RESEARCH ARTICLE

Zebrafish modeling of intestinal injury, bacterial exposures andmedications defines epithelial in vivo responses relevant to humaninflammatory bowel diseaseLing-shiang Chuang1,2, Joshua Morrison1,3, Nai-yun Hsu1,2, Philippe Ronel Labrias1, Shikha Nayar1,Ernie Chen1,2, Nicole Villaverde1, Jody Ann Facey1, Gilles Boschetti4, Mamta Giri1, Mireia Castillo-Martin5,Tin Htwe Thin5, Yashoda Sharma1, Jaime Chu3 and Judy H. Cho1,2,*

ABSTRACTGenome-wide association studies have identified over 200 genomicloci associated with inflammatory bowel disease (IBD). High-effectrisk alleles define key roles for genes involved in bacterial responseand innate defense. More high-throughput in vivo systems are requiredto rapidly evaluate therapeutic agents. We visualize, in zebrafish, theeffects on epithelial barrier function and intestinal autophagy of one-course and repetitive injury. Repetitive injury induces increasedmortality, impaired recovery of intestinal barrier function, failure tocontain bacteria within the intestine and impaired autophagy.Prostaglandin E2 (PGE2) administration protected against injury byenhancing epithelial barrier function and limiting systemic infection.Effects of IBD therapeutic agents were defined: mesalamine showedprotective features during injury, whereas 6-mercaptopurine displayedmarked induction of autophagy during recovery. Given the highlyconserved nature of innate defense in zebrafish, it represents an idealmodel system with which to test established and new IBD therapiestargeted to the epithelial barrier.

This article has an associated First Person interview with the firstauthor of the paper.

KEY WORDS: Crohn’s disease, DSS injury model, Epithelial barrier,IBD, Lysosome-rich enterocytes

INTRODUCTIONIBD is classified into two subtypes: Crohn’s disease (CD) andulcerative colitis (UC). While UC affects the superficial layers ofthe intestine, CD often affects deeper layers of the bowel wall. Themost significant loci identified in genome-wide association studies

(GWAS) for IBD include protein-altering alleles in IL23R (interleukin23 receptor), NOD2 (nucleotide oligomerization domain 2),ATG16L1 (autophagy-related 16-like 1) (Huang et al., 2017),LRRK2 (leucine-rich repeat kinase 2) (Hui et al., 2018) andCSF2RB (colony stimulating factor 2 receptor) (Chuang et al.,2016), as well as non-coding risk alleles nearPTGER4 (prostaglandinE receptor 4), that increase gene expression (Libioulle et al., 2007;Jostins et al., 2012; Liu et al., 2015). The NOD2 risk alleles showimpaired intracellular sensing of bacterial peptidoglycan (Bonen et al.,2003), resulting in impaired NF-κB activation (Ogura et al., 2001).TheATG16L1 alanine risk allele at codon 300 increases its proteolyticcleavage by caspase 3 and 7 (Lassen et al., 2014;Murthy et al., 2014),implicating impaired autophagy in CD.

The striking overlap between genes implicated in CD andmycobacterial susceptibility highlights a key role for innate defenseand containment of microbes (Jostins et al., 2012). Recent literaturehas defined the particular value of zebrafish in modeling innatedefense and immunity (Madigan et al., 2017; Cambier et al., 2017;Cronan et al., 2016). Because of their transparency over the initialweeks of life, zebrafish serve as a powerful tool for performing livein vivo imaging of intestinal macrophages and epithelial responsesto injury and bacterial exposure.

RESULTSRepeated DSS treatments prevent recovery ofacidified-lysosome and mucin lossTo model the chronicity of IBD, we first compared the effects ofsingle- and three-course dextran sodium sulfate (DSS) injury(Fig. 1A, Fig. S1A) in zebrafish. Similar to DSS injury in mice(Chassaing et al., 2014), the gut length is significantly shortened inDSS-injured zebrafish, with no difference in the total lengthobserved (Fig. S1B,C). Using doses as previously reported (Oehlerset al., 2012, 2013), we observed a dose-dependent induction ofmortality with repeated injury (Fig. 1A; 68% for 0.25% DSS, 29%for 0.1%). With both single and repeated DSS injury, the gobletcells were observed by H&E histology (Fig. 1B).

We next sought to examine altered epithelial phenotypescontributing to the high mortality with repetitive DSS injury.Neutral Red is a dye that can diffuse across cellular membranes atphysiological pH (pH=7), but is protonated under acidic conditions,at which point the dye appears red and is unable to leave the acidiccompartments it has entered. Owing to the acidic nature oflysosomes, Neutral Red will accumulate in properly acidifiedlysosomes (Fig. S2A,C) (Oehlers et al., 2012; Chazotte, 2011), witha marked enrichment in lysosome-rich enterocytes (LREs) in theposterior mid-intestine (Fig. S2D,E) (Rodriguez-Fraticelli et al.,Received 12 October 2018; Accepted 11 July 2019

1Department of Genetics and Genomic Sciences, Icahn School of Medicine atMount Sinai, New York, NY 10029, USA. 2The Charles Bronfman Institute forPersonalized Medicine, Icahn School of Medicine at Mount Sinai, New York, NY10029, USA. 3Department of Pediatrics, Icahn School of Medicine at Mount Sinai,New York, NY 10029, USA. 4Department of Oncological Science, Icahn School ofMedicine at Mount Sinai, New York, NY 10029, USA. 5Departments of Pathology,Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

*Author for correspondence ( judy.cho@mssm.edu)

L.-s.C., 0000-0001-5054-1305; N.-y.H., 0000-0003-3321-6388; P.R.L., 0000-0001-7281-9339; S.N., 0000-0001-8519-4569; E.C., 0000-0003-1573-7577; N.V.,0000-0002-1421-8305; G.B., 0000-0003-4039-7607; M.G., 0000-0002-9019-515X;T.H.T., 0000-0002-7891-1459; J.C., 0000-0002-9291-8630; J.H.C., 0000-0002-7959-0466

This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use,distribution and reproduction in any medium provided that the original work is properly attributed.

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© 2019. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2019) 12, dmm037432. doi:10.1242/dmm.037432

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Fig. 1. Impaired recovery of acidic-lysosome function and mucin production after repeated DSS injury in zebrafish intestines. (A) Dose-dependentmortality observed after repeated DSS injury (N=1739 from 11 clutches). Compared to the single DSS injury, which has a 98-100% survival rate (day 6), repeatedDSS injury induces a high mortality rate of 63% in 0.25% DSS and 29% in 0.1% DSS. (B) Hematoxylin and eosin (H&E) staining of a longitudinal section ofzebrafish larvae intestine. A total of 20 larvae per experimental condition were embedded in paraffin and sectioned at 10 μm per slide. Scale bars: 100 μm.(C,D) Quantification of Neutral Red images (top panels) and Neutral Red accumulation (bottom panel) for single (C) and repeated (D) DSS-injured zebrafish.Neutral Red accumulation in the intestine indicates normally functioning acidic lysosomes. The damage to lysosomal function is fully recovered within 2 days withthe single DSS injury (C; N=253 from four clutches) but is impaired with the repeated DSS injury (D; N=137 from three clutches; day 2 N=5 out of 13 from singleclutch due to high mortality rate of 61.5%). Neutral Red images for single (C) and repeated (D) injury are shown as control, and 0.1% and 0.25% (w/v) DSStreatments at treatment times of 1 day and 2 days after injury. Scale bars: 100 µm. ***P<0.001, **P<0.01, *P<0.05. Error bars, mean±s.e.m. (E) Images (left panel)and quantification (right panel) of Alcian Blue staining of intestine with single and repeated injury with control and 24 h after removal from DSS treatment.The quantification of total Alcian Blue intensity from single (N=160 from three clutches) and repeated (N=154 from three clutches) injury is shown. There wasan increase in mucin production after single DSS injury, but mucin production was impaired after repeated DSS injury. Scale bars: 100 µm.

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2015; Oehlers et al., 2011; Farber et al., 2001; Wallace et al., 2005;Rombout et al., 1985). LREs are a population of highly endocyticenterocytes in the zebrafish posterior midgut (Rodriguez-Fraticelliet al., 2015). The intensity of Neutral Red staining indicates theamount of healthy lysosomes with acidic pH (Chazotte, 2011).Therefore, a decrease in Neutral Red intensity indicates a depletionof healthy, properly acidified lysosomes. We observed a markeddecrease in Neutral Red intensity with a single course of DSS thatwas significantly restored 1 day after DSS removal and largelyrecovered by 2 days after DSS removal (Fig. 1C, Fig. S2F). Incontrast, with repetitive DSS injury, the reduction in NeutralRed staining observed after the third course of DSS did not recoverat 1 or 2 days following DSS removal (Fig. 1D). Moreover, byquantitative real-time PCR (qPCR), we show that expression of tnfaand Il1b, encoding proinflammatory cytokines, was predominatelyinduced in the intestine with single DSS injury, but their expressionis also present in non-intestinal tissues after repeated injury(Fig. S3A,B). Similarly, Alcian Blue staining, which measuresepithelial mucus production (Fig. S4A,B) (Oehlers et al., 2012),demonstrated a normal amount of mucus with no changes in gobletcell number with single-course DSS injury. However, repeatedinjury resulted in decreased mucus levels that did not recover whencompared to controls (Fig. 1E, Fig. S5A-D).

DSS injury with bacterial exposure impacts LRE autophagyand bacterial protein uptakeThe uptake of bacterial proteins by gut-associated specializedenterocytes and possibly mononuclear phagocytes serves to containbacterial contaminants to the intestine, a critical protective function.One of the main functions of LREs in zebrafish is endocytosis ofproteins from the lumen of the intestine (Rodriguez-Fraticelli et al.,2015). Using fluorescent and confocal microscopy, we sought tovisualize the local containment of bacteria in the absence andpresence of DSS. We used pHrodo-labeled, heat-killed Escherichiacoli, which fluoresces at pH levels below 5.5. The amine of E. coliproteins was conjugated by activated succinimidyl ester to pHrododye. After 1 hour of E. coli incubation, we observed markedlyincreased uptake of E. coli proteins with DSS treatment (Fig. 2A).Cyto-ID fluorescently stains autophagosomes in live cells, butdoes not stain lysosomes (Marx, 2015). To confirm the specificity ofCyto-ID staining for autophagy in the zebrafish intestine,Bafilomycin A1 treatment was combined with both Cyto-ID andNeutral Red staining. Bafilomycin A1 is a late-stage autophagyinhibitor that blocks both starvation-induced and starvation-independent autophagy (Hundeshagen et al., 2011). In the larvalzebrafish, inhibiting autophagy leads to loss of Cyto-ID staining butnot Neutral Red staining of LREs, indicating depleted autophagylevels but normal levels of functional lysosomes (Fig. S6A,B).Colocalization of labeled E. coli proteins and autophagy as measuredby Cyto-ID was observed in LREs (Fig. 2B, Movies 1-4). Thefluorescence from E. coli proteins was most intense in the posteriormidgut (Fig. 2A), with pHrodo signals localizing to the regionenriched for LREs as shown by LysoSensor-labeled lysosomes(Fig. S7A). Similar to recovery trends seen with Neutral Redstaining, we observed markedly decreased Cyto-ID staining witheither single or repeated DSS injury (Fig. 2C). The single-DSS-injury finding was further validated with p62 immunoblot (Fig. S8)(Schiebler et al., 2015; Wu et al., 2016). Outside of the posteriormidgut, Cyto-ID staining of autophagy is present and not limited tothe regionwith ingestedE. coli proteins (Fig. S7B).With andwithout0.25% DSS treatment, we showed the decrease of Cyto-ID intensityin the LRE region of the posterior midgut (Fig. S9). One course of

DSS injury demonstrated a marked recovery of autophagy 1 day afterremoval of DSS (Fig. 2D); however, similar to trends observed withNeutral Red staining, three courses of DSS injury did notdemonstrate recovery of Cyto-ID staining.

Endocytosis of proteins is an essential function of LREs in thezebrafish intestine (Rodriguez-Fraticelli et al., 2015). Plasmolipin(Pllp), a cellular marker for LREs, is also functionally necessary forproper apical endocytosis (Rodriguez-Fraticelli et al., 2015). Takentogether, it follows that LREs may have the ability to endocytose theproteins of immotile pathogens. Treatment with high-dose, but notlow-dose, DSS followed by E. coli exposure results in markedmortality (high-dose: Fig. 2E left panel; low-dose: Fig. S10). Usingthe zebrafish model, the role of bacterial invasion, an initial step ofsepsis and systemic infection, is dramatically demonstrated in vivo(Fig. 2E right panel, Movies 6,7) and quantified (Fig. S11). Theintensities of fluorescently labeled E. coli proteins are high in theintestine (Movies 6,7), which suggests that the initiation of bacterialinvasion originates from the intestine. However, further analysiswill be needed to exclude the possibility of DSS injury allowingE. coli invasion via extra-intestinal surfaces of the zebrafish.

PGE2 and common IBD medications impact barrier function,bacterial containment and LRE functionWe next sought to examine mechanisms modulating the mucuslayer, with a particular focus on the effects of treatment withprostaglandin E2 (PGE2). The CD-predominant association to thechromosome 5p13 region containing PTGER4 is the third mostsignificant association in Europeans, with the PTGER4 risk allelescorrelating with increased expression (Libioulle et al., 2007; Gettleret al., 2019). Contrary to this, however, PGE2 is known to play acritical role in intestinal mucosal cytoprotection mediated byPTGER4 (Takeuchi, 2010). Blockade of PGE2 through the use ofCOX2 (cyclo-oxygenase 2) inhibitors (e.g. non-steroidal anti-inflammatory agents) impairs mucosal cytoprotection, frequentlyresulting in intestinal ulceration (Miyoshi et al., 2016). Using ourin vivo model, we observed a dose-dependent increase in mucusexpression by Alcian Blue staining with PGE2 treatment for 24 h(Fig. 3A,B). This increasewas evenmoremarkedwhen confining theAlcian Blue quantification to the secreted, luminal mucus staining inthe distal hindgut region (Fig. 3C; Oehlers et al., 2012). With DSSinjury, we observed the same level of increase ofAlcianBlue stainingwith PGE2 treatment (Fig. S12A). Similar results were obtained withenteroids from human intestinal epithelium, with a marked inductionof Alcian Blue staining (Fig. 3D; images: Fig. S12B).

We next sought to define the time-course dependencies ofPGE2 treatment relative to DSS injury and bacterial exposure.DSS injury followed by E. coli exposure results in high levels ofmortality (Figs 2E and 3F, green line). With one course of DSSinjury, followed by PGE2, and then E. coli treatment (Fig. 3E), weobserved a significant decrease in mortality (Fig. 3F, purplecompared to green line); concomitant treatment of PGE2 duringtheDSS injury period, followed by a 5 h recovery period, thenE. colitreatment, resulted in an even further decrease in mortality (Fig. 3F,blue line). With DSS administration (Fig. 3E-G, green, purple andblue lines), the relative mortality (Fig. 3F) parallels the fraction ofingestedE. coli proteins at 1 and 2 h post-DSS treatment asmeasuredby pHrodo intensities (Fig. 3G); after 3 h of E. coli exposure, amongthe surviving individuals, all three of the DSS-treated conditionslevel off to equivalent amounts of ingested pHrodo-labeled E. coliproteins. In the absence of DSS treatment (Fig. 3F,G, black and redlines), E. coli treatment alone or with antecedent PGE2 treatmentbefore E. coli exposure (Fig. 3E, black and red bars), no mortality is

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detected (Fig. 3F); successively increasing amounts of ingestedpHrodo-labeled E. coli proteins were observed 1, 2 and 3 h afterE. coli exposure (Fig. 3G, black and red lines), in amounts that farsurpass the DSS-treatment groups (Fig. 3G, green, purple and bluelines). This indicates that, in the absence of DSS treatment, with lessabrupt bacterial exposure, robust uptake and containment of bacterialproteins by LREs limits mortality.

Finally, we sought to model the effects of PGE2 as well as drugscommonly utilized in IBD treatment on intestinal LRE phenotypesand function. Administration of either PGE2 or 6-mercaptopurineduring DSS injury did not result in enhancement of Neutral Red orCyto-ID staining compared to DSS treatment alone (Fig. 3H, light-green points). Interestingly, co-treatment of mesalamine duringDSS injury resulted in increased Neutral Red staining at both the 13

Fig. 2. See next page for legend.

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and 65 µM doses, with an increase in Cyto-ID observed at the65 µM dose (Fig. 3H, light-green points). Neutral Red and Cyto-IDintensities were higher 5 h after DSS removal (Fig. 3H, light-bluepoints) compared to measurements taken immediately after DSSremoval (Fig. 3H, light-green points). Treatment with mesalamineadministered for 5 h immediately after removal of DSS (Fig. 3H,light-blue points, recovery phase) did not result in enhancement ofNeutral Red or Cyto-ID intensities compared to untreated recoverycontrols. In contrast, 5 h of PGE2 treatment in the recovery phaseresulted in significant induction of Neutral Red and Cyto-IDintensities (Fig. 3H, light-blue points), presumably reflecting themucus cytoprotective effects of PGE2 (Fig. 3A-D). Importantly,6-mercaptopurine treatment resulted in increased Neutral Red andCyto-ID intensities at both the 66 and 330 µM doses (Fig. 3H, light-blue points), demonstrating a surprisingly rapid in vivo induction ofautophagy in the recovery period following DSS injury.

DISCUSSIONTraditionally, the most commonly utilized models in IBD involvemurine models, including IL10-pathway knockouts, the T-celltransfer model, DSS treatment and, more recently, the abcb1aknockout (mdr1a−/−), which accurately predicted human responsesto blockade of IL12/23 and IL17 as being beneficial and harmful,respectively (Keubler et al., 2015; Ostanin et al., 2009; Chassainget al., 2014; Wilk et al., 2005). However, long-term remission rates,as measured by mucosal healing at week 4, indicated thatdevelopment of agents that address the epithelial alterations inIBD are needed (Holleran et al., 2017). In particular, given theknown role that decreased autophagy in epithelial cells plays indriving CD (Ke et al., 2016; Iida et al., 2017), development ofin vivomodels that can rapidly scale testing of new agents is needed.Zebrafish models of IBD provide substantive advantages in

modeling repetitive intestinal injury and repair. In this regard, themost broadly utilized models of IBD have used DSS, which resultsin epithelial injury and inflammation, followed by prompt recoveryfollowing DSS removal. It has been reported that DSS injury resultsfrom complex formation with intraluminal fatty acids during DSSadministration, resulting in impaired barrier function. Chronicmodels of DSS in mice have been reported, but take months todevelop (Wirtz et al., 2017). In the present models, we observe a

striking failure to fully recover from repetitive DSS injury asmanifested by increased mortality, impaired mucus production andimpaired autophagy, all within 2 weeks; although the inflammatoryresponse is predominant in the intestine, given the mode ofadministration, extraintestinal effects may occur. We observed theincomplete recovery with repetitive injury both by measurements ofNeutral Red, which measures lysosomal pH, as well as by CytoID,which measures autophagosomes.

The high mortality observed with repetitive DSS injury wasassociated with a striking, systemic presence of microbial products(Fig. 2F). Both in vivo and in vitro (Fig. 3A-G), we observed thatPGE2 potently induces mucus expression, which has previouslybeen reported to be mediated by prostaglandin E2 receptor EP4subtype (PTGER4)-mediated mucin exocytosis in a humancarcinoma cell line (Belley and Chadee, 1999). Furthermore, micedeficient in PTGER4 in the intestinal epithelial layer did notdevelop wound-associated epithelial cells, resulting in impairedtissue repair (Miyoshi et al., 2016). Our data highlight the criticalrole that PGE2 plays in protecting against intestinal-barrier-associated bacterial invasion through increased mucin production,blocking uptake of luminal E. coli proteins (Fig. 4). These findingsare consistent with a critical role of the PGE2-PTGER4 pathway inmediating intestinal barrier function, and administration of PGE2after removal of DSS resulted in enhanced recovery of Cyto-IDstaining (Fig. 3H). The complex actions of PGE2-PTGER4 inintestinal barrier function, epithelial repair and innate immunefunctions have yet to be fully elucidated. The presence of CD-predominant PTGER4 associations that increase PTGER4 geneexpression (Libioulle et al., 2007) would appear to contradict theoverall protective actions of PGE2-PTGER4 in intestinal barrier andepithelial barrier function. Future studies evaluating pathogeniceffects of increased PTGER4 expression, especially in CD, are ofthe highest priority.

During DSS injury, only mesalamine resulted in enhanced Cyto-ID staining; this finding is consistent with prior literature thatmesalamine acts by blocking pro-inflammatory-cytokine-mediatedinduction of NF-κB, thereby enhancing epithelial barrier function(Kaiser et al., 1999; Di Paolo et al., 1996). In our study, mesalamineexerts its protective effects only during injury and not duringthe recovery period following DSS removal. In contrast,6-mercaptopurine enhances autophagy solely after DSS removal,consistent with prior literature of direct effects of thiopurines inenhancing autophagy (Chaabane and Appell, 2016). Whileblockade of pro-inflammatory cytokines such as anti-TNF or anti-IL12/23 (Sandborn et al., 2012) represents major mechanisms fortreating moderate to severe CD, it is still an open question as towhether management combining pro-inflammatory cytokineblockade (Abraham et al., 2017) with 6-mercaptopurine providesadditional benefit (Colombel et al., 2010). As with many highlyutilized, older medications, the precise mechanism of action of 6-mercaptopurine is incompletely understood and likely multifactorialin nature (Ordás et al., 2012; Poppe et al., 2006). Its use has beensupplanted by more potent pro-inflammatory blockade in moderateto severe disease. While the CD-predominant associations toautophagy represent some of the highest effect and previouslyunappreciated pathways implicated by genome-wide associationstudies, given the overall lower efficacy of 6-mercaptopurine inmoderate to severe CD (Colombel et al., 2010), our studies provide acautionary note regarding the potency of CD-based therapeuticdevelopment focused solely on enhancing autophagy in theintestine. In the era of precision medicine, more rapid scalingof a sophisticated understanding of time course factors, disparate

Fig. 2. Lysosome-rich enterocyte-mediated bacterial protein uptake andautophagy with injury and bacterial exposure. (A) Bright-field, fluorescentand merged images of pHrodo-labeled heat-killed E. coli K12MG1655 within azebrafish larval intestine with and without DSS treatment. A total of 20 larvafrom each experimental condition were treated with the pHrodo-labeled E. coliproteins. The pHrodo dye is a pH-sensing dye indicating ingested E. coliproteins. Scale bars: 50 μm. The quantification is shown at the 0 h time point ofFig. 3G. (B) Uptake of E. coli proteins (red) colocalized with Cyto-ID-positiveautophagosomes (green) in intestine. Scale bars: 20 μm. (C) Quantification ofCyto-ID intensities immediately following single (top panel) and repeated DSSinjury (bottom panel). DSS [0.05% and 0.1% (w/v)] was applied at thebeginning of treatment. ***P<0.001. Error bar, mean±s.e.m. (D) Cyto-IDintensities measured 1 day after DSS treatment shows recovery and non-recovery after single (top panel) and repeated (bottom panel) DSS injury.Altogether for C and D, N=544 from six clutches. ***P<0.001. **P<0.01. Errorbar, mean±s.e.m. (E) High levels of mortality with E. coli treatment followingsingle DSS injury. The left panel plots mortality rates at 1, 2 and 3 h afterexposure to heat-killed E. coli labeled with Alexa Fluor 488 (greenfluorescence) following single DSS injury. *P<0.05, comparing mortality ratesat each time point with and without DSS treatment. N=341 from three clutches.Error bar, mean±s.e.m. The right panel shows systemic penetration ofE. coli inthe dorsal aorta (upper arrow) and posterior cardinal vein (lower arrow) withDSS treatment imaged 90 min after heat-killed E. coli treatment. Scale bars:20 µm. Full bacterial invasion videos are shown in Movies 6 and 7.

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cellular effects and mechanisms of action of established and newdrugs will be required. While mammalian systems will remain alinchpin, the superior speed, sample size, visualization (e.g.

microbes, autophagy) and key role of the epithelial barrier makeshigh-throughput studies in the zebrafish an important component ofthe IBD therapeutic development pipeline.

Fig. 3. See next page for legend.

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MATERIALS AND METHODSZebrafish maintenanceAdult zebrafish were maintained on a 14:10 h light:dark cycle at 28°C.Wild-type (WT; AB andTab 14) and Tg(mfap4:turquoisext27) (Oehlers et al., 2015;Walton et al., 2015) fish were used. Fertilized embryos collected followingnatural spawning were cultured at 28.5°C in egg water (0.6 g/l Instant Ocean;Blacksburg, VA, USA) containing Methylene Blue (0.002 g/l) and 75 μM1-phenyl 2-thiourea. The Mount Sinai School of Medicine Institutional

Animal Care and Use Committee approved all protocols. For fluorescentimaging, Methylene Blue was removed within 24 h of embryo collection.Larvae and adults were fed once a day with Hikari First Bites (Hikari) at 6days post-fertilization and Zeigler Zebrafish Diet with Hatching BrineShrimp Eggs (Pentair Aquatic Eco-Systems, FL, USA), respectively.

Enteroid culture establishment and differentiationHuman biopsy collection was approved by the Institutional Review Board ofthe Mount Sinai School of Medicine. Human enteroid lines were generatedaccording to the method from VanDussen et al. (2015) with modifications.Briefly, biopsies were obtained from healthy and IBD individuals duringroutine colonoscopy visit. Biopsies were dissociated by collagenase I(Thermo Fisher Scientific) incubation at 37°C for 30 min and vigorouspipetting of mixtures every 5 min. Biopsy mixtures were passed through a70-µm cell strainer (BD Biosciences) followed by two washes with washmedium (DMEM/F12 medium with 15 mM HEPES, 2 mM L-glutamine,100 U/ml of penicillin and 0.1 mg/ml of streptomycin). The crypt mixtureswere suspended in Matrigel (Corning) and cultured in 50% L-WRN (L-cellexpressing Wnt3a, Rspondin3 and noggin) conditioned medium with10 µM of Y-27632 (ROCK inhibitor; R&D Systems) and 10 µMSB-431542 (TGF-βR inhibitor; R&D Systems). L-WRN conditionedmedia was prepared as described (Miyoshi and Stappenbeck, 2013). Cellswere passaged every 4-6 days by trypsinization, and fresh 50% L-WRNwasreplaced every 2 days. After five passages, cells were frozen in 8.3%DMSOin wash media and kept in liquid nitrogen. Prior to differentiation, cells werethawed and expanded in 50% L-WRN with 10 µM of Y-27632 and 10 µMSB-43154. To induce differentiation, 2.5×105 of trypsinized spheroids wereseeded in 6.5-mm PET Transwell inserts (Corning) precoated with 1%gelatin and cultured in 5% L-WRN with 10 µM of Y-27632 for 3 days.For mucus quantification studies, 1 µM of PGE2 or the same volume ofethanol control was supplement in 5% L-WRN during the last 24 h of thedifferentiation.

Single and repeated DSS injury modelThe single-injury protocol was adapted and modified from Oehlers et al.(2013). Batches of 60 larvae were segregated into Petri dishes in 50 ml ofegg water. Phenylthiourea (75 μM; Acros Organics) was added 24 h post-fertilization to prevent pigment-cell formation. To induce intestinal injury,3 days post-fertilization (dpf) larvae were placed in freshly prepared 0.1 or0.25% (w/v) colitis grade DSS (36,000-50,000 MW, MP Biomedical) for3 days. In the repeated-injury protocol, larvae were treated with 0.05, 0.1 or0.25% DSS at 5, 8 and 11 dpf for 24 h, followed by removal of DSS. Larvaewere not fed for the duration of the single DSS treatment experiments(6 dpf); the larvae were fed after 7 dpf with repeated DSS. Feeding wasskipped on the DSS treatment day.

Automated hematoxylin and eosin (H&E) stainingAn Autostainer XL (ST5010, Leica Biosystems, Nussloch, Germany) wasused for H&E staining. The automated staining protocol consisted of thefollowing steps: three changes of xylenes (2, 2 and 1 min), rehydration in anethanol/water gradient (100%, 100%, 95% ethanol for 2, 1 min and 20 s,respectively), followed by washing with deionized (DI) water (2 min). Therehydrated slides were stained with Gill 3 Hematoxylin (12 min; Sigma-Aldrich), washed with DI water (2 min), followed by 0.17% acid alcohol(1 min), washed with DI water (1 min), ammonia water (20 s), washed withDI water (1 min), and 95% ethanol (10 s). Staining with eosin (45 s; Sigma-Aldrich) was completed, followed by four washes with 100% ethanol (1 mineach), and four washes with xylene (1 min each). H&E-stained slides werecoverslipped with Cytoseal XYL (Thermo Fisher Scientific) and dried priorto imaging.

Neutral Red and Alcian Blue staining and image quantificationThe Neutral Red and Alcian Blue staining protocols were adapted andoptimized fromOehlers et al. (2013). Zebrafish larvae were stained live with2.5 μg/ml Neutral Red (ACROS, Bridgewater, NJ, USA) in egg water for5 h. The intensity of Neutral Red staining is non-saturated and consistentbetween 4 and 6 h (Fig. S2A). After moving stained larvae to fresh 50 mlegg water, larvae were anesthetized in Tricaine-S (Western Chemical),

Fig. 3. Effects of PGE2 and commonly utilized IBDmedications on barrierfunction, bacterial containment and LRE function. (A-C) PGE2 inducesmucin expression and release in a dose-dependent manner. (A) The images ofAlcian Blue staining with treatments of 0.1, 1 and 10 μM PGE2. Thequantification of the whole gut region is shown in B, and the red boxes indicatethe quantification areas in C. Scale bars: 100 μm. Alcian Blue intensities of thefull intestine (B) and the intestinal lumen (C) are shown (N=100 from threeclutches). **P<0.01. ***P<0.001. Error bar, mean±s.e.m. (D) Quantification ofrelative Alcian Blue intensity in a human enteroid differentiated epithelialmonolayer with and without 1 μM PGE2 treatment. ***P<0.001. N=3 patientswith three biopsies each. (E-G) Experiment design of DSS and PGE2treatment (E) for mortality assays (F) or ingested pHrodo-labeledE. coli proteinintensity (G). *P<0.05. **P<0.01. N=1145 from nine clutches. Error bar, mean±s.e.m. (G) Fluorescent intensity of ingested pHrodo-labeled E. coli proteins 1,2 and 3 h after removal from DSS. N=240 from three clutches. Error bar, mean±s.e.m. (H) Applying PGE2 (left panels), mesalamine (middle panels) and6-mercaptopurine (right panels) to the DSS injury model. The relative NeutralRed (top panels) and Cyto-ID (bottom panels) intensities with treatment alone(dark blue), with treatment during DSS injury for 24 h (light green) and with 5 hof treatment after DSS removal (light blue) are reported relative to untreatedcontrols. **P<0.01. ***P<0.001. Neutral-Red–PGE2, N=505 from threeclutches. Neutral-Red–mesalamine, N=695 from six clutches. Neutral-Red–6-mercaptopurine, N=705 from three clutches. Cyto-ID–PGE2, N=196 fromthree clutches. Cyto-ID–mesalamine, N=189 from three clutches. Cyto-ID–6-mercaptopurine, N=213 from three clutches. Error bar, mean±s.e.m.

Fig. 4. Effects of PGE2 on mucus production and barrier protection. Inhealthy intestine, the mucus layer prevents microbes/microbial products frommaking contact with the epithelial barrier. With DSS injury, the mucus isdepleted and the epithelial barrier is damaged, allowing microbes/microbialproducts access to the barrier. Escherichia coli incubation following DSStreatment yields increased E. coli in the intestine and increased bacterialcontact with the epithelial cells. PGE2 rescues loss of the mucus layer due toDSS injury, preventing E. coli from reaching the epithelial barrier.

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followed immediately by live imaging. For the zebrafish intestinal injurymodel, mucin was stained as the quantifiable metric. Larvae were fixed in4% paraformaldehyde at room temperature for 2 h, rinsed twice with acidalcohol (70% ethanol with 1% hydrochloric acid) and stained in 0.01%Alcian Blue (w/v, in 80% ethanol with 20% glacial acetic acid) for 3 hours atroom temperature. The Alcian Blue intensities were linear and non-saturatedat 2-4 h of staining (Fig. S4A,B). After rinsing with acid alcohol three times,fixed larvae were imaged on a thin layer of 5% agarose gel.

For Alcian Blue staining of differentiated human enteroids, cells werefixed in cold methanol-Carnoy solution (60% methanol, 30% drychloroform, 10% glacial acetic acid; Sigma) overnight and washed with1× phosphate-buffered saline (PBS; 10 mM PO4

3−, 137 mM NaCl and2.7 mM KCl) three times. Then, cells were kept in 1× PBS with 0.02%acetic acid solution for an additional 16 h at 4°C. Prior to Alcian Bluestaining, cells were kept in 3% acetic acid for 3 min at room temperature.Cells were stained with 1% Alcian Blue (in 3% acetic acid solution;ACROSOrganics) for 30 min at room temperature followed by threewashesin 1× PBS solution. Transwell membranes were cut and mounted influoromount G (Electron Microscopy Sciences) and visualized.

For both Neutral Red and Alcian Blue staining, the images of full gutand/or lumen were collected under 100× total magnification with theEVOS XL core microscope (Thermo Fisher Scientific). For AlcianBlue images, we extracted intensity from the blue channel before weperformed quantification. For both Neutral Red and Alcian Blue staining,we inverted the color of the image, traced the gut and/or lumen, ormonolayer cells from differentiated enteroids, and measured the meanintensity per pixel using ImageJ software. To remove background signals,we subtracted the mean intensity per pixel of images from the anal end of thegut. The relative expressions (%) were calculated by normalizing to theuntreated group. Statistical tests were done with two-tailed Wilcoxon-testin RStudio.

Bacterial infection and autophagyHeat-killed E. coli (K-12 strain) were conjugated either with BioParticle–Alexa-Fluor-488 conjugates or pHrodo Red E. coli BioParticlesConjugate for uptake studies (ThermoFisher, Waltham, MA, USA).Bacterial concentrations were titrated and optimized based on intestinalfluorescent intensity. Zebrafish larvae were incubated in 2 μg/μl (6×107

CFU/ml) for 1 hour. Larvae were rinsed and moved to egg water, withmortality and fluorescence measured at 0, 1, 2 and 3 h.

We used a Cyto-ID Autophagy detection kit 2.0 (Enzo Biochem) toanalyze autophagy with DSS and/or bacterial infection in live zebrafish.Zebrafish were incubated in 1 μl CYTO-ID per 500 μl egg water for 1 hourwith or without E. coli or DSS and washed with 50 ml egg water beforeimaging. The fluorescent images for quantification were collected afterE. coli treatment with the EVOS FL Cell Imaging System. Using ImageJsoftware, we measured the mean intensity per pixel for the gut andsubtracted this background for individual images. Auto-fluorescence wascorrected by comparing to the unstained fish gut. The relative expressions(%) were calculated by normalizing to the untreated group. Statistical testswere done with Wilcoxon-test in RStudio.

The confocal images of colocalization analysis and movies weregenerated with the ZEISS LSM 880 with Airyscan. For colocalization,Tg(mfap4:turquoise) fish were treated with pHrodo Red E. coli and stainedfor autophagy with CytoID. The confocal z-stack movies (Movies 1-4) andcell migration movies (Movie 5) were collected after 5 h of E. coli treatment.Systemic infection movies (Movies 6,7) were imaged with BioParticle–Alexa-Fluor-488 conjugate at 90 min after the end of E. coli treatment.

PGE2 and drugs commonly utilized in IBD treatmentPGE2 and IBD drugs were added directly to zebrafish media either with orwithout DSS at 5 dpf for co-treatment as a therapy. In the recovery groups,drugs were added after removing DSS, and imaging was performed 5 h later.The doses of drugs were tested and the highest doses without fish deathwere chosen. Final concentrations of PGE2 were 0.1 and 1 µM with equalamounts of ethanol used for controls. Mesalamine (5-aminosalicyclic acid)was dissolved in egg water at a concentration of 10 mg/ml and adjustedto pH 7 with dilute sodium hydroxide. 6-Mercaptopurine was at a

concentration of 35 mg/ml dissolved and diluted to the finalconcentrations with egg water, with pH adjusted to 7.

Gene expression analysis of zebrafish intestinesFrom anesthetized 6-dpf zebrafish larvae, whole intestines weremicrodissected and separated from the remaining carcasses, with eachcollected in 30 µl RLTBuffer (Qiagen). Total RNAwas isolated fromN=15-25 intestines or carcasses per sample by TRIzol extraction (LifeTechnologies). cDNA libraries were generated by reverse transcriptionusing the SuperScript cDNA synthesis kit (Quanta). Quantitative real-timePCR (qRT-PCR) was performed using PerfeCTa SYBR Green Fast Mix(Quanta) with the LightCycler 480 (Roche). Gene expression levels werenormalized to ribosomal protein large P0 (rppo) using the comparativethreshold cycle (ΔΔCt) method. Primer sequences are listed in Table S1.

Single-cell suspension of zebrafish intestines for flow cytometryAt 6 dpf, zebrafish were stained with LysoTracker Green DND-26(Invitrogen) at a 1:100 dilution for 1 h. Whole intestines were thendissected from anesthetized zebrafish (N=25 per group) and collected in1 ml MACS Rinsing Solution (Miltenyi Biotech) with 0.5% BSA (FisherScientific), followed by the single-cell suspension protocol (Bresciani et al.,2018). In short, samples were rocked at room temperature for 30 min beforebeing centrifuged at 700 g for 5 min. The cell pellet was suspended in PBSwith 1 mg/ml DNase I (Sigma Aldrich) and 1 mg/ml collagenase IV (SigmaAldrich), followed by harsh pipetting and incubation at room temperaturefor 5 min. Samples were centrifuged at 700 g for 5 min and the supernatantwas removed. The cell pellet was resuspended in DPBS 1× with 0.5% BSA(Gibco) before being strained through a 70-µm mesh. Single-cellsuspensions were flow sorted in the Mount Sinai Flow Cytometry CoREusing the BD influx cell sorter (BD Biosciences). RNAwas extracted usingthe RNAqueous kit (Invitrogen). cDNA synthesis and qPCR wereconducted as described in the Gene Expression Analysis section.

Immunoblot analysesAt 6 dpf, whole zebrafish larvae were homogenized in lysis buffer [20 mMTris pH 7.5, 150 mM NaCl, 1% NP-40, 2 mM EDTA, 10% glycerol, 1%BME, and protease inhibitors (Roche Complete)]. From each sample, 10 µgof protein was separated on a Mini-Protean TGX 4-15% (Bio-Rad) gel andtransferred to a PVDF membrane using a Bio-Rad electrophoretic transferapparatus (Trans-Blot Turbo, Bio-Rad). After blocking with 5% BSA, themembrane was probed with the primary antibodies anti-p62 (PM045,polyclonal, MBL; 1:1000) and anti-tubulin (12G10, DSHB; 1:2000). Asecondary incubation of either 1:40,000 dilution of horseradish peroxidase(HRP)-conjugated goat anti-mouse or 1:20,000 dilution goat anti-rabbitsecondary antibody was applied to the membrane. The immunoblots weredeveloped with SuperSignal West Pico (Life Technologies).

Colocalization analysis of LysoSensor and Neutral RedThe confocal images (Fig. S2B, top panel) of zebrafish intestine stained withLysoSensor and Neutral Red were taken with a Zeiss LSM 880 confocalmicroscope using 20× air lens (NA=0.8). The zoomed-in images (Fig. S2B,bottom panel) were generated from the non-saturated area in intestine ofthe original images using Zen 2.3 Lite (Zeiss). Colocalization analysisof both LysoSensor- and Neutral-Red-positive cells was performed usingImageJ Fiji software. The 2D intensity histogram and Pearson correlationcoefficient were generated by the Coloc2 plugin of Fiji.

Quantitation of E. coli intensity in cells of zebrafish dorsal aortaand cardinal veinZebrafish were treated in the presence or absence of 0.25% DSS for 24 hbefore being subjected to Alexa-Fluor-488-labeled heat-killed E. coliincubation (for 60 min). Movies were taken at 90 min post E. coliincubation. The E. coli fluorescence intensity of 170 of zebrafish cells werequantified using Zen 2.3 Lite software from Zeiss (38 cells for control-aorta:control treatment in dorsal aorta; 53 cells for DSS-aorta: DSS treatment indorsal aorta; 38 cells for control-vein: control treatment in posterior cardinalvein; 41 cells for DSS-vein: DSS treatment in posterior cardinal vein).P-values were tested by the Mann–Whitney test.

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Chemical inhibitor for autophagyBafilomycin A1 (Sigma Aldrich) was dissolved in DMSO to create a100 μM stock solution and then diluted to a concentration of 10 and 20 nMin egg water. Zebrafish larvae at 5 dpf were treated for 24 h, followed byCyto-ID and Neutral Red staining.

AcknowledgementsMicroscopy, intestinal sections and H&E staining were performed at the Microscopy,Biorepository and Pathology CoRE at the Icahn School of Medicine at Mount Sinaiwith assistance from Nikos Tzavaras, Lisette Conde and Alan Soto, respectively.Zebrafish were maintained and cultivated at the Zebrafish Shared Research Facility(Z-SRF) at the Icahn School of Medicine at Mount Sinai with assistance fromCharlesDerossi and Kathryn Bambino. The mfap4 transgenic zebrafish line Tg(mfap4:turquoise) was a generous gift from David Tobin at Duke University.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: L.-s.C., N.V., J.H.C.; Methodology: L.-s.C., N.-y.H., P.R.L., J.M.,S.N., G.B., M.C.M., T.H.T., J.C.; Software: L.-s.C., M.G.; Validation: L.-s.C., N.-y.H.,P.R.L., J.M.; Formal analysis: L.-s.C., P.R.L., J.M., M.G.; Investigation: L.-s.C.,N.-y.H., P.R.L., J.M., S.N., E.C.; Resources: L.-s.C., P.R.L., J.M., S.N., E.C., N.V.,J.A.F., G.B., M.C.M., T.H.T., J.C.; Data curation: L.-s.C., N.-y.H., P.R.L., J.M., S.N.,J.A.F.; Writing - original draft: L.-s.C., J.H.C.; Writing - review & editing: L.-s.C., J.M.,Y.S., J.C., J.H.C.; Visualization: L.-s.C., N.-y.H., P.R.L.; Supervision: L.-s.C., J.H.C.;Project administration: L.-s.C., P.R.L., Y.S., J.H.C.; Funding acquisition: L.-s.C.,J.H.C.

FundingThis study was supported by research grants from the Dr Sanford J. Grossman Trustfor Integrative Studies in IBD, National Institutes of Health [DK062429, DK062422,K08 DK101340, DK106593], Crohn’s and Colitis Foundation Visiting IBD ResearchFellowship, Gilead Sciences Liver Research Scholars Award, and TheMindich ChildHealth and Development Institute at the Icahn School of Medicine at Mount Sinai.

Supplementary informationSupplementary information available online athttp://dmm.biologists.org/lookup/doi/10.1242/dmm.037432.supplemental

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RESEARCH ARTICLE Disease Models & Mechanisms (2019) 12, dmm037432. doi:10.1242/dmm.037432

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