Flowcytometric Immune

Transkript

1 Flowcytometric Flowcytometric Immune Immune Profiling Profiling in in the the Seminal Seminal Plasma Plasma Christoph ChristophHudemann Hudemann Institute Instituteof oflaboratory LaboratoryMedicine Medicineand andpathobiochemistry Pathobiochemistry

2 T-cell differentiation Polarization signal Naive Th0 cell IL12, IFNg TNF, IL-6 IL-4 TH2 TH1 Tbet - Intracellular pathogens - Apoptosis of tissue cells IFN-γ GATA3 - Allergic inflammation - Eosinophilia - IgE IL-4, 5, 9, 13 IL-25, 31, 33 TGF-b + IL-6 TTHH99 PU.1 TH17 T H17 pstat3 - Mucus production - Tissue inflammation - necessary for protective immunity against extracellular pathogens IL-9, 10 IL-6, 8, 17, 22 TH22 Ahr - Pro and antiinflammatory effects IL-22 TFH Bcl-6 - Intracellular pathogens - Apoptosis of tissue cells IFN-γ adapted from Akdis et al JACI

3 Major types and function of cytokines Tumor Necrosis Factor activation of CD8 Tcells Chemokines chemotaxis, leukocyte recruitment Interleukins leukocyte development Colony-Stimulating Factor proliferation and differentiation of hematopoietic stem cells Interferons regulation of adaptive immunity, antiviral functions

4 Th1 / TH17 driven disease exemplified in the immune pathogenesis COPD Emphysema Fibrosis Mucus hypersecretion Tubby et al Clinical Science 121,

5 Th2 driven disease exemplified in the immune pathogenesis of asthma bronchiale APC B-Zelle IgE MHC-II Allergen Mastzelle IL-4 TCR IL-5 GATA-3 GM-CSF TH0-Zelle Eosinophiler Granulozyt TH2-Zelle IL-2 T-Zellproliferation TH2-Zellen

6 Th2 driven disease exemplified in the immune pathogenesis of asthma bronchiale

7 Biomarker analysis in seminal plasma Drabovich et al. Nat Rev Urol 2014

8 Why seminal plasma? 1) High concentrations of organ-specific proteins allow accurate quantification. 2) Male reproductive system disorders result in altered composition of the seminal plasma proteome. 3) Cancer-specific proteins can appear earlier in seminal plasma than in blood serum, specific and sensitive seminal plasma analysis could facilitate the early diagnosis of prostate and testicular cancers. Drabovich et al. Nat Rev Urol 2014

9 Cytokine quantification methods

10 Platform for Immune Profiling qpcr high specificity, but not necessarily representative for actual protein concentration

11 Platform for Immune Profiling Western blotting hard to quantify and low sensitivity, high sample volume

12 Platform for Immune Profiling ELISA high sample volume, high costs

13 Platform for Immune Profiling BD Accury C6 / BioRad Bioplex Cytometric Bead Array flex sets (CBAs)

14 Platform for Immune Profiling Polystyrol Beads bound to antigenspecific capture Antibodies two dyes directly bound to the beads allows detection of seperate populations

15 Platform for Immune Profiling each bead population bound to specific capture AB maximum simultaneous antigen-detection 35 (BD) and 200 (Biorad)

16 Platform for Immune Profiling Applications - simultaneous and quantitative measurement of a broad range of proteins (e.g. cytokines, chemokines, growth factors, IgGs, signal transduction proteins) Source - cell culture supernatant, serum, plasma, cell lysate Species - kits available for human, mouse, rat, NHP

17 Platform application in Cytokine analysis of seminal plasma Pilatz & Hudemann et al submitted

18 Platform application in Cytokine analysis of blood serum only IL-6 and IL-10 different between control and MetS subjects Pilatz & Hudemann et al submitted

19 Platform application in Cytokine analysis of blood serum only IL-6 and IL-10 different between control and MetS subjects Pilatz & Hudemann et al submitted

20 Platform application in Pilatz & Hudemann et al submitted

21 Function of IL-6 Physiology synthesized by monocytes, fibroblasts, endothelial cells, T-cells main source are after antigen contact activated monocytes/macrophages 3-4 hrs after activation (viral, bacterial, fungal antigens) up to > 1000 pg/ml secreted Function pro-inflammatory cytokine in the innate immune response differentiation factor for B- and T-cells & activator for T-cells

22 Function of IL-6 Physiology synthesized by monocytes, fibroblasts, endothelial cells, T-cells main source are after antigen contact activated monocytes/macrophages 3-4 hrs after activation (viral, bacterial, fungal antigens) up to > 1000 pg/ml secreted Function pro-inflammatory cytokine in the innate immune response differentiation factor for B- and T-cells & activator for T-cells differentiation switch of the M1-macrophage to the pro-inflammtory M2-macrophage Reilly & Saltiel Nature Immunology (2014)

23 Function of IL-6 Pathophysiology Increased concentrations arise during sepsis, autoimmune diseases, AIDS, Infections obesity characterized by chronic low-grade inflammation (e.g. IL-6, TNF) relationship of IL-6 with semen quality is controversial IL-6 concentration in seminal plasma of infertile men is significantly higher than that of fertile men depending on the study and patient cohort, relations between IL-6 and sperm parameters differed (e.g. migration testing and IL-6 concentration) Eder K et al. Inflamm Res Camejo st al. Arch. Androl Castiglione et al. Andrologia 2013

24 Function of IL-10 Physiology anti-inflammatory cytokine primarily produced by monocytes and macrophages after PRR binding to lesser extend by Th2 and Th1 CD4 T-cells O'Garra & Paulo Vieira. Nat Rev Immun 2007

25 Function of IL-10 Physiology anti-inflammatory cytokine primarily produced by monocytes and macrophages after PRR binding to lesser extend by Th2 and Th1 CD4 T-cells Function suppresses macrophage activation suppresses pro-inflammatory cytokine production (e.g. TNF, IL-6) modulates heamatopoetic cells (T cells, B cell, neutrophils) Hawrylowicz & O'Garra. Nat Rev Imun 2005

26 Function of IL-10 Pathophysiology IL-10-secreting regulatory T-cell populations in patients following exposure to allergen are upregulated anti-inflammatory effects useful in SIT against allergic diseases HCV suppression of antigen-specific PBMC proliferation and IFNg production mycobacterium tuberculosis suppression of allogenic CD4 T-cell proliferation Fungi (e.g. Pneumocystis carinii) IL-10ko mouse displayed red. fungal growth, but increased inflammatory pathology Parasites (e.g. Schistosoma mansoni) suppression of T-cell proliferation and increased survival increased in seminal plasma from patients with chronic prostatitis and chronic pelvic pain syndrome increase during clamydia infection appears to impair immune responses

27 Summary CBA analytical evaluation demonstrated high reproducibility, sensitivity and good applicability for small volume samples CBA analytical evaluation provides a perfect and quick platform for large biological sample population analysis immune system polarization due to viral or bacterial infections, obesity, vasectomy etc. results in a characteristic cytokine and chemokine pattern function of cytokines in numerous male reproductive system related disorders has been subject of evaluation seminal plasma promising biological fluid as a clinical sample for noninvasive urogenital diagnostics

28 Acknowledgment Institute of Laboratory Medicine and Pathobiochemistry Renz all members Pilatz Weidner Schuppe Fijak Financial Support