Transient receptor potential canonical 5 (TRPC5) protects against pain and vascular inflammation in arthritis and joint inflammation

Abstract

Objective: Transient receptor potential canonical 5 (TRPC5) is functionally expressed on a range of cells including fibroblast-like synoviocytes, which play an important role in arthritis. A role for TRPC5 in inflammation has not been previously shown in vivo. We investigated the contribution of TRPC5 in arthritis.

Methods: Male wild-type and TRPC5 knockout (KO) mice were used in a complete Freund's adjuvant (CFA)-induced unilateral arthritis model, assessed over 14 days. Arthritis was determined by measurement of knee joint diameter, hindlimb weightbearing asymmetry and pain behaviour. Separate studies involved chronic pharmacological antagonism of TRPC5 channels. Synovium from human postmortem control and inflammatory arthritis samples were investigated for TRPC5 gene expression.

Results: At baseline, no differences were observed. CFA-induced arthritis resulted in increased synovitis in TRPC5 KO mice assessed by histology. Additionally, TRPC5 KO mice demonstrated reduced ispilateral weightbearing and nociceptive thresholds (thermal and mechanical) following CFA-induced arthritis. This was associated with increased mRNA expression of inflammatory mediators in the ipsilateral synovium and increased concentration of cytokines in synovial lavage fluid. Chronic treatment with ML204, a TRPC5 antagonist, augmented weightbearing asymmetry, secondary hyperalgesia and cytokine concentrations in the synovial lavage fluid. Synovia from human inflammatory arthritis demonstrated a reduction in TRPC5 mRNA expression.

Conclusions: Genetic deletion or pharmacological blockade of TRPC5 results in an enhancement in joint inflammation and hyperalgesia. Our results suggest that activation of TRPC5 may be associated with an endogenous anti-inflammatory/analgesic pathway in inflammatory joint conditions.

Keywords: Arthritis; Inflammation; Synovitis.

Figure 1

Transient receptor potential canonical 5 (TRPC5) mRNA expression is regulated in the arthritic synovium; evidence that deletion exacerbates complete Freund's adjuvant (CFA)-induced arthritis. (A) Real-time PCR analysis of TRPC5 expression in the synovial membrane of wild-type (WT) mice 14 days following saline or CFA, normalised to hypoxanthine-guanine phosphoribosyltransferase (HPRT), β-actin and PLA₂; n=7. (B) Representative double immunofluorescence staining of CD55 (green) and TRPC5 (red) in normal and arthritic synovium, scale bars represent 50 µm. (C) Time-course analysis of weightbearing asymmetry following induction of arthritis in TRPC5 WT (n=9) and TRPC5 knockout (KO) (n=8) mice. *p<0.05, **p<0.01, ***p<0.001 vs control; ###p<0.001 vs WT by two-tailed Student's test (A) and two-way analysis of variance+Bonferroni post hoc test (C); values are mean±SEM.

Figure 2

Nociceptive parameters assessment in complete Freund's adjuvant (CFA)-induced arthritis in wild-type (WT) and transient receptor potential canonical 5 (TRPC5) knockout (KO) mice. (A) Time-course analysis of primary hyperalgesia of the hindlimb in TRPC5 WT (n=9) and KO (n=8) mice. (B) Secondary thermal hyperalgesia assessed before and weekly following CFA-induced arthritis in TRPC5 WT and KO mice. (C) Secondary mechanical hyperalgesia in the hindpaw assessed before and weekly following CFA-induced arthritis in TRPC5 WT and KO mice. (D) Real-time quantitative PCR analysis of the expression of inflammatory mediators: nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB), monocyte chemoattractant protein-1 (MCP-1), tumour necrosis factor α (TNFα), cluster of differentiation 3 (CD3) in the contralateral and ipsilateral synovium of TRPC5 WT (n=8) and KO (n=7) mice, normalised to hypoxanthine-guanine phosphoribosyltransferase (HPRT), β-actin and PLA₂. *p<0.05, **p<0.01, ***p<0.001 vs control; #p<0.05, ##p<0.01 vs WT by two-way analysis of variance +Bonferroni post hoc test; values are mean±SEM.

Figure 3

Genetic deletion of transient receptor potential canonical 5 (TRPC5) enhances synovitis and cellular infiltration following complete Freund's adjuvant (CFA)-induced arthritis. (A) Histological assessment of synovitis assessed by H&E staining; top panel shows the tibiofemoral junction (×4) 14 days following saline or CFA injection, inset shows higher power magnification (×20) below. Each knee shown is a representative for a group of mice (n=5–6); scale bars (×4) represents 200 µm, (×20) represents 50 µm; T, tibia; F, femur; S, synovium. (B) Cytospin preparations of synovial lavage fluid of wild-type (WT) and TRPC5 knockout (KO) mice illustrating cellular infiltration 14 days following CFA-induced arthritis and leucocyte population analysis. (C) Real-time quantitative PCR analysis of the expression of matrix metalloproteinases (MMP-2, MMP-3, MMP-13) in the ipsilateral synovium compared with the contralateral synovium in TRPC5 WT (n=8) and KO (n=7) mice. (D) Expression of tissue inhibitors of MMP (TIMP-2, TIMP-3, TIMP-4) in the ipsilateral synovium compared with the contralateral synovium in TRPC5 WT (n=8) and KO (n=7) mice, normalised to hypoxanthine-guanine phosphoribosyltransferase (HPRT), β-actin and PLA2..*p<0.05, **p<0.01, ***p<0.001 vs control; #p<0.05, ##p<0.01 vs WT by two-way analysis of variance+Bonferroni post hoc test; values are mean±SEM.

Figure 4

Chronic pharmacological blockade of transient receptor potential canonical 5 (TRPC5) exacerbates complete Freund's adjuvant (CFA)-induced arthritis in wild-type (WT) mice. (A) Time-course analysis of weightbearing asymmetry in vehicle (2% dimethyl sulfoxide (DMSO) in saline, intraperitoneally)-treated WT and TRPC5 knockout (KO) mice (n=5), ML204-treated WT and TRPC5 KO mice (2 mg/kg, intraperitoneally, daily n=6). (B) Secondary thermal hyperalgesia assessed before and weekly following CFA-induced arthritis in TRPC5 WT and KO mice treated with vehicle or ML204. (C) Secondary mechanical hyperalgesia in the hindpaw assessed before and weekly following CFA-induced arthritis in TRPC5 WT and KO mice treated with vehicle or ML204. (D) Cytokine concentrations in the synovial lavage fluid 14 days following CFA-induced arthritis in TRPC5 WT and KO mice treated with vehicle or ML204; interferon-γ (IFN-γ), tumour necrosis factor-α (TNFα), interleukin (IL)-6, IL-10 and IL-1β, keratinocyte chemoattractant (KC). *p<0.05, **p<0.01, ***p<0.001 vs baseline (A–C) and versus control (E); #p<0.05, ##p<0.01, ###p<0.001 vs WT vehicle by two-way analysis of variance+Bonferroni post hoc test; values are mean±SEM.

Figure 5

Augmented synovial vascularity and swelling in transient receptor potential canonical 5 (TRPC5) knockout (KO) and antagonist-treated wild-type (WT) mice. (A) Top panel: representative flux/blood flow image obtained by full-field laser perfusion imaging of blood flow in the synovial membrane day 14 following complete Freund's adjuvant (CFA)-induced arthritis in TRPC5 WT (n=7) and TRPC5 KO (n=7) mice with graphical representation of the results. (B) Synovial blood flow (top panel) assessed on day 14 following CFA-induced arthritis in vehicle (2% dimethyl sulfoxide (DMSO) in saline, intraperitoneally)-treated WT (n=5) and ML204-treated mice (2 mg/kg, intraperitoneally, daily n=6) on day 14 following CFA-induced arthritis with graphical representation of the results. (C) Time course of knee diameter swelling following CFA-induced arthritis in TRPC5 WT (n=10) and KO (n=10) mice and (E) delta (Δ) change in knee joint swelling. (D) Time course of joint swelling following CFA-induced arthritis in vehicle (2% DMSO in saline, intraperitoneally)-treated WT (n=5) and ML204-treated mice (2 mg/kg, intraperitoneally, daily n=6) following CFA-induced arthritis and (F) delta (Δ) change in knee joint diameter. *p<0.05, **p<0.01, ***p<0.001 vs control (A and B) versus baseline (C and D); ##p<0.01 vs WT by two-way analysis of variance+Bonferroni post hoc test; values are mean±SEM.

Figure 6

Expression of transient receptor potential canonical 5 (TRPC5) in human synovium under normal and inflammatory arthritis conditions. Real-time quantitative PCR analysis of the expression of (A) TRPC5, (B) tumour necrosis factor receptor 1 (TNF-R1) and (C) vascular cell adhesion molecule (VCAM-1) in the synovium of postmortem (PM) control, rheumatoid arthritis (RA) and osteoarthritis (OA) normalised to β-actin, B₂M, and RPL13A. *p<0.05 vs control, as determined by Kruskal–Wallis test followed by Dunn's post hoc comparison; values are mean±SEM.