 6 for each group on each day). *P < 0.05, **P < 0.01, unpaired 2-tailed t test (A–E and G); **P < 0.01, log-rank test (F). (H–K) Following irradiation, B6 mice received 5.0 × 10⁶ BM cells plus 0 × 10⁶ to 5.0 × 10⁶ T cells, as indicated, from C3H.SW donors. Samples were analyzed on day +7 after BMT (n = 4 for each group). (H) Body weight measurement. (I) Clinical GVHD score. (J) Serum REG3γ levels. (K) Reg3g mRNA expression in the ileum. *P < 0.05, **P < 0.01, 1-way ANOVA. Data are expressed as mean ± SEM.

Figure 3. Serum REG3α levels and correlates with nonrelapse mortality in patients with GI GVHD.

Patients with GI GVHD (n = 145) who were treated with systemic steroids for at least 7 days provided blood samples for biomarker analysis and were divided according to biomarker probabilities into low (blue) and high (pink) groups as previously published (16). Twelve-month nonrelapse mortality and REG3α serum levels are shown for test (n = 71) (A) and validation (n = 74) (B) cohorts. **P < 0.01, unpaired 2-tailed t test. Data are expressed as box-and-whisker plots.

Figure 4. REG3γ absence increases GVHD severity.

WT B6 and B6-Reg3g^–/– (KO) mice underwent BMT from B6-Ly5.1 donors (GVHD –: WT, white circles; KO, white squares) or C3H.SW donors (GVHD +: WT, black circles; KO, red squares). (A–C) Serial body weight measurement (A), clinical GVHD score (B), and survival (C) of mice after BMT (WT, white circles, n = 7; KO, white squares, n = 7; WT, black circles, n = 18; KO, red squares, n = 16). (D–F) On day +7 after BMT, WT or KO recipients (Lgr5-EGFP⁺) were euthanized. Small intestine crypt cells were isolated and analyzed by flow cytometry. Small intestine sections were stained by immunochemistry. (D and E) Quantification of Paneth cells (D) and quantification of Paneth cells undergoing early apoptosis by annexin V⁺ staining (E) (WT, white circles, n = 6; KO, white squares, n = 6; WT, black circles, n = 8; KO, red squares, n = 8). (F) Quantification of cleaved caspase-3⁺ cells per crypt of ileum sections in recipients (each group, n = 5). *P < 0.05, **P < 0.01, ***P < 0.001, unpaired 2-tailed t test (A, B, and D–F); **P < 0.01, log-rank test (C). Data are expressed as mean ± SEM. (G–I) Microbiome composition of fecal samples from WT and KO mice 1 day before BMT (Pre-BMT) (WT, white circles, n = 12; KO, white squares, n = 13) or day +7 after BMT (Post-BMT) (WT, black circles, n = 12; KO, red squares, n = 13). (G) Principal component analysis was computed from the Bray-Curtis β-diversity matrix among all samples. (H and I) Bray-Curtis compositional distances were measured from the centroid of WT pre-BMT samples (H) or post-BMT samples (I). NS, P > 0.05; *P < 0.05, **P < 0.01, Wilcox test.

Figure 5. Prophylactic administration of IL-22 reduces GVHD.

B6D2F1 mice received PBS or IL-22 injections from day +1 after BMT from B6 donors (GVHD, +) or B6D2F1 donors (No GVHD, –). (A and B) Clinical GVHD score (A) and survival (B) of mice after BMT (GVHD +: PBS, black circles, n = 18; IL-22, blue circles, n = 16; GVHD –, white circles, n = 7). (C−E) Samples were analyzed on day +7 after BMT. (C) Serum REG3γ levels. (D) Ileal tissue Reg3g mRNA expression. *P < 0.05, **P < 0.01, unpaired 2-tailed t test (A), 1-way ANOVA (C and D); *P < 0.05, log-rank test (B). (E) H&E (top) staining and REG3γ (bottom) staining by immunohistochemistry in ileum from individual mice with or without IL-22 treatment. Naive B6D2F1 mice were used as controls. Images were taken with an Olympus BX51 with a ×20 objective. Scale bars: 100 μm. Data are expressed as mean ± SEM.

Figure 6. IL-22 treatment reverses GVHD.

B6 mice received BM and T cells from C3H.SW donors (GVHD +) treated with PBS (black circles) or IL-22 (blue circles) from day +7. As a control, mice received BM only (No GVHD, –, white circles). (A and B) Clinical GVHD score (A) and survival (B) of mice after BMT (PBS, n = 14; IL-22, n = 13; no GVHD, n = 5). Samples were analyzed on day +10. (C) Serum REG3γ protein. (D) Small intestine tissue Reg3g mRNA. *P < 0.05, **P < 0.01, unpaired 2-tailed t test (A), 1-way ANOVA (C and D); **P < 0.01, log-rank test (B). Data are expressed as mean ± SEM.

Figure 7. IL-22 requires REG3γ to reduce GVHD.

(A) Survival of WT B6 and B6-Reg3g^–/– (KO) mice received PBS or IL-22 injections from day +1 after BMT from C3H.SW donors (WT-PBS, black circles, n = 9; WT–IL-22, blue circles, n = 8; KO-PBS, red squares, n = 8; KO–IL-22, blue squares, n = 7). (B) Survival of WT B6 and B6-Reg3g^–/– (KO) mice received PBS or IL-22 injections from day +1 after BMT from BALB/c donors (WT-PBS, black circles, n = 10; WT–IL-22, blue circles, n = 11; KO-PBS, red squares, n = 10; KO–IL-22, blue squares, n = 10). *P < 0.05, log-rank test. (C and D) WT B6 and B6-Reg3g^−/− (KO) mice (Lgr5-EGFP⁺) received PBS or IL-22 injections from day +1 after BMT from B6-Ly5.1 donors (GVHD –) or C3H.SW donors (GVHD +). On day +7 after BMT, small intestine crypt cells were analyzed by flow cytometry. (C) Quantification of Paneth cells (GVHD –: WT-PBS, empty black circles, n = 8; WT–IL-22, empty blue circles, n = 4; KO-PBS, empty red squares, n = 8; KO–IL-22, empty blue squares, n = 6; GVHD +: WT-PBS, filled black circles, n = 13; WT–IL-22, filled blue circles, n = 9; KO-PBS, filled red squares, n = 16; KO–IL-22, filled blue squares, n = 10). (D) Quantification of Paneth cells undergoing early apoptosis by annexin V⁺ staining (GVHD –: WT-PBS, empty black circles, n = 5; WT–IL-22, empty blue circles, n = 5; KO-PBS, empty red squares, n = 6; KO–IL-22, empty blue squares, n = 5; GVHD +: WT-PBS, filled black circles, n = 7; WT–IL-22, filled blue circles, n = 9; KO-PBS, filled red squares, n = 10; KO–IL-22, filled blue squares, n = 12). NS, P > 0.05; *P < 0.05, **P < 0.01, unpaired 2-tailed t test. Data are expressed as mean ± SEM.

Figure 8. REG3γ protects ISCs from apoptosis.

(A–D) WT B6 and B6-Reg3g^–/– (KO) mice (Lgr5-EGFP⁺) received PBS or IL-22 injections from day +1 after BMT from B6-Ly5.1 donors (GVHD –) or C3H.SW donors (GVHD +). On day +7 after BMT, small intestine crypt cells were analyzed by flow cytometry, and Reg3g mRNA was measured by qPCR. (A) Quantification of ISCs from mice in Figure 7. (B) Quantification of ISCs undergoing early apoptosis by annexin V⁺ staining in the mice from Figure 7. Data are combined from 3 separate experiments. (C) Quantification of EdU⁺ ISCs in syngeneic recipients (GVHD –: WT-PBS, empty black circles, n = 7; WT–IL-22, empty blue circles, n = 4; KO-PBS, empty red squares, n = 9; KO–IL-22, empty blue squares, n = 5), allogeneic recipients (GVHD +: WT-PBS, filled black circles, n = 8; WT–IL-22, filled blue circles, n = 9; KO-PBS, filled red squares, n = 13; KO–IL-22, filled blue squares, n = 11), and naive WT (downward triangles, n = 5) and KO (upward triangles, n = 5) controls. (D) Correlation of ISCs and Reg3g mRNA expression in WT mice from 2 of the 3 experiments shown in A (n = 25), as calculated by the Pearson correlation coefficient. (E–G) HT-29 cells were cultured with recombinant REG3α protein and apoptotic stimuli as described in Methods. Data are representative of 3 independent experiments. (E and F) Cell lysates were collected after 16-hour cell culture for Western blot analysis. Quantification of cleaved caspase-3 (E) and cleaved caspase-8 (F) expression. (G) Quantification of cell viability by CellTiter-Glo 2.0 assay after 24-hour cell culture. *P < 0.05, **P < 0.01, unpaired 2-tailed t test. Data are expressed as mean ± SEM. ND, not detectable.