Mitochondrial antiviral signaling protein (MAVS) monitors commensal bacteria and induces an immune response that prevents experimental colitis

Abstract

RIG-I-like receptors (RLRs) activate host innate immune responses against virus infection through recruiting the mitochondrial adaptor protein MAVS (also known as IPS1, VISA, or CARDIF). Here we show that MAVS also plays a pivotal role in maintaining intestinal homeostasis. We found that MAVS knockout mice developed more severe mortality and morbidity than WT animals in an experimental model of colitis. Bone marrow transplantation experiments revealed that MAVS in cells of nonhematopoietic origin plays a dominant role in the protection against colitis. Importantly, RNA species derived from intestinal commensal bacteria activate the RIG-I-MAVS pathway to induce the production of multiple cytokines and antimicrobial peptides, including IFN-β and RegIIIγ. These results unveil a previously unexplored role of MAVS in monitoring intestinal commensal bacteria and maintaining tissue homeostasis.

Fig. 1.

Mavs^−/− mice are highly susceptible to DSS-induced colitis. Four groups of mice—WT, Mavs^−/−, MyD88^−/−, and MyD88^−/−Mavs^−/−—were given 2% DSS in drinking water for 7 d, followed by normal drinking water for another 14 d. (A) Increased mortality in Mavs-deficient mice. Statistical analysis was performed using Mantel-Cox test. Compared with WT: Mavs^−/− P = 0.0045, MyD88^−/− P < 0.0001, MyD88^−/−Mavs^−/− P < 0.0001. Compared with MyD88^−/−: MyD88^−/−Mavs^−/− P = 0.0002. (B) Weight change following DSS administration. Error bars represent SEMs. Statistics of the weight changes on day 8 was determined using the Student's test, Mavs^−/− P = 0.0015, MyD88^−/− P = 0.0022, MyD88^−/−Mavs^−/− P < 0.0001. (C) Gross morphological changes of colon and spleen. On day 8 after 7 d of 2% DSS and 1 d drinking water, colons and spleens were excised and photographed. Representative photos of colon and spleen are shown on the left. Colon lengths are shown on the right. The numbers of mice used in the experiments are as follows: WT: n = 10; Mavs^−/−: n = 11; MyD88^−/−: n = 5; MyD88^−/−Mavs^−/−: n = 5. (D) Colons were cut longitudinally, fixed, and then stained with H&E. Representative histological images are shown on the left and pathological scores on the right. WT: n = 10; Mavs^−/−: n = 11; MyD88^−/−: n = 8; MyD88^−/−Mavs^−/−: n = 10.

Fig. 2.

MAVS in cells of nonhematopoietic origin plays a dominant role in preventing DSS-induced colitis. Bone marrow transplantation experiments were carried out and the efficiency of transplantation was verified by FACS analysis of leukocyte surface markers, as described in Methods. (A) All animals were treated with 2% DSS in drinking water before switching back to normal water, and mortality was monitored until the 21st day. Mavs^−/− mice reconstituted with WT bone marrow show slightly better survival than those reconstituted with Mavs^−/− bone marrow (P = 0.0448). (B) Weight loss of the mice after DSS treatment was monitored, as described in Fig. 1B. The results are representative of three independent experiments.

Fig. 3.

RNA derived from mouse feces induces IFN-β through the RIG-I–MAVS pathway. (A) Feces RNA was treated with or without SAP or RNase III, then transfected into HEK293T IFN-β-luciferase reporter cells. Luciferase activity was measured 24 h after transfection. (B) RIG-I–dependent induction of IFN-β mRNA by feces RNA. Feces RNA was transfected into WT or RIG-I–deficient MEF cells, then IFN-β mRNA was measured by qRT-PCR 8 h after transfection. (C) One-microgram of RNA from the feces of conventional, antibiotics-treated, or germ-free mice was transfected into BMDM. IFN-β mRNA was measured by qRT-PCR 8 h after transfection. (D) Feces RNA from conventionally raised mice was transfected into BMDM from mice of the indicated genotypes, and the secretion of IFN-β protein in the media was measured by ELISA 24 h after transfection. (E) Feces RNA or poly(I:C) was transfected into HT-29 cells, then IFN-β RNA was measured by qRT-PCR 8 h after transfection. LPS was added to the culture media without transfection reagent. (F) RNA isolated from L. salivarius (LAB) was treated with RNase V1 or not treated before transfection into Raw264.7 cells (Left) and the induction of IFN-β RNA was measured by qRT-PCR. (Right) LAB or liver RNA was transfected into BMDM from WT or Mavs^−/− mice, followed by measurement of IFN-β RNA. Error bars represent the variation range of duplicate experiments (A) or SD of triplicate experiments (B–F).

Fig. 4.

(A–D) Feces RNA induces multiple cytokines through MAVS and MyD88. Feces RNA was transfected into BMDM cells from WT, Mavs^−/−, MyD88^−/−, and MyD88^−/−Mavs^−/− mice, and the indicated cytokines were measured by qRT-PCR 8 h after transfection. LPS was added to the culture media without transfection reagent.

Fig. 5.

MAVS-dependent expression of RegIIIγ in gut mucosa. (A) Feces RNA and poly(I:C) were transfected into HT-29 cells and RegIIIγ was measured by qRT-PCR 8 h after transfection. LPS was added to the culture media without transfection reagent. (Right) Feces RNA was treated with RNase V1 or SAP before transfection into HT-29 cells. (B) Tissue samples were prepared from ileums of WT and mutant mice, as described in Methods, and the protein extracts were analyzed by immunoblotting with antibodies against RegIIIγ, chaperone proteins BiP and Hsp70, and GAPDH. (C) RegIIIγ mRNA in the small intestines from DSS-treated mice was measured by qRT-PCR. The result represents two independent experiments.