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

• Results in a rapid response aimed at eliminating the threat

• Stimulates expression of genes encoding molecules that potentiate the response

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

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

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

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

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)

BAEC Real-time PCR Analysis

0

10

20

30

40

50

60

70

NI A13a A13c NI A13a A13c NI A13a A13c

A20 E-sel P-sel

NT

LPS

dsRNA

TNFa

Bte Real-time PCR Analysis

0

5

10

15

20

25

NI A13a NI A13a NI A13a

A20 IL-6 IL-8

NT

LPS

dsRNA

TNFa

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 - 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

Luciferase Assay of Stimulate Bte Cells

0

10

20

30

40

50

60

NT LPS dsRNA TNF

Min

NI

A13a

A13c

15

45

36 60

Conclusions1. Stimulation of BAE and Bte cells with

TLR and TNFR ligands increased specific gene expression

2. BVDV2 infection negatively affected NF-B activation

3. Acute BVDV infection more effective at inhibition of NF-B activation

4. TLR3 (dsRNA) appears to be inhibited to greater degree