Gene expression in NTS infection in HIV positive patients February 2009

1. Gene expression in NTS infection in HIV positive patientsFebruary 2009

2. Protocol • Sample collection: whole blood on PaxGene tubes • RNA purification using PaxGene kits • RNA QC • Hybridization/scanning (Illumina HumanWG-6_V3_0_R2_11223189_A arrays ) • Data analysis: GeneSpring, InnateDB

3. Gene expression during acute infection and recovery. Samples taken at admission (acute) and patients were asked to come back for a follow up 1-3 months after. Control groups of HIV positive and HIV negative. • Stimulation assay. Samples were stimulated for 4 hours at 37C with PBS, S. Typhimurium LPS or flagellin, before transfering to Paxgene tubes. HIV positive control matched with follow up cases (CD4 T cell count < 200)

4. Samples * three tubes per sample

5. Hierarchical clustering by groups A O N P FU

6. Hierarchical clustering all samples (no follow ups)

7. Differential gene expression during acute infection HIVposvsHIVneg 668 genes Acute vsHIVneg 5499 genes 419 153 3413 43 53 1624 1391 Acute vsHIVpos 3111 genes FC cut-off: 1.5 Corrected p-value cut-off: 0.05 (FDR)

8. Differential gene expression after recovery from infection HIVposvsHIVneg 668 genes FU vsHIVneg 589 genes 221 440 359 0 7 9 19 FU vsHIVpos 35 genes FC cut-off: 1.5 Corrected p-value cut-off: 0.05 (FDR)

9. Differential gene expression during acute non NTS infection HIVposvsHIVneg 668 genes Other vsHIVneg 5182 genes 364 215 2838 59 30 1921 758 Other vsHIVpos 2768 genes FC cut-off: 1.5 Corrected p-value cut-off: 0.05 (FDR)

10. Differential gene expression during acute infection (bacterial effect) NTS infection 3015 genes Other bacterial infection 2679 genes 2008 671 2344 19 FC cut-off: 1.5 Corrected p-value cut-off: 0.05 (FDR)

11. GO HIV

12. Apoptosis/anti-apoptosis Translation Cell differentiation IL2 biosynthesis Positive regulation of mast cell degranulation Immunoglobulin secretion B cell differentiation Isotype switching Lymphocyte proliferation Immune response Response to virus Innate immune response Humoral immune response Cell-cell signalling B cell costimulation Monocyte differentiation Positive regulation of germinal centre formation Positive regulation of NK cell cytotoxicity Complement activation DNA damage response (p53) Interferon type I biosynthesis Cell proliferation Negative regulation of viral replication Antigen presentation T cell mediated immunity Negative regulation of IL2 biosynthetic process Negative regulation of JAK-STAT cascade

13. Pathway analysis

14. GO Acute (compared against HIV positive control)

15. IL8 biosyntesis Maintenance of apical/basal cell polarity NK cell proliferation Transcription Cell differentiation NK T cell proliferation Negative regulation of chemokinebiosyntesis Negative regulation of anti-apoptosis Negative regulation of LPS-mediated signaling Positive regulation of IL4 production Immune response Translation DNA replication Cell cycle Mitosis Regulation of defense response to virus Neutrophil homeostasis Negative regulation of IL2, IL6 and IL8 production Positive regulation of IL10 and IL13 biosyntesis Regulation of viral genome replication Oxigen an reactive oxigen species metabolism Regulation of T helper cell differentiation Lymph node development Caspase activation via cytochrome C

16. Pathways Immune-related pathways IL22 receptor signaling pathway IL10 signaling pathway (JAK1 TYK2 STAT3) TGFbetasuperfamilysignaling pathway (canonical) Gene expression of SOCS by STAT dimer Notch signaling pathway Role of mef2d in t-cell apoptosis IL7 signaling pathway (JAK1 JAK3 STAT5) IL7 transduction IL7 signaling pathwaygata3 participate in Th2 cytokine gene expression Vif-mediated degradation of APOBEC3G Proteasome Negative feedback regulation of TGF beta superfamilysiganilng pathway by R-smad degradation TGF beta receptor I degradation signaling p53-dependent G1 DNA damage response Vpu mediated degradation of CD4 Aurora B signaling p53 signaling pathway Classical complement pathway

17. Metabolic pathways

18. A O N P FU Genes that are differentially regulated in NTS and Non-NTS (other) infections Most involved in signal transduction in immune related pathways

19. Genes differentially expressed in NTS vs other bacterial infections: pathway analysis Up in NTS Up in “others” Viral mRNA translation Proteasome Cell cycle TGF beta degradation signal Proteasome complex Translationapoptoticsignaling in response to DNA damage p53 signaling pathway Cytokine receptor degradation signaling IL4 TNF alpha TRAIL signaling pathway TNFR2 signaling pathway IFNgsignaling pathway IL1 signaling events TLR5 cascade Fassignaling pathway

20. Donw in NTS Down in “others” Gene expression by SOCS1, SOCS3 and STAT p38 MAPK signaling pathway IL22 soluble receptor signaling pathway IL7 and IL7 signaling pathway IL2 Jak-STAT pathway MAPK signaling pathway NK cell mediated cytotoxicity BMP receptor signaling HIV-1 elongation and recovery Nef mediated-CD4 down regulation Rassignaling pathway Lck and fyn tyrosine kinases in initiation of TcR activation Tat-mediated elongation of the HIV transcript Tat-mediated HIV-1 elongation and recovery TLR receptor signaling pathway TCR Downstream TcRsignaling Nef-mediated CD8 down-regulation IL12 signaling mediated by STAT4

21. Stimulation assay

23. Differential gene expression Comparison in between groups, by stimulant: Comparison between stimulants, by group: FC cut-off: 1.5 Corrected p-value cut-off: 0.05 (FDR)

24. By stimulant comparison: • Stimulation does not have a big impact in HIV control groups (HIV positive versus HIV negative) • PBS stimulation has a similar effect than the non stimulation study (differentially expressed gene number) • LPS fails to stimulate gene expression in Follow up group (FU vs HIV positive), but flagellin stimulation induces gene expression • By group comparison: • No differences in gene expression between flagellin or LPS stimulated groups • Flagellin stimulated follow up cells showed a reduce gene expression than the other groups

25. Differentially regulated pathways in FU stimulated with flagellinvs mock-stimulation with PBS

26. But... When comparing FU against HIV positive, both stimulated with flagella we get the opposite effect: immune related pathway are downregulated, e.gIL12 and 23 signaling pathways, TNF, NFkB, IFNg, IL4 and TLR pathways

27. And... Only flagellin stimulation induces viral replication