PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of jimmunolThe Journal of ImmunologyThe JI Author ChoiceCurrent IssueThis ArticleSubmit a Manuscript
 
J Immunol. 2017 April 1; 198(7): 2661–2670.
Published online 2017 February 27. doi:  10.4049/jimmunol.1403088
PMCID: PMC5357783

Metformin Suppresses Systemic Autoimmunity in Roquinsan/san Mice through Inhibiting B Cell Differentiation into Plasma Cells via Regulation of AMPK/mTOR/STAT3

Abstract

Circulating autoantibodies and immune complex deposition are pathological hallmarks of systemic lupus erythematosus (SLE). B cell differentiation into plasma cells (PCs) and some T cell subsets that function as B cell helpers can be therapeutic targets of SLE. Mechanistic target of rapamycin (mTOR) signaling is implicated in the formation of B cells and germinal centers (GCs). We assessed the effect of metformin, which inhibits mTOR, on the development of autoimmunity using Roquinsan/san mice. Oral administration of metformin inhibited the formation of splenic follicles and inflammation in kidney and liver tissues. It also decreased serum levels of anti-dsDNA Abs without affecting serum glucose levels. Moreover, metformin inhibited CD21highCD23low marginal zone B cells, B220+GL7+ GC B cells, B220CD138+ PCs, and GC formation. A significant reduction in ICOS+ follicular helper T cells was found in the spleens of the metformin-treated group compared with the vehicle-treated group. In addition, metformin inhibited Th17 cells and induced regulatory T cells. These alterations in B and T cell subsets by metformin were associated with enhanced AMPK expression and inhibition of mTOR–STAT3 signaling. Furthermore, metformin induced p53 and NF erythroid-2–related factor-2 activity in splenic CD4+ T cells. Taken together, metformin-induced alterations in AMPK–mTOR–STAT3 signaling may have therapeutic value in SLE by inhibiting B cell differentiation into PCs and GCs.

Introduction

Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease encompassing a variety of manifestation and outcomes. It mainly affects women. SLE is characterized by circulating autoantibodies to components of nucleus and immune complex deposition, thus inducing damage to target organs, such as skin, kidney, and brain. Approximately 50–80% of patients with SLE have lupus nephritis (LN) (1). Renal involvement, the most serious organ involvement, is the strongest predictor of a poor outcome for patients with SLE.

Accumulating evidence clearly indicated that autoantibodies produced by B cells play critical roles in SLE pathogenesis. Anti-dsDNA Abs that directly deposit in the kidney of LN patients (2) and renal tissue of murine lupus (3) can inflict inflammatory damage to renal tissues and deteriorate renal function in affected subjects. Together with autoreactive pathologic Abs, autoantibody-producing plasma cells (PCs) and their helper cells should be major treatment targets for LN.

Metformin, originally introduced as a biguanide antibiotic medication, has an anti-inflammatory effect via activating AMP-activated protein kinase (AMPK), a major sensor that modulates lipid and glucose metabolism (4). The mechanistic target of rapamycin (mTOR) and AMPK pathways play critical and opposing roles in immunity and metabolism. mTOR is one of the downstream targets of AMPK that functions as an intracellular nutrient sensor to control protein synthesis, cell growth, metabolism, and autophagy (5).

It was reported that mTOR kinase activities of T cells are increased in SLE patients compared with matched healthy controls (6). Such enhanced mTOR activities could be reversed by rapamycin treatment (6). Suppression of mTOR activity with rapamycin treatment can markedly prolong survival, decrease anti-dsDNA Ab production, and ameliorate nephritis activity in MRL/lpr lupus-prone mice (7). With regard to the pathophysiological roles of T cell subsets in SLE, it was suggested that the development of SLE involves IL-17–producing Th17 immunity (8). Regulatory T cells (Tregs) have indispensable roles in maintaining peripheral tolerance. In active SLE patients, the immunoregulatory function of Tregs was decreased compared with controls or patients with inactive SLE (9), suggesting the defective function of Tregs in active SLE. Furthermore, the frequency of Tregs was reported to be reduced in a mouse model of SLE (10) and SLE patients (11). mTOR signaling proceeds via two complexes: mTOR complex (mTORC)1 and mTORC2. mTORC1 is essential for Th17 differentiation (12). It suppresses Treg differentiation by inhibiting Foxp3 expression (13). One recent study showed that mTORC1 activity is increased in SLE T cells, whereas mTORC2 activity is decreased (11). In that study, rapamycin, which has mTORC1-inhibiting properties, can promote Treg expansion in untouched T cells from SLE patients, suggesting that the therapeutic target is mTORC1 in SLE (11). Furthermore, rapamycin treatment is effective in SLE patients who are refractory to conventional treatment (14). N-acetylcysteine (NAC) is an antioxidant that can inhibit mTOR activity in vitro (15). Interestingly, a pilot study showed that NAC treatment improves SLE disease activity by reducing the activity of mTOR (16).

There is plenty of evidence regarding mTOR signaling and T cell activation. However, there is a relative paucity of data concerning the role of mTOR in the function and differentiation of B cells, a major component of adaptive immunity. Recent studies indicated that mTOR activity plays a critical role in B cells. Conditional deletion of the mTOR gene in B cells can strongly impair B cell proliferation and germinal center (GC) differentiation (17). Suppressing mTOR activity by rapamycin can markedly inhibit cell proliferation and Ab responses in mouse and human B cells (18, 19).

With regard to mTOR signaling in immunity, mTOR activation can lead to inhibited activity of STAT3. In addition to its crucial role during Th17 cell differentiation (20, 21), STAT3 activation is required for the differentiation of B lymphocytes into PCs in response to IL-21, CD154, and other stimuli (22, 23). Given the fact that metformin can suppress STAT3 activation via AMPK–mTOR signaling and that STAT3 is involved in the differentiation of B cells into Ab-producing PCs, it is plausible to assume that metformin can suppress LN by inhibiting pathogenic autoantibody production.

Therefore, the objective of this study was to determine whether oral administration of metformin can suppress autoimmunity developed in Roquinsan/san mice, which is a new murine model of SLE. We verified that metformin inhibited systemic autoimmunity in Roquinsan/san mice by suppressing marginal zone B (MZB) cell and B lymphocyte differentiation into PCs associated with a significant reduction in GC formation. With regard to T cells, the populations of follicular helper T (Tfh) and Th17 cells in Roquinsan/san mice were significantly decreased by metformin treatment, whereas the population of Tregs was increased. AMPK activities in splenic CD19+ B cells and CD4+ T cells were increased in metformin-treated Roquinsan/san mice, whereas the expression levels of its downstream mTOR–STAT3 signals were attenuated. Our results implicated that an AMPK-inducing strategy might be a novel therapeutic intervention for LN.

Materials and Methods

Animals and administration of metformin

Twenty-week-old male Roquinsan/san mice (Jackson Laboratory, Bar Harbor, ME) were maintained in groups (five per group) in polycarbonate cages in a specific pathogen–free environment. They were provided ad libitum access to mouse chow (Ralston Purina) and water. All experimental procedures were reviewed and approved by the Animal Research Ethics Committee at the Catholic University of Korea. To examine the effect of metformin on Roquinsan/san mice, metformin (5 mg per mouse) or saline was administered orally daily for 3 wk.

Measurement of Ig and glucose concentrations

Sera were collected from mice of each group at 5 wk after treatment with metformin or saline. Serum concentrations of IgG and IgG1 were measured using mouse IgG and IgG1 ELISA quantitation kits (Bethyl Laboratories, Montgomery, TX). Total serum glucose and triglyceride levels were measured using commercial kits from Asan Pharmaceutical (Hwaseong-si Gyeonggi-do, Korea).

Autoantibody and dsDNA measurement

To measure dsDNA in serum, mouse dsDNA (100 μg/ml; R&D Systems, Minneapolis, MN) was coated onto 96-well plates (Nunc, Roskilde, Denmark) and incubated at 37°C overnight, followed by a blocking step for 30 min at room temperature. Serum samples were diluted 1:50 in Tris-buffered saline (pH 8) containing 1% chicken serum albumin and 0.5% Tween-20. Samples were incubated at room temperature for 4 h. Plates were washed and incubated with HRP-conjugated anti-mouse IgG Ab (Bethyl Laboratories). The absorbance values were measured on an ELISA plate reader at 450 nm.

Isolation of CD19+ B cells and CD4+ T cells

Cell pellets were prepared from the spleens of normal C57BL/6, Roquinsan/san, or metformin-treated Roquinsan/san mice. Anti-mouse CD19 or CD4 MicroBeads were used, as recommended by the manufacturer (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, cells were resuspended in 100 μl of MACS buffer (1% BSA, 5 mM EDTA, and 0.01% sodium azide). CD19 or CD4 MicroBeads (1 × 107 cells per 10 μl) were added and incubated for 10 min at 4°C. Cells were diluted in 10 μl of MACS buffer, pelleted, resuspended in 500 μl, and separated magnetically in an AutoMACS magnet fitted with a MACS MS column (Miltenyi Biotec).

In vitro assay

Isolated splenocytes (5 × 105) from Roquinsan/san mice were incubated with 100 ng/ml LPS and metformin (0.1, 1, or 5 mM; both from Sigma-Aldrich, St. Louis, MO) for 3 d. Culture supernatant was subjected to IgG measurement. Cells were subjected to flow cytometry analysis.

Flow cytometry analysis

Splenocytes were isolated 5 wk after treatment with metformin. Various B cell populations were identified with specific Abs from eBioscience (San Diego, CA). To examine the population of B cells, splenocytes were stained anti-B220–allophycocyanin, anti-CD21–FITC, anti-CD23–PE, anti-CD138–PE, and anti-GL7–FITC. Populations of Th17 cells and Tregs expressing the splenocytes were stained anti-CD4– PerCP complex, anti-IL-17–PE, anti-CD25–allophycocyanin, or anti-Foxp3–PE and analyzed. Flow cytometry was performed using a FACSCalibur cytometer (BD Biosciences, San Diego, CA). Splenocytes were stained with anti-mouse B220-allophycocyanin (eBioscience) and anti-mouse CD138-PE (eBioscience) for 30 min at 4°C. Cells were permeabilized and fixed with Cytofix/Cytoperm (BD Biosciences), according to the manufacturer’s protocol, stained further with anti-mouse p-STAT3 705-PE (BD Biosciences) and anti-mouse p-STAT3 727-PE (BD Biosciences), and subjected to flow cytometric analysis (FACSCalibur; BD Biosciences).

Confocal microscopy of immunostaining

Spleen tissues were obtained from mice at 15 wk after primary immunization. Various cell populations were identified with specific Abs from eBioscience. To examine the populations of B and T cells, tissues were stained with anti-CD4–PerCP, anti-B220–allophycocyanin (or PE), anti-GL7–FITC, anti-CD138–PE, anti-CD4–PE (or FITC), anti-CD25–allophycocyanin, anti-Foxp3–FITC, and anti-IL-17–PE. To analyze the populations of cells expressing STAT, tissues were stained with anti-CD19–PE (or FITC), anti-CD4–FITC (or allophycocyanin), anti-p-AMPK–FITC (or PE), anti-mTOR–FITC, anti-p-STAT3 727–FITC (or PE), anti-p53–FITC, anti-NrF2–FITC (or PE), and anti-p-STAT3 705–PE. Stained sections were analyzed using a confocal microscopy system (LSM 510 Meta; Carl Zeiss).

CD4 T cell and CD19 B cell isolation

Cell pellets were prepared from the spleens of Roquinsan/san mice or metformin-treated Roquinsan/san mice. Anti-mouse CD4 or anti-mouse CD19 MicroBeads were used, as recommended by the manufacturer (Miltenyi Biotec). Briefly, cells were resuspended in 100 μl of MACS buffer (1% BSA, 5 mM EDTA, and 0.01% sodium azide). CD4 or CD19 MicroBeads (10 μl per 1 × 107 cells) were added and incubated for 10 min at 4°C. Cells were diluted in 10 μl of MACS buffer, pelleted, resuspended in 500 μl, and separated magnetically in an AutoMACS magnet fitted with a MACS MS column (Miltenyi Biotec).

Western blot analysis

CD4+ T cell or CD19+ B cell lysates were prepared from ~3 × 106 cells by homogenization in lysis buffer and were centrifuged (14,000 rpm, 15 min). The protein concentration in the supernatant was determined using the Bradford method (Bio-Rad, Hercules, CA). Protein samples were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Uppsala, Sweden). For Western blotting, the membrane was preincubated with 0.5% skim milk in 0.1% Tween 20 in TBS (TTBS) buffer at room temperature for 1 h. Primary Abs to p-AMPK, p-mTOR, p-STAT3 705, p-STAT3, p-Raptor (p-Rictor, AMPK, mTOR, STAT3; all from Cell Signaling Technologies, Danvers, MA), or β-actin (Sigma-Aldrich) were diluted 1:1000 in 5% BSA and TTBS. After overnight incubation at 4°C, the membrane was washed three times with TTBS. Next, the HRP-conjugated secondary Ab was added and incubated for 1 h at room temperature. After washing with TTBS, signals were detected using an ECL detection kit and Hyperfilm-ECL reagents (Amersham Pharmacia Biotech).

Immunohistopathological analysis of spleen, liver, and kidney

Mouse spleen, liver, and kidney tissues were obtained at 5 wk after treatment with metformin. They were fixed in 4% paraformaldehyde, decalcified in EDTA bone decalcifier, and embedded in paraffin. Tissues were sectioned at 7 μm thickness, dewaxed using xylene, dehydrated through a gradient of alcohol, and stained with H&E.

Statistical analysis

Statistical analysis was performed using IBM SPSS Statistics 20 for Windows (IBM, Armonk, NY). Statistical significance of data with multiple groups was calculated using one-way ANOVA; if a significant difference was observed among groups, the Bonferroni post hoc test was used to assess the statistical difference between specific groups. Comparisons of numerical data between two groups were performed with a nonparametric Mann–Whitney U test (two-tailed). The p values <0.05 were considered significant. Data are presented as mean ± SD.

Results

Metformin regulates IgG expression and pathogenic B cell subpopulations

First, we investigated the effect of metformin on Ab production of LPS-activated B cells. LPS-induced IgG production in B cells from C57BL/6 and Roquinsan/san mice was inhibited significantly by metformin in a dose-dependent manner compared with cell cultures stimulated with LPS alone (Fig. 1A). Cells were tested for viability using an MTT assay, to determine whether reduced IgG production was the result of decreased cell viability. No change in cell viability was observed following treatment with metformin (up to 5 mM; data not shown). We next analyzed the effect of metformin on the generation of B220CD138+ PCs and B220+CD138 GC B cells. The addition of metformin to LPS-stimulated cultures also led to decreased differentiation into these pathologic B cell subpopulations (Fig. 1B).

FIGURE 1.
Treatment with metformin in murine B cells inhibits IgG production and the differentiation of B cells into PCs and GC B cells. Murine B cells isolated from normal C57BL/6 mice and Roquinsan/san mice were stimulated with LPS (100 ng/ml), with or without ...

Metformin suppresses induction of the autoimmune phenotype of Roquinsan/san mice and inhibits autoantibody production

Roquinsan/san mice were introduced to demonstrate the lupus-like phenotype and pathology associated with spontaneous GC formation and expansion of the Tfh subset (24). To investigate the therapeutic potential of metformin for SLE, Roquinsan/san mice were orally administered metformin (5 mg per mouse) or saline daily for 3 wk (Fig. 2A). Two weeks after the last administration of metformin, sera were obtained from mice of each group. Serum levels of anti-dsDNA Ab, total IgG, and serum IgG1 were increased in Roquinsan/san mice compared with wild-type mice; however, the serum levels of these Abs were decreased significantly in the metformin-treated group (Fig. 2B). Although metformin has been widely used to control blood glucose levels in patients with type 2 diabetes, it was reported that it has no effect on blood glucose levels in nondiabetic human subjects (25). Consistent with that report, the mean glucose level in saline-treated Roquinsan/san mice was not significantly different from that in metformin-treated Roquinsan/san mice (Fig. 2B). The administration of metformin significantly inhibited the formation of splenic follicles (Fig. 2C). Also, inflammation in the kidney and liver tissues of Roquinsan/san mice also was reduced after treatment with metformin (Fig. 2C). The nephritis score was significantly attenuated by metformin treatment (Fig. 2C, right panel), implying a potential for metformin as a novel treatment strategy for LN.

FIGURE 2.
Therapeutic potential of metformin in Roquinsan/san mice. (A) Schematic diagram of the experimental protocols used for metformin treatment of Roquinsan/san mice (n = 5). The blue arrows denote Metformin oral administration days. (B) Ab (anti-dsDNA Abs, ...

Metformin suppresses the differentiation of B cells into PCs and GC B cells, as well as GC formation, in Roquinsan/san mice

The spontaneous formation of GCs as an important source of autoreactive PCs is the hallmark of SLE. The population of autoreactive MZB cells was reported to be increased in lupus mice, with implications for the development of autoimmunity via the production of pathologic anti-dsDNA Abs (26). Therefore, we investigated whether systemic administration of metformin could alter the differentiation and development of B cell subsets or GC formation. Flow cytometric analysis was used to analyze altered B cell subsets following metformin administration in Roquinsan/san mice. Our results revealed that the population and absolute number of CD21highCD23low MZB cells were significantly inhibited by metformin treatment, whereas those of CD21midCD23high follicular B (FOB) cells were increased (Fig. 3A). B220+GL7+ GC B cells and B220CD138+ PCs were significantly inhibited by metformin treatment (Fig. 3A). Furthermore, the numbers (Fig. 3B) and size (represented by yellow dotted line in Fig. 3C) of spontaneously developed GCs in the spleens of Roquinsan/san mice were also decreased in the metformin-treated group. These results suggested that metformin administration in murine lupus could alter B cell subsets and inhibit the differentiation of B cells into PCs by decreasing the number and size of GCs, the main source of long-lived PCs.

FIGURE 3.
Altered distribution of B cell subsets in metformin-treated Roquinsan/san mice. (A) Splenocytes were isolated from each group of mice. The populations of CD21highCD23low MZB cells and CD21midCD23high FOB cells among B220+ B cells were analyzed by flow ...

Metformin activates AMPK and decreases mTOR and STAT3 phosphorylation in Roquin B cells

To discover the signaling pathway involved in the metformin-induced alteration in pathological B cell subsets that underlie autoimmunity development, confocal microscopy analysis was performed. Metformin is known to affect AMPK–mTOR–mediated signaling and to suppress STAT3 activity in vitro (27). In this study, we found that splenic CD19+ B cells from metformin-treated Roquinsan/san mice exhibited a significant increase in the expression of p-AMPK but a significant decrease in the expression of mTOR and STAT3 phosphorylation at tyrosine 705 (pSTAT3 Y705) and STAT3 phosphorylation at serine 727 (pSTAT3 S727) compared with vehicle-treated mice (Fig. 4A). AMPK activation can induce apoptosis in multiple cancer cells by inducing p53 (28). Interestingly, the loss of p53 can lead to an increase in total B220+ B cells, supporting a role for p53 in B cell expansion (29). As expected, p53 expression in splenic CD19+ B cells was markedly induced in metformin-treated Roquinsan/san mice (Fig. 4A). Next, we aimed to determine the specific effect of metformin treatment on B cell differentiation into PCs in Roquinsan/san mice. Splenic PCs (B220CD138+) that were isolated from each group of mice were analyzed by flow cytometry. The results indicated a decreased population of pSTAT3 Y705– or pSTAT3 S727–expressing PCs in metformin-treated Roquinsan/san mice compared with the vehicle-treated group (Fig. 4B). Western blot analysis was performed to quantify the changes in activities of AMPK, STAT3, mTOR, Raptor, and Rictor in B cells from metformin-treated Roquinsan/san mice. Metformin treatment augmented the expression of p-AMPK and inhibited the expression of pSTAT3 S727, p-mTOR, and p-Raptor in CD19+ B cells in a murine model of lupus (Fig. 4C). In summary, metformin treatment of Roquinsan/san mice induced p-AMPK activity and attenuated mTOR and STAT3 phosphorylation in splenic B cells. Such an effect of metformin on signaling molecules might have contributed to its suppression on pathological B cell subsets, such as PCs and MZB cells.

FIGURE 4.
Altered activities of AMPK, mTOR, p-STAT3 (Y705 and S727), and p53 induced by metformin in CD19+ B cells from Roquinsan/san mice. (A) To determine how metformin treatment in Roquinsan/san mice was attained by alteration of AMPK and its downstream signals, ...

Metformin inhibits the differentiation of Tfh cells, Th17 cells, and Tregs in vivo

Tfh cells are specialized providers of B cell help. They are important for the formation of GCs. Once GCs are formed, Tfh cells maintain them and regulate GC B cell differentiation into PCs and memory B cells. Tfh cells express high levels of ICOS and CXCR5 (28). GL-7 was demonstrated as a GC marker on GC B cells, thymocytes, and some neuronal cell types (30). The spleens of metformin-treated Roquinsan/san mice showed decreased populations of GL-7–expressing GC B cells, with a marked reduction in ICOS-expressing Tfh cells compared with vehicle-treated Roquinsan/san mice (Fig. 5A). Based on the inhibitory effect of metformin on STAT3 activity, confocal staining and flow cytometry were used to analyze the proportions of Th17 cells and Tregs. Th17 cells and Foxp3+ Tregs were found to have opposing effects on the autoimmune response (31). Metformin treatment of Roquinsan/san mice resulted in a significantly increased number of CD4+CD25+Foxp3+ Tregs and a decreased number of Th17 cells (Fig. 5B).

FIGURE 5.
Regulation of Tfh cells, Th17 cells, and Foxp3+ Tregs in spleens of Roquinsan/san mice. Spleen tissues were obtained from Roquinsan/san mice on day 35 after the initiation of metformin or vehicle (saline) administration. (A) Tfh cells were stained with ...

Metformin inhibits the mTOR/STAT3 signaling pathway on splenic CD4+ T cells of Roquinsan/san mice

Because the pharmacologic effects of metformin are largely dependent on AMPK, the activities of AMPK, mTOR, and STAT3 in splenic CD4+ T cells were analyzed by confocal microscopy. Our results revealed that metformin treatment in Roquinsan/san mice resulted in enhanced AMPK activity (Fig. 6A); however, it inhibited the activities of mTOR and STAT3 (Fig. 6A). Our group recently revealed that p53, one of the most well-known tumor-suppressor proteins, can directly suppress Th17 differentiation via its direct interaction with STAT3 (32). In addition, p53 can skew Treg differentiation through interacting with STAT5 (32). Therefore, we examined whether the population of p53-expressing splenic CD4+ T cell was affected by metformin administration. Interestingly, p53 expression in CD4+ T cells from metformin-treated Roquinsan/san mice was markedly increased compared with vehicle-treated mice (Fig. 6A). Emerging evidence showed that AMPK activation can decrease oxidative stress (33). A recent in vitro study showed that pharmacological induction of AMPK can induce the activation of NF erythroid-2–related factor-2 (Nrf2) (34), a key transcriptional factor necessary for antioxidant and anti-inflammatory responses. A few studies suggested that oxidative stress may be implicated in the pathogenesis of LN (35). Based on these findings, we determined the expression level of Nrf2 in the splenic CD4+ T cells of each group of mice. Our results showed that Nrf2 activity in CD4+ T cells was significantly induced by treatment with metformin. Western blot analysis confirmed that metformin treatment in Roquinsan/san mice significantly attenuated in vivo expression of p-mTOR, p-STAT3 (both Y705 and S727), and p-Rictor in splenic CD4+ T cells (Fig. 6B). These altered activities of the mTOR signaling pathway were associated with enhanced activity of AMPK by metformin treatment (Fig. 6B). These results, together with those shown in Fig. 4, indicated that AMPK activation by metformin appeared to be working upstream of Nrf2, therefore exerting anti-inflammatory properties in a murine model of autoimmunity. Furthermore, the inhibitory properties of metformin on mTOR–STAT3 signaling were associated with reciprocal regulation of CD4+ T cells and B cell subsets.

FIGURE 6.
Altered activities of AMPK, mTOR, p-STAT3 (Y705 and S727), p53, and Nrf2 induced by metformin in splenic CD4+ T cells from Roquinsan/san mice. (A) To determine how metformin treatment in Roquinsan/san mice was attained by alteration of AMPK and its downstream ...

Discussion

This study revealed that oral administration of metformin inhibited the differentiation of B cells into autoreactive PCs, as well as GC formation, in a murine model of SLE. In addition, serum levels of anti-dsDNA Abs, IgG, and IgG1 that were profoundly increased in Roquinsan/san mice were markedly decreased after systemic administration of metformin, without inducing hypoglycemia. The pathologic changes in the kidney and liver of Roquinsan/san mice were diminished after the treatment with metformin. Metformin treatment altered B cell subsets, suppressing the formation of MZB cells, GC B cells, and PCs. The total count and size of GCs were significantly decreased in metformin-treated Roquinsan/san mice compared with the vehicle-treated group. AMPK-induced inhibition of mTOR–STAT3 signaling following metformin treatment might be implicated in the inhibition of excessive GC formation and decreased differentiation into mature PCs. These results were associated with Tfh cells responsible for the development of systemic autoimmunity in Roquinsan/san mice. To strengthen the therapeutic potential of metformin in lupus, other lupus-prone animals, such as MRL, MRL/lpr, and NZB/WF1 mice, should be used to confirm our findings.

Despite the advances that have been made in management strategies, 5–20% of LN patients eventually progress to end-stage renal disease (ESRD) within 10 y after the diagnosis of LN and require dialysis or kidney transplantation (36). The potential therapeutic options (e.g., mycophenolate mofetil, belimumab, and ocrelizumab) for the treatment of LN have increased; however, it is unknown whether these treatments will reduce the development of ESRD in patients with LN. Interestingly, the incidences of ESRD secondary to LN in the United States and the U.K. have not decreased (37, 38). Our previous study suggested that it will be a difficult and complicated task to prevent the occurrence of ESRD due to LN without the development of novel drugs (39).

Although the pathogenesis of SLE is still poorly understood, loss of B cell tolerance in the pathogenesis of SLE is well established. The breakdown of B cell tolerance may precede SLE because SLE patients have autoantibodies several years prior to any clinical manifestations of the disease (40), suggesting that ongoing B cell activities contribute to the onset of SLE. Despite theoretical evidence supporting the pathophysiological significance of B cells in SLE pathogenesis, depletion of B cells through an anti-CD20 mAb, such as rituximab, fails to eliminate autoantibodies in SLE patients (41). This implies that CD20 long-lived PCs that are resistant to rituximab are the main source of cells producing Abs. Indeed, recent studies revealed that autoantibody-secreting PCs are enriched in the kidney of lupus-prone mice, as well as in SLE patients (4244).

Autoantibody production and clinical severity of disease in lupus-prone mice (NZB/NZW mice) are dependent on aid from CD4+ T cells (45). Activated B cells recruit T cells in T cell zones from secondary lymphoid organs. This is followed by the differentiation of B cells extrafollicularly into plasmablasts with low-affinity Ab-producing capacity or by the move of B cells along the follicular route, where they form GCs and are differentiated into long-lived PCs (46). Tfh cells in GCs can promote somatic hypermutation and isotype switching, resulting in the generation of long-lived PCs that can produce high-affinity IgG Abs (47). A correlation between an increased number of Tfh cells and the development of autoimmunity was demonstrated in a murine model of SLE (48, 49), indicating that defects in positive selection by Tfh cells might lead to systemic autoimmunity, such as lupus. In addition to Tfh cells, CD4+CD25+Foxp3+ Tregs play a role in the regulation of lupus-associated Abs and anti-dsDNA Abs (50). The percentage of Tregs in the peripheral blood of SLE patients is reported to be significantly decreased, with decreased migratory capacity to CCR4 ligand, compared with that in healthy controls (51, 52). Furthermore, there is a significant negative correlation between Tregs and anti-dsDNA Ab levels (51). Although the exact role of Tregs in the pathogenesis of SLE remains to be elucidated, the strategy for expanding Tregs could present a future challenge for SLE treatment with respect to B cell tolerance. Th17 cells were suggested to be a distinct T cell subset. They might be responsible for the aberrant selection of autoreactive GC B cells and the humoral response in vivo (53). Taken together, inhibition of Tfh cell, Th17 cell, and B cell differentiation into PCs, as well as the expansion of Tregs, could be considered novel treatment options for SLE.

In summary, this study tested the therapeutic potential of metformin in SLE. The regulatory properties of metformin on AMPK–mTOR–STAT3 signaling inhibited B cell differentiation into PCs and spontaneous GC formation. This, in turn, reduced the production of autoantibodies and the infiltration of inflammatory cells into target tissues, including kidney and liver. These effects might be dependent, at least in part, on the inhibition of Th17 cell differentiation, as well as the induction of Tregs. Collectively, these data suggested that pharmacologic induction of AMPK activity might be a beneficial treatment for SLE and other autoantibody-mediated diseases.

Acknowledgments

We thank the Institutional Animal Care and Use Committee, School of Medicine, The Catholic University of Korea, for help with animal care and study.

This work was supported by Grant HI14C3417 from the Korean Health Technology Research and Development Project, which is funded by the Ministry for Health and Welfare, Republic of Korea. This work was also supported by Institute of Clinical Medicine Research of Bucheon St. Mary’s Hospital, Research Fund Grant BCMC14YA11 and by Grant NRF-2014R1A6A3A01009173 from the National Research Foundation of Korea, which is funded by the Korean Government.

Abbreviations used in this article:

AMPK
AMP-activated protein kinase
ESRD
end-stage renal disease
FOB
follicular B
GC
germinal center
LN
lupus nephritis
mTOR
mechanistic target of rapamycin
mTORC
mTOR complex
MZB
marginal zone B
NAC
N-acetylcysteine
Nrf2
NF erythroid-2–related factor-2
PC
plasma cell
pSTAT S727
STAT3 phosphorylation at serine 727
pSTAT Y705
STAT3 phosphorylation at tyrosine 705
SLE
systemic lupus erythematosus
Tfh
follicular helper T
Treg
regulatory T cell
TTBS
0.1% Tween 20 in TBS.

Disclosures

The authors have no financial conflicts of interest.

References

1. Sada K. E., Makino H. 2009. Usefulness of ISN/RPS classification of lupus nephritis. J. Korean Med. Sci. 24 Suppl.: S7–S10. [PMC free article] [PubMed]
2. Winfield J. B., Faiferman I., Koffler D. 1977. Avidity of anti-DNA antibodies in serum and IgG glomerular eluates from patients with systemic lupus erythematosus. Association of high avidity antinative DNA antibody with glomerulonephritis. J. Clin. Invest. 59: 90–96. [PMC free article] [PubMed]
3. Lambert P. H., Dixon F. J. 1968. Pathogenesis of the glomerulonephritis of NZB/W mice. J. Exp. Med. 127: 507–522. [PMC free article] [PubMed]
4. Zhou G., Myers R., Li Y., Chen Y., Shen X., Fenyk-Melody J., Wu M., Ventre J., Doebber T., Fujii N., et al. 2001. Role of AMP-activated protein kinase in mechanism of metformin action. J. Clin. Invest. 108: 1167–1174. [PMC free article] [PubMed]
5. Inoki K., Kim J., Guan K. L. 2012. AMPK and mTOR in cellular energy homeostasis and drug targets. Annu. Rev. Pharmacol. Toxicol. 52: 381–400. [PubMed]
6. Fernandez D. R., Telarico T., Bonilla E., Li Q., Banerjee S., Middleton F. A., Phillips P. E., Crow M. K., Oess S., Muller-Esterl W., Perl A. 2009. Activation of mammalian target of rapamycin controls the loss of TCRzeta in lupus T cells through HRES-1/Rab4-regulated lysosomal degradation. J. Immunol. 182: 2063–2073. [PMC free article] [PubMed]
7. Warner L. M., Adams L. M., Sehgal S. N. 1994. Rapamycin prolongs survival and arrests pathophysiologic changes in murine systemic lupus erythematosus. Arthritis Rheum. 37: 289–297. [PubMed]
8. Shah K., Lee W. W., Lee S. H., Kim S. H., Kang S. W., Craft J., Kang I. 2010. Dysregulated balance of Th17 and Th1 cells in systemic lupus erythematosus. [Published erratum appears in 2010 Arthritis Res. Ther. 12: 402.] Arthritis Res. Ther. 12: R53. [PMC free article] [PubMed]
9. Valencia X., Yarboro C., Illei G., Lipsky P. E. 2007. Deficient CD4+CD25high T regulatory cell function in patients with active systemic lupus erythematosus. J. Immunol. 178: 2579–2588. [PubMed]
10. Wu H. Y., Staines N. A. 2004. A deficiency of CD4+CD25+ T cells permits the development of spontaneous lupus-like disease in mice, and can be reversed by induction of mucosal tolerance to histone peptide autoantigen. Lupus 13: 192–200. [PubMed]
11. Kato H., Perl A. 2014. Mechanistic target of rapamycin complex 1 expands Th17 and IL-4+ CD4−CD8− double-negative T cells and contracts regulatory T cells in systemic lupus erythematosus. J. Immunol. 192: 4134–4144. [PMC free article] [PubMed]
12. Delgoffe G. M., Pollizzi K. N., Waickman A. T., Heikamp E., Meyers D. J., Horton M. R., Xiao B., Worley P. F., Powell J. D. 2011. The kinase mTOR regulates the differentiation of helper T cells through the selective activation of signaling by mTORC1 and mTORC2. Nat. Immunol. 12: 295–303. [PMC free article] [PubMed]
13. Delgoffe G. M., Kole T. P., Zheng Y., Zarek P. E., Matthews K. L., Xiao B., Worley P. F., Kozma S. C., Powell J. D. 2009. The mTOR kinase differentially regulates effector and regulatory T cell lineage commitment. Immunity 30: 832–844. [PMC free article] [PubMed]
14. Fernandez D., Bonilla E., Mirza N., Niland B., Perl A. 2006. Rapamycin reduces disease activity and normalizes T cell activation-induced calcium fluxing in patients with systemic lupus erythematosus. Arthritis Rheum. 54: 2983–2988. [PMC free article] [PubMed]
15. O’Loghlen A., Pérez-Morgado M. I., Salinas M., Martín M. E. 2006. N-acetyl-cysteine abolishes hydrogen peroxide-induced modification of eukaryotic initiation factor 4F activity via distinct signalling pathways. Cell. Signal. 18: 21–31. [PubMed]
16. Lai Z. W., Hanczko R., Bonilla E., Caza T. N., Clair B., Bartos A., Miklossy G., Jimah J., Doherty E., Tily H., et al. 2012. N-acetylcysteine reduces disease activity by blocking mammalian target of rapamycin in T cells from systemic lupus erythematosus patients: a randomized, double-blind, placebo-controlled trial. Arthritis Rheum. 64: 2937–2946. [PMC free article] [PubMed]
17. Zhang S., Pruitt M., Tran D., Du Bois W., Zhang K., Patel R., Hoover S., Simpson R. M., Simmons J., Gary J., et al. 2013. B cell-specific deficiencies in mTOR limit humoral immune responses. J. Immunol. 191: 1692–1703. [PMC free article] [PubMed]
18. Janes M. R., Limon J. J., So L., Chen J., Lim R. J., Chavez M. A., Vu C., Lilly M. B., Mallya S., Ong S. T., et al. 2010. Effective and selective targeting of leukemia cells using a TORC1/2 kinase inhibitor. Nat. Med. 16: 205–213. [PMC free article] [PubMed]
19. Aagaard-Tillery K. M., Jelinek D. F. 1994. Inhibition of human B lymphocyte cell cycle progression and differentiation by rapamycin. Cell. Immunol. 156: 493–507. [PubMed]
20. Powell J. D., Pollizzi K. N., Heikamp E. B., Horton M. R. 2012. Regulation of immune responses by mTOR. Annu. Rev. Immunol. 30: 39–68. [PMC free article] [PubMed]
21. Hirahara K., Ghoreschi K., Laurence A., Yang X. P., Kanno Y., O’Shea J. J. 2010. Signal transduction pathways and transcriptional regulation in Th17 cell differentiation. Cytokine Growth Factor Rev. 21: 425–434. [PMC free article] [PubMed]
22. Diehl S. A., Schmidlin H., Nagasawa M., van Haren S. D., Kwakkenbos M. J., Yasuda E., Beaumont T., Scheeren F. A., Spits H. 2008. STAT3-mediated up-regulation of BLIMP1 is coordinated with BCL6 down-regulation to control human plasma cell differentiation. J. Immunol. 180: 4805–4815. [PMC free article] [PubMed]
23. Kwon H., Thierry-Mieg D., Thierry-Mieg J., Kim H. P., Oh J., Tunyaplin C., Carotta S., Donovan C. E., Goldman M. L., Tailor P., et al. 2009. Analysis of interleukin-21-induced Prdm1 gene regulation reveals functional cooperation of STAT3 and IRF4 transcription factors. Immunity 31: 941–952. [PMC free article] [PubMed]
24. Linterman M. A., Rigby R. J., Wong R. K., Yu D., Brink R., Cannons J. L., Schwartzberg P. L., Cook M. C., Walters G. D., Vinuesa C. G. 2009. Follicular helper T cells are required for systemic autoimmunity. J. Exp. Med. 206: 561–576. [PMC free article] [PubMed]
25. Sambol N. C., Chiang J., O’Conner M., Liu C. Y., Lin E. T., Goodman A. M., Benet L. Z., Karam J. H. 1996. Pharmacokinetics and pharmacodynamics of metformin in healthy subjects and patients with noninsulin-dependent diabetes mellitus. J. Clin. Pharmacol. 36: 1012–1021. [PubMed]
26. Grimaldi C. M., Michael D. J., Diamond B. 2001. Cutting edge: expansion and activation of a population of autoreactive marginal zone B cells in a model of estrogen-induced lupus. J. Immunol. 167: 1886–1890. [PubMed]
27. Deng X. S., Wang S., Deng A., Liu B., Edgerton S. M., Lind S. E., Wahdan-Alaswad R., Thor A. D. 2012. Metformin targets Stat3 to inhibit cell growth and induce apoptosis in triple-negative breast cancers. Cell Cycle 11: 367–376. [PubMed]
28. Lee D. H., Lee T. H., Jung C. H., Kim Y. H. 2012. Wogonin induces apoptosis by activating the AMPK and p53 signaling pathways in human glioblastoma cells. Cell. Signal. 24: 2216–2225. [PubMed]
29. Schmidt N. W., Mayo L. D., Donner D. B., Kaplan M. H. 2006. p53 regulates Btk-dependent B cell proliferation but not differentiation. J. Leukoc. Biol. 79: 852–859. [PubMed]
30. Cervenak L., Magyar A., Boja R., László G. 2001. Differential expression of GL7 activation antigen on bone marrow B cell subpopulations and peripheral B cells. Immunol. Lett. 78: 89–96. [PubMed]
31. Fletcher J. M., Lonergan R., Costelloe L., Kinsella K., Moran B., O’Farrelly C., Tubridy N., Mills K. H. 2009. CD39+Foxp3+ regulatory T Cells suppress pathogenic Th17 cells and are impaired in multiple sclerosis. J. Immunol. 183: 7602–7610. [PubMed]
32. Park J. S., Lim M. A., Cho M. L., Ryu J. G., Moon Y. M., Jhun J. Y., Byun J. K., Kim E. K., Hwang S. Y., Ju J. H., et al. 2013. p53 controls autoimmune arthritis via STAT-mediated regulation of the Th17 cell/Treg cell balance in mice. Arthritis Rheum. 65: 949–959. [PubMed]
33. Price N. L., Gomes A. P., Ling A. J., Duarte F. V., Martin-Montalvo A., North B. J., Agarwal B., Ye L., Ramadori G., Teodoro J. S., et al. 2012. SIRT1 is required for AMPK activation and the beneficial effects of resveratrol on mitochondrial function. Cell Metab. 15: 675–690. [PMC free article] [PubMed]
34. Mo C., Wang L., Zhang J., Numazawa S., Tang H., Tang X., Han X., Li J., Yang M., Wang Z., et al. 2014. The crosstalk between Nrf2 and AMPK signal pathways is important for the anti-inflammatory effect of berberine in LPS-stimulated macrophages and endotoxin-shocked mice. Antioxid. Redox Signal. 20: 574–588. [PMC free article] [PubMed]
35. Wu T., Ye Y., Min S. Y., Zhu J., Khobahy E., Zhou J., Yan M., Hemachandran S., Pathak S., Zhou X. J., et al. 2014. Prevention of murine lupus nephritis by targeting multiple signaling axes and oxidative stress using a synthetic triterpenoid. Arthritis Rheumatol. 66: 3129–3139. [PMC free article] [PubMed]
36. Mojcik C. F., Klippel J. H. 1996. End-stage renal disease and systemic lupus erythematosus. Am. J. Med. 101: 100–107. [PubMed]
37. Ward M. M. 2009. Changes in the incidence of endstage renal disease due to lupus nephritis in the United States, 1996–2004. J. Rheumatol. 36: 63–67. [PMC free article] [PubMed]
38. Croca S. C., Rodrigues T., Isenberg D. A. 2011. Assessment of a lupus nephritis cohort over a 30-year period. Rheumatology (Oxford) 50: 1424–1430. [PubMed]
39. Moon S. J., Park H. S., Kwok S. K., Ju J. H., Kim H. Y., Park S. H. 2013. Predictors of end-stage renal disease and recurrence of lupus activity after initiation of dialysis in patients with lupus nephritis. Clin. Exp. Rheumatol. 31: 31–39. [PubMed]
40. Arbuckle M. R., McClain M. T., Rubertone M. V., Scofield R. H., Dennis G. J., James J. A., Harley J. B. 2003. Development of autoantibodies before the clinical onset of systemic lupus erythematosus. N. Engl. J. Med. 349: 1526–1533. [PubMed]
41. Townsend M. J., Monroe J. G., Chan A. C. 2010. B-cell targeted therapies in human autoimmune diseases: an updated perspective. Immunol. Rev. 237: 264–283. [PubMed]
42. Lacotte S., Dumortier H., Décossas M., Briand J. P., Muller S. 2010. Identification of new pathogenic players in lupus: autoantibody-secreting cells are present in nephritic kidneys of (NZB×NZW)F1 mice. J. Immunol. 184: 3937–3945. [PubMed]
43. Hutloff A., Büchner K., Reiter K., Baelde H. J., Odendahl M., Jacobi A., Dörner T., Kroczek R. A. 2004. Involvement of inducible costimulator in the exaggerated memory B cell and plasma cell generation in systemic lupus erythematosus. Arthritis Rheum. 50: 3211–3220. [PubMed]
44. Cassese G., Lindenau S., de Boer B., Arce S., Hauser A., Riemekasten G., Berek C., Hiepe F., Krenn V., Radbruch A., Manz R. A. 2001. Inflamed kidneys of NZB/W mice are a major site for the homeostasis of plasma cells. Eur. J. Immunol. 31: 2726–2732. [PubMed]
45. Liu Z., Bethunaickan R., Huang W., Lodhi U., Solano I., Madaio M. P., Davidson A. 2011. Interferon-α accelerates murine systemic lupus erythematosus in a T cell-dependent manner. Arthritis Rheum. 63: 219–229. [PMC free article] [PubMed]
46. MacLennan I. C., Toellner K. M., Cunningham A. F., Serre K., Sze D. M., Zúñiga E., Cook M. C., Vinuesa C. G. 2003. Extrafollicular antibody responses. Immunol. Rev. 194: 8–18. [PubMed]
47. Jacob J., Kelsoe G., Rajewsky K., Weiss U. 1991. Intraclonal generation of antibody mutants in germinal centres. Nature 354: 389–392. [PubMed]
48. Subramanian S., Tus K., Li Q. Z., Wang A., Tian X. H., Zhou J., Liang C., Bartov G., McDaniel L. D., Zhou X. J., et al. 2006. A Tlr7 translocation accelerates systemic autoimmunity in murine lupus. Proc. Natl. Acad. Sci. USA 103: 9970–9975. [PubMed]
49. Vinuesa C. G., Cook M. C., Angelucci C., Athanasopoulos V., Rui L., Hill K. M., Yu D., Domaschenz H., Whittle B., Lambe T., et al. 2005. A RING-type ubiquitin ligase family member required to repress follicular helper T cells and autoimmunity. Nature 435: 452–458. [PubMed]
50. Seo S. J., Fields M. L., Buckler J. L., Reed A. J., Mandik-Nayak L., Nish S. A., Noelle R. J., Turka L. A., Finkelman F. D., Caton A. J., Erikson J. 2002. The impact of T helper and T regulatory cells on the regulation of anti-double-stranded DNA B cells. Immunity 16: 535–546. [PubMed]
51. Lee H. Y., Hong Y. K., Yun H. J., Kim Y. M., Kim J. R., Yoo W. H. 2008. Altered frequency and migration capacity of CD4+CD25+ regulatory T cells in systemic lupus erythematosus. Rheumatology (Oxford) 47: 789–794. [PubMed]
52. Liu M. F., Wang C. R., Fung L. L., Wu C. R. 2004. Decreased CD4+CD25+ T cells in peripheral blood of patients with systemic lupus erythematosus. Scand. J. Immunol. 59: 198–202. [PubMed]
53. Hsu H. C., Yang P., Wang J., Wu Q., Myers R., Chen J., Yi J., Guentert T., Tousson A., Stanus A. L., et al. 2008. Interleukin 17-producing T helper cells and interleukin 17 orchestrate autoreactive germinal center development in autoimmune BXD2 mice. Nat. Immunol. 9: 166–175. [PubMed]

Articles from The Journal of Immunology Author Choice are provided here courtesy of The American Association of Immunologists, Inc.