Inflammatory pattern of the infrapatellar fat pad in dogs with canine cruciate ligament disease

Abstract

Background: Despite the importance of inflammation during the pathogenesis of cranial cruciate ligament disease (CCLD) in dogs and despite the latest knowledge suggesting a significant role of adipose tissue in osteoarthritis, the infrapatellar fat pad (IFP) was up to now mostly disregarded in veterinary investigations. In the present study, the inflammatory activity of the IFP, the main adipose structure within the stifle joint, was thoroughly investigated to evaluate its potential impact in the pathogenesis of this common disease of our canine companions. Samples of IFP, subcutaneous adipose tissue (ScAT) of the thigh and synovial fluid in both diseased (n = 36) and healthy control (n = 23) dogs were tested for their immune cell composition but also for interleukins (IL-1β, IL-6, IL-8, IL-10), degradative enzymes (MMP-1, MMP-3, MMP-13, TIMP-2, iNOS) and adipokines (leptin and adiponectin). Characterization of the immune cell composition was ascertained by fluorescence activated cell sorting. Gene expression and protein release of the inflammatory markers was determined by real RT-qPCR and ELISA.

Results: IFPs of dogs with CCLD had a significantly increased immune cell count with T cells (CD3) as the most abundant immune cells. T cells and macrophages (CD14) were significantly increased compared to healthy controls or corresponding ScAT. In addition, IFPs of dogs with CCLD demonstrated a significant increase on gene as well as protein level of multiple inflammatory indicators (IL-1β, IL-6, MMP-1, MMP-13) compared to the other tissues. TNFα was only increased on gene expression. Adipokine analysis showed higher secretion of adiponectin and lower leptin secretion in IFP from dogs with CCLD than from controls. In the synovial fluid from dogs with CCLD concentrations of IL-1β, MMP-1, MMP-13 as well as leptin were significantly increased compared to the synovial fluid from healthy control dogs.

Conclusions: The present study indicates that the IFP is a potential contributory factor in the pathogenesis of CCLD, due to its inflammatory phenotype and the proximity within the stifle joint. To determine the extent of this possible inter-relationship, further studies need to be undertaken.

Keywords: CCLD; Cranial cruciate ligament disease; Dogs; Inflammatory pattern; Infrapatellar fat pad; Osteoarthritis.

Fig. 1

Immune cell composition of the ScAT and IFP. Cells were isolated from the stromal vascular fraction of ScAT and IFP of control dogs (white boxes) and CCL diseased dogs (grey boxes). Immune cells were characterized by flow cytometry and shown as percentage of total stromal vascular cells. * denotes statistically significant differences with p < 0.005, ** p < 0.0005

Fig. 2

Gene expression and protein release of ScAT and IFP and cytokine concentration in synovial fluid. mRNA was quantified by real time RT PCR (RT qPCR). Values of IL-1β, IL-6, IL-8 and IL-10 were normalized to the housekeeping gene (β-actin) and presented as the fold increase compared to control ScAT on the left y-axis. Protein concentration of IL-1β and IL-6 secreted by ScAT and IFP as well as in synovial fluid were measured by ELISA and presented as pg/ mL on the right y-axis. * denotes statistically significant differences with p < 0.05, ** p < 0.01, and *** p < 0.001. White boxes demonstrate control group and grey boxes samples from dogs with CCLD

Fig. 3

Gene expression and protein release of ScAT and IFP and cytokine concentration in synovial fluid. mRNA was quantified by real time RT PCR (RT qPCR). Values for MMP-1, MMP-3, MMP-13 and TIMP-2 were normalized to the housekeeping gene (β-actin) and presented as the fold increase compared to control ScAT on the left y-axis. Protein concentration of MMP-1, MMP-3 and MMP-13 secreted by ScAT and IFP as well as in synovial fluid were measured by ELISA and presented as ng/ ml on the right y-axis. * denotes statistically significant differences with p < 0.05 and ** p < 0.01. White boxes demonstrate control group and grey boxes samples from dogs with CCLD

Fig. 4

Gene expression and protein release of ScAT and IFP and cytokine concentration in synovial fluid. mRNA was quantified by real time RT PCR (RT qPCR). Values for TNFα and iNOS were normalized to the housekeeping gene (β-actin) and presented as the fold increase compared to control ScAT (upper 2 boxplots). Concentration of adiponectin and leptin secreted by ScAT and IFP as well as in synovial fluid were measured by ELISA and presented as ng/ ml (lower 2 boxplots). * denotes statistically significant differences with p < 0.05, ** p < 0.01, and *** p < 0.001. White boxes demonstrate control group and grey boxes samples from dogs with CCLD