A systems immunology approach identifies the collective impact of five miRs in Th2 inflammation Supplementary information Ayşe Kılıç, Marc Santolini, Taiji Nakano, Matthias Schiller, Mizue Teranishi,Pascal Gellert, Yuliya Ponomareva, Thomas Braun, Shizuka Uchida, Scott T. Weiss, Amitabh Sharma and Harald Renz Corresponding Author: Harald Renz, MD Institute of Laboratory Medicine Philipps University Marburg 35043 Marburg, Germany Phone +49 6421 586 6235 [email protected] 1 Kılıç A et. al. 2018 A B C D PBS OVA chronic PAS PAS (cm H2O*s/ml (cm Raw Sirius Red Sirius Supplementary figure 1: Characteristic phenotype of the acute and chronic allergic airway inflammatory response in the OVA-induced mouse model. (a) Total and differential cell counts determined in BAL and cytospins show a dominant influx of eosinophils. (b) Serum titers of OVA-specific immunoglobulins are elevated. (c) Representative measurement of airway reactivity to increasing methacholine (Mch) responsiveness measured by head-out body plethysmography. (d) Representative lung section staining depicting airway inflammation and mucus production (PAS, original magnification x 10) and airway remodeling (SiriusRed; original magnification x 20). Data are presented as mean ± SEM and are representative for at least 3 independent experiments with n=8-10 animals per group. Statistical analyses have been performed with one-way ANOVA and Tukey’s post-test and show *p<0.05, **p<0.01 and ***p<0.001. 2 Kılıç A et. al. 2018 lymphocytes A lung activated CD4+-cells CD4+-T-cell-subpopulations SSC FSC ST2 live CD69 CD4 CXCR3 B spleen Naive CD4+- T cells DAPI FSC single cells SSC CD62L CD4 CD44 FSC FSC-W Supplementary figure 2. Gating strategy for the isolation of live CD4+ T cell subsets using FACS-sorting. Representative dot-plots, depicting the gating strategy for the isolation of activated Th2 cells as CD4+ CD69+ T1/ST2+ CXCR3- from inflamed lung tissue. (A) Activated/tissue resident Th1 and Th2 cells were sorted from the CD4+CD69+ fraction and based on surface expression of CXCR3+ (Th1) and T1/ST2+ (Th2). (B) Naïve CD4+ T cells were isolated from spleens of sham immunized and sham challenged mice by sorting CD4+CD62L+CD44- cells. (C) 3 Kılıç A et. al. 2018 A B mRNA-mRNA network miRNA-mRNA network PLAUR CTNNA1 CDK6 KIF3A MAP3K5 LTB ETV6 GNB4 LILRB4 DMXL2 DDX26B EZH2 CTSH EPAS1 NAV2 ARHGAP29 CENPE PICALM CYP11A1 FURIN RORA TYMS RHOQ CNN3 DSTN BUB1 TNFSF10 LNX2 hsa-mir-921hsa-mir-181a ECT2 TNFRSF1A SEC24D NCAPG CD44 PER1 PTPN4 IPCEF1 PTPRJ HIST2H2AA3 AHR NPNT hsa-mir-182 BCL9 PITPNM2 TIGIT CCR8 E2F8 IKZF2 TIPARP LFNG TPX2 NEB CD38 IPMK FRMD4B hsa-mir-509-3p ERN1 DAPK1 RAB31 hsa-mir-363hsa-mir-374b hsa-mir-181c ACTN1 ID2 TEC HIST1H2AG TMEM62 PDE2A ALDOA CREB3L2 GNPTAB hsa-mir-595 hsa-mir-603 ACP2 CAMK2G MPZL1 LEF1 CTLA4 PBX3 TUBA4A KIF23 ATRNL1 DUSP2 CD9 AKAP8 RACGAP1 TANK PPP4R1 hsa-mir-520e IMPA2 hsa-mir-665 hsa-mir-17 ZBTB32 CREM LZTFL1 NCKAP1 MTMR10 IL2RB PTGS2 ARL14 PDLIM5 hsa-mir-302c PALLD HIST1H4J hsa-mir-30b CRIM1 HSF2 HIST2H3A HIST1H2BO MFNG MAP3K5 ARMCX2 PRDM1 BCAR3 IL4 DNAJB14 ELL2 GPM6B ACVR2A HIST1H2AA GNG2 CD2AP IL10 BTG2 GAD1 EVI5 TAF7 AHR BCL3 ANLN GEM GNPDA1 hsa-mir-25 hsa-mir-24 hsa-mir-146a DGKZ RPL13A BACH2 CD80 KLF12 hsa-mir-96 hsa-mir-93 hsa-mir-148b PTPN3 STAMBPL1 hsa-mir-943 MYO1Dhsa-mir-604 PCGF5 PTPN6 CYTH3 hsa-mir-593hsa-let-7b OLR1 hsa-mir-578 hsa-mir-18a BCL2L11 RALGDS NCKAP1 COBLL1 MKI67 hsa-mir-32 USP28 ID3 HIST1H3A hsa-let-7d PLP2 SVIL CNN3 PCYT1A EPAS1 VCL IL13 ARL5A HSF2 hsa-mir-136CD2AP HMMR NFKBIZ hsa-mir-23b ALDH3A2 GRPhsa-mir-98 OSBPL3 BCL3 PRDM1 ITGB8 hsa-mir-181dMPZL1 IL1R2 SAMSN1HIST1H3C CHKA IL6 IL13 APBB1IP HIST2H2BF CDK1 PIP4K2A PLAGL1 ELMO2 USP24 PHF20L1 RPL13 STX11 HIST1H3E CSF1 ATXN1 hsa-mir-504 PLEKHA1 IKZF3 TIAM1 hsa-mir-657 TNFRSF1B ACTN1 UBE2D1 TNFSF11 FYN MBP ETFB S100A11 INHBA H2AFZ PLCD4 SLC7A8 PDE5A hsa-mir-335 TNFRSF1B MMP13 MCM6 UBE2D1 SLCO3A1 ATP2B4 IL24 HLF SPP1 RAB11FIP1 PHF20 CBLB HIST1H2BF INPP5A CYCS IGF2R DUSP4 PLCL1 hsa-mir-19a TIMP1 IL1R1 hsa-mir-150 RAPH1 DYNLT3 TRPS1 AQP9 hsa-mir-92a hsa-mir-27b PLP2 INPP4A RAB39B hsa-mir-20b CCNB1 ANLN PDLIM5 ALDOA STRBP PACSIN1 HIST1H4C GOT1HIST1H2AC IL10RA ZBTB10 LEF1 CYFIP1 MYLIP KIAA1598 DGCR2 IKBKE CD200R1 HIST1H2BC HIST1H3F RACGAP1 IFNG TXNDC17 hsa-mir-874PTPN4 BCAR3 hsa-mir-380 TAF4B GSN TRIM35 NCF4 ARHGEF12 GAD1 hsa-mir-29b hsa-mir-22 CDKN2C HIST1H2BI MYO1C ELK3 hsa-let-7c KSR1 INADL hsa-mir-498 F2R GNPTAB HIST1H2BN INHBA C14orf1 IER3 ANXA11 AURKB VDR FRMD4B hsa-mir-378 ADAM19STAT5A hsa-mir-422a SLC20A1 HIST1H2BL VCL ARHGEF3 HIST1H2AM GEM RGS1 CEP68 CA5B ECT2 CRTAM ADAM9 hsa-mir-545 NOTCH1 MYLIP ATP1B1 HIST1H2BK TBX21 ICOS CAMK2G ADARB1 hsa-mir-484 SETX INPP5A EBI3 HIST1H3B PPARG CASC5 GZF1 hsa-mir-138 BOLL HIST4H4 CCR5 PLEK CBX7 hsa-mir-581 RRM2 HIST1H3J NT5E IL6 ENTPD1 STAT5A hsa-mir-7 MID2 hsa-mir-554hsa-mir-26b NRP1 PTP4A3 LY9 ANXA2 hsa-mir-200c PTH RAPGEF6 NR3C1 hsa-let-7i N4BP1 PRKCH FHL3 RNF19A EGLN3 HMGB2 GRAMD3 HIST1H2AE IFI16 HIST1H3H ACP2 PTPN13 hsa-mir-106a UTRN ACLY HIST1H4F IRF8 BACH2 hsa-mir-31 C14orf1 FAM46A GLRX SEC23A hsa-mir-29a NFIL3 MID2 GAPDH hsa-mir-200bhsa-mir-557 FNDC3A GLUD1 RNF128 RYK hsa-mir-21 PTPRS IRF5 MAF AP1S2 IGF1R LPXN RPS6KA5 RAB39B LY75 GNG2 HIST1H2BB CTNNA1 EZH2 ARNT2 hsa-mir-198 ATP1B1 GNAQ CTSB LTB4R CD44 ACVR2A CAPG DGKD ARHGEF12 NFIL3 hsa-let-7g hsa-mir-200a MAF FOXP3 CCR1 EVI5 hsa-mir-221 hsa-mir-141 RNF130 NCAPG2 CREM ATP2B4 NFKBIZ C21orf91 SEC23A NEB hsa-mir-203 HIST1H4D RAB9A SOCS2 hsa-mir-505 FAM46Ahsa-mir-29c HSPA8 STAT3 DLG2 CD200R1 TASP1 hsa-mir-20a HIST2H4B GATA3 TOP2A hsa-mir-154 IGFBP7 CDK6 PBX3 PLEKHA1 HIST1H2AH hsa-mir-27a RUNX2 CASP6 CASP4 GNA15 SNX9 hsa-mir-185 PLEKHB2 ELK3 GRIPAP1 LNX2 UHRF2 KLF12 PLEK TMEM62 PDE5A PLCD4 BMPR2 DUSP16 DSTN HIST1H2BG PLOD2 SNX9 DDX11 PTGER2 DLGAP5 FUNDC2 HIST1H2AJ MYO1E PHF20 PTPRC HLF PLCXD2 PTPN3 RUNX2 FLNB PLCL1 NDUFV2 hsa-mir-610 hsa-mir-520h IGFBP7 N4BP2 RIOK3hsa-mir-132 PALLD IKZF4 MAD2L1 S100A6 HIST1H2AB CCNA2 RPS6KA5 hsa-mir-493 HAVCR2 NOTCH1 CBLB GALNT3 ARNT2 CCNE2 BATF TPI1 MAML3 hsa-mir-487a PRICKLE3 HIST1H2BM H2AFZ CLCN5 ITPR1 DLG4 CCL1 hsa-mir-520f ADAM9 BMPR2 ADAM19 HMGB2 PTPLAD1 CCNE2 TNFSF11 ATXN7L1 HIST1H3D SEC24D STAT3 NEK6 hsa-mir-26a hsa-mir-383 KIF23 RPLP2 INADL AHNAK CSF1 GATA3 NEK6 hsa-let-7a ZDHHC23 HIC1 TAF4B hsa-mir-500 ZNF318 IL10hsa-mir-30e RGS1 UBASH3B ARL14 NCAPH ETV6 FFAR2 GALC CASP1 IKZF2 OTUB2 CYP1A1 AMMECR1 ERO1L DAPK1 FNTB IL5 IL1RN ATP2B1 NR3C1 HIST2H2AC LDHB hsa-mir-16 IKZF4 CLDND1 BZW2 RNH1 KIF3A NDUFB1 HIST1H2BE CHKA LAMC1 hsa-mir-922 SMURF1 ZDHHC21 hsa-mir-520g BMPR1A SVIL TIAM1 ZFYVE16 RNF128 FES GNAQ hsa-mir-650 hsa-mir-100 target ATXN1 hsa-mir-575 BAZ1A PPARG CLCF1 ERF HIST1H2AD HIST1H3I hsa-mir-30a HIST2H4A hsa-mir-187 DLG2 KIF11 hsa-mir-891b ITPR1 AGPAT4 IGF1R HS3ST3B1 ANXA4 FURIN ETV5 hsa-mir-34a INTS8hsa-mir-149 miRNA PTPN13 DAPP1 RNF130 hsa-mir-192hsa-mir-15b hsa-mir-23a SCN1B hsa-mir-617hsa-mir-744hsa-mir-95hsa-mir-133bhsa-mir-585 hsa-mir-620 hsa-mir-99b hsa-mir-639hsa-mir-663 hsa-mir-652 hsa-mir-637 hsa-mir-638 hsa-mir-765hsa-mir-220b hsa-mir-675 hsa-mir-181b hsa-mir-625 COBLL1 FES hsa-mir-597 t t es es h mRNA w ig Lo H miRNA Degree Supplementary Figure 3. (A) The connectivity of mRNA/genes coding proteins in interactome in early Th2 cells. (B) The connectivity of miRNA and their targets in miRNA-mRNA network (mirDIP). We applied force-directed layout in Cytoscape for visualization of the networks. 4 Kılıç A et. al. 2018 mRNA-mRNA network miRNA-mRNA network RAD50 TRIM35 CYP1B1 CHRAC1 EZR hsa-mir-922 RAB8A PTPN3 GLB1 CYP1A1 hsa-mir-30b A CAPN2 SPN STK25 RXRB XRN2 CUL4A NCOR2 B TAF4 hsa-mir-551b BACE2 VCL BRPF1 PSIP1 FOXN2 SRRT IL5 PRMT8 MAPKAPK2 DYRK2 CHCHD3 ACIN1 hsa-mir-138 PITPNM2 STUB1 CSNK1G3 PUM1 U2AF2 VEZT CHRM2 ATG16L1CCDC22 KIF2A PTPRC ATRIP GAPVD1 GATA3 LPHN2 hsa-mir-203 RBM28 FRMD4B NFIL3 NR3C1 CRY2 USP28 RNF130 SERINC3 FOXP4 NEDD8 HMGXB3 RRAS2 LCOR SDC1 ENAH TARS2 DYRK1B PCYT1A hsa-mir-649 IL1R2 ELL2 PACSIN1 hsa-mir-891b GRM5 FOXJ3 TIMP1 KLC1 SIAH2 PPARG GPR45 hsa-mir-575 TERF2 CDON DYRK2 ABCC1 RALY DUSP4 RAB31 CHKA ESYT2 DCAF6 FGF2 SRF SLC25A27 CD79A RANBP3 SLC48A1 OVGP1 TPBG BACH2 CD84 MAPK8IP3 hsa-mir-607MLF1 hsa-mir-593 RPS15A RALGPS2 PTK2B hsa-mir-665 PTPLAD1 GNPDA1 THOC1 ELF2 ZNF277 hsa-mir-363 FOXJ3 MVP GLUD1 DSTN HPSE TOM1 ITPR1 ABL2 XRCC4 ELK4 FUNDC2MYO1E FBXW11 IL22 FURIN SLK PEX13 MKNK1 DVL1 NCOA3 GRN MAPRE1 hsa-mir-27a hsa-mir-548a-3p IL10RA BRF1 CLSTN1 RPS5 RBPJ H2AFZ IPPK DENND4A RTN3 PEX10 ITPR1 TSC1 ZFAND3 CNOT10 SETD7 GAPDH CA13 PTPN12 PIP4K2A JPH1 TCF12 HIPK2 CWC15 KIF16B SP1 KSR1 PTPN4 hsa-mir-30ePPP1R2 ITGB8 GJB3 hsa-mir-516a-5p hsa-mir-195 PCID2 RNLS PLCD1 C1orf94 IMPA2 PTPN9 HLA-A ADAM9 ME1 hsa-mir-220c KLRD1 hsa-mir-520hPRDM2 POGZ ID2 SUFU CPSF3 RHOQ ID3 IL9R hsa-mir-610 hsa-mir-452 SFN RGS13 IL12RB1RPL18 GOT1 ARMC8 EPN2 BCAR3 MIPOL1 PRICKLE3 TREX1 hsa-mir-24 ZNF653 KLHL6 MYO1D SVIL GMNN EFR3A RUNX2 INHBA SMAD3 RPS13 GSN REV1 ATR SAP130 CTNNA1 GATA3 SUV420H1hsa-mir-22 IER3PRKCD PYGM BCL2L11 PHF20L1 CAMKK2 BNC1 RYK ERO1L CRMP1 PLEKHO1 IL6ST SLC4A2 PROS1 UTRN TAF8 PRDX6 RPL13 GNAQ BMPR1A CLDND1 IGFBP7 MAF CD2AP PDCD10 ACBD5 IL1RL1 IGF1R IRF3 hsa-mir-378 PTK2B UBE2D1 GTF2I RBM6 COIL SERINC3 TASP1 IFI16 RAB11FIP1 TNFRSF1A AKAP8L MED1 FURIN SDC4 N4BP1 hsa-mir-383 IL24 PLK2 MSC INADL SLC44A1 DDX19B MKRN2 LRP12 MEF2A ERAP1 TESK1 USP15MYLIP ITGAD TPI1 PLOD2 PHF3 SERTAD2 TSC22D1 SUCLG2 CHD7PCNA TXK RPL19 AMPH PLCD4 SMAD3 R3HDM1 PALLDMYD88 LY75RPL31 NCKAP1 INPPL1 CTSH hsa-mir-181b ATP2B1 PRDM1 FBXW11 TRIM8 VASP MAPKAPK5 TSC2 EMP1 hsa-mir-21 NDUFB1 PKD2 ARNT2 USP7 ATG4B IGF2R IPO9 EIF3KSMARCD1 CA5B LY75