Oral GABA treatment downregulates inflammatory responses in a mouse model of rheumatoid arthritis

Abstract

Current treatments for rheumatoid arthritis (RA) have long-term side effects such that new treatments are needed that can safely help manage the disease. There is a growing appreciation that GABA receptors (GABA-Rs) on immune cells provide new targets that can be used to modulate immune cell activity. Here, we show for the first time that activation of peripheral GABA-Rs can inhibit the development of disease in the collagen-induced arthritis (CIA) mouse model of RA. Mice that received oral GABA had a reduced incidence of CIA, and those mice that did develop CIA had milder symptoms. T cells from GABA-treated mice displayed reduced proliferative responses to collagen and their APC had a reduced ability to promote the proliferation of collagen-reactive T cells. Thus, GABA downregulated both T-cell autoimmunity and APC activity. Collagen-reactive T cells from GABA-treated mice displayed reduced recall responses in the presence of GABA ex vivo, indicating that GABA consumption did not desensitize these cells to GABA. GABA-treated mice had reduced collagen-reactive IgG2a, but not IgG1 antibodies, consistent with reduced Th1 help. The levels of serum anti-collagen IgG2a antibodies were correlated significantly with the CIA disease scores of individual mice. Our results suggest that activation of peripheral GABA-Rs may provide a new modality to modulate T cell, B cell, and APC activity and help ameliorate RA and other inflammatory diseases.

Figure 1

GABA treatment does not alter mouse consumption of water or food, or their weight. DBA/1j mice were immunized with collagen and fed water (control, open circles) or water containing GABA (black circles). Their (A) water, (B) food consumption, and (C) weights were measured longitudinally. Data shown are mean ± SEM. N = 8 mice/group.

Figure 2

GABA treatment inhibits the development of CIA. The development of CIA in collagen-immunized mice that were given plain water (open circles), or water containing GABA (black circles), was monitored longitudinally. The CIA scores were recorded as mean of four paw joints of individual mice at each time point. 0, no evidence of erythema or swelling; 1, erythema or mild swelling in the midfoot (tarsals) or ankle joint; 2, erythema and mild swelling extending from ankle to the midfoot; 3, erythema and moderate swelling extending from the ankle to metatarsal joints; and 4, erythema and severe swelling encompassing the ankle, foot, and digits. Graphs show longitudinal mean disease severity scores from two separate studies (N = 8 mice/group for panel A, N = 10–12 mice/group for panel B).

Figure 3

T cells from GABA-treated mice display reduced proliferative responses to collagen and are still sensitive to GABA-mediated inhibition in vitro. Splenic mononuclear cells from control (circles) and GABA-treated (triangles) mice were challenged in triplicate with the indicated concentrations of bCII peptide in the absence (open symbols) or presence (black symbols) of 1mM of GABA. Antigen-stimulated T-cell proliferation was determined by ³H-thymidine incorporation. Data are expressed as mean CPM ± SEM of each group of mice (N = 5 mice per group). *p < 0.05 vs. GABA-treated mice, ^#p < 0.05 vs. GABA-treated in vitro by Student’s t-test.

Figure 4

GABA treatment modulates APC function. Mice were immunized with bCII peptide and given plain water or water with GABA. Nine days later, their popliteal lymph node T cells and splenic APC were purified. The purified T cells from control mice (C-T), or GABA treated (G-T), were co-cultured with purified splenic APC from control (C-APC) or GABA-treated (G-APC) mice and challenged with the indicated concentrations of bCII peptide to assess T-cell proliferation. Data shown are mean CPM ± SEM for each group of mice (N = 5 per group). The cells in medium alone had 580–820 CPM and stimulated with anti-CD3 had 22,000–28,000 CPM. *p < 0.05 vs. the C-T/G-APC group, ^#p < 0.05 vs. the G-T/C-APC group by Student’s t-test.

Figure 5

Analysis of serum antibodies against collagen. (A) Eight weeks after the initial immunization, the levels of serum IgG, IgG1, and IgG2a responses to bCII in individual control (open circles) and GABA-treated (black circles) mice were determined by ELISA. Data shown are the mean values from individual mice. The background values of OD arranged from 0.102 to 0.114. *p < 0.001. (B) The levels of serum anti-bCII IgG2a were plotted against the CIA scores at the end of the 8-week observation period for individual control and GABA-treated mice. The association was determined by Spearman’s correlation test.