Hyaluronic Acid Induces Radioprotection in the Intestine by a TLR-4 and COX-2 Dependent Mechanism

Background: Hyaluronic acid (HA), a glycosaminoglycan that forms a part of the extracellular matrix, affects inflammation and wound repair through binding to its receptors CD44, TLR2 and TLR4. HA expression is increased in dextran sodium sulfate (DSS) colitis and exogenous HA is protective in DSS colitis through a mechanism that was both MyD88 and COX-2 dependent. PGE2 produced by COX-1 and COX-2 is radioprotective in the intestine. Here we sought to determine if radiation induces HA expression and if exogenous HA induces radioprotection in the intestine. Methods: All experiments were done in wild type, MyD88-/, TLR4-/-, and COX-2-/mice on a C57Bl/6 background. To determine if radiation induces HA expression, mice were irradiated (12Gy), six hours later they were sacrificed. Small intestinal HA expression was assessed by immunohistochemistry. Plasma HA was assessed by ELISA. To determine if exogenous HA is radioprotective, mice were given either vehicle or HA (30mg/kg) intraperitoneally and were irradiated (12Gy). Six hours later the mice were sacrificed and radiation-induced apoptosis was assessed by histology. Mice were given vehicle or HA and irradiated, 84 hours later they were sacrificed and crypt survival was assessed. Using immunohistochemistry for surface markers we identified a population of constitutively COX-2 positive lamina propria mononuclear cells as mesenchymal stem cells (MSCs). We followed the effects of HA on the number and distribution of the MSCs. Results: Radiation increased HA expression in the small intestine with all the expression in the lamina propria. Plasma HA levels six hours after radiation increased six fold (p<.001). Administration of HA prior to irradiation induced a 60% decrease in radiation induced apoptosis at positions 4-12 (p<.01). Crypt survival was 27 crypts per cross section in the mice receiving HA compared with 15 crypts per cross section in mice receiving vehicle (p<.001). The effects of HA on radiation induced apoptosis and crypt survival were MyD88, TLR4, and COX-2dependent. The total number of intestinal MSCs in Wild Type mice changed little after HA treatment, but the distribution changed from 20% in crypts and 80% in villi in controls to 40% in crypts and 60% in villi at 8 hours after HA treatment. In TLR4-/mice, the total number of cells COX-2 expressing decreased after HA treatment, but the distribution remained unchanged with 32% in crypts and 68% in villi. Conclusion: Radiation induces HA expression in the small intestine and increases plasma HA. Intraperitoneal HA induces radioprotection in the small intestine through a COX-2, MyD88, and TLR4 dependent pathway. Intraperitoneal HA induces the migration of COX-2 positive MSCs from the lamina propria in the villi to the lamina propria near the crypt epithelial cells. This migration may relate to the radioprotective effects of HA.