Inflammatory Mediators in the Mesenteric Lymph Nodes, Site of a Possible Intermediate Phase in the Immune Response to Feline Coronavirus and the Pathogenesis of Feline Infectious Peritonitis?

Abstract

Feline infectious peritonitis (FIP) is an almost invariably fatal feline coronavirus (FCoV)-induced disease thought to arise from a combination of viral mutations and an overexuberant immune response. Natural initial enteric FCoV infection may remain subclinical, or result in mild enteric signs or the development of FIP; cats may also carry the virus systemically with no adverse effect. This study screened mesenteric lymph nodes (MLNs), the presumed first site of FCoV spread from the intestine regardless of viraemia, for changes in the transcription of a panel of innate immune response mediators in response to systemic FCoV infection and with FIP, aiming to identify key pathways triggered by FCoV. Cats with and without FIP, the latter with and without FCoV infection in the MLN, were compared. Higher expression levels in FIP were found for toll-like receptors (TLRs) 2, 4 and 8. These are part of the first line of defence and suggest a response to both viral structural proteins and viral nucleic acid. Expression of genes encoding inflammatory cytokines and chemokines, including interleukin (IL)-1β, IL-6, IL-15, tumour necrosis factor (TNF)-α, CXCL10, CCL8, interferon (IFN)-α, IFN-β and IFN-γ, was higher in cats with FIP, consistent with inflammatory pathway activation. Expression of genes encoding transcription factors STAT1 and 2, regulating signalling pathways, particularly of the interferons, was also higher. Among cats without FIP, there were few differences between virus-positive and virus-negative MLNs; however, TLR9 and STAT2 expression were higher with infection, suggesting a direct viral effect. The study provides evidence for TLR involvement in the response to FCoV. This could open up new avenues for therapeutic approaches.

Keywords: cytokines; feline coronavirus; mesenteric lymph nodes; toll-like receptors.

Fig. 1

Boxplots demonstrating relative levels of FCoV transcription in G1+ and G2. The amount of FCoV was calculated by 2^−ΔΔCq, using fGAPDH as the internal reference gene and expressed as an n fold difference relative to the G1+ mean as a calibrator. The boxes depict the median and interquartile (IQ) range with whiskers extending to the highest and lowest values, which are within 1.5× the IQ range. Outliers beyond this are individually marked. The three columns of individual crosses within G2 depict the three variations in the viral S protein at codons 1,048 and 1,050, respectively. From left to right: L, leucine at 1,048 (‘systemic’ virus); M&A, methionine and alanine (‘systemic’ virus); M&S, methionine and serine (‘enteric’ virus). 2E+, 2E–, 2L+ and 2L– represent relative FCoV levels among MLN of cats with and without effusions/lesions.

Fig. 2

Examples of MLNs with and without lesions from cats with FIP. (a, b) Case G2.5. (a) Focal pyogranulomatous inflammation with central necrosis (*). HE. (b) Viral antigen expression is seen in abundant intact lesional macrophages. IHC. (c, d) Case G2.19. (c) Reactive hyperplasia with expansion of the marginal sinus by macrophages (*). HE. (d) Some of the latter are FCoV antigen positive. IHC.

Fig. 3

Boxplots of relative levels of TLR gene expression in each group. The amount of target was calculated by 2^−ΔΔCq, using fGAPDH as the internal reference gene and expressed as an n fold difference relative to the G1 mean as a calibrator. The boxes depict the median and interquartile (IQ) range with whiskers extending to the highest and lowest values, which are within 1.5 × the IQ range. Outliers beyond this are individually marked. * marks significant differences between individual groups (P ≤ 0.05) or, where joined by a bar, between G1 as a whole and G2. 2E+, 2E–, L2+ and L2– represent relative gene expression levels among MLNs of cats with and without effusions/lesions.

Fig. 4

Boxplots of relative levels of cytokine and chemokine gene expression in each group. The amount of target was calculated by 2^−ΔΔCq, using fGAPDH as the internal reference gene and expressed as an n fold difference relative to the G1 mean as a calibrator. The boxes depict the median and interquartile (IQ) range with whiskers extending to the highest and lowest values, which are within 1.5 × the IQ range. Outliers beyond this are individually marked. * marks significant differences between individual groups (P ≤ 0.05) or, where joined by a bar, between G1 as a whole and G2. 2E+, 2E–, 2L+ and 2L– represent relative gene expression levels among MLNs of cats with and without effusions/lesions.

Fig. 5

Boxplots of relative levels of STAT gene expression in each group. The amount of target was calculated by 2^−ΔΔCq, using fGAPDH as the internal reference gene and expressed as an n fold difference relative to the G1 mean as a calibrator. The boxes depict the median and interquartile (IQ) range with whiskers extending to the highest and lowest values, which are within 1.5 × the IQ range. Outliers beyond this are individually marked. * marks significant differences between individual groups (P ≤ 0.05) or, where joined by a bar, between G1 as a whole and G2. 2E+, 2E–, 2L+ and 2L– represent relative gene expression levels among MLNs of cats with and without effusions/lesions.