MUC2 Mucin and Butyrate Contribute tothe Synthesis of the AntimicrobialPeptide Cathelicidin in Response toEntamoeba histolytica- and DextranSodium Sulfate-Induced Colitis

Eduardo R. Cobo,* Vanessa Kissoon-Singh, France Moreau, Ravi Holani,Kris ChadeeDepartment of Microbiology, Immunology and Infectious Diseases, Gastrointestinal Research Group, SnyderInstitute for Chronic Diseases, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada

ABSTRACT Embedded in the colonic mucus are cathelicidins, small cationic peptidessecreted by colonic epithelial cells. Humans and mice have one cathelicidin-relatedantimicrobial peptide (CRAMP) each, LL-37/hCAP-18 and Cramp, respectively, withrelated structure and functions. Altered production of MUC2 mucin and antimicro-bial peptides is characteristic of intestinal amebiasis. The interactions between MUC2mucin and cathelicidins in conferring innate immunity against Entamoeba histolyticaare not well characterized. In this study, we quantified whether MUC2 expressionand release could regulate the expression and secretion of cathelicidin LL-37 in co-lonic epithelial cells and in the colon. The synthesis of LL-37 was enhanced with bu-tyrate (a product of bacterial fermentation) and interleukin-1� (IL-1�) (a proinflam-matory cytokine in colitis) in the presence of exogenously added purified MUC2. TheLL-37 responses to butyrate and IL-1� were higher in high-MUC2-producing cellsthan in lentivirus short hairpin RNA (shRNA) MUC2-silenced cells. Activation of cyclicadenylyl cyclase (AMP) and mitogen-activated protein kinase (MAPK) signaling path-ways was necessary for the simultaneous expression of MUC2 and cathelicidins. InMuc2 mucin-deficient (Muc2�/�) mice, murine cathelicidin (Cramp) was significantlyreduced compared to that in Muc2�/� and Muc2�/� littermates. E. histolytica-induced acute inflammation in colonic loops stimulated high levels of cathelicidin inMuc2�/� but not in Muc2�/� littermates. In dextran sodium sulfate (DSS)-inducedcolitis in Muc2�/� mice, which depletes the mucus barrier and goblet cell mucin,Cramp expression was significantly enhanced during restitution. These studies dem-onstrate regulatory mechanisms between MUC2 and cathelicidins in the colonic mu-cosa where an intact mucus barrier is essential for expression and secretion ofcathelicidins in response to E. histolytica- and DSS-induced colitis.

KEYWORDS Entamoeba histolytica, MUC2, antimicrobial peptides, mucin

The innate defenses of the colon and first barrier against pathogens include glyco-sylated MUC2 mucin secreted by goblet cells that forms an inner sterile layer firmly

attached to the epithelium and an outer layer with an expanded volume and that iscolonized by bacteria (1). Embedded in colonic mucus are cathelicidins, small cationicpeptides (23 to 37 amino acids) secreted by neutrophils and epithelial cells, includingcolonic epithelium, with broad antimicrobial activity (2, 3). The only cathelicidin de-scribed in humans is LL-37/hCAP-18, which is homologous to mouse cathelicidin-related antimicrobial peptide (CRAMP) (2, 3). Both LL-37 and CRAMP have relatedstructure and antimicrobial and chemotactic functions (2, 3). Sodium butyrate pro-

Received 24 October 2016 Returned formodification 18 November 2016 Accepted21 December 2016

Accepted manuscript posted online 9January 2017

Citation Cobo ER, Kissoon-Singh V, Moreau F,Holani R, Chadee K. 2017. MUC2 mucin andbutyrate contribute to the synthesis of theantimicrobial peptide cathelicidin in response toEntamoeba histolytica- and dextran sodiumsulfate-induced colitis. Infect Immun 85:e00905-16. https://doi.org/10.1128/IAI.00905-16.

Editor John H. Adams, University of SouthFlorida

Copyright © 2017 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Kris Chadee,kchadee@ucalgary.ca.

* Present address: Eduardo R. Cobo,Department of Production Animal Health,Faculty of Veterinary Medicine, University ofCalgary, Calgary, Alberta, Canada.

FUNGAL AND PARASITIC INFECTIONS

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duced by commensal bacteria in the colon functions as a histone deacetylase inhibitorto induce cathelicidin mRNA and protein expression in the colonic epithelium (4).

The intestinal mucus barrier is critical in innate host defense against protozoaninfections. Infectious colitis caused by Entamoeba histolytica, a protozoan that causesamebic dysentery and/or liver abscesses (5), is a good model to study the relevance ofinnate factors in the colon in response to an enteric pathogen. The virulence factorcysteine proteinase 5 of E. histolytica (EhCP5) is a potent mucus secretagogue (6) thatcan also dissolve the mucus barrier by degrading MUC2 (7–9). In contact with the gutmucosa, EhCP5 binds macrophages/dendritic cells to evoke a potent proinflammatoryresponse dominated by interleukin-1� (IL-1�) and tumor necrosis factor alpha (TNF-�)(10, 11). In Muc2�/� mice, E. histolytica inoculated in colonic loops elicited robustsecretory and acute proinflammatory responses, aberrant protein secretions, and al-tered tight junction permeability (12). Prolonged contact of E. histolytica with thecolonic epithelium induced the synthesis of cathelicidins in humans and mouse, whichwere degraded by E. histolytica cysteine proteinases (13). Even though E. histolytica isresistant to killing by LL-37 (13), one report showed that shortened peptides derivedfrom cleaved LL-37 affected E. histolytica integrity and viability (14). Altered productionof colonic mucin and antimicrobial peptides is also characteristic of chronic inflamma-tory bowel diseases (IBDs). In ulcerative colitis (UC), the thickness of the adherentmucus gel is reduced, even to the point of denudation, in areas with acute inflamma-tion (15, 16) and the number of mature goblet cells is decreased in the upper third ofthe crypts (17). In UC, the expression of cathelicidin is increased, mostly in the inflamedmucosa (18). Likewise, colitis induced by dextran sodium sulfate (DSS), an animal modelthat mimics human UC, downregulates Muc2 mucin genes with depletion of gobletcells and adherent mucin in rats (19), while it increases CRAMP protein expression in thecolonic epithelium and macrophages in mice (20). In Crohn’s disease, the mucin layerappears normal or even thicker than that in controls (15, 16) with no increase in theexpression of cathelicidin (18).

These studies indicate that the innate immune factors MUC2 mucin and cathelici-dins may act in concert at the colonic mucosa to support the microbiota and to prevententry of pathogens. In support of this, we have recently established that defectivecolonic MUC2 mucin leads to deficient stimulation of �-defensin 2 produced by thecolonic epithelium (21). In the present study, we interrogated the innate host defensemechanisms at the colonic mucosa by determining the cooperative or regulatoryexpression of MUC2 mucin and cathelicidins under homeostatic conditions and inresponse to E. histolytica-induced inflammation and chronic colitis induced by dextransodium sulfate (DSS). Our results demonstrate that MUC2 gene expression and mucusrelease and butyrate were necessary for the expression and secretion of cathelicidin inhuman cultured colonic epithelial cells. Moreover, in the absence of the colonic mucusbarrier or in its altered state, there was an impaired local cathelicidin response towardE. histolytica and DSS-induced colitis in mice.

RESULTSButyrate and MUC2 mucin regulate the expression and production of catheli-

cidin LL-37 peptide in human colonic epithelial cells. Previous studies have shownthat parental HT-29 cells pretreated with butyrate promoted the synthesis of catheli-cidins (4, 22) and MUC2 (23). We used these cells to investigate the effect of IL-1�, ahallmark proinflammatory cytokine observed in amebic colitis (11) and IBD (24), in thesynthesis of cathelicidins in the presence/absence of butyrate and MUC2 mucin. Whenparental HT-29 cells were stimulated with graded doses of IL-1�, there was a modestincrease in LL-37 expression only with the highest concentration used (20 ng/ml) (Fig.1A). Thus, to determine whether the high-MUC2 mucin phenotype was necessary forthe expression of cathelicidin under inflammatory conditions, we next determined ifthis concentration of IL-1� promoted a differential cathelicidin response in MUC2 H/Lcells. To do this, MUC2 H/L cells were first stimulated with graded doses of butyratealone that significantly induced LL-37 gene expression (P � 0.05) (Fig. 1B). MUC2 H/L

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cells primed with butyrate were then stimulated with IL-1� (20 ng/ml). Under theseconditions, IL-1� induced a 10-fold increase in LL-37 mRNA expression over that withbutyrate induction alone in MUC2 H but not MUC2 L cells (Fig. 1B). Increasing doses ofbutyrate (0 to 4 mM; 72 h) induced a modest stepwise increase in MUC2 mRNAexpression in MUC2 H/L cells (Fig. 1C). However, in the presence of butyrate, IL-1�

enhanced MUC2 gene expression 8-fold in MUC2 H cells (Fig. 1C). Neither butyrate norIL-1� alone or in combination had a significant effect on MUC2 gene expression inMUC2 L cells.

As LL-37 responses to inflammatory stimulus were more prominent in the presenceof butyrate and MUC2, we next determined if exogenously added MUC2 mucin couldpromote cathelicidin synthesis in parental HT-29 cells. HT-29 cells primed with butyrateand then supplemented with increasing doses of exogenous purified MUC2 showedincreased LL-37 gene expression in the presence of IL-1� (Fig. 1D). Immunofluorescencestudies showed that prestimulation with butyrate (4 mM; 72 h) followed by IL-1� (20ng/ml; 16 h) induced strong immunoreactive LL-37 peptide in the cytoplasm of MUC2H cells with punctate accumulation at the periphery of the cells (Fig. 2A). Butyrate aloneinduced expression of LL-37 peptide diffused throughout the cytoplasm in MUC2 Hcells (Fig. 2A). The accumulation of LL-37 after stimulation with butyrate or IL-1� wasobserved to a lesser extent in MUC2 L cells (Fig. 2A). Likewise, confocal studies inparental HT-29 cells showed that butyrate and MUC2 mucin induced the accumulationof intracellular cathelicidin LL-37 exposed to IL-1� (Fig. 2B). Taken together, these datashow that both butyrate and MUC2 mucin mRNA expression and biosynthesis ofmature mucin were critical for the induction and synthesis of cathelicidins in colonicepithelial cells. Moreover, cathelicidin expression was augmented in an inflammatorymilieu characterized here by the presence of the proinflammatory cytokine IL-1�.

FIG 1 Cathelicidin expression in human colonic epithelial cells. (A) The expression of LL-37 mRNA was quantified inparental HT-29 cells pretreated with butyrate (4 mM for 2 days) with increasing doses of IL-1� (0 to 20 ng/ml; 16 h). (B andC) Expression of MUC2 and cathelicidin LL-37 mRNA was quantified in MUC2 H and MUC2 L cells. (D) Expression of LL-37mRNA in parental HT-29 cells supplemented with purified human colonic MUC2. (B to D) Colonic cells were pretreated withbutyrate and then stimulated with IL-1� (20 ng/ml; 16 h). MUC2 and LL-37 mRNA expression was quantified by qPCR andexpressed as fold change relative to untreated cells. P values for all significant comparisons with control group arerepresented (*, P � 0.05).

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Activation of cAMP and mitogen-activated protein kinase (MAPK) signalingpathways is necessary for the simultaneous expression of MUC2 and cathelicidinsin colonic epithelial cells. To evaluate common intracellular signaling mechanisms inthe regulation of MUC2 and cathelicidins, we focused on the adenylyl cyclase pathwaythat was shown to transcriptionally regulate cathelicidin (25) and newly synthesizedmucin (26) in intestinal epithelial cells. Butyrate activates cyclic AMP (cAMP) responseelement binding protein (CREB) and increases the activity of cAMP response element(CRE) (27), both of which have been implicated in the activation of the cathelicidinpromoter (25). Similarly, IL-1� mediates mucin regulation through activation of CREB inepithelial cells (28). Consistently with these findings, we observed that the cAMPagonist forskolin (1 to 20 �M) significantly (P � 0.05) increased MUC2 and LL-37 mRNAexpression in MUC2 H cells (Fig. 3A and B). MUC2 L cells also showed the capacity toproduce cathelicidins when stimulated with forskolin (Fig. 3B). These findings wereconfirmed by immunofluorescence studies that showed that forskolin increased intra-cytoplasmic accumulation of MUC2 mucin and LL-37 peptides in the cytoplasm ofMUC2 H cells, although it was not evident in MUC2 L cells (Fig. 4A). To explore themechanism by which cAMP regulated the induction of MUC2 and cathelicidins, weinterrogated two families of downstream signaling proteins, MAPK kinase and phos-phatidylinositol 3-kinase. To do this, MUC2 H/L cells were incubated with specificinhibitors of MAPK kinase (PD98059, 20 �M, 1 h) and phosphatidylinositol 3-kinase

FIG 2 Intracellular localization of cathelicidin is increased in human colonic epithelial cells stimulated by butyrate and MUC2 mucin. (A)Cathelicidin LL-37 was detected with primary mouse monoclonal antibody anti-LL-37 (green) in HT-29 MUC2 H/L cells. Cells wereprestimulated with butyrate (4 �M; 72 h) and then stimulated with IL-1� (20 ng/ml; 16 h). (B) Cathelicidin LL-37 was detected with primarymouse monoclonal antibody anti-LL-37 (yellow) in parental HT-29 cells prestimulated with butyrate (4 �M; 72 h) and then stimulated withpurified MUC2 mucin (10 �g/ml) and IL-1� (20 ng/ml) for 16 h. Nuclei were stained with DAPI (blue). IgG was used as antibody control.Image is from 1 of 5 independent experiments. Bars, 10 �m. *, P � 0.05. Quantification of confocal images was performed by measuringthe intensity of LL-37 immunostaining within the cell and represented in the contiguous histograms.

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(LY294002, 20 �M, 1 h) before incubation with forskolin (10 �M, 3 h) (Fig. 3C and D).Inhibiting MAPK kinase significantly (P � 0.05) reduced cAMP-induced MUC2 and LL-37mRNA expression (Fig. 3C and D) whereas the phosphatidylinositol 3-kinase inhibitordid not (Fig. 3C and D). These results suggest that the cAMP signaling pathway largelycontributed to the expression of both MUC2 and LL-37 genes.

Butyrate priming enhances cathelicidin expression in response to E. histolytica.E. histolytica is a model enteric pathogen that degrades components of innate hostdefenses, including the mucin polymer network and cathelicidins (9, 13), followed bydisruption and invasion of the colonic epithelium (7, 12). To evaluate the importance ofMUC2 mucin in aiding epithelial cathelicidin defenses against E. histolytica, we evalu-ated the expression of cathelicidin in butyrate-primed MUC2 H/L cells exposed to IL-1�

or E. histolytica trophozoites. Treatment with butyrate (72 h) significantly (P � 0.05)induced LL-37 mRNA expression in MUC2 H cells (Fig. 5) while stimulation with IL-1�

(20 ng/ml) following butyrate exposure did not induce a further increase in LL-37 mRNAexpression in MUC2 H cells (Fig. 5). The butyrate effect was replicated in cells lackingMUC2 (Fig. 5). Exposure to E. histolytica trophozoites (30 min) did not induce furtherincreases in LL-37 mRNA expression in either MUC2 H or MUC2 L cells beyond theincrease induced by butyrate alone in MUC2 H cells (Fig. 5). The importance of butyratewas demonstrated by microscopy studies that showed higher LL-37 immunostaining incolonic cells treated with butyrate (with or without E. histolytica) (Fig. 4B). Moreover,confocal microscopy studies showed synthetic LL-37 bound to the surface of fixed E.histolytica (Fig. 4C). These results suggest that cathelicidin responses in colonic epithe-

FIG 3 Forskolin activation of the adenylyl cyclase pathway induces simultaneously MUC2 and cathelicidin mRNA in colonic epithelial cells.MUC2 and cathelicidin LL-37 mRNA was quantified in colonic epithelial cells by qPCR and expressed as fold change relative tounstimulated controls. (A and B) MUC2 H/L cells were stimulated with forskolin (10 �M; 3 h). (C and D) Parental HT-29 cells were incubatedwith forskolin (10 �M; 3 h) before pretreatment with specific inhibitors of MAPK/ERK kinase (PD98059; 20 �M; 1 h), phosphatidylinositol3-kinase inhibitors (LY294002; 20 �M; 1 h), or DMSO, the organic solvent used to dissolve the inhibitors, as control. P values for allsignificant comparison with control group are represented (*, P � 0.05).

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lial cells expressing MUC2 mucin are mainly induced by butyrate and that thesepeptides can bind the membrane of the parasite. Short exposure of colonic epithelialcells to E. histolytica trophozoites did not augment cathelicidin gene expression.

Muc2�/� littermates express reduced cathelicidin in the colon. We have recentlyshown that Muc2�/� mice are deficient in �-defensins in the colon (21). Muc2�/� micedevelop low-grade spontaneous colitis that mimics lesions similar to those in UC. Todetermine if the absence of a mucin barrier or Muc2 biosynthesis could also affect theproduction of other host defense peptides such as cathelicidin, we quantified theexpression of mouse cathelicidin Cramp in Muc2�/�, Muc2�/�, and Muc2�/� litter-mates. As predicted, Muc2�/� mice showed lower basal expression of Cramp mRNA inthe colon than did Muc2�/� and Muc2�/� littermates (Fig. 6A). By confocal microscopy,Muc2�/� mice showed almost no Cramp immunoreactive peptide in the colonicmucosa compared to Muc2�/� and Muc2�/� mice, which showed Cramp immunore-active peptide distributed throughout the cytoplasm in colonic epithelial cells andintense expression in the gut lumen within the mucus layer (Fig. 7A).

FIG 4 (A) The activator of adenylyl cyclase forskolin induces intracellular localization of MUC2 mucin and LL-37 cathelicidin in human colonic epithelial cells.MUC2 H/L cells were pretreated with butyrate (72 h) and then stimulated with forskolin (10 �M; 3 h). LL-37 was detected with primary mouse monoclonalantibody anti-LL-37 (red), and mucin was detected with primary rabbit polyclonal antibody anti-human LS174T mucin (yellow). (B) Intracellular localization ofcathelicidin is increased in human colonic epithelial cells stimulated by butyrate and not by E. histolytica. Cathelicidin LL-37 was detected with primary mousemonoclonal antibody anti LL-37 (orange) in parental HT-29 cells prestimulated with butyrate (4 �M; 72 h) and then stimulated with live E. histolyticatrophozoites (1 � 104/ml; 1 h). (C) Cathelicidins bound to E. histolytica trophozoites. Glutaraldehyde-fixed E. histolytica trophozoites (3 � 105) were incubatedwith synthetic LL-37 (5 �M) for 1 h, and bound LL-37 was detected with primary monoclonal murine IgG antibodies (OSX12; Novus Bio) (yellow). Nuclei werestained with DAPI (blue). IgG was used as antibody control. Image is from 1 of 5 independent experiments. Bar, 10 �m. *, P � 0.05. Quantification of confocalimages was performed by measuring the intensity of LL-37 and MUC2 immunostaining within the cell, or bound on the surface of trophozoites, and representedin the contiguous histograms. DIC, differential inference contrast; Eh, E. histolytica.

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Even though Cramp is basally expressed, it is not known if its expression could bealtered in response to E. histolytica. To study this, we used a murine model of acuteinflammation induced by inoculating E. histolytica trophozoites in closed colonic loops(12). We observed that in the absence of mucin in Muc2�/� mice, Cramp mRNAexpression was low, whereas in Muc2�/� littermates, Cramp expression in response toE. histolytica was significantly enhanced compared to that in control loops (Fig. 6B). Byimmunofluorescence, intense Cramp immunoreactive staining was evident in the co-

FIG 5 Butyrate pretreatment enhances the expression of cathelicidins and is increased in response toIL-1� and E. histolytica. The expression of LL-37 mRNA was quantified in MUC2 H/L cells by qPCR andexpressed as fold change relative to control MUC2 H cells. Colonic cells (1 � 105/ml) were preincubatedwith butyrate (4 �M, 72 h) and stimulated with IL-1� (20 ng/ml; 16 h) followed by live E. histolyticatrophozoites (1 � 104/ml; 1 h). P values for all significant comparisons with control group are represented(*, P � 0.05). Eh, E. histolytica.

FIG 6 Cathelicidin expression in the colon is impaired in animals deficient in Muc2 and in response to E. histolyticaand DSS-induced colitis. Expression of Cramp mRNA was quantified in the colonic mucosa of Muc2�/�, Muc2�/�,and Muc2�/� mice by qPCR and expressed as fold change relative to control Muc2�/� mice. Mice remained underuntreated conditions (A), were challenged with live E. histolytica trophozoites in closed colonic loops for 3 h (B), orwere provided DSS in drinking water (3% for five consecutive days for Muc2�/� mice and 1% for 3 days for Muc2�/�

littermates) followed by water and euthanized at days 7 and 15 posttreatment (C). P values for all significantcomparisons with control Muc2�/� group (*, P � 0.05) are represented. Eh, E. histolytica.

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lonic epithelium in control and E. histolytica-inoculated colon in Muc2�/� but notMuc2�/� mice (Fig. 7B). Importantly, cathelicidin peptides appeared as distinct punctathat were bound to E. histolytica trophozoites in close proximity to the colonic epithe-lium. No dead E. histolytica cells were observed that contacted and/or ingested thecathelicidin peptides. In Muc2�/� mice, E. histolytica was not observed close to theepithelial surface (Fig. 7B) and colonic loops had intense fluid-filled exudates. Theseresults suggest that an intact Muc2 mucin barrier was necessary for the colonicepithelium to produce and secrete cathelicidins basally and in response to E. histolyticainfection.

The role of cathelicidins in the intestine is restricted not only to acute amebic colitis,as cathelicidins have been shown to ameliorate chronic DSS-induced colitis (20, 29).However, at present, it is not known if the cathelicidin response observed in amebiccolitis is comparable to that in chronic colitis producing an altered mucus barrier. Thismechanism is of importance to understand cathelicidin defenses against E. histolytica,as the mucus barrier is degraded by E. histolytica followed by depletion during theprogressive development of amebic colitis (6–8, 21). To explore this, we studiedDSS-induced colitis in animals deficient in Muc2. We observed that there was a modest

FIG 7 Intracellular localization of murine cathelicidin in the colonic mucosa in response to E. histolytica (Eh) or DSS. (A) Murine cathelicidin was detected withprimary rabbit polyclonal antibody anti-Cramp (orange) in untreated Muc2�/�, Muc2�/�, and Muc2�/� littermates. (B) Colonic cathelicidin response to E.histolytica is decreased in Muc2�/� mice. Muc2�/� and Muc2�/� littermates were challenged with live E. histolytica trophozoites in closed colonic loops for 3h. (C) Colonic cathelicidin response in dextran sodium sulfate (DSS) colitis is decreased in mice with an impaired mucin barrier. Mice received DSS in drinkingwater (3% for five consecutive days for Muc2�/� mice and 1% for 3 days for Muc2�/� mice) and were euthanized at days 7 and 15 posttreatment. Murinecathelicidin was detected with anti-Cramp primary rabbit polyclonal antibody (red) in the colonic mucosa. Nuclei were stained with DAPI (blue). Arrows indicateE. histolytica trophozoites in close contact with the epithelium. Image is from 1 of 5 independent experiments. Bar, 15 �m. *, P � 0.05. Quantification of confocalimages was performed by measuring the intensity of Cramp immunostaining within the cell and represented in the contiguous histograms.

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similar increase in Cramp mRNA expression in Muc2�/� and Muc2�/� littermates at day7 (Fig. 6C) during severe colitis where the mucus layer and goblet cells are depleted. Asexpected, during restitution on day 15, when the mucus layer and goblet cells returnedto normal, Cramp mRNA expression was significantly enhanced in Muc2�/� but notMuc2�/� littermates. These results were confirmed by immunofluorescence assaysdemonstrating that during peak colitis at day 7, Cramp immunoreactivity staining wasreduced in the cytoplasm of colonic epithelial cells and in the outer lumen in Muc2�/�

littermates as no mucus layer was present (Fig. 7C). At restitution on day 15, intenseCramp-immunoreactive peptide was present in colonic epithelial cells and abundant inthe outer mucus layer of Muc2�/� but not Muc2�/� littermates (Fig. 7C). These dataclearly indicate that cathelicidin innate responses toward colonic injury and restitutiondepend on a functional and established Muc2 mucin barrier.

DISCUSSION

The colonic mucus barrier is essential in mucosal health to protect the host andindigenous microbiota while avoiding invasion by pathogens. Colonic MUC2 mucin isthe first line of the innate host defense to prevent E. histolytica-induced acute proin-flammatory responses (12) and invasion of the epithelium (7, 9). Embedded within themucus layer are host defense peptides, but it is unclear whether MUC2 mucin iscovalently bound to those peptides or whether MUC2 mucin and the peptides needeach other to exert their protective functions. The colon is constantly exposed tobutyrate produced by bacterial fermentation of dietary fibers that regulates cathelicidingene expression in mucosal epithelial cells (4). In this study, we describe regulatoryinteractions between MUC2 and butyrate-induced cathelicidins in the colonic lumenunder homeostatic and inflammatory conditions induced by E. histolytica. Our dataindicate that defects or depletion in MUC2 mucin in the colon, a hallmark of amebiccolitis and IBD, severely affect innately constitutive expression of naturally occurringcathelicidins in mice and human colonic cells. Impaired production of MUC2 mucin andthus cathelicidin degrades intestinal epithelial defenses and will likely negativelyimpact the outcome of infectious processes.

In amebic colitis, epithelial responses to E. histolytica involve a complex interplaybetween its virulence factors and a variety of innate factors. Cysteine proteinasesreleased by E. histolytica could convert human pro-IL-1� into biologically active IL-1�

(30). Thus, in E. histolytica colitis, mucosal epithelial cells theoretically could be exposedto high levels of IL-1� to stimulate proinflammatory mediators (30). However, activationof cathelicidins seems to be unresponsive to IL-1�. Previous studies (4) have shown thatIL-1� alone did not alter cathelicidin levels in colonic epithelial cells. In agreement, weshowed that high doses of IL-1� (20 ng/ml) moderately induced cathelicidins in colonicepithelial cells (3-fold at gene level). In addition, IL-1� did not further augment LL-37responses in colonic cells already exposed to butyrate and E. histolytica. Thus, weconclude that E. histolytica alone did not induce the expression of cathelicidin in colonicepithelial cells and that IL-1� by itself is not the main component responsible forexpression of cathelicidin. Butyrate is the principal inducer of cathelicidins as it primescolonic epithelial cells to be more responsive to other stimuli, such as E. histolytica orIL-1�. From the host perspective, the cathelicidin response in the colonic mucosa maylimit amebic colitis. We found that cathelicidin peptides bound fixed E. histolytica,suggesting the physical binding of these cationic peptides to anionic parasite mem-brane structures. This coincides with our observation of cathelicidin bound to E.histolytica trophozoites in the colonic lumen of infected mice. The actual effect ofcathelicidins on the survival of E. histolytica is still elusive. Synthetic cathelicidin-derivedpeptides were shown to impair E. histolytica integrity and viability (14). Moreover,although E. histolytica-secreted cysteine proteinases degrade cathelicidins, the frag-mented cathelicidin peptides have potent bactericidal effects over enteric bacteria thatcould control E. histolytica colonization (13). Thus, the absence of mucin and/or theinsufficient butyrate that leads to deficient cathelicidin responses weakens epithelialinnate defenses and likely induces colonic dysbiosis that could play a role the patho-

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genesis of E. histolytica. In agreement, the roles of cathelicidins and MUC2 in intestinaldefense against other enteric pathogens have been well demonstrated. For example,Clostridium difficile or toxin A was shown to induce early protective cathelicidin CampmRNA and peptide in the intestinal epithelium of mice (31). In addition, exogenouscathelicidins reduced histological damage, macrophage infiltration, myeloperoxidaseactivity, and TNF-� expression in experimentally induced C. difficile colitis (31). Theinterconnected protective effects of MUC2 and cathelicidins in the intestine are appar-ently not exclusively dependent on the presence of pathogens. Mice with an impairedMuc2 mucin barrier also showed an accompanying failure in the cathelicidin responsein DSS-induced colitis. However, in DSS-induced inflammation in intact animals, theincrease in cathelicidin was shown to be protective in the mucosal epithelium, whereascathelicidin-deficient mice developed more severe colitis (20, 29). Our studies havefound that other innate host defenses such as MUC2 and butyrate are criticallyimportant to elicit high cathelicidin levels toward E. histolytica and chemically inducedcolitis.

At present, it is not known how MUC2 mucin regulates cathelicidin. Colonic colum-nar epithelial cells and goblet cells produce and store MUC2 mucin and host defensepeptides, including cathelicidins (4, 32). However, the regulatory signaling pathwaysbetween MUC2 and cathelicidins in the intestinal epithelium remain largely unknown.In our study, we found that the synthesis of cathelicidins was restricted to humancolonic epithelial cells and to mouse models genetically capable of producing Muc2.Mechanistically, we showed that the accumulation of intracellular cAMP-activatingprotein kinase and activation of MAPK concurrently induced both MUC2 and LL-37 andrestored the MUC2 effect in colonic cells deprived of MUC2. We have previously shownthat cAMP agonist stimulated the secretion of preformed and newly synthesized mucin(26, 33). Moreover, cAMP signaling regulates cathelicidin expression by binding CREBand activator protein 1 (AP-1) to the promoter region (25). Thus, we speculate thatMUC2 functions as a cAMP ligand to induce the production of cathelicidins from thecolonic epithelium. Colonic epithelial cells deficient in MUC2 would have the signalingreceptors to produce cathelicidins but lack the ligand (i.e., MUC2). In the intrinsicsignaling pathways between MUC2 and cathelicidins in the colonic epithelium, we alsodemonstrated the importance of dietary compounds such as butyrate on colon celldifferentiation for the production of MUC2 and cathelicidins. Other studies have shownindividually the effects of butyrate on innate intestinal epithelial defenses. Butyrateincreases MUC2 mRNA via AP-1 and acetylation/methylation of histones at the MUC2promoter (34) and cathelicidin LL-37 in colonic epithelial cells via MEK/extracellularsignal-regulated kinase (ERK) and p38/MAPK signaling pathways (35). Several othermechanisms of cooperation between MUC2 and cathelicidin and the involvement ofother signaling pathways beyond cAMP may figure in intestinal defenses.

Mucus might function as a reservoir for the accumulation of antimicrobial defensinsas seen in the immunofluorescence images. Recently, it was shown that rectal mucinreversibly binds and retains several antimicrobial peptides, including human �-defensin1 and cathelicidin, that display antimicrobial activity against Gram-positive bacteria,including Staphylococcus aureus, and Gram-negative bacteria, including Bacteroidesfragilis and Escherichia coli, and the yeast Candida albicans (36). Alternatively, MUC2mucin may promote the induction of cathelicidins through the activation of Toll-likereceptors (TLRs). LL-37 was shown to be upregulated via stimulation of TLR-2, TLR-4,and TLR-9 in monocyte-derived macrophages (37). It is also possible that MUC2 andcathelicidins might establish certain types of physical interactions; LL-37 was shown tobind O-glycosylated MUC2 membrane mucin (38). However, at the functional level,MUC2 may reduce the antimicrobial effect of cathelicidins. We have recently shownthat MUC2 mucin inhibits the in vitro antimicrobial effect of human �-defensin 2against enteropathogenic and nonpathogenic E. coli (21). Even though MUC2 regulatesthe expression and function of host defense peptides, including cathelicidins and�-defensins in the colon (21), cathelicidins may also promote the synthesis of colonicmucin. Intrarectal administration of synthetic cathelin-related peptide or mCRAMP-

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expressing plasmid ameliorated DSS-induced colitis in mice genetically deficient incathelicidin by preserving mucus thickness and inducing the expression of Muc1, Muc3,Muc4, and, in particular, Muc2 mucin genes (29, 39). Further studies showed that LL-37stimulated mucus synthesis and the expression of MUC1 and MUC2 via the MAPKpathway (40, 41).

The complex interplay between innate host defense molecules at the mucosalsurface is an emerging field, and we are now beginning to appreciate significantcooperation between these molecules to exert their protective functions. The intestinalepithelium is sheltered by MUC2 mucin and commensal bacteria that release a varietyof compounds, including butyrate, that limit exposure to threats to the epithelium. Ourstudies clearly show that intracellular and exogenous MUC2 mucin and butyrate arecritical to promote the synthesis of cathelicidins and their response to infection andchemically induced colitis. Thus, defects in mucin biosynthesis or alterations in themucus barrier accompanied by deficient production of butyrate predispose to adeficient cathelicidin response and intestinal defenses against enteropathogenic bac-teria.

MATERIALS AND METHODSEntamoeba histolytica. E. histolytica (HM-1:IMSS) was grown axenically in TYI-S-3 medium with 100

U ml�1 penicillin and 100 �g/ml streptomycin sulfate at 37°C in sealed 15-ml borosilicate glass tubes andregularly passaged through gerbil livers to maintain high virulence (32). Trophozoites were harvestedduring the logarithmic growth phase (48 to 72 h) by being chilled on ice for 10 min and pelleted bycentrifugation at 200 � g (5 min at 4°C).

Purification of human colonic mucin. Secreted MUC2 mucin was purified from human LS174Tgoblet cells as previously described (21, 32). We used secreted rather than intracellular MUC2 mucinbecause it is native polymeric mucin and best represents the physiological mucus layer. LS174T cellsconstitutively express and secrete high levels of MUC2 mRNA (42), have low expression of MUC1 with noMUC3 or MUC4 (43), and secrete high-density hexose-rich fractions rich in serine, threonine, proline,N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), and galactose (Gal) typical of nativesecreted human colonic mucin (32). In brief, cell-free supernatants derived from 80% confluent cells werecentrifuged (300 � g) for 5 min at 4°C, dialyzed against deionized water (12,000- to 14,000-molecular-weight cutoff [MWCO]; SpectraPor; Spectra Labs), and lyophilized. Crude lyophilized mucus (100 mg) wasdissolved in Tris-HCl buffer (0.01 M) containing 0.001% sodium azide (pH 8.0; Sigma) and fractionated(100 mg/column) on an equilibrated Sepharose 4B gel filtration column (2.5 by 50 cm; Bio-Rad) (columnflow rate, 12 ml/h). High-molecular-weight mucin fractions in the void volume (V0) (fractions 25 to 30)were pooled, dialyzed against deionized water (4°C), and concentrated (SpinX UF, 10,000 MW; Corning).The V0 mucin (2 mg) were digested with DNase (1 mg) and RNase (2 mg) and subjected to CsCl densitygradient ultracentrifugation (starting density, 1.41 g/ml) to purify MUC2 (�1.46 g/ml). Fractions were runon a 1.2% agarose gel under reducing conditions and Western blotted with affinity-purified rabbit IgGanti-CsCl purified LS174T mucin antibody (32). To validate the presence of MUC2 only, purified MUC2CsCl fractions (1 �g) were run on a 1.2% agarose gel and Western blotted with affinity-purified rabbitpolyclonal anti-MUC2, mouse monoclonal anti-MUC5AC (Santa Cruz), and goat polyclonal anti-MUC5B(Santa Cruz) antibodies (21). Protein concentration was estimated using the bicinchoninic acid (BCA)protein assay (Pierce, Thermo Scientific) (21).

Human colonic epithelial cells. The parental human adenocarcinoma epithelial cell line HT-29was used. This cell line does not produce substantial amounts of MUC2 but produces cathelicidinsunder inductive stimuli (4). HT-29 cells were maintained in Dulbecco’s modified Eagle’s medium(DMEM) (catalog no. 12430; Gibco) with 10% fetal bovine serum (BenchMark; Gemini), sodiumpyruvate (1 mM) (catalog no. 11360; Gibco), penicillin (100 U/ml), and streptomycin (100 �g/ml;HyClone Thermo) in a humidified environment of 95% air and 5% CO2. To determine if MUC2 couldalter the synthesis of cathelicidins, we used a high-mucin-producing clone (44) of HT-29 (clone HT-29C16) obtained by prolonged treatments with butyrate designated MUC2 H. In addition, we used aderivate clone from MUC2 H silenced by lentivirus short hairpin RNA (shRNA) designated MUC2 L aspreviously described (21). Briefly, hairpin MUC2 shRNA sequences comprising a 21-bp stem and a6-bp loop were cloned into pLKO.1 viral vectors and cotransfected into HEK293T packaging cellsalong with an envelope and a packaging plasmid (Invitrogen). Viral particles containing thetransfected shRNA plasmid were collected, concentrated by ultracentrifugation, and titrated. Toachieve maximal MUC2 silencing, the viral particles were transduced/infected in colonic epithelialcells in the presence of Polybrene. The transduced cells were then selected with puromycin. Usingthis procedure, the virus integrated into the host genome along with the MUC2 shRNA, and only celllines with low MUC2 expression (MUC2 L) were generated. MUC2 shRNA-silenced MUC2 L cellsshowed low MUC2 gene expression (less than 10%) with no significant compensatory effects on thelow basal levels of MUC5AC and MUC5B (21).

Colonic epithelial cells were grown in tissue culture plates with 12 or 24 wells (Corning Costar) until80% confluent, washed twice with phosphate-buffered saline (PBS), and stimulated with the followingagonists: butyrate (catalog no. B5887; Sigma) for up to 72 h with replenishment every 24 h with the same

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medium; IL-1� (catalog no. 200-01A; Peprotech), forskolin (FSK) (catalog no. 3828; Cell Signaling) (10 �M),or phorbol 12-myristate 13-acetate (PMA) (catalog no. P1585; Sigma) for 3 h; or purified MUC2 mucin (upto 10 �g/ml) or E. histolytica trophozoites (1 � 104/ml) for 1 h. In replicate experiments, HT-29 cells werepreincubated with the MAPK kinase inhibitor (MAPK/ERK kinase; PD98059; 25 �M for 1 h). This MAPK/ERKinhibitor specifically inhibits binding to the ERK-specific MAPK MEK, therefore preventing phosphoryla-tion of ERK1/2 (p44/p42 MAPK) by MEK1/2. In addition, cells were preincubated with a morpholine-containing chemical inhibitor of phosphoinositide 3-kinases (PI3Ks; LY294002) (50 �M; 1 h) dissolved indimethyl sulfoxide (DMSO).

Mice. All studies were approved by the University of Calgary Animal Care Committee. Male 10- to12-week-old C57BL/6 wild-type mice from Charles River and Muc2�/� (44) mice of the same geneticbackground were bred in-house, and F1 Muc2�/� mice were backcrossed to generate F2 Muc2�/�,Muc2�/�, and Muc2�/� littermates. Mice were maintained in sterilized and filter-top cages underspecific-pathogen-free conditions and provided food and water ad libitum.

Models of colitis. Colonic loops were used as a model for short acute infection with E. histolytica inmice (33). Animals were subjected to fasting overnight and anesthetized by intraperitoneal injection withsodium pentobarbital dissolved in sterile water (4.2 mg/kg of body weight) (Ceva Santé Animale). Alaparotomy was performed to exteriorize the large intestine, and a loop (3 cm) was made at the proximalportion of the colon immediately after the cecum by two ligations with 3.0 black silk sutures (Ethicon Inc.,Peterborough, Ontario, Canada). E. histolytica trophozoites (106) in log phase suspended in 100 �lphosphate-buffered saline (PBS), pH 7.3, were inoculated into the loop. Care was taken to keep themesenteries, blood vessels, and nerves intact. Mice were maintained under surveillance for up to 4 h andthen sacrificed by cervical dislocation.

To investigate the role of Muc2 and cathelicidins in chronic colitis, dextran sulfate sodium (DSS) (MPBiochemicals; 36,000 to 50,000 MW) dissolved in drinking tap water was given to Muc2�/� mice (3% DSS)for five consecutive days or Muc2�/� mice (1% DSS) for 3 days (12). This differential DSS treatment wasnecessary to obtain similar histological damage scores between Muc2�/� and Muc2�/� mice (12). Micewere then maintained on tap water and sacrificed by cervical dislocation on days 7 and 15 (day 0 is thebeginning of the DSS treatment). The amounts of water drunk and weight were examined daily, and thedisease activity index (DAI) was calculated based on weight loss, stool consistency, blood loss, andappearance. In all mice, the entire colon was externalized aseptically and fresh samples up to 2 cm indiameter were homogenized in liquid nitrogen for quantitative PCR (qPCR) studies. In parallel, fresh colontissue was fixed in Carnoy’s fixative (60% ethanol 100%, 30% chloroform, 10% glacial acetic acid) for 3h to preserve the mucus layers, transferred to ethanol 100% at 4°C, and then embedded in paraffin blocksfor confocal immunofluorescence studies.

Expression of cathelicidin and MUC2 mRNA in human colonic epithelial cells and murine colon.MUC2 and cathelicidin (LL-37 from human colonic epithelial cells and Cramp from murine colonicmucosa) gene expression was examined by quantitative real-time PCR (qRT-PCR). Total RNA wasextracted from human goblet cells and homogenized frozen murine colon by TRIzol reagent (Invitrogen),and RNA purity and yield were assessed with a spectrophotometer (NanoDrop; Thermo Scientific). cDNAwas prepared from 1 �g of total RNA using Moloney murine leukemia virus (MMLV) reverse transcriptase(catalog no. 95048; qScript cDNA SuperMix; Quanta Biosciences). Absence of contaminating genomicDNA from RNA preparations was checked using a minus-reverse-transcriptase control (i.e., a sample withall the RT-PCR reagents except reverse transcriptase). Reactions were performed in a Rotor-Gene 3000real-time PCR system (Corbett Research). Each reaction mixture (total, 25 �l) contained 100 ng of cDNA(10 �l), 2� Rotor-Gene SYBR green PCR master mix (catalog no. 204072; Qiagen) (12.5 �l), and 1 �Mprimers (2.5 �l). The primers used were as follows: for human LL-37, CAMP, PPH09430A, GenBankaccession no. NM_004345.3; MUC2, forward, 5=-CAGCACCGATTGCTGAGTTG-3=, and reverse, 5=-GCTGGTCATCTCAATGGCAG-3=; and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH), sense, 5=-TGCACCACCAACTGCTTAGC-3=, and antisense, 5=-GGCATGGACTGTGGTCATGAG-3=. For mouse, we used Cramp(Camp; PPM25023A; GenBank accession no. NM_009921.2; RT2 qPCR primer assay [Qiagen]) and murineactin (sense, 5=-CTACAATGAGCTGCGTGTG-3=; antisense, 5=-TGGGGTGTTGAAGGTCTC-3=). The primersused (RT2 qPCR primer assay sourced from Qiagen) were experimentally verified for specificity andefficiency. Amplification of a single product of the correct size with SYBR green was guaranteed to havehigh PCR efficiency (�90%). The reaction mixtures were incubated for 95°C for 5 min, followed bydenaturation for 5 s at 95°C and combined annealing/extension for 10 s at 60°C for a total of 40 cycles.Target mRNA values were corrected relative to the housekeeping genes coding for human GAPDH andmurine actin, respectively. Two housekeeping genes, GAPDH and �-actin, were initially tested, andbecause both showed no alterations under our treatments, we proceeded using the former gene. Allexperiments were done in triplicates (i.e., on three different independent occasions, with independentsamples and treatments), and in each experiment, treatments were done in triplicates. Data wereanalyzed using the threshold cycle (2�ΔΔCT) method and expressed as fold change (mean � standarderror [SE]). For comparison of MUC2 H with MUC2 L, untreated cells from the same type were used ascontrols. Likewise, data from nonchallenged Muc2�/� mice served as the control for Muc2�/� challengedwith E. histolytica while nonchallenged Muc2�/� mice served as the control for Muc2�/� mice challengedwith E. histolytica.

Confocal immunofluorescence microscopy. Location of cathelicidin CRAMP was studied in thecolon of Muc2�/� and Muc2�/� mice, and that of cathelicidin LL-37 was studied in colonic epithelial cells.Colonic tissues fixed in Carnoy’s fixative were sectioned (7 �m) and deparaffinized with a xylenesubstitute (Neo-Clear; Millipore), followed by decreasing concentrations of ethanol and running tapwater (3 min each). Slides were boiled in 10 mM sodium citrate (catalog no. S4641; Sigma) plus 0.05%

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Tween 20 (pH 6) for 20 min in a microwave oven for antigen retrieval and then cooled at roomtemperature (RT) and rinsed in PBS. Free aldehydes were blocked by 0.1 M glycine in PBS for 10 min atRT. Human colonic epithelial cells were grown in 12-well plates and pretreated with butyrate (4 mM; 72h) and purified MUC2 mucin (20 �g/ml), IL-1� (20 ng/ml), or forskolin (10 �M) or exposed to E. histolytica(1 � 104 trophozoites/ml) for 30 min. Cells were rinsed with PBS and fixed in 4% paraformaldehyde with150 mM NaCl, 4 mM NaH2PO4, and 5 mM KCl (pH 7.4) for 10 min at RT. Sections were rinsed in cold PBSplus Tween 0.05% (pH 7.2) (PBS-Tw), permeabilized with PBS-Tw plus 0.25% Triton X-100 for 10 min atRT, and rinsed in cold PBS-Tw. Histologic sections from murine colon and human colonic cells wereblocked with PBS-Tw, 1% bovine serum albumin (BSA), 10% goat serum, 0.3 M glycine, and 0.05%saponin for 2 h at RT and rinsed with PBS-Tw. Sections from murine colon were exposed to affinity-purified rabbit polyclonal IgG anti-mouse Cramp antibodies (Ab74868; Abcam), and HT-29 cells wereexposed to monoclonal murine IgG anti-human LL-37 (Ab87701, OSX12; Abcam) and affinity-purifiedgoat IgG anti-human MUC2 (Santa Cruz) antibodies. Primary antibodies were diluted 1:100 in PBS-Twwith 0.05% saponin and incubated at 4°C overnight in a humid chamber. As secondary antibodies, HT-29cells were blotted with DyLight 647 affinity-purified donkey anti-mouse IgG F(ab=)2 fragment-specificantibodies and DyLight 594 affinity-purified donkey anti-goat IgG F(ab=)2 fragment-specific antibodies,and murine tissues were blotted with DyLight 647 affinity-purified donkey anti-rabbit IgG F(ab=)2

fragment-specific antibodies. Secondary antibodies were diluted 1:1,000 in PBS-Tw plus 1% bovine serumalbumin and incubated for 1 h at RT. Sections were rinsed in cold PBS-Tw, and nuclei were counter-stained with 4=,6-diamidino-2-phenylindole (DAPI). Sections were rinsed with cold PBS-Tw and mountedwith FluorSave reagent (Calbiochem). Slides were examined using a FluoView FV1000 confocal immu-nofluorescence microscope (Olympus).

To define the interaction between cathelicidin peptides and E. histolytica, confocal microscopystudies were conducted on E. histolytica (3 � 105 trophozoites) previously fixed with glutaraldehyde (1%)incubated with synthetic cathelicidin LL-37 (5 �M) for 1 h in a 100-�l reaction mixture in microtubes.Trophozoites were rinsed in cold PBS-Tw, permeabilized with PBS-Tw plus 0.25% Triton X-100 for 10 minat RT, and rinsed in cold PBS-Tw. Cells were blocked with PBS-Tw, 1% bovine serum albumin (BSA), 10%goat serum, 0.3 M glycine, and 0.05% saponin for 2 h at RT and rinsed with PBS-Tw. Then, cells wereexposed to monoclonal murine IgG anti-LL-37 antibodies (OSX12; Novus Bio) (1:100 in PBS-Tw) andincubated at RT for 2 h in a humid chamber. As secondary antibodies, cells were blotted with DyLight594 affinity-purified donkey anti-mouse IgG F(ab=)2 fragment-specific antibodies (1:1,000 in PBS-Tw plus1% bovine serum albumin) and incubated for 1 h at RT. Sections were rinsed with cold PBS-Tw andmounted with FluorSave reagent (Calbiochem). Slides were examined using a FluoView FV1000 confocalimmunofluorescence microscope (Olympus). Quantitative statistical comparison of staining fluorescenceintensity between groups was performed using Image Processing and Analysis in Java (Image J; NIH).Image files were processed as TIFF file formats, and cell areas were selected to determine fluorescenceindex. Areas of the image with similar numbers of cells and uniform background were randomly selected.Data represented in a histogram are means and standard errors of the means from at least three imagesfrom independent experiments.

Statistical analysis. Results were normally distributed and reported as means and standard errors ofthe means from at least three independent experiments. Statistical difference comparisons were con-ducted between treated and untreated/control groups. Data from qPCR and fluorescence intensity wereanalyzed by a nonpaired two-tailed Student t test or analysis of variance (ANOVA) versus the control,followed by a post hoc Bonferroni posttest (GraphPad Prism 5.0 Mac; GraphPad Software). Graphsrepresent two to three independent experiments, and error bars represent means � standard errors.Differences in values were considered significant when P was �0.05.

ACKNOWLEDGMENTSThis work was supported by a grant from the Canadian Institute of Health Research

(CIHR) to K.C. E.R.C. was supported by an Alberta Innovates-Health Solutions FellowshipAward. The Live Cell Imaging Facility is funded by an equipment and infrastructuregrant from the Canadian Foundation Innovation (CFI) and the Alberta Science andResearch Authority.

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