Supplementary Figure 1. Microarray data mining and validation
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1 Supplementary Figure 1. Microarray data mining and validation Microarray (>47,000 probes) Raw data Background subtraction NanoString (124 genes) Set cut-off and threshold values Raw data Normalized to median of all samples Normalized to baseline Normalized to positive controls Filter by flags (100% present) Filter by flags (100% present) Normalized to housekeeping genes Fold change >2 Fold change >2 MWU test uncorrected p<0.05 MWU test corrected for BH-FDR p<0.05 MWU test corrected for BH-FDR p<0.05 Week 2 = 76 genes Week 0.5 = 20 genes Week 2 = 125 genes Week 0.5 = 50 genes Week 2 = 281 genes 43 genes common to all 3 approaches at week 2 Two analytical approaches were used to extract differentially abundant transcripts from microarray. The classical approach is by normalization to median of all samples and by our alternative approach, normalization to individual baseline values. Genes from the classical approach were then validated by a technically independent method, the NanoString assay. At 2 weeks, 43 transcripts were consistently differentially abundant irrespective of platform and analytic method. MWU = Mann Whitney U test; BH-FDR = Benjamini-Hochberg corrected False Discovery Rate.
2 Supplementary Table 1. Clinical characteristics of 63 patients with HIV-associated TB Variables Microarray Non-microarray Non-IRIS (n = 15) IRIS (n = 17) Non-IRIS (n = 15) IRIS (n = 16) P-value Male sex 6 (35) 6 (40) 7 (44) 7 (47) Age (years) 28.8 (27-37) 31 (27-36) 32.6 (24-43) 32.9 (26-44) TB treatment prior to ART (days) HIV viral load (10 4 copies/ml) CD4 baseline (cells/mm 3 ) 41 (34-76) 38 ( ) 26 (22-43) 54 (27-62) (6.1-72) 32 ( ) 49 (27-250) 102 (15-158) (27-156) 80 (35-151) 82 (50-205) 88 ( ) TB Diagnosis Clinical & Radiological 5 (33) 4 (24) 4 (27) 5 (31) Sputum smear 0 (0) 0 (0) 0 (0) 1 (6) Cultured AFB/MTB 10 (67) 13 (76) 11 (73) 10 (63) Type of TB PTB 2 (13) 3 (18) 2 (13) 2 (12) EPTB 5 (33) 1 (6) 2 (13) 3 (19) PTB + EPTB 8 (63) 13 (76) 11 (73) 11 (69) WHO stage III 2 (13) 2 (12) 2 (13) 3 (19) IV 13 (87) 15 (88) 13 (87) 13 (81) Pre-ART corticosteroid therapy 2 (13) 2 (12) 4 (27) 2 (13) TB-IRIS and non-iris patients included in the analysis were found to be matched for clinical variables in subsequent analysis. Numbers in parentheses are percentage for contingency data or median plus interquartile range for continuous data.
3 Supplementary Table 2. Differentially abundant transcripts identified at week 0.5 Week 0.5 (Normalized to median) Week 0.5 (Normalized to baseline) Gene Regulation Fold change Gene Regulation Fold change BATF2 up AIM2 up CEACAM3 up ANP32A up DHRS13 up APOL2 up FCGR1A up APOL6 up FCRL2 down BATF2 up FUT6 up BCL2A1 up HIST1H2BD up BST2 up HIST1H3D up CARD16 up HS up CEACAM1 up IER3 up CEACAM3 up LOC up DDAH2 up LOC up DDX60L up LRG1 up DHRS9 up MAFF up FCGR3A up OSM up FCHO2 up PID1 down GBP3 up SERPING1 up GRAMD1B up SOCS1 up HIST1H3D up SOCS3 up HIST1H4E up TIMM10 up HIST1H4H up HLX up HSPA1B up IFI35 up IFIH1 up IFIT2 up IFIT3 up IL18R1 up IL1RN up IRF7 up LOC up LOC up MDK up NCF1B up NCOA1 up NT5C3 up OAS1 up OASL up PLSCR1 up RAB33B up RSAD2 up RTP4 up SAMD9L up SEPT4 up SERPING1 up SIPA1L2 up SOCS1 up SP140 up TNFSF13B up USP25 up ZBP1 up Using two different normalization approaches, we identified 20 (normalized to median) and 50 (normalized to baseline) genes that were differentially abundant in TB-IRIS. Five genes overlapped in both approaches: BATF2, CEACAM3, HIST1H3D, SERPING1 and SOCS1.
4 Supplementary Table 3. Differentially abundant transcripts identified at week 2 (normalization to median) Week 2 (Normalized to median) Gene Regulation Fold change Gene Regulation Fold change ACSL1 up LILRA5 up ADCY3 up LIMK2 up ADM up LIN7A up AGPAT9 up LOC up AIG1 up LOC up ANXA3 up LOC up APOB48R up LOC up AQP10 up LOC up ASPRV1 up LOC up B3GNT8 up LOC down BAMBI up LOC up BASP1 up LOC down BCL2A1 up LOC up C19orf35 up LOC up C19orf59 up LOC up CAMKK2 up LRG1 up CARD17 up LRPAP1 up CASP5 up MAFF up CATSPER1 up MANSC1 up CDK5RAP2 up MAPK13 up CEACAM1 up MAPK14 up CEACAM3 up MIAT down CKAP4 up MIIP up CLC down MMP25 up COL4A3BP up MRPL40 up CR1 up NAMPT up CSF3R up NBN up CYP1B1 up NLRC4 up DHRS13 up OAS1 up DKFZp761E198 up PAG1 up DOK3 up PFKFB3 up DSC2 up PGS1 up ECE1 up PHAX up EEF1D up PPP2R3A up ELF2 up PRDX6 up ERI1 up PSG9 up FCGR1A up RAB20 up FCGR1C up RASGRP4 up FCRL2 down RBM47 up FLJ20273 up RHOBTB1 up FLJ35801 down RP2 up FLJ43093 down SEMA6B down FUT6 up SERPINA1 up GK up SERPING1 up GLT1D1 up SIGLEC9 up GNB4 up SIPA1L2 up GPR141 up SMARCD3 up GPR160 up SPRYD3 down GPR175 down SPTY2D1 up GPR97 up TDRD9 up H2AFJ up TLR10 up HECW2 up TLR2 up HIST1H2BD up TLR4 up HIST1H4H up TLR5 up HIST2H2AB up TP53I3 up HIST2H2AC up TPST1 up IFIT3 up TRIB1 up IFITM3 up TUFT1 up IGSF6 up VNN1 up IL18R1 up ZAK up IL1R2 up ZBTB16 up KCNJ15 up KIAA0232 up KLHL2 up Using the normalization to median approach, 125 genes were identified to be differentially abundant in TB-IRIS at week 2 (median time of symptom onset).
5 Supplementary Table 4. Differentially abundant transcripts identified at week 2 (normalization to baseline) Week 2 (Normalized to baseline) Gene Regulation Fold change Gene Regulation Fold change Gene Regulation Fold change Gene Regulation Fold change ACSL1 up FCHO2 up LOC down RAB33B up ADCY3 up FCRL2 down LOC up RANBP10 down ADORA1 down FKBP4 up LOC down REPS2 up AGPAT9 up FKBP5 up LOC down RHOBTB1 up AIG1 up FLJ20273 up LOC up RNF187 down AIM2 up FLJ22662 up LOC down RP11-529I10.4 down AK1 down FLJ43093 down LOC up RPL12P6 down ALAS2 down FOXO3 down LOC down RSAD2 up ALDOB down FTHL2 up LOC up RTP4 up ALPL up FYB up LOC down S100A11 up ANP32A up GBP3 up LOC up S100A12 up ANXA3 up GFOD2 down LOC down S100P up APOB48R up GNB4 up LOC up SAMD9L up ARHGAP18 up GPR141 up LOC down SAMSN1 up ASPRV1 up GPR160 up LOC up SDF2 up AVL9 up GPR97 up LOC up SELS up AXUD1 up GYG1 up LOC down SERPINB8 up B3GNT8 up H2AFJ up LOC down SERPING1 up B4GALT5 up HDC down LOC up SIGLEC7 up BAMBI up HECW2 up LOC up SIGLEC9 up BASP1 up HERC5 up LOC up SIPA1L2 up BATF up HIST1H1C up LRIG1 down SLC11A1 up BCL11B down HIST1H2BD up LRPAP1 up SLC14A1 down BCL2A1 up HIST1H3D up LY6E up SLC1A5 down BCL6 up HIST1H4E up LY96 up SLC25A45 down BSG down HIST1H4H up MAL down SLC26A8 up BST2 up HIST1H4K up MANSC1 up SMARCD3 up C10orf119 up HIST2H2AA3 up MAP2K3 down SOAT1 up C14orf45 down HIST2H2AA4 up MAPK14 up SP100 up C19orf35 up HIST2H2AB up MARCH8 down SPAST up C19orf59 up HIST2H2AC up MAZ up SPATS2L up C1orf128 down HIST2H4A up MFF up SPTLC2 up CARD16 up HPS1 down MIA3 up SRRD down CARD17 up HPSE up MICALCL down SSBP3 down CARS up HSPA1B up MIIP up STRADB down CASP5 up IDO2 down MIR877 down STX3 up CATSPER1 up IFI44 up MMP9 up SULT1B1 up CCNJL up IFI44L up MOSC1 up SYTL4 up CDK5RAP2 up IFI6 up MSI2 down TCP11L2 down CEACAM1 up IFIT1 up MX1 up TDRD9 up CLEC1B up IFIT1L down NACC2 up TESC down CLTC up IFIT2 up NBN up TFDP2 down COL4A3BP up IFIT3 up NCOA1 up TIFA up CSTA up IFITM3 up NFIA down TIMM10 up CXCL1 up IL18R1 up NFIL3 up TLR2 up DCAF6 down IL1RN up NFIX down TLR5 up DDAH2 up IRAK3 up NINJ2 down TNFSF10 up DDX60L up IRF7 up NSUN7 up TNS1 down DENND1A up ISG15 up NT5C3 up TP53I3 up DHRS13 up JUN up OAS1 up TPST1 up DHRS9 up KCNJ10 down OAS2 up TRAM2 up DHX58 up KCNJ15 up OAS3 up TREML2 up DKFZp761E198 up KIAA0319L up OASL up TRIB1 up DNM1L up KIAA1033 up OMA1 up TRIM10 down DOCK5 up KIAA1618 up OPLAH up TRIM25 up DOK3 up KIF1B up OR2W3 down TSPAN5 down DPM2 down KLC3 down PACSIN2 up TSPAN7 down DYSF up KLF1 down PCSK1N down TTC25 down E2F2 down KLHL2 up PDZK1IP1 down TTC33 up ECE1 up KLHL8 up PFKFB3 up TTRAP up EEF1D up LAMC1 up PHC2 up TUFT1 up EIF2AK2 up LAMP3 up PHF3 up UBE2R2 up EPB41 down LEF1 down PIP5K2A down UBL5 up EPB42 down LGALS9 up PLSCR1 up USP48 up EXOSC4 up LILRA3 up PMAIP1 up VNN1 up F5 up LILRA6 up PPP2R3A up ZFP36L1 up FBXO30 up LIN7A up PRDX2 down ZNF23 down FBXO7 down LMNB1 up PRDX6 up ZNF683 down FBXO9 down LOC up PSG9 up FCER1A down LOC up PSMF1 down FCGR3A up LOC up PTGDR down Using the normalization to baseline approach, 281 genes were identified to be differentially abundant in TB-IRIS at week 2 (median time of symptom onset).
6 Supplementary Table 5. Upstream activity prediction of week 2 signature Upstream Regulator Molecule Type Predicted Activation State Activation z-score p-value of overlap IRF7 transcriptional regulator Activated E-06 OSM cytokine Activated E-05 IFNG cytokine Activated E-05 IRF3 transcriptional regulator Activated E-04 TNF cytokine Activated E-04 CSF3 cytokine Activated E-03 HIP1A transcriptional regulator Activated E-03 IL-5 cytokine Activated E-02 CEBPA transcriptional regulator Activated E-02 IL1B cytokine Activated E-02 Upstream regulator prediction was performed using the transcriptomic signatures identified at week 2 (normalization to median method). An array of proinflammatory cytokines and transcription factors that regulate interferon and other cytokines were predicted to be activated (see also Figure 3). The activation z-score was calculated by matching observed and predicted up/down-regulation patterns based on the signatures. The p-value of overlap measures overlap of observed and predicted regulated gene sets using the Fisher s exact test.
7 Supplementary Table 6. Validation of microarray data by NanoString assay Gene Mean (non-iris) Mean (TB-IRIS) P value Significance Gene Mean (non-iris) Mean (TB-IRIS) P value Significance ACSL ** KLHL ** ADCY * LILRA ** ADM ** LIMK * AGPAT * LIN7A AIG ** LOC * ANXA ** LOC * APOB48R * LOC * AQP * LOC ASPRV *** LOC B3GNT ** LOC BAMBI ** LOC BASP ** LOC BCL2A * LOC C19orf ** LOC C19orf LOC CAMKK * LOC * CARD *** LRG * CASP ** LRPAP CATSPER MAFF CDK5RAP * MANSC ** CEACAM MAPK CEACAM * MAPK ** CKAP MIAT ** CLC MIIP COL4A3BP MMP ** CR * MRPL CSF3R * NAMPT *** CYP1B ** NBN DHRS * NLRC * DKFZp761E * OAS * DOK * PAG DSC * PFKFB * ECE ** PGS * EEF1D PHAX ELF * PPP2R3A ERI PRDX FCGR1A * PSG ** FCGR1C * RAB * FCRL RASGRP * FLJ RBM ** FLJ RHOBTB * FUT RP *** GK * SEMA6B GLT1D *** SERPINA ** GNB ** SERPING * GPR SIGLEC * GPR * SIPA1L ** GPR SMARCD ** GPR * SPRYD H2AFJ SPTY2D HECW ** TDRD HIST1H2BD TLR ** HIST1H4H TLR *** HIST2H2AB TLR ** HIST2H2AC TLR ** IFIT * TP53I IFITM *** TPST * IGSF ** TRIB ** IL18R TUFT * IL1R VNN KCNJ ** ZAK *** KIAA ZBTB In a technically independent setup using probes with sequences different from the microarray, all but one of the 125 differentially abundant transcripts identified by the microarray at week 2 were validated by NanoString (see also Figure 3b). The FLJ20273 gene was excluded due to poor probe specificity. Seventy-six transcripts were found to be significantly differentially abundant in TB-IRIS using MWU test with BH-FDR. * p 0.05, ** p 0.01, *** p 0.001
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