Nature Immunology: doi:10.1038/ni.3745

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 S100a8/a9 pre-treatment and sepsis induction in S100a9-/-

neonates.

Nature Immunology: doi:10.1038/ni.3745

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.

Nature Immunology: doi:10.1038/ni.3745

Nature Immunology: doi:10.1038/ni.3745

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 log2 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 < 0.0005 (unpaired t-test).

Nature Immunology: doi:10.1038/ni.3745

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 < 0.005 (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 < 0.0005 (unpaired t-test.).

Nature Immunology: doi:10.1038/ni.3745

Supplementary Figure 5

S100a9–/–

neonates show a hyperinflammatory response to microbial challenges compared with wild-type neonates.

(a) Plasma cytokine levels in WT and S100a9-/-

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 S100a9

-/- 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).

Nature Immunology: doi:10.1038/ni.3745

Supplementary Figure 6

S100A8/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 S100A8/A9 or 100 ng/ml S100A8 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..

Nature Immunology: doi:10.1038/ni.3745

Supplementary Table 2 Reactome pathway enrichment analysis.

P  value1Mapping number of genes

Total number of genes in this pathway

Name of pathway P  value1Mapping number of genes

Total number of genes in this pathway

Name of pathway

1.40E‐09 14 55 Chemokine receptors bind chemokines 3.31E‐18 50 358 Cytokine signaling1.91E‐09 19 110 Signaling by interleukins 1.06E‐13 36 249 Interferon signaling4.45E‐08 33 358 Cytokine signaling 6.45E‐11 23 130 Interferon alpha/beta signaling1.40E‐07 12 55 NLR signaling pathways 2.51E‐10 9 15 ISGylation of host proteins2.14E‐07 6 10 Receptor CCBP2 binds most inflammatory CC chemokines 1.42E‐08 18 105 Expression of interferon‐induced genes2.26E‐06 75 1397 Immune system 2.87E‐08 13 55 Chemokine receptors bind chemokines2.34E‐06 14 96 Activated TLR4 signaling 3.11E‐08 84 1397 Immune system3.41E‐06 14 99 Toll‐like receptor 4 (TLR4) cascade 3.67E‐08 7 12 Regulation of protein ISGylation by ISG15 deconjugating USP184.03E‐06 5 9 Phosphorylation of I‐kappaB by activated IKK 2.62E‐06 18 147 Interferon‐gamma signaling5.68E‐06 7 23 Growth hormone receptor signaling 4.81E‐06 15 110 Signaling by interleukins7.74E‐06 9 43 Interleukin‐1 signaling 6.75E‐06 4 5 ISGylation of RIG‐I7.85E‐06 5 10 SCF‐beta‐TrCPs bind NF‐kappaB;phospho‐I‐kappakB 6.75E‐06 4 5 Interaction of E3 ligase with ISG15:E2 complex1.21E‐05 12 82 TRIF‐mediated TLR3/TLR4 signaling  1.03E‐05 5 10 Receptor CCBP2 binds most inflammatory CC chemokines1.26E‐05 8 35 NOD1/2 Signaling Pathway 1.97E‐05 4 6 ISGylation of IRF31.37E‐05 12 83 MyD88‐independent cascade  1.97E‐05 4 6 ISGylation of viral protein NS11.37E‐05 12 83 Toll‐like receptor 3 (TLR3) cascade 1.97E‐05 4 6 ISGylation of host protein filamin B2.01E‐05 14 115 Toll‐like receptors cascades 1.97E‐05 4 6 ISGylation of protein phosphatase 1 beta (PP2CB)4.01E‐05 11 78 MyD88:Mal cascade initiated on plasma membrane 1.97E‐05 4 6 ISGylation of protein translation regulator 4EHP4.01E‐05 11 78 Toll‐like receptor TLR1:TLR2 cascade 4.07E‐05 3 3 NF‐kappaB p105, TPL2 (COT) and ABIN2 form a stable complex4.01E‐05 11 78 Toll‐like receptor TLR6:TLR2 cascade 4.47E‐05 4 7 ISGylation of E2 conjugating enzymes4.01E‐05 11 78 Toll‐like receptor 2 (TLR2) cascade 7.30E‐05 5 14 Regulation of interferon‐gamma signaling5.47E‐05 6 22 TAK1 activates NF‐kappaB by phosphorylation of IKKs 8.76E‐05 10 67 RIG‐I/MDA5 mediated induction of IFN‐alpha/beta pathways5.60E‐05 5 14 Regulation of interferon‐gamma signaling 9.15E‐05 8 43 Interleukin‐1 signaling5.60E‐05 5 14 Ubiquitination of I‐kappaB by SCF‐beta‐TrCP 9.80E‐05 6 23 Growth hormone receptor signaling9.12E‐05 10 71 Toll‐like receptor 10 (TLR10) cascade9.12E‐05 10 71 Toll‐like receptor 5 (TLR5) cascade9.12E‐05 10 71 TRAF6‐mediated induction of NF‐kappaB and MAP kinases9.12E‐05 10 71 MyD88 cascade initiated on plasma membrane

1 One‐tailed Fisher's exact testMyD88‐dependent pathwayTRIF‐dependent pathway

Adult Mo Neonatal Mo

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Supplementary Table 4List of co-expressed genes basal differentially expressed between adult and neonatal Mo.

Expression of LPS‐inducible genes neonatal > adult adult > neonatalADORA2A ABTB2ALCAM AIM2ASAP2 APOBEC3ABCAT1 APOL3BCL2A1 APOL6CCL2 BCL2L14CCL20 C1QTNF1CCL3 CCL5CXCL1 CCR7CXCL3 CD38CXCL6 CFBCXCL8 CKBDFNA5 CYP27B1DNAJB9 DDX60DNAJC3 DHX58DUSP1 DUSP5EHD1 EBI3EZH2 EMR1F3 FEZ1FAM65B FSCN1FPR2 GBP1G0S2 GBP2HCAR3 HERC5HES1 HERC6IL1A HIVEP2IL1R1 HS3ST3B1IL2RA IDO1INHBA IFIT5KYNU IL15LIMK2 IL15RALOC100129518 IL7RLRIG1 IRF7MAFF ISG15MAP3K8 ISG20MAPKAPK2 LAMP3MSANTD3‐TMEFF1 LSSMST4 MAMLD1MYC MX2NABP1 NFKB2NBN OAS2PDGFA OAS3PDSS1 OASLPHLDA2 OPTNPKIG PANX1PLD1 PDGFRLPMAIP1 RSAD2PTGS2 SAMD9PTPN2 SERPINB7PTX3 SERPINB9RAB33A SLAMF1RIPK2 SP110RND1 STAT4SEMA3C STEAP1SLC1A3 TNFAIP2SPRED2 TNIP3TBC1D30 TRIM22TCF7L2 XAF1TNCTNFTNFRSF9TPD52WNT5AZBTB10

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Supplementary Table 5 Clinical characteristics of the study group of healthy term infants.

All 1 ‐ 3 4 ‐ 10 11 ‐ 30 31 ‐ 180 181 ‐ 365Number of infants 127 30 30 26 19 22Vaginal delivery [%] 42 37 45 44 40 41Gestational age at birth [weeks] 1 39.5 (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] 1 3395 (482) 3350 (501) 3407 (430) 3430 (503) 3461 (309) 3482 (294)Gender, female [%] 53 61 58 46 46 51WBC x 103/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 103/µl 1 7.4 (4.5) 8.1 (4.5) 8.5 (4.8) 4.2 (2.7) 4.6 (1.6) 3.6 (2.2)

Lymphs x 103/µl 1 4.4 (2.4) 4.8 (2.3) 3.9 (2.4) 4.1 (2.3) 3.7 (2.3) 3.9 (2.7)

Monos x 103/µl 1 1.2 (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] 1 2.1 (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 countPMN: polymorphnuclear leukocyteCRP: C‐reaktive protein

Age group (day of life)

Nature Immunology: doi:10.1038/ni.3745

Supplementary Table 6 Birth characteristics of the group of preterm and term infants analyzed for S100A8/A9 level in cord blood.

LONS 2 w/o LONSNumber of infants 31 49 13 36Vaginal delivery [%] 65 18 23 17Gestational age at birth [weeks] 1 39.1 (1.0) 29.7 (4.0) 28.3 (4.2) 30.2 (3.9)

Birth weight [g] 1 3607 (469) 1568 (831) 1360 (960) 1650 (776)Gender, female [%] 40% 39% 31% 42%Apgar index at min 10 1 9.9 (0.4) 8.8 (0.8) 8.5 (0.7) 8.9 (0.9)

umbilical arterial pH 1 7.28 (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 diagnosisLONS: late‐onset neonatal sepsis

Term PretermPreterm

Nature Immunology: doi:10.1038/ni.3745

Supplementary Table 7Distribution of S100A8/S100A9 cord blood levels and associated risk of LONS in preterm infants.

Categories of S100A8/A9 cord blood level (ng/ml)

Controls         (n  = 36)

LONS            (n  = 13) P  value 1 OR 95% CI P  value 2

Quartile 1                     (0‐330 ng/ml)

 6 (16.7%) 6 (46.2%) 1.00

Quartile 2                    (331‐1,100 ng/ml)

9 (25.0%) 4 (30.8%) 0.44 0.087 ‐ 2.276 0.333

Quartile 3               (1,111‐2,000 ng/ml)

9 (25.0%) 3 (23.1%) 0.33 0.059 ‐ 1.878 0.213

Quartile 4               (2,001‐10,000 ng/ml)

12 (33.3%)  0 (0.0%) 0.049 0.04 0.002 ‐ 0.827 0.0371 Chi‐Quadrat‐test, 2 reference: quartile 1LONS = late‐onset neonatal sepsisOR = odds ratioCI = confidence intervals

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Supplementary Note 1 Uncropped blots and gels

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