Host Response and Inflammation

Dietary Chitin Particles Called Mimetic Fungi Ameliorate Colitis in Toll-Like Receptor 2/CD14- and Sex-Dependent Manners

Patricia Louis, Brian Mercer, Aiko M. Cirone, Christina Johnston, Zachary J. Lee, Nwadiuto Esiobu, Zhongwei Li, Jianning Wei, C. Kathleen Dorey, Yoshimi Shibata, Changlong Nan
George S. Deepe, Editor
DOI: 10.1128/IAI.00006-19

ABSTRACT

Chitin is a natural N-acetylglucosamine polymer and a major structural component of fungal cell walls. Dietary chitin is mucoadhesive; anti-inflammatory effects of chitin microparticles (CMPs; 1- to 10-μm diameters) have been demonstrated in models of inflammatory bowel disease (IBD). The goals of this study were to assess (i) whether CMPs among various chitin preparations are the most effective against colitis in male and female mice and (ii) whether host chitin-binding Toll-like receptor 2 (TLR2) and CD14 are required for the anti-inflammatory effect of chitin. We found that colitis in male mice was ameliorated by CMPs and large chitin beads (LCBs; 40 to 70 μm) but not by chitosan (deacetylated chitin) microparticles, oligosaccharide chitin, or glucosamine. In fact, LCBs were more effective than CMPs. In female colitis, on the other hand, CMPs and LCBs were equally and highly effective. Neither sex of TLR2-deficient mice showed anti-inflammatory effects when treated with LCBs. No anti-inflammatory effect of LCBs was seen in either CD14-deficient males or females. Furthermore, an in vitro study indicated that when LCBs and CMPs were digested with stomach acidic mammalian chitinase (AMC), their size-dependent macrophage activations were modified, at least in part, suggesting reduced particle sizes of dietary chitin in the stomach. Interestingly, stomach AMC activity was greater in males than females. Our results indicated that dietary LCBs were the most effective preparation for treating colitis in both sexes; these anti-inflammatory effects of LCBs were dependent on host TLR2 and CD14.

INTRODUCTION

Inflammatory bowel disease (IBD) is a chronic relapsing colitis, associated with risk for obstruction, fistula, and colorectal cancer, that affects approximately 1.4 million individuals in the United States (1, 2). IBD is manifested by an exaggerated recognition of gut microbiota by host immune cells, allowing aberrant microbial adhesion and internalization into colonic epithelial cells; this results in damaged epithelia, protracted episodes of diarrhea, and abdominal pain (3–6). While gut bacterial dysbiosis clearly plays a pathogenic role, recent studies suggest that the fungal microbiota is equally important in human physiology and various inflammatory diseases, including IBD (7). The inflammatory response to oral challenges by Candida albicans has been linked to its secretion of the toxic protein candidalysin, causing epithelial damage (8, 9). In contrast, anti-inflammatory effects are induced in mouse IBD by oral challenge with other fungi, such as Saccharomyces boulardii, or β-glucan, a major component of fungal cell walls (10, 11). The anti-inflammatory effects of chitin, another essential structural component of fungal cell walls, have also been documented in murine IBD (12–16), but further studies are needed to understand how hosts respond to chitin “mimetic fungi.”

Chitin is an N-acetylglucosamine polymer that also occurs in crustaceans, insects, and chordates but not in bacteria or mammals. Its role in gut protection may have ancient origins; recently, Dishaw et al. demonstrated that the gut mucosa of the protochordate Ciona intestinalis contains an extensive chitin matrix that binds host chitin-binding proteins bearing immunoglobulin type V domains and microbes and may facilitate barrier functions (17). Dextran sodium sulfate (DSS)-induced gut inflammation in Ciona decreased mucosal chitin; reconstitution of mucosal chitin by exogenously administered chitin resulted in repair of the inflammatory damage (18). Therefore, chitin has a strong mucoadhesive and barrier property, which may contribute to its protective action. Mammals do not synthesize chitin, but dietary chitin binds the gut mucosa and ameliorates inflammation; studies have tested the possible therapeutic effects of various chitin preparations, including chitin microparticles and glucosamine, as well as chitosan oligosaccharides, nanoparticles, and nanofibrils (12–16).

Parenteral administration of chitin particles has been explored to understand the roles of chitin in host responses to chitin-containing pathogens and allergens (19–21). Intraperitoneal and intranasal administrations of chitin particles activate peritoneal and alveolar macrophages, respectively, resulting in a wide range of polarized macrophage activations that seem to be dependent on particle size (22, 23). For instance, phagocytosis of chitin microparticles (CMPs; 1- to 10-μm diameters) induces classically activated macrophages (M1), capable of killing intracellular microbes but also damaging nearby tissues (24–28). This M1 activation by CMPs is initiated by Toll-like receptor 2 (TLR2)- and CD14-mediated phagocytosis, followed by recruitment of adaptor myeloid differentiation primary response gene 88 and TIR-containing adapter proteins to the phagosomal CMP/TLR2/CD14 cluster, generating M1-specific NF-κB and activating protein 1 signals (29). In contrast, large chitin beads (LCBs; 40- to 70-μm diameters), which are larger than macrophages and difficult to phagocytose, tend to induce alternatively activated macrophages (M2) that contribute to repair of tissue damage but also enhance allergic responses (19, 30, 31).

Unlike resident peritoneal and alveolar macrophages, macrophages in the colon are derived from persistent migration of blood monocytes. They maintain phagocytic and bactericidal capacities but are immunologically anergic, at least in part due to high concentrations of transforming growth factor β (TGF-β) and interleukin-10 (IL-10) in the gut mucosa (32). Recently, Leonardi et al. demonstrated that intestinal CX3C chemokine receptor 1 (fractalkine receptor) (CX3CR1)-positive macrophages recognize luminal fungi and control antifungal immunity; IBD patients frequently show a missense mutation in the gene encoding CX3CR1 (33). Nagatani and coworkers demonstrated significant anti-inflammatory effects of dietary CMPs, mimetic fungi, in DSS-elicited acute colitis (termed DSS-colitis) in C57BL/6 mice and in chronic colitis in T-cell receptor alpha (TCRα)-deficient (knockout [KO]) mice (12). In this study, using both male and female mice, we determined which preparation of dietary chitin was the most effective in ameliorating colitis and whether anti-inflammatory effects of chitin were dependent on host TLR2 and CD14. We recognized that sex-dependent activities of stomach acidic mammalian chitinase (AMC) reduced the particle sizes of dietary chitin and modified their anti-inflammatory effects.

RESULTS

Effectiveness of CMPs and other chitin derivatives against colitis in male mice.Disease progression in DSS-elicited colitis is evident in weight loss and higher clinical scores for diarrhea, bloody stool, and hunching positions; animals given oral CMPs demonstrated improvement in these clinical parameters as well as in colon histological scores (12). We therefore determined whether dietary CMPs produce the most effective anti-IBD activity among several types of chitin preparations. To minimize effects of potential sex-biased gene expression, we used male mice for the initial screening approach. As shown in Fig. 1, we confirmed that oral CMPs protected against the disease-associated weight loss (Fig. 1A) and improved the clinical scores (Fig. 1B). However, disease parameters were not improved by soluble forms of chitin or glucosamine (Fig. 1C and D) or de-CMPs (Fig. 1E and IF). Interestingly, mice given LCBs were protected from acute colitis, as indicated by reduced weight loss and lower clinical scores; in fact, the LCB treatment offered greater protection on these parameters than the CMP treatment (Fig. 1A and B).

FIG 1
FIG 1

LCBs are better than CMPs in treating DSS-induced colitis in male mice. Groups of six C57BL/6 males, 7 weeks old, were given oral gavage with 2 mg each chitin preparation or 0.5 ml saline (vehicle control) daily, starting 7 days prior to DSS treatment. To induce colitis, mice were orally inoculated with DSS through drinking water containing 4% DSS (5 days, days 1 to 5) and then regular drinking water (4 days, days 6 to 9). Body weight (BW) changes and clinical scores were determined daily in mice treated with CMPs and LCBs (A and B), with soluble chitin and glucosamine (C and D), or with de-CMPs (E and F). The panels show 100% as the body weight and clinical score zero on day 0. Data are expressed as means ± SD from six independent animals per group; error bars are displayed in one direction for clarity. *, P < 0.05 compared with the saline group; #, P < 0.05 compared with the CMP group.

Colon inflammation.We next determined that oral LCBs as well as CMPs reduced the histology scores on day 9, improving the colon inflammation (Fig. 2A). To further assess the colon inflammation improved by LCBs and CMPs, we measured inflammatory mediators, which play key roles in colitis. Tumor necrosis factor alpha (TNF-α) and IL-6, both proinflammatory cytokines in DSS-induced colitis (34), were slightly increased in colitis compared to their levels in the noncolitis group (Fig. 2B). However, neither LCBs nor CMPs reduced these proinflammatory cytokine levels. DSS-elicited colitis reduced gamma interferon (IFN-γ), which, together with TNF-α, induces epithelial barrier dysfunction (35) but also activates anti-inflammatory indoleamine-pyrrole 2,3-dioxygenase 1 (IDO1) and IL-18bp genes in intestinal epithelial cells (12, 36); neither LCBs nor CMPs increased the IFN-γ levels. In contrast, there was no effect of DSS-colitis on IL-1β, which propagates inflammation in DSS-colitis (37) but induces group 3 innate lymphoid cells to produce anti-inflammatory IL-22 (38). Treatment with LCBs, but not CMPs, slightly increased IL-1β levels. Figure 2B further shows that relative to a group without colitis or chitin treatment, colitis mice with vehicle treatment had significantly lower colon IL-10 and IL-22 levels, both of which are anti-inflammatory in DSS-colitis (39). IL-22 is reported not only to heal mucosal inflammation but also to maintain barrier integrity (38). These IL-10 and IL-22 levels were restored to normal levels in LCB-treated mice. However, CMP treatment did not restore the levels of these anti-inflammatory cytokines (Fig. 2B). Chitinase 3-like 1 (CHI3L1), which is a mucosal chitin-binding lectin regulating a chitin-mediated interaction between epithelial cells and microbes (4) and anti-inflammatory in DSS-colitis (40), was increased by LCB and CMP treatments. Overall, our results indicated that anti-inflammatory cytokines, such as IL-10, IL-22, and IL-1β, and chitin-binding CHI3L1, were increased by LCB treatment, whereas proinflammatory cytokines TNF-α and IL-6 were not significantly reduced by LCBs.

FIG 2
FIG 2

Dietary LCBs and CMPs changed inflammatory mediators in the colon in male colitis. (A) Histology scores of the middle part of the colon of DSS-elicited male WT mice (day 9) that received saline, LCBs, or CMPs (same mice as those shown in Fig. 1A). Data are expressed as means ± SD from five to six independent samples per group. *, P < 0.05 compared to the vehicle control group. (B) Indicated inflammatory mediators were measured by ELISAs in the proteins extracted from colon samples obtained on day 9 from mice treated with saline (blue), LCB (red), or CMP (green). Data are expressed as means ± SD from five to six independent samples per group; error bars are shown in one direction. *, P < 0.05 compared to the vehicle control group.

We then measured these mediator levels in serum samples harvested on day 9. We detected IL-6 and CHI3L1 in the saline-treated colitis group (see Fig. S1 in the supplemental material). Interestingly, serum IL-6 levels were significantly reduced more than 70% by LCB and CMP treatments (Fig. S1). CHI3L1 levels were not modified by LCBs or CMPs. However, other mediators (TNF-α, IL-1β, IFN-γ, IL-10, and IL-22) were difficult to detect (data not shown); analysis at an earlier or later time point might be necessary for consistent results.

LCB treatment modifies bacterial translocation.A previous study (4) suggested that dietary CMPs suppress an interaction between colonic mucosa and microbes important in the systemic invasion of local bacteria in DSS-elicited colitis. We confirmed that the total number of bacteria associated with the colon (Fig. 3A) as well as the liver (Fig. 3B) and spleen (Fig. 3C) was significantly reduced in the CMP-treated group. LCB treatment also reduced the number of bacteria translocated into these tissues (Fig. 3A to C).

FIG 3
FIG 3

Dietary LCBs and CMPs reduced gut microbiota in male colitis. Colons, livers, and spleens were isolated from the saline, LCB, and CMP treatment groups of mice on day 9 (same mice as those shown in Fig. 1A). Bacterial numbers in the colon (A), liver (B), and spleen (C) in each group were examined. Data are expressed as means ± SD from five to six independent samples per group. *, P < 0.05 compared to the vehicle control group.

TLR2 and CD14 are responsible for anti-IBD effects of LCBs.Previously, we identified that TLR2 and CD14 are essential for CMP-induced classical macrophage (M1) activation of peritoneal macrophages (29). TLR2 KO and CD14 KO mice are known to develop more severe colitis than wild-type (WT) mice in response to DSS treatments, indicating that both TLR2 and CD14 play protective roles (41, 42). Therefore, we used 3%, instead of 4%, DSS and explored whether these chitin-binding proteins were responsible for anti-inflammatory effects of LCBs using male mice deficient in TLR2 and male mice deficient in CD14.

In TLR2 KO mouse colitis, body weight loss was not prevented (Fig. 4A) and the clinical score was not improved by dietary LCBs (Fig. 4B). The saline group in TLR2 KO colitis appeared to express relatively higher levels of colon TNF-α, IFNγ, IL-10, and IL-22 than those in WT colitis (Fig. 4C versus Fig. 2), although it is unclear whether the increased cytokine levels were due to lower DSS concentration and/or TLR2 deficiency. However, it is particularly important that these inflammatory mediator levels were not modified by dietary LCBs in TLR2 KO colitis (Fig. 4C), implying that dietary LCB-induced production of anti-inflammatory cytokines is TLR2 dependent.

FIG 4
FIG 4

No anti-IBD effect of LCBs in TLR2 KO male colitis. Groups of six C57BL/6 background TLR2-deficient male mice, 7 weeks old, were given oral gavage with 2 mg LCBs or 0.5 ml saline (vehicle control) daily, starting 7 days prior to DSS treatment. To induce colitis, mice were given 3% DSS in their drinking water for 5 days (days 1 to 5), followed by 4 days (days 6 to 9) on regular drinking water. The mice were euthanized on day 9. Body weight changes (A), clinical scores (B), and colon cytokine levels (C) were measured. Data are expressed as means ± SD from six independent samples per group.

Similarly, LCBs did not improve any disease parameters in the CD14 KO colitis. In fact, LCB treatment worsened the disease parameters (Fig. 5A and B), although LCB treatment increased levels of anti-inflammatory IL-10, IL-22, IFN-γ, and IL-1β (Fig. 5C). It is unclear why dietary LCBs worsened CD14 KO colitis. In this regard, Buchheister et al. (42) demonstrated that the intestinal barrier function is not affected by CD14 deficiency but is reduced in CD14 KO colitis. Alternatively, Wang et al. (43) demonstrated that increased inflammatory cytokines, including IFN-γ, induce intestinal epithelial barrier dysfunction by upregulating myosin light-chain kinase expression. Further studies are needed to elucidate how colon cytokines induced by dietary LCBs regulate CD14-mediated tight-junction permeability.

FIG 5
FIG 5

No anti-IBD effect of LCBs in CD14 KO male colitis. Groups of 6 C57BL/6 background CD14 KO male mice, 7 weeks old, were given oral gavage with 2 mg LCBs or 0.5 ml saline (vehicle control) daily, starting 7 days prior to DSS treatment. To induce colitis, mice were given 3% DSS through drinking water for 5 days (days 1 to 5) and then regular drinking water for 4 days (days 6 to 9). The mice were euthanized on day 9. Body weight changes (A), clinical scores (B), and colon cytokine levels (C) were measured. Data are expressed as means ± SD from six independent samples per group. *, P < 0.05 compared to the vehicle control group.

Anti-inflammatory effects of LCBs and CMPs in female mice with colitis.In male colitis, anti-inflammatory activities of chitin preparations addressed above showed that dietary LCBs resulted in better anti-inflammatory effects than CMPs and that the effects were dependent on host CD14 and TLR2. We next examined whether LCBs are more effective than CMPs in treating female colitis. Figure 6 shows that CMPs and LCBs had comparable anti-inflammatory effects, as detected by significantly reducing both body weight loss (Fig. 6A) and clinical scores (Fig. 6B) in colitis in WT female mice. Furthermore, neither CD14 KO (Fig. 6C and D) nor TLR2 KO (Fig. 6E and F) female mice showed any anti-inflammatory effects of LCBs. Taken together, our results indicated that LCBs were fully effective for TLR2/CD14-dependent anti-inflammatory activity in both sexes, whereas CMPs were fully effective in females but less effective in males.

FIG 6
FIG 6

Both LCBs and CMPs ameliorated female colitis in WT but not in TLR2 KO or CD14 KO mice. Groups of six female WT, TLR2 KO, and CD14 KO mice, 7 weeks old, were given oral gavage with 2 mg LCBs or 0.5 ml saline (vehicle control) daily, starting 7 days prior to DSS treatment. WT mice were also treated with 2 mg CMPs. To induce colitis, mice were given drinking water containing 4% DSS for WT mice and 3% DSS in TLR2 KO and CD14 KO mice (days 1 to 5) and then regular drinking water (days 6 to 9). Body weight changes (A, C, and E) and clinical scores (B, D, and F) in WT, TLR2 KO, and CD14 KO mice were monitored daily. The panels show 100% as the body weight on day 0. The mice were euthanized on day 9. Data are expressed as means ± SD from six animals per group. *, P < 0.05 compared with the vehicle control group.

Stomach AMC.The anti-inflammatory effects of LCBs and CMPs addressed above suggested that the activity of orally administered LCBs and CMPs can be influenced by additional gastrointestine (GI)-specific factors that are possibly sex dependent. We hypothesized that orally administered LCBs and CMPs are digested in the mouse GI tract by chitinases, specifically AMC, and that the resulting reduction in particle sizes facilitates phagocytosis of LCB remnants; stomach AMC activity is sex dependent. To test this hypothesis, we extracted AMC from mouse stomach and colon using a method previously described (44). As shown in Fig. 7A, levels of stomach AMC preparations isolated from saline- and LCB-treated colitis groups were greater in males than in females. Moreover, AMC levels in the colon samples in these two groups were also higher in males than in females (Fig. 7B). Serum AMC levels in the male groups were higher than those in female groups (Fig. 7C). These sex-dependent AMC levels in the stomach, colon, and serum samples were found in mice without colitis or LCB treatment (Fig. 7A to C).

FIG 7
FIG 7

Bioactivities of AMC-digested chitin. (A) Stomach AMC levels. Stomachs were isolated from the indicated groups. Proteins were extracted as indicated in Materials and Methods. AMC levels were measured by ELISA in three to six independent samples per group. Data are expressed as means ± SD. *, P < 0.05 compared to the male counterpart. (B) Colon AMC levels. Data are expressed as means ± SD from three to six independent samples per group. *, P < 0.05 compared to the male counterpart. (C) Serum AMC levels. Data are expressed as means ± SD from six independent samples per group. *, P < 0.05 compared to the male counterpart. (D) AMC-digested LCBs induced TNF-α production by RAW 264.7 macrophages. LCBs and CMPs in 0.1 M glycine-HCl buffer (pH 2.0) were incubated with 10 ng/ml AMC at 37°C for 60 min. Bisdionin F (BF), an AMC inhibitor, at 10 mM was also added. RAW 264.7 cells (5 × 105 cells/ml) were stimulated with digested LCBs and CMPs at the indicated doses for 18 h at 37°C. TNF-α in the culture supernatants was measured by ELISA in three independent cultures. Data are expressed as means ± SD. *, P < 0.05 compared to the corresponding AMC/BP group. (E and F) AMC-digested LCBs and CMPs induced TNF-α production by peritoneal macrophages isolated from WT, TLR2 KO, and CD14 KO mice. Peritoneal macrophages (2 × 105 cells/ml) were stimulated with digested LCBs and CMPs at the indicated doses for 18 h at 37°C. TNF-α in the culture supernatants was measured by ELISA in three independent cultures. Data are expressed as means ± SD. *, P < 0.05 compared to untreated LCBs or CMPs; #, P < 0.05 compared to the corresponding group of WT macrophages.

CMPs are known to induce phagocytosis-mediated M1 activation in a TLR2/CD14-dependent manner; LCBs, larger than macrophages and difficult to phagocytose, induce no M1 activation (29). We therefore determined whether AMC-digested LCBs induce M1 and whether CMP-induced M1 activation is reduced by AMC treatment. Figure 7D shows that, as demonstrated previously (29), LCBs induced no TNF-α production by RAW 264.7 cells. AMC digestion increased LCB-induced TNF-α production (Fig. 7D). In contrast, untreated CMPs (buffer control) are known to induce higher production of TNF-α (29); the production of TNF-α by CMPs was slightly but significantly reduced when CMPs were digested by AMC (Fig. 7D). All these effects of AMC were reduced by bisdionin F (BF), an AMC inhibitor. Furthermore, the mechanism inducing TNF-α production by digested LCBs was also dependent on TLR2 and CD14, since peritoneal macrophages isolated from TLR2 KO and CD14 KO mice did not respond to the digested LCBs (Fig. 7E). Taken together, the results suggested that stomach AMC digested dietary chitin particles, which became smaller but were still recognized by cells expressing TLR2 and CD14.

DISCUSSION

Oral administration of mucoadhesive chitin, an essential fungal cell wall component, is known to ameliorate mouse colitis (12–16). The present study was conducted to answer questions regarding what rationale guides preparation of the most effective chitin for IBD treatment, how chitin ameliorates colitis, and whether anti-inflammatory chitin is effective in both sexes. Important findings are that oral administration of chitin particles, larger than macrophages (LCBs; 40 to 70 μm), provides a significant anti-inflammatory effect in both males and females, whereas phagocytosable particles (CMPs; 1 to 10 μm) are more effective in females than males. Treatments with other forms of chitin, including oligosaccharide chitin, glucosamine, and deacetylated chitin (chitosan) microparticles (de-CMPs; 1 to 10 μm), are less effective for colitis in males. Our results demonstrated, for the first time, that female colitis was effectively ameliorated by a wider range of particle sizes of chitin than male colitis.

Data regarding sex differences in the incidence, disease severity, response to medications, and mortality of IBD patients are limited and inconclusive (45, 46). However, the presence of sex differences in IBD in patients is indicated by several lines of evidence. Risk for development of IBD in patients is modulated by endogenous sex hormones; pregnancy reduces future disease flare-ups (47), and postmenopausal women without hormonal supplementation have a worse prognosis than women taking hormonal replacement therapy (48). Similarly, an estrogen receptor agonist protected rats against colitis (49). Women with IBD were found to suffer from adverse effects of anti-inflammatory agents more often than men (50). Therefore, further research elucidating the basis of sex-dependent effects of chitin on IBD is essential to support clinical studies on implementation of chitin as an intervention to decrease risk for IBD.

Our study revealed a unique possibility that stomach AMC activities contribute to the size-dependent anti-inflammatory effects of chitin particles. Digestion of orally administered chitin by GI tract chitinases, including AMC, was characterized by Suzuki et al. (44) and by Ohno et al. (51). In particular, AMC levels in stomachs, colons, and sera appear to be higher in males than in females. Prodan et al. (52) found that salivary chitinase activities in young human adults were much lower in females than in males, although their measurement was not specific for AMC. Lower levels of AMC in females, therefore, should give the CMPs a longer half-life and therefore greater efficacy in females. In addition to enzymatic digestion of chitin by stomach AMC, colon AMC would protect epithelial cells from inflammatory damage. Protection of epithelial cells by AMC may also occur via a chitinolytic-independent mechanism, which is well documented in the lungs (53).

Provocative findings are that host chitin-binding TLR2 and CD14, protective in mouse colitis elicited by DSS (41, 42), are both responsible for anti-inflammatory effects of LCBs. In general, these receptors are expressed by mucosal epithelial cells as well as myeloid cell populations; host immune responses mediated through TLR2 and/or CD14 signals seem to be regulated at least in part under sex chromosomes and be influenced by sex hormones (55–58). In this study, cytokine profiles in colitis in TLR2 KO and CD14 KO mice exhibit some differences, implying that these two receptors, which closely interact in phagocytosis-mediated M1 activation by CMPs in peritoneal macrophages (29), have independent roles in their LCB-induced anti-inflammatory activity. However, the contributions of both receptors to the anti-inflammatory effects of LCBs are sex independent.

Medina-Contreras et al. demonstrated that intestinal CX3CR1+ macrophages, taking up luminal antigens and influenced under bacterial metabolites, play a role in mucosal homeostasis, bacterial translocation, and colitogenic Th17 responses in mice (59). Leonardi et al. further demonstrated that these macrophages appear to phagocytose luminal fungi through Dectin-1 and Dectin-2, both β-glucan receptors, and regulate mucosal immunity against fungi (33). It remains to be studied, however, whether CX3CR1+ or other intestinal macrophages phagocytose luminal chitin particles, mimetic fungi, through their TLR2 and CD14 and contribute to the production of mucosal anti-inflammatory cytokines listed above. In this regard, we demonstrated that M1 cytokine production by peritoneal macrophages requires phagocytosis of CMPs through TLR1/TLR2 heterodimer and CD14 coreceptor but not through Dectin-1 or Dectin-2 (29).

The literature indicates that chitin binds pathogenic gut microbes, including adherent invasive Escherichia coli (60) and C. albicans (61), through their chitin-binding domains, suggesting that dietary LCBs/CMPs block epithelial damage caused by the pathogens. However, our preliminary result failed to show reduced gut colonization of C. albicans by LCBs (see Fig. S2 in the supplemental material). Since DSS-colitis in mice has shown no increased gut colonization of C. albicans (8), further studies, utilizing C. albicans-challenged IBD animal models, are needed to determine whether dietary LCBs inhibit C. albicans colonization.

In summary, data presented here demonstrated that LCB treatment offered greater protection than CMP treatment against IBD, showing beneficial effects on weight loss, clinical score, and histology scores in male mice, but that LCBs and CMPs were equally effective in female mice. The anti-inflammatory effects of LCBs are dependent on both TLR2 and CD14 and are associated with increased colon expression of anti-inflammatory cytokines, reduced serum levels of proinflammatory IL-6, and reduced numbers of bacteria in the colon, liver, and spleen. Finally, we found that stomach AMC appears to play a rate-limiting and sex-dependent role in the anti-inflammatory effects of dietary chitin particles. Further studies are needed to determine whether dietary chitin particles, modified by stomach AMC digestion, localize at mucosal intestinal sites forming a chitin matrix and are phagocytosed by mucosal macrophages for ameliorating colitis.

MATERIALS AND METHODS

Preparation of CMPs, LCBs, and de-CMPs.CMPs (1 to 10 μm) were prepared as previously described (29). The quality of CMP preparations was evaluated on the basis of the following criteria: (i) the capacity to induce splenic macrophages in vitro to produce TNF-α and IL-12 but not IL-10 (24–26); (ii) more than 95% of particles had 1- to 10-μm diameters, determined by flow cytometry calibrated by 1-, 10-, and 48-μm latex beads (Polysciences) (24, 27); (iii) no detectable endotoxin (<0.03 endotoxin units/10 mg chitin/ml) determined by the Limulus amebocyte lysate assay; and (iv) minimal surface deacetylation of chitin particles determined by attenuated total reflection Fourier transform infrared (ATR-FTIR) difference spectroscopy (29). LCBs were prepared as a by-product of CMP preparation (24) and met criteria for size, endotoxin content, and surface deacetylation. To prepare de-CMP, CMPs were deacetylated with 1 N sodium hydroxide for 4 h at 37°C, followed by neutralization and washing (29). Oligosaccharide chitin and glucosamine (both from YSK, Yaizu, Japan) were also used for this study.

Mice.C57BL/6 WT, TLR2-deficient (KO), and CD14 KO mice were purchased from the Jackson Laboratory (Bar Harbor, ME). These mice were maintained under specific Helicobacter-free conditions at Florida Atlantic University. Animal care and procedures of the experiments in this study were approved by the IACUC at FAU, protocol number A15-34.

DSS-elicited colitis and dietary chitin treatment.For the DSS-induced colitis model, acute colitis was elicited after mouse body weight reached 19 to 21 g (around 8 weeks old) by administration in drinking water of 4% DSS (molecular mass, 35 to 45 kDa; MP Biomedicals, Irvine, CA) for WT mice and 3% DSS for CD14 KO and TLR2 KO mice; after 5 days, they were switched to untreated drinking water. For chitin treatments, groups of 6 mice were given oral gavage with 0.5 ml saline containing 2 mg each chitin preparation listed above daily for 15 days, starting 7 days before DSS treatment. Vehicle control groups were given 0.5 ml saline. Mice were euthanized 9 days after DSS treatment; blood, spleens, livers, and colons were harvested. The colon was cut open longitudinally along the main axis, luminal contents were collected, and the remaining colon was washed with ice-cold saline.

Measurements of disease parameters.(i) Body weight and clinical score. Colitis mice were weighed and monitored daily for appearance and signs of bloody stools (score of 0 or 1), diarrhea with soft stools (0 or 1), and hunching posture (0 or 1) as clinical parameters previously described (4).

(ii) Histologic score. Histological evaluation of colitis was performed using hematoxylin-and-eosin-stained sections of colon tissue as previously described (4). A total histologic score ranging from 0 to 6 represents the sum of the epithelial nonulcerative region alteration score and the infiltration score. Epithelium nonulcerative region alteration scores were the following: 0, normal morphology; 1, loss of goblet cells in small areas; 2, loss of goblet cells in large areas. Infiltration scores were the following: 0, no infiltration; 1, infiltration around crypt basis; 2, infiltration reaching the lamina muscularis mucosae; 3, extensive infiltration reaching the lamina muscularis mucosae and thickening of the mucosa with abundant edema; 4, infiltration of the lamina submucosa.

(iii) Colon cytokine measurement. Proteins were extracted from colons, and levels of inflammatory mediators IL-1β, IL-6, IL-10, IL-22, IFNγ, TNF-α, and CHI3L1 were measured by specific enzyme-linked immunosorbent assays (ELISAs) (BioLegend, San Diego, CA, and BD, Franklin Lakes, NJ). Protein levels were measured by Pierce protein bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Rockford, IL).

(iv) Bacterial CFU. Bacterial translocation assay was performed as previously described (4). Tissues were homogenized in Hanks’ balanced salt solution. Serial dilutions of the tissue homogenates were plated on Luria-Bertani (LB) agar plates and incubated at 37°C overnight. Numbers of CFU were counted.

Extraction of AMC from mouse stomachs.Stomach tissue isolated from normal WT mice was homogenized in 10 volumes (one stomach in 3 ml) of ice-cold Tris buffer (20 mM Tris-HCl [pH 7.6], 150 mM NaCl, 1:500 protease inhibitor cocktail; P8340; Sigma-Aldrich) using a Teflon/glass homogenizer (51). The homogenates were centrifuged at 12,000 rpm for 20 min at 4°C, and the supernatants were filtered through a 0.22-μm filter (Mustang E-membrane; Pall, Nottingham, MD) to remove endotoxin, collected, and kept at −80°C. Mouse AMC and protein levels were measured by ELISA (Nordic Biosite, Plymouth Meeting, PA) and Pierce protein BCA assay.

M1 activation by LCBs and CMPs after digestion with AMC.LCBs at 1 mg/ml in 0.1 M glycine-HCl buffer, pH 2.0, were incubated with stomach AMC at 10 ng/ml. Some groups treated with AMC were further supplemented with BF, an AMC inhibitor (Sigma), at 10 mM. After incubation at 37°C for 60 min, digested LCBs were washed with saline. CMPs were also digested in an identical manner. To measure M1 induction by digested LCBs and CMPs, RAW 264.7 macrophages (5 × 105 cells/ml) were stimulated with digested LCBs and CMPs at selected doses for 18 h at 37°C. Peritoneal macrophages (2.5 × 105 cells/ml), isolated from WT, TLR2 KO, and CD14 KO mice (29), were also stimulated with the digested LCBs and CMPs. TNF-α in the culture supernatants was measured by ELISA (BioLegend).

Statistics.Statistical significance was evaluated by Student’s t test for parametric data, and results are shown as the means ± standard deviations (SD). Nonparametric data (e.g., clinical data) were analyzed by using the Mann-Whitney U test. Differences were considered statistically significant at a P value of ≤0.05.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health R15 AT008252-01, RO1 HL71711, DOD DAMD 17-03-1-0004, and Bankhead-Coley Cancer Research Program 06BS-04-9615.

We thank Yaizu Suisankagaku (YSK), Yaizu, Japan, for kindly providing chitin products, Emiko Mizoguchi, Massachusetts General Hospital and Harvard Medical School, and now Kurume University School of Medicine, Kurume, Japan, for consulting on the initial approach.

Authors made the following contributions: designed and performed studies, P.L., A.M.C., C.J., Z.J.L., C.K.D., B.M., N.E., Z.L., J.W., Y.S., and C.N.; analyzed and interpreted results, P.L., A.M.C., C.J., Z.J.L., C.K.D., B.M., N.E., Z.L., J.W., Y.S., and C.N.; prepared the manuscript, P.L., C.K.D., Y.S., and C.N. We have no conflicts of interest to declare.

FOOTNOTES

    • Received 3 January 2019.
    • Returned for modification 25 January 2019.
    • Accepted 5 February 2019.
    • Accepted manuscript posted online 19 February 2019.

  • Supplemental material for this article may be found at https://doi.org/10.1128/IAI.00006-19.

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KEYWORDS

Candida albicans
acidic mammalian chitinase
chitin binding protein
dysbiosis
inflammatory bowel disease
large chitin beads

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