TREM-1 regulates macrophage polarization in ureteral obstruction

Transcription

1 sic reserch & 1 Interntionl Society of Nephrology regultes mcrophge polriztion in ureterl ostruction Tzu-Hn Lo 1,1, Ki-Yu Tseng,1, Wen-Shn Tso, Chih-Y Yng,3, Shie-Ling Hsieh,3,,5,,7, Allen Wen-Hsing Chiu 8, Toshiyuki Tki 9, Tk W. Mk 1, Der-Cherng Trng 1,,5,,11 nd Nien-Jung Chen,5 1 Institute of Physiology, School of Medicine, Ntionl Yng-Ming University, Tipei, Tiwn (ROC); Institute of Microiology nd Immunology, School of Life Sciences, Ntionl Yng-Ming University, Tipei, Tiwn (ROC); 3 Genomic Reserch Center, Acdemi Sinic, Tipei, Tiwn (ROC); Institute of Clinicl Medicine, School of Medicine, Ntionl Yng-Ming University, Tipei, Tiwn (ROC); 5 Inflmmtion nd Immunity Reserch Center, Ntionl Yng-Ming University, Tipei, Tiwn (ROC); Immunology Center, Tipei Veterns Generl Hospitl, Tipei, Tiwn (ROC); 7 Institute for Cncer Biology nd Drug Discovery, College of Medicl Science nd Technology, Tipei Medicl University, Tipei, Tiwn; 8 Deprtment of Urology, School of Medicine, Ntionl Yng-Ming University, Tipei, Tiwn (ROC); 9 Deprtment of Experimentl Immunology, Institute of Development, Aging nd Cncer, Tohoku University, Sendi, Jpn; 1 The Cmpell Fmily Institute for Brest Cncer Reserch, Ontrio Cncer Institute, University Helth Network nd Deprtment of Medicl Biophysics, University of Toronto, Toronto, Ontrio, Cnd nd 11 Division of Nephrology, Deprtment of Medicine, Tipei Veterns Generl Hospitl, Tipei, Tiwn (ROC) Chronic kidney disese (CKD) is n emerging worldwide pulic helth prolem. Inflmmtory cell infiltrtion nd ctivtion during the erly stges in injured kidneys is common pthologic feture of CKD. Here, we determined whether n importnt inflmmtory regultor, triggering receptor expressed on myeloid cells (TREM)-1, is upregulted in renl tissues collected from mouse ureterl ostruction induced nephritis. is crucil for modulting mcrophge polriztion, nd hs pivotl role in mediting tuulr injury nd interstitil collgen deposition in ostructive nephritis. Lystes from nephritic kidneys triggered -dependent M1 polriztion ex vivo, consistent with the oservtion tht grnulocytemcrophge colony-stimulting fctor (GM-CSF)-derived M1 mcrophges express higher levels of in comprison with M-CSF-derived cells. Moreover, gonistic crosslink significntly strengthens the inductions of inos nd GM- CSF in M1 cells. These oservtions re vlidted y strong clinicl correltion etween infiltrting -expressing/ inos-positive mcrophges nd renl injury in humn ostructive nephropthy. Thus, my e potentil dignostic nd therpeutic trget in humn kidney disese. Correspondence: Tk W. Mk, The Cmpell Fmily Institute for Brest Cncer Reserch, Ontrio Cncer Institute, University Helth Network nd Deprtment of Medicl Biophysics, University of Toronto, University Avenue, Room 7-7, Toronto, Ontrio, Cnd M5G C1. E-mil: tmk@uhnreserch.c or Der-Cherng Trng, Deprtment nd Institute of Physiology, School of Medicine, Ntionl Yng-Ming University, No.155, Sec., LinongStreet, Tipei 11, Tiwn (ROC). E-mil: dctrng@vghtpe.gov.tw or Nien-Jung Chen, Institute of Microiology nd Immunology, School of Life Sciences, Ntionl Yng-Ming University, No.155, Sec., Linong Street, Tipei 11, Tiwn (ROC). E-mil: njchen@ym.edu.tw 1 These uthors contriuted eqully to this work. Received April 13; revised 1 April 1; ccepted April 1; pulished online 11 June 1 Kidney Interntionl (1) 8, ; doi:1.138/ki.1.5; pulished online 11 June 1 KEYWORDS: chronic kidney disese; firosis; immunology nd pthology; mcrophges; ostructive nephropthy Chronic kidney disese (CKD) is n emerging helth prolem tht poses growing socioeconomic urden for societies in North Americ nd the Asi-Pcific regions. 1 5 A common pthologic feture of CKD is inflmmtory cell infiltrtion occurring t erly stges in the injured kidneys, followed y tuulointerstitil firosis t lter stges of disese progression. Among CKD, upper urinry trct ostruction resulting in renl dysfunction is significnt clinicl prolem in oth dult nd peditric popultions. Pthologic mnifesttions of renl ostructive injury include tuulr diltion, tuulr cell poptosis, 5 s well s progressive interstitil firosis nd mssive mcrophge infiltrtion in the ostructed kidney. Clssiclly ctivted mcrophges (M1), chrcterized y high mjor histocomptiility complex clss II expression with strong interleukin-1 (IL-1) nd IL-3 production, produce nitric oxide, rective oxygen species, nd proinflmmtory cytokines, such s IL-1, IL-, nd tumor-necrosis fctor (TNF),,,7 leding to tuulr cell poptosis nd renl tissue injury. In contrst, lterntively ctivted mcrophges (M) typiclly hve immune-suppressive ctivity nd express rginse (Arg)-1, promoting cell prolifertion nd collgen production. Besides, YM-1, 8,9 Mnnose receptor, 1 glectin-3, 11 nd trnsforming growth fctor- 1 lso hve een reported to e preferentilly expressed on M cells. Microenvironmentl cytokines, glucocorticoid hormones, nd surrounding pthogens nd poptotic cells regulte M1/M differentition. 1,13,1 117 Kidney Interntionl (1) 8,

2 T-H Lo et l.: modultes M1 polriztion in nephritis sic reserch Triggering receptor expressed on myeloid cells (TREM) is n immunogloulin-like fmily whose memers hve criticl role in modulting infection-induced inflmmtion. 15,1 A common feture of TREM downstrem signling is the link with dptor DNAX ctivtion protein (DAP)-1. 1,17 Activtion of TREM leds to the production of M1 proinflmmtory cytokines. 1,18 is the est-chrcterized memer in the TREM fmily. Tretment with solule decreses inflmmtion nd mortlity in high-dose lipopolyscchride-medited septic shock model. 15 Cliniclly, the levels of solule in serum nd ronchiolveolr lvge fluid hve een suggested s sensitive nd specific predictor of cteril pneumoni in humns. 19 Previously, we lso reported tht -medited cteril clernce in the smll intestine is n importnt immune response ginst K. pneumonie. A puttive pthogenic role of ws reported in rheumtoid rthritis 1 nd inflmmtory owel disese. However, the role of in the nephropthy of CKD hs not yet een chrcterized. A mouse model of experimentl unilterl ureterl ostruction () is chrcterized y infiltrting mcrophges nd the different stges of ostructed nephropthy, which ccelertes with disese progression. 3 In this study, we exmine expression nd function in kidneys y using Trem-1 knockout () mice. Our results demonstrte novel role of in regulting grnulocytemcrophge colony-stimulting fctor (GM-CSF)/iNOS nd M1 polriztion. In ddition, correltions of expression, mcrophge profiling, nd severity of renl injury in kidney specimens from ptients of ostructive nephropthy further support tht is criticl fctor in ostructive nephritis. RESULTS Depleting meliortes the renl pthology mrna is not detectle in shm control kidneys ut is significntly upregulted in the ostructed kidneys hrvested from wild-type () C57BL/ mice t dy 7 nd dy 1 fter unilterl ureterl ostruction (; Figure 1), nd protein is minly detected in supopultion of F/8 þ cells within the renl tuulointerstitium fter (Figure 1c nd d). To ssess the in vivo role of in, we compred the histologicl renl chnges etween nd Trem-1 mice (Supplementry Figure S1 online). deletion in kidneys is confirmed y quntittive reverse trnscriptse-pcr (Figure 1) nd immunohistochemicl nlysis (Figure 1). Histologiclly, induces mrked renl injury in mice (chrcterized y corticl tuulr diltion with tuulr epithelil cell necrosis, rush order loss, intrtuulr cst formtion, nd interstitil firosis), wheres Trem-1 mice show significntly less renl dmge fter (Supplementry Figure S online). Anlyses y PAS nd Msson s trichrome stining lso show drstic tuulr injuries t dy 7 (5±9%) nd dy 1 (81±%) in kidneys fter in comprison with the shm group (Figure nd ), ccompnied y incresed interstitil collgen deposition fter t dy 7 (9.1±.) nd dy 1 (3.9±5.7)(Figure c nd d).in contrst, deficiency results in significntly less tuulr injuries (3±7%; 5±13%) t dy 7 nd dy 1 fter, nd lower interstitil firosis (1.8±5.) t dy 1 fter (Figure d). Tuulr cell injury ws further exmined y using terminl deoxynucleotidyl trnsferse medited dutp nickend leling nd Ki7 stining methods. In Trem-1 kidneys, the numers of poptotic nd proliferting cells re significntly lower thn those in ones (Supplementry Figure S3 nd online). In ddition, mssive -SMA þ myofirolsts ccumulte in kidneys ut re reltively less in Trem-1 kidneys isolted from dy-1- mice (Figure e nd f). is criticl for regulting mcrophge polriztion ut not recruitment Renl inflmmtory cell infiltrtion is prominent feture ssocited with the pthogenesis of. Renl mcrophge recruitment ws ccessed y determining the distriution of F/8 þ mcrophges. Interestingly, similr levels of F/8 þ cells re found in nd Trem-1 -ostructed kidneys (Supplementry Figure S c online). A recent report suggests tht hs n importnt role in regulting neutrophil trfficking to mplify inflmmtion ginst cteril infection, leding us to exmine the neutrophil infiltrtion fter. However, only very few LyG þ neutrophils re oserved in the ostructed kidneys, nd no significnt difference cn e found in the numers of infiltrting neutrophils etween nd Trem-1 groups (Supplementry Figure Sd nd e online). Thus, is dispensle for mcrophge nd neutrophil renl recruitment upon. M1 polriztion hs een suggested s key step in medited renl dmge. Whether regultes the lnce of clssicl nd lterntive ctivtion of mcrophges in remins to e investigted. M1 cells were evluted y inos expression, which is presented in F/8 þ infiltrting cells nd tuulr epithelil cells on renl sections, prticulrly t dy 1 fter. By contrst, it is mrkedly ttenuted in the cortex nd medull of Trem-1 kidneys (Figure 3 nd ). Accordingly, the mrnas of inos, TNF, IL-1, nd IL- (M1 mrkers) re highly expressed in kidneys, ut the expression is significntly lower in Trem-1 kidneys (Figure 3c). Furthermore, the expression of inos, TNF, nd IL-1 mrna is significntly lower in Trem-1 renl mcrophges freshly isolted from kidneys compred with cells (Supplementry Figure S5 online). In prllel, M differentition ws evluted y Arg-1 stining on renl sections. Unlike in smples, which only show mild stining, Arg-1 is highly induced nd significntly elevted in renl F/8 þ infiltrting cells in Trem-1 kidneys t dy 7 nd dy 1 fter (Figure 3d nd e). The mrna expression levels of Arg-1, YM-1, IL-1, nd Mnnose receptor (M mrkers) re significntly higher in Trem-1 kidneys in Kidney Interntionl (1) 8,

3 sic reserch T-H Lo et l.: modultes M1 polriztion in nephritis 1 P<.1 P<.1 Shm 7 1 Shm Dy 7 Dy 1 c F/8 IHC IHC kidney from mouse t dy 1. ( 1) d F/8 DAPI Merge Figure 1 is upregulted nd coloclized with mcrophges in kidneys. Unilterl ureterl ostruction () surgery ws performed on wild-type () nd Trem-1 knockout () mice nd they were killed t dy 7 nd dy 1. () Quntittive reverse trnscriptse-pcr nlysis nd () immunohistochemicl (IHC) stining of expression in the ostructed kidney fter, compred with shm control. (c) F/8 nd IHC nlyses on renl sections hrvested from mice t 1 dys fter (1). (d) Renl tissue ws otined t 1 dys fter nd stined for -dimidino--phenylindole (DAPI; lue), F/8 (red), nd (green). Representtive merge imges re shown t n originl mgnifiction of. () Representtive results were shown s men±s.d. ( nd d) Scle rs ¼ 5 mm. (, n ¼ 9; Trem-1, n ¼ in ech group.) comprison with ones (Figure 3f), wheres the expressions of IL- nd trnsforming growth fctor- re comprle etween the two groups (dt not shown). However, the increse of M mrker mrna expression is not oserved in isolted Trem-1 renl mcrophges (Supplementry Figure S5 online), suggesting tht complete renl microenvironment is crucil to mintin the M-relted mrker gene expression. The renl protein levels of M1 nd M mrkers were lso determined y western lotting. Trem- 1 kidneys contin lower inos nd TNF ut higher Arg-1 nd IL-1 proteins in comprison with smples t dy 7 nd dy 1 fter (Supplementry Figure S online). -injured kidney lystes trigger -dependent M1 differentition The fctor(s) in kidney tht cn trigger therey further mediting the downstrem M1 polriztion remins unknown. Homogentes prepred from kidneys were used for ex vivo stimultion of one mrrow derived mcrophges (BMMs) from or Trem-1 mice (Figure ). Treting BMMs with dy-1- kidney lyste (KL) significntly ugments higher expression thn shm lyste does (Figure ). We next evlute the role of GM-CSF, well-known M1-like mcrophge differentition fctor, 5 8 in -medited cell polriztion in response to KL tretment. ut not Trem-1 BMMs show higher level of GM-CSF induction in response to dy-7- nd dy-1- KLs (Figure c). Accordingly, M1-ssocited inos is highly induced in dy-1- KL treted cells ut not in the shm lyste treted ones. In contrst, dy-1- KL treted Trem-1 BMMs only express lower level of inos (Figure d) ccompnied y significnt upregultion of Arg-1 while stimulte cells with dy-7- KL (Figure e), supporting tht -lystes contin stimuli tht trigger -medited regultion on M1/M differentition. 117 Kidney Interntionl (1) 8,

4 sic reserch T-H Lo et l.: modultes M1 polriztion in nephritis Shm Dy 7 Dy 1 Tuulr injury (%) P<.1 1 P<.1 8 Shm 7 1 d c Interstitil collgen deposition (%) P<.1 5 Shm Dy 7 Dy Shm 7 1 e ( ) f α-sma positive re (%) Shm Dy 7 Dy 1 P < Shm 7 1 ( ) Figure hs n importnt role in the progression of renl pthogenesis fter. Unilterl ureterl ostruction () surgery ws performed on wild-type () nd Trem-1 knockout () mice nd they were killed t dy 7 nd dy 1. Representtive kidney tissue sections stined with ( nd ) periodic cid Schiff, (c nd d) Msson s trichrome, nd (e nd f) immunohistochemicl stining of the -smooth muscle ctin locted in the tuulointerstitium t dy 7 nd dy 1 fter, compred with shm control. Quntittive positive re of () tuule injury, (c) interstitil collgen deposition, nd (e) interstitil firosis (-SMA positive) were shown s men±s.d. (, n ¼ 9; Trem-1, n ¼ in ech group). (, d, nd f) Scle rs ¼ 5 mm. () Blck rrowhed, rush order; white rrowhed, loss of rush order. GM-BMMs preferentilly express, which medites inos nd proinflmmtory cytokine induction M1-like mcrophges cn e in vitro differentited y treting one mrrow cells with GM-CSF (GM-BMMs).5,7,8 Unexpectedly, we oserved tht GM-BMMs preferentilly express higher thn M-CSF-derived mcrophges (M-BMMs) (Figure 5 nd ). To evlute the contriution of in the plsticity of mcrophges, differentited M-BMMs were treted with GM-CSF to convert them into the inflmmtory stge, nd the inflmmtory GM-BMMs Kidney Interntionl (1) 8, were then reversed ck to the resting stge y replcing them in M-CSF culture condition. Consistently, GM-CSF tretment induces higher levels of in M-BMMs (Figure 5c), wheres replcing GM-BMMs in M-CSF culture ttenutes their expression (Figure 5d). The effects of triggering on the induction of inflmmtion cytokines nd M1/M mrkers were investigted y cross-linking with plte-ond-specific gonistic ntiody. Cross-linked lone triggers TNF secretion from ut not from GM-BMMs 1177

5 sic reserch T-H Lo et l.: modultes M1 polriztion in nephritis inos IHC F/8 inos DAPI Dy 1 inos Dy 7 F/8 Shm c mrna (reltive fold) inos TNF IL-1β P<.1 1 P<.1 P<.1 IL- 1 P<.5 d Arg-1 IHC e F/8 Arg-1 DAPI Dy 1 Arg-1 Dy 7 F/8 Shm f mrna (reltive fold) Arg-1 YM-1 IL-1 P<.1 MR P<.1 3 P< P< P<.5 P<.5 Figure 3 is crucil in modulting mcrophge polriztion upon. ( nd d) Renl sections were otined t dy 1 fter unilterl ureterl ostruction () nd stined for -dimidino--phenylindole (lue), F/8 (red), nd () inos (green) or (d) Arg-1 (green). Representtive merge imges re shown for oth genotypes t n originl mgnifiction of 1. Scle rs ¼ 1 mm. n ¼ in ech group. ( nd e) Representtive renl sections were otined t dy 7 nd dy 1 fter nd immunohistochemiclly nlyzed for () inos nd (e) Arg-1 expression (). Scle rs ¼ 5 mm., n ¼ 9; Trem-1, n ¼ in ech group. (c nd f) Expressions of (c) M1 mrkers (inos, TNF, IL-1, nd IL-) nd (d) M mrkers (Arg-1, YM-1, IL-1, nd Mnnose receptor) in kidneys t dy 7 nd dy 1 were determined y quntittive reverse trnscriptse-pcr (, n ¼ 9; Trem-1, n ¼ in ech group). IL, interleukin;, knockout; TNF, tumor-necrosis fctor Kidney Interntionl (1) 8,

6 T-H Lo et l.: modultes M1 polriztion in nephritis sic reserch or BM cells d Dy Tretment with KL inos P<.5 P<.1 Tretment with KL M-CSF ( ng/ml) P<.1 c e GM-CSF P<.5 P<.1 P<.1 P<.5 Tretment with KL Tretment with KL (Supplementry Figure S7 online), nd it synergisticlly oosts the lipopolyscchride-medited secretion of TNF nd IL- in GM-BMMs, ut not in Trem-1 ones (Supplementry Figure S7 nd online). Similr results re lso otined from mrna nlyses of TNF nd inos expressions (Figure 5e nd f). By contrst, Arg-1 is slightly induced in GM-BMMs under stimultion (Figure 5g) in much milder wy. Protein levels of inos nd Arg-1 were further checked y immunolotting. Accordingly, crosslink only induced inos induction in ut not in Trem-1 GM-BMMs. Arg-1 induction is not oserved in oth groups of GM-BMMs, nd neither inos nor Arg-1 protein re detected in nd Trem-1 M-BMMs (Figure 5h) Shm KLs 7-dy KLs 1-dy KLs Arg-1 P<.1 Dy 5 Dy P<.1 qrt-pcr Figure -injured kidneys contin n unidentified lignd tht triggers M1 mcrophge differentition. () Experimentl design of ex vivo kidney lyste induced mcrophge polriztion. Homogentes of kidney (KL) in phosphteuffered sline were isolted nd used s stimuli for the susequent ex vivo BMM stimultion. M-BMMs derived from nd Trem-1 mice were incuted with KLs from shm, dy-7, nd dy-1 injured kidneys for h. Expression levels of (), (c) GM-CSF, (d) inos, nd (e) Arg-1 mrna in stimulted M-BMMs were determined y quntittive reverse trnscriptse-pcr nlysis. Representtive results of three independent experiments re shown s men±s.d. BMM, one mrrow derived mcrophge; KL, kidney lyste;, knockout;, unilterl ureterl ostruction;, wild-type. The induction of inos under crosslink in M-BMMs with M-CSF, GM-CSF, or dy-1 -KL stimultion ws next exmined. As predicted, triggers more inos in M-BMMs cultured with GM-CSF thn M-CSF (Figure 5i), nd it lso further ugments inos induction in KL treted M-BMMs (Figure 5j). HMGB1 ws recently reported to e puttive lignd for. 9 We investigted whether treting GM-BMMs with recominnt HMGB1 is sufficient to induce inos through. However, neither group of cells showed significnt inos induction fter HMGB1 tretment (dt not shown), suggesting tht recominnt HMGB1 lone is not sufficient to trigger inos vi. More investigtions re needed to identify the lignd contriuting to M1 cell polriztion in. The role of DAP1 in -medited M1 polriztion ws lso determined y compring the inos nd Arg-1 mrna expression in, Dp1, 3 nd Trem-1 GM-BMMs with crosslink. Although expression is comprle in nd Dp1 GM-BMMs (Supplementry Figure S8 nd online), the inductions of inos nd Arg-1 medited y re completely olished in Dp1 GM-BMMs (Supplementry Figure S8c nd d online), supporting tht DAP1 is crucil for mediting -triggered cell ctivtion. GM-CSF is crucil fctor downstrem of for M1 induction M-BMMs could e polrized into M1 under -KL tretment through -dependent process (Figure d). Accordingly, crosslink of triggers the induction of GM-CSF (Figure ). To determine the role of GM-CSF in KL medited M1 polriztion, ntgonist ntiody ginst GM-CSF ws dded in the culture. Depletion of GM- CSF significntly reduces KL triggered M1 differentition (Figure ). Neutrliztion of GM-CSF lso suppresses KL induced nd GM-CSF expression in BMMs (Figure ). Thus, modultion of GM-CSF ctivity in -KL stimultion ffects -medited GM-CSF induction nd locks the positive feedck of enhncement nd M1 differentition. Adoptive trnsfer of GM-BMMs into Trem-1 mice deteriortes pthogenesis To verify the importnce of expressed on M1-like mcrophges in mediting the pthogenesis in vivo, we dptively trnsferred croxyfluorescein succinimidyl ester-leled or Trem-1 GM-BMMs into treted Trem-1 mice (Figure 7). 31,3 Reconstitution of croxyfluorescein succinimidyl ester-leled cells, which re þ (Figure 7) nd F/8 þ (dt not shown), cn e oserved in the kidneys of Trem-1 mice tht received the GM-BMMs. In greement with our previous findings, trnsferring GM-BMMs, ut not Trem-1 ones, into Trem-1 recipient mice cn intensify the tuulr injury, Kidney Interntionl (1) 8,

7 sic reserch T-H Lo et l.: modultes M1 polriztion in nephritis c e f g h P<.1 8 GM-BMM M-BMM P<.1 M-CSF GM-CSF TNF 1 M-BMM P<.1 Ctrl nti- Crosslink Counts GM-BMM inos 8 P<.1 Ctrl nti- Crosslink GM-BMM M-BMM Arg-1 8 M-BMM d P<.1 GM-CSF M-CSF GM-BMM Ctrl nti- Crosslink TR1 TR1 A IgG TR1 IgG TR1 TR1 TR1 inos Arg-1 α-tuulin i inos P <.5 nti- + + M-CSF GM-CSF j Fold (crosslink / KLS lone) inos induction P <.5 KLs Shm 1-dy Figure 5 is preferentilly expressed in GM-BMMs nd modultes mcrophge polriztion towrd M1. ( nd ) expression in M-BMMs nd GM-BMMs ws determined y () quntittive reverse trnscriptse-pcr nd () fluorescence-ctivted cell sorting nlyses (gry filled: nonstined; dshed line: isotype control; lck line: ). (c nd d) mrna expression in nd Trem-1 (c) M-BMMs nd (d) GM-BMMs treted with M-CSF or GM-CSF for h. (e) TNF, (f) inos, nd (g) Arg-1 mran expression levels in nd Trem-1 GM-BMMs were left untreted or stimulted with nti- gonistic ntiody for h. (h) inos nd Arg-1 expression levels were determined y immunolotting nlyses on nd Trem-1 GM-BMMs nd M-BMMs. -tuulin served s loding control. Representtive results of three independent experiments re shown. (i) inos expression in nd Trem-1 M-BMMs (mintined in M-CSF or treted with GM-CSF) were left untreted or costimulted with nti- gonistic ntiody for h. The expression levels of indictive genes were determined y quntittive reverse trnscriptse-pcr. (j) inos induction in M-BMMs (left untreted or treted with KLs from shm nd dy-1 smples) costimulted with or without nti- gonistic ntiody for h. The fold induction of inos ws clculted y normlizing with results from cells without crosslink. (, c g, i, nd j) Representtive results of three independent experiments re shown s men±s.d. BMM, one mrrow derived mcrophge; GM-CSF, grnulocyte-mcrophge colony-stimulting fctor; KL, kidney lyste;, knockout;, unilterl ureterl ostruction;, wild-type. 118 Kidney Interntionl (1) 8,

8 T-H Lo et l.: modultes M1 polriztion in nephritis sic reserch interstitil firosis, nd myofirolst ccumultion in kidneys of Trem-1 mice (Figure 7c nd d). Assocition of expression, inos expression, nd renl injury in humn ostructive nephropthy To vlidte the pthogenic role of in humn noninfectious nephritis, nephrectomized specimens of the ffected kidneys were otined from nondietic, nonhypertensive ptients with unilterl prtil (n ¼ 3) nd complete (n ¼ ) ureterl ostruction (Tle 1). The cuses of ostruction re renl stone nd ureterl cncer. The norml tissues of nephrectomized kidneys from renl cell crcinom ptients served s controls (n ¼ ). There re no differences in ge nd gender distriution mong the three groups. However, seline glomerulr filtrtion rte (GFR) mesured y the technetium-99m-diethylene-pentcette method is significntly lower in the ffected kidneys thn in the contrlterl nonffected kidneys. On the sis of the histopthologic nlysis, tuulr injury score nd collgen deposition in the interstitium re positively correlted with the severity of ureterl ostruction (Supplementry Figure S9 online), nd negtively correlted with glomerulr filtrtion rte of the ffected kidneys efore surgery (Tle 1). Consistent with the results from the mouse model, numers of CD8-positive mcrophges nd - positive cells re the highest in the specimens of completely ostructed kidneys, followed y prtilly ostructed kidneys nd then norml renl tissues (Figure 8 nd nd Tle 1). -positive cells in ostructed kidneys re minly oserved in the interstiti, which re lso positively stined for the mcrophge cell mrker CD8 y immunofluorescent stining (Figure 8c). The -stining profile is lso highly correlted with the profile of inos in smples of completely ureterl ostructed kidneys (Figure 8d). By contrst, very few nd inos-stined cells re oserved in norml renl tissues. Doule stining demonstrtes tht the numers of infiltrting corticl interstitil mcrophges positive for oth nd inos re positively ssocited with tuulr injury nd interstitil firosis, nd negtively with glomerulr filtrtion rte in the ureterl ostructed kidneys (Tle 1). Tken together, our results support tht hs crucil role in mediting M1-medited pthogenesis of renl injury in ostructive nephropthy. DISCUSSION is crucil in infection-ssocited inflmmtion, ut its role in sterile inflmmtion remins to e determined. Deletion of in mice ttenutes diethylnitrosmineinduced heptocellulr crcinogenesis. Loss of ttenutes Kupffer cell ctivities including inflmmtory cytokine production nd signl induction, resulting in diminished liver injury fter diethylnitrosmine exposure. 9 In the present study, we report n independent Trem-1-deficient mouse model nd revel novel role of in the pthogenesis of nephritis. Similr to previous model,,9 the sic immune prmeters, such s immune cell counts, WBC popultion, nd the weight of lymphoid orgns, in our Trem-1 mice re comprle with mice. In ddition, we lso show tht is dispensle for renl mcrophge nd neutrophil recruitment upon. Our finding regrding the involvement of / DAP1 in nephritis ws further supported y recent report tht shows tht DAP1 (prtly through /3) is involved in renl inflmmtion during. 33 However, in contrst to our results tht Trem-1 deficiency meliortes oth renl injury/inflmmtion nd firosis in mice with, Tmmro A. et l. reported tht the renl firosis ws comprle in nd Trem-1/3 doule mice. Of note, there re out DAP1-ssocited receptors expressed on vrious types of cells. 18 Some of these receptors hve opposite roles in modulting inflmmtion nd tissue firosis (such s vs. TREM). Whether TREM-3 hs inflmmtory 8 GM-CSF P<.1 Ctrl nti- Crosslink 3 inos GM-CSF P<.1 P<.1 1 P<.1 1-dy KLs Blocking with isotype Ctrl Blocking with nti-gm-csf 3 1-dy KLs 1-dy KLs Figure Neutrliztion of GM-CSF inhiits -medited M1 polriztion. GM-CSF mrna expression in nd Trem-1 () GM-BMMs left untreted or stimulted with nti- gonistic ntiody for h. () inos,, nd GM-CSF expressions in M-BMMs stimulted with dy-1- KLs in the presence of GM-CSF-neutrlizing ntiodies (1 mg/ml, R&D systems) or isotype control (1 mg/ml) for h. The expressions of indicted genes were determined y quntittive reverse trnscriptse-pcr. Representtive results of three independent experiments were shown s men±s.d. BMM, one mrrow derived mcrophge; GM-CSF, grnulocyte-mcrophge colony-stimulting fctor; KL, kidney lyste;, knockout;, unilterl ureterl ostruction;, wild-type. Kidney Interntionl (1) 8,

9 sic reserch T-H Lo et l.: modultes M1 polriztion in nephritis or nti-inflmmtory role in modulting tissue firosis during remins to e investigted. Thus, the controversy oserved etween Trem-1 nd Trem-1/3 doule mice my e result of functionl compenstion etween - nd TREM-3. We hve exmined the Trem-3 expression in nd Trem-1 mice y / GM-BMM (CFSE) trnsfer / GM-BMM (CFSE) trnsfer Scrifice Dy Sline +/+ Sline / +/+ / / / Merge CFSE (1 dys) c Sline +/+ Sline / +/+ / / / α-sma Msson trichrome PAS (1 dys) d e f Tuulr injury (%) P<.5 S S +/+ +/+ / / / / Positive msson s trichrome stin re (%) 5 P< S S +/+ / +/+ / / / Positive α-sma stin re (%) 3 P<.1 1 S S +/+ / +/+ / / / 118 Kidney Interntionl (1) 8,

10 T-H Lo et l.: modultes M1 polriztion in nephritis sic reserch quntittive reverse trnscriptse-pcr, nd found tht the expression of Trem-3 is comprle in nd Trem-1 GM-BMMs nd kidney isolted mcrophges (dt not shown), which further supports the plusiility of this hypothesis. Infiltrted M1 nd M mcrophges cn medite renl injury nd tissue repir, respectively.,3 Initilly, infiltrting mcrophges re predominntly in M1 type to secrete proinflmmtory cytokines nd induce the deth of tuulr Tle 1 Bseline clinicl chrcteristics, pthology index, nd cell popultion nlyses mong ptients without or with ostructive nephropthy due to prtil nd complete ureterl ostruction No ureterl ostruction (n ¼ ) Prtil ureterl ostruction (n ¼ 3) Complete ureterl ostruction (n ¼ ) Clinicl chrcteristics Age (yers) 59±5 58±9 ±3 Gender (M/F) / /1 / Cuses Stone, n (%) (.7) (.7) Ureterl cncer, n (%) 1 (33.3) (33.3) GFR y Tc-99m-DTPA (ml/min per 1.73 m ) Affected kidney 31.± ±3., Nonffected kidney 53.5±.5 5.±1.7 5.±.5 Renl pthology index Tuulr injury (%).5±3. 5.±. c 8.3±1.,d Interstitil firosis score.1±. 1.±. c 3.±.,d Cell popultion CD8 þ (cells/hpf).±..1±.3 c 18.9±.8,d þ (cells/hpf).±. 1.±.1 c 13.5±3.,d inos þ (cells/hpf).±..8±.8 1.7±.3,d CD8 þ, þ.±. 1.±. c 13.3±3.,d (cells/hpf) þ,inos þ (cells/hpf).±..±.1 9.8±1.7,d Arevitions: F, femle; GFR; glomerulr filtrtion rte; HPF, high-powered field; M, mle; Tc-99m-DTPA, technetium-99m-diethylene-pentcette; TREM, triggering receptor expressed on myeloid cells. Vlues re mens ± s.d. of positive cells/ high-powered field. The sttisticl nlysis ws performed y ANOVA with post hoc pirwise comprison. Po.5, versus nonffected kidney in prtil nd complete ureterl ostruction, respectively. Po.1, versus prtil ureterl ostruction. c Po.5, versus no ureterl ostruction. d Po.1, versus no ureterl ostruction. cells, leding to tissue injury nd ultimtely to the development of scrring nd firosis. Intriguingly, the sence of meliortes M1-medited tissue injury ut induces switch in infiltrted mcrophges from M1 to M. -deficient mcrophges in ostructive kidneys express Arg-1 nd led to tissue remodeling nd repir (Supplementry Figure S1 online). Our dt demonstrte tht criticlly modultes M1 polriztion oth in vivo nd ex vivo. Accordingly, is preferentilly expressed y M1-like GM-BMMs, nd crosslink of significntly strengthens the expression of inos, TNF, nd GM-CSF. Cliniclly, hs een identified s novel iomrker in the ssessment of peditric mlri 3 nd pneumoni disese severity. 19 In the present study, the histologicl nlyses of kidney specimens from ptients with ostructive nephropthy prove tht the frequency of expression in infiltrting mcrophges is significntly correlted with the disese progression from prtil to complete ostruction, demonstrting tht my serve s new locl dignostic surrogte of humn ostructive nephropthy. The identity of the nturl lignd(s) remins mystery. 35 The genes encoding the puttive lignds expressed on pltelets nd mcrophges remin unchrcterized. 3,37 HMGB1 ws recently found s -intercting protein. 9 However, treting cells with recominnt HMGB1 lone is not sufficient to medite - regulted M1 polriztion. It cnnot e ruled out tht the ctivity of recominnt HMGB1 is different from ntive HMGB1 (with vrious posttrnsltion modultion), nd treting cells with isolted HMGB1 from inflmed tissues my e required for inducing -medited M1 polriztion. Further frctiontion nd chrcteriztion on KLs will enle us to identify the novel - triggering dnger-ssocited moleculr pttern(s). In summry, novel positive feedck cycle medited y nd GM-CSF ws reveled to enhnce M1 polriztion under (Supplementry Figure S1 online). Upon, injured tissue-derived GM-CSF initilly induces the expression of, followed y -medited ugmenttions of GM-CSF nd other M1-promoting fctors, nd finlly mplifies the polriztion of M1 responses leding to kidney dmges. This mplifiction cycle medited y /GM-CSF my e puttive trget for CKD therpeutic drug design. Figure 7 Histopthologic nlyses of kidney tissues in Trem-1 mice receiving croxyfluorescein succinimidyl ester (CFSE)- leled or Trem-1 GM-BMMs. () Experimentl design for doptive trnsfer of CFSE-leled GM-BMMs to Trem-1 mice. After surgery, cultured or Trem-1 GM-BMMs leled with CFSE were intrvenously injected (31 GM-BMMs/mouse) into Trem-1 mice t dy 1 nd dy 8 (the sme mount of sline ws injected in the first two groups of mice s controls). Mice were killed t dy 1, nd renl tissues were hrvested for nlyses. () Renl sections were stined for (red) nd -dimidino--phenylindole (lue). Representtive merge imges re shown t n originl mgnifiction of. (c) Representtive results of renl sections stined with PAS, Msson s trichrome, nd -smooth muscle ctin. () (d f) Quntittive positive re of (d) tuule injury, (e) interstitil collgen deposition, nd (f) interstitil firosis. Results were shown s men±s.d. Scle rs ¼ 5 mm (n ¼ in ech group). BMM, one mrrow derived mcrophge; GM-CSF, grnulocyte-mcrophge colony-stimulting fctor;, knockout;, unilterl ureterl ostruction;, wild-type. Kidney Interntionl (1) 8,

11 sic reserch T-H Lo et l.: modultes M1 polriztion in nephritis CD8 IHC IHC c DAPI CD8 DAPI CD8 Isotype ctrl Complete ostruction Prtil ostruction Norml d DAPI inos DAPI inos Isotype ctrl Complete ostruction Prtil ostruction Norml ( ) Figure 8 Immunohistochemicl nd immunofluorescene stining of kidney specimens in ptients with or without ostructive nephropthy. expression ws chrcterized y renl interstitil mcrophges in the nephrectomized kidney tissues from ptients with prtil nd complete ureterl ostruction. () Accumultion of infiltrted CD8 þ mcrophges, s well s () the expression of, in the renl interstitium of kidney tissues ws determined y immunohistochemistry. Doule immunofluorescence stining reveled the (c) colocliztion of CD8 þ mcrophges (green) nd -expressing cells (red), nd (d) colocliztion of inos-expressing mcrophges (green) nd þ cells (red) in the tuulointerstitium of ptients with prtil nd complete ureterl ostruction. (c nd d) Nuclei were stined with the DNA-interclting dye -dimidino--phenylindole (lue); isotype Ctrl mens the control stining using control ntiodies with the sme isotype. Scle rs ¼ 5 mm. MATERIALS AND METHODS Animls Age-mtched (8- to 1-week-old) mle C57BL/J, Trem-1 (B.19P-Trem-1 tm1mk ), nd Dp1 (generted in the 19/SvJ nd C57BL/ hyrid ckground s descried 9 nd ckcrossed in C57BL/J ckground) were mintined in the niml center of NYMU in ccordnce with the IACUC guidelines. Experimentl niml model surgery ws performed y mking mid-dominl incision under intrperitonel nesthesi with pentoritl (5 mg/kg). The left ureter ws dissected free nd ligted with 5. silk. Age-mtched nd Trem-1 mice, incision nd suture of the mid-dominl wll without kidney ligtion were used s shm control. Pthologicl exmintions of the kidneys The kidney ws cut prt. Hlf of the tissue ws frozen in liquid nitrogen for protein nd RNA nlyses. The other hlf ws fixed in % prformldehyde for h nd emedded in -mm-thick prffin sections. After deprffiniztion, the sections were stined with hemtoxylin nd eosin, periodic cid Schiff (PAS) regent (Sigm-Aldrich, St Louis, MO), nd Msson s Trichrome (Sigm-Aldrich) stins. 118 Kidney Interntionl (1) 8,

12 T-H Lo et l.: modultes M1 polriztion in nephritis sic reserch Immunohistochemicl stining Sections were treted with.3% hydrogen peroxide to quench the endogenous peroxidse ctivity. Titrted primry ntiodies ginst the following ntigens were used: for mouse, F/8, LyG (ebioscience, Sn Diego, CA), (R&D Systems, Minnepolis, MN), Ki7 (BioLegend, Sn Diego, CA), inos, Arg-1, nd -SMA (Snt Cruz Biotechnology, Snt Cruz, CA); for humns, (R&D Systems), CD8 (KP1, DA-CD8, Denmrk), nd inos (Snt Cruz Biotechnology). Immunorectivity ws detected with the Envision vidin-iotin-free HRP-leled polymer (Dko Cytomtion) nd visulized using the diminoenzidine kit (L Vision, Fremont, CA). Sttisticl nlysis Descriptive sttistics included men±s.d. The comprisons etween nd shm groups (or nd groups) were performed y independent Student s t-test or n indictive method for difference using the Sttisticl Pckge of Socil Science (SPSS 1., 3; SPSS, Chicgo, IL) softwre. DISCLOSURE All the uthors declred no competing interests. ACKNOWLEDGMENTS This work ws supported y grnts to NJC from the Ntionl Science Council (NSC97-3-B1-3-MY; NSC99-3-B1-3-MY3; NSC1-3-B -1-15)), Tipei Veterns Generl Hospitl (V97S5-1; V98S5-8; V99S5-; V1E-3), Yen Tjing Ling Medicl Foundtion (CI-1-1), nd grnt (98A-C-D117) from the Ministry of Eduction, Aim for the Top University Pln in Tiwn. DCT is supported y grnts from the Ntionl Science Council (NSC B-1--MY3; NSC 1-31-B-1--MY3), Tipei Veterns Generl Hospitl (V11E-1; V1E-1), nd Bureu of Helth Promotion, Deprtment of Helth (DOH98-HP-111). TWM is supported y grnts from the Cndin Institute of Helth Reserch, the Leukemi Lymphom Society, the Cndin Institute of Helth Reserch, nd the Terry Fox Cncer Reserch Foundtion. SUPPLEMENTARY MATERIAL Tle S1. Primer sequences for rel-time polymerse chin rection. Figure S1. Genertion of Trem-1 mice. Figure S. Depletion of in mice meliortes the progression of renl pthogenesis fter. Figure S3. Deletion of protects mice from -medited renl tuulr cell poptosis nd prolifertion. Figure S. is dispensle in mcrophge nd neutrophil recruitment upon. Figure S5. M1 nd M mrker mrna expression levels in or Trem-1 mcrophges isolted from 7-dy or 1-dy- kidneys. Figure S. Renl protein levels of M1/M mrkers t dy 7 nd dy 1 fter. Figure S7. contriutes to M1 proinflmmtory cytokine production in GM-BMMs. Figure S8. DAP1 hs essentil roles in -medited inos induction in GM-BMMs. Figure S9. Histologicl nlysis on specimens otined from the nephrectomized kidneys of ptients with unilterl prtil nd complete ureterl ostruction nd the surrounded norml renl tissues of ptients with renl cell crcinom. Figure S1. hs criticl role in the regultion mcrophge polriztion in kidney. Supplementry mteril is linked to the online version of the pper t REFERENCES 1. Lopez-Novo JM, Mrtinez-Slgdo C, Rodriguez-Pen AB et l. Common pthophysiologicl mechnisms of chronic kidney disese: therpeutic perspectives. Phrmcol Ther 1; 18: Ricrdo SD, vn Goor H, Eddy AA. Mcrophge diversity in renl injury nd repir. J Clin Invest 8; 118: Schiffer L, Bethunickn R, Rmnujm M et l. Activted renl mcrophges re mrkers of disese onset nd disese remission in lupus nephritis. J Immunol 8; 18: Vielhuer V, Kulkrni O, Reichel CA et l. Trgeting the recruitment of monocytes nd mcrophges in renl disese. Semin Nephrol 1; 3: Klhr S, Morrissey J. Ostructive nephropthy nd renl firosis. Am J Physiol Renl Physiol ; 83: F81 F875.. Benoit M, Desnues B, Mege JL. Mcrophge polriztion in cteril infections. J Immunol 8; 181: McMicking J, Xie QW, Nthn C. Nitric oxide nd mcrophge function. Annu Rev Immunol 1997; 15: Chng NC, Hung SI, Hw KY et l. A mcrophge protein, Ym1, trnsiently expressed during inflmmtion is novel mmmlin lectin. J Biol Chem 1; 7: Res G, Noel W, Beschin A et l. FIZZ1 nd Ym s tools to discriminte etween differentilly ctivted mcrophges. Dev Immunol ; 9: Stein M, Keshv S, Hrris N et l. Interleukin potently enhnces murine mcrophge mnnose receptor ctivity: mrker of lterntive immunologic mcrophge ctivtion. J Exp Med 199; 17: Dumont P, Berton A, Ngy N et l. Expression of glectin-3 in the tumor immune response in colon cncer. L Invest 8; 88: Tsunwki S, Sporn M, Ding A et l. Dectivtion of mcrophges y trnsforming growth fctor-et. Nture 1988; 33:. 13. Svill J, Drnsfield I, Gregory C et l. A lst from the pst: clernce of poptotic cells regultes immune responses. Nt Rev Immunol ; : Gordon S. Alterntive ctivtion of mcrophges. Nt Rev Immunol 3; 3: Bouchon A, Fcchetti F, Weignd MA et l. mplifies inflmmtion nd is crucil meditor of septic shock. Nture 1; 1: Klesney-Tit J, Turnull IR, Colonn M. The TREM receptor fmily nd signl integrtion. Nt Immunol ; 7: McVicr DW, Tylor LS, Gosselin P et l. DAP1-medited signl trnsduction in nturl killer cells. A dominnt role for the Syk proteintyrosine kinse. J Biol Chem 1998; 73: Lnier LL. DAP1- nd DAP1-ssocited receptors in innte immunity. Immunol Rev 9; 7: Giot S, Crvoisy A, Levy B et l. Solule triggering receptor expressed on myeloid cells nd the dignosis of pneumoni. N Engl J Med ; 35: Lin YT, Tseng KY, Yeh YC et l. promotes survivl during Klesiell pneumonie liver scess in mice. Infect immun 1; 8: Kui J, Gregory B, Hill A et l. expression is incresed in the synovium of rheumtoid rthritis ptients nd induces the expression of pro-inflmmtory cytokines. Rheumtology (Oxford) 9; 8: Schenk M, Bouchon A, Seiold F et l. expressing intestinl mcrophges crucilly mplify chronic inflmmtion in experimentl colitis nd inflmmtory owel diseses. J Clin Invest 7; 117: Chevlier RL, Fores MS, Thornhill BA. Ureterl ostruction s model of renl interstitil firosis nd ostructive nephropthy. Kidney Int 9; 75: Klesney-Tit J, Keck K, Li X et l. Trnsepithelil migrtion of neutrophils into the lung requires. J Clin Invest 13; 13: Hmilton JA. Colony-stimulting fctors in inflmmtion nd utoimmunity. Nt Rev Immunol 8; 8: Verreck FA, de Boer T, Lngenerg DM et l. Humn IL-3-producing type 1 mcrophges promote ut IL-1-producing type mcrophges suvert immunity to (myco)cteri. Proc Ntl Acd Sci USA ; 11: Fleetwood AJ, Lwrence T, Hmilton JA et l. Grnulocyte-mcrophge colony-stimulting fctor (CSF) nd mcrophge CSF-dependent mcrophge phenotypes disply differences in cytokine profiles nd trnscription fctor ctivities: implictions for CSF lockde in inflmmtion. J Immunol 7; 178: Kidney Interntionl (1) 8,

13 sic reserch T-H Lo et l.: modultes M1 polriztion in nephritis 8. Wu MF, Chen ST, Yng AH et l. CLEC5A is criticl for dengue virusinduced inflmmsome ctivtion in humn mcrophges. Blood 13; 11: Wu J, Li J, Slcedo R et l. The proinflmmtory myeloid cell receptor controls Kupffer cell ctivtion nd development of heptocellulr crcinom. Cncer Res 1; 7: Kifu T, Nkhr J, Inui M et l. Osteopetrosis nd thlmic hypomyelinosis with synptic degenertion in DAP1-deficient mice. J Clin Invest 3; 111: Wng Y, Co Q, Zheng G et l. By homing to the kidney, ctivted mcrophges potently excerte renl injury. Am J Pthol 8; 17: Co Q, Wng Y, Zheng D et l. Filed renoprotection y lterntively ctivted one mrrow mcrophges is due to prolifertion-dependent phenotype switch in vivo. Kidney Int 1; 85: Tmmro A, Stroo I, Rmpnelli E et l. Role of TREM1-DAP1 in renl inflmmtion during ostructive nephropthy. PLoS One 13; 8: e te Witt R, vn Wolfswinkel ME, Petit PL et l. Neopterin nd proclcitonin re suitle iomrkers for exclusion of severe Plsmodium flciprum disese t the initil clinicl ssessment of trvellers with imported mlri. Mlr J 1; 9: Shrif O, Knpp S. From expression to signling: roles of nd TREM- in innte immunity nd cteril infection. Immunoiology 8; 13: Giot S, Buonsnti C, Mssin F et l. Modultion of the triggering receptor expressed on the myeloid cell type 1 pthwy in murine septic shock. Infect Immun ; 7: Hselmyer P, Grosse-Hovest L, von Lndenerg P et l. lignd expression on pltelets enhnces neutrophil ctivtion. Blood 7; 11: Kidney Interntionl (1) 8,