Retinoic acid suppresses intestinal mucus production and ...numbers of goblet cells and reduced...

INTRODUCTIONAltered goblet cell physiology is a hallmark of inflammatory boweldisease (IBD) pathology (Kaser et al., 2010). Individuals withCrohn’s disease (CD) or ulcerative colitis (UC) have decreasednumbers of goblet cells and reduced mucus thickness atpresentation; however, goblet cells are replaced during activeinflammation in individuals with CD but not in those with UC,suggesting an etiological role for goblet cell abnormalities in UC(Trabucchi et al., 1986; Gersemann et al., 2009; Zheng et al., 2011).The most commonly used chemically induced animal models ofIBD utilize dextran sodium sulfate (DSS) or trinitrobenzene sulfonicacid (TNBS) to induce colitis (Wirtz et al., 2007). These chemicallyinduced models share a conspicuous loss of goblet cells with agenetic model of spontaneous colitis, the Winnie mouse,demonstrating a conserved role for mucin production inmaintaining intestinal barrier function (Heazlewood et al., 2008;Eri et al., 2011).

Treatment with retinoids increases the risk of developing IBDin humans (Crockett et al., 2010). The best-explored hypothesisto explain this effect is that retinoic acid (RA) in the intestineaffects the production of regulatory dendritic and T cells, leadingto immune dysregulation (Eksteen et al., 2009; Iliev et al., 2009;Westendorf et al., 2009). Administration of exogenous RA is

known to stimulate the release of mucin in cells of the upperairways and the addition of vitamin A to growing rats inhibitsthe differentiation of intestinal goblet cells (Gadzhieva and Kon,1984). However, to our knowledge, the effects of exogenous RAon intestinal mucus secretion in response to inflammation havenot been reported.

Intestinal development and physiology is highly conservedbetween mammals and zebrafish. Larval zebrafish intestinalphysiology is amenable to relatively simple chemical-geneticinterrogation by immersion of larvae in solutions of drugs andmicroinjection of morpholinos (modified oligonucleotides to knockdown gene function) (Hama et al., 2009). Of importance to themodeling of IBD, the antimicrobial roles of NOD1 and NOD2 areconserved in zebrafish (Oehlers et al., 2011c; Oehlers et al., 2011b),as are many aspects of the host response to microbial colonization,including intestinal alkaline phosphatase expression and NFBactivation (Bates et al., 2007; Kanther et al., 2011). Furthermore,zebrafish models of IBD-like enterocolitis have been recentlydescribed (Brugman et al., 2009; Fleming et al., 2010; Oehlers etal., 2011a). Because zebrafish adaptive immunity is only functionalafter 4-6 weeks of development (Lam et al., 2004), larvae in thefirst week of development were used to investigate the effects ofexogenous RA on intestinal physiology in the absence of adaptiveimmunity.

RESULTSDSS-induced enterocolitis is similar, but not identical, to TNBS-induced enterocolitisA range of DSS doses were initially investigated to determine themaximum non-lethal dose that could be continuously tolerated bylarvae at 3 days postfertilization (dpf). A dose of 0.5% (w/v) wasestablished to be the highest concentration that did not causesignificant mortality (data not shown). DSS-exposed larvae at 6 dpf

Disease Models & Mechanisms 457

Disease Models & Mechanisms 5, 457-467 (2012) doi:10.1242/dmm.009365

1Department of Molecular Medicine and Pathology, School of Medical Sciences,The University of Auckland, Auckland 1142, New Zealand*Author for correspondence ([email protected])

Received 7 December 2011; Accepted 15 March 2012

© 2012. Published by The Company of Biologists LtdThis is an Open Access article distributed under the terms of the Creative Commons AttributionNon-Commercial Share Alike License (http://creativecommons.org/licenses/by-nc-sa/3.0), whichpermits unrestricted non-commercial use, distribution and reproduction in any medium providedthat the original work is properly cited and all further distributions of the work or adaptation aresubject to the same Creative Commons License terms.

SUMMARY

Exposure to retinoids for the treatment of acne has been linked to the etiology of inflammatory bowel disease (IBD). The intestinal mucus layer isan important structural barrier that is disrupted in IBD. Retinoid-induced alteration of mucus physiology has been postulated as a mechanism linkingretinoid treatment to IBD; however, there is little direct evidence for this interaction. The zebrafish larva is an emerging model system for investigatingthe pathogenesis of IBD. Importantly, this system allows components of the innate immune system, including mucus physiology, to be studied inisolation from the adaptive immune system. This study reports the characterization of a novel zebrafish larval model of IBD-like enterocolitis inducedby exposure to dextran sodium sulfate (DSS). The DSS-induced enterocolitis model was found to recapitulate several aspects of the zebrafishtrinitrobenzene-sulfonic-acid (TNBS)-induced enterocolitis model, including neutrophilic inflammation that was microbiota-dependent andresponsive to pharmacological intervention. Furthermore, the DSS-induced enterocolitis model was found to be a tractable model of stress-inducedmucus production and was subsequently used to identify a role for retinoic acid (RA) in suppressing both physiological and pathological intestinalmucin production. Suppression of mucin production by RA increased the susceptibility of zebrafish larvae to enterocolitis when challenged withenterocolitic agents. This study illustrates a direct effect of retinoid administration on intestinal mucus physiology and, subsequently, on the progressionof intestinal inflammation.

Retinoic acid suppresses intestinal mucus productionand exacerbates experimental enterocolitisStefan H. Oehlers1, Maria Vega Flores1, Christopher J. Hall1, Kathryn E. Crosier1 and Philip S. Crosier1,*

RESEARCH ARTICLED

iseas

e M

odel

s & M

echa

nism

s

DM

M

manifested liver discoloration reminiscent of TNBS-exposed larvaebut otherwise appeared morphologically normal (Fig. 1A).

Enumeration of bacteria from whole larval homogenates revealeda significant (P<0.0001, Student’s t-test, n7) increase in culturablebacterial load in DSS-, but not TNBS-, treated larvae comparedwith control larvae (Fig. 1B).

A leukocyte response that can be monitored visually has beenrecognized as a useful readout of inflammation in neutrophilicinflammation models (Hall et al., 2009; d’Alencon et al., 2010;Oehlers et al., 2011a). The location and number of neutrophils inDSS-exposed larvae was characterized by live imaging of 6-dpfTg(mpx:EGFP)i114 larvae and by fluorescence activated cell sorting(FACS). Exposure to DSS resulted in a microbiota-dependentdistribution of neutrophils away from the caudal hematopoietictissue towards the intestine and epidermis, and an overall increase

in neutrophil numbers, reminiscent of TNBS-induced enterocolitis(Fig. 1C). Consistent with the previously described TNBS-inducedenterocolitis model, the DSS-induced enterocolitis protocol wasstandardized to include addition of DSS at 3 dpf and assay ofoutcome at 6 dpf (3 days post-exposure).

Zebrafish larvae produce nitric oxide in response toinflammatory stress, and this can be live-imaged in larvae incubatedwith a 4-amino-5-methylamino-2�,7�-difluorofluorescein diacetate(DAF-FM-DA) probe (Grimes et al., 2006; Lepiller et al., 2007).Larvae exposed to DSS manifested identical patterns and intensityof nitric oxide to control larvae, whereas TNBS exposure inducedexcess nitric oxide production in the region of the cleithrum andnotochord (Fig. 1D). To confirm the fidelity of the nitric oxidereadout of TNBS-induced inflammation, nitric oxide productionwas assessed in TNBS-exposed larvae in which inflammation had

dmm.biologists.org458

Retinoic acid, colitis and intestinal mucusRESEARCH ARTICLE

Fig. 1. DSS exposure causes a distinct enterocolitis in zebrafish larvae when compared with TNBS exposure. (A)Whole-mount live imaging of control, DSS-and TNBS-exposed larvae. Red arrows indicate liver. (B)Comparison of recovered microbiota from homogenates of control, DSS- and TNBS-exposed larvae (n7).(C)Characterization of neutrophilic inflammation in zebrafish larvae by: (i) live imaging of Tg(mpx:EGFP)i114 larvae; (ii) enumeration of neutrophils by FACS (n5);and (iii) enumeration of intestinal neutrophils (n≥30 per group; three biological replicates). (D)Comparison of DAF-FM-DA staining in wild-type larvae treated asindicated. Red arrow indicates heart, blue arrow indicates cleithrum and yellow arrow indicates notochord. (E)Quantitative PCR analysis of pro-inflammatorycytokine and proliferating cell nuclear antigen gene expression in untreated and DSS-treated 6 dpf larvae (n4). (F)Characterization of cellular proliferation inzebrafish larvae by: (i) live imaging of resazurin-stained larvae; (ii) quantification of fluorescence from live imaging (n19); and (iii) manual counting of BrdU-positive cells in the gut and trunk (n7). White arrowhead indicates otic vesicle and blue arrowhead indicates intestine in live images. Error bars indicate s.e.m.;***P<0.0001, **P<0.01 and *P<0.05 as determined by ANOVA (B,C) or Student’s t-tests (E,F). Scale bars: 1 mm.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

been modified by the administration of antibiotics and anti-inflammatory drugs (Oehlers et al., 2011a). The production of nitricoxide in these larvae was identical to control samples(supplementary material Fig. S1). Interestingly, the production ofnitric oxide was increased in larvae treated with the non-steroidalanti-inflammatory drug 5-aminosalicylic acid. This observationsuggests that administration of this drug alone induces a nitric oxideresponse in zebrafish larvae while not affecting other measures ofinflammation (Oehlers et al., 2011a).

To examine other measures of inflammation, quantitative PCR(qPCR) was undertaken. Exposure to DSS caused an appreciableupregulation of ccl20, il1b, il23, il8, mmp9 and tnfa transcription(Fig. 1E). Interestingly, the analysis of proliferating cell nuclearantigen (pcna) transcription revealed significantly reducedtranscription of this marker of cellular proliferation (P<0.01).

Further analysis of cellular proliferation was performed byadapting the resazurin reduction assay to enable fluorescent whole-mount live quantification, with confirmation bybromodeoxyuridine (BrdU) labeling of proliferative cells. Resazurinstaining was most prominent in the otic vesicle and gut of 6-dpflarvae (Fig. 1F). Comparison of fluorescence from control and DSS-exposed samples indicated reduced proliferation in DSS-exposedsamples. This observation was confirmed by manual counting ofBrdU-positive cells in the gut and trunk of control and DSS-exposedsamples. There was no evidence of induction of cell death, asassessed by acridine orange or terminal deoxynucleotidyltransferase-mediated deoxyuridinetriphosphate nick end-labeling(TUNEL) staining (data not shown).

DSS induces the accumulation of acidic mucins in the intestinalbulbTo examine changes to intestinal goblet cells in the DSS exposuremodel, alcian blue staining for acidic mucins was performed onwhole-mount specimens. A striking feature of DSS-exposedsamples was the appearance of alcian blue staining in the intestinalbulb (Fig. 2A). We termed this accumulation of acidic mucins inthe intestinal bulb the ‘mucosecretory phenotype’. Sectioningrevealed the stained substance to be in the lumen of the intestinalbulb, with no evidence of staining in intestinal bulb epithelial cells(Fig. 2B). Further examination of transverse intestinal sections didnot reveal overt histological changes at 6 dpf (Fig. 2C,D).

Because there was no evidence to suggest transdifferentiation ofintestinal bulb cells and a similar number of alcian-blue-stained gobletcells were observed in the mid intestine of untreated, DSS- andTNBS-exposed larvae at 6 dpf (Fig. 2E), it was hypothesized that thestained substance was mucus produced by esophageal goblet cells.However, examination of alcian-blue-stained transverse esophagealsections did not reveal any changes in alcian blue staining within theesophageal goblet cells or in the thickness of mucus lining theesophageal–intestinal-bulb junction of DSS-exposed larvae whencompared with controls (supplementary material Fig. S2).

To examine the effect of DSS or TNBS treatment on mucin geneexpression, quantitative PCR analysis of muc (a putative zebrafishortholog of the human esophagus-expressed MUC5 gene family)expression was carried out on control, DSS- and TNBS-exposedlarvae. Exposure to TNBS, but not to DSS, increased the expressionof muc (Fig. 2F).


Retinoic acid, colitis and intestinal mucus RESEARCH ARTICLE

Fig. 2. DSS exposure induces a mucosecretory phenotype in zebrafish larvae. (A)Whole-mount control and DSS-exposed larvae stained with alcian blue.Blue arrows indicate intestinal bulb staining, red arrows indicate cartilage staining. Scale bar: 1 mm. (B)Longitudinal sections of intestinal bulb from control andDSS-exposed larvae, stained with alcian blue. Scale bar: 50m. (C)Longitudinal sections of the anterior-mid intestinal junction at the posterior edge of the swimbladder from control and DSS-exposed larvae, stained with alcian blue. Scale bar: 50m. (D)Longitudinal sections of the mid-distal intestinal junction fromcontrol and DSS-exposed larvae, stained with alcian blue. Scale bar: 50m. (E)Enumeration of mid intestinal goblet cells by manual counting (n≥28 per group;two biological replicates). (F)Quantitative PCR analysis of muc expression in control, DSS- and TNBS-exposed larvae (n4). (G; left) Live image of 6-dpfTg(kita:GAL4, UAS:EGFP) larvae; fin fold was defined as epidermis immediately ventral to the intestine. Scale bar: 1 mm. (G; right) Enumeration of GFP-expressingskin goblet cells on the fin fold of individual larvae (n≥12 per group; two biological replicates). Error bars represent s.e.m.; *P<0.05, ***P<0.0001 as determined byANOVA.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

To determine whether DSS-induced changes in goblet cellfunction were restricted to the esophagus, the fin fold mucusproducing cells marked in Tg(kita:GAL4, UAS:EGFP) larvae wereenumerated by live imaging (Feng et al., 2010; Santoriello et al.,2010). Exposure to DSS, but not to TNBS, was found to cause asmall but statistically significant increase in the number of fin foldmucus-secreting cells (Fig. 2G).

Because Kit expression has been observed in zebrafish mast cells(Dobson et al., 2008), crosses with the Tg(lyzC:dsRed)nz50 line wereperformed to determine whether the Tg(kita:GAL4-EGFP)construct drives expression in mast cells, because expression of lyzChas also been observed in zebrafish mast cells (Hall et al., 2007;Dobson et al., 2008). Live imaging of untreated and TNBS-challenged compound transgenic larvae by confocal microscopyfailed to detect any transgene coexpression, indicating that thekita:GAL4-EGFP double transgenic line does not label mast cells(supplementary material Fig. S3).

To investigate the possibility that DSS-induced mucusaccumulation in the intestinal bulb is caused by reduced peristalsis,peristalsis in untreated, DSS- and TNBS-exposed larvae wasobserved. However, the rate of peristalsis did not differ betweencontrol (mean 3.577, n26) and DSS-exposed (mean 3.448, n29)larvae (95% CI of difference –0.6085 to 0.8658, determined byANOVA), or between control and TNBS-exposed (mean 3.571,n21) larvae (95% CI of difference –0.7953 to 0.8063, determinedby ANOVA).

The mucosecretory phenotype is microbiota-dependent butindependent of neutrophilic inflammationTo investigate the role of inflammation in the mucosecretoryphenotype, larvae were co-treated with DSS and the anti-

inflammatory glucocorticoid dexamethasone to suppressinflammation. Dexamethasone co-treatment at a dose of 50 g/mlprevented DSS-induced neutrophilic inflammation inTg(mpx:EGFP)i114 larvae (Fig. 3A). However, dexamethasonetreatment had no effect on the mucosecretory phenotype (Fig. 3B).These data suggested that the DSS-induced mucosecretoryphenotype was not dependent on inflammatory signaling.

The role of the microbiota as a trigger for the mucosecretoryphenotype was investigated by co-administration of broad-spectrum antibiotics with DSS (Oehlers et al., 2011a). Depletionof the microbiota prevented the appearance of the DSS-inducedmucosecretory phenotype (Fig. 3C).

DSS exposure is protective against further chemical-inducedenterocolitisBecause increased mucin secretion protects against TNBS-inducedcolitis in mice (Krimi et al., 2008), it was hypothesized that DSS-induced intestinal bulb mucin accumulation might protect larvaeagainst TNBS challenge. To control for possible direct interactionbetween DSS and TNBS, exposure to 0.25% dextran was used asan additional control for this experiment (Fig. 4A). Larvae that werecoexposed to DSS and TNBS had less neutrophilic inflammationthan larvae exposed to dextran and TNBS or TNBS alone (Fig.4B,C).

To complement this experiment, larvae were exposed to eitherdextran or DSS for 3 days and then challenged with a high doseof TNBS (Fig. 4D). Analysis of larval survival and neutrophilicinflammation following high-dose TNBS treatment confirmed a protective effect of DSS pretreatment against TNBS-induced mortality and neutrophilic inflammation, respectively(Fig. 4E,F).



Fig. 3. The mucosecretoryphenotype is microbiota-dependent but independent ofneutrophilic inflammation. (A)Liveimaging of neutrophil distribution inTg(mpx:EGFP)i114 larvae co-treatedwith dexamethasone and DSS, andenumeration of intestinalneutrophils (n≥23 per group; twobiological replicates). Scale bar: 1mm. Error bars represent s.e.m.;***P<0.0001 as determined byANOVA. (B)Whole-mount larvae co-treated with dexamethasone andDSS, stained with alcian blue. Bluearrows indicate intestinal bulbstaining; red arrows indicatecartilage staining. Scale bar: 1 mm.(C)Longitudinal sections of intestinalbulb from larvae co-treated with acocktail of broad-spectrumantibiotics and DSS, stained withalcian blue. Scale bar: 50m.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

Retinoic acid suppresses the mucosecretory phenotypeRA plays an important role in the epithelial transformationassociated with Barrett’s esophagus, whereby esophageal epithelialcells adopt intestinal phenotypes (Chang et al., 2007; Cooke et al.,2008). Preliminary investigations by our group have indicatedexogenous RA can induce in vivo transformation of zebrafishesophageal epithelial cells into intestinal bulb cells (M.V.F.,unpublished data). RA is known to be a conserved modulator ofintestinal epithelial cell differentiation in zebrafish throughactivation of Hoxc8 (Nadauld et al., 2004). Initial experimentssuggested that coexposure to RA could suppress the DSS-inducedmucosecretory phenotype (data not shown). Titration ofmicromolar concentrations of RA established a dose-dependentrelationship between exogenous RA and suppression of the DSS-induced mucosecretory phenotype (Fig. 5A and supplementarymaterial Fig. S4). Co-administration of 1 M RA to 3-dpf larvaewas found to profoundly suppress the DSS-induced mucosecretoryphenotype, while maintaining a normal morphology.

To examine the reversibility of the DSS-induced mucosecretoryphenotype, larvae were exposed to DSS from 3 dpf andadministered 1 M RA at 3, 4 or 5 dpf. DSS-induced mucosecretionwas assayed at 6 dpf, revealing an exposure-duration-dependentrelationship between exogenous RA and suppression of the DSS-induced mucosecretory phenotype (Fig. 5B).

To examine the effect of RA on esophageal muc expression,whole-mount in situ hybridization was carried out. Although the

intestinal bulb staining for muc expression varied widely withinuntreated and RA-treated groups, esophageal expression of mucwas profoundly reduced in larvae treated with RA (Fig. 5C).

Histological examination of alcian-blue-stained esophagealsections of RA co-treated larvae revealed a decrease in theintensity of acidic mucin staining in the anterior esophagus inlarvae that were co-treated with DSS and RA (Fig. 5D).Examination of posterior esophagus sections also revealed astriking RA-induced loss of mucin staining in the pneumatic duct. However, the thickness of the mucus lining theesophageal–intestinal-bulb junction did not seem to be alteredfollowing administration of RA.

To further examine the effects of RA treatment on intestinalglycobiology, isolectin GS-IB4 was used to stain theesophagus–intestinal-bulb junction. The staining pattern ofisolectin GS-IB4 was unchanged following exposure to RA,confirming the results from the examination of alcian-blue-stainedesophageal–intestinal-bulb junction sections (supplementarymaterial Fig. S5A). Furthermore, expression of RFP in theTg(ifabp:RFP)as200 transgenic line and neutral red uptake wasunchanged, indicating normal intestinal bulb and mid intestinalspecification (supplementary material Fig. S5B and S5C,respectively) (Flores et al., 2008; Oehlers et al., 2011a). Together,these data indicate that the dose and exposure parameters of RAused in this study selectively suppressed mucin production byesophageal cells but did not alter esophageal cell fate.



Fig. 4. DSS-induced mucus secretionprotects against further chemical-induced enterocolitis. (A)Schematicdescribing the experimental procedureanalyzed in panels B and C. (B)Liveimaging of neutrophil distribution inTg(mpx:EGFP)i114 larvae co-treated asindicated. Scale bar: 1 mm.(C)Enumeration of intestinal neutrophils(n≥22 per group; two biological replicates).Error bars indicate s.e.m.; ***P<0.0001 asdetermined by ANOVA. (D)Schematicdescribing the experimental procedurereported in panels E and F. (E)Survivalanalysis of larvae treated as indicated from3 dpf and exposed to 75g/ml TNBS from6 dpf (n≥10 per group; six biologicalreplicates). Error bars indicate 95%confidence interval. DSS vs control, DSS vsdextran: P<0.0001 as determined by log-rank test. (F)Enumeration of intestinalneutrophils in larvae exposed to 75g/mlTNBS from 6 dpf and assayed at 24 hourspost-TNBS exposure (n≥12 per group;three biological replicates). Error barsindicate s.e.m.; ***P<0.0001, **P<0.01 asdetermined by ANOVA.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

To determine whether RA administration affected neutrophilicinflammation in the DSS model, live imaging of Tg(mpx:EGFP)i114

larvae was performed (supplementary material Fig. S5D).Administration of RA did not suppress DSS-induced neutrophilicinflammation.

Retinoic acid exposure exacerbates zebrafish enterocolitisBecause TNBS-induced enterocolitis increased the expression ofmuc, it was hypothesized that suppression of muc expression byexogenous RA would exacerbate TNBS-induced enterocolitis. Totest this hypothesis, larvae were coexposed to RA and TNBS from3 dpf (Fig. 6A). Analysis of larval survival revealed a marked

decrease in the survival of larvae coexposed to RA and TNBScompared with larvae exposed to TNBS alone (Fig. 6B).Furthermore, neutrophilic inflammation was increased in larvaethat were coexposed with RA and TNBS compared with controlor mono-exposed larvae (Fig. 6C).

Because pre-exposure to DSS caused mucus secretion thatseemed to protect against TNBS-induced mortality andneutrophilic inflammation, it was hypothesized that suppressionof DSS-induced mucosecretion by exogenous RA would negate theprotective effects of DSS pre-exposure. To test this hypothesis,larvae were coexposed to combinations of RA and DSS from 3 dpfand subsequently challenged with TNBS at 6 dpf (Fig. 6D). Analysisof larval survival following TNBS challenge showed that, althoughcoexposure to RA did not affect the survival of untreated ordextran-exposed larvae, the protective effect of DSS pre-exposurewas suppressed by coexposure with RA (Fig. 6E). Furthermore,coexposure to RA increased neutrophilic inflammation in larvaethat were pre-exposed to DSS compared with larvae that wereexposed to only DSS (Fig. 6F).

DISCUSSIONThis paper presents a novel model of enterocolitis with increasedmucin production. This model adds a useful tool for investigatinginflammatory processes in zebrafish larvae. Furthermore, by usinglarval zebrafish, in which key cell types involved in enterocolitiscan be studied in isolation from the adaptive immune system,exogenous RA has been shown to both suppress intestinal mucinproduction and exacerbate experimental inflammation.

The inflammatory parameters of the DSS-induced enterocolitismodel seem to be largely similar to the TNBS-induced enterocolitismodel (see Table 1). However, the difference in nitric oxideproduction between DSS- and TNBS-exposed larvae is strikingbecause increased nitric oxide production has been previouslyobserved as a core zebrafish inflammatory response (Lepiller et al.,2007). We speculate that the stark difference in the cellularproliferation response following exposure to TNBS (increasedcellular proliferation) and DSS (decreased cellular proliferation) isa manifestation of the opposing stimulatory effects of TNBShaptenization and erosive effects of DSS on epithelial surfaces(Oehlers et al., 2011a). Further study of the zebrafish DSS- andTNBS-induced enterocolitis models will lead to insights intoinnate-immunity-mediated intestinal inflammation.

Comparison of alterations to mucus physiology between animalmodels of intestinal inflammation reveals a striking differencebetween phenotypes, with a reduced number of goblet cellsobserved in adult mice and an increased number of goblet cells oraccumulation of mucus observed in zebrafish larvae exposed to ahigh dose of TNBS or DSS, respectively (see Table 1). From thelimited number of reports using zebrafish models of intestinalinflammation, the dichotomy in responses seems to be a functionof the age of the organism. Whereas goblet cells seem to besusceptible to chemically induced cell death in adult animals, gobletcells in the larval zebrafish intestine seem to both be resistant tochemically induced cell death and increase their function inresponse to chemically induced intestinal damage. We speculatethat the heightened resilience of goblet cells in the developingintestine is due to higher levels of growth factors associated withorganogenesis and maturation.



Fig. 5. Retinoic acid suppresses the DSS-induced mucosecretoryphenotype. (A)Dose-response curve of the percentage of DSS-exposedlarvae positive for the mucosecretory phenotype after the administration of arange of doses of RA (n≥20 per group; two biological replicates). (B)Proportionof DSS-exposed larvae positive or negative for the mucosecretory phenotypeat 6 dpf after administration of RA at the day indicated. Fisher’s exact test P-values vs untreated controls: 3 dpf<0.0001, 4 dpf0.0002, 5 dpf0.0011; n≥20per group, two biological replicates. (C)Whole-mount in situ hybridizationdetection of muc expression in the esophagus in control and RA-treatedlarvae. Blue arrows indicate the location of the esophagus. (D)Alcian-blue-stained esophageal sections of larvae treated with 1M RA and exposed toDSS. Arrows indicate the pneumatic duct. Scale bar: 50m.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

Comparison by electron microscopy of the ultrastructuralmorphology of the intestinal epithelium in chemically inducedenterocolitis zebrafish models is necessary to complete ourunderstanding of these models (Fleming et al., 2010). Furthercomparison between adult and larval zebrafish models couldfurther our understanding of the intestinal response to damage atdifferent stages of development.

From the results of the microbiota depletion experiments, it hasbeen hypothesized that DSS-induced mucin is produced inresponse to bacterial products penetrating the intestinal epithelium

as a result of the erosive effects of DSS (Iger et al., 1994). Twozebrafish mutants with an accumulation of intestinal mucus, stuffedand stuffy, were characterized in a gynogenetic screen (Mohideenet al., 2003). It is possible that these mutants might phenocopyaspects of the DSS-induced mucosecretory phenotype, and furthercharacterization of these mutants could yield insight into intestinalmucosecretory diseases such as cystic-fibrosis-associated smallintestinal bacterial overgrowth (De Lisle et al., 2006).

The recent characterization of Fusobacterium nucleatum-stimulated MUC2 expression in humans (Dharmani et al., 2011)has demonstrated that specific components of the microbiotastimulate intestinal mucus production. Because the increase in totalbacterial load following exposure to DSS could either be a causeor effect of the mucosecretory phenotype, it will be intriguing tostudy the compositional changes to the microbiota of inflamedlarval zebrafish with molecular techniques. Additionally, becausethe larval zebrafish is amenable to gnotobiotic techniques (Phamet al., 2008), it provides a tractable model for the study ofmicrobiota-induced mucin production.

Important limitations of chemically induced zebrafish larvalmodels of intestinal inflammation for the modeling of IBD are theextraintestinal effects of immersion exposure on the epidermis andthe lack of T cell involvement or a chronic disease stage with IBD-like histopathology (see Table 1). Conversely, the zebrafish larvalplatform offers the ability to perform high resolution live imaging,rapid genetic manipulation including the targeting of IBDsusceptibility genes, and a level of experimental complexity andthroughput necessary for performing high content disease modifierscreens that are relevant to IBD. We postulate that the mostimportant factors that influence an IBD model are thatinflammation is microbiota-dependent and responsive to existingIBD therapies.

Quantification of intestinal neutrophilic inflammation bystereomicroscopy as defined in our methodology is an attractivereadout for chemical and/or genetic disease modifier screening. Itis important to acknowledge that this readout is complicated bythe recruitment of neutrophils to the epidermis, including theepidermis of the larval intestinal tract, following exposure to eitherDSS or TNBS. However, as evidenced by our previous work andthis paper, there is a strong correlation between the level ofinflammation [as determined by cytokine expression, neutrophilnumber, nitric oxide production (in the case of TNBS model) andbona fide intestinal epithelium-associated neutrophils (asdetermined by confocal microscopy)] and the number of intestinalneutrophils as defined by our methodology (Oehlers et al., 2011a).

Increased expression of MUC5AC is observed in IBD and otherforms of colonic inflammation (Shaoul et al., 2004; Fyderek et al.,2009). Both the DSS and TNBS exposure models of zebrafishenterocolitis investigated in this report demonstrated increasedmucus production, albeit by different mechanisms. Our finding thatthe increased mucus secretion caused by exposure to DSS protectslarvae from TNBS-induced pathologies suggests a cytoprotectiverole for DSS-induced intestinal mucus accumulation.

Because intestinal mucus is known to be an important scaffoldfor antimicrobial activity in the mammalian intestine (Meyer-Hoffert et al., 2008), it is likely that zebrafish intestinal mucus alsohas an antimicrobial function. The zebrafish larval intestinaldamage models presented in this paper and elsewhere should be



Fig. 6. Retinoic acid exposure exacerbates zebrafish enterocolitis.(A)Schematic describing the experimental procedure reported in panel B.(B)Survival analysis of larvae co-treated with 1M RA and 75g/ml TNBS from3 dpf (n≥20 per replicate; three biological replicates). Error bars indicate 95%confidence interval; P<0.0001 as determined by log-rank test. (C)Enumerationof intestinal neutrophils in larvae co-treated with 1M RA and 25g/ml TNBSfrom 3 dpf (n≥22 per group; three biological replicates). Error bars indicates.e.m.; *P<0.05, ***P<0.0001 as determined by ANOVA. (D)Schematicdescribing the experimental procedure reported in panel E. (E)Survivalanalysis of larvae pre-treated as indicated from 3 dpf and exposed to 75g/mlTNBS at 6 dpf (n≥20 per group; four biological replicates). Error bars indicate95% confidence interval; control vs RA: P0.1815; dextran vs dextran + RA:P0.2272; DSS vs DSS + RA: P<0. 0001 as determined by log-rank test.(F)Enumeration of intestinal neutrophils in larvae pre-treated as indicatedfrom 3 dpf and exposed to 75g/ml TNBS at 6 dpf and assayed at 24 hourspost-TNBS exposure (n≥18 per group; three biological replicates). Error barsindicate s.e.m.; **P<0.01 as determined by ANOVA.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

tractable platforms to explore intestinal mucus function (Oehlerset al., 2011b; Oehlers et al., 2011a).

The association between retinoid treatment for dermatologicalconditions and the development of IBD is stronger for UC than itis for CD (Popescu and Popescu, 2011). Interestingly, UC, but notCD, is characterized by a deficient goblet cell phenotype(Gersemann et al., 2009). Our observations of RA-inducedexacerbation of TNBS-induced enterocolitis, as analyzed by survivaland neutrophilic inflammation, was surprising because RA has beenlargely demonstrated to have an anti-inflammatory effect by alteringdendritic cell function (Eksteen et al., 2009; Iliev et al., 2009;Westendorf et al., 2009). We hypothesize that this unexpected resultis largely due to RA-induced suppression of intestinal mucusproduction rather than an effect on dendritic cells, because few

dendritic cells are present in larval zebrafish (Lugo-Villarino et al.,2010; Wittamer et al., 2011).

Retinoid-containing drugs are primarily prescribed toadolescents for the treatment of acne; however, few studies haveexamined the effect of retinoid treatment on the developingintestine. A sole study on young rats has shown thathypervitaminosis A reduces the number of small intestine gobletcells and induces an inflammatory cell infiltrate (Gadzhieva andKon, 1984). Our study adds to these findings by demonstrating thatexogenous RA suppresses protective DSS-induced mucus secretion,subsequently leading to increased mortality and neutrophilicinflammation upon further enterocolitic challenge. Collectively,these data suggest that retinoids deliver a ‘first hit’ in a multistepetiology by suppressing intestinal MUC expression in adolescents.



Table 1. Comparison of zebrafish models of intestinal inflammation with mouse chemically induced colitis models

Model

Leukocytic

inflammation

Involvement

of adaptive

immunity

Patho-

histological

damage to

intestine

Alterations

to goblet

cells

Can induce

chronic

disease?

Extra-

intestinal

effects

Accessible

to high-

resolution

live

imaging

Availability of

rapid gene

manipulation

tools

Accessibility to

chemical

or genetic

screening

Larval DSS

immersion (this

paper)

Yes; microbiota-

dependent and

responsive to

dexamethasone

No Yes; mucus

accumulation

No

quantitative

change to

mid intestinal

goblet cells

No Yes; liver

discoloration,

epidermal

recruitment

of neutrophils

and increased

number of

skin goblet

cells

Yes Yes Highly accessible

Larval TNBS

immersion

(Fleming et al.,

2010; Oehlers et

al., 2011a)

Yes; microbiota-

dependent and

responsive to anti-

inflammatory

drugs

No Yes; mid

intestinal

shortening,

brush border

erosion and

reduced

vascularization

Variable; no

effect at low

to medium

doses, high

doses

increase

number of

mid intestinal

goblet cells

No Yes; liver

discoloration,

epidermal

recruitment

of neutrophils

and, at high

doses,

epidermal

damage

Yes Yes;

morpholino-

mediated

depletion of

MyD88

Highly accessible

Larval lipopoly-

saccharide (LPS)

immersion

(Bates et al.,

2007)

Yes No Not observed Unknown No Yes; organ

failure and

recruitment

of neutrophils

to liver

Yes Yes;

morpholino-

mediated

depletion of

Intestinal

alkaline

phosphatase,

MyD88 and

Tnfr1

Accessible but

vulnerable to

variable potency

of LPS

Larval bacterial

immersion

(Oehlers et al.,

2010; Oehlers et

al., 2011b)

Not observed No Not observed Unknown No Yes;

epidermal

colonization

Yes Yes;

morpholino-

mediated

depletion of

Nod1 and Nod2

Not readily

accessible; weak

phenotypes

Adult intrarectal

administration

of oxazolone

(Brugman et al.,

2009)

Yes; microbiota-

dependent

Unknown but

possible

Yes; disruption

of epithelial

architecture

Yes; depletion

of goblet cells

No Not observed No No Not readily

accessible

Mouse models

(Wirtz et al.,

2007)

Yes; microbiota-

dependent and

responsive to anti-

inflammatory

drugs

Yes Yes; disruption

of epithelial

architecture

Yes; depletion

of goblet cells

Yes Yes;

considered

secondary to

intestinal

inflammation

No No Not readily

accessible

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

In a subset of already susceptible individuals, reduced mucusproduction and impaired mucus layer restoration could thencombine with other genetic and environmental factors to triggerUC. Further study is warranted to examine the manifestation ofthese effects in patients undergoing treatment with retinoids.

METHODSZebrafish manipulationsZebrafish (Danio rerio) embryos were obtained from naturalspawnings and raised until 1 dpf at 28.5°C in E3 embryo mediumsupplemented with methylene blue. E3 embryo medium used after1 dpf did not contain methylene blue. Induction of enterocolitis byDSS exposure was carried out essentially as described for TNBS(Oehlers et al., 2011a), with the substitution of DSS, or dextran as acontrol (MP Biomedicals), dissolved in E3 embryo medium forsolutions of TNBS. Briefly, 10% (w/v) stock solutions of DSS ordextran were prepared in E3 at room temperature with gentlerocking. All analyses were performed at 6 dpf unless otherwise noted.Dexamethasone (Sigma-Aldrich) was dissolved in dimethyl sulfoxide(DMSO). All-trans RA (Sigma-Aldrich) was dissolved in DMSO anddiluted to working concentrations with water.

ImagingWhole-mount imaging was carried out as previously describedusing a Nikon SMZ1500 stereomicroscope equipped with a DS-U2/L2 camera or a Nikon D-Eclipse C1 confocal microscope(Oehlers et al., 2011a). Neutrophilic inflammation was analyzed as previously described (Oehlers et al., 2011a). Briefly,Tg(mpx:EGFP)i114 larvae were anesthetized in tricaine and mountedin 3% methylcellulose for imaging; ‘intestinal neutrophils’ weredefined as EGFP-expressing cells within the two-dimensionalboundary of the intestine from the intestinal bulb to the anus (seesupplementary material Fig. S6 for illustration).

Bacterial enumerationEnumeration of bacteria from larval zebrafish was carried out aspreviously described with the modification that overnight growthwas carried out at 28.5°C to recover commensal microbiota(Oehlers et al., 2011b).

Fluorescence activated cell sortingCell sorting experiments were carried out as previously described(Oehlers et al., 2011a). Briefly, larvae were dissociated in 0.25%trypsin-EDTA (Invitrogen) and analyzed using a BD LSRII.

Nitric oxide detectionLive imaging detection of nitric oxide production was carried outas described (Lepiller et al., 2007). Briefly, larvae were incubatedin a 10 M solution of DAF-FM-DA (Molecular Probes) for 2 hours,rinsed in E3, anesthetized and imaged by epifluorescent microscopy.

Resazurin reduction assayThe resazurin reduction assay was adapted from the manufacturer’sinstructions. Briefly, larvae were incubated in a 1� solution ofalamar blue (Invitrogen; final concentration 1 mg/ml resazurin) for60 minutes, anesthetized and individually imaged by epifluorescentmicroscopy at fixed zoom and excitation intensity settings.

Fluorescence intensity was quantified in ImageJ software Version1.43 (National Institutes of Health).

Cell proliferation and deathAnalysis of cell proliferation and death was performed as previouslydescribed using the In Situ Cell Proliferation Kit, FLUOS (Roche),the In Situ Cell Death Kit, TMR red (Roche) and acridine orangestaining (Oehlers et al., 2011a). Gut proliferation was enumeratedby manual counting along the length of the gut, and trunkproliferation was enumerated by manual counting of a single fieldof view immediately posterior to the swim bladder.

Quantitative PCRThe Ambion MicroPoly(A)Purist kit was used to purify RNA frompools of whole larvae for reverse transcription (Kerr et al., 2011).Reverse transcription and quantitative PCR was carried out aspreviously described (Oehlers et al., 2011c). Additional primers foramplifying il23 were previously described (Holt et al., 2011).Primers to amplify muc (5�-TGTGGACCCGAGCAAAAATTA-3�and 5�-AGCTAACTCGAATCCCACATCAC-3�) were designed inPrimer Express (Applied Biosystems).

Histological analysisLarvae were fixed in paraformaldehyde, rinsed in acidic ethanoland stained with alcian blue. Unbound stain was removed byrepeated rinsing with acidic ethanol prior to whole-mount imagingor preparation for histological sectioning. Specimens for sectioningwere embedded in agarose, infiltrated with paraffin, cut into 7 msections, rehydrated and counterstained with nuclear fast red(Vector Labs). Histological imaging was carried out on a Leica DMRcompound microscope with a DFC420C camera.

Whole-mount in situ hybridizationPCR with gene-specific primers (5�-CCTACACCTCCTCC -AGTTATCTGC-3� and 5�-CACTCGCCTTTTTGTTCCACG-3�)for muc (CR854881.2-201; NCBI accession number: 100148804)were used to amplify a 908 bp product from larval cDNA. ThisPCR product was cloned and transcribed to create riboprobes.Whole-mount in situ hybridization was carried out as described(Thisse and Thisse, 2008), and no staining was observed in sensestrand control riboprobes.

Lectin stainingLectin staining was carried out as described (Bates et al., 2006).Briefly, larvae were fixed for 2 hours at room temperature orovernight at 4°C, rinsed with PBST, blocked in PBST supplementedwith 2% (w/v) blocking solution (Roche), incubated in PBSTsupplemented with 2% blocking solution and 10 g/ml lectin, rinsedwith PBST and imaged by epifluorescent microscopy.

Statistical analysesAll statistical tests were carried out with GraphPad Prism version5.0a for Mac (GraphPad Software). Multiple comparisons withinANOVA tests were carried out by Tukey test.ACKNOWLEDGEMENTSWe thank Alhad Mahagaonkar for expert running of the zebrafish facility;Kazuhide Okuda and Jonathan Astin for helpful discussions regardingphenotyping; and members of the zebrafish community for reagents.


Retinoic acid, colitis and intestinal mucus RESEARCH ARTICLED

iseas

e M

odel

s & M

echa

nism

s

DM

M

COMPETING INTERESTSThe authors declare that they do not have any competing or financial interests.

AUTHOR CONTRIBUTIONSS.H.O.: data collection, manuscript preparation. S.H.O., M.V.F., C.J.H., K.E.C., P.S.C.:data analysis and study design. All authors edited the manuscript prior tosubmission.

FUNDINGThis work was supported by the Ministry of Science and Innovation (New Zealand)[UOAX0813 to P.S.C.]; and the Tertiary Education Commission (New Zealand)[UOAX07049 to S.H.O.].

SUPPLEMENTARY MATERIALSupplementary material for this article is available athttp://dmm.biologists.org/lookup/suppl/doi:10.1242/dmm.009365/-/DC1

REFERENCESBates, J. M., Mittge, E., Kuhlman, J., Baden, K. N., Cheesman, S. E. and Guillemin,

K. (2006). Distinct signals from the microbiota promote different aspects of zebrafishgut differentiation. Dev. Biol. 297, 374-386.

Bates, J. M., Akerlund, J., Mittge, E. and Guillemin, K. (2007). Intestinal alkalinephosphatase detoxifies lipopolysaccharide and prevents inflammation in zebrafish inresponse to the gut microbiota. Cell Host Microbe 2, 371-382.

Brugman, S., Liu, K. Y., Lindenbergh-Kortleve, D., Samsom, J. N., Furuta, G. T.,Renshaw, S. A., Willemsen, R. and Nieuwenhuis, E. E. (2009). Oxazolone-inducedenterocolitis in zebrafish depends on the composition of the intestinal microbiota.Gastroenterology 137, 1757-1767.

Chang, C. L., Lao-Sirieix, P., Save, V., De La Cueva Mendez, G., Laskey, R. andFitzgerald, R. C. (2007). Retinoic acid-induced glandular differentiation of theoesophagus. Gut 56, 906-917.

Cooke, G., Blanco-Fernandez, A. and Seery, J. P. (2008). The effect of retinoic acidand deoxycholic acid on the differentiation of primary human esophagealkeratinocytes. Dig. Dis. Sci. 53, 2851-2857.

Crockett, S. D., Porter, C. Q., Martin, C. F., Sandler, R. S. and Kappelman, M. D.(2010). Isotretinoin use and the risk of inflammatory bowel disease: a case-controlstudy. Am. J. Gastroenterol. 105, 1986-1993.

d’Alencon, C. A., Pena, O. A., Wittmann, C., Gallardo, V. E., Jones, R. A., Loosli, F.,Liebel, U., Grabher, C. and Allende, M. L. (2010). A high-throughput chemicallyinduced inflammation assay in zebrafish. BMC Biol. 8, 151.

De Lisle, R. C., Roach, E. A. and Norkina, O. (2006). Eradication of small intestinalbacterial overgrowth in the cystic fibrosis mouse reduces mucus accumulation. J.

Pediatr. Gastroenterol. Nutr. 42, 46-52.Dharmani, P., Strauss, J., Ambrose, C., Allenvercoe, E. and Chadee, K. (2011).

Fusobacterium nucleatum infection of colonic cells stimulates MUC2 mucin andtumor necrosis factor-{alpha}. Infect. Immun. 79, 2597-2607.

Dobson, J. T., Seibert, J., Teh, E. M., Daas, S., Fraser, R. B., Paw, B. H., Lin, T. J. andBerman, J. N. (2008). Carboxypeptidase A5 identifies a novel mast cell lineage in thezebrafish providing new insight into mast cell fate determination. Blood 112, 2969-2972.

Eksteen, B., Mora, J. R., Haughton, E. L., Henderson, N. C., Lee-Turner, L.,Villablanca, E. J., Curbishley, S. M., Aspinall, A. I., von Andrian, U. H. and Adams,D. H. (2009). Gut homing receptors on CD8 T-cells are retinoic acid dependent andnot maintained by Liver dendritic or stellate cells. Gastroenterology 137, 320-329.

Eri, R. D., Adams, R. J., Tran, T. V., Tong, H., Das, I., Roche, D. K., Oancea, I., Png, C.W., Jeffery, P. L., Radford-Smith, G. L. et al. (2011). An intestinal epithelial defectconferring ER stress results in inflammation involving both innate and adaptiveimmunity. Mucosal. Immunol. 4, 354-364.

Feng, Y., Santoriello, C., Mione, M., Hurlstone, A. and Martin, P. (2010). Live imagingof innate immune cell sensing of transformed cells in zebrafish larvae: parallelsbetween tumor initiation and wound inflammation. PLoS Biol. 8, e1000562.

Fleming, A., Jankowski, J. and Goldsmith, P. (2010). In vivo analysis of gut functionand disease changes in a zebrafish larvae model of inflammatory bowel disease: Afeasibility study. Inflamm. Bowel Dis. 16, 1162-1172.

Flores, M. V., Hall, C. J., Davidson, A. J., Singh, P. P., Mahagaonkar, A. A., Zon, L. I.,Crosier, K. E. and Crosier, P. S. (2008). Intestinal differentiation in zebrafish requiresCdx1b, a functional equivalent of mammalian Cdx2. Gastroenterology 135, 1665-1675.

Fyderek, K., Strus, M., Kowalska-Duplaga, K., Gosiewski, T., Wedrychowicz, A.,Jedynak-Wasowicz, U., Sladek, M., Pieczarkowski, S., Adamski, P., Kochan, P. etal. (2009). Mucosal bacterial microflora and mucus layer thickness in adolescentswith inflammatory bowel disease. World J. Gastroenterol. 15, 5287-5294.

Gadzhieva, Z. M. and Kon, I. (1984). [Changes in the rat intestinal mucosa inhypervitaminosis A]. Voprosy pitaniia, 47-50.

Gersemann, M., Becker, S., Kubler, I., Koslowski, M., Wang, G., Herrlinger, K. R.,Griger, J., Fritz, P., Fellermann, K., Schwab, M. et al. (2009). Differences in gobletcell differentiation between Crohn’s disease and ulcerative colitis. Differentiation 77,84-94.

Grimes, A. C., Stadt, H. A., Shepherd, I. T. and Kirby, M. L. (2006). Solving an enigma:arterial pole development in the zebrafish heart. Dev. Biol. 290, 265-276.

Hall, C., Flores, M. V., Storm, T., Crosier, K. and Crosier, P. (2007). The zebrafishlysozyme C promoter drives myeloid-specific expression in transgenic fish. BMC Dev.

Biol. 7, 42.Hall, C., Flores, M. V., Crosier, K. and Crosier, P. (2009). Live cell imaging of zebrafish

leukocytes. Methods Mol. Biol. 546, 255-271.Hama, K., Provost, E., Baranowski, T. C., Rubinstein, A. L., Anderson, J. L., Leach, S.

D. and Farber, S. A. (2009). In vivo imaging of zebrafish digestive organ functionusing multiple quenched fluorescent reporters. Am. J. Physiol. Gastrointest. Liver

Physiol. 296, G445-G453.Heazlewood, C. K., Cook, M. C., Eri, R., Price, G. R., Tauro, S. B., Taupin, D.,

Thornton, D. J., Png, C. W., Crockford, T. L., Cornall, R. J. et al. (2008). Aberrantmucin assembly in mice causes endoplasmic reticulum stress and spontaneousinflammation resembling ulcerative colitis. PLoS Med. 5, e54.

Holt, A., Mitra, S., van der Sar, A. M., Alnabulsi, A., Secombes, C. J. and Bird, S.(2011). Discovery of zebrafish (Danio rerio) interleukin-23 alpha (IL-23alpha) chain, asubunit important for the formation of IL-23, a cytokine involved in thedevelopment of Th17 cells and inflammation. Mol. Immunol. 48, 981-991.

Iger, Y., Lock, R. A., van der Meij, J. C. and Wendelaar Bonga, S. E. (1994). Effects ofwater-borne cadmium on the skin of the common carp (Cyprinus carpio). Arch.

Environ. Contam. Toxicol. 26, 342-350.Iliev, I. D., Mileti, E., Matteoli, G., Chieppa, M. and Rescigno, M. (2009). Intestinal

epithelial cells promote colitis-protective regulatory T-cell differentiation throughdendritic cell conditioning. Mucosal. Immunol. 2, 340-350.

Kanther, M., Sun, X., Muhlbauer, M., Mackey, L. C., Flynn, E. J., 3rd, Bagnat, M.,Jobin, C. and Rawls, J. F. (2011). Microbial colonization induces dynamic temporaland spatial patterns of NF-kappaB activation in the zebrafish digestive tract.Gastroenterology 141, 197-207.

Kaser, A., Zeissig, S. and Blumberg, R. S. (2010). Inflammatory bowel disease. Annu.

Rev. Immunol. 28, 573-621.Kerr, T. A., Ciorba, M. A., Matsumoto, H., Davis, V. R., Luo, J., Kennedy, S., Xie, Y.,

Shaker, A., Dieckgraefe, B. K. and Davidson, N. O. (2011). Dextran sodium sulfateinhibition of real-time polymerase chain reaction amplification: A poly-A purificationsolution. Inflamm. Bowel Dis. 18, 344-348.



TRANSLATIONAL IMPACT

Clinical issueRetinoids, such as isotretinoin, are indicated for use in treating severe acne butare recognized to cause a number of serious side effects. Epidemiologicalstudies have linked isotretinoin exposure to the etiology of inflammatorybowel disease (IBD). Mechanisms by which retinoids have been proposed tocause IBD include dysregulation of the adaptive immune system and alterationof intestinal mucus physiology. Retinoids are known to dry out epithelialbarriers, including the skin and airways, but there is little biological evidencethat exposure to retinoids modulates IBD-relevant intestinal mucus physiology.

ResultsThis research examined a novel zebrafish model of IBD induced by immersingzebrafish larvae in dextran sodium sulfate (DSS), a chemical that is commonlyused to induce colitis in rodent models of IBD. Analysis of this model revealeda protective mucosecretory response to enterocolitis that was similar to thatseen in individuals with IBD. Administration of exogenous retinoic acid wasfound to suppress both homeostatic and DSS-induced mucus production bythe intestinal epithelium. Suppression of either homeostatic or DSS-inducedmucus production was correlated with increased neutrophilic inflammationand larval mortality following further enterocolitic challenge.

Implications and future directionsThis study provides direct evidence that exposure to retinoids disruptsintestinal mucus physiology in a whole animal. The data suggest that drugssuch as isotretinoin might contribute to IBD pathogenesis by compromisingboth the physiological production of intestinal barrier mucus and the stress-induced production of protective mucus.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

Krimi, R. B., Kotelevets, L., Dubuquoy, L., Plaisancie, P., Walker, F., Lehy, T.,Desreumaux, P., Van Seuningen, I., Chastre, E., Forgue-Lafitte, M. E. et al. (2008).Resistin-like molecule beta regulates intestinal mucous secretion and curtails TNBS-induced colitis in mice. Inflamm. Bowel Dis. 14, 931-941.

Lam, S. H., Chua, H. L., Gong, Z., Lam, T. J. and Sin, Y. M. (2004). Development andmaturation of the immune system in zebrafish, Danio rerio: a gene expressionprofiling, in situ hybridization and immunological study. Dev. Comp. Immunol. 28, 9-28.

Lepiller, S., Laurens, V., Bouchot, A., Herbomel, P., Solary, E. and Chluba, J. (2007).Imaging of nitric oxide in a living vertebrate using a diamino-fluorescein probe. FreeRadic. Biol. Med. 43, 619-627.

Lugo-Villarino, G., Balla, K. M., Stachura, D. L., Banuelos, K., Werneck, M. B. andTraver, D. (2010). Identification of dendritic antigen-presenting cells in the zebrafish.Proc. Natl. Acad. Sci. USA 107, 15850-15855.

Meyer-Hoffert, U., Hornef, M. W., Henriques-Normark, B., Axelsson, L. G.,Midtvedt, T., Putsep, K. and Andersson, M. (2008). Secreted enteric antimicrobialactivity localises to the mucus surface layer. Gut 57, 764-771.

Mohideen, M. A., Beckwith, L. G., Tsao-Wu, G. S., Moore, J. L., Wong, A. C., Chinoy,M. R. and Cheng, K. C. (2003). Histology-based screen for zebrafish mutants withabnormal cell differentiation. Dev. Dyn. 228, 414-423.

Nadauld, L. D., Sandoval, I. T., Chidester, S., Yost, H. J. and Jones, D. A. (2004).Adenomatous polyposis coli control of retinoic acid biosynthesis is critical forzebrafish intestinal development and differentiation. J. Biol. Chem. 279, 51581-51589.

Oehlers, S. H., Flores, M. V., Hall, C. J., O’Toole, R., Swift, S., Crosier, K. E. andCrosier, P. S. (2010). Expression of zebrafish cxcl8 (interleukin-8) and its receptorsduring development and in response to immune stimulation. Dev. Comp. Immunol.34, 352-359.

Oehlers, S. H., Flores, M. V., Okuda, K. S., Hall, C. J., Crosier, K. E. and Crosier, P. S.(2011a). A chemical enterocolitis model in zebrafish larvae that is dependent onmicrobiota and responsive to pharmacological agents. Dev. Dyn. 240, 288-298.

Oehlers, S. H., Flores, M. V., Hall, C. J., Swift, S., Crosier, K. E. and Crosier, P. S.(2011b). The inflammatory bowel disease (IBD) susceptibility genes NOD1 and NOD2have conserved anti-bacterial roles in zebrafish. Dis. Model. Mech. 4, 832-841.

Oehlers, S. H., Flores, M. V., Chen, T., Hall, C. J., Crosier, K. E. and Crosier, P. S.(2011c). Topographical distribution of antimicrobial genes in the zebrafish intestine.Dev. Comp. Immunol. 35, 385-391.

Pham, L. N., Kanther, M., Semova, I. and Rawls, J. F. (2008). Methods for generatingand colonizing gnotobiotic zebrafish. Nat. Protoc. 3, 1862-1875.

Popescu, C. M. and Popescu, R. (2011). Isotretinoin therapy and inflammatory boweldisease. Arch. Dermatol. 147, 724-729.

Santoriello, C., Gennaro, E., Anelli, V., Distel, M., Kelly, A., Koster, R. W., Hurlstone,A. and Mione, M. (2010). Kita driven expression of oncogenic HRAS leads to earlyonset and highly penetrant melanoma in zebrafish. PLoS ONE 5, e15170.

Shaoul, R., Okada, Y., Cutz, E. and Marcon, M. A. (2004). Colonic expression of MUC2,MUC5AC, and TFF1 in inflammatory bowel disease in children. J. Pediatr.

Gastroenterol. Nutr. 38, 488-493.Thisse, C. and Thisse, B. (2008). High-resolution in situ hybridization to whole-mount

zebrafish embryos. Nat. Protoc. 3, 59-69.Trabucchi, E., Mukenge, S., Baratti, C., Colombo, R., Fregoni, F. and Montorsi, W.

(1986). Differential diagnosis of Crohn’s disease of the colon from ulcerative colitis:ultrastructure study with the scanning electron microscope. Int. J. Tissue React. 8, 79-84.

Westendorf, A. M., Fleissner, D., Groebe, L., Jung, S., Gruber, A. D., Hansen, W. andBuer, J. (2009). CD4+ Foxp3+ regulatory T cell expansion induced by antigen-driveninteraction with intestinal epithelial cells independent of local dendritic cells. Gut 58,211-219.

Wirtz, S., Neufert, C., Weigmann, B. and Neurath, M. F. (2007). Chemically inducedmouse models of intestinal inflammation. Nat. Protoc. 2, 541-546.

Wittamer, V., Bertrand, J. Y., Gutschow, P. W. and Traver, D. (2011). Characterizationof the mononuclear phagocyte system in zebrafish. Blood 117, 7126-7135.

Zheng, X., Tsuchiya, K., Okamoto, R., Iwasaki, M., Kano, Y., Sakamoto, N.,Nakamura, T. and Watanabe, M. (2011). Suppression of hath1 gene expressiondirectly regulated by hes1 via notch signaling is associated with goblet celldepletion in ulcerative colitis. Inflamm. Bowel Dis. 17, 2251-2260.


Retinoic acid, colitis and intestinal mucus RESEARCH ARTICLED

iseas

e M

odel

s & M

echa

nism

s

DM

M

Retinoic acid suppresses intestinal mucus production and ...numbers of goblet cells and reduced...

Documents

Transcript of Retinoic acid suppresses intestinal mucus production and ...numbers of goblet cells and reduced...