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
). 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
). 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
). 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
). 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
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.
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
). 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
), 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
) and C. albicans
), 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
), further studies, utilizing C. albicans
-challenged IBD animal models, are needed to determine whether dietary LCBs inhibit C. albicans
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
); (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.
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).
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.
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.B., 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.B., 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.