SUPPLEMENTARY INFORMATION doi:.8/nture89 4 4 Ilr -/- Ilr -/- Ilr -/- Cspse- -/- As -/- Nlrp -/- Il8 -/- Ilr -/- Supplementl figure. Inresed severity of NASH in inflmmsome-defiient mie, ut not in Ilr-defiient nimls. To indue NASH, mie were fed with MCDD for 4 d. Their serum ALT nd AST tivities mesured nd NAFLD histologil tivity sores were determined. (-) Comprison of ALT, AST, nd NAFLD tivity, plus histologil sores for stetosis nd inflmmtion etween singly-housed wild-type () mie nd Ilr -/- nimls. () Representtive hemtoxylin nd eosin (H&E)-stined setions of livers from, spse- -/-, As -/-, Nlrp -/-, Il8 -/-, nd Ilr -/- mie. Inflmmtory foi re highlighted with n rrowhed. Dt represent two independent experiments (n=7-9 mie/tretment group). Error rs represent the SEM of smples within group. Sle rs = µm (K). p.5, p., p. (Student s t test). WWW.NATURE.COM/NATURE
RESEARCH SUPPLEMENTARY INFORMATION ell numer.! 7 8.! 6 MCDD 6.! 6 (As -/- ) MCDD As -/- () MCDD 4.! 6.! 6 4.! 6.! 6 ell numer CD45+ B ells NK ells NKT ells T ells totl CD4 T ells CD8 T ells dendriti ells mono/mroph neutrophils d 4 4 As -/- ;Rg -/- As -/- ;Rg -/- As -/- ;Rg -/- Supplementl figure. Chnges in liver ellulrity in MCDD-fed As-defiient mie nd ohoused nimls. Singly-housed, o-housed As -/- (), nd o-housed (As -/- ) nimls were fed MCDD for 4 d to indue NASH, nd hemtopoieti ell susets in liver were quntified y FACS. () Totl numers of CD45+ ells, B ells (B + ), T ells (TCR! + ), CD4+ T ells, CD8+ T ells, NK ells (NK. + TCR! - ), NKT ells (NK. + TCR! + ), dendriti ells (CD + CD - ), mononuler mrophges (MHCII + CD + ), nd neutrophils (Gr + ). p".5, p"., p". etween the single housed group nd the o-housed (As -/- ) nimls (Student s t test). Dt is prepresenttive of two independent experiments. (-d) Comprison of serum ALT, serum AST, plus NAFLD tivity histologil sores for stetosis nd inflmmtion in nd ompound homozygous knokout As -/- ;Rg -/- mie. Error rs represent the SEM of smples within group. p".5, p"., p". (Student s t test). WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH 6 4 4 > Nlrp -/- > > Nlrp -/- > > Nlrp -/- > d e f 4 > As -/- > > As -/- > > As -/- > g 4 h i 4 ;Cd-re NlrpKI;Cd-re ;Cd-re NlrpKI;Cd-re WT;CD+-Cre ;Cd-re NLRPKI;CD NlrpKI;Cd-re + -Cre j k l 5 4 ;Alumin-re NlrpKI;Alumin-re 5 4 ;Alumin-re NlrpKI;Alumin-re ;Alumin-re NlrpKI;Alumin-re Supplementl figure. Ativtion of the NLRP inflmmsome in hemtopoieti ells nd heptoytes does not influene NASH severity. To indue NASH, mie were given MCDD for 4 d, nd their serum ALT nd AST tivities, nd NAFLD histologil tivity sores were determined. (-f) Comprison of ALT, AST, nd NAFLD tivity histologil sores for stetosis nd inflmmtion etween himeri mie generted with ( > ) nd Nlrp -/- (Nlrp -/- >) one mrrow (BM) (-) or As -/- (As -/- >) BM (d-f). (g-l) Comprison of serum ALT nd AST tivities, nd NAFLD tivity histologil sores for stetosis nd inflmmtion etween ;CD+-Cre nd NlrpKI;CD-Cre mie (g-i) or ;lumin-cre nd NlrpKI;lumin-Cre mie (j-l). Error rs represent the SEM of smples within group. p.5, p., p.. (Student s t test) WWW.NATURE.COM/NATURE
RESEARCH SUPPLEMENTARY INFORMATION d 8 8, 6 4 6 4 8 6 4 8 6 4 (Cspse- -/- ) (Cspse- -/- ) (Nlrp -/- ) (Nlrp -/- ) e f g h 6 4 5 4 4 8 6 4 (Nlrp6 -/- ) (Nlrp6 -/- ) (Nlrp4 -/- ) (Nlrp4 -/- ) i j k l 4 4 6 6 4 4 (Nlr4 -/- ) (Nlr4 /- ) (Nlrp -/- ) (Nlrp -/- ) Supplementl figure 4. Inresed severity of NASH in spse--, Nlrp-, nd Nlrp6-defiient mie is trnsmissile to o-housed wild-type nimls. Singly-housed () mie nd mie o-housed with spse- -/- nimls ( (spse- -/- )) (,), Nlrp -/- nimls ((Nlrp -/- )) (,d), Nlrp6 -/- nimls ((Nlrp6 -/- )) (e,f), Nlrp4 -/- mie ((Nlrp4 -/- )) (g,h), Nlr4 -/- mie ((Nlr4 -/- )) (i,j), nd Nlrp -/- mie ((Nlrp -/- )) (k,l) were given MCDD for 4 d to indue NASH. Comprison of serum ALT nd AST tivities in nd (spse- -/- ) (,), (Nlrp -/- ) (,d), (Nlrp6 -/- ) (e,f), (Nlrp4 -/- ) (g,h), (Nlr4 -/- ) (i,j), nd ((Nlrp -/- )) nimls (k,l). Error rs represent the SEM of smples within group (n=-8 mie/group). p.5, p., p.. (Student s t test) 4 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH 5 5 5 Antiiotis 4 5 (As -/- ) As -/- () (As -/- ) As -/- () (As -/- ) As -/- () (As -/- ) As -/- () 5 Antiiotis d (As -/- ) As -/- () (As -/- ) As -/- () (As -/- ) As -/- () (As -/- ) As -/- () Antiiotis Antiiotis Supplementl figure 5. The inresed severity of NASH in As nd Il8-defiient mie nd o-housed wildtype nimls is olished with ntiioti tretment. (-d) Comprison of serum ALT () nd AST (), plus NAFLD tivity histologil sores for stetosis () nd inflmmtion (d) of (As -/- ) nd As -/- () mie tht were untreted or treted orlly with omintion of metronidzole nd iprofloxin for 4 weeks. Inflmmtory foi re highlighted with n rrowhed. Dt re represents two independent experiments (n=5-7 mie/tretment group). Error rs represent the SEM of smples within group. p.5, p., p.. (ANOVA) WWW.NATURE.COM/NATURE 5
RESEARCH SUPPLEMENTARY INFORMATION 8 8 6 6 4 4 e 8 6 4 Myd88 -/- ;Trif -/- () Myd88 -/- ;Trif -/- (As -/- ) Myd88 -/- ;Trif -/- () Myd88 -/- ;Trif -/- (As -/- ) 4 Tlr4 -/- Tlr4 -/- (As -/- ) d f NAFLD NAFLD tivity tivity sore sore TLR4-/- Tlr4 TLR4-/-(ASC-/-) Tlr4 (As ) Tlr9 -/- Tlr9 -/- (As -/- ) g 6 4 Tlr9 -/- Tlr9 -/- (As -/- ) Tlr5 -/- Tlr5 -/- (As -/- ) h TLR5-/- Tlr5 TLR5-/-(ASC-/-) Tlr5 (As ) H Supplementl figure 6. Inresed severity of NASH in As-defiient mie nd o-housed wild-type nimls is medited y TLR4, TLR9. As -/- mie were o-housed with, Myd88 -/- ;Trif -/-, Tlr4 -/-, Tlr9 -/-, or Tlr5 -/- mie for 4 weeks, fter whih time mie were fed MCDD for 4 dys to indue NASH. (-) Comprison of serum ALT nd AST tivities from MCDD-fed (As -/- ) nd Myd88 -/- ;Trif -/- (As -/- ) mie. Dt re representtive of two independent experiments. (-h) Comprison of serum AST levels nd NAFLD tivity histologil sores for stetosis nd inflmmtion from MCDD-fed Tlr4 -/- (As -/- ) (-d), Tlr9 -/- (As -/- ) (e-f), nd Tlr5 -/- (As -/- ) nimls (g-h) nd their singly-housed ounterprts. Dt represent two independent experiments. Error rs represent the SEM of smples within group. p.5, p., p. (Student s t test). 6 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH, 6 Tlr gonists (pg/ml) 8 6 4 Tlr9 gonists (pg/ml) 5 4 - (As -/- ) As -/- - (As -/- ) As -/- As -/- Supplementl figure 7. Inresed severity of NASH in As-defiient mie nd o-housed wild-type nimls is medited y TLR gonist influx into portl irultion. As -/- mie were o-housed with mie for 4 weeks, fter whih time mie were fed MCDD for 4 dys to indue NASH. (-) Comprison of TLR () nd TLR9 () gonists in portl vein ser otined t the time of srifie of singly-housed MCDD-fed mie, o-housed (As -/- ) nimls nd singly-housed As -/- nimls. Dt represent two independent experiments. () Representtive trnsmission eletron mirosopy imges tken from oloni setions prepred from (top X8) nd As -/- nimls (ottom left X65). Error rs represent the SEM of smples within group. p!.5, p!., p!.. (ANOVA) WWW.NATURE.COM/NATURE 7
RESEARCH SUPPLEMENTARY INFORMATION mrna (fold indution).5..5..5. As -/- mrna (fold indution).5..5..5 Il8 -/-.. Tnfα Il6 Ilβ Tnfα Il6 Ilβ Tnfα Il6 Ilβ mrna (fold indution).5..5..5 Ilr -/- mrna (fold indution) d 6 4 Tnfα Il6 Ilβ e (As -/- ) mrna (fold indution) 8 6 4 Tnfα Il6 Ilβ (Il8 -/- ) f 4 Tnfα -/- Tnfα -/- (As -/- ) (As -/- ) g Tnfα -/- Tnfα -/- (As -/- ) (As -/- ) Supplementl figure 8. Inresed Tnfα expression in As -/-, Il8 -/-, ut not in Ilr -/- mie during NASH. (-) Comprison of hepti Tnfα, Il6, nd Ilβ mrna levels in singly-housed nd As -/- (), Il8 -/- (), or Ilr -/- () mie. (d-e) Comprison of hepti Tnfα, Il6, nd Ilβ mrna levels in singly-housed mie () versus mie tht were previously o-housed with As -/- nimls ((As -/- )) (d), or Il8 -/- nimls ((Il8 -/- ))(e) for four weeks prior to NASH indution. (f-g) AST serum levels (f) nd representtive H&E-stined setions of livers from singly-housed Tnfα -/- mie, nd o-housed mie ((As -/- ) or Tnfα -/- mie o-housed with As -/- nimls (Tnfα -/- (As -/- ). Sle rs = µm. Dt re representtive for two independent experiments. Error rs represent the SEM of smples within group. p.5, p., p.. (Student s t test) 8 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH d Tlr4 gonists (pg/ml) WT CCL5-/- Cl5 WT CCL5-/- Cl5 WT CCL5-/- Cl5 8 6 6 4 e Tlr9 gonists (pg/ml) 4 f sore Tlr gonists (pg/ml), 8 6 4 (As -/- ) Cl5 -/- (As -/- ) (As -/- ) Cl5 -/- (As -/- ) (As -/- ) Cl5 -/- (As -/- ) Supplementry figure 9. Intestinl inflmmtion ssoited with n As -/- gut miroiot inreses the influx of TLR gonists into the portl irultion. To indue NASH, mie were given MCDD for 4 d, nd their serum ALT nd AST tivities, nd NAFLD histologil tivity sores were determined. (-) Comprison of ALT (), AST (), nd NAFLD tivity histologil sores for stetosis nd inflmmtion () etween seprtely housed nd Cl5 -/- mie. (n=8 nimls surveyed/group). (d-f) or Cl5 -/- mie were o-housed with As -/- mie for 4 weeks fter whih time mie were fed MCDD for 4 d to indue NASH. Comprison of TLR4 (d), TLR9 (e), nd TLR (f) gonists in portl vein ser olleted from MCDD-treted, o-housed (As -/- ) nd Cl5 -/- (As -/- ) mie. Error rs represent the SEM of smples within group (n=6 nimls surveyed/group). p!.5, p!., p!. (Student s t test). WWW.NATURE.COM/NATURE 9
RESEARCH SUPPLEMENTARY INFORMATION Weight (g) weight (g) 5 4 WT Nlr4 NLRC4-/- 4 5 6 7 8 9 Weeks weeks on on HFD HFD Gluose (mg/dl) 8 6 4 5 45 6 75 9 minutes minutes fter fter gluose gluose i.p i.p d Gluose (mg/dl) 5 5 Insulin (ng/ml) WT NLRC4-/- Nlr4 WT NLRC4-/- WT NLRC4-/- Nlr4 Nlr4 Insulin (ng/ml) 5 4 Supplementry figure. Nlr4-defiient mie hve norml weight gin rte nd glyemi ontrol on HFD. Age-mthed mle Nlr4 -/- mie nd mie were fed 6% HFD. Body weights were monitored weekly (), gluose tolerne tests were performed in mie nd Nlr4 -/- mie fter weeks of HFD (). Fsting (4h) lood gluose () nd insulin (d) levels were mesured fter 8 weeks on the HFD, (n=8- mie/group). Error rs represent the SEM of smples within group. WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH (As -/- ) As -/- Supplementl figure. As-defiient mie o-housed mie develop inresed stetosis on HFD. () Representtive hemtoxylin nd eosin (H&E)-stined setions of livers from, (As -/- ), nd As -/- mie. Sle rs = µm. WWW.NATURE.COM/NATURE
RESEARCH SUPPLEMENTARY INFORMATION Supplementl figure. Antiioti tretment leds to redution in tx ssoited with HFD. () As-/- nd WT mie were or were not treted with iprofloxin nd metronidzole for 4 weeks efore eing swithed to high ft diet. Time points were tken fter eing fed HFD for nd 8 weeks. (-e) PCoA nd r grphs showing redution in Prevotellee, Porphyromondee nd Bteroidee fter ntiioti tretment. Enterooee were noted to inrese in representtion fter ntiioti tretment. WWW.NATURE.COM/NATURE