Efficacy of the Combination of Metformin and CTLA4Ig in the (NZB × NZW)F1 Mouse Model of Lupus Nephritis

The combination of CTLA4Ig and metformin reduces renal pathology after a 4-wk treatment.

Mice were treated with 2DG, CTLA4Ig, metformin, or the combination of metformin and CTLA4Ig for 1 mo. The metformin alone and metformin and CTLA4Ig treatments represent three cohorts along with their controls. Serum autoantibodies were compared before and after treatment for anti-dsDNA IgG (A) and ANA (original magnification ×20) (B) as fold change. Fold change values after 4 wk of treatment with 2DG for mice that were sacrificed after 8 wk of treatment are shown for reference. Representative ANA images (original magnification ×20) are shown for one control and two mice treated with metformin and CTLA4Ig. (C–E) Immune complex deposition in the glomeruli of control mice and mice treated with metformin or metformin and CTLA4Ig. (C) Representative images of C3 and IgG2a stains (original magnification ×10). Quantitation of C3 (D) and IgG2a (E) with each symbol representing a glomerulus from three control mice and five from each group of treated mice. (F) Mean glomerulus size in one coronal section per mouse. (G) GN pathology scores. (H) Terminal proteinuria. Mean ± SEM compared with one-way Brown–Forsythe and Welch ANOVA, t test (G), or χ² test (H) *p < 0.05. Each symbol represents a mouse except in (D) and (E) (n = 21 for controls in four cohorts, n = 15 for 2DG in three cohorts, n = 13 for metformin in three cohorts, n = 10 for CTLA4Ig in two cohorts, and n = 13 for metformin and CTLA4Ig in three cohorts). *p < 0.05, **p < 0.01.

The combination of CTLA4Ig and metformin alters CD4⁺ T cells after a 4-wk treatment.

Frequency of B220⁺ B cells (A), CD38⁺ memory B cells (B), GL7⁺CD95⁺ GC B cells (C), CD138⁺ B220^low plasma cells (D), CD4⁺ T cells (E), CD62L⁺ CD44⁻ Tn cells (F), CD62L⁻ CD44⁺ Tem cells (G), Tem/Tn ratio (H), frequency of Tfh (I), and Treg (J) cells. Mean ± SEM compared by one-way Brown–Forsythe and Welch ANOVA with Dunnett T3 multiple comparisons tests. Each symbol represents a mouse (n = 13 for controls in three cohorts, n = 9 for metformin in two cohorts, n = 4 for CTLA4Ig in one cohort, and n = 9 for metformin and CTLA4Ig in two cohorts). *p < 0.05, **p < 0.01, ***p < 0.001.

Metabolic effects of the 4-wk treatments.

B cell glucose uptake (A) and GLUT1 expression (B). S473 pAKT expression in total B cells (C), CD38⁺ memory B cells (D), and GC B cells (E). CD4⁺ T cell glucose uptake (F) and GLUT1 expression (G). S473 pAKT expression in total CD4⁺ T (H), Tem (I), and Treg (J) cells. (K–N) B cell mitochondrial stress test. OCR time course (K) and basal OCR (L); ECAR time course (M) and basal ECAR (N). (O–R) CD4⁺ T cell mitochondrial stress test. OCR time course (O) and basal OCR (P); ECAR time course (Q) and basal ECAR (R). In the time-course plots, the arrows on the x-axes correspond to the injection of oligomycin, trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP), and antimycin A and rotenone, in that order. Basal OCR and ECAR correspond to the average values before the addition of oligomycin. For clarity, the metformin and CTLA4Ig plots are not shown. Mean ± SEM compared by one-way Brown–Forsythe and Welch ANOVA with Dunnett T3 multiple comparisons tests. Each symbol represents a mouse (n = 13 for controls in three cohorts, n = 5 for metformin, n = 4 for CTLA4Ig, and n = 5 for metformin and CTLA4Ig, each in one cohort). *p < 0.05, **p < 0.01, ***p < 0.001.

The combination of CTLA4Ig and metformin reduces renal pathology after an 8-wk treatment.

(A) Terminal serum anti-dsDNA IgG. Fold changes between the terminal and initial values for serum anti-dsDNA IgG (B) and ANA (C) with representative images of ANA (original magnification ×20) for each treatment (D). (E) Representative images (original magnification ×20) of C3 (top) and IgG2a (bottom) glomerular deposits with corresponding quantification, with each symbol representing the average of four glomeruli from four to nine mice (F and G). (H) Representative periodic acid Schiff–stained kidney sections from a control and metformin and CTLA4Ig–treated mouse. Scale bars, 200 μm. (I) Mean glomerulus size in one coronal section per mouse. (J) GN pathology scores. (K) Terminal proteinuria. Mean ± SEM compared by one-way Brown–Forsythe and Welch ANOVA with Dunnett T3 multiple comparisons tests. Each symbol represents a mouse (n = 14 for controls in three cohorts; n = 5 for 2DG, metformin, and CTLA4Ig, each in one cohort; and n = 10 for metformin and CTLA4Ig in two cohorts). *p < 0.05, **p < 0.01, ***p < 0.001.

The combination of CTLA4Ig and metformin reduces plasma cell and effector CD4⁺ T cell differentiation after an 8-wk treatment.

(A) Splenocyte numbers. Frequency of total B cells (B), GC B cells (C), and plasma cells (D). Frequency of total CD4⁺ T cells (E), Tem cells (F), Tfh cells (G), and Treg cells (H) with CD25 expression on Treg cells (I). Mean ± SEM compared by one-way Brown–Forsythe and Welch ANOVA with Dunnett T3 multiple comparisons tests. Each symbol represents a mouse (n = 14 for controls in three cohorts; n = 5 for 2DG, metformin, metformin and 2DG, and CTLA4Ig, each in one cohort; and n = 10 for metformin and CTLA4Ig in two cohorts). *p < 0.05, **p < 0.01, ***p < 0.001.