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Original Articles: Gastroenterology

Hemin Exerts Multiple Protective Mechanisms and Attenuates Dextran Sulfate Sodium–induced Colitis

Zhong, Wenwei*; Xia, Zhenwei*; Hinrichs, David; Rosenbaum, James T; Wegmann, Keith W; Meyrowitz, Jeffery§; Zhang, Zili§

Author Information
Journal of Pediatric Gastroenterology and Nutrition: February 2010 - Volume 50 - Issue 2 - p 132-139
doi: 10.1097/MPG.0b013e3181c61591
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Abstract

Inflammatory bowel disease (IBD) is a common disorder characterized by recurrent and serious inflammation of the gastrointestinal tract (1,2). Crohn disease and ulcerative colitis represent 2 major clinical subtypes of IBD. It is generally believed that Crohn disease can affect any part of the gastrointestinal tract, whereas ulcerative colitis involves only the large intestine. The common histological changes associated with IBD include ulceration and inflammation of the intestinal mucosa with leukocyte infiltration (3,4). However, transmural inflammation and granuloma formation are the pathological hallmarks of Crohn disease, and the intestinal mucosa layer is the target of ulcerative colitis. The diversity of IBD clinical presentation unveils the complex interplay between various host genetics and environmental influences. Recent genomewide association studies have identified multiple susceptibility genes related to IBD. Among them, a number of genes such as ATG16L1, NOD2, and interleukin (IL)-23 receptor (IL23R) are implicated in autophagy and innate or adaptive immunity, and provide an insight into the pathogenesis of IBD (4–6). For instance, IL23R is a critical receptor transmitting the signal essential for TH17 lymphocyte development (7,8). Well documented in both human patients with IBD and animal colitis models, IL-17 produced by TH17 cells drives the intestinal inflammatory response (9–12). To counterbalance inflammation, our immune system develops several defense mechanisms. One of the extensively studied anti-inflammatory measures is regulatory T cells (Treg), which have been shown to play an essential role in controlling intestinal inflammation (13,14). A well-documented subpopulation of Treg cells comprises CD4 + CD25 + Foxp3+ lymphocytes. The expression of Foxp3, a master regulatory gene, is highly correlated with the anti-inflammatory function of Treg cells (15). In fact, some patients with IBD display a defect in their anti-inflammatory Treg function (16,17). Upregulation of Treg cells and the suppression of inflammation are primary therapeutic goals in the treatment of IBD.

Heme oxygenase-1 (HO-1) is a rate-limiting enzyme for heme metabolism. It catalyzes heme into biliverdin, carbon monoxide, and free iron. Since the original discovery of its role in heme metabolism, numerous studies have shown that HO-1 is implicated in diverse biological processes such as antioxidation, anti-inflammation, antiapoptosis, and antismooth muscle proliferation (18–20). Thus, not surprisingly, induction of HO-1 has been shown to ameliorate many inflammatory conditions including experimental colitis. This protective effect of HO-1 is mediated in part by biliverdin and carbon monoxide. These metabolites protect cells and tissues by interfering with mitogen-activated protein kinase and cyclic guanosine monophosphate pathways (21). HO-1 is an acute phase reactant, and often serves as a naturally occurring immunomodulator under inflammatory conditions. Recent studies have shown that HO-1 is involved in the downstream effect of Treg cells (22,23). Furthermore, we reported that induction of HO-1 can reduce asthmatic airway inflammation by functioning as an upstream regulator of Treg cells (24). Therefore, HO-1 has emerged as a novel anti-inflammatory protein, and holds a promise of potential clinical application in various inflammatory diseases. A major aim of this study was to determine whether the upregulation of HO-1 by hemin could attenuate dextran sulfate sodium (DSS)–induced colitis, a murine IBD model. In addition, we further characterized the effects of hemin on CD4 + CD25 + Foxp3+ Treg cell proliferation and IL-17 production.

MATERIALS AND METHODS

Mice

Female BALB/c mice were purchased from Jackson Laboratories (Bar Harbor, ME). All of the mice used in the study were between 6 and 8 weeks of age. The animals had free access to water and standard chow. All of the experiments were performed with the approval of the IACUC at the Oregon Health & Science University and VA Medical Center, Portland, OR.

Induction of Colitis

Experimental colitis was induced by oral administration of DSS (MP Biochemicals, Solon, OH). The mice were fed with 4% (w/v) DSS in drinking water ad libitum. The severity of colitis was evaluated by daily monitoring of weight change, loose stools, and rectal bleeding. At the end of the experiment, the colon and spleen were harvested for the various assays as indicated.

Splenocyte Culture

After the mice were sacrificed, their spleens were removed. Single-cell suspensions were prepared by passing the tissue through a 70-μm cell strainer (BD Biosciences, Mountain View, CA). Cells were washed twice with phosphate-buffered saline (PBS) and 5% fetal calf serum (FCS), and then digested at 37°C for 30 minutes in RPMI 1640, 5% FCS, 1 mmol/L MgCl2, 1 mmol/L CaCl2, and 150 U/mL collagenase 1A (Sigma-Aldrich, St Louis, MO). These cells were incubated in the presence or absence of anti-CD3 and anti-CD28 T cell–activating antibodies (eBioscience, San Diego, CA) for 24 hours.

Histology

For histological evaluation, colonic samples were fixed in 3% paraformaldehyde. Then, the tissues were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Colonic inflammation was assessed by light microscopy according to the degree of epithelial erosion, ulceration, and cellular infiltration.

Real-time Polymerase Chain Reaction (PCR) and TH17 Pathway-focused PCR Array

Total RNA from colonic homogenates was isolated with RNAeasy Mini kit (Qiagen, Valencia, CA). First-strand cDNA synthesis was accomplished with oligo (dT)-primed Omniscript reverse transcriptase kit (Qiagen). Gene-specific cDNA was amplified by PCR using mouse-specific primer pairs (HO-1 sense, 5′-CCC ACC AAG TTC AAA CAG CTC-3′, and HO-1 antisense, 5′-AGG AAG GCG GTC TTA GCC-3′; β-actin (sense, 5′-ATG CCA ACA CAG TGC TGT CT-3′, and antisense, 5′-AAG CAC TTG CGG TGC ACG AT-3′). The real-time PCR was performed using a RT2 Real-Time PCR Master Mix (SABiosciences, Frederick, MD) and run for 40 cycles at 95°C for 15 seconds and 55°C for 40 seconds. The mRNA level of HO-1 in each sample was normalized to β-actin mRNA and quantified using a formula: 2 [(Ct/β-actin – Ct/gene of HO-1)]. The result was expressed as fold difference in DSS and hemin treatment groups compared to controls.

Total RNA from splenocytes was isolated with the RNAeasy Mini kit as discussed above. RNA samples from 3 animals were pooled in the same experimental group. TH17 for Autoimmunity and Inflammation PCR Array was performed independently 3 times according to the manufacturer's protocol (SABiosciences), and GEArray Expression Analysis Suite software was used for data extraction and analysis.

Enzyme-linked Immunosorbent Assay (ELISA)

Colonic tissues were homogenized in a lysis buffer containing PBS, 1% Triton X-100, and a proteinase inhibitor mixture (Roche Molecular Biochemicals, Indianapolis, IN). The tissue lysates and splenocyte culture supernatants were collected, and ELISA was performed to measure the production of IL-6 and IL-17 according to the manufacturer's protocols (R&D Systems, Minneapolis, MN).

Enzyme-linked Immunosorbent Spot (ELISPOT) Assay for IL-17 Production

IL-17 production by splenocytes was quantitated by the ELISPOT assay. Isolated cells were washed once and seeded at a density of 5 × 105 cells per well in triplicate into 96-well sterile 0.45-μm MultiScreen-HA filter plates (Millipore, Billerica, MA) coated with 5 μg/mL anti-IL-17 monoclonal antibody (eBioscience). These cells were incubated in the presence or absence of anti-CD3 and anti-CD28 monoclonal antibodies (eBioscience) for 24 hours at 37°C in 5% CO2. Then the cells were washed, IL-17-producing cells were detected as positive spots by addition of a second biotin-conjugated anti-IL-17 antibody (eBioscience), followed by streptavidin alkaline phosphatase and 5-bromo-4-chloro-3-indolyl phosphate toluidine p-nitro blue tetrazolium chloride substrate (KPL, Gaithersburg, MD). Spots were analyzed by an AID ELISPOT high-resolution reader system ELISPOT 04 HR (AID ELISPOT, Strassberg, Germany).

Intracellular Foxp3 Staining and Flow Cytometry

Cells were suspended in PBS containing 1% FCS, 0.5 mmol/L ethylenediaminetetraacetic acid, and 0.1% sodium azide. After staining of surface markers of anti-CD4 and anti-CD25 antibodies (eBioscience) conjugated with phycoerythrin and allophycocyanin, respectively, splenocytes were incubated with brefeldin A for 5 hours. The cells were intracellularly stained with fluorescein isothiocyanate-conjugated antibody against Foxp3 (eBioscience) for 12 hours at 4°C using the Cytofix/Cytoperm kit (BD Biosciences). Data acquisition was performed on a FACSCalibur flow cytometer, and data were analyzed using CellQuest software (BD Biosciences).

Terminal Deoxynucleotidyl Transferase Deoxyuridine Triphosphate Nick End-labeling (TUNEL) Staining

TUNEL staining was performed using the DeadEnd Colorimetric TUNEL System (Promega, Madison, WI) according to the manufacturer's instruction. Briefly, 4-μm colonic tissue sections were mounted on poly-L-lysine–coated slides, deparaffinized, hydrated, and treated for 10 minutes with proteinase K (20 μg/mL). After rinsing, terminal deoxynucleotidyl transferase (TdT) reaction mix was added to the samples. Slides were incubated in a humidified chamber for 60 minutes at 37°C, rinsed, and incubated with streptavidin horseradish peroxidase. Then, 3,3′-diaminobenzidine was added, and slides were incubated until brown color appeared. Apoptotic cells were identified by the presence of a distinct brown staining of the nucleus. Hematoxylin was used for a counterstaining.

Statistics

Data are expressed as the average ± SEM, and a representative experiment is shown for each figure. The difference between 2 groups was evaluated by Student t test, and analysis of variance with Fisher follow-up testing was used to analyze multiple group comparisons. P < 0.05 was considered significant.

RESULTS

Induction of HO-1 Expression in Intestinal Epithelium After DSS Challenge

It is well documented that HO-1, an acute phase reactant, is upregulated in many inflammatory and stress conditions. The induction of HO-1 theoretically plays a protective role in controlling tissue damage caused by harmful stimuli. Thus, we first evaluated whether the expression of HO-1 is enhanced in the intestinal epithelium during DSS-induced colitis, a widely used murine IBD model. BALB/c mice were administered daily with 4% DSS in their drinking water or water alone as a control. On day 9, the colon was harvested, and HO-1 expression was assessed by immunohistochemistry. As shown in Figure 1, control mice did not express inducible HO-1 in the intestine; however, DSS treatment upregulated HO-1 production in the epithelial cells. The induction of endogenous HO-1 is a stress and compensatory reaction in response to colonic inflammation.

FIGURE 1
FIGURE 1:
Representative image of HO-1 expression in the colon epithelium of BALB/c mice. The mice were administered with 4% DSS in their drinking water for 9 days. Some mice received intraperitoneal injection of hemin. Then, the colon was harvested, and HO-1 expression was assessed by immunohistochemistry (n = 6). Note: DSS induced endogenous HO-1 in the colon, and hemin markedly enhanced HO-1 expression in the colonic epithelium of the mice challenged with DSS. DSS = dextran sulfate sodium; HO-1 = heme oxygenase-1.

Hemin Enhances Colonic HO-1 Expression

Hemin is a potent inducer of HO-1 expression. Moreover, it has been shown to upregulate the biological activity of HO-1. To further study the role of HO-1 in colitis, we examined whether hemin augmented the expression of HO-1 in the colon challenged with DSS. BALB/c mice received intraperitoneal injection of 75 μmol/kg hemin or a control vehicle solution on days 0, 1, and 6 of DSS challenge. On day 9 after initial DSS treatment, the colon was harvested, and HO-1 expression was assessed by immunohistochemistry. Compared to the control group and the mice treated with DSS alone, hemin treatment resulted in a more substantial upregulation of HO-1 in colonic epithelium (Fig. 1).

This immunohistochemistry finding is further consistent with the data of quantitative PCR. On day 9 after the induction of colitis, real-time PCR showed an approximately 5-fold increase of colonic HO-1 transcription compared to the control group (Fig. 2). Hemin treatment significantly enhanced HO-1 expression in the colon of the mice challenged with DSS (Fig. 2). The induction of HO-1 by hemin laid a foundation for the next experiment defining the protective effect of HO-1 on colitis.

FIGURE 2
FIGURE 2:
Hemin enhanced HO-1 gene expression in the colon of the mice challenged with DSS. The mice were administered with 4% DSS in their drinking water. Some mice received intraperitoneal injection of hemin. Nine days later, the colon was harvested from all of the mice and total RNA was collected. Real-time PCR analysis revealed upregulation of IL-17 in DSS-challenged colon, and the expression of HO-1 was further augmented by hemin. Data represent the mean ± SD of 3 mice (*P < 0.05). DSS = dextran sulfate sodium; HO-1 = heme oxygenase-1; IL = interleukin; PCR = polymerase chain reaction.

Hemin Attenuated DSS-induced Colitis

In light of the above finding, we investigated whether further upregulation of HO-1 by hemin could ameliorate DSS-induced colitis. Similar to human IBD, we demonstrated that the DSS-treated colon displayed monocytic leukocyte infiltration and focal epithelial ulceration on day 9 of the experiment (Fig. 3). Nevertheless, we administered 75 μmol/kg hemin (a HO-1 inducer) intraperitoneally to the mice on days 0, 1, and 6 of DSS challenge. Compared to DSS alone, hemin significantly attenuated DSS-induced colitis as evidenced by reducing inflammatory cell infiltration and maintaining epithelial integrity.

FIGURE 3
FIGURE 3:
Representative histology of DSS-induced colonic inflammation of the mice with and without HO-1 upregulation. These mice were administered with 4% DSS in their drinking water for 9 days, and some mice received intraperitoneal injection of hemin or SnPP. The intestine was harvested for H&E staining (n = 6). Intestinal inflammation was evaluated by light microscopy. Note: Hemin treatment markedly attenuated colonic inflammation compared to other experimental groups. DSS = dextran sulfate sodium; H&E = hematoxylin and eosin; HO-1 = heme oxygenase-1; SnPP = Sn-protoporphyrin.

To further attest to this protective effect of inducible HO-1, BALB/c mice were administered with 75 μmol/kg Sn-protoporphyrin (SnPP) on days 0, 1, and 6 of DSS treatment. SnPP has been shown to upregulate HO-1 expression. It is a potent inhibitor by suppressing the enzymatic activity of HO-1 (25). As shown in Figure 3, SnPP treatment exaggerated DSS-induced colonic inflammation. This histological change is further correlated with systemic inflammation. Both control mice and the mice that received only SnPP maintained a normal steady weight gain during the course of experimentation. Nevertheless, enteral administration of 4% DSS caused the mice to lose body weight on day 6. By the end of the study (day 9), they generally lost 15% of their total weight. Administration of SnPP into DSS-challenged mice resulted in more marked rectal bleeding and weight loss (Fig. 4), suggesting that inhibition of endogenous HO-1 accelerated DSS-induced colitis. These results suggest that enhancement of HO-1 functional activity is implicated in controlling intestinal inflammation.

FIGURE 4
FIGURE 4:
Inhibition of HO-1 resulted in more weight loss of mice with DSS-induced colitis. The mice were administered with 4% DSS in their drinking water. Some mice received intraperitoneal injection of SnPP. Their weight change was monitored daily, and the value is expressed as percentage of original body weight. Note: SnPP alone did not affect the weight, whereas SnPP significantly potentiated the weight loss caused by DSS colitis. Data represent the mean ± SEM of 6 mice (*P < 0.05, DSS vs control; #P < 0.05, DSS with SnPP vs DSS). DSS = dextran sulfate sodium; HO-1 = heme oxygenase-1; SnPP = Sn-protoporphyrin.

Hemin Treatment Increases CD4 + CD25 + Foxp3+ Regulatory T Cell Proliferation

Previously, we demonstrated that hemin inhibited airway inflammation in part by upregulating CD4 + CD25 + Foxp3+ Treg cells (18). Therefore, we examined whether hemin enhanced Treg cells in the intestinal draining lymph nodes as a potential anti-inflammatory mechanism. Control and DSS-challenged BALB/c mice were treated with and without hemin as described above. Then the mesenteric lymph nodes were harvested for flow cytometry analysis of CD25 and Foxp3 expression in the CD4+ T cell population. Foxp3 is a critical transcriptional factor promoting Treg cell development and widely used as a marker of Treg activation. As shown in Figure 5, DSS challenge slightly increased the frequency of CD4 + CD25 + Foxp3+ Treg cells in the mesenteric lymph nodes compared to the control group. This mild elevation of Foxp3+ Treg cells indicates a compensatory immune modulatory response to local inflammation induced by DSS. Treatment of mice with 75 μmol/kg hemin significantly increased Foxp3 expression in both control and DSS-challenged mice (Fig. 5). This suggests that hemin is implicated in the modulation of peripheral Treg population.

FIGURE 5
FIGURE 5:
Flow cytometry analysis of Foxp3+ Treg T cells in the mesenteric lymph nodes of control and DSS-treated mice. Lymphocytes were harvested from the mice treated with and without hemin. After surface labeling of CD4 and CD25, these cells were intracellularly stained and analyzed for Foxp3 expression by flow cytometry. Note: Hemin markedly increased Foxp3+ Treg population. Data represent the mean ± SD of 3 mice (*P < 0.05).

Hemin Suppresses IL-17 and IL-6 Production

Recently, numerous studies demonstrate the importance of IL-17 and TH17 cells in the pathogenesis of various autoimmune diseases. Moreover, we and others have reported that IL-17 is a critical cytokine implicated in human IBD and animal colitis models (11,26,27). Therefore, we examined whether hemin treatment exerted an inhibitory effect on IL-17 production. The mice received intraperitoneal injection of hemin and/or SnPP as previously described. On day 9 after DSS challenge, both spleen and colon were collected. The splenocytes were cultured for an additional 24 hours in the presence of anti-CD3– and anti-CD28–activating antibodies. Then IL-17 production was measured by ELISPOT assay. As shown in Figure 6, hemin significantly ameliorated splenic IL-17 production, and this inhibitory effect was specifically reversed by SnPP. To further assess the biological relevance of hemin on TH17 and Treg cells, we used ELISA to determine the levels of IL-17 and IL-6 in the colon challenged with DSS. IL-6 is a potent inflammatory mediator that plays a critical role in the development of colitis. Moreover, IL-6 is a downstream cytokine of IL-17. As shown in Figures 7 and 8, hemin significantly suppressed colonic IL-17 and IL-6 production in DSS-induced colitis. Conversely, SnPP enhanced IL-6 expression. The inhibitory effect of hemin on IL-6 is consistent with the histological change and IL-17 suppression. Taken together, these data revealed a novel anti-inflammatory mechanism of hemin.

FIGURE 6
FIGURE 6:
ELISPOT analysis of IL-17 production in the spleen of DSS-treated mice. The mice were intraperitoneally administered with or without hemin or SnPP, and further received 4% DSS in their drinking water for 9 consecutive days. Then, the splenocytes were harvested from these mice. These cells were further stimulated in the presence or absence of anti-CD3– and anti-CD28–activating antibodies for 24 hours. IL-17 production activity was measured by ELISPOT (n = 6). Data represent the mean ± SEM of 6 mice. Note: DSS-treated groups displayed a significantly higher IL-17 response than controls (*P < 0.05). However, hemin significantly attenuated IL-17 production (#P < 0.05).
FIGURE 7
FIGURE 7:
ELISA analysis of IL-17 level in the colon of DSS-treated mice. The mice were administered with 4% DSS in their drinking water for 9 days, and some of them also received hemin or SnPP intraperitoneally. Then, their colons were harvested and homogenized for ELISA of IL-17. The data represent the mean of 3 independent experiments. Note: DSS challenge significantly increased IL-17 level in the colon (*P < 0.05), whereas hemin attenuated IL-17 production in DSS-induced colitis (#P < 0.05).
FIGURE 8
FIGURE 8:
ELISA analysis of IL-6 level in the colon of DSS-treated mice. The mice were administered with 4% DSS in their drinking water for 9 days, and some of them also received hemin or SnPP intraperitoneally. Then, their colons were harvested and homogenized for ELISA of IL-6. The data represent the mean of 2 independent experiments. Note: DSS treatment increased IL-6 level in the colon (*P < 0.05). Nevertheless, hemin significantly inhibited IL-6 production in DSS-induced colitis (#P < 0.05).

Hemin Inhibits IL-17 and Its Related Gene Expression

Because our data demonstrated the inhibition of IL-17 production by hemin, we explored the effect of hemin on other IL-17-related gene expression in the hope of better elucidating the mechanism of action for hemin. Thus, using a TH17-specific real-time PCR microarray (SABiosciences, Frederick, MD), we examined transcriptional changes of IL-17–related genes in the splenocytes of the BALB/c mice after 24-hour in vitro anti-CD3 and anti-CD28 antibody stimulation in the presence and absence of hemin. Recent studies indicate that IL-6 and transforming growth factor-β induce mouse TH17 cells differentiated from naïve T cells, where IL-23 maintains TH17 lineage with memory function (28). Once differentiated, TH17 cells produce signature cytokines IL-17F, IL-21, and IL-22 in addition to IL-17 (28). IL-21 further exerts a propagating effect on TH17 cells in an autocrine fashion (7). As shown in Figure 7 and Table 1, hemin attenuated the expression of TH17 unique receptor (IL-23R) and IL-17 effector molecules (IL-17F, IL-21, and IL-22). However, hemin virtually did not alter the expression of these IL-17–related genes in the cells cotreated with SnPP (data not shown). This result suggests that upregulation of HO-1 by hemin has a regulatory effect on TH17 differentiation cascade.

TABLE 1
TABLE 1:
Expression of selected IL-17-related genes in the splenocytes at 24 hours after hemin treatment in vitro

Hemin Attenuates Epithelia Apoptosis in DSS-induced Colitis

The anti-inflammatory action of hemin is evident; however, its inhibition of TH17 response appears to be moderate. Although the role of IL-17 is increasingly appreciated in the pathogenesis of IBD, the etiology of IBD is complex and multifactorial. Therefore, we went on to examine whether hemin prevented epithelial apoptosis in DSS-induced colitis. The mice received DSS, hemin, and SnPP as described previously. On day 9, colonic specimens were collected for TUNEL staining. As illustrated in Figure 9, there was a clear increase of apoptotic cells in the mice challenged with DSS. TUNEL-positive cells were mainly situated in the epithelial layer of the colon. Thus, it is feasible to speculate that intestinal inflammation and stress result in epithelial apoptosis, which in turn leads to further gut barrier disruption and damage. Interestingly, a marked reduction of apoptotic epithelial cells was observed in the mice treated with hemin, whereas the colon of SnPP-treated group appeared to have stronger TUNEL staining (Fig. 9). This suggests that HO-1 also plays an antiapoptotic role during the process of intestinal inflammation.

FIGURE 9
FIGURE 9:
The effect of hemin on colonic epithelial apoptosis by the TUNEL assay. Mice with DSS-induced colitis received hemin, SnPP, or control vehicle. Nine days later, colonic sections were used for TUNEL staining (n = 3). Representative histology revealed a substantial number of epithelial cells undergoing apoptosis (brown) after DSS treatment. Note: Hemin markedly reduced apoptotic cells, whereas SnPP augmented epithelial apoptosis in DSS-challenged colon.

DISCUSSION

IBD is a common and serious gastrointestinal disease for which our society bears significant burdens that are economic as well as health related. However, current potent anti-inflammatory medications nonspecifically inhibit the host immune system, thereby increasing the risk of infection and other severe side effects. Thus, it is imperative to identify novel therapeutic target(s) to develop a new generation of anti-inflammatory medications with fewer suppressive effects on the host defense.

HO-1 is a rate-limiting enzyme of ferroprotoporphyrin metabolism. Recent studies show that HO-1 is a stress-induced protein. It is known to be protective for tissue injury through multiple mechanisms including antioxidation, anti-inflammation, and antiapoptosis. HO-1 catalyzes ferroprotoporphyrin to generate biliverdin, bilirubin, and carbon monoxide. These enzymatic products are in part responsible for HO-1 action. The anti-inflammatory effect of HO-1 is well studied in several disease models ranging from asthma, multiple sclerosis, and organ transplant rejection to colitis (24,29–31). Hemin is a substrate and inducer of HO-1, whereas SnPP is an inhibitor of HO-1 widely used to study HO-1 function. According to our previous study, we chose optimal doses of hemin and SnPP for this experiment. Consistent with published findings, here we demonstrated the upregulation of HO-1 in inflamed intestinal epithelium. Furthermore, augmentation of HO-1 expression by hemin significantly attenuated DSS-induced colitis, whereas inhibition of endogenous HO-1 by SnPP exaggerated the intestinal inflammation.

It is well documented that Treg cells are essential for regulating effector immune cells. They play a pivotal role in controlling both autoimmune and inflammatory diseases. CD4 + CD25+ cells are a well-characterized Treg population (32,33). These CD4+ lymphocytes express a high level of CD25 on the cell surface and possess potent immunosuppressive functions. Transcription factor Foxp3 is not only highly expressed in Treg cells but also correlated with their activation (15). Thus, Foxp3 is widely regarded as a marker of active Treg cells. Previously, we found that upregulation of HO-1 by hemin ameliorates asthmatic airway inflammation in part by augmenting Foxp3+ Treg activation (24). Likewise, we observed a tangible effect of hemin at studied dose on the peripheral CD4 + CD25 + Foxp3+ Treg proliferation in the DSS-induced colitis model. These results indicate that HO-1 is implicated in Treg activation. Moreover, upregulation of HO-1 is an upstream event of Treg activation.

TH17 cells, featured by IL-17 and other unique cytokine production, are a newly categorized lymphocyte subpopulation. Recently, they have received intense research interest due to their pivotal role in the pathogenesis of many autoimmune diseases (34–37). Indeed, IL-17 has been shown as a driving force of intestinal inflammation in a number of published (11,12,38). IL-17 activates NF-κB, thereby inducing IL-1β, IL-6, TNF-α, neutrophilic chemokines, ICAM-1, and nitric oxide synthase (39). All of these IL-17 downstream events are well reported in IBD and contribute to gastrointestinal inflammation. Our study showed that hemin not only reduced DSS-induced colitis but also attenuated IL-17 production. This suppression of TH17 cells is likely a novel anti-inflammatory mechanism of hemin. Furthermore, the real-time PCR array study showed that hemin affected several key molecules of TH17 cells. This regulation of the upstream events of TH17 cell activation could be due to direct or indirect effect of hemin and its enzymatic products. Presently, several lines of evidence suggest an interplay between Treg and TH17 cells (40–42). Treg cells have been shown to inhibit TH17 activity through a novel anti-inflammatory cytokine IL-35 (43). In addition, IL-2, IL-27, and retinoic acid are found to promote Treg cells and suppress TH17 lymphocytes (44–46). Nevertheless in this study, it is unclear whether hemin directly inhibits IL-17 production or indirectly suppresses TH17 cells by enhancing Treg activity.

DSS-induced colitis is a widely used IBD research model. DSS is a chemical irritant that causes epithelial damage. However, the immunological mechanism(s) that lead to the clinical stage of this disease have not been fully defined (47). Several studies showed that CD4+ and CD8+ T cells are implicated in the progression of DSS-induced colitis, especially in the subacute stage (48,49). Our unpublished study found that the initiation of DSS-induced colitis is not dependent on CD4+ cells. However, equilibrium between Treg and IL-17-producing cells significantly influences the outcome of DSS-induced colitis severity. Although most recent studies focus on CD4+ TH17 cells, CD8+ as well as NK T cells also have been shown to produce IL-17 (50,51). Moreover, γδ T cells, major intestinal intraepithelial lymphocytes, are a major source of IL-17 production (52–54). Unpublished data from our laboratory showed that depletion of CD4+ T cells by GK1.5 antibody did not abolish IL-17 production in DSS-induced colitis, suggesting that IL-17–producing CD8+ and γδ T cells are likely targets of hemin action seen in this study.

Although TH17 cells and IL-17 are implicated in IBD, other pathogenic lymphocytes and cytokines also participate in the process of intestinal inflammation. Thus, the moderate effect of hemin on IL-17 expression indicates that hemin has multiple protective mechanisms. Recent studies have demonstrated that a disturbance of epithelial barrier function is associated with the development of IBD (55,56). Inflammatory cytokines induce epithelial cell apoptosis. The loss of epithelial viability in turn causes further epithelial disruption, leading to bacterial leak and more inflammation. In this study, we showed that hemin treatment substantially reduced epithelial apoptosis in DSS colitis as a part of its protective mechanism. Interestingly, a recent study showed that dendritic cells that ingest infected apoptotic cells facilitate TH17 cell differentiation (57). Moreover, after the encounter of apoptotic blebs containing autoantigens, dendritic cells promote T cells to produce IL-17 (58). Because our intestinal tact hosts an enormous amount of microorganisms and antigens, it is inevitable that the gut dendritic cells will process a large number of apoptotic cells along with bacteria and various antigens during intestinal inflammation. Hence, this sets up a perfect environment for the induction and activation of TH17 cells. However, our study has not determined whether antiapoptosis by hemin leads to suppression of IL-17 production or vice versa. Further study is needed to elucidate the role of HO-1 in the reciprocal relation between apoptosis and TH17 differentiation.

In summary, our data are consistent with other reports that upregulation of HO-1 by hemin attenuates experimental colitis (59–61). This study suggests that modulation of Treg and IL-17 as well as prevention of apoptosis is likely a novel anti-inflammatory action of hemin. These results help us to further understand the protective mechanism of HO-1 in IBD. In addition, this study elucidates the merit of further exploration into the application of a HO-1 inducer as a novel therapeutic strategy in IBD, ultimately allowing for a more specific anti-inflammatory effect with less compromise of host defense. Thus, this may lead to the transition from basic scientific finding to true clinical application.

REFERENCES

1. Bouma G, Strober W. The immunological and genetic basis of inflammatory bowel disease. Nat Rev Immunol 2003; 7:521–533.
2. Podolsky DK. Inflammatory bowel disease. N Engl J Med 2002; 347:417–429.
3. Carpenter HA, Talley NJ. The importance of clinicopathological correlation in the diagnosis of inflammatory conditions of the colon: histological patterns with clinical implications. Am J Gastroenterol 2000; 95:878–896.
4. Duerr RH, Taylor KD, Brant SR, et al. A genome-wide association study identifies IL23R as an inflammatory bowel disease gene. Science 2006; 314:1461–1463.
5. Van Limbergen J, Russell RK, Nimmo ER, et al. The genetics of inflammatory bowel disease. Am J Gastroenterol 2007; 102:2820–2831.
6. Cho JH, Abraham C. Inflammatory bowel disease genetics: Nod2. Annu Rev Med 2007; 58:401–416.
7. Nurieva R, Yang XO, Martinez G, et al. Essential autocrine regulation by IL-21 in the generation of inflammatory T cells. Nature 2007; 441:231–234.
8. Zhou L, Ivanov II, Spolski R, et al. IL-6 programs Th-17 cell differentiation by promoting sequential engagement of the IL-21 and IL-23 pathways. Nat Immunol 2007; 8:967–974.
9. Fujino S, Andoh A, Bamba S, et al. Increased expression of interleukin 17 in inflammatory bowel disease. Gut 2003; 52:65–70.
10. Nielsen OH, Kirman I, Rüdiger N, et al. Upregulation of interleukin-12 and -17 in active inflammatory bowel disease. Scand J Gastroenterol 2003; 38:180–185.
11. Zhang Z, Zheng M, Bindas J, et al. Critical role of IL-17 receptor signaling in acute TNBS-induced colitis. Inflamm Bowel Dis 2006; 12:382–388.
12. Zhang Z, Hinrichs DJ, Lu H, et al. After interleukin-12p40, are interleukin-23 and interleukin-17 the next therapeutic targets for inflammatory bowel disease? Int Immunopharmacol 2007; 7:409–416.
13. Allez M, Mayer L. Regulatory T cells: peace keepers in the gut. Inflamm Bowel Dis 2004; 10:666–676.
14. Huibregtse IL, Van Lent AU, Van Deventer SJ. Immunopathogenesis of IBD: insufficient suppressor function in the gut? Gut 2007; 56:584–592.
15. Tang Q, Bluestone JA. The Foxp3+ regulatory T cell: a jack of all trades, master of regulation. Nat Immunol 2008; 9:239–244.
16. Yu QT, Saruta M, Avanesyan A, et al. Expression and functional characterization of FOXP3+ CD4+ regulatory T cells in ulcerative colitis. Inflamm Bowel Dis 2007; 13:191–199.
17. Maul J, Loddenkemper C, Mundt P, et al. Peripheral and intestinal regulatory CD4+ CD25(high) T cells in inflammatory bowel disease. Gastroenterology 2005; 128:1868–1878.
18. Xia ZW, Zhong WW, Meyrowitz JS, et al. The role of heme oxygenase-1 in T cell-mediated immunity: the all encompassing enzyme. Curr Pharm Des 2008; 14:454–464.
19. Pae HO, Lee YC, Chung HT. Heme oxygenase-1 and carbon monoxide: emerging therapeutic targets in inflammation and allergy. Recent Pat Inflamm Allergy Drug Discov 2008; 2:159–165.
20. Exner M, Minar E, Wagner O, et al. The role of heme oxygenase-1 promoter polymorphisms in human disease. Free Radic Biol Med 2004; 37:1097–1104.
21. Ryter SW, Otterbein LE, Morse D, et al. Heme oxygenase/carbon monoxide signaling pathways: regulation and functional significance. Mol Cell Biochem 2002; 234–235:249–263.
22. Vignali DA, Collison LW, Workman CJ. How regulatory T cells work. Nat Rev Immunol 2008; 8:523–532.
23. George JF, Braun A, Brusko TM, et al. Suppression by CD4+CD25+ regulatory T cells is dependent on expression of heme oxygenase-1 in antigen-presenting cells. Am J Pathol 2008; 173:154–160.
24. Xia ZW, Zhong WW, Xu LQ, et al. Heme oxygenase-1-mediated CD4+CD25high regulatory T cells suppress allergic airway inflammation. J Immunol 2006; 177:5936–5945.
25. Sardana MK, Kappas A. Dual control mechanism for heme oxygenase: tin(IV)-protoporphyrin potently inhibits enzyme activity while markedly increasing content of enzyme protein in liver. Proc Natl Acad Sci 1987; 84:2464–2468.
26. Maloy KJ. The interleukin-23/interleukin-17 axis in intestinal inflammation. J Intern Med 2008; 263:584–590.
27. Ahern PP, Izcue A, Maloy KJ, et al. The interleukin-23 axis in intestinal inflammation. Immunol Rev 2008; 226:147–159.
28. Korn T, Bettelli E, Oukka M, et al. IL-17 and Th17 cells. Annu Rev Immunol 2009; 27:485–517.
29. Bach FH. Heme oxygenase-1 and transplantation tolerance. Hum Immunol 2006; 67:430–432.
30. Soares MP, Bach FH. Heme oxygenase-1 in organ transplantation. Front Biosci 2007; 12:4932–4945.
31. Chora AA, Fontoura P, Cunha A, et al. Heme oxygenase-1 and carbon monoxide suppress autoimmune neuroinflammation. J Clin Invest 2007; 117:438–447.
32. Chatila TA. Role of regulatory T cells in human diseases. J Allergy Clin Immunol 2005; 116:949–959.
33. Bluestone JA, Tang Q. How do CD4+CD25+ regulatory T cells control autoimmunity? Curr Opin Immunol 2005; 17:638–642.
34. Nakae S, Nambu A, Sudo K, et al. Suppression of immune induction of collagen-induced arthritis in IL-17-deficient mice. J Immunol 2003; 171:6173–6177.
35. Nakae S, Saijo S, Horai R, et al. IL-17 production from activated T cells is required for the spontaneous development of destructive arthritis in mice deficient in IL-1 receptor antagonist. Proc Natl Acad Sci U S A 2003; 100:5986–5990.
36. Albanesi C, Cavani A, Girolomoni G. IL-17 is produced by nickel-specific T lymphocytes and regulates ICAM-1 expression and chemokine production in human keratinocytes: synergistic or antagonist effects with IFN-gamma and TNF-alpha. J Immunol 1999; 103:1345–1352.
37. Pène J, Chevalier S, Preisser L, et al. Chronically inflamed human tissues are infiltrated by highly differentiated Th17 lymphocytes. J Immunol 2008; 180:7423–7430.
38. Andoh A, Ogawa A, Bamba S, et al. Interaction between interleukin-17-producing CD4+ T cells and colonic subepithelial myofibroblasts: what are they doing in mucosal inflammation? J Gastroenterol 2007; 42(Suppl 17):29–33.
39. Kolls JK, Lindén A. Interleukin-17 family members and inflammation. Immunity 2004; 21:467–476.
40. Zhou L, Lopes JE, Chong MM, et al. TGF-beta-induced Foxp3 inhibits T(H)17 cell differentiation by antagonizing RORgammat function. Nature 2008; 453:236–240.
41. Lochner M, Peduto L, Cherrier M, et al. In vivo equilibrium of proinflammatory IL-17+ and regulatory IL-10+ Foxp3+ ROR{gamma}t+ T cells. J Exp Med 2008; 205:1381–1393.
42. Du J, Huang C, Zhou B, et al. Isoform-specific inhibition of ROR{alpha}-mediated transcriptional activation by human FOXP3. J Immunol 2008; 180:4785–4792.
43. Yang J, Yang M, Htut TM, et al. Epstein-Barr virus-induced gene 3 negatively regulates IL-17, IL-22 and RORgamma t. Eur J Immunol 2008; 38:1204–1214.
44. Yoshida H, Yoshiyuki M. Regulation of immune responses by interleukin-27. Immunol Rev 2008; 226:234–247.
45. Mucida D, Park Y, Cheroutre H. From the diet to the nucleus: vitamin A and TGF-beta join efforts at the mucosal interface of the intestine. Semin Immunol 2009; 21:14–21.
46. Ivanov II, Zhou L, Littman DR. Transcriptional regulation of Th17 cell differentiation. Semin Immunol 2007; 19:409–417.
47. Elson CO, Cong Y, McCracken VJ, et al. Experimental models of inflammatory bowel disease reveal innate, adaptive, and regulatory mechanisms of host dialogue with the microbiota. Immunol Rev 2005; 206:260–276.
48. Yoshihara K, Yajima T, Kubo C, et al. Role of interleukin 15 in colitis induced by dextran sulphate sodium in mice. Gut 2006; 55:334–341.
49. Shintani N, Nakajima T, Okamoto T, et al. Involvement of CD4+ T cells in the development of dextran sulfate sodium-induced experimental colitis and suppressive effect of IgG on their action. Gen Pharmacol 1998; 31:477–481.
50. He D, Wu L, Kim HK, et al. CD8+ IL-17-producing T cells are important in effector functions for the elicitation of contact hypersensitivity responses. J Immunol 2006; 177:6852–6858.
51. Michel ML, Keller AC, Paget C, et al. Identification of an IL-17-producing NK1.1(neg) iNKT cell population involved in airway neutrophilia. J Exp Med 2007; 204:995–1001.
52. O'Brien RL, Roark CL, Born WK. IL-17-producing gammadelta T cells. Eur J Immunol 2009; 39:662–666.
53. Shibata K, Yamada H, Hara H, et al. Resident Vdelta1+ gammadelta T cells control early infiltration of neutrophils after Escherichia coli infection via IL-17 production. J Immunol 2007; 178:4466–4472.
54. Shibata K, Yamada H, Nakamura R, et al. Identification of CD25+ gamma delta T cells as fetal thymus-derived naturally occurring IL-17 producers. J Immunol 2008; 181:5940–5947.
55. Edelblum KL, Yan F, Yamaoka T, et al. Regulation of apoptosis during homeostasis and disease in the intestinal epithelium. Inflamm Bowel Dis 2006; 12:413–424.
56. Schulzke JD, Ploeger S, Amasheh M, et al. Epithelial tight junctions in intestinal inflammation. Ann N Y Acad Sci 2009; 1165:294–300.
57. Torchinsky MB, Garaude J, Martin AP, et al. Innate immune recognition of infected apoptotic cells directs Th17 cell differentiation. Nature 2009; 458:78–82.
58. Fransen JH, Hilbrands LB, Ruben J, et al. Mouse dendritic cells matured by ingestion of apoptotic blebs induce T cells to produce interleukin-17. Arthritis Rheum 2009; 60:2304–2313.
59. Hegazi RA, Rao KN, Mayle A, et al. Carbon monoxide ameliorates chronic murine colitis through a heme oxygenase 1-dependent pathway. J Exp Med 2005; 202:1703–1713.
60. Berberat PO, A-Rahim YI, Yamashita K, et al. Heme oxygenase-1-generated biliverdin ameliorates experimental murine colitis. Inflamm Bowel Dis 2005; 11:350–359.
61. Wang WP, Guo X, Koo MW, et al. Protective role of heme oxygenase-1 on trinitrobenzene sulfonic acid-induced colitis in rats. Am J Physiol Gastrointest Liver Physiol 2001; 281:G586–G594.
Keywords:

colitis; inflammatory bowel disease; interleukin-17; regulatory T cells

© 2010 Lippincott Williams & Wilkins, Inc.