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BVDV Infection Alters Toll-like and TNF-  Receptor Signaling in Bovine Cells

BVDV Infection Alters Toll-like and TNF-  Receptor Signaling in Bovine Cells. J.D. Neill, R. Zuerner and J.F. Ridpath. Innate Immunity. First line of defense against invading pathogens Pathogens detected by sensors that are present in most cells

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BVDV Infection Alters Toll-like and TNF-  Receptor Signaling in Bovine Cells

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  1. BVDV Infection Alters Toll-like and TNF- Receptor Signaling in Bovine Cells J.D. Neill, R. Zuerner and J.F. Ridpath

  2. Innate Immunity • First line of defense against invading pathogens • Pathogens detected by sensors that are present in most cells • Results in a rapid response aimed at eliminating the threat • Stimulates expression of genes encoding molecules that potentiate the response

  3. Innate Immunity • Toll-like receptors and intracellular soluble receptors • These receptors recognized pathogen-associated molecular patterns (PAMPs) • Stimulate expression of important response regulators: • Type I interferons, inflammatory cytokines (TNF-), chemokines, etc. • Induction of inflammatory response • Characterized by activation of NF-B

  4. Toll-Like and TNF- Receptors • Toll-like receptors (TLR): • Transmembrane proteins - 12 known • Detect specific PAMPS • TLR3 - dsRNA, Type I interferon, NF-B • TLR4 - lipopolysaccharide (LPS), NF-B • TNF- receptor • Ligated by TNF-, NF-B

  5. Activation of NF-B and Inflammation • Activation of NF-B results in expression of large number of proteins: • P- and E-selectins in endothelial cells • A20 • IL6, IL8

  6. Inhibition of Innate Immunity by BVDV • Examined effect of BVDV infection on responses to LPS, dsRNA and TNF- • Bovine aortic endothelial cells (BAEC) and primary fetal testicular cells (Bte) • ncpBVDV 2 strain A13 isolated from commercial endothelial cells • Non-infected, 24-48 hr acute, chronic

  7. Indirect Assay of NF-B Activation • Used real-time PCR to examine genes known to be upregulated by NF-B • P-selectin, E-selectin, A20, IL6 and IL8 • Stimulated cells with LPS, dsRNA or TNF- for 4 hours • RNA isolated with TRIzol reagent • Real-time PCR using Superscript III SYBR green qt-PCR kit • Normalized to 2-microglobulin • Expression levels calculated by Liu and Saint (Anal. Biochem. 302:52)

  8. BAEC Real-time PCR Analysis

  9. Bte Real-time PCR Analysis

  10. Direct Assay of NF-B Activation • Plasmid containing minimal CMV promoter fused to NF-Bresponse elements upstream from luciferase gene • Plasmid introduced into Bte cells using lipofectin-mediated transfection - n=6 • Cells stimulated with LPS, dsRNA or TNF- at 24 hours post-transfection for 4 hours • Cells were lysed and assayed for luciferase activity - relative light units

  11. Luciferase Assay of Stimulate Bte Cells 36 60 45 15

  12. Conclusions • Stimulation of BAE and Bte cells with TLR and TNFR ligands increased specific gene expression • BVDV2 infection negatively affected NF-B activation • Acute BVDV infection more effective at inhibition of NF-B activation • TLR3 (dsRNA) appears to be inhibited to greater degree

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