Inhibition of Glycolysis Reduces Disease Severity in an Autoimmune Model of Rheumatoid Arthritis

Abstract

The K/BxN mouse is a spontaneous model of arthritis driven by T cell receptor transgenic CD4⁺ T cells from the KRN strain that are activated by glucose-6-phosphate isomerase (GPI) peptides presented by the H-2^g7 allele from the NOD strain. It is a model of autoimmune seropositive arthritis because the production of anti-GPI IgG is necessary and sufficient for joint pathology. The production of high levels of anti-GPI IgG requires on the expansion of CD4⁺ follicular helper T (Tfh) cells. The metabolic requirements of this expansion have never been characterized. Based on the therapeutic effects of the combination of metformin and 2-deoxyglucose (2DG) in lupus models that normalized the expansion of effector CD4⁺ T cells. We showed that the CD4⁺ T cells and to a lesser extent, the B cells from K/BxN mice are more metabolically active than the KRN controls. Accordingly, preventive inhibition of glycolysis with 2DG significantly reduced joint inflammation and the activation of both adaptive and innate immune cells, as well as the production of pathogenic autoantibodies. However, contrary to the lupus-prone mice, the addition of metformin had little beneficial effect, suggesting that glycolysis is the major driver of immune activation in this model. We propose that K/BxN mice are another model in which autoreactive Tfh cells are highly glycolytic and that their function can be limited by inhibiting glucose metabolism.

Keywords: arthritis; follicular helper T cells; glycolysis; metabolism; mouse.

Figure 1

KBN lymphocytes have a high metabolism. Mitochondrial stress test in B cells (A) and CD4⁺ T cells (B) purified from 6 week old KRN and KBN females (N = 3) with one B6 female shown as control. ECAR plots were compared between KRN and KBN mice by 2-way ANOVA. (C). Basal OCR and SRC as well as maximum ECAR in CD4⁺ T cells and B cells calculated from data shown in (A,B). (D). pS6, pE4-BP, CD98 and pAKT in CD4⁺ T cells and B cells (N = 5). t-tests were used in C and D. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2

Glycolysis inhibition reduced joint inflammation in KBN mice. Time course of joint thickness (A) and clinical scores (B) in mice treated or not with 2DG (female controls N = 11; female 2DG N = 12; male controls N = 7; male 2DG N = 9). (C). Percent change in joint thickness between d 35 and d 61 in these mice (t-tests). (D). Time course of joint thickness in mice in which treatment with 2DG started after severe joint inflammation was established, at 51 d old for females and 61 d old for males (as indicated by arrows). (E). Changes in joint thickness after 2DG treatment shown in (D) in individual mice (initial and terminal measurements, paired t-tests). (F). Serum anti-GPI IgG in mice treated preventively or not with 2DG as shown in (A,B) (geometric means ± 95% confidence intervals). (G). Joint thickness in KRN mice after transfer of serum from KBN mice treated with 2DG or controls (N = 6 each). An uninjected KRN mouse is included as control. Plots in (A,B,G) were compared by 2-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3

Glycolysis inhibition reduced lymphoid expansion as well as GC B cells and plasma cell differentiation. Mice treated with 2DG showed a reduced number of splenocytes (A) and frequency of GL7⁺CD95⁺ GC B cells (B), both of which were correlated with disease severity, as well as a reduction of the frequency of CD138⁺B220^lo/neg plasma cells, which was not correlated with disease activity (C). 2DG also reduced the number of cells (D) and the frequency of GC B cells (E) in the JDLN. Mean values between the treated and control mice were evaluated with t-tests. *p < 0.05; **p < 0.01; ***p < 0.001. Correlations between joint thickness and immune variables were computed by pooling the 4 groups of mice and evaluated with a Pearson test as indicated for each graph.

Figure 4

Glycolysis inhibition reduced Tfh and Th17 cell expansion. Mice treated with 2DG showed a reduced frequency of Tfh (A), CD4⁺ T cells expressing the activation marker CD69 (B), Th17 (C) and Treg (D) cells, all of which were correlated with disease severity. The frequency of T_EM cells was not decreased by the 2DG treatment and T_EM frequency was no correlated to disease severity (E). Mean values between 2DG-treated and control mice were evaluated with t-tests. Correlations were evaluated with the Pearson test. **p < 0.01; ***p < 0.001.

Figure 5

Glycolysis inhibition reduced the frequency of neutrophils and monocytes in the JDLN and altered their activation. The frequency of monocytes (A) and neutrophils (B) was reduced by 2DG treatment, and the number of these cells in the joint draining lymph node correlated with disease severity. Mean values between the treated and control mice were evaluated with t-tests. Correlations were evaluated with the Pearson test. **p < 0.01; ***p < 0.001.

Figure 6

Metformin does not improve the protective effect of 2DG: (A). Time course of joint thickness in KBN mice treated with Met+2DG (8 females and 7 males), 2DG (9 females and 6 males) or untreated controls (5 females and 2 males). The 2DG treated and control mice were contemporaneous to the Met+2DG treated mice, and were a different cohort from the mice presented in Figure 2. The results were pooled between males and females. Treatments were compared to controls by a 2-way ANOVA. (B). Joint thickness in KRN mice after transfer of serum from KBN mice treated with Met+2DG, 2DG, or controls (N = 8 recipient each). Mean terminal values between treated and control mice were evaluated with t-tests. For both A and B, results were expressed as percentage change from the first day of treatment or serum transfer. OCR (C) and ECAR (D) from a mitochondrial stress test in CD4⁺ T cells purified from the KBN mice at the end of the treatment with Met+2DG, 2DG or control. Statistical analyses shown for the mean values of basal ECOR and ECAR (left of the graphs) and SRC or maximal ECAR (right of the graphs) between the treated and control mice evaluated with t-tests. The first row indicates comparison between 2DG and control, and the second row between 2DG and controls. **p < 0.01; ***p < 0.001.

Figure 7

Differential effects of Met+2Dg and 2DG alone in vivo treatment on the expansion of immune subsets. Frequency of Tfh cells (A), CD4⁺ T cells expressing the activation marker CD69 (B), IL-17A producing CD4⁺ T cells (C), Treg cells (B), Tem cells (E), GC B cells (F), and plasma cells (D) in the spleen. Numbers of monocytes (H), neutrophils (I), pDCs (J), CD11b⁺ (K) and CD11⁻ (L) in the DCs JDLN. There was not significant difference between males and females, and their samples were pooled. Mean values between treated and control mice were evaluated with one-way ANAOVA and multiple comparison tests. *p < 0.05, **p < 0.01; ***p < 0.001.