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

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
(No Transcript)
8
(No Transcript)
9
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. 30252)

10
BAEC Real-time PCR Analysis
11
Bte Real-time PCR Analysis
12
Direct Assay of NF-?B Activation

• Plasmid containing minimal CMV promoter fused to
  NF-?B response elements upstream from luciferase
  gene
• Plasmid introduced into Bte cells using
  lipofectin-mediated transfection - n6
• 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

13
Luciferase Assay of Stimulate Bte Cells
36
60
45
15
14
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