Steven Russo

An Anti-Inflammatory Role for Carbon Monoxide and Heme Oxygenase-1 in Chronic Th2-Mediated Murine Colitis

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.

Carbon monoxide and heme oxygenase-1 prevent intestinal inflammation in mice by promoting bacterial clearance.

BACKGROUND & AIMS Heme oxygenase-1 (HO-1) and its metabolic by-product, carbon monoxide (CO), protect against intestinal inflammation in experimental models of colitis, but little is known about their intestinal immune mechanisms. We investigated the interactions among CO, HO-1, and the enteric microbiota in mice and zebrafish. METHODS Germ-free, wild-type, and interleukin (Il)10(-/-) mice and germ-free zebrafish embryos were colonized with specific pathogen-free (SPF) microbiota. Germ-free or SPF-raised wild-type and Il10(-/-) mice were given intraperitoneal injections of cobalt(III) protoporphyrin IX chloride (CoPP), which up-regulates HO-1, the CO-releasing molecule Alfama-186, or saline (control). Colitis was induced in wild-type mice housed in SPF conditions by infection with Salmonella typhimurium. RESULTS In colons of germ-free, wild-type mice, SPF microbiota induced production of HO-1 via activation of nuclear factor erythroid 2-related factor 2-, IL-10-, and Toll-like receptor-dependent pathways; similar observations were made in zebrafish. SPF microbiota did not induce HO-1 in colons of germ-free Il10(-/-) mice. Administration of CoPP to Il10(-/-) mice before transition from germ-free to SPF conditions reduced their development of colitis. In Il10(-/-) mice, CO and CoPP reduced levels of enteric bacterial genomic DNA in mesenteric lymph nodes. In mice with S typhimurium-induced enterocolitis, CoPP reduced the numbers of live S typhimurium recovered from the lamina propria, mesenteric lymph nodes, spleen, and liver. Knockdown of HO-1 in mouse macrophages impaired their bactericidal activity against E coli, E faecalis, and S typhimurium, whereas exposure to CO or overexpression of HO-1 increased their bactericidal activity. HO-1 induction and CO increased acidification of phagolysosomes. CONCLUSIONS Colonic HO-1 prevents colonic inflammation in mice. HO-1 is induced by the enteric microbiota and its homeostatic function is mediated, in part, by promoting bactericidal activities of macrophages.

IL-10 Regulates Il12b Expression via Histone Deacetylation: Implications for Intestinal Macrophage Homeostasis

To prevent excessive inflammatory responses to commensal microbes, intestinal macrophages, unlike their systemic counterparts, do not produce inflammatory cytokines in response to enteric bacteria. Consequently, loss of macrophage tolerance to the enteric microbiota plays a central role in the pathogenesis of inflammatory bowel diseases. Therefore, we examined whether the hyporesponsive phenotype of intestinal macrophages is programmed by prior exposure to the microbiota. IL-10, but not in vivo exposure to the microbiota, programs intestinal macrophage tolerance, because wild-type (WT) colonic macrophages from germ-free and specific pathogen-free (SPF)-derived mice produce IL-10, but not IL-12 p40, when activated with enteric bacteria. Basal and activated IL-10 expression is mediated through a MyD88-dependent pathway. Conversely, colonic macrophages from germ-free and SPF-derived colitis-prone Il10−/− mice demonstrated robust production of IL-12 p40. Next, mechanisms through which IL-10 inhibits Il12b expression were investigated. Although Il12b mRNA was transiently induced in LPS-activated WT bone marrow-derived macrophages (BMDMs), expression persisted in Il10−/− BMDMs. There were no differences in nucleosome remodeling, mRNA stability, NF-κB activation, or MAPK signaling to explain prolonged transcription of Il12b in Il10−/− BMDMs. However, acetylated histone H4 transiently associated with the Il12b promoter in WT BMDMs, whereas association of these factors was prolonged in Il10−/− BMDMs. Experiments using histone deacetylase (HDAC) inhibitors and HDAC3 short hairpin RNA indicate that HDAC3 is involved in histone deacetylation of the Il12b promoter by IL-10. These results suggest that histone deacetylation on the Il12b promoter by HDAC3 mediates homeostatic effects of IL-10 in macrophages.

Innate PI3K p110δ Regulates Th1/Th17 Development and Microbiota-Dependent Colitis

The p110δ subunit of class IA PI3K modulates signaling in innate immune cells. We previously demonstrated that mice harboring a kinase-dead p110δ subunit (p110δKD) develop spontaneous colitis. Macrophages contributed to the Th1/Th17 cytokine bias in p110δKD mice through increased IL-12 and IL-23 expression. In this study, we show that the enteric microbiota is required for colitis development in germfree p110δKD mice. Colonic tissue and macrophages from p110δKD mice produce significantly less IL-10 compared with wild-type mice. p110δKD APCs cocultured with naive CD4+ Ag-specific T cells also produce significantly less IL-10 and induce more IFN-γ– and IL-17A–producing CD4+ T cells compared with wild-type APCs. Illustrating the importance of APC–T cell interactions in colitis pathogenesis in vivo, Rag1−/−/p110δKD mice develop mild colonic inflammation and produced more colonic IL-12p40 compared with Rag1−/− mice. However, CD4+CD45RBhigh/low T cell Rag1−/−/p110δKD recipient mice develop severe colitis with increased percentages of IFN-γ– and IL-17A–producing lamina propria CD3+CD4+ T cells compared with Rag1−/− recipient mice. Intestinal tissue samples from patients with Crohn’s disease reveal significantly lower expression of PIK3CD compared with intestinal samples from non–inflammatory bowel disease control subjects (p < 0.05). PIK3CD expression inversely correlates with the ratio of IL12B:IL10 expression. In conclusion, the PI3K subunit p110δ controls homeostatic APC–T cell interactions by altering the balance between IL-10 and IL-12/23. Defects in p110δ expression and/or function may underlie the pathogenesis of human inflammatory bowel disease and lead to new therapeutic strategies.

NFIL3-Deficient Mice Develop Microbiota-Dependent, IL-12/23–Driven Spontaneous Colitis

NFIL3 is a transcription factor that regulates multiple immunologic functions. In myeloid cells, NFIL3 is IL-10 inducible and has a key role as a repressor of IL-12p40 transcription. NFIL3 is a susceptibility gene for the human inflammatory bowel diseases. In this article, we describe spontaneous colitis in Nfil3−/− mice. Mice lacking both Nfil3 and Il10 had severe early-onset colitis, suggesting that NFIL3 and IL-10 independently regulate mucosal homeostasis. Lymphocytes were necessary for colitis, because Nfil3/Rag1 double-knockout mice were protected from disease. However, Nfil3/Rag1 double-knockout mice adoptively transferred with wild-type CD4+ T cells developed severe colitis compared with Rag1−/− recipients, suggesting that colitis was linked to defects in innate immune cells. Colitis was abrogated in Nfil3/Il12b double-deficient mice, identifying Il12b dysregulation as a central pathogenic event. Finally, germ-free Nfil3−/− mice do not develop colonic inflammation. Thus, NFIL3 is a microbiota-dependent, IL-10–independent regulator of mucosal homeostasis via IL-12p40.

In Vitro and In Vivo Evaluation of a Water-in-Oil Microemulsion System for Enhanced Peptide Intestinal Delivery

Peptide and protein drugs have become the new generation of therapeutics, yet most of them are only available as injections, and reports on oral local intestinal delivery of peptides and proteins are quite limited. The aim of this work was to develop and evaluate a water-in-oil (w/o) microemulsion system in vitro and in vivo for local intestinal delivery of water-soluble peptides after oral administration. A fluorescent labeled peptide, 5-(and-6)-carboxytetramethylrhodamine labeled HIV transactivator protein TAT (TAMRA-TAT), was used as a model peptide. Water-in-oil microemulsions consisting of Miglyol 812, Capmul MCM, Tween 80, and water were developed and characterized in terms of appearance, viscosity, conductivity, morphology, and particle size analysis. TAMRA-TAT was loaded and its enzymatic stability was assessed in modified simulated intestinal fluid (MSIF) in vitro. In in vivo studies, TAMRA-TAT intestinal distribution was evaluated using fluorescence microscopy after TAMRA-TAT microemulsion, TAMRA-TAT solution, and placebo microemulsion were orally gavaged to mice. The half-life of TAMRA-TAT in microemulsion was enhanced nearly three-fold compared to that in the water solution when challenged by MSIF. The treatment with TAMRA-TAT microemulsion after oral administration resulted in greater fluorescence intensity in all intestine sections (duodenum, jejunum, ileum, and colon) compared to TAMRA-TAT solution or placebo microemulsion. The in vitro and in vivo studies together suggested TAMRA-TAT was better protected in the w/o microemulsion in an enzyme-containing environment, suggesting that the w/o microemulsions developed in this study may serve as a potential delivery vehicle for local intestinal delivery of peptides or proteins after oral administration.