Gene expression in NTS infection in HIV positive patients

1. Gene expression in NTS infection in HIV positive patients

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 * one is E. Coli ** three tubes per sample

5. Hierarchical clustering by groups A O FU N P

6. Hierarchical clustering all samples

7. Differential gene expression during acute infection HIVpos vs HIVneg 353 genes Acute vs HIVneg 3540 genes 241 61 2109 21 30 1379 1759 Acute vs HIVpos 2824 genes FC cut-off: 2.0 Corrected p-value cut-off: 0.05 (FDR)

8. Differential gene expression after recovery from infection HIVpos vs HIVneg 353 genes FU vs HIVneg 288 genes 130 219 156 0 4 2 25 FU vs HIVpos 31 genes FC cut-off: 2.0 Corrected p-value cut-off: 0.05 (FDR)

9. GO HIV

10. Apoptosis Translation Cell differentiation Positive regulation of IL3 Positive regulation of mast cell degranulation Immunoglobulin secretion B cell differentiation Isotype switching Lymphocyte proliferation Lymph node development Immune response Response to virus Innate immune response Humoral immune response Cell-cell siganling B cell costimulation Monocyte differentiation Positive regulation of germinal centre formation Positive regulation of NK cell cytotoxicity Complement activation DNA damage response (p53) Inflammatory response Negative regulation of T cell activation Interferon type I biosynthesis Cell prolifereation Negative regulation of viral replication Infected cell apoptosis Negative regulation of caspase activation

12. GO Acute (compared against HIV positive control)

13. Negative regulation of cell adhesion Cell motility Positive regulation of type I hypersensitivity Chemotaxis Negative regulation of apoptosis MAPK activity activation C-C chemokine receptor activity Endocytosis Response to stimulus Neutrophil differentiation Positive regulation of endothelial cell migration Positive regulation of fatty acid oxidation Positive regulation of IL3 biosynthesis Positive regulation of mast cell degradation Tolerance induction Negative regulation of blood cell coagulation Translation DNA replication Cell cycle Mitosis NFkB import to nucleus Anti-apoptosis Phagocytosis Fatty acid catabolism Negative regulation of myeloid cell differentiation Regulation of viral genome replication Oxigen an reactive oxigen species metabolism Response to LPS Complement activation

14. Pathways Immune-related pathways

15. HIV infection-related pathways

16. Metabolic pathways

17. IL12 pathway

18. IL23 pathway

19. TLR signaling pathway

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

22. Stimulation assay

24. Differential gene expression

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 This effect however, is not seen when stimulating with LPS (looking at p-values instead of corrected p-values)

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