An anti-inflammatory role for carbon monoxide and heme oxygenase-1 in chronic Th2-mediated murine colitis

Abstract

Cigarette smoking is a significant environmental factor in the human inflammatory bowel diseases, remarkably, conferring protection in ulcerative colitis. We previously demonstrated that a prominent component of cigarette smoke, CO, suppresses Th17-mediated experimental colitis in IL-10(-/-) mice through a heme oxygenase (HO)-1-dependent pathway. In this study, homeostatic and therapeutic effects of CO and HO-1 were determined in chronic colonic inflammation in TCR-α-deficient ((-/-)) mice, in which colitis is mediated by Th2 cytokines, similar to the cytokine milieu described in human ulcerative colitis. TCRα(-/-) mice exposed to CO or treated with the pharmacologic HO-1 inducer cobalt protoporphyrin demonstrated amelioration of active colitis. CO and cobalt protoporphyrin suppressed colonic IL-1β, TNF, and IL-4 production, whereas IL-10 protein secretion was increased. CO induced IL-10 expression in macrophages and in vivo through an HO-1-dependent pathway. Bacterial products regulate HO-1 expression in macrophages through MyD88- and IL-10-dependent pathways. CO exposure and pharmacologic HO-1 induction in vivo resulted in increased expression of HO-1 and IL-10 in CD11b(+) lamina propria mononuclear cells. Moreover, induction of the IL-10 family member IL-22 was demonstrated in CD11b(-) lamina propria mononuclear cells. In conclusion, CO and HO-1 induction ameliorated active colitis in TCRα(-/-) mice, and therapeutic effects correlated with induction of IL-10. This study provides further evidence that HO-1 mediates an important homeostatic pathway with pleiotropic anti-inflammatory effects in different experimental models of colitis and that targeting HO-1, therefore, is a potential therapeutic strategy in human inflammatory bowel diseases.

FIGURE 1

CO ameliorates Th2-mediated colitis in TCRα^−/− mice. TCRα^−/− mice were housed in ambient air or a chamber maintaining a constant concentration of CO at 250 ppm (n = 10 each) from 12–16 wk of age. A, CO-exposed mice gained more weight than did air-exposed mice. B, Colitis scores were significantly decreased in CO-exposed mice compared with control mice. Results are presented as the sum total of four averaged scores from five regions of the large intestine graded by a pathologist blinded to the groups using a standard scoring system. Bars represent mean ± SEM of 10 mice/group. C, Spontaneous protein secretion determined in 24-h supernatants from colonic explants from CO-exposed (black bars) and air-exposed (white bars) TCRα^−/− mice. Spontaneous IL-10 and IL-4 were measured by cytokine-specific ELISA. D, IL-1β, TNF, and IL-17 secretion were assessed by Linco 16-multiplex cytokine assay. Each result represents the mean ± SEM of triplicate assays. E, Ten-week-old TCRα^−/− mice were divided into three groups: exposed to CO (250 ppm; black bar) for 4 wk, exposed to air for 4 wk (white bar), or exposed to CO for 2 wk and then transferred to ambient air housing conditions for 2 wk (gray bar). Spontaneous IL-10 secretion was measured in full-length colonic cell-free supernatants using cytokine-specific IL-10 ELISA. Results represent mean ± SEM from three experiments (n = 3 mice/group). *p < 0.05, **p < 0.01 versus air-exposed mice.

FIGURE 2

CO upregulates Hmox1 and Il10 expression in colonic CD11b⁺ LPMCs from TCRα^−/− mice. LPMCs were isolated from colons of TCRα^−/− mice treated with iALF186 (n = 4) or ALF186 (n = 4). LPMCs were further separated into CD11b⁻ and CD11b⁺ cells and analyzed for Hmox1 (A), Il10 (B), and Il22 (C) expression by real-time RT-PCR. Results were normalized to β-actin. Bars represent mean ± SEM of triplicate cultures from pooled LPMCs from four mice per group. *p < 0.05 versus iALF186-treated CD11b⁺ LPMCs.

FIGURE 3

CO induces IL-10 in murine macrophages via the HO-1 pathway. BMMs from WT (A), TCRα^−/− (B), or Hmox1^−/− (C) mice were cultured in CO (250 ppm, black bars) or ambient air (white bars). Following activation with LPS (1 μg/ml), IL-10 protein secretion was assayed in 24-h supernatants by ELISA. WT BMMs were incubated with iALF186 (100 μg/ml) or ALF186 (100 μg/ml) for 3 h, and Hmox1 (D) and Il10 (E) expression was analyzed by real-time RT-PCR. Results were normalized to β-actin and represent the mean ± SEM of triplicate assays from three independent experiments. *p < 0.05 versus air-exposed or iALF186-treated BMMs.

FIGURE 4

The HO-1 inducer CoPP ameliorates colitis in TCRα^−/− mice. Twelve-week-old TCRα^−/− mice were treated with i.p. injection of CoPP (5 mg/kg, twice a week for 2 wk) (n = 8), and control mice were treated with DMSO vehicle i.p. (n = 12). A, CoPP-injected mice had less severe colitis. B, Spontaneous protein secretion determined in 24-h supernatants from colonic explants from CoPP-treated (5 mg/kg) and vehicle-treated (DMSO) TCRα^−/− mice. C, Spontaneous IL-4 was determined by cytokine-specific ELISA, and IL-1β, TNF, and IL-17 secretion were determined by Linco 16-multiplex cytokine assay. Bars represent mean ± SEM from 12 mice per group. D, LPMCs were isolated from colons of TCRα^−/− mice treated with CoPP (black bars) and vehicle (white bars), separated into CD11b⁻ and CD11b⁺ cells, and analyzed for Hmox1 expression by real-time RT-PCR, with results normalized to β-actin. E, IL-10 secretion was determined by ELISA. Each result represents the mean ± SEM of triplicate assays from four mice/treatment group. *p < 0.05 versus vehicle-treated mice.

FIGURE 5

Hmox1 function is required for amelioration of colitis and Il10 and Il22 upregulation by CO in TCRα^−/− mice. A, Colitis scores were significantly higher in iALF-186 (n = 6) ALF186⁺ SnPP (50 μM/kg twice/weekly for 2 wk) (n = 6) treated mice compared with mice treated with ALF186 alone (n = 6). B and C, LPMCs were isolated from colons of TCRα^−/− mice treated with ALF186 (n = 6) or ALF186+SnPP (n = 6). They were separated into CD11b⁻ and CD11b⁺ cells and analyzed for Il10 (B) and Il22 (C) expression by real-time RT-PCR. Results were normalized to β-actin. Bars represent mean ± SEM triplicate cultures from pooled LPMCs from six mice per group. *p < 0.05 versus iALF186-treated TCRα^−/− mice and CD11b⁺ LPMCs.

FIGURE 6

Regulation of HO-1 in macrophages is IL-10 and MyD88 dependent. A, WT and IL-10⁻ BMMs were stimulated with LPS alone (100 ng/ml) or with LPS plus IL-10 (10 ng/ml) for 12 h. Total RNA was isolated and analyzed for Hmox1, and β-actin mRNA expression was detected by real-time RT-PCR. Results are expressed as mean ± SEM from three independent experiments. *p < 0.05 versus LPS-treated WT BMMs. B, WT and IL-10^−/− BMMs were stimulated with LPS (100 ng/ml) in the presence of IL-10 (10 ng/ml) for 24 h after initial preincubation with anti–IL-10 Ab (10 μg/ml) for 1 h. HO-1 protein was analyzed by Western blotting. Data are representative of five independent experiments with similar results. C, WT and MYD88^−/− BMMs were stimulated with LPS (100 ng/ml), CpG (1 μM), sBLP (100 ng/ml), flagellin (10 ng/ml), or IL-10 (10 ng/ml) for 24 h. HO-1 and Nrf2 protein was analyzed by Western blotting. Data are representative of three independent experiments.