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

Davidson and Laurence MorelCaleb Cornaby, Ahmed S. Elshikha, Xiangyu Teng, Seung-Chul Choi, Yogesh Scindia, Anne

http://www.immunohorizons.org/content/4/6/319https://doi.org/10.4049/immunohorizons.2000033doi:

2020, 4 (6) 319-331ImmunoHorizons 

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Efficacy of the Combination of Metformin and CTLA4Ig in the(NZB 3 NZW)F1 Mouse Model of Lupus Nephritis

Caleb Cornaby,*,1,2 Ahmed S. Elshikha,*,†,2 Xiangyu Teng,* Seung-Chul Choi,* Yogesh Scindia,‡ Anne Davidson,§ andLaurence Morel**Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 32610; †Department of Pharmaceutics,

Zagazig University, Zagazig, Sharkia 44519, Egypt; ‡Department of Medicine, University of Florida, Gainesville, FL 32610; and §Feinstein Institutes for

Medical Research, Manhasset, NY 11030

ABSTRACT

CTLA4Ig, a reagent that inhibits CD28 signaling, has shown therapeutic efficacy in mouse models of lupus nephritis (LN) when

combined with several other biologics or standard of care drugs. Unfortunately, clinical trials treating LN patients with CTLA4Ig

(abatacept) have not met endpoints. Metformin, a drug used to control hyperglycemia that inhibits mitochondrial metabolism,

lowered the effective dose of glucocorticoids and prevented major flares when added on to the standard of care treatment of lupus

patients with low disease activity. Metformin combined with inhibition of glycolysis by 2-deoxyglucose showed therapeutic efficacy

in multiple mouse models of LN. Because CD28 signaling triggers glucose metabolism in T cells, we hypothesized that combining

CTLA4Ig treatment with metformin would have the same effect. In this study, we showed that the combination of metformin and

CTLA4Ig decreased the development of LN in (NZB 3 NZW)F1 mice treated at the early stage of disease. This preventive effect was

associated with a decreased expansion of CD4+ T cell effector subsets. However, contrary to the combination with 2-deoxyglucose,

metformin combined with CTLA4Ig did not alter autoantibody production, suggesting different mechanisms of symptom mitigation.

Overall, this study shows therapeutic efficacy of the combination of metformin and CTLA4Ig, two drugs with established safety

records, in a preclinical mouse model of LN. ImmunoHorizons, 2020, 4: 319–331.

INTRODUCTION

Systemic lupus erythematosus (SLE) is an autoimmune diseasewhose complexity and heterogeneity have impeded the develop-ment of alternative therapies to glucocorticoids and immunosup-pressive drugs despite intense preclinical research and numerousclinical trials (1). CTLA4 is a negative regulator of CD4+ T cellactivation that competes with coreceptor CD28 for binding toCD80 and CD86. Based on the obligate involvement of CD4+

T cells in SLE (2), preclinical studies have tested CTLA4Ig, asoluble fusion protein that blocks CD28 interactions with CD80/86, in mouse models of lupus. Although CTLA4Ig alone did notshow any clinical efficacy in established nephritis, lasting diseaseremission was achieved in combination with cyclophosphamide(3, 4), anti-CD40L (5), TACI-Ig (6), or BAFF-R (7). Interest-ingly, the inhibition of T cell costimulation appeared to have along-lasting effect on end-organ injury rather than immuneactivation and autoantibody production (4). These studies have

Received for publication May 7, 2020. Accepted for publication May 27, 2020.

Address correspondence and reprint requests to: Dr. Laurence Morel, University of Florida, Box 100275, Gainesville, FL 32610-0275. E-mail address: morel@ufl.edu

ORCIDs: 0000-0003-1098-1378 (C.C.); 0000-0003-3759-6832 (A.S.E.); 0000-0001-6230-9087 (S.-C.C.); 0000-0001-6382-6289 (Y.S.).1Current address: McLendon Clinical Laboratories, University of North Carolina at Chapel Hill Hospitals, Chapel Hill, NC.2C.C. and A.S.E. contributed equally to this work.

This work was supported by grants from the Lupus Research Alliance (541570) (to L.M.) and from Merck Serono (to A.D.).

Abbreviations used in this article: ANA, anti-nuclear Ab; BWF1, (NZB 3 NZW)F1; 2DG, 2-deoxyglucose; ECAR, extracellular acidification rate; GC, germinal center; GN,glomerulonephritis; LN, lupus nephritis; OCR, oxygen consumption rate; SLE, systemic lupus erythematosus; TC, triple congenic B6.Sle1.Sle2.Sle3; Tem, effectormemory T; Tfh, follicular helper T; Tn, naive T; Treg, regulatory T.

The online version of this article contains supplemental material.

This article is distributed under the terms of the CC BY-NC 4.0 Unported license.

Copyright © 2020 The Authors

https://doi.org/10.4049/immunohorizons.2000033 319

RESEARCH ARTICLE

Clinical and Translational Immunology

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provided a rationale for a randomized clinical trial in which activeclass III or IV lupus nephritis (LN) patients treatedwithCTLA4Ig(abatacept) were compared with standard of care alone (8). Theprimary endpoint, whichwas defined as a sustained improvementof LN clinical parameters, was not met. However, a modest im-provement was observed in the patients with the most severedisease, as well as a reduction in serum anti-DNA Abs and anincrease in C3 and C4 levels, indicating that the treatment had abiological effect. This modest clinical effect was independentlyconfirmed in a small cohort (9). A post hoc analysis of the PBMCsshowed a heterogeneous response to the treatment based on theactivation of specific immune cell types inwhich themost respon-sive patients shared a distinctive transcriptional signature ofplasma cells, activated dendritic cells, NK cells, and neutrophils(10). In addition, structural variants in CTLA4g and/or its ligandsmay also affect the response to treatment (11). Moreover, despitethe efficacy of CTLA4Ig in combination therapies in lupus mice(12), the outcome of abatacept treatment in LN patients was notimproved by its combination with low-dose cyclophosphamideand azathioprine in the ACCESS trial (13).

Cellular metabolic programs (i.e., the type and amount ofmetabolic substrates used by a cell, aswell as themanner bywhichthey are used), control the effector phase of the immune system.Targeting T cell metabolism has been proposed as a novelapproach tomanipulate the immune response toward therapeuticoutcomes in immune mediated diseases (14). CD4+ T cells frommousemodels and patientswith SLEpresent numerousmetabolicabnormalities, including an increased glycolysis and oxidativephosphorylation (15). We have shown that 2-deoxyglucose (2DG)and metformin, which are inhibitors of glycolysis and the mito-chondrial electron transport chain, respectively, normalized thefunctions of human and murine lupus CD4+ T cells. Furthermore,treatment with the combination of these two drugs reversedautoimmunepathology inmultiplemousemodels of the disease (16,17). Moreover, these inhibitors have little impact on the non-autoreactive functionsof the immune system, including its responseto immunization and infection (18). These findings suggest thatmetabolic inhibitorscan improveclinicaloutcomes in lupusperhapswith less systemic side effects than currently used therapies.

In SLE patients, metformin can enhance the efficacy of existingtherapies as a glucocorticoid-sparing add-on treatment (19) and byreducing the rate of major flares in SLE patients with low diseaseactivity (20). Inmice,metformin treatment expands the regulatory T(Treg) cell subset while inhibiting the expansion of Th1 and Th17effector cells, reducing the severity ofmultiple autoimmunediseases,such as experimental autoimmune encephalomyelitis (21), insulitis(22), and scleroderma (23). In lupus-prone mice, metformindecreases the production of IFN-g and the expansion of memoryCD4+ T cells while increasing the production of IL-2 (16, 17).Metforminhas, however, little effect on follicular helperT (Tfh) cellsor autoantibody production, which are targeted by glycolysisinhibition with 2DG (18). Therefore, the combination of metforminand 2DG has the potential to provide an optimal effect in reversingearlydisease in lupusmiceby targetingdifferentarmsofautoimmuneactivation.Becauseof the limitation andpotential side effects of using

2DG clinically, we investigated whether metformin could enhancethe efficacy of another treatment in a preclinical model of lupus.

Theconcepttosimultaneouslytargetaselect immunepathwayandcellularmetabolismhasbeenvalidatedbycombiningaPI3Kg inhibitorwith checkpoint blockade in multiple preclinical tumor models(24). Furthermore, there is evidence that blocking specific immunepathways affects the metabolism of the targeted immune cells. Wefocused on CTLA4Ig because it is a safe biologic with documentedmodest effects inLNpatients. Furthermore, checkpoint blockadewithAbsagainstCTLA4restoredTcell glycolysis andeffector functions inamouse tumor xenograft model (25). Because CD28 signaling activatesglycolysis (26), CTLA4Ig should inhibit glycolysis in T cells.

Thegoal of this studywas to test thehypothesis thatmetforminwould improve the efficacy of CTLA4Ig in (NZB 3 NZW)F1(BWF1)mice treated at a preclinical stage of the disease, amodel oflupus in which a short course of CTLA4Ig has shown efficacy incombination with other drugs (12). We showed that althoughneither metformin nor CTLA4Ig alone were effective, the com-bination of metformin with CTLA4Ig reduced renal pathologyto a similar extent as the combination ofmetforminwith 2DG.Themechanisms of these two combination therapies are, however,different because the CTLA4Ig combination minimally affectsthe production of autoantibodies, which is eliminated by thecombination with 2DG. Given the safety record of both CTLA4Igand metformin in SLE patients, these results suggest that theircombination may offer an effective venue to treat LN.

MATERIALS AND METHODS

MiceBWF1 females were purchased from The Jackson Laboratory at6 wk of age. Treatments started when the mice were 27–32 wk ofage. All age-matched cohorts included single treatments and a no-treatment control. CTLA4Ig was administered as previouslydescribed (5). Briefly, 100 mg of CTLA4Ig was given via i.p.injection six times over a 2-wk period. 2DG (6 mg/ml; Sigma-Aldrich) or metformin (3 mg/ml; Sigma-Aldrich) was adminis-tered as previously described in drinking water (16), eitherseparately or in combination with another treatment for 4 or8 wk. Mice were sacrificed at week 4 or 8. Serum for Abmeasurementswascollectedon theday the treatmentstarted, thenat 4 and 8 wk. Peripheral blood was collected at wk 2 for flowcytometry. Proteinuria was measured semiquantitatively withAlbustix strips. C57BL/6J (B6) mice as well as the lupus-pronetriple congenic B6.Sle1.Sle2.Sle3 (TC) (27) mice were bred andmaintained at the University of Florida. All experiments wereconducted according to protocols approved by the University ofFlorida Institutional Animal Care and Use Committee.

Autoantibody measurementFor the detection of anti-nuclear Abs (ANAs), indirect immunos-taining of Hep-2 slides (Bio-Rad Laboratories) with Alexa Fluor488–conjugated goat anti-mouse IgG (BD Biosciences) wasperformed with sera diluted 1:40 as previously described (16).

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Fluorescence intensity was analyzed using the ImageJ program.Detection of anti-dsDNA IgG was performed with sera diluted1:100 as previously described (16). Results were normalized to aserial dilution of a pool of titer TC sera in which the 1:100 dilutionwas set to 100 U.

Flow cytometryPeripheral blood was collected in heparinized tubes. Single-cellsuspensions were prepared using standard procedures fromspleens. Cells were stained in FACS staining buffer (2.5% FBSand 0.05% sodium azide in PBS). Fluorochrome-conjugated Absagainst B220 (RA3-6B2), CD25 (PC61.5), CD38 (90/CD38), CD4(RM4-5), CD44 (IM7), CD62L (MEL-14), CD95 (15A7), CD138(281.2), CXCR5 (2G8), Foxp3 (FJK-16S), GL-7, S473P-AKT(SDRNR), PD-1 (RPMI-30), and P-S6 (D57.2.2E) were purchasedfrom BD Biosciences, eBioscience, BioLegend, and Cell SignalingTechnology. Follicular T cellswere stained in a three-step processusing purified CXCR5, followed with biotinylated anti-rat IgG(Jackson ImmunoResearch Laboratories) and PerCP-Cy5 strep-tavidin in FACS on ice. GLUT1 expression was measured with arabbit anti-mouse Ab (EPR3915; Abcam), followed by an AlexaFluor 640–conjugated goat anti-rabbit IgG–(H + L chain), F(ab9)2fragment (Cell Signaling Technology). Intracellular staining wasperformed with a Fixation/Permeabilization kit (eBioscience).Dead cells were excluded with fixable viability dye (eFluor780;Thermo Fisher Scientific). All samples were acquired on anLSRFortessa flow cytometer (BD Biosciences) and analyzed withthe FlowJo software (Tree Star) as previously described (28).

HistologyRenal pathology was scored on periodic acid Schiff–stainedsections as previously described on a 0–4 scale (4). The score foreachmousewascalculatedas theaverageof20glomeruli in eachoftwo sections (40 glomeruli per mouse). The surface area of everyglomerulus from one coronal section per mouse was measuredon scanned slides with the Aperio ImageScope software (LeicaBiosystems), then averaged to give one value per mouse. Thepresence of IgG2a/C3 immune complex in glomeruliwas detectedin frozen sections with FITC-tagged anti-IgG2a and anti-C3 Abs,as previously reported (16), and quantitated with ImageJ.

Metabolic analysisOxygen consumption rate (OCR) and extracellular acidificationrate (ECAR), a measure of glycolysis, were assessed on splenicB cells and CD4+ T cells purified by negative selection (MiltenyiBiotec) fromtreatedmiceduringamitochondrial stress testusingaSeahorse XFe96 instrument (Agilent Technologies) as previouslyreported (16). One age-matched B6 mouse was included in eachassay for normalization. We also performed mitochondrial stresstests onBcells andCD44+CD4+Tcells purified from2-mo-oldB6,TC, andBWF1mice. Tomeasure glucose uptake, splenocyteswereincubated with FITC-labeled 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2NBDG) (Sigma-Aldrich) at 37°C for30 min. FITC mean fluorescence intensity was evaluated by flowcytometry in the B220+ B cell and CD4+ T cell gates.

StatisticsStatistical analyses were performed with the GraphPad Prism 8.0software. Differences between the control and treatment groupswere evaluated by one-way Brown–Forsythe and Welch ANOVAwith Dunnett T3 multiple comparisons tests with individualvariances computed for each comparison. Additional tests forspecific datasets were used as described in the figure legends. Toreduce instrument-induced variations between cohorts, meanfluorescence intensity results were normalized for each cohort tothe mean value of the control group set as 1. The results areexpressed as means 6 SEM. The levels of statistical significancewere set at *p, 0.05, **p, 0.01, and ***p, 0.001.

RESULTS

Effect of the single treatments on circulating lymphocytesWe evaluated the effect of a 2-wk treatment with CTLA4Igcompared with the metabolic inhibitors metformin and 2DG onPBLs.We observed no change in the frequency of total ormemoryB cells with any of the treatments (Fig. 1A, 1B), but 2DG decreasedthe number of plasma cells (Fig. 1C). Both metformin and 2DGdecreased the frequency of circulating CD4+ T cells (Fig. 1D). 2DGdecreased the frequency of memory CD44+CD4+ T cells (Fig. 1E),whereasmetformin increased the frequency ofTreg cells (Fig. 1F),as reported by others (21). Overall, short-term treatmentswith metformin and 2DG resulted in specific alterations incirculating lymphocytes, whereas treatment with CTLA4Ig hadno effect on these phenotypes.

The combination of CTLA4Ig and metformin reducedautoimmune pathology after a 4-wk treatmentThe efficacy of a treatmentwithmetformin andCTLA4Igwasfirstevaluated compared with CTLA4Ig, 2DG, or metformin alone inmice sacrificed4wk after the treatmentwas started. Only the 2DGtreatment reduced both anti-dsDNA IgG and ANA production(Fig. 2A, 2B). There was a modest, but variable, decrease in ANAlevels inmice treatedwithmetformin andCTLA4Ig (Fig. 2B). Theefficacy of 2DG indicated that a 4-wk treatment is long enough toeliminate anti-DNA–producing cells in this lupus model, whichhas a large contribution of short-lived autoreactive plasmablasts(29). The amount of IgG2a/C3 immune complexes deposited inglomeruli of the metformin and CTLA4Ig–treated mice wassimilar to that of control mice (Fig. 2C–E), confirming a modesteffect, at best, of thecombinationtreatmentonautoantibodies.Thecombined treatment showed, however, a reduction of renalpathology. The size of glomeruli, which is directly correlated withthe severity of glomerulonephritis (GN) (30),was decreasedby themetformin and CTLA4Ig treatment (Fig. 2F). The GN severityscores were also decreased in mice that received the combinationtreatment (Fig. 2G). Finally, only 1/8 mice treated with metforminand CTLA4Ig presentedwith 2000mg/dl terminal proteinuria, ascompared with 5/8 controls (Fig. 2H). Metformin alone showedonly a trend in reducing renal pathology, consistent across ourthreemeasurements of glomerular size,GNscore, andproteinuria,

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which confirmed previous findings with another lupus-pronemodel (17). CTLA4Ig alone had no effect on renal pathology,assessed either with immune complex deposition, GN scores, orproteinuria (data not shown). Overall, these results showed that ashort treatment combining metformin and CTLA4Ig reducedrenal pathology as compared with untreated mice, whereas thesingle treatments showed either only a trend or no effect ascompared with untreated mice. This effect on renal pathologyoccurred without affecting the production of autoantibodies inBWF1 mice.

The combination of metformin and CTLA4Ig reduced CD4+

T cell activation after a 4-wk treatmentThe total number of splenocytes was not affected by any of thetreatments (data not shown). We evaluated the distribution ofB and CD4+ T cell effector subsets in the spleen. Globally, theB cell populations were not affected, as shown by the frequencyof total B cells, memory B cells, germinal center (GC) B cells,and plasma cells (Fig. 3A–D). However, both metformin andmetformin and CTLA4Ig increased the frequency of totalCD4+ T cells (Fig. 3E).Metformin and CTLA4Ig also increasedthe frequency of naive CD4+ T cells (Fig. 3F), and it decreasedthe ratio of effector memory T (Tem) cells over naive T (Tn)cells (Fig. 3H) as well as the frequency of Tfh cells (Fig. 3I).Metformin alone did not change the frequency of effectorT cells, and CTLA4Ig alone increased the frequency of

Tem cells (Fig. 3G). Finally, there was no effect on the Tregpopulation (Fig. 3J).

We next assessed whether changes in the metabolism of B andCD4+ T cells of treated mice corresponded to their immuneactivation. B cells of mice treated with metformin, either alone orin combination with CTLA4Ig, increased their glucose uptakemeasured with the glucose analogue 2NBDG (Fig. 4A). This is anexpected response to the inhibition of respiration induced bymetformin, but itwasnot associatedwith an increased expressionofGLUT1, the main glucose transporter in lymphocytes (Fig. 4B) (31).Metformin alone or in combination with CTLA4Ig also decreasedS473 pAKT expression in total B cells and memory B cells (Fig. 4C,4D),butnot inGCBcells (Fig.4E).AKTisphosphorylatedatS473byPI3K in response to BCR and TCR/coreceptor stimulation, whichthen initiates mTORC1 activation (32). mTOR activation, however,was unchanged when measured by pS6 levels (data not shown). Asfor B cells, CD4+ T cells from mice treated with metformin with orwithoutCTLA4Ig increased their glucose uptake (Fig. 4F), althougha small decrease inGLUT1 expressionwas observedonCD4+Tcellsfrom mice treated with metformin alone (Fig. 4G). Unlike B cells,however,S473pAKTexpressiononCD4+Tcellswasnotchangedbyany of the treatments (Fig. 4H–J).

We have previously shown that CD4+ T cells from TC micepresent higher respiration and glycolysis than B6 controls evenbeforeautoimmunemanifestations (16), but it isunknownwhetherit is also the case in BWF1 mice. In this study, we compared the

FIGURE 1. Effect of treatment with single inhibitors on peripheral blood B and CD4+ T cells.

The percentage of total B cells (A), memory B cells (B), number of plasma cells (C), frequency of total CD4+ T cells (D), CD44+ memory CD4+ T cells

(E), and Treg cells (F) were evaluated 2 or 4 wk (2DG) after treatment. Means 6 SEM compared with one-way Brown–Forsythe and Welch ANOVA.

Each symbol represents a mouse (n = 16 for controls in three cohorts, n = 5 for 2DG, n = 10 for metformin in two cohorts, and n = 5 for CTLA4Ig).

*p , 0.05, **p , 0.01, ***p , 0.001.

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FIGURE 2. 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 magnification320) (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 320) 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 (Continued)(Continued)

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metabolism of total B cells and CD44+ memory CD4+ T cells in2-mo-old B6, TC, and BWF1 mice before they produced auto-antibodies. B cells showed higher respiration (SupplementalFig. 1A, 1B) and glycolysis (Supplemental Fig. 1C, 1D) in BWF1mice,withTCBcells showing intermediate values.Unexpectedly,the metabolism of activated CD4+ T cells was similar between B6and BWF1 mice, whereas we confirmed a higher metabolism inCD44+ T cells from TC mice (Supplemental Fig. 1E—H). Similarresults were obtained with naive CD442 CD4+ T cells of old TCandBWF1mice aswell aswith untreated control oldermice (datanot shown). The results suggest that global metabolic alterations

affect B cells to a greater extent than CD4+ T cells in the BWF1model. This finding may, at least in part, explain why thetreatments reduced pAKT levels in B cells, but not in CD4+

T cells. With these results as a baseline, we next evaluated theeffect of the 4-wk treatments on respiration and glycolysis.Metformin reduced respiration and glycolysis in both B andCD4+

T cells (Fig. 4K–R). CTLA4Ig treatment reduced respiration andglycolysis in CD4+ T cells (Fig. 4O–R), consistent with CD28signaling being the gatekeeper of glucose metabolism in T cells(26) and glucose being the main substrate for respiration in CD4+

T cells of lupus-pronemice (17). Interestingly, the combination of

FIGURE 3. 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+ B220low plasma cells (D), CD4+ T cells (E), CD62L+

CD442 Tn cells (F), CD62L2 CD44+ Tem cells (G), Tem/Tn ratio (H), frequency of Tfh (I), and Treg (J) cells. Mean 6 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.

treated with metformin or metformin and CTLA4Ig. (C) Representative images of C3 and IgG2a stains (original magnification 310). 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 6 SEM compared with one-way

Brown–Forsythe and Welch ANOVA, t test (G), or x2 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.

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FIGURE 4. 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 6 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.

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FIGURE 5. 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 repre-

sentative images of ANA (original magnification320) for each treatment (D). (E) Representative images (original magnification320) 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 andG). (H) Representative periodic acid Schiff–stained kidney sections from a control andmetformin and CTLA4Ig–treatedmouse. Scale bars, 200 mm.

(I) Mean glomerulus size in one coronal section per mouse. (J) GN pathology scores. (K) Terminal proteinuria. Mean 6 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.

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metformin and CTLA4Ig had nometabolic effect in pAKT levels,respiration, or glycolysis in either B or T cells. Although oursample size for these assays was small, it was sufficient to yieldsignificant resultswith the single treatments in this studyandwithother metabolic inhibitors in previous studies (16, 17). Theseresults suggest that the efficacy of this treatment on renalpathology is not associated with a global metabolic reprogram-ming of total B and CD4+ T cells.

The combination of CTLA4Ig and metformin reducedautoimmune pathologyThe 4-wk treatment with the combination of metformin andCTLA4Ig resulted in a significant, but modest, therapeutic effecton renal pathology.Wehave shown that the potent combination ofmetformin and2DGrequired an8-wk treatment for full effect (16).To better assess the efficacy of a treatment combining CTLA4Igandmetformin, we prolonged the treatment with metformin for atotal of 8 wk and compared the results to treatments withmetformin alone, as well as with 2DG and 2DG and metformin,which are two effective treatments in the BWF1model (16, 18). Asexpected, 2DG either alone or in combination with metformineliminated the production of anti-dsDNA IgG (Fig. 5A, 5B). Thecombination of metformin and CTLA4Ig had variable effects onthese Abs with a reduction in a small number of mice, but overallno significant effect (Fig. 5A–D). It is possible that a larger samplesize would clarify this issue; however, the 8-wk treatmentconfirmed the results obtained with the shorter treatment

(Fig. 2). Accordingly, treatments with either 2DG or 2DG andmetformin reduced the amount of C3 and IgG2a immunecomplexes in the kidneys (Fig. 5E–G). The treatment withmetformin and CTLA4Ig reduced C3 deposits, and there was atrend for IgG2a (Fig. 5E–G). These results suggest that theautoantibodies produced by metformin and CTLA4Ig–treatedmice are not reduced in quantity but may have a lower pathogeniceffect. This was confirmed by reduced GN severity (Fig. 5H),glomerular size (Fig. 5I), GN score (Fig. 5J), and proteinuria to asimilar extent as the treatmentwithmetforminand2DG (Fig. 5K).It should be noted that every 8-wk treatment, except CTLA4Igalone, reduced the size of glomeruli (Fig. 5I). These resultsvalidated the findings obtained with a 4-wk treatment thatmetformin and CTLA4Ig reduced renal pathology without amajor effect on autoantibody production, differing from themetformin and 2DG treatment, which reduced both.

The combination of CTLA4Ig and metformin reduced CD4+

T cell activation after an 8-wk treatmentContrary to the 4-wk treatment,mice treatedwithCTLA4Ig aloneor in combinationwithmetforminpresentedwith a lower count oftotal splenocytes 8 wk after the treatment began, suggesting thatthe 4-wk treatment was too short to be effective. The same resultswere observed in mice treated with either 2DG or 2DG andmetformin (Fig. 6A). The treatments with 2DG or 2DG andmetformin also reduced the frequency of B cells (Fig. 6B), GCBcells (Fig. 6C), andplasma cells (Fig. 6D).However, similar to the

FIGURE 6. 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). Mean6 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.

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results observed after the 4-wk treatment (Fig. 3), metforminandCTLA4Ig only reduced the frequency of plasma cells (Fig. 6D).The combination treatments of metformin with either 2DG orCTLA4Ig had no effect on the frequency of total CD4+ T cells(Fig. 6E), but they reduced the frequency of TemandTfh cells (Fig.6F, 6G). Each of the single treatments was effective at reducing thefrequency of Tem cells, and both 2DG and metformin reduced thefrequency of Tfh cells (Fig. 6G). Finally, the frequency of Treg cellswas also decreased by the metabolic treatments (Fig. 6H), as we

have previously reported in TCmice (16), with a modest increaseof CD25 expression on these Treg cells in mice treated withmetformin and 2DG (Fig. 6I). This suggests that the clinicalimprovement associated with any of the treatments was not linkedto improved Treg suppression.

Last, we compared the metabolic effects of metformincombined with either 2DG or CTLA4Ig. The metformin and2DG treatment decreased glucose uptake andGLUT1 expressionin B cells, where it had no effect on S473 pAKT expression but

FIGURE 7. Comparative effects of the 8-wk treatments combining metformin with 2DG or CTLA4Ig on B and CD4+ T cell metabolism.

Glucose uptake, GLUT1, S473 pAKT, and pS6 expression in total B cells (A–D) and CD4+ T cells (E–H). Mitochondrial stress test basal OCR and basal

ECAR in B cells (I and J) and CD4+ T cells (K and L). Mean 6 SEM compared by one-way Brown–Forsythe and Welch ANOVA with Dunnett T3

multiple comparisons tests. Each symbol represents a mouse (n = 7 for controls in two cohorts, n = 3 for metformin and 2DG in one cohort, and

n = 5 for metformin and CTLA4Ig in one cohort). *p , 0.05, **p , 0.01, ***p , 0.001.

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reducedmTORactivation (Fig. 7A–D). In contrast, themetforminand CTLA4Ig treatment did not change any of these parametersinB cells. Similar resultswere obtained forCD4+Tcells,with theexceptions that metformin and 2DG decreased S473 pAKTexpression (Fig. 7G) and that metformin and CTLA4Ig decreasedmTOR activation in CD4+ T cells (Fig. 7H). Neither treatmentchanged themetabolism of B cells (Fig. 7I, 7J), butmetformin andCTLA4Ig increased both respiration and glycolysis in CD4+ T cells(Fig. 7K, 7L). The results suggest that the initial metabolic effect ofCTLA4Ig was lost 6 wk after that short-course treatment wasstopped. It also confirms in this preclinical model that clinicalimprovement by metformin and CTLA4Ig is uncoupled fromsplenic B and CD4+ T cell metabolism. Although the sample size inthese metabolic studies was small, it was sufficient to observe aneffectwith the single treatments, suggesting that their combinationmay cancel each other’s effect.

DISCUSSION

This study used BFW1 mice, a classical model of spontaneouslupus, to test the hypothesis that the combination of metforminand CTLA4Ig would have a beneficial clinical outcome whentreatment is initiatedat the early clinical stage of disease before theonset of proteinuria. These two drugs have an established safetyprofile in SLE patients. There is considerable evidence for theefficacy of CTLA4Ig in preclinical models of lupus, with durableremission achieved in early stages of LN when administered incombinationwith either a brief inductionwith cyclophosphamideor with other biologics (12). Unfortunately, these promising re-sults have not been translated into clinical success, with the failureof abatacept clinical trials to meet endpoints, although somemodest biological activity was observed in the patients withactive disease (8, 9). Metformin prevents or delays the develop-ment of disease in lupus-prone mice when treatment startedbefore they develop autoantibodies (17) but showed littletherapeutic efficacy (16). Metformin used as add-on treatment tostandard of care showed steroid-sparing activity (19), and theability to reduce the appearance of major flares in patients withlow disease activity (20).

Beyond their safety and biological activity records in preclinicalmodels andSLEpatients, the rationale forcombiningmetforminandCTLA4Ig is that a treatment combining metformin with 2DG, aglycolysis inhibitor, is very effective in multiple mouse models oflupus (16). Because CD28 signaling induces glycolysis in T cells (26),we predicted that CTLA4Ig could at least partially substitute for2DG.We combined a short 2-wk CTLA4Ig treatment with either a4- or 8-wkmetformin treatment, andwe found significantly reducedrenal pathology at both time points with little, if any, effect onautoantibody production. This result is consistent with a previousstudy showing that the therapeutic effect of CTLA4Ig was largelydownstreamof immune complex deposition in the kidneys, possiblybypreventingdendritic cells’ability to recruit inflammatorycells (4).Therefore, although we confirmed our hypothesis that CTLA4Iginhibits glucosemetabolism inCD4+Tcells, its effect in combination

with metformin is different from that of 2DG, which diminishesautoantibody production (Refs. 16 and 18 and this study). Our studyconfirmed that monotherapy with CTLA4Ig is not therapeutic (33).Although our sample size was small for this group, we observedin these mice physiological effects that were consistent with thefunction of CTLA4Ig. This indicates that the combination ofmetformin and CTLA4Ig results in a unique therapeutic effectthat is not achieved by either monotherapies.

We have reported that T-dependent B cell responses are verysensitive to CTLA4Ig, which limits both class-switching andsomatic hypermutations in young (,20 wk old) BWF1 mice thatare treated before autoantibodies arise (33). In this study of oldermicewithearly clinical disease, however, theonlyBcell subset thatwas affected by the metformin and CTLA4Ig treatment wasplasma cells. Because this was not associated with a reduction ofautoantibody levels, the significance of this finding is unclear. Inthe short term, this treatment decreased pAKT phosphorylation,but no major changes in B cell metabolism were observed.

Consistent with its inability to induce remission when ad-ministered alone to nephritic BWF1 mice, it has been reportedthat CTLA4Ig does not affect the expansion of memory CD4+

T cells (33). In this study, we even found an expanded Tem cellpopulation in mice treated with CTLA4Ig alone. The combina-tion of metformin and CTLA4Ig, however, effectively reducedthe expansion of Tem cells, which are tightly associated withlupus pathogenesis. This reduction in inflammatory effectorphenotypes was, however, not associated with a reduction inCD4+ T cell metabolism. It is possible that metabolic changesoccurred earlier in the treatment andweremissed at the terminalendpoints or that they occurred in specific subsets rather totalCD4+ T cells.

CTLA4Ig inhibits the development and homeostasis of Tregcells, which depend on CD28 signaling (34). This potentiallyproinflammatory side effect, however, has not been reported inmultiplemodels of lupus (12).Weobserveda reduced frequency ofTreg cells in all treated BWF1 mice, with only a modest increasedCD25 expression in the metformin and 2DG treatment, supportingour previous findings that the clinical efficacy ofmetabolic inhibitorsin lupus-prone mice does not involve Treg cells (16).

Overall, this preclinical study supports the hypothesis thatmetformin is an old drug that can be repurposed to treatautoimmune diseases such as lupus (35), and it can be an effectiveand simple approach to boost the efficacy of CTLA4Ig, whichdespite great promise, did not meet endpoints in clinical trials.

DISCLOSURES

The authors have no financial conflicts of interest.

ACKNOWLEDGMENTS

We thank the Morel Laboratory members for technical assistance anddiscussion and the University of Florida Department of PathologyMolecular Pathology Core for assistance with histology.

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