Nature Immunology: doi: /ni.3745
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2 Supplementary Figure 1 Experimental settings. (a) Experimental setup and bioinformatic data analysis of transcriptomic changes induced in neonatal and adult monocytes by stimulation with 10 ng/ml LPS for 4h. QC/QA = quality control/quality assurance. (b) Workflow for blood sampling from healthy infants (n = 127) and adults (n = 20). (c) Timeline and workflow for Sa8/a9 pre-treatment and sepsis induction in Sa9 -/- neonates.
3 Supplementary Figure 2 GOEA of LPS-induced changes in expression in human adult and neonatal monocytes. Network visualization of GOEAs based on DE genes upon LPS treatment in adult (a) and neonatal (b) monocytes. Enriched GO-terms based on up-regulated DE genes are depicted by red nodes, based on down-regulated DE genes by blue borders. Color and size represent the corresponding FDR-adjusted enrichment P value (Q value). Overlap of genes between nodes is indicated by an edge.
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5 Supplementary Figure 3 Hierarchical clustering (HC) and differences in protein expression between adult and neonatal monocytes. (a-f) HC of genes enriched in the indicated GO clusters defined by GOEA of DE genes between the basal conditions. The z-scored log 2 expression is displayed as heat map. (g) Extra- and intracellular flow cytometric analysis of protein expression of indicated MyD88- and TRIF-dependent genes in adult and neonatal monocytes 16h cultured with PBS (Ctrl) or LPS (each group n = 4). Bars represent means ± s.e.m. of mean fluorescence intensity (MFI). *P < 0.05, **P < 0.01, ***P < (unpaired t-test).
6 Supplementary Figure 4 Differences in MyD88- and TRIF-dependent gene expression between adult and neonatal mice. (a) Relative basal gene expression of MyD88- and TRIF-dependent genes in murine adult and neonatal monocytes, determined by qrt-pcr. Bars represent means ± s.e.m. (n = 3). **P < 0.01, ***P < (unpaired t-test). (b) LPS-induced fold changes (FC) of MyD88- and TRIF-dependent gene expression in murine adult and neonatal monocytes determined by qrt-pcr. Bars represent means ± s.e.m. (n = 3). *P < 0.05, **P < 0.01 (unpaired t-test.). (c) Plasma cytokine levels in adult and neonatal mice treated s.c. for 2h and 16h with 20 µg/g LPS or PBS (Ctrl). Bars represent means ± s.e.m. (adult: n = 8 (Ctrl), 6 (2h LPS) and 3 (16h LPS); neonatal: n = 6 (Ctrl), 4 (2h LPS) and 3 (16h LPS)). *P < 0.05, **P < 0.005, ***P < (unpaired t-test.).
7 Supplementary Figure 5 Sa9 / neonates show a hyperinflammatory response to microbial challenges compared with wild-type neonates. (a) Plasma cytokine levels in WT and Sa9 -/- neonates 12h and 24h after infection with S. aureus. Bars represent means ± s.e.m (each group n = 5). *P < 0.05 (unpaired t-test). (b) Plasma cytokine levels in WT and Sa9 -/- neonates 1.5h and 4h after s.c. injection of heat-inactivated S. aureus. Bars represent means ± s.e.m (each group n = 6 (1.5h), 5 (4h)). *P < 0.05, **P < 0.01 (unpaired t-test).
8 Supplementary Figure 6 SA8/A9 induces inflammatory hyporesponsiveness and improves bacterial killing in human monocytes. (a-c) Human adult and neonatal monocytes were isolated and cultured overnight in medium and, in case of adult monocytes, also in the presence of 10 µg/ml SA8/A9 or ng/ml SA8 followed by challenges with PBS (Ctrl) or S. aureus (SA) (n = 4 in each group). Bars represent means ± s.e.m.. (a) IL-6 and TNF levels were determined in Ctrl and SA (1 MOI) supernatants 6h p.i.. * P < 0.05 (unpaired t-test). (b) Survival of SA in monocytes 3h (left panel) and 6h (right panel) after infection with 0.1 and 1.0 MOI SA. * P < 0.05, ** P < 0.01 (unpaired t-test). (c) Proportion of monocytes surviving 6h p.i. with 0.1 and 1.0 MOI SA. (d) Phagocytosis rate of adult and neonatal monocytes (each group n = 4) 30 min and 60 min after challenge with heat-killed SA. Bars represent means ± s.e.m..
9 Supplementary Table 2 Reactome pathway enrichment analysis. Adult Mo P value 1 Mapping number of genes Total number of genes in this pathway Neonatal Mo Name of pathway P value 1 number of Mapping genes Total number of genes in this pathway Name of pathway 1.40E Chemokine receptors bind chemokines 3.31E Cytokine signaling 1.91E Signaling by interleukins 1.06E Interferon signaling 4.45E Cytokine signaling 6.45E Interferon alpha/beta signaling 1.40E NLR signaling pathways 2.51E ISGylation of host proteins 2.14E Receptor CCBP2 binds most inflammatory CC chemokines 1.42E Expression of interferon induced genes 2.26E Immune system 2.87E Chemokine receptors bind chemokines 2.34E Activated TLR4 signaling 3.11E Immune system 3.41E Toll like receptor 4 (TLR4) cascade 3.67E Regulation of protein ISGylation by ISG15 deconjugating USP E Phosphorylation of I kappab by activated IKK 2.62E Interferon gamma signaling 5.68E Growth hormone receptor signaling 4.81E Signaling by interleukins 7.74E Interleukin 1 signaling 6.75E ISGylation of RIG I 7.85E SCF beta TrCPs bind NF kappab;phospho I kappakb 6.75E Interaction of E3 ligase with ISG15:E2 complex 1.21E TRIF mediated TLR3/TLR4 signaling 1.03E Receptor CCBP2 binds most inflammatory CC chemokines 1.26E 05 8 NOD1/2 Signaling Pathway 1.97E ISGylation of IRF3 1.37E MyD88 independent cascade 1.97E ISGylation of viral protein NS1 1.37E Toll like receptor 3 (TLR3) cascade 1.97E ISGylation of host protein filamin B 2.01E Toll like receptors cascades 1.97E ISGylation of protein phosphatase 1 beta (PP2CB) 4.01E MyD88:Mal cascade initiated on plasma membrane 1.97E ISGylation of protein translation regulator 4EHP 4.01E Toll like receptor TLR1:TLR2 cascade 4.07E NF kappab p105, TPL2 (COT) and ABIN2 form a stable complex 4.01E Toll like receptor TLR6:TLR2 cascade 4.47E ISGylation of E2 conjugating enzymes 4.01E Toll like receptor 2 (TLR2) cascade 7.30E Regulation of interferon gamma signaling 5.47E TAK1 activates NF kappab by phosphorylation of IKKs 8.76E RIG I/MDA5 mediated induction of IFN alpha/beta pathways 5.60E Regulation of interferon gamma signaling 9.15E Interleukin 1 signaling 5.60E Ubiquitination of I kappab by SCF beta TrCP 9.80E Growth hormone receptor signaling 9.12E Toll like receptor 10 (TLR10) cascade 9.12E Toll like receptor 5 (TLR5) cascade 9.12E TRAF6 mediated induction of NF kappab and MAP kinases 9.12E MyD88 cascade initiated on plasma membrane 1 One tailed Fisher's exact test MyD88 dependent pathway TRIF dependent pathway
10 Supplementary Table 4 List of co-expressed genes basal differentially expressed between adult and neonatal Mo. Expression of LPS inducible genes neonatal > adult adult > neonatal ADORA2A ABTB2 ALCAM AIM2 ASAP2 APOBEC3A BCAT1 APOL3 BCL2A1 APOL6 CCL2 BCL2L14 CCL20 C1QTNF1 CCL3 CCL5 CXCL1 CCR7 CXCL3 CD38 CXCL6 CFB CXCL8 CKB DFNA5 CYP27B1 DNAJB9 DDX60 DNAJC3 DHX58 DUSP1 DUSP5 EHD1 EBI3 EZH2 EMR1 F3 FEZ1 FAM65B FSCN1 FPR2 GBP1 G0S2 GBP2 HCAR3 HERC5 HES1 HERC6 IL1A HIVEP2 IL1R1 HS3ST3B1 IL2RA IDO1 INHBA IFIT5 KYNU IL15 LIMK2 IL15RA LOC IL7R LRIG1 IRF7 MAFF ISG15 MAP3K8 ISG20 MAPKAPK2 LAMP3 MSANTD3 TMEFF1 LSS MST4 MAMLD1 MYC MX2 NABP1 NFKB2 NBN OAS2 PDGFA OAS3 PDSS1 OASL PHLDA2 OPTN PKIG PANX1 PLD1 PDGFRL PMAIP1 RSAD2 PTGS2 SAMD9 PTPN2 SERPINB7 PTX3 SERPINB9 RAB33A SLAMF1 RIPK2 SP110 RND1 STAT4 SEMA3C STEAP1 SLC1A3 TNFAIP2 SPRED2 TNIP3 TBC1D30 TRIM22 TCF7L2 XAF1 TNC TNF TNFRSF9 TPD52 WNT5A ZBTB10
11 Supplementary Table 5 Clinical characteristics of the study group of healthy term infants. Age group (day of life) All Number of infants Vaginal delivery [%] Gestational age at birth [weeks] (1.4) 39.8 (1.1) 39.4 (1.5) 39.3 (1.6) 39.2 (1.3) 39.1 (1.5) Birth weight [g] (482) 30 (501) 3407 (430) 3430 (503) 3461 (309) 3482 (294) Gender, female [%] WBC x 10 3 /µl 1 14 (6.9) 16.3 (8.2) 14.3 (6.5) 11.6 (4.2) 10.5 (2.2) 9.8 (2.6) PMN x 10 3 /µl (4.5) 8.1 (4.5) 8.5 (4.8) 4.2 (2.7) 4.6 (1.6) 3.6 (2.2) Lymphs x 10 3 /µl (2.4) 4.8 (2.3) 3.9 (2.4) 4.1 (2.3) 3.7 (2.3) 3.9 (2.7) Monos x 10 3 /µl (0.8) 1.2 (0.8) 1.3 (0.8) 1.1 (0.7) 1.2 (0.5) 0.9 (0.6) CRP [mg/l] (3.3) 0.4 (1.0) 3.7 (3.5) 2.3 (2.6) 2.7 (2.9) 2.5 (2.3) 1 Mean (± s.d.) WBC: white blood count PMN: polymorphnuclear leukocyte CRP: C reaktive protein
12 Supplementary Table 6 Birth characteristics of the group of preterm and term infants analyzed for SA8/A9 level in cord blood. Term Preterm Preterm LONS 2 w/o LONS Number of infants Vaginal delivery [%] Gestational age at birth [weeks] (1.0) 29.7 (4.0) 28.3 (4.2) 30.2 (3.9) Birth weight [g] (469) 1568 (831) 1360 (960) 1650 (776) Gender, female [%] 40% 39% 31% 42% Apgar index at min (0.4) 8.8 (0.8) 8.5 (0.7) 8.9 (0.9) umbilical arterial ph (0.09) 7.32 (0.07) 7.33 (0.06) 7.31 (0.08) 1 Mean (± s.d.), 2 day 9 ± 5 of life at diagnosis LONS: late onset neonatal sepsis
13 Supplementary Table 7 Distribution of SA8/SA9 cord blood levels and associated risk of LONS in preterm infants. Categories of SA8/A9 cord blood level (ng/ml) Quartile 1 (0 330 ng/ml) Quartile 2 (331 1, ng/ml) Quartile 3 (1,111 2,000 ng/ml) Quartile 4 (2,001 10,000 ng/ml) Controls (n = 36) 1 Chi Quadrat test, 2 reference: quartile 1 LONS = late onset neonatal sepsis OR = odds ratio CI = confidence intervals LONS (n = 13) 6 (16.7%) 6 (46.2%) 1.00 P value 1 OR 95% CI P value 2 9 (25.0%) 4 (30.8%) (25.0%) 3 (23.1%) (33.3%) 0 (0.0%)
14 Supplementary Note 1 Uncropped blots and gels Ad Figure 3e 70 adult RelB neonatal RelB Ad Figure 3d adult neonatal 70 pp65 pp65 70 HDAC1 HDAC1 Ad Figure 3g 70 p65 p65 adult neonatal pirf3 dimer pirf3 monomer Ad Figure 3f adult neonatal IRF5 dimer IRF3 monomer IRF3 pirf3 dimer IRF5 monomer IRF5 monomer Ad Figure 3h adult pstat1 neonatal pstat1 GAPDH GAPDH STAT1 STAT1
15 Ad Figure 4b min pp min pirf min pirf min pstat1 70 p65 GAPDH IRF3 STAT1 Ad Figure 4c
16 Ad Figure 4e Ad Figure 4f
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