Collagen-induced arthritis: severity and immune response attenuation using multivalent N-acetyl glucosamine

Abstract

Rheumatoid arthritis is an autoimmunity leading to considerable impairment of quality of life. N-acetyl glucosamine (GlcNAc) has been described previously as a potent modulator of experimental arthritis in animal models and is used for osteoarthritis treatment in humans, praised for its lack of adverse effects. In this study we present a comprehensive immunological analysis of multivalent GlcNAc-terminated glycoconjugate (GC) application in the treatment of collagen-induced arthritis (CIA) and its clinical outcome. We used immunohistochemistry and FACS to describe conditions on the inflammation site. Systemic and clinical effects were evaluated by FACS, cytotoxicity assay, ELISA, cytometric bead array (CBA), RT-PCR and clinical scoring. We found reduced inflammatory infiltration, NKG2D expression on NK and suppression of T, B and antigen-presenting cells (APC) in the synovia. On the systemic level, GCs prevented the activation of monocyte- and B cell-derived APCs, the rise of TNF-α and IFN-γ levels, and subsequent type II collagen (CII)-specific IgG2a formation. Moreover, we detected an increase of anti-inflammatory IL-4 mRNA in the spleen. Similar to the synovia, the GCs caused a significant reduction of NKG2D-expressing NK cells in the spleen without influencing their lytic function. GCs effectively postponed the onset of arthritic symptoms, reduced their severity and in 18% (GN8P) and 31% (GN4C) of the cases completely prevented their appearance. Our data prove that GlcNAc glycoconjugates prevent the inflammatory response, involving proinflammatory cytokine rise, APC activation and NKG2D expression, leading to the attenuation of clinical symptoms. These results support the glycobiological approach to the treatment of collagen-induced arthritis/rheumatoid arthritis (CIA/RA) as a way of bringing new prospects for more effective therapeutic interventions.

Keywords: CIA; GlcNAc glycoconjugates; clinical scoring; cytokines; humoral response.

Fig. 1

Chemical structures of N-acetyl glucosamine (GlcNAc) and glycodendrimers. 2-(acetylamino)-2-deoxy-D-glucose or GlcNAc (a) is the simple monosaccharide derivative of glucose. GN8P (b) is a glycodendrimer bearing eight GlcNAc moieties on polyamidoamine (PAMAM) scaffold. GN4C (c) is a glycodendrimer bearing four GlcNAc moieties on calix[4]arene core providing a more rigid structure than PAMAM.

Fig. 2

Immune cell infiltration in the synovium. Representative images of the synovial infiltrate in hind paw joints of healthy controls (HC) (a), untreated collagen-induced arthritis (CIA) (b), and GN8P-treated CIA (c). Inflammatory infiltrate of the synovial and perisynovial tissue was stained by haematoxylin. Magnification ×40.

Fig. 3

Immunohistochemistry of NKG2D⁺ cell infiltration in the synovium. Representative images of the synovial infiltrate in hind paw joints of healthy controls (HC) (a), untreated collagen-induced arthritis (CIA) (b), and GN8P-treated CIA (c). NKG2D-positive cells in the synovial and perisynovial tissue were stained by anti-NKG2D antibody. NKG2D-positive cells are marked with arrows. Counterstained with haematoxylin. Magnification ×40.

Fig. 4

Immunohistochemistry of CD11b⁺ infiltration in the synovium. Representative images of the synovial infiltrate in hind paw joints of healthy controls (HC) (a), untreated collagen-induced arthritis (CIA) (b) and GN8P-treated CIA (c). CD11b-positive cells in the synovial and perisynovial tissue were stained by anti-CD11b antibody. Counterstained with haematoxylin. Magnification ×40.

Fig. 5

Cytometric (FACS) analysis of synovial fluid. NKG2D-expressing cells in the synovial fluid (a), NKG2D expression [mean fluorescence intensity (MFI)] on NK cells (mean ± standard deviation) (b), relative distribution of CD11b⁺CD11c⁺ antigen-presenting cells (APCs) (c), CD45R/B220⁺ B cells (d) and activated CD86⁺ antigen-presenting B cells (e) in the synovia were measured by flow cytometry (n = 5–10 mice per group, *P < 0·05; ***P < 0·001). Box-plots represent median, 25% and 75% percentiles, minimum and maximum.

Fig. 6

Serum levels of TNF-α and IFN-γ, mRNA expression of IL-4 in spleen mononuclear cells (SMC) and type II collagen (CII)-specific IgG2a level. The inflammatory cytokines TNF-α (a) and IFN-γ (b) were measured in sera of healthy and collagen-induced arthritis (CIA) mice by cytometric bead array. Relative fold expression of IL-4 mRNA (c) was measured in SMC by real-time RT–PCR and normalized to β2-microglobulin [healthy controls (HC) = 1)]. Levels of CII-specific IgG2a (d) were determined by ELISA (n = 5–10 mice per group, sample dilution ×100). Data are expressed as mean ± standard deviation. *P < 0·05; **P < 0·01; ***P < 0·001.

Fig. 7

Glycoconjugate-induced changes of relevant cell populations in the lymph nodes. Cells were isolated from the lymph nodes and analysed by flow cytometry (FACS). The relative distribution of CD45R/B220⁺ B cells (a), activated CD86⁺ antigen-presenting B cells (b) and myeloid CD11b⁺CD11c⁺ antigen-presenting cells (APCs) (c) are shown. Data represent results of three experiments performed (n = 5–10 mice per group) *P < 0·05; **P < 0·01; ***P < 0·001. Box -plots represent median, 25% and 75% percentiles, minimum and maximum.

Fig. 8

Function, distribution and phenotype of splenic NK cells. Gradual impairment of splenic NK cytotoxicity during collagen-induced arthritis (CIA) development expressed as a percentage of NK cell-mediated cytotoxicity of healthy mice stated as 100% (specific cytotoxicity = 46·8 ± 5·1%) and the effect of presymptomatic and symptomatic glycoconjugate (GC) treatment (a). Effect of CIA and GC treatment on the relative distribution of NK cells in the spleen (b) and the subset of NKG2D⁺ NK cells on day 37 (c). Results of three experiments performed (n = 5–10 mice per group) **P < 0·01; ***P < 0·001. Box-plots represent median, 25% and 75% percentiles, minimum and maximum.

Fig. 9

Effects of glycoconjugate treatment on disease onset, incidence and clinical symptoms. Average time of onset of the collagen-induced arthritis (CIA) symptoms in untreated (CIA), GN4C and GN8P-treated groups (a). The percentage of ill animals in each group at the end of the experiment (day 37) (b). The progression of the disease severity was evaluated by clinical scoring (mean score per CIA-affected limb – c) and by the percentage of affected limbs per experimental group (d). Data represent results of three experiments performed (n = 5–10 mice per group), ***P < 0·001.