Expression of Toll-like receptors 3, 7, 9 and cytokines in feline infectious peritonitis virus-infected CRFK cells and feline peripheral monocytes

Abstract

Background: The role of Toll-like receptors (TLRs) in a feline infectious peritonitis virus (FIPV) infection is not completely understood.

Objectives: This study examined the expression of TLR3, TLR7, TLR9, tumor necrosis factor-alpha (TNF-α), interferon (IFN)-β, and interleukin (IL)-10 upon an FIPV infection in Crandell-Reese feline kidney (CRFK) cells and feline monocytes.

Methods: CRFK cells and monocytes from feline coronavirus (FCoV)-seronegative cats and FCoV-seropositive cats were infected with type II FIPV-79-1146. At four, 12, and 24 hours post-infection (hpi), the expression of TLR3, TLR7, TLR9, TNF-α, IFN-β, and IL-10, and the viral load were measured using reverse transcription quantitative polymerase chain reaction. Viral protein production was confirmed using immunofluorescence.

Results: FIPV-infected CRFK showed the upregulation of TLR9, TNF-α, and IFN-β expression between 4 and 24 hpi. Uninfected monocytes from FCoV-seropositive cats showed lower TLR3 and TLR9 expression but higher TLR7 expression compared to uninfected monocytes from FCoV-seronegative cats. FIPV-infected monocytes from FCoV-seropositive cats downregulated TLR7 and TNF-α expression between 4 and 24 hpi, and 4 and 12 hpi, respectively. IFN-β was upregulated early in FIPV-infected monocytes from FCoV-seropositive cats, with a significant difference observed at 12 hpi compared to FCoV-seronegative cats. The viral load in the CRFK and FIPV-infected monocytes in both cohorts of cats was similar over time.

Conclusion: TLR7 may be the key TLR involved in evading the innate response against inhibiting TNF-α production. Distinct TLR expression profiles between FCoV-seronegative and FCoV-seropositive cats were observed. The associated TLR that plays a role in the induction of IFN-β needs to be explored further.

Keywords: Feline infectious peritonitis (FIP); Toll-like receptor (TLR); cytokine; monocytes.

Fig. 1. Relative expression of TLRs and cytokines in CRFK cells infected with FIPV 79-1146 at 4, 12, and 24 hpi. The relative expression was calculated using the 2^-ΔΔCt method using GAPDH and β-2-microglobulin as reference genes for normalization and the expression of uninfected CRFK cells at the same time point as calibrator (horizontal dashed line). The data were reported as mean (top of the bar) ± SD (error bar) and analyzed using Kruskal–Wallis test with Dunn’s post hoc test. The p values, p^a and p^b for Kruskal–Wallis and Dunn’s post hoc tests, respectively, were considered significant among the different time points (4, 12, and 24 hpi) if p ≤ 0.05.

TLR, Toll-like receptor; TNF-α, tumor necrosis factor-alpha; IFN, interferon; IL, interleukin; CRFK, Crandell-Reese feline kidney; FIPV, feline infectious peritonitis virus.

Fig. 2. Relative expression of TLRs and cytokines in uninfected monocytes from FCoV-seronegative (white) and FCoV-seropositive (grey) cats cultured for 24 h. The relative expression was calculated using the 2^-ΔΔCt method using GAPDH and β-2-microglobulin as reference genes for the normalization and expression of uninfected monocytes at 4 hpi as calibrator (horizontal dashed line). The data were reported as the mean (top of the bar) ± SD (error bar) and analyzed using Mann–Whitney test where the p value, p^c ≤ 0.05 was considered significant between the different groups of cats.

TLR, Toll-like receptor; FCoV, feline coronavirus; TNF-α, tumor necrosis factor-alpha; IFN, interferon; IL, interleukin.

Fig. 3. Relative expression of TLRs and cytokines in monocytes from FCoV-seronegative (white) and FcoV-seropositive (grey) cats infected with FIPV 79-1146 at 4, 12, and 24 hpi. The relative expression was calculated using the 2^-ΔΔCt method using GAPDH and β-2-microglobulin as reference genes for normalization and the expression of uninfected cells at the same time point, as calibrator (horizontal dashed line). The data were represented as mean (top of the bar) ± SD (error bar) and analyzed using the Kruskal-Wallis test with Dunn’s post hoc test. The p values, p^a and p^b for Kruskal-Wallis and Dunn’s post hoc tests, respectively, p ≤ 0.05 was considered significant between the different time points (4, 12, and 24 hpi). The data was also analyzed using Mann–Whitney test where the p value, p^c of ≤ 0.05 was considered significant between the different groups of cats.

TLR, Toll-like receptor; FCoV, feline coronavirus; TNF-α, tumor necrosis factor-alpha; IFN, interferon; IL, interleukin.