Heme oxygenase-1 and carbon monoxide regulate intestinal homeostasis and mucosal immune responses to the enteric microbiota

Abstract

Heme oxygenase-1 (HO-1) and its enzymatic by-product carbon monoxide (CO) have emerged as important regulators of acute and chronic inflammation. Mechanisms underlying their anti-inflammatory effects are only partially understood. In this addendum, we summarize current understanding of the role of the HO-1/CO pathway in regulation of intestinal inflammation with a focus on innate immune function. In particular, we highlight our recent findings that HO-1 and CO ameliorate intestinal inflammation through promotion of bacterial clearance. Our work and that of many others support further investigation of this global homeostatic pathway in the human inflammatory bowel diseases (IBDs).

Keywords: :

• Crohn disease
• ulcerative colitis
• macrophage
• heme oxygenase
• carbon monoxide

This article refers to:

Introduction

The maintenance of immune homeostasis in the intestine requires a complex interplay between host and environmental factors that regulate immune responses directed against the enteric microbiota. Loss of tolerance to the enteric microbiota is a unifying pathogenic hypothesis in the initiation and perpetuation of chronic intestinal inflammation.¹ Our studies were conceived to understand mechanistically the impact of clinically relevant environmental factors on host-microbiota interactions in IBD. An important epidemiologic observation in the human IBDs is that cigarette smoking is protective against development of ulcerative colitis (UC).² Immunomodulatory components in cigarette smoke that may explain this phenomenon remain poorly defined. Cigarette smoke is a complex mixture of over 5000 known compounds, many of which may exert immunologic effects. Despite this complexity, immunologic and clinical studies to address this issue in IBD have focused on nicotine. However, clinical trial experience in UC patients with nicotine gum and transdermal nicotine has been inconclusive.^3,4 Carbon monoxide (CO) is a prominent component of cigarette smoke and has been shown to have anti-inflammatory effects in multiple experimental models of acute inflammation.^5-8 The heme oxygenase (HO) group of enzymes, of which HO-1 is inducible in immune cells, mediate heme degradation, generating equimolar amounts of iron, biliverdin, and CO. We and others have demonstrated that the HO-1/CO pathway can regulate intestinal inflammation in experimental models of acute and chronic intestinal inflammation.^9-13 Indeed, in our work, anti-inflammatory effects of CO required the induction of HO-1. CO/HO-1 affect multiple immunologic cell types including T cells, B cells, epithelial cells, neutrophils, mast cells, dendritic cells, and macrophages.^9,14-19 Our studies have focused on the role of these pathways in macrophage biology. We recently reported that intestinal HO-1 is induced by the enteric microbiota and modulates macrophage bactericidal activity, suggesting its importance in maintaining homeostasis.¹² Our work leads to a model where, in the local microenvironment of the intestine, CO and HO-1 are components of an evolutionarily conserved homeostatic pathway modulated in health and disease by cross-talk between the microbiota and the mucosal immune compartment.

HO-1 is Induced by the Microbiota

The enteric microbiota induce intestinal HO-1, with a requirement for the homeostatic cytokine IL-10.¹² To define this pathway, germ free (GF) wild-type (WT) and colitis prone Il10^−/− mice, were colonized with a conventional microbiota. HO-1 was significantly induced post-colonization in WT but not in Il10^−/− mice relative to respective GF controls. Induction of HO-1 was mediated by the transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2) that is activated under cellular stress and inflammation and is largely responsible for activation of heme oxygenase gene (Hmox1) transcription in mice and humans.²⁰ Additionally, Hmox1 expression in bone marrow derived macrophages (BMDM) was induced in a MyD88 and IL-10 dependent fashion upon stimulation with Toll-like receptor (TLR) ligands.¹¹ The protective effect of HO-1 induction on enteric microbiota-driven colitis was further supported by experiments where GF Il10^−/− mice were treated with a small molecule HO-1 inducer, cobalt protoporphyrin IX (CoPP), prior to colonization with a conventional microbiota, resulting in attenuation of colitis.¹² Hence, although in the absence of IL-10, the microbiota cannot induce Hmox1 expression, pharmacologic induction of Hmox1 can overcome this defect and protect against colitis development. Induction of HO-1 was also observed in GF zebrafish colonized with a conventional microbiota, demonstrating evolutionary conservation of this central homeostatic pathway among vertebrate hosts.¹²

HO-1/CO Ameliorate Colitis through Altered Cytokine Expression

The HO-1/CO pathway can profoundly alter the balance of pro-inflammatory and anti-inflammatory cytokine expression in the intestine.^9-11,14 Importantly, CO exposure and HO-1 induction have been shown to ameliorate colitis in numerous experimental models of IBD with different immunopathogeneses, highlighting the importance of this pathway as a homeostatic checkpoint in intestinal immune responses.^{9-11,13,14,21} Using Il10^−/− mice as a model of microbiota dependent, IL-12/23-mediated chronic intestinal inflammation, we demonstrated that exposure to CO ameliorated colitis in an HO-1 dependent manner.¹⁴ T cell receptor α deficient (TCRα^−/−) mice develop spontaneous, Th2-mediated chronic colitis, driven by IL-4 and IL-1β.^22,23 Exposure of mice to CO or treatment with CoPP ameliorated active colitis. Decreased levels of intestinal pro-inflammatory TNF, IL-1β, IL-4 and IL-17 cytokines were seen with increased colonic levels of the anti-inflammatory cytokines IL-10 and IL-22.¹¹ Similarly, CO exposure in 2, 4, 6-trinitrobenzine sulfonic acid (TNBS) and dextran sulfate sodium (DSS) experimental colitis was associated with attenuation of colitis and decreased intestinal TNF expression.^9,10 Furthermore, modulation of HO-1 transcriptional regulation alters development of experimental colitis with consequent changes in cytokine expression. Mice deficient in broad complex, tramtrack, bric-a-brac domain (BTB), and cap’n’collar (CNC) homolog 1 (Bach1), a transcriptional repressor of HO-1, are protected against TNBS colitis.^24,25 This effect is mediated by macrophages and is associated with HO-1 induction and decreased expression of TNF and interferon-γ.²⁴ Nrf2^−/− mice have impaired HO-1 induction and demonstrate increased susceptibility to DSS induced colitis with increased levels of IL-1β, TNF, and IL-6.²⁶

HO-1 and CO Promote Bactericidal Activity in Innate Immune Cells

There is also emerging evidence that pleiotropic effects of the HO-1/CO pathway include significant antimicrobial activities. Defects in innate immune responses to enteric bacteria have been implicated in the pathogenesis of human IBDs through genetic associations,^1,27 including the confirmation of synonymous IBD susceptibility single nucleotide polymorphisms (SNPs) in autophagy genes encoding ATG16L1 and IRGM and a SNP in the phagosomal gene NCF4.^28-31 The HO-1/CO pathway has been shown to augment responses to systemic bacterial infection through enhanced phagocytosis.^32,33 In a mouse model of bacterial sepsis, Hmox1^−/− mice demonstrated significantly decreased survival compared with WT controls. Likewise, systemic overexpression of human HO-1 in mice resulted in improved clearance of bacteria and survival.³² In these HO-1 overexpressing mice, there was an increase in intestinal nuclear binding oligomerization domain containing 2 (NOD2) expression as well as increased phagocytosis by peritoneal macrophages, mediated by CO and NOD2. Single nucleotide polymorphisms in the intracellular pattern recognition receptor NOD2 provide the strongest genetic risk for Crohn disease. Moreover, NOD2 has been linked to autophagy signaling and anti-microbial responses.^34-36 Additionally, HO-1 has been identified as a regulator of autophagy signaling in LPS exposed macrophages.³⁷ HO-1 activity is also important for bacterial clearance in a Salmonella typhimurium model of peritonitis in mice.³⁸ We demonstrated that CO exposure of colitis-prone Il10^−/− mice resulted in improved clearance of intestinal bacteria from extraintestinal sites.¹² Additionally, induction of HO-1 by CoPP led to increased bacterial clearance from the intestinal mucosa and systemic sites in an enteric bacterial infectious colitis model with S. typhimurium in WT mice. Given that macrophages are critical for Salmonella eradication,³⁹ we further examined the effect of the HO-1/CO pathway in augmenting macrophage responses to enteric bacteria. Knock-down of HO-1 expression in BMDMs using Hmox1 small interfering RNA resulted in decreased killing of Escherichia coli, S. typhimurium, and Enterococcus faecalis. Augmented bactericidal activity was also demonstrated in BMDMs exposed to CO. Possible mechanisms for enhanced bactericidal activity include HO-1 augmentation of autophagy via TLR signaling as well as increased phagolysosomal formation/activation.^12,37,40

Hence, the HO-1/CO pathway regulates intestinal immune homeostasis through pleiotropic mechanisms. In intestinal macrophages, the HO-1/CO pathway is induced by the enteric microbiota (Fig. 1). TLR stimulation leads to MyD88 and IL-10 mediated induction of Hmox1, via Nrf2 transcriptional control. Subsequently, increased CO production leads to augmented phagocytosis, promotion of bacterial killing, and increased anti-inflammatory and decreased pro-inflammatory cytokine production (Fig. 2). However, whether HO-1 has effects on microbicidal activity and cytokine expression that are independent of CO are unaddressed possibilities. Indeed, another byproduct of heme metabolism by HO, biliverdin (metabolized in the liver to bilirubin), has been shown to directly inhibit inflammatory cytokine production in macrophages.⁴¹

Figure 1. Regulation of intestinal Hmox1 by the enteric microbiota. TLR signaling activates Nrf2 mediated Hmox1 transcription via MyD88 and IL-10 dependent mechanisms. Nrf2 and Bach1 bind to cis-acting antioxidant response elements (ARE) with Bach1 acting as a transcriptional repressor.

Figure 2. Functional consequences of HO-1 induction. CO is produced as a product of HO-1 metabolism of heme. The HO-1/CO pathway augments phagocytosis and bacterial killing while promoting anti-inflammatory cytokine expression.

CO, HO-1, and the Enteric Microbiota

Recent work has demonstrated that alterations in the enteric microbiota, e.g., dysbiosis, plays a role in the development of colitis.⁴² Environmental factors such as cigarette smoking have been associated with changes in the microbiota in human IBD.⁴³ We recently found that exposure to CO in mice can alter the microbiota and induce bacterial species that express HO homologs involved in iron acquisition (N. Maharshak et al., manuscript submitted). The proximity of the mucosal immune compartment, including the epithelium, to the enteric microbiota leads to signaling cross-talk that can potentially be harnessed therapeutically. The existence of enteric bacterial HO homologs leads us to speculate that the enteric microbiota may actually provide a significant source of CO in the gut, involved in the maintenance of homeostasis. These findings also raise the possibility that enteric bacterial HO may be utilized as a therapeutic strategy in IBD. Our unpublished results suggest that bacterial HO homologs can modulate innate immune responses, possibly through elaboration of CO (N. Maharshak et al., manuscript submitted). Indeed, another group has reported that genetically engineered enteric bacteria that express mammalian HO-1 have therapeutic potential in a mucosal injury model.⁴⁴

Future Directions

CO and HO-1 define a central homeostatic pathway in the intestine, affecting critical innate immune processes, such as cytokine expression and macrophage bactericidal activity, linked to the genetics and immunopathology of the human IBDs. Consequently, these investigations underscore a therapeutic potential of the HO-1/CO pathway for human IBDs. Progress has been made in translating these findings into future therapies through development of methodologies to finely control CO release to target organs of interest such as CO-releasing small molecules which may eventually merit testing in humans.^45,46 We are currently exploring approaches to deliver CO locally to the intestine via enteric bacteria that express HO. Likewise, therapeutic strategies to induce intestinal HO-1 could be a powerful approach to correct fundamental defects and ameliorate inflammation in human IBD. A more complete understanding of the HO-1 regulatory pathway will further elucidate novel approaches and compounds to treat human disease.

Abbreviations:
CO	=	carbon monoxide
CoPP	=	cobalt protoporphyrin
GF	=	germ free
HO-1	=	heme oxygenase 1
IL	=	interleukin
LPS	=	lipopolysaccharide

Disclosure of Potential Conflicts of Interest

No potential conflict of interest was disclosed.

Acknowledgments

We thank all contributors to the original article. This work was supported in part by National Institutes of Health grants R01 DK54452, Gastroenterology Research Training grant T32 DK007737, National Research Service Award F32 DK083186, and a Crohn’s and Colitis Foundation of America Research Fellowship Award.

10.4161/gmic.27290