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1、ArticleMacrophages Maintain Epithelium Integrity byLimiting Fungal Product AbsorptionGraphical AbstractHighlightsdEpithelium in the distal colon requires macrophages(Ns)for survivaldDistal colon Ns insert balloon-like protrusions(BLPs)inthe epitheliumdBLPs sense fungal toxins within the fluids absor
2、bed throughepithelial cellsdBLP+Nspreventtheabsorptionoffungaltoxins,protectingthe barrier integrityAuthorsAleksandra S.Chikina,Francesca Nadalin,Mathieu Maurin,.,Iliyan D.Iliev,Danijela Matic Vignjevic,Ana-Maria Lennon-Dume nilCorrespondencedanijela.vignjeviccurie.fr(D.M.V.),ana-maria.lennoncurie.f
3、r(A.-M.L.-D.)In BriefProtrusions on distal colonicmacrophages orchestrate fluid sampling,which is critical to protect epithelial cellsfrom absorbing fluids enriched in fungitoxins/metabolites.Chikina et al.,2020,Cell 183,118October 15,2020 2020 Elsevier Inc.https:/doi.org/10.1016/j.cell.2020.08.048l
4、lArticleMacrophages Maintain EpitheliumIntegrity by Limiting Fungal Product AbsorptionAleksandra S.Chikina,1,2Francesca Nadalin,2,10,11Mathieu Maurin,2Mabel San-Roman,2Thibault Thomas-Bonafos,2Xin V.Li,3,4Sonia Lameiras,5Sylvain Baulande,5Sandrine Henri,6Bernard Malissen,6,7Livia Lacerda Mariano,8Jo
5、rge Barbazan,1J.Magarian Blander,4Iliyan D.Iliev,3,4Danijela Matic Vignjevic,1,9,*and Ana-Maria Lennon-Dume nil2,9,12,*1Institut Curie,PSL Research University,CNRS UMR 144,F-75005 Paris,France2Institut Curie,PSL Research University,INSERM U932,F-75005 Paris,France3Gastroenterology and Hepatology Div
6、ision,Joan and Sanford I.Weill Department of Medicine,Weill Cornell Medicine,Cornell University,New York,NY 10021,USA4The Jill Roberts Institute for Research in Inflammatory Bowel Disease,Weill Cornell Medicine,Cornell University,New York,NY 10021,USA5Institut Curie,PSL Research University,Next Gene
7、ration Sequencing Facility,F-75005 Paris,France6Centre dImmunologie de Marseille-Luminy,Aix Marseille Universite,INSERM,CNRS,13288 Marseille,France7Centre dImmunophe nomique,Aix Marseille Universite,INSERM,CNRS,13288 Marseille,France8Department of Immunology,INSERM U1223,Institut Pasteur,75015 Paris
8、,France9These authors contributed equally10Present address:European Molecular Biology Laboratory,European Bioinformatics Institute(EMBL-EBI),Wellcome Trust GenomeCampus,Hinxton,Cambridge CB10 1SD,UK11Present address:Center for Genomic Science of IITSEMM,Instituto Italiano di Tecnologia(IIT),20139 Mi
9、lan,Italy12Lead Contact*Correspondence:danijela.vignjeviccurie.fr(D.M.V.),ana-maria.lennoncurie.fr(A.-M.L.-D.)https:/doi.org/10.1016/j.cell.2020.08.048SUMMARYThe colon is primarily responsible for absorbing fluids.It contains a large number of microorganismsincluding fungi,which are enriched in its
10、distal segment.The colonic mucosa must therefore tightly regulatefluidinfluxtocontrolabsorptionoffungalmetabolites,whichcanbetoxictoepithelialcellsandleadtobarrierdysfunction.How this is achieved remains unknown.Here,we describe a mechanism by which the innate im-mune system allows rapid quality che
11、ck of absorbed fluids to avoid intoxication of colonocytes.This mech-anism relies on a population of distal colon macrophages that are equipped with balloon-like protrusions(BLPs)inserted in the epithelium,which sample absorbed fluids.In the absence of macrophages or BLPs,epithelial cells keep absor
12、bing fluids containing fungal products,leading to their death and subsequentloss of epithelial barrier integrity.These results reveal an unexpected and essential role of macrophages inthe maintenance of colon-microbiota interactions in homeostasis.INTRODUCTIONThe intestinal tract provides a unique e
13、nvironment because it iscontinuously exposed to food antigens as well as to thecommensal microbiota.It comprises different regions withdistinct anatomies and physiological roles.Maintenance of theintestinal barrier is critical given that it controls the absorptionof nutrients,electrolytes,and water
14、from the gut lumen into theblood circulation and further prevents the absorption of toxicluminalsubstances(Al-Bahranietal.,2010).Accordingly,disrup-tion of the intestinal barrier leads to multiple pathological situa-tions ranging from nutrient deprivation(Fishman et al.,2014)and inflammatory bowel d
15、iseases(Anbazhagan et al.,2018;Michielan and DInca,2015)to sepsis(Yoseph et al.,2016)andmulti-organ failure(Al-Bahrani et al.,2010;de Haan et al.,2009;Swank and Deitch,1996).Among the different regions of the intestine,the distal colonis particularly important for fluid absorption.On the one hand,th
16、e distal colon epithelium is exposed to the elevated osmoticpressure needed to induce water absorption by epithelial cellsto dehydrate and solidify feces(Naftalin,1994).On the otherhand,the distal colon faces a tremendous amount of microor-ganisms,including bacteria,fungi,archaea,and viruses(Do-nald
17、son et al.,2016;Flynn et al.,2018;Leonardi et al.,2018;Scupham et al.,2006).In particular,fungi are moreabundant in the distal colon and can produce metabolitesthat trigger apoptosis of intestinal epithelial cells(Uppermanet al.,2003).The colonic mucosa must therefore tightly regu-late fluid absorpt
18、ion to avoid potential entry of toxic fungalmetabolites into epithelial cells and further into the blood cir-culation.The mechanisms underlying such regulation remainunknown.llCell 183,118,October 15,2020 2020 Elsevier Inc.1Please cite this article in press as:Chikina et al.,Macrophages Maintain Epi
19、thelium Integrity by Limiting Fungal Product Absorption,Cell(2020),https:/doi.org/10.1016/j.cell.2020.08.048(legend on next page)ll2Cell 183,118,October 15,2020Please cite this article in press as:Chikina et al.,Macrophages Maintain Epithelium Integrity by Limiting Fungal Product Absorption,Cell(202
20、0),https:/doi.org/10.1016/j.cell.2020.08.048ArticleFluid absorption in the distal colon is achieved by a single-layered epithelium displaying a selective permeability.Perme-ability is controlled by the presence of(1)an abundant mucus,which consists of net-like polymers(Matsuo et al.,1997)andacts as
21、a physical mesh separating the colon luminal contentfrom the epithelium(Kamphuis et al.,2017),and(2)junctionalcomplexes,which seal the space between epithelial cells(Bal-lardetal.,1995).Fluidabsorptionthroughepithelialcellsistightlycoordinated by sodium pumps,ionic channels,transporters(e.g.,ENac,Na
22、/K-ATPase),and Aquaporins(Masyuk et al.,2002).Altogether,these components control the selective ab-sorption of nutrients,electrolytes,and water.This process,referredtoasintestinalpermeability,continuouslyadaptstofluc-tuations in the availability of nutrients and electrolytes and to thebody salt/wate
23、r balance(Mart nez et al.,2012;De Santis et al.,2015).Intestinal permeability is also regulated by the microbiota(Yu,2018;Martinez-Guryn et al.,2018)and immune cells(Daltonet al.,2006;Groschwitz et al.,2009;Musch et al.,2002).How thedialog between intestinal epithelial cells,microbes,and immunecells
24、isorchestratedtomaintainintestinalpermeabilityinhomeo-stasis remains unclear.In homeostatic conditions,one of the most abundant types ofintestinal immune cells are macrophages(Ms),which mainlydifferentiate from monocytes in response to local cues.IntestinalMs reside either within the lamina propria
25、or the muscle layer,where they participate in a variety of biological processes,including the degradation of microorganisms(Smith et al.,2011),silent clearance of apoptotic bodies(Cummings et al.,2016;Sisirak et al.,2016),tissue repair(Pull et al.,2005),andgastrointestinal motility(Muller et al.,201
26、4).Intestinal Ms alsolimit inflammation(Shouval et al.,2017;Ueda et al.,2010;Zig-mond et al.,2014)and facilitate the survival of local FOXP3+Tregulatory cells and tolerance to food(Brockmann et al.,2017;Mazzini et al.,2014).The function of Ms is tightly regulated bythe microbiota(Schulthess et al.,2
27、019)and,accordingly,theyare more abundant in the colon than in the small intestine(Den-ning et al.,2011;Nagashima et al.,1996).Interestingly,in the co-lon,Ms can be found in close association with epithelial cells(Kang et al.,2020;Nagashima et al.,1996).Disruption of Mssub-epithelial localization pa
28、rticipates in the loss of intestinalbarrier integrity observed in inflammatory bowel diseases suchas ulcerative colitis and Crohns disease(Nalle and Turner,2015).Ms are,therefore,ideally positioned to orchestrateepithelial cell-microbiota interactions for maintenance of colonhomeostasis.Here,we inve
29、stigated the role of Ms in the integrity and func-tion of the colon epithelium in homeostasis.We show that sub-epithelial Ms perform a rapid quality check of the fluids ab-sorbed through distal colon epithelial cells,protecting themfrom being poisoned by luminal fungal metabolites.RESULTSM Control E
30、pithelial Cell Survival and Barrier Integrityin the Distal ColonThe gut immune system is highly compartmentalized,withdifferent cell types distributed in gradients along the intestine(Mowat and Agace,2014).This particularly concerns Ms,whichare present in greater amounts in the colon as compared to
31、thesmall intestine.Colonic Ms display an anergic phenotype(Bainet al.,2013;Smythies et al.,2005)and their loss correlates withthe development of ulcerative colitis and Crohns disease(Rubioet al.,2018),suggesting a contribution to the maintenance ofepithelium integrity.However,the mechanism(s)by whic
32、h Msachieve such a homeostatic function in vivo remains unknown.To address this question,we depleted Ms using the CD64DTRmouse model(Baranska et al.,2018)and evaluated the stateof the epithelium(Figure 1A).We analyzed both proximal anddistal colons as they are known to exhibit differences both inphy
33、siology(Dolman and Edmonds,1975;Foster et al.,1986;Hardin et al.,1999)and microbiota composition(Flynn et al.,2018;Leonardi et al.,2018).The efficient depletion of colonicMs was verified by flow cytometry and immunostaining inboth colon segments(Figure S1A).Unexpectedly,we foundthat M depletion led
34、to massive apoptosis of epithelial cells inthe distal but not the proximal colon(Figures 1B and 1C).DeathFigure 1.Ms Are Required for Epithelial Cell Survival in the Distal Colon and Form Balloon-like Protrusions Inserted in betweenEpithelial Cells(A)Scheme of Nv depletion.CD64WTor CD64DTRlittermate
35、s received two injections of diphtheria toxin(DT)24 h apart.(B)Maximum z-projection(30 mm)of proximal and distal colon transversal sections 44 h after the first DT injection.Apoptotic cells were revealed with cleavedcaspase 3 staining(red),F-actin(green).Scale bar:50 mm.(C)Numberofapoptoticepithelia
36、l cellspercryptinthedistalorproximalcolon.Pooled datafromthreeindependent experiments;dotsrepresent averagenumberper individual mouse.Mean SEM,multiple comparison Kruskal-Wallis test,*p 0.05.(D)Serum fluorescence intensities 510 min after intra-rectal administration of hypotonic solution of hydrazid
37、e-AlexaFluor633.All mice were injected with DT.Pooled data from two independent experiments;dots represent average number per individual mouse.Mean SEM,Mann-Whitney test,*p 0.05.(E)Morphological differences of Nvs in the proximal and distal colon.Whole-mount staining of the distal and proximal colon
38、 of CD11c:Cre/R26mTmGmice.mGFP(green),CD11b(blue),CD103(red),membranetdTomato(gray).BLPsareindicatedwitharrows,theborderbetweenepitheliumandthestromaisindicatedwith the dashed line.Z-projections of 2040 mm;scale bars:50 mm.(F)Single Mv forming BLPs(left)or thin extensions(right).Yellow star:cell bod
39、ies;green arrows:BLPs;green arrowheads:extensions.Maximum z-projection of1015 mm;scale bar:2 mm.(G)Number of BLPs,normalized per crypt(left)or per Mv(right).Dots represent average number per individual mouse;left:pooled data from seven independentexperiments;right:pooled data from another two indepe
40、ndent experiments.(H)Number of Mvs in the proximal and distal colon,analyzed by imaging(F4/80+MHCII+CD103?cells per crypt;each dot represents average number per in-dividual mouse;data pooled from three independent experiments)and by flow cytometry(presented as percentage of CD45+cells;dots represent
41、 individualmouse;data pooled from four independent experiments).(I)Number of extensions,normalized per Mv.Dots represent average number per individual mouse;four independent experiments.In(GI),mean SEM,Mann-Whitney test,*p 0.05,*p 0.01,*p 0.0001.See also Figure S1 and S2,Video S1.llCell 183,118,Octo
42、ber 15,20203Please cite this article in press as:Chikina et al.,Macrophages Maintain Epithelium Integrity by Limiting Fungal Product Absorption,Cell(2020),https:/doi.org/10.1016/j.cell.2020.08.048Article(legend on next page)ll4Cell 183,118,October 15,2020Please cite this article in press as:Chikina
43、et al.,Macrophages Maintain Epithelium Integrity by Limiting Fungal Product Absorption,Cell(2020),https:/doi.org/10.1016/j.cell.2020.08.048Articleof epithelial cells in the distal colon was also observed whenMswere depleted by injecting anti-CSF1 receptor antibodies inC57BL/6J mice(Figures S1B and S
44、1C).Of note,if the presenceof apoptotic cells merely resulted from the lack of scavenging byMs,equal numbers of dead cells should be detected in distaland proximal colons(Figure 1C).Our results,therefore,suggestthatMsfacilitate the survival of epithelial cells in the distal colonspecifically.To eval
45、uate the impact of epithelial cell death on epitheliumpermeability,we infused CD64WTand CD64DTRmice intra-rectally with a hypotonic solution containing the small fluores-cent molecule hydrazide and measured its appearance in theblood.We found that hydrazide was more abundant in the bloodof M-deplete
46、d mice compared to control animals(Figure 1D),indicative of an increase in barrier permeability in these animals.Altogether,these results show that Ms are needed for epithelialcell survival and intestinal barrier integrity in the distal colon.Distal Colon M Insert Balloon-like Protrusions inbetween
47、Epithelial CellsThese results prompted us to investigate distribution of Ms andtheir physical interaction with epithelial cells in the proximal anddistalcolon.Toaddressthisquestion,weperformedwhole-mountimmunostainingofproximalanddistalcolonsectionsisolatedfromCD11c-Cre/R26mTmGmice(Caton et al.,2007
48、;Muzumdar et al.,2007).In this mouse model,all cells that express CD11cthroughout their differentiation,which include intestinal Ms(anddendritic cells,DCs),switch from membrane-tomato to mem-brane-GFP expression,allowing better visualization of thesephagocytes in the tissue.Using these animals,we ob
49、served a phagocyte population inthe distal colon that physically interacted with epithelial cellsthrough membrane protrusions shaped as balloons(Figures 1Eand 1F),which we will refer to as balloon-like protrusions(BLPs)hereafter.The cell bodies of these cells were localizedaround the opening of the
50、crypts(Video S1).On average,eachcell displayed three BLPs that crossed the basement membraneand contacted the base of epithelial cells.Of note,BLPs do notcorrespondtothetransepithelialdendritespreviouslydescribed in the small intestine(Rescigno et al.,2001;Vallon-Eberhard et al.,2006)as they do not