Regulation of Colonic Epithelial Repair in Mice by Toll-Like Receptors and Hyaluronic Acid  Ling Zheng, Terrence E. Riehl, William F. Stenson  Gastroenterology  Volume 137, Issue 6, Pages 2041-2051 (December 2009) DOI: 10.1053/j.gastro.2009.08.055 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 HA expression is increased in the DSS model of colitis. (A) Immunofluorescence staining of HA in distal colons of wild type and MyD88−/− mice during 7 days of treatment with 2.5% DSS. In WT mice, DSS treatment induced a marked and progressive increase in intestinal HA in the lamina propria of the distal colons, but no increase occurred in MyD88−/− mice. (B) Mice were given DSS in the same schedule as in A, and plasma was collected at 1, 3, and 7 days. Plasma HA levels were measured by enzyme-linked immunosorbent assay. n = 6, *P < .01 compared with untreated mice. (C) DSS treatment increased the mRNA levels of HAS-2 and HAS-3 by 10-fold in the distal colons of WT mice compared with untreated mice but had no effect in MyD88−/− mice. The mRNA levels of HAS1 and CD44 did not change with DSS treatment. Data are the means ± SE for 4–7 mice, *P < .001 for HA-treated compared with untreated mice. (D) Immunofluorescence staining for HAS-3 in mouse distal colon. There were only scattered HAS-3–expressing cells in untreated mice, and the DSS treatment over 7 days increased the number of HAS-3–expressing stromal cells. (E) Immunofluorescence for F4/80 (green) and HAS-3 (red). Original magnification 400×. Gastroenterology 2009 137, 2041-2051DOI: (10.1053/j.gastro.2009.08.055) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 (A) WT and MyD88−/− mice were given intraperitoneal HA at time 0, and plasma HA levels were measured at 6, 24, 48, and 72 hours. n = 4, *P < .001 compared with MyD88−/− mice. (B) WT mice were given intraperitoneal HA on days 1, 3, and 5. The mice were killed on day 7, and mRNA was extracted from the distal colon. Reverse-transcription polymerase chain reaction was performed for HAS-1, HAS-2, and HAS-3. n = 4, *P < .01, **P < .001 compared with untreated mice. (C) TNF-α production in the distal colon increased over 3-fold in WT mice treated with HA (30 mg/kg), as measured by enzyme-linked immunosorbent assay. Pretreating with PEP-1 (40 mg/kg) for 1 hour blocked HA induction of TNF-α production. The data are the means ± SE for 9 mice. *P < .001 compared with HA-treated mice. (D) Immunofluorescence for MIP-2 (green) and F4/80 (red) in distal colons of WT mice shows that most of the MIP-2–positive cells were macrophages, as indicated by MIP-2 colocalization (arrows) with the macrophage marker F4/80 (original magnification 400×). (E) Western blot analysis shows that administration of HA induced 3-fold increases in the expression of COX-2 and MIP-2 in the distal colons of WT but not MyD88−/− mice. PEP-1 blocked the induction of COX-2 and MIP-2 by HA in WT but not MyD88−/− mice. Gastroenterology 2009 137, 2041-2051DOI: (10.1053/j.gastro.2009.08.055) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 (A) Immunofluorescence for COX-2 (red) in the lamina propria of the distal colon of WT mice. Mice were untreated or received HA on days 1, 3, and 5 or received HA and PEP-1 on days 1, 3, and 5. Mice were killed on day 7 (original magnification 400×). (B) The number of COX-2–expressing cells. Data are the means ± SE for 4 mice. *P < .001 comparing HA-treated with untreated or HA + PEP-1–treated mice. (C) Immunofluorescence for COX-2 (red) and TLR4 (green) in mouse distal colons. Mice were untreated or treated with HA on days 1, 3, and 5 and killed on day 7 (original magnification 400×). (D) Immunofluorescence for COX-2 (red) and F4/80 (green) in the colons of untreated and HA-treated mice (arrow: yellow cell with colocalization of COX-2 and F4/80). Gastroenterology 2009 137, 2041-2051DOI: (10.1053/j.gastro.2009.08.055) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 HA administration reduced colitis in the DSS model in WT but not MyD88−/− mice. (A, upper panel) HA (30 mg/kg) prevented weight loss in DSS-treated WT mice. *P < .001 comparing DSS-treated with DSS + HA–treated WT mice. (A, lower panel) HA had no effect in MyD88−/− mice. Data are means ± SE for 9 mice. (B, upper panel) Colonic disease severity scores of DSS-treated WT mice were improved with the addition of HA. Administration of PEP-1 (40 mg/kg) to DSS-treated WT mice resulted in more severe scores than mice treated with DSS alone. *P < .001 comparing DSS-treated with DSS + HA– or DSS + PEP-1–treated WT mice. (B, lower panel) HA and PEP-1 had no effect on colonic disease severity scores in DSS-treated MyD88−/− mice. The data are means ± SE for 9 mice. (C) In WT mice, the addition of HA to DSS reduced histologic scores in the distal colon compared with DSS alone. The addition of PEP-1 to DSS increased histologic scores. HA and PEP-1 had no effect in DSS-treated MyD88−/− mice. Data are the means ± SE for 9 mice. *P < .01 comparing DSS-treated with DSS + HA– or **P < .001 with DSS + PEP-1–treated mice on day 7. Gastroenterology 2009 137, 2041-2051DOI: (10.1053/j.gastro.2009.08.055) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 HA treatment of preexisting DSS-induced colitis. WT mice were treated with oral DSS for 7, 14, or 21 days. HA therapy was initiated after 7 days of oral DSS administration. HA therapy consisted of intraperitoneal administration of HA on alternate days (arrows). (A) Weight change. n = 4, *P < .001 comparing DSS + HA–treated mice with mice treated with DSS alone. **P < .001 comparing untreated mice with DSS-treated mice. (B) Colonic disease severity score. n = 4, *P < .01, **P < .001 compared with DSS alone for 14 days. n = 4, **P < .001 compared with DSS alone for 21 days. (C) Histologic score. n = 4, *P < .001 compared with DSS alone for 21 days. Gastroenterology 2009 137, 2041-2051DOI: (10.1053/j.gastro.2009.08.055) Copyright © 2009 AGA Institute Terms and Conditions

Figure 6 HA rescues DSS-induced colitis in CD44−/− but not TLR4−/− mice. (A) Weight loss in CD44−/− mice. n = 4, **P < .001 compared with untreated mice. (B) Weight loss in TLR4−/− mice. n = 4, **P < .001 compared with untreated mice. (C) Colonic disease severity score. n = 4, **P < .01 or *P < .05 compared with DSS alone. (D) Histologic scores. n = 4, **P < .01 compared with DSS alone. (E) Plasma HA levels. n = 6, **P < .001. Gastroenterology 2009 137, 2041-2051DOI: (10.1053/j.gastro.2009.08.055) Copyright © 2009 AGA Institute Terms and Conditions

Figure 7 HA treatment failed to rescue the DSS-induced weight loss and histologic score in COX2−/− mice. (A) Weight loss in COX2−/− mice. *P < .01 compared with untreated mice. (B) Histologic scores in COX-2−/− mice. *P < .001 compared with untreated mice. Gastroenterology 2009 137, 2041-2051DOI: (10.1053/j.gastro.2009.08.055) Copyright © 2009 AGA Institute Terms and Conditions