Rituximab exerts its anti-arthritic effects via inhibiting NF-κB/GM-CSF/iNOS signaling in B cells in a mouse model of collagen-induced arthritis

Abstract

Rheumatoidarthritis (RA) is an autoimmune disease characterized by uncontrolled joint inflammation and damage to bone and cartilage. B cells are known to play a crucial role in the pathogenesis and development of arthritis. Previous studies have found that B cells may be a potential target for treating RA. Rituximab, a monoclonal antibody targeting B cells, has induced long-term clinical responses in RA. Collagen-induced arthritis (CIA) mouse model is a widely studied autoimmune model of RA. CIA mouse model was used to investigate the effect of rituximab on the RA severity in the mice. Following induction of CIA, animals were treated with rituximab (250 mg/kg/week) intraperitoneally on the days 28, 35, 42, 49, 56, and 63 after collagen induction. We investigated the effect of rituximab on NF-κB p65, IκBα, GM-CSF, MCP-1, iNOS, TNF-α, and IL-6 cells in splenic CD19⁺ and CD45R⁺ B cells using flow cytometry. We also assessed the effect of rituximab on NF-κB p65, GM-CSF, IκBα, MCP-1, iNOS, TNF-α, and IL-6 at mRNA levels using RT-PCR analyses of knee tissues. Rituximab treatment significantly decreased CD19⁺NF-κB p65⁺, CD45R⁺NF-κB p65⁺, CD19⁺GM-CSF⁺, CD45R⁺GM-CSF⁺, CD19⁺MCP-1⁺, CD45R⁺MCP-1⁺, CD19⁺TNF-α⁺, CD45R⁺TNF-α⁺, CD19⁺iNOS⁺, CD45R⁺iNOS⁺, CD19⁺IL-6⁺, and CD45R⁺IL-6⁺, and increased CD45R⁺IκBα⁺ in spleen cells of CIA mice. We further observed that rituximab treatment downregulated NF-κB p65, GM-CSF, MCP-1, iNOS, TNF-α, and IL-6, whereas it upregulated IκBα, mRNA level. All these findings suggest that rituximab may be a novel therapeutic target for the treatment of RA.

Keywords: Autoimmune disease; B cells; Collagen-induced arthritis; NF-κB/GM-CSF/iNOS signaling; Rituximab.

Fig. 1

A, B, and C. Therapeutic effect of rituximab on NF-κB- and IκBα-expressing CD19⁺ and CD45R⁺ B cells were analyzed through flow cytometry in the spleen. D and E The expression levels of NF-κB and IκBα mRNA were analyzed by RT-PCR in the knee tissues. F Representative flow cytometry dot plots of one mouse from each group. The cells were gated on FSC-SSC, and then the lymphocytes were gated for analyzing the percentage of CD19⁺NF-κB⁺, CD45R⁺NF-κB⁺, CD19⁺IκBα⁺, and CD45R⁺IκBα⁺ B cells. Normal control (NC) mice received saline and rituximab (250 mg/kg/week) intraperitoneally (ip). CIA mice were treated with rituximab (250 mg/kg/week) ip starting from day 28 until day 63 after collagen induction. The significance level was set at *p < 0.05 compared with the CIA untreated mice. The data present the mean ± SD (n = 6).

Fig. 2

A and B. Therapeutic effect of rituximab on GM–CSF–expressing CD19⁺ and CD45R⁺ B cells was analyzed through flow cytometry in the spleen. C The expression level of GM-CSF mRNA was analyzed by RT-PCR in the knee tissues. D Representative flow cytometry dot plots of one mouse from each group. The cells were gated on FSC-SSC, and then the lymphocytes were gated to analyze the percentage of CD19⁺GM-CSF⁺ and CD45R⁺GM-CSF⁺ B cells. Normal control (NC) mice received saline and rituximab (250 mg/kg/week) intraperitoneally (ip). CIA mice were treated with rituximab (250 mg/kg/week) ip starting from day 28 until day 63 after collagen induction. The significance level was set at *p < 0.05 compared with the CIA untreated mice. The data present the mean ± SD (n = 6).

Fig. 3

A and B. Therapeutic effect of rituximab on MCP-1-expressing CD19⁺ and CD45R⁺ B cells was analyzed through flow cytometry in the spleen. C The expression level of MCP-1 mRNA was analyzed by RT-PCR in the knee tissues. D and E Representative flow cytometry dot plots of one mouse from each group. The cells were gated on FSC-SSC, and then the lymphocytes were gated to analyze the percentage of CD19⁺MCP-1⁺ and CD45R⁺MCP-1⁺ B cells. Normal control (NC) mice received saline and rituximab (250 mg/kg/week) intraperitoneally (ip). CIA mice were treated with rituximab (250 mg/kg/week) ip starting from day 28 until day 63 after collagen induction. The significance level was set at *p < 0.05 compared with the CIA untreated mice. The data present the mean ± SD (n = 6).

Fig. 4

A and B. Therapeutic effect of rituximab on iNOS-expressing CD19⁺ and CD45R⁺ B cells was analyzed through flow cytometry in the spleen. C The expression level of iNOS mRNA was analyzed by RT-PCR in the knee tissues. D Representative flow cytometry dot plots of one mouse from each group. The cells were gated on FSC-SSC, and then the lymphocytes were gated to analyze the percentage of CD19⁺iNOS⁺ and CD45R⁺iNOS⁺ B cells. Normal control (NC) mice received saline and rituximab (250 mg/kg/week) intraperitoneally (ip). CIA mice were treated with rituximab (250 mg/kg/week) ip starting from day 28 until day 63 after collagen induction. The significance level was set at *p < 0.05 compared with the CIA untreated mice. The data present the mean ± SD (n = 6).

Fig. 5

A and B. Therapeutic effect of rituximab on TNF-α-expressing CD19⁺ and CD45R⁺ B cells was analyzed through flow cytometry in the spleen. C The expression level of TNF-α mRNA was analyzed by RT-PCR in the knee tissues. D Representative flow cytometry dot plots of one mouse from each group. The cells were gated on FSC-SSC, and then the lymphocytes were gated to analyze the percentage of CD19⁺TNF-α⁺ and CD45R⁺TNF-α⁺ B cells. Normal control (NC) mice received saline and rituximab (250 mg/kg/week) intraperitoneally (ip). CIA mice were treated with rituximab (250 mg/kg/week) ip starting from day 28 until day 63 after collagen induction. The significance level was set at *p < 0.05 compared with the CIA untreated mice. The data present the mean ± SD (n = 6).

Fig. 6

A and B. Therapeutic effect of rituximab on IL-6-expressing CD19⁺ and CD45R⁺ B cells was analyzed through flow cytometry in the spleen. C The expression level of IL-6 mRNA was analyzed by RT-PCR in the knee tissues. D Representative flow cytometry dot plots of one mouse from each group. The cells were gated on FSC-SSC, and then the lymphocytes were gated to analyze the percentage of CD19⁺IL-6⁺ and CD45R⁺IL-6⁺ B cells. Normal control (NC) mice received saline and rituximab (250 mg/kg/week) intraperitoneally (ip). CIA mice were treated with rituximab (250 mg/kg/week) ip starting from day 28 until day 63 after collagen induction. The significance level was set at *p < 0.05 compared with the CIA untreated mice. The data present the mean ± SD (n = 6).