Effect of M3 muscarinic acetylcholine receptor deficiency on collagen antibody-induced arthritis

Abstract

Background: There is increasing evidence that the non-neuronal cholinergic system might be of importance for the pathology of rheumatoid arthritis. The role of M3 muscarinic acetylcholine receptor (M3R) in this regard has, however, not been investigated to date. Thus, in the present study we analyzed if M3R deficiency might have a protective effect on experimentally induced arthritis.

Methods: Collagen antibody-induced arthritis (CAIA) was evoked in M3R-deficient (M3R(-/-)) mice and wild-type (WT) littermates. Severity of arthritis was assessed by scoring of paw swelling. The joints of arthritic and nonarthritic animals were analyzed for histopathological changes regarding synovial tissue, cartilage degradation and bone destruction. Further, gene expression analysis of respective markers was performed. Systemic and local inflammatory response was determined by flow cytometry and immunohistochemistry for leukocytes as well as mRNA and protein measurements for pro-inflammatory cytokines and chemokines.

Results: In arthritic M3R(-/-) mice the number of leukocytes, specifically neutrophils, was enhanced even though clinical arthritis score was not significantly different between WT and M3R(-/-) mice with CAIA. In M3R(-/-) mice, levels of neutrophil chemoattractant chemokine C-X-C-motif ligand 2 (CXCL2) as well as the pro-inflammatory cytokine interleukin-6 were already strongly increased in mice with low arthritis score, whereas WT mice only showed prominent expression of these markers when reaching high arthritis scores. Furthermore, arthritic M3R(-/-) mice displayed a stronger degradation of collagen II in the articular cartilage and, most strikingly, histopathological evaluation revealed more severe bone destruction in arthritic mice with M3R deficiency compared to WT littermates. Moreover, in M3R(-/-) mice, gene expression of markers for bone degradation (matrix metalloproteinase 13, cathepsin K and receptor activator of nuclear factor-κB ligand) was already increased in mice with low arthritis score.

Conclusions: Taken together, the present study shows that while M3R(-/-) mice were not protected from CAIA, they had a tendency toward a higher inflammatory response after arthritis induction than WT mice. Further, arthritis-induced joint destruction was significantly stronger in mice with M3R deficiency, indicating that stimulation of M3R might have protective effects on arthritis.

Fig. 1

Mice deficient for M3R were not protected from clinical signs of arthritis. a Clinical arthritis score for WT and M3R^−/− mice with CAIA. Male (n = 6 for each genotype) and female (n = 4 for each genotype) mice of the respective CAIA group were taken for analysis. The cumulative arthritis score (CAS) on each day of experiment is represented as mean ± SEM. b Incidence of arthritis development in male (n = 6 for each genotype) and female (n = 4 for each genotype) WT and M3R^−/− mice with CAIA. Incidence is given as percentage of mice per group that reached a CAS ≥ 4 on the indicated day of experiment. c Total physical condition score of male LPS control (n = 5 for each genotype) and arthritic (CAS ≥ 4) WT (n = 4) and M3R-deficient (n = 5) mice. The total condition score for every day of experiment is given as mean ± SEM. Asterisks indicate statistical significance (*P < 0.05: CAIA vs. respective LPS control; ^# P < 0.01: M3R^−/−-CAIA vs. WT-CAIA). d Average body weight of male LPS control and arthritic M3R^−/− and WT mice. For each group the average percentage of body weight throughout the experiment compared to experimental day 0 is given as mean ± SEM. *P < 0.05: CAIA vs. respective LPS control; ^# P < 0.05: M3R^−/−-CAIA vs. WT-CAIA. CAIA collagen antibody-induced arthritis, LPS lipopolysaccharide, M3R M3 muscarinic acetylcholine receptor, WT wild-type

Fig. 2

Synovial changes in WT and M3R-deficient mice with CAIA. a–g Hematoxylin and eosin (H&E) staining of knee joint sections from male WT-LPS (a), M3R^−/−-LPS (b, e), WT-CAIA (c, f) and M3R^−/−-CAIA (d, g) mice. Original magnification: 25× (a–d) and 200× (e–g). h Quantification of histopathological changes in knee joint synovial tissue of male arthritic WT and M3R^−/− mice. Data are given as mean histopathological score ± SEM. i–p Transmission electron microscopy of synovial macrophages (i–l) and fibroblasts (m–n) of male WT-LPS (i and m), M3R^−/−-LPS (j and n), WT-CAIA (k and o) and M3R^−/−-CAIA (l and p) mice. Original magnification: 5000×. CAIA collagen antibody-induced arthritis, LPS lipopolysaccharide, M3R M3 muscarinic acetylcholine receptor, WT wild-type

Fig. 3

Analysis of α-SMA positive blood vessels in arthritic and nonarthritic WT and M3R^−/− mice. a–g Immunohistochemical staining for α-SMA in knee joints of male WT-LPS (a, e), M3R^−/−-LPS (b, f), WT-CAIA (c) and M3R^−/−-CAIA (d, g) mice. Original magnification: 200× (a–d) and 1000× (e–g). h Histopathological score for α-SMA staining. Data are given as mean histopathological score ± SEM. *P < 0.05 CAIA vs. respective LPS control. CAIA collagen antibody-induced arthritis, LPS lipopolysaccharide, M3R M3 muscarinic acetylcholine receptor, WT wild-type, α-SMA alpha-smooth muscle actin

Fig. 4

Higher number of pro-inflammatory cells in circulation and joints of M3R-deficient mice. a, b Quantification of FACS analysis of total CD45+ (a) and CD45 + Ly6C + GR1+ (b) cells in whole blood from male LPS control and arthritic M3R^−/− and WT animals. Data are given as box plots with the median indicated by a solid line within the box. *P < 0.05 CAIA vs. respective LPS control; ^# P < 0.05: M3R^−/−-CAIA vs. WT-CAIA; ^§ P < 0.05: M3R^−/−-LPS vs. WT-LPS. c–f Immunohistochemical staining for neutrophils in knee joints of male WT-LPS (c), M3R^−/−-LPS (d), WT-CAIA (e), M3R^−/−-CAIA (f) mice. Original magnification: 200×. g Quantification of neutrophil staining with data given as average number of positive cells per high-power field (hpf) ± SEM. *P < 0.05 CAIA vs. respective LPS control. h Real-time RT-PCR analysis of Cxcl2 mRNA expression in paws of male WT and M3R^−/− mice with CAIA and respective LPS-treated control mice, normalized to β-actin mRNA. Data are given as –dCT values presented as box plots. *P < 0.05 and **P < 0.01 CAIA vs. respective LPS control. i Correlation of Cxcl2 mRNA expression with the arthritis score of the respective paw in arthritic WT and M3R^−/− mice. Dotted lines indicate the mean Cxcl2 mRNA expression of the corresponding LPS-treated control mice. CAIA collagen antibody-induced arthritis, LPS lipopolysaccharide, M3R M3 muscarinic acetylcholine receptor, WT wild-type

Fig. 5

Effect of M3R-deficiency on pro-inflammatory cytokine IL-6 in mice with CAIA. a Enzyme-linked immunosorbent assay (ELISA) measurement of IL-6 concentration in plasma of male arthritic WT and M3R^−/− mice and respective LPS-treated control mice. b Correlation of circulating IL-6 abundance with the respective cumulative arthritis score (CAS) of arthritic WT and M3R^−/− mice. Dotted lines indicate the mean IL-6 concentration of the respective LPS-treated control mice. c Ratio of plasma IL-6 level and CAS in arthritic male WT and M3R^−/− mice given as mean ± SEM. ^# P < 0.05: M3R^−/−-CAIA vs. WT-CAIA. d Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis of Il6 mRNA expression in paws of male WT and M3R^−/− mice with CAIA and respective LPS-treated control mice, normalized to β-actin mRNA. **P < 0.01 CAIA vs. respective LPS control. e ELISA measurement of IL-6 concentration in paw homogenates of male LPS-treated and arthritic WT and M3R^−/− mice. Data were normalized to total protein concentration. *P < 0.05 and **P < 0.01 CAIA vs. respective LPS control. CAIA collagen antibody-induced arthritis, CAS cumulative arthritis score, IL-6 interleukin-6, LPS lipopolysaccharide, M3R M3 muscarinic acetylcholine receptor, WT wild-type

Fig. 6

Analysis of cartilage destruction in arthritic WT and M3R^−/− mice. a–g Alcian blue/periodic acid-Schiff (PAS) staining on knee joint sections from male WT-LPS (a, e), M3R^−/−-LPS (b), WT-CAIA (c, f), and M3R^−/−-CAIA (d, g) mice. Original magnification: 100× (a–d) and 1000× (e–g). Arrows indicate regions of interest. h Histopathological quantification of cartilage destruction in knee joints of male arthritic WT and M3R^−/− mice. Data are given as mean of histopathological score ± SEM. i–l Immunohistochemical staining for collagen II (original magnification 200×) in sections from knee joints of male WT-LPS (i), M3R^−/−-LPS (j), WT-CAIA (k) and M3R^−/−-CAIA (l) mice. m–p Transmission electron microscopy of chondrocytes in knee joints of male WT-LPS (m), M3R^−/−-LPS (n), WT-CAIA (o) and M3R^−/−-CAIA (p) mice. Original magnification: 5000×. CAIA collagen antibody-induced arthritis, LPS lipopolysaccharide, M3R M3 muscarinic acetylcholine receptor, WT wild-type

Fig. 7

Stronger effect of CAIA on bone destruction in M3R-deficient mice. a–d Toluidine blue staining on sections from knee joints of male WT-LPS (a), M3R^−/−-LPS (b), WT-CAIA (c), and M3R^−/−-CAIA (d) mice. Original magnification: 200×. e Histopathological quantification of bone destruction in knee joints of male arthritic WT and M3R^−/− mice. Data are given as mean of histopathological score ± SEM. ^# P < 0.05: M3R^−/−-CAIA vs. WT-CAIA. f–h Toluidine blue staining (f) and immunohistochemistry for tartrate-resistant acidic phosphatase (TRAP) (g and h) on section from knee joint of representative male M3R^−/−-CAIA mouse. Original magnification: 200× (f, g) and 1000× (h) The square in g indicates the area shown in h. i–l Transmission electron microscopy of osteocytes in knee joints of male WT-LPS (i), M3R^−/−-LPS (j), WT-CAIA (k) and M3R^−/−-CAIA (l) mice. Original magnification: 6300×. CAIA collagen antibody-induced arthritis, LPS lipopolysaccharide, M3R M3 muscarinic acetylcholine receptor, WT wild-type

Fig. 8

Analysis of markers for joint destruction in arthritic WT and M3R^−/− mice. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis of Mmp13 (a), Rankl (e), and Ctsk (i) mRNA expression in paws of male WT and M3R^−/− mice with CAIA and respective LPS-treated control mice, normalized to β- actin mRNA. *P < 0.05 and **P < 0.01 CAIA vs. respective LPS control. Correlation of Mmp13 (b), Rankl (f) and Ctsk (j) mRNA expression with the arthritis score of the respective paw in male arthritic WT and M3R^−/− mice. Dotted lines indicate the mean mRNA expression of the respective LPS-treated control mice. Immunohistochemical staining for matrix metalloproteinase 13 (MMP13) (c–d), receptor activator of nuclear factor-κB ligand (RANKL) (g–h) and cathepsin K (k–l) in knee joint sections from male M3R^−/−-LPS (c, g and k) and M3R^−/−-CAIA (d, h and l) mice. Original magnification: 100× (c–d) and 400× (g–h and k–l). CAIA collagen antibody-induced arthritis,  Ctsk cathepsin K, LPS lipopolysaccharide, M3R M3 muscarinic acetylcholine receptor, Mmp13 matrix metalloproteinase 13, Rankl receptor activator of nuclear factor-κB ligand, WT wild-type