Gastrointestinal microbiota contributes to the development of murine transfusion-related acute lung injury

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1 REGULAR ARTICLE Gastrontestnal mcrobota contrbutes to the development of murne transfuson-related acute lung njury Rck Kapur, 1 Mchael Km, 2,3 Johan Rebetz, 1 Björn Hallström, 4 Jonas T. Björkman, 5 Alsa Takabe-French, 2,3 Noel Km, 2,3 Jonathan Lu, 2,3 Shanjeevan Shanmugabhavananthan, 2,3 Stefan Mlosevc, 2,3 Mark J. McVey, 2,3,6-9 Edwn R. Speck, 2,3 and John W. Semple 1-3,6, Dvson of Hematology and Transfuson Medcne, Lund Unversty, Lund, Sweden; 2 Keenan Research Centre for Bomedcal Scence and 3 The Toronto Platelet Immunobology Group, St. Mchael s Hosptal, Toronto, ON, Canada; 4 Center for Translatonal Genomcs, Lund Unversty, Lund, Sweden; 5 Center for Molecular Dagnostcs, Skåne Unversty Hosptal, Lund, Sweden; 6 Canadan Blood Servces, Toronto, ON, Canada; 7 Department of Anesthesa and 8 Department of Physology, Unversty of Toronto, Toronto, ON, Canada; 9 Department of Anesthesa and Pan Medcne, Hosptal for Sck Chldren, Toronto, ON, Canada; and 10 Department of Pharmacology, 11 Department of Medcne, and 12 Department of Laboratory Medcne and Pathobology, Unversty of Toronto, Toronto, ON, Canada Key Ponts Gastrontestnal flora contrbutes to development of antbody-medated murne TRALI. Depleton of gastrontestnal flora prevents TRALI by nhbtng MIP-2 secreton and pulmonary neutrophl accumulaton. Transfuson-related acute lung njury (TRALI) s a syndrome of respratory dstress upon blood transfuson and s the leadng cause of transfuson-related fataltes. Whether the gut mcrobota plays any role n the development of TRALI s currently unknown. We observed that untreated barrer-free (BF) mce suffered from severe antbody-medated acute lung njury, whereas the more sterle housed specfc pathogen-free (SPF) mce and gut flora-depleted BF mce were both protected from lung njury. The preventon of TRALI n the SPF mce and gut flora depleted BF mce was assocated wth decreased plasma macrophage nflammatory proten-2 levels as well as decreased pulmonary neutrophl accumulaton. DNA sequencng of amplcons of the 16S rbosomal RNA gene revealed a varyng gastrontestnal bacteral composton between BF and SPF mce. BF fecal matter transferred nto SPF mce sgnfcantly restored TRALI susceptblty n SPF mce. These data reveal a lnk between the gut flora composton and the development of antbody-medated TRALI n mce. Assessment of gut mcrobal composton may help n TRALI rsk assessment before transfuson. Introducton Transfuson-related acute lung njury (TRALI) s a serous complcaton of blood transfusons and s characterzed by the onset of acute respratory dstress resultng from pulmonary edema wthn 6 hours after transfuson. 1 At present, TRALI s the leadng cause of transfuson-related fataltes and, apart from supportve measures such as oxygen or ventlaton, no specfc therapes are avalable. The pathogeness of TRALI s ncompletely understood but generally, TRALI s hypotheszed to occur because of a 2-ht model. 2 The frst ht s recpent-predsposng factors, such as systemc nflammaton, whch can manfest tself as ncreased nterleukn (IL)-6, 3,4 IL or C-reactve proten (CRP) levels. 6,7 The second ht s subsequently conveyed by pathogenc antleukocyte antbodes or other bologcal response modfers present n the transfused donor blood. 2,8 In the majorty of the cases, human neutrophl antgen or HLA-specfc antbodes n the transfused blood are nvolved. 2 Usng anmal models of antbodymedated TRALI, t has become clear that neutrophls (polymorphonuclear cells [PMNs]) are the major effector cells n TRALI. 6,9-13 Moreover, usng a novel C57BL/6 knockout (KO) model of antbodymedated TRALI, we recently demonstrated that PMN-reactve oxygen speces are crtcally essental for nducng the acute lung njury n TRALI. 12 In addton, CD4 1 T regulatory cells and dendrtc cells were dentfed as mportant suppressor cells of antbody-medated TRALI and ths protectve response was assocated wth producton of the ant-nflammatory cytokne IL Smlarly, we recently found that Submtted 22 March 2018; accepted 22 May DOI / bloodadvances Presented at the 59th annual meetng of the Amercan Socety of Hematology, Atlanta, GA, 9-12 December The full-text verson of ths artcle contans a data supplement by The Amercan Socety of Hematology 10 JULY 2018 x VOLUME 2, NUMBER

2 plasma IL-10 levels were sgnfcantly lower n patents undergong TRALI reactons compared wth, for example, patents who suffered from septc acute lung njury. 14 In the current paper, we have further nvestgated the pathogeness of antbody-medated TRALI usng the murne C57BL/6 TRALI model based on CD4 1 T-cell depleton and followed by nfuson of ant-major hstocompatblty complex (MHC) class I antbodes. 12 We hypotheszed that the gut mcrobota composton may be a factor drvng the secreton of the PMN-chemoattractant macrophage nflammatory proten-2 (MIP-2) and pulmonary PMN recrutment durng antbody-medated TRALI n mce. To nvestgate ths, we housed mce n a barrer-free (BF) settng compared wth a specfc pathogen-free (SPF) envronment. SPF s a term used for laboratory anmals that are guaranteed free of partcular pathogens. The populaton s therefore checked for the presence of antbodes aganst the specfed pathogens. BF housng, on the other hand, also ensures the health of the anmals but provdes a less strngent pathogen-free envronment and consequently reflects a less sterle housng envronment compared wth SPF housng. Ths may, for nstance, be reflected by the strct adherence to closed, nternally ventlated cages and/or wearng more protectve clothng n an SPF settng compared wth open cages and/or less protectve covers n a BF settng. We demonstrate that SPF mce as well as BF mce that were depleted of ther gastrontestnal (GI) flora by broad spectrum antbotcs are protected from antbody-medated TRALI development by mparment of plasma MIP-2 secreton and pulmonary PMN recrutment and have a dfferent GI mcrobota composton compared wth BF mce, whch are susceptble to TRALI. These results suggest that the GI mcrobota composton contrbute to the susceptblty to murne antbody-medated TRALI. Methods Mce BF-housed C57BL/6 (H-2b, C57BL/6NCrl) mce were obtaned from Charles Rver Laboratores (Montreal, QC, Canada) and SPF-housed C57BL/6 (H-2b, C57BL/6NCrl) mce were obtaned from Charles Rver Laboratores (Sulzfeld, Germany). IL-10 KO mce (B6.129P2-Il10tm1Cgn/J, background stran C57BL/6) were obtaned from The Jackson Laboratory (Bar Harbor, ME) and housed n an SPF settng. All mce were males, 8 to 10 weeks of age, and housed for at least 1 week n the desgnated anmal faclty before ntatng experments. All anmal studes were approved by the St. Mchael s Hosptal Anmal Care Commttee, Toronto, and the Anmal Ethcs Commttee of Lund Unversty, Lund, Sweden. Antbodes and reagents TRALI nducton antbodes: s (monoclonal mouse IgG2a that reacts wth murne H-2Kd and H-2Dd MHC class I molecules) and AF (monoclonal mouse IgG2a that reacts to murne MHC class I H-2Kb), as well as the n vvo CD4 1 Tcell depletng antbody: GK1.5 (ant-cd4, rat IgG2b) were all purchased from Bo X Cell (West Lebanon, NH); RM4-5 (ant-mouse CD4-PE, ant-mouse CD4-FITC) was purchased from BD Pharmngen, San Dego, CA. Antbotc drnkng water: sucrose, vancomycn, ampclln sodum salt, neomycn trsulfate salt, and metrondazole were all purchased from Sgma-Aldrch, Oakvlle, ON, Canada. Pretestng and the antbody-medated murne TRALI model Mce were randomzed nto the ndcated groups and rectal temperatures were taken drectly upon arrval as well as after 1 week of housng n the ndcated anmal faclty. TRALI was nduced as prevously descrbed. 12 Brefly, 18 hours before TRALI nducton, mce were njected ntrapertoneally wth the monoclonal antbody GK1.5 (4.5 mg/kg) to deplete CD4 1 T cells. On the day of experment, the CD4-depleted mce were njected IV wth 600 ml ofamxtureof the TRALI-nducng antbodes s (45 mg/kg) and AF (4.5 mg/kg). Subsequently, rectal temperatures, ndcatve of systemc shock, were recorded every 30 mnutes (up to 90 mnutes) usng an RET-3 rectal probe for mce (ADInstruments Lmted, Oxford, Unted Kngdom) connected to a traceable dgtal thermometer (VWR Internatonal, Stockholm, Sweden). Nnety mnutes after TRALI ntaton, mce were anesthetzed wth a mxture of Ketamnol and Rompun and exsangunated by cardac puncture wth blood drawn nto 100 ml of PBS/ctrate phosphate dextrose adenne. The study desgn, ncludng a schematc overvew of the antbody-medated TRALI model, s descrbed n the supplemental Methods. In ndcated experments, mce were prmed 18 hours before TRALI nducton wth 0.1 mg/kg lpopolysaccharde (LPS) ntrapertoneally (from Eschercha col O55:B5, Sgma-Aldrch Sweden AB). In the ndcated experments, SPF mce were prmed twce a day, 2 days before TRALI nducton, ntrarectally wth 100 ml of BF fecal matter dssolved n sterle PBS. Antbotc treatment and bacteral depleton Selected mouse groups were depleted of both aerobc and anaerobc bactera by admnstraton of a combnaton of broad spectrum antbotcs (vancomycn, ampclln, neomycn, and metrondazole, all at 1 mg/ml) n drnkng water (contanng 1.75% weght-to-volume sucrose) that was changed every 48 hours for 1 week. Further detals on assessment of bacteral depleton are descrbed n the supplemental Methods. Tssue processng See supplemental Methods. Lung W/D weght ratos Lung wet/dry (W/D) weght ratos are a measure of pulmonary edema and were determned as prevously descrbed. 6,10-13 Brefly, the rght lung of each mouse was removed and weghed to determne the wet weght and then dred n an oven at 60 C for 48 hours. The dred samples were reweghed to obtan the dry weght. The lung W/D weght rato was calculated by the formula: net wet weght/net dry weght. Pulmonary PMN enumeraton The red blood cell lysed lung cell suspensons were mounted on mcroscope sldes (VWR Frosted Economy sldes) usng a Shandon Cytospn 4 (ThermoFsher Scentfc, Nepean, ON, Canada) followed by stanng wth a hematoxyln and eosn kt (Harleco-Hemacolor; EMD Chemcals, Darmstadt, Germany). The sldes were examned under Permount (Fsher Scentfc Company, Ottawa, ON, Canada) usng a Nkon Eclpse E800 mcroscope equpped wth a 340/0.75 objectve lens, n a blnded manner. Pulmonary PMN were evaluated by countng the total number of nucleated cells n 4 randomly selected and nonoverlappng felds and PMN numbers were 1652 KAPUR et al 10 JULY 2018 x VOLUME 2, NUMBER 13

3 enumerated by usng ImageJ software n a blnded manner. The pulmonary PMN percentage was determned by the formula: average number of observed PMN (n 4 felds)/average number of nucleated cells (n 4 felds) Lung tssue hstology A segment of the left lung was fxed n 10% formaln soluton and the fxed sample was embedded nto paraffn and sectoned wth a mcrotome. The sectons were staned wth hematoxyln and eosn usng a Leca AutoStanerXL (Leca Bosystems, Nussloch, Germany). The staned sldes were examned under Permount (Fsher Scentfc Company) usng a Nkon Eclpse E800 mcroscope equpped wth a 320 and a 340/0.75 objectve lens. Cytokne measurements Collected blood was centrfuged twce at 2500g for 15 mnutes (wthout brake) and plasma was collected, alquoted, and stored at 280 C for subsequent batch analyss. The thawed plasma was analyzed for murne MIP-2 and IL-10 usng sold phase sandwch enzyme-lnked mmunosorbent assay (ELISA) kts (Mouse CXCL2/MIP-2 Quantkne ELISA Kt and Mouse IL-10 Quantkne ELISA Kt; R&D Systems, Mnneapols, MN), accordng to the manufacturer s protocol. Rectal temperature (ºC) Arrval NS * 1 week housng 1 week housng Antbotcs NS Arrval 1 week housng Mcrobal proflng usng 16S rbosomal RNA gene amplcon sequencng DNA was solated from fecal matter collected from ndcated mouse groups, accordng to the manufacturer s protocol (QIAamp DNA stool mn kt; Qagen, GmbH, Hlden, Germany). Sequencng was performed accordng to the Illumna 16S metagenomc sequencng lbrary preparaton gude (Illumna part # Rev. B), as descrbed n the supplemental Methods. Statstcal analyss The specfc statstcal tests used are lsted n each of the fgure legends. Statstcal sgnfcance was set at P,.05 and statstcal analyss was performed usng GraphPad Prsm 7.03 software for Wndows (GraphPad Software, San Dego, CA). Results BF mce dsplay elevated body temperatures ndcatve of a dfferng GI bacteral composton It has been descrbed that GI flora can upregulate body temperature n mce 15 ; therefore, we assessed temperatures n the ndcated mouse groups. Compared wth the SPF-housed mce, after 1 week of housng, the temperatures of BF mce sgnfcantly ncreased (Fgure 1). Moreover, the rectal temperatures of BF mce were sgnfcantly hgher compared wth the SPF mce after 1 week of housng ( C vs C, respectvely; Fgure 1, column 2 vs 5). The rectal temperatures remaned elevated n BF mce for multple weeks after arrval, even up to 28 weeks after arrval (supplemental Fgure 1). To further confrm f ths ncreased temperature was related to ther GI bacteral composton, we treated the BF mce wth antbotcs on the day of ther arrval. The 1-week rectal temperatures n the antbotc-treated BF mce dd not elevate compared wth the untreated BF mce (Fgure 1, column 1 vs 3). The antbotc-related statc temperatures were assocated wth at least a 75% depleton of both aerobc and anaerobc GI flora (supplemental BF-housng SPF-housng Fgure 1. Rectal temperatures are ncreased n BF mce compared wth SPF mce and BF mce wth antbotcs-nduced gut flora depleton. Rectal temperatures of C57BL/6 mce upon arrval and after 1 week of housng n a BF or SPF envronment. BF-housed mce that were housed for a week were also subsequently treated wth broad spectrum antbotcs for a week. For the statstcal analyses, only sgnfcant comparsons of nterest are shown. Statstcal analyss was performed wth a 1-way analyss of varance (ANOVA) wth Dunn multple comparson s test. Each dot represents 1 mouse; error bars represent standard devaton (SD). Fgure shows combnatoral data from 3 experments. *P,.05, P,.01, P, NS, nonsgnfcant. Fgure 2). These data ndcate that hgher body temperatures n BF mce compared wth antbotc-treated BF mce and SPF mce were due to ther GI bacteral composton. We then nvestgated whether ths dfference n GI flora could affect TRALI dsease-susceptblty. GI bacteral composton determnes TRALI ntaton Based on a prevously establshed murne TRALI model, 12 SPF mce and BF mce (ether treated or not wth antbotcs) were CD4 1 T cell depleted and exposed to the TRALI nducng antbodes. In a tme course experment, at both 60 and 90 mnutes after antbody nfuson, the rectal temperatures of untreated BF mce were sgnfcantly lower compared wth SPF mce or BF mce treated wth antbotcs (Fgure 2A). The low body temperatures n the untreated BF mce correlated wth a sgnfcant ncrease n lung W/D ratos, ndcatng acute lung njury (Fgure 2B; lung W/D, 5.77) compared wth SPF mce (lung W/D, 4.62) or BF mce treated wth antbotcs (lung W/D, 4.49) or mce from the same suppler as used n the BF settng (vendor A) housed n an SPF settng (lung W/D, 4.71). Hstologcal analyses confrmed sgnfcant acute lung njury n BF-housed mce ncludng ncreased alveolar septal thckenng, acute nflammatory nfltrates, and alveolar exudates ndcatve of pulmonary edema (Fgure 3B-). Furthermore, IL-10 KO mce that 10 JULY 2018 x VOLUME 2, NUMBER 13 GASTROINTESTINAL FLORA DETERMINES TRALI RESPONSES 1653

4 A B Rectal temperature (ºC) Lung W/D weght rato BF SPF BF Antbotcs 0' 30' 60' 90' Tme post antbody njecton (mn) NS NS NS Groups BF vs SPF BF vs BF Antbotcs BF vs SPF BF vs BF Antbotcs Tme P value Fgure 2. SPF and BF mce wth antbotcs-nduced gut flora depleton are protected from antbody-medated TRALI. (A) Rectal temperatures, (B) lung W/D weght ratos upon CD4 1 T-cell depleton, and ant-mhc class I antbody (clones s and AF ) nfuson nto ndcated mouse groups. Vendor A and B ndcate 2 vendors for laboratory mce. For the statstcal analyses, only sgnfcant comparsons of nterest are shown. Statstcal analyss was performed wth 1-way ANOVA wth a Tukey post hoc test at 60 and at 90 mnutes, as depcted n the accompanyng table n panel A and 1-way ANOVA wth a Tukey s post hoc test (B). Data are based on 5, 13, and 10 mce per tme pont for BF, SPF, and BF antbotc mce, respectvely. (B) Each dot represents 1 mouse; error bars represent SD. Fgure represents combnatoral data from 3 experments. P,.01, P, BF (Vendor A) SPF (Vendor B) BF Antbotcs (Vendor A) SPF (Vendor A) were prevously found to be hypersenstve to antbody-medated TRALI nducton n a BF settng 12 were resstant to TRALI nducton when housed n an SPF settng (Fgures 3F- and 4). TRALI protecton from decreased GI flora s assocated wth reduced MIP-2 chemokne producton and pulmonary PMN accumulaton Plasma levels of MIP-2, a potent PMN chemoattractant (murne equvalent of human IL-8), were analyzed because t was been prevously shown that MIP-2 s essental for antbody-medated TRALI nducton. 11 Compared wth TRALI-protectedSPFmceandantbotctreated BF mce, only the untreated BF mce undergong TRALI reactons had sgnfcantly hgher plasma levels of MIP-2 (Fgure 5A; pg/ml). Smlarly, low levels of MIP-2 were also found n the plasma of the TRALI-protected SPF-housed IL-10 KO mce (Fgure 5A). When the numbers of pulmonary PMN were enumerated, the baselne pulmonary PMN were found to be 23% on average n BF-housed mce vs 6% n SPF-housed mce. Subsequently, sgnfcant ncreases n pulmonary PMN were observed only n the TRALIresponsve BF mce ( %) compared wth TRALI-protected SPF mce (28 6 9%) and antbotc-treated BF mce (25 6 5%) (Fgure 5B). Smlar fndngs were observed n TRALI-protected SPF IL-KO mce ( %) (Fgure 5B). Pathogenc TRALI response can be sgnfcantly restored by prmng SPF mce wth LPS before nducng TRALI To nvestgate f we could restore the pathogenc TRALI response n SPF-housed mce, we frst prmed the mce wth a low dose of LPS (0.1 mg/kg), a gram-negatve bacteral endotoxn that has been used n other TRALI mouse models as a prmng factor. 16 When the SPF mce were prmed wth low-dose LPS, depleted of ther CD4 1 cells, and nfused wth TRALI antbodes, the pathogenc TRALI reacton was sgnfcantly restored, as s demonstrated by ncreased lung W/D weght ratos, ncreased MIP-2 levels (supplemental Fgure 3), ncreased pulmonary PMN, and sgns of acute lung njury on hstology (Fgure 6). TRALI-susceptble BF mce dsplay a dfferent GI mcrobome composton than TRALI-resstant SPF mce To further confrm f the dfferences n TRALI susceptblty between BF and SPF mce may ndeed be due to the GI flora, we characterzed the gut mcrobome by solatng DNA from fecal samplesfrombfaswellasspfmceandperformed16srbosomal RNA gene amplcon sequencng The phylogenetc tree of the gut 1654 KAPUR et al 10 JULY 2018 x VOLUME 2, NUMBER 13

5 Fgure 3. SPF and BF mce wth antbotc-nduced gut flora depleton do not dsplay sgns of severe antbody- A medated acute lung njury on lung tssue hstology. Lung hstology from ndcated mouse groups. Panels A-F and A-F represent lung tssue mages taken at orgnal WT-BF magnfcaton 320 and 340, respectvely (hematoxyln and eosn stan). Representatve mages of each ndcated group are shown. Scale bars represent 100 mm (A-F) and 50 mm (A-F). WT, wld type. B CD4-depleton s AF BF C CD4-depleton s AF BF Antbotcs D WT-SPF E CD4-depleton s AF SPF F IL-10 KO mce s AF SPF mcrobota shows a dfferent clusterng pattern of fecal bactera n BF mce that are susceptble to TRALI compared wth SPF mce (and BF antbotc-treated mce), whch were resstant to TRALI development (Fgure 7A). Heat maps and pe charts of famly 10 JULY 2018 x VOLUME 2, NUMBER 13 (Fgure 7B-C), genus (Fgure 7D), and speces levels (Fgure 7E) llustrate the presence of a dfferent bacteral envronment n the gut of BF vs SPF mce. Further analyses at the level of bacteral speces ndcated that the anaerobc gram-negatve bacterum GASTROINTESTINAL FLORA DETERMINES TRALI RESPONSES 1655

6 5.5 NS A Lung W/D weght rato MIP-2 (pg/ml) Nave IL-10 KO TRALI-antbodes Fgure 4. SPF IL-10 KO mce are protected from antbody-medated TRALI. Lung W/D weght ratos upon ant-mhc class I antbody (clones s and AF ) nfuson nto IL-10 KO mce. IL-10 KO status was confrmed by performng an IL-10 ELISA usng collected plasma, as descrbed n the Methods. Statstcal analyss was performed usng an unpared Student t test. Each dot represents 1 mouse. Fgure shows combnatoral data from 2 experments. Bacterodes caccae s sgnfcantly ncreased n the TRALI-susceptble BF-housed mce whereas, n contrast, ths bacterum was not present n TRALI-resstant SPF-housed mce (supplemental Fgure 4). Fecal transfer from BF mce to SPF mce sgnfcantly restores TRALI susceptblty To drectly nvestgate f BF fecal transfer could restore TRALI susceptblty n SPF mce, SPF mce were prmed wth fecal matter from BF mce. When the prmed SPF mce where depleted of ther CD4 1 T cells and nfused wth TRALI antbodes, the pathogenc TRALI reacton was sgnfcantly restored, as s demonstrated by ncreased lung W/D weght ratos (Fgure 7F) and ncreased pulmonary PMN (Fgure 7G). Dscusson The GI flora has been shown to be altered n many dsease states, such as cardovascular dsease, 17 colts, 18 obesty, 19 malgnancy, 20 type 2 dabetes, 21 psychatrc dsorders (anxety, depresson, schzophrena), 22,23 neurodegeneraton (murne model of Parknson dsease), 24 neurodevelopmental abnormaltes n murne offsprng, 25,26 and asthma. 27 In the current study, we therefore specfcally nvestgated f the GI flora may also be of mportance for the pathogenc responses n antbody-medated TRALI. We descrbe an assocaton between the GI mcrobota and the onset of TRALI usng our prevously establshed TRALI model n C57BL/6 mce. 12 We drectly compared mce housed n an SPF settng (restrcted pathogen envronment) vs those housed n a BF settng (less restrcted pathogen envronment). Strkngly, mce housed n a BF settng dsplayed hgher baselne rectal temperatures and hgher baselne pulmonary PMN levels compared wth mce housed n the SPF envronment (Fgures 1 and 5B, respectvely). Ths dfference n temperature was not related to the temperature n the anmal rooms (all housed at 21 C room temperature) but was found to be related to the GI mcrobota n BF mce, whch was n accordance wth a prevous study. 15 In that study, Kluger and colleagues demonstrated B Pulmonary neutrophls (%) 0 BF-housng SPF-housng CD4-depleton: s AF : Antbotcs: IL-10 KO: BF-housng SPF-housng CD4-depleton: s AF : Antbotcs: IL-10 KO: Fgure 5. Protecton from TRALI s assocated wth decreased levels of plasma MIP-2 and decreased pulmonary PMN n both SPF and BF mce wth antbotcnduced gut flora depleton. (A) Plasma MIP-2 and (B) pulmonary PMN levels n ndcated mouse groups. For the statstcal analyses, only sgnfcant comparsons of nterest are shown. Statstcal analyss was performed wth 1-way ANOVA wth a Tukey post hoc test. Each dot represents 1 mouse; error bars represent SD. Fgure panels show combnatoral data from 3 experments. *P,.05, P,.01, P, that GI flora n BF-rased mce had a stmulatory effect on body temperature (unrelated to physcal actvty) compared wth germfree mce. 15 In addton to the untreated SPF mce, we examned BF mce that were frst treated wth broad spectrum antbotcs, a wdely establshed technque to sgnfcantly deplete the gut flora (supplemental Fgure 2) We observed that after depleton of the GI flora n BF mce (supplemental Fgure 2), ther rectal temperatures remaned statc after housng for 1 week (Fgure 1). Antbotcs are known to have a sgnfcant mpact on GI flora by decreasng ts densty and modfyng ts composton n a long-lastng fashon. 32 Ths can cause reduced sgnalng to the GI mucosa and perpheral organs and mpar the functon of the mmune system. * * 1656 KAPUR et al 10 JULY 2018 x VOLUME 2, NUMBER 13

7 A C * Lung W/D weght rato SPF-C57BL/6 mce WT LPS CD4-depleton v 4.3 LPS: CD4-depleton: Isotype Mouse IgG2a: s AF : LPS CD4-depleton Isotype Mouse IgG2a v v B Pulmonary neutrophls (%) SPF-C57BL/6 mce * LPS CD4-depleton s AF v v 0 LPS: CD4-depleton: Isotype Mouse IgG2a: s AF : Fgure 6. Prmng wth low-dose LPS restores TRALI n SPF mce. (A) Lung W/D weght ratos, (B) pulmonary PMN numbers, and (C) lung tssue hstology analyses n LPS-prmed mce (0.1 mg/kg) upon CD4 1 T-cell depleton and ant-mhc class I antbody (clones s and AF ) nfuson nto ndcated mouse groups. (C) Lung tssue mages taken at orgnal magnfcaton 320 (,,v,v) and 340 (,v,v,v) (hematoxyln and eosn stan). Representatve mages of each ndcated group are shown. Scale bars represent 100 mm (,,v,v) and 50 mm (,v,v,v). For the statstcal analyses, only sgnfcant comparsons of nterest are shown. Statstcal analyss was performed wth 1-way ANOVA wth a Tukey post hoc test. (A-B) Each dot represents 1 mouse; all error bars represent SD. Fgure panels show combnatoral data from 3 experments. *P,.05, P,.01, *P,.001, P, Wth regard to how gut flora affects the lungs, lttle s known about underlyng nflammatory dsorders of the gut that coexst wth nflammatory pulmonary dsorders. For nstance, t was reported that 33% of 133 patents sufferng from rrtable bowel syndrome had respratory problems and that 16% of these patents suffered from asthma. 33 In addton, patents wth chronc obstructve pulmonary dsorder were found to have a 3 tmes hgher rsk of developng Crohn dsease and ulceratve colts compared wth healthy controls. 34 Because of the heterogeneous composton of the GI flora, ts general depleton may cause ether benefcal or adverse effects dependng on the specfc clncal scenaro. For example, t was reported that compared wth untreated mce, 20% of gut flora depleted mce ded of pneumona nfecton wthn 50 hours. 35 Ths suggests that the gut mcrobota elcts a protectve effect for pneumona and that perhaps specfc antbotc therapy may be able to treat specfc lung nfectons whle leavng commensal GI flora ntact. On the other hand, t was recently reported that the lung mcrobota n both sepss and acute respratory dstress syndrome (ARDS) s enrched for bactera that are usually found n the lower GI tract. 36 Moreover, the abundance of gut bactera n human ARDS samples correlated wth dsease severty, ndcatng the gut s mcrobota plays a pathogenc role. 36 We found that n contrast to the BF mce, untreated SPF mce or BF mce treated wth antbotcs (gut flora depleted) were protected from antbody-medated TRALI (Fgures 2 and 3). Ths pathogenc TRALI response was not mousevendor dependent, but was determned by the housng condtons (Fgure 2B). Interestngly, IL-10 KO mce housed n a BF envronment were prevously found to be hypersenstve to antbodymedated TRALI. 12 In contrast, we found that f the IL-10 KO mce were housed n an SPF envronment, they became resstant to TRALI development (Fgure 4). Ths suggests that the gut flora, more mportantly than IL-10 levels, contrbute to the pathogenc 10 JULY 2018 x VOLUME 2, NUMBER 13 GASTROINTESTINAL FLORA DETERMINES TRALI RESPONSES 1657

8 A BF - TRALI Color Key BF - Antbotcs NO TRALI B SPF - NO TRALI Column Z-score Lachnospraceae (Unassgned) Rumnococcaceae Porphyromonadaceae Bacterodaceae Eryspelotrchaceae Rkenellaceae Lactobacllaceae Prevotellaceae Corobacteraceae Desulfovbronaceae Eubacteraceae Sutterellaceae Rhodosprllaceae Peptostreptococcaceae Enterobacteraceae Helcobacteraceae Bfdobacteraceae Anaeroplasmataceae Verrucomcrobaceae Mycoplasmataceae Deferrbacteraceae Clostrdaceae_1 C BF-arrval BF-1 week housng SPF-arrval SPF-1 week housng 50 % 23 % 14 % 11 % 3 % Lachnospraceae Bacterodaceae Rumnococcaceae Others Porphyromonadaceae 39 % 26 % 12 % 12 % 11 % Lachnospraceae Bacterodaceae Rumnococcaceae Others Porphyromonadaceae 43 % 15 % 11 % 9 % 7 % 6 % 5 % 5 % 0 % Porphyromonadaceae Lachnospraceae Prevotellaceae Others Rkenellaceae Bacterodaceae Rumnococcaceae Verrucomcrobaceae Helcobacteraceae 29 % 16 % 13 % 12 % 11 % 10 % 5 % 3 % 1 % Lachnospraceae Bacterodaceae Rumnococcaceae Porphyromonadaceae Helcobacteraceae Others Rkenellaceae Prevotellaceae Verrucomcrobaceae Fgure 7. (Contnued) KAPUR et al 10 JULY 2018 x VOLUME 2, NUMBER 13

9 D Clostrdum_XIVa Oscllbacter Bacterodes Clostrdum_IV Clostrdum_XIVb Flavonfractor Alstpes Lactobacllus Acetatfactor Rosebura Parabacterodes Rumnococcus Saccharbactera_genera_ncertae_seds Paraprevotella Anaerotruncus Eubacterum Eryspelotrchaceae_ncertae_seds Parasutterella Clostrdum_lll Enterorhabdus Helcobacter Prevotella Romboutsa Coprococcus Desulfovbro Parvbacter Turcbacter Blophla Clostrdum_XVIII Bfdobacterum Akkermansa Anaeroplasma Clostrdum_ sensu_ strcto Mycoplasma Odorbacter Mucsprllum Eschercha/Shgella Lachnospracea_ncertae_seds E Oscllbacter_valercgenes Flavonfractor_plaut Clostrdum_lactatfermentans Acetatfactor_murs Clostrdum_vrde Anaerotruncus_colhomns Paraprevotella_clara Parabacterodes_dstasons Clostrdum_colnum Clostrdum_leptum Bacterodes_xylansolvens Eubacterum_coprostanolgenes Parasutterella_excrementhomns Bacterodes_unforms Clostrdum_scndens Eubacterum_sraeum Alstpes_onderdonk Coprococcus_comes Lactobacllus_tawanenss Rumnococcus_brom Lactobacllus_anmals Enterorhabdus_mucoscola Eubacterum_dolchum Clostrdum_aerotolerans Bacterodes_acdfacens Clostrdum_methylpentosum Parabacterodes_goldsten Parabacterodes_merdae Alstpes_shah Mycoplasma_sualv Anaeroplasma_bactoclastcum Alstpes_ndstnctus Rumnococcus_champanellenss Rumnococcus_flavefacens Turcbacter_sanguns Alstpes_fnegold Parvbacter_caeccola Akkermansa_mucnphla Mucsprllum_schaedler Eubacterum_desmolans Bacterodes_nord Bacterodes_fragls Clostrdum_cocleatum Eschercha_ferguson Blophla_wadswortha Helcobacter_typhlonus Bacterodes_caccae Lactobacllus_ntestnals Eubacterum_ventrosum Bacterodes_dore Helcobacter_mastomyrnus Fgure 7. (Contnued). 10 JULY 2018 x VOLUME 2, NUMBER 13 GASTROINTESTINAL FLORA DETERMINES TRALI RESPONSES 1659

10 F 4.7 * G 60 Lung W/D weght rato Pulmonary neutrophls (%) BF-fecal transfer: CD4-depleton: s AF : - 0 BF-fecal transfer: CD4-depleton: s AF : - Fgure 7. BF and SPF mce demonstrate a dfferent gastrontestnal mcrobome composton and prmng wth BF fecal matter restores TRALI-susceptblty n SPF mce. (A) Phylogenetc tree of BF mce, whch are susceptble to TRALI (BF TRALI); SPF mce, whch do not suffer from TRALI (SPF NO TRALI); and BF mce treated wth antbotcs, whch are also resstant to TRALI (BF antbotcs NO TRALI). (B-C) Gastrontestnal bacteral heat maps and pe charts on famly level, respectvely. (D-E) Gastrontestnal bacteral heat maps on genus and speces levels, respectvely. (F-G) Data from BF fecal transfer nto SPF mce: (A) lung W/D weght ratos, (B) pulmonary PMN numbers upon CD4 1 T-cell depleton and ant-mhc class I antbody (clones s and AF ) nfuson nto ndcated mouse groups. Heat map row numbers 1-3, BF mce upon arrval; 4-6, BF mce after 1 week of housng; 7-9, SPF mce upon arrval; and 10-12, SPF mce after 1 week of housng. (F-G) Analyzed by a 1-taled unpared Student t test and represent combnatoral data from 2 experments. Each dot represents 1 mouse; error bars represent SD. *P,.05, P,.01. TRALI response. It was prevously shown that ncreased levels of MIP-2 and pulmonary PMN accumulaton n mce are key early processes that are responsble for ntatng antbody-medated TRALI reactons. 6,10-13 Ths was underlned by experments llustratng that mce were fully protected from antbody-medated TRALI when ther PMN were depleted n vvo. 12 We prevously found that the ant-mhc class I antbody bndng to ts cognate antgen on blood monocytes s a requred trgger for MIP-2 chemokne producton durng murne TRALI. 11 In lne wth ths, we observed that GI flora depleton or SPF housng appears to block the secreton of the PMN-chemoattractant MIP-2 and subsequently nhbts PMN nflux nto the lungs (Fgure 5), thereby preventng TRALI nducton (Fgures 2 and 3). Prmng of the SPF mce wth LPS, however, was able to sgnfcantly restore antbody-medated TRALI (Fgure 6). Ths may reflect that stmulatory sgnalng pathways may be dfferent n an LPS settng vs a more complex gut mcrobal settng. LPS, for nstance, stmulates Toll-lke receptor-4 and nduces a pathogenc release of pronflammatory cytoknes actvatng mmune responses. 37 In contrast, nteracton of commensal bactera wth Toll-lke receptors occurs under normal steady-state condtons, whch plays a role n mantanng ntestnal homeostass. 29 Ths suggests that the GI flora n untreated BF mce somehow enables pathogenc antbody-medated TRALI responses; ndeed, we observed a dfferent GI mcrobome composton n BF vs SPF mce when performng 16S rbosomal RNA gene amplcon sequencng (Fgure 7A-E). Although a combnaton of specfc gut bactera s most lkely to form the bass for TRALI susceptblty, we found B caccae to be at least 1 of the promsng canddates because t was sgnfcantly ncreased n the TRALI-susceptble BF-housed mce but not present n TRALI-resstant SPF-housed mce (supplemental Fgure 4). Ths bacterum has been suggested to have a pathogenc role n nflammatory bowel dsease 38 ; ths may potentally also be the case n antbody-medated TRALI, although further studes wll be requred to more drectly nvestgate ths. We dd, however, prme TRALI-resstant SPF mce wth fecal matter from TRALI-susceptble BF mce. The fecal transfer sgnfcantly restored the TRALI reacton, wth ncreased levels of pulmonary edema and pulmonary PMN accumulaton n SPF mce (Fgure 7F-G). Notably, the degree of antbody-medated acute lung njury was sgnfcant, but was not as severe as observed n the BF mce. Ths may be due to the already present gut mcrobes n the SPF mce, ndcatng a complex nterplay between dfferent gut mcrobota. The ntestne s the most densely bacterally colonzed surface n the human body (10 14 bactera), 39 and the lower respratory tract s 1 of the least colonzed surfaces n the human body (wth bactera per 1000 human cells). 40 Both gut and lung compartments, nonetheless, are largely colonzed by Bacterodetes and Frmcutes and t has been suggested that they are n constant communcaton wth each other and that fluctuatons n the abundance of bacteral dversty occurs n an dentcal manner n both compartments. 44 It s therefore plausble that the changes we now descrbe n gut flora composton (Fgure 7) are dentcally present n the lung flora. Addtonally, studes have reported correlatons between lung mcrobota and dsease severty n murne LPS-nduced lung njury 45 and n ARDS. 46 Unfortunately, we were not able to obtan suffcent amounts of DNA for murne lung mcrobota characterzaton (supplemental Fgure 5), therefore not allowng us to nvestgate f the lung mcrobota may also contrbute to the onset of murne TRALI. Of nterest, IL-8, the human equvalent of murne MIP-2, s a known rsk factor for human TRALI 3-5 ; pulmonary PMN accumulaton has also been reported n autopsy reports of TRALI patents. 47 As we have prevously shown, usng the same establshed murne model of TRALI, PMN are key effector cells n nducng TRALI. 12 Our current data suggest that the gut mcrobota nfluences the ablty of PMN to mgrate toward the lungs, at least va regulaton of plasma MIP-2 levels, upon nducton of antbody-medated TRALI. Other studes have also shown the ablty of gut mcrobota to drve PMN mgraton toward stes of njury. 48,49 Interestngly, resdent mcrobota were shown to also modfy the nteracton of dendrtc cells wth T-regulatory 1660 KAPUR et al 10 JULY 2018 x VOLUME 2, NUMBER 13

11 cells (both found to be major protectve cells n TRALI 12 ), whch ncreased the susceptblty to nflammatory dsorders such as colts. 29,50 How ths s exactly occurrng n TRALI wll need to be further dssected n subsequent studes. Addtonally, treatment wth antbotcs may perhaps be an effcent treatment strategy for human TRALI. Regardng rsk assessment of TRALI development, patents treated wth antbotcs before transfuson may perhaps be protected aganst TRALI. Ths may, for nstance, apply to orthopedc surgery patents that are prophylactcally treated wth antbotcs because of hgh nfecton rsk. For example, a prevously descrbed cohort of antbotc-treated orthopedc surgery patents dd not develop TRALI. 7 These results wll, however, need to be further valdated. On the other hand, t cannot be excluded that crtcally ll patents recevng antbotcs may stll suffer from TRALI. Cauton s advsed, because antbotcs treatment may also skew the gut mcrobal balance n a manner that may stll enable TRALI. It wll therefore be mportant to further nvestgate ths as well as to see f TRALI patents dsplay alteratons n ther gut mcrobota composton. In summary, we have dentfed a prevously unestablshed lnk between the GI flora and the onset of antbody-medated murne TRALI. Only BF mce were susceptble to TRALI because of ther GI bacteral composton compared wth SPF mce or antbotc-treated BF mce. The depleted GI flora n these mce protected them from antbody-medated TRALI nducton and TRALI could be restored n SPF mce by pretreatng the mce wth a low dose of LPS. Sequencng of the gut mcrobome confrmed a varyng GI bacteral composton n BF vs SPF mce, and BF fecal transfer nto SPF restored TRALI susceptblty. Addtonal studes are warranted to further dssect the communcaton lnk between the gut and the lungs durng TRALI. Acknowledgments Ths work was supported by grants from Lund Unversty, Crafoordska Stftelsen (# ), Vetenskapsrådet (Swedsh Research Councl, VR, # ), Avtal om Läkarutbldnng och Forsknng, and the Health Canada and Canadan Blood Servces (#340668) (J.W.S.). R.K. was the recpent of a young nvestgator award by the Royal Physographc Socety of Lund and was prevously the recpent of a postdoctoral fellowshp from Canadan Blood Servces. Authorshp Contrbuton: R.K. desgned all research, performed experments, collected data, analyzed and nterpreted data, performed statstcal analyses, made the fgures, and wrote and edted the paper; M.K., J.R., A.T.-F., N.K., S.S., S.M., M.J.M., and E.R.S. performed experments and collected data; B.H. and J.T.B. performed 16S rrna mcrobal proflng; and J.W.S. provded fnancal resources and edted the manuscrpt. Conflct-of-nterest dsclosure: The authors declare no competng fnancal nterests. ORCID profles: R.K., X; J.R., ; J.W.S., Correspondence: John W. 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