RIG-I/MAVS and STING signaling promote gut integrity during irradiation- and immune-mediated tissue injury

Abstract

The molecular pathways that regulate the tissue repair function of type I interferon (IFN-I) during acute tissue damage are poorly understood. We describe a protective role for IFN-I and the RIG-I/MAVS signaling pathway during acute tissue damage in mice. Mice lacking mitochondrial antiviral-signaling protein (MAVS) were more sensitive to total body irradiation- and chemotherapy-induced intestinal barrier damage. These mice developed worse graft-versus-host disease (GVHD) in a preclinical model of allogeneic hematopoietic stem cell transplantation (allo-HSCT) than did wild-type mice. This phenotype was not associated with changes in the intestinal microbiota but was associated with reduced gut epithelial integrity. Conversely, targeted activation of the RIG-I pathway during tissue injury promoted gut barrier integrity and reduced GVHD. Recombinant IFN-I or IFN-I expression induced by RIG-I promoted growth of intestinal organoids in vitro and production of the antimicrobial peptide regenerating islet-derived protein 3 γ (RegIIIγ). Our findings were not confined to RIG-I/MAVS signaling because targeted engagement of the STING (stimulator of interferon genes) pathway also protected gut barrier function and reduced GVHD. Consistent with this, STING-deficient mice suffered worse GVHD after allo-HSCT than did wild-type mice. Overall, our data suggest that activation of either RIG-I/MAVS or STING pathways during acute intestinal tissue injury in mice resulted in IFN-I signaling that maintained gut epithelial barrier integrity and reduced GVHD severity. Targeting these pathways may help to prevent acute intestinal injury and GVHD during allogeneic transplantation.

Fig. 1. Endogenous RIG-I/MAVS signaling reduces intestinal tissue damage in mice

(A) Representative images of tissue damage in hematoxylin and eosin–stained small intestine biopsies from mice after an 11-Gy TBI conditioning regimen. White asterisks, villus stunting; black arrowhead, crypt apoptosis; black arrows, granulocyte infiltration. (B) Histopathological score from (A); pooled data from two independent experiments. (C) Number of leukocytes infiltrating the mouse gut lamina propria after TBI (11 Gy) analyzed by flow cytometry. Pooled data from four independent experiments. Survival (D) and weight loss (E) in mouse allo-HSCT recipients transplanted with 5 × 10⁶ BM ± 2 × 10⁶ T cells (donor BALB/c WT and C57BL/6 MAVS^+/+ or MAVS^−/− recipients). Pooled data from four independent experiments. (F) FITC-dextran concentrations in plasma after allo-HSCT [described in (D) and (E)]. Pooled data from two independent experiments. (G) Survival of Rig-I^−/+ and Rig-I^−/− mouse recipients after transplant with 5 × 10⁶ BM and 1 × 10⁶ T cells (donor C57BL/6 WT, recipient 129/sv Rig-I^−/+ or Rig-I^−/−). Animal numbers per group (n) are depicted in the figure panels. Survival was analyzed using the log-rank test. All other experiments were analyzed using two-tailed unpaired t test. *P < 0.05, **P < 0.01, and ***P < 0.001. Data are means ± SEM. n.s., not significant.

Fig. 2. MAVS signaling in nonhematopoietic cells maintains intestinal barrier function

(A) Average relative abundance of bacterial genera in the intestinal microbiota of cohoused Mavs^+/+ (n = 5) and Mavs^−/− (n = 5) litter-mates. One representative experiment of two independent experiments. (B) Survival of C57BL/6 BM chimeric mice, which were Mavs-deficient either in the hematopoietic or in the nonhematopoietic compartment. These mice were analyzed after a second allo-HSCT with BM and T cells from B10.BR WT donor mice. (C) GVHD histopathological score for small intestine biopsies from C57BL/6 BM chimeric mice, which were Mavs-deficient either in the hematopoietic or in the nonhematopoietic compartment. These mice were analyzed after a second allo-HSCT with BM and T cells from BALB/c WT donor mice. Pooled data from two independent experiments. (D) Quantitative polymerase chain reaction (qPCR) of Itgb6 and RegIIIγ expression in the small intestine after allo-HSCT with BM and T cells from BALB/c WT mouse donors into C57BL/6 MAVS^+/+ or MAVS^−/− recipients. Pooled data from five independent experiments. Animal numbers per group (n) are depicted above panels. Survival was analyzed using the log-rank test. Other experiments were analyzed using ordinary one-way analysis of variance (ANOVA) for multiple comparisons or two-tailed unpaired t test. *P < 0.05, **P < 0.01, and ***P < 0.001. Data are means ± SEM.

Fig. 3. RIG-I/MAVS pathway activation protects from intestinal tissue damage after TBI

Survival (A) and weight loss (B) after allo-HSCT involving transplant of 5 × 10⁶ BM ± 1 × 10⁶ T cells from C57BL/6 WT donor mice into BALB/c WT recipients with or without 3pRNA treatment on day −1. Pooled data from four independent experiments. (C) Histopathological score of small intestine tissue after allo-HSCT from C57BL/6 WT donor mice to BALB/c WT mouse recipients with or without 3pRNA treatment on day −1. Pooled data from two independent experiments. (D) Weight loss in mouse recipients from (C) after allo-HSCT with or without 3pRNA treatment on day 0 (d0) or day +1 (d+1). Pooled data from three independent experiments. (E) FITC-dextran concentrations in plasma after allo-HSCT from C57BL/6 WT donor mice to BALB/c WT recipient mice with or without 3pRNA treatment on day −1. (F) qPCR of RegIIIγ expression in mouse gut epithelial cells after allo-HSCT from C57BL/6 WT donor mice into BALB/c WT recipient mice with or without 3pRNA treatment on day −1. Pooled data from three independent experiments. (G) Bacterial colony-forming units (CFU) in sera from mouse recipients after allo-HSCT [described in (F)]. Pooled data from three independent experiments. (H) Leukocytes in the small intestine lamina propria of BALB/c mice after TBI (9 Gy) analyzed by flow cytometry. Pooled data from two independent experiments. (I) Leukocytes in the small intestine lamina propria of C57BL/6 mice after treatment with chemotherapy (doxorubicin) analyzed by flow cytometry. Pooled data from three independent experiments. (J) Weight loss in C57BL/6 mice that received doxorubicin (20 mg/kg). Pooled data from six independent experiments. (K) FITC-dextran concentrations in plasma from C57BL/6 mice after doxorubicin treatment (20 mg/kg). One representative experiment of four independent experiments is shown. Animal numbers per group (n) are depicted above figure panels. 3pRNA treatment was always performed on day −1 in indicated groups except for (D). All experiments were analyzed using one-tailed (G) or two-tailed unpaired t test or ordinary one-way ANOVA for multiple comparisons. Survival was analyzed using the log-rank test. *P < 0.05, **P < 0.01, and ***P < 0.001. Data are means ± SEM.

Fig. 4. RIG-I–induced type I IFN signaling mediates intestinal tissue protection

(A) Survival and percent of initial weight after allo-HSCT. BM cells (5 × 10⁶) plus T cells (1 × 10⁶) from C57BL/6 WT mice were transplanted into BALB/c recipient mice with or without 3pRNA treatment on day −1 before allo-HSCT and treatment with IFNAR1-blocking antibody (α-IFNAR1) or immunoglobulin G1 (IgG1) isotype control on day −2 before allo-HSCT. Pooled data from three independent experiments. (B) Weight loss after allo-HSCT. BM cells (5 × 10⁶) plus T cells (1 × 10⁶) from C57BL/6 WT mice were transplanted into BALB/c recipient mice with or without 3pRNA treatment on day −1 before allo-HSCT and treatment with IFNAR1-blocking antibody (α-IFNAR1) 48 hours before allo-HSCT (−48 h before TX), at the time of allo-HSCT (0 h before TX), or 24 hours after allo-HSCT (24 h after TX). (C) FITC-dextran concentrations in plasma of BALB/c mouse recipients after allo-HSCT, with or without 3pRNA treatment on day −1 before allo-HSCT, and treatment with IFNAR1-blocking antibody (α-IFNAR1) or IgG1 isotype control (IC) on day −2 before allo-HSCT. Pooled data from three independent experiments. (D and E) qPCR of RegIIIγ and Itgb6 expression in small intestine biopsies from BALB/c recipient mice after allo-HSCT, with or without 3pRNA treatment on day −1 before allo-HSCT, and treatment with IFNAR1-blocking antibody (α-IFNAR1) or IgG1 isotype control on day −2 before allo-HSCT. Pooled data from three (D) and six (E) independent experiments. (F) Flow cytometry analysis of leukocyte infiltration into the small intestine lamina propria of BALB/c mice after TBI(9 Gy), with or without 3pRNA treatment on day −1 before TBI, and treatment with IFNAR1-blocking antibody (α-IFNAR1) or IgG1 isotype control on day −2 before TBI. Pooled data from two independent experiments. (G) Survival and weight loss in BALB/c mouse recipients lacking IL-22 (Il-22^−/−) after allo-HSCT with BM and T cells from C57BL/6 WT mice with or without 3pRNA treatment on day −1 before allo-HSCT. Pooled data from two independent experiments. Animal numbers per group (n) are depicted above figure panels. Treatment time points for 3pRNA and α-IFNAR1 antibody are indicated. All experiments were analyzed using two-tailed unpaired t test or ordinary one-way ANOVA for multiple comparisons. Survival was analyzed using the log-rank test. *P < 0.05, **P < 0.01, and ***P < 0.001. Data are means ± SEM.

Fig. 5. RIG-I–induced type I IFN signaling in nonhematopoietic cells promotes regeneration of the ISC compartment

(A) Weight loss after allo-HSCT. BM cells (5 × 10⁶) plus T cells (5 × 10⁶) from BALB/c donor mice transplanted into C57BL/6 recipient mice (genotypes are indicated in the figure) with or without 3pRNA treatment on day −1. Pooled data from two independent experiments. (B) Number of organoids after 5 days in culture with or without the addition of 3pRNA (2 μg/ml) on day 1 of culture; organoids were derived from C57BL/6 IFNAR1^+/+ or IFNAR1^−/− mice. One representative experiment of three independent experiments. (C) Representative images of organoids derived from C57BL/6 WT mice after 5 days in culture with or without the addition of 3pRNA (2 μg/ml) on day 1 of culture. Organoid area is shown. (D) Measurement of organoids from C57BL/6 WT mice after 5 days in culture with or without the addition of 3pRNA (2 μg/ml) or a-IFNAR1–blocking antibody (10 μg/ml) on day 1 of culture. One representative experiment of three independent experiments. (E) Size of organoids derived from C57BL/6 WT mice after 7 days in culture with or without the addition of recombinant murine IFN-b (20 U/ml) on day 1 of culture. One representative experiment of three independent experiments. (F) qPCR of RegIIIγ expression in organoids 24 hours after stimulation with indicated combinations of 3pRNA and α-IFNAR1–blocking antibody or IgG1 isotype control. Pooled data from three independent experiments. All experiments were analyzed using two-tailed unpaired t test or ordinary one-way ANOVA for multiple comparisons. Survival was analyzed using the log-rank test. *P < 0.05, **P < 0.01, and ***P < 0.001. Data are means ± SEM.

Fig. 6. RIG-I activation protects ISCs after allo-HSCT

(A) qPCR expression of Lysozyme P and Lgr5 in small intestine biopsies from MAVS^+/+ or MAVS^−/− C57BL/6 recipient mice after allo-HSCT with 5 × 10⁶ BM cells and 2 × 10⁶ T cells from BALB/c WT donor mice. Pooled data from five independent experiments. (B) Analysis of allo-HSCT BALB/c recipients in the presence or absence of 3pRNA treatment on day −1. Immunohistochemistry showing lysozyme staining of the small intestine of BALB/c recipients 8 days after allo-HSCT. Lysozyme-positive gut Paneth cells are indicated by black arrowheads. Histogram shows number of Paneth cells per crypt. Representative images and pooled data from three independent experiments. (C) qPCR showing expression of Lysozyme P and Lgr5 in IECs from the small intestine of BALB/c recipient mice 8 days after allo-HSCT with or without 3pRNA treatment on day −1 before allo-HSCT. Pooled data from three independent experiments. (D) qPCR of Lysozyme P and Lgr5 expression in the small intestine of BALB/c recipient mice after allo-HSCT with or without 3pRNA treatment on day +1. Data from one experiment. (E) Number of organoids derived from C57BL/6 recipient mice on day 8 after allo-HSCT with or without 3pRNA treatment on day −1 before allo-HSCT. Pooled data from four independent experiments. Animal numbers per group (n) are depicted above figure panels. All experiments were analyzed using two-tailed unpaired t test or ordinary one-way ANOVA for multiple comparisons. *P < 0.05, **P < 0.01, and ***P < 0.001. Data are means ± SEM.

Fig. 7. STING pathway protects mouse recipients from GVHD after allo-HSCT

(A) Survival of cohoused C57BL/6 WT or Sting^gt/gt mice that received 5 × 10⁶ BM cells plus1× 10⁶ T cells from WT B10.BR donor mice. Pooled data from two independent experiments. (B) Relative abundance of bacterial genera in the intestinal microbiota of cohoused WT and Sting^gt/gt mice. One representative experiment of two independent experiments. Survival (C) and weight loss (D) for C57BL/6 mouse recipients after allo-HSCT from B10.BR donor mice with or without IFN-stimulatory DNA (ISD) treatment on day −1. Pooled data of two (C) or three (D) independent experiments. (E) FITC-dextran concentrations in plasma of BALB/c mouse recipients after allo-HSCT from C57BL/6 donors with or without calf thymus DNA (CT DNA) or IFN-stimulatory DNA treatment on day −1. Pooled data from two independent experiments. (F) Number of organoids derived from WT or Sting^gt/gt C57BL/6 mouse small intestine after 5 days in culture with or without the addition of IFN-stimulatory DNA (2 μg/ml) on day 1 of culture. Pooled data from three independent experiments. (G) Organoids derived from C57BL/6 mouse small intestine. Measurement of organoid size after 5 days in culture with or without the addition of IFN-stimulatory DNA (2 μg/ml), α-IFNAR1–blocking antibody (10 μg/ml), or IgG1 isotype control on day 1 of culture. The experiment was performed three times, and images of one representative experiment are shown. (H) qPCR of RegIIIγ expression in organoids derived from C57BL/6 WT mice 24 hours after stimulation with indicated combinations of IFN-stimulatory DNA, α-IFNAR1–blocking antibody, or IgG1 isotype control. Pooled data from three independent experiments. (I) DNA in plasma of BALB/c mice 24 hours after TBI (9 Gy) or allo-HSCT. Pooled data from three independent experiments. Animal numbers per group (n) are depicted above figure panels. All experiments were analyzed using two-tailed unpaired t test or ordinary one-way ANOVA for multiple comparisons. Survival was analyzed using the log-rank test. *P < 0.05, **P < 0.01, and ***P < 0.001. Data are means ± SEM.