rheumatoid arthritis ( ra ) is a multisystem autoimmune disease of unknown etiology . the characteristic pathology of affected joints is the inflammation of hyperplastic synovial membrane that can result in destruction of adjacent cartilage and bone .
though the exact molecular pathogenesis of ra remains elucidated , evidence suggests that interleukin- ( il- ) 17-producing t cell , th17 , is a central player .
il-17 is known to act synergistically with tumor necrosis factor- ( tnf- )  and il-1 , which are abundant proinflammatory cytokines found in arthritic joints enhancing the activation of fibroblasts , chondrocytes , and osteoclasts .
in contrast , regulatory t ( treg ) cell has been shown to have an anti - inflammatory role where its regulatory function is known to be deranged in ra patients . th17 and treg have been reported to have plasticity and can be converted to each other according to the cytokine milieu when the cells encounter .
therefore , a therapeutic approach that can increase treg cell while diminishing th17 cell simultaneously may be very promising in ra treatment .
earlier studies have suggested that the majority of pharmacologic effect of metformin is dependent on its ability to activate amp - activated protein kinase ( ampk ) , a major cellular regulator of lipid and glucose metabolism .
metformin inhibits mitochondrial respiratory chain resulting in energy deficiency noted as an increase in amp , activating ampk and protein kinase a ( pka ) .
ampk activation also promotes glucose uptake and fatty acid oxidation in muscle , enhancing the lowering effect of glucose .
in addition to the glucose lowering effect , metformin has been reported to exert an anti - inflammatory effect , which is also mediated by metformin - activated ampk .
metformin - activated ampk suppresses mammalian target of rapamycin ( mtor ) , which regulates t cell effector differentiation in vitro and in vivo .
ampk has been shown to be associated with th17 cell suppression by inhibiting mtor and signal transducer and activator of transcription 3 ( stat3 ) , suggesting the therapeutic potential of ampk agonist . while th17 cells are responsible for perpetuating chronic inflammation in ra , osteoclasts resorb bone resulting in subsequent joint destruction .
the role of mtor in osteoclastogenesis has been reported where a downstream molecule of the mtor pathway ( s6k ) conveys cell survival signal in osteoclasts .
recently , indo et al . reported that ampk - mediated inhibition of mtor and hypoxia - induced factor ( hif)-1 negatively regulated osteoclastogenesis .
these regulatory effects of ampk on th17 cells and osteoclasts have prompted the investigation on the effect of the ampk agonist , metformin , on autoimmune arthritis . in the present study ,
the suppression of metformin in a collagen - induced arthritis ( cia ) mouse model was demonstrated by focusing on the suppression of th17 differentiation and enhancing treg differentiation .
phosphorylation of stat3 was diminished by metformin while phosphorylation of stat5 increased , which seems to contribute to the alteration of th17/treg population .
in addition , osteoclastogenesis was suppressed via inhibition of mtor and ampk activation by metformin .
12-week - old male c57bl/6 mice ( orient bio , korea ) were maintained under specific pathogen - free conditions and fed standard laboratory mouse chow ( ralston purina , st .
all experimental procedures were examined and approved by the animal research ethics committee of the catholic university of korea , which conforms to all national institutes of health of the usa guidelines .
all surgeries were performed under isoflurane anesthesia and all efforts were made to minimize suffering .
mice were immunized into the base of the tail with 100  g of chicken cii ( chondrex inc . ,
redmond , wa , usa ) in complete freund 's adjuvant ( chondrex inc . ) .
100  g of chicken cii in incomplete freund 's adjuvant ( chondrex inc . ) was injected at tail and one foot on day 14 .
cia mice were orally fed 3 times a week for 9 weeks with 5  mg / mouse metformin ( sigma - aldrich ) or saline as a control beginning on day 7 after first immunization ( permit number : cumc-2013 - 0128 - 01 ) .
arthritis in these mice was examined visually two times per week for the appearance of arthritis in the peripheral joints .
the severity of arthritis was recorded using the mean arthritis index on scale of 04 , as previously reported , as follows : ( 0 ) , no evidence of erythema and swelling ; ( 1 ) , erythema and mild swelling confined to the midfoot ( tarsals ) or ankle joint ; ( 2 ) , erythema and mild swelling extending from the ankle to the midfoot ; ( 3 ) , erythema and moderate swelling extending from the ankle to the metatarsal joints ; ( 4 ) , erythema and severe swelling encompassing the ankle , foot , and digits .
the severity of arthritis was analyzed by the sum of scores from all legs , assessed by two independent observers with no knowledge of the experimental groups .
joints of each mouse were fixed in 10% formalin , decalcified in 10% edta , and embedded in paraffin wax .
the sections were stained with hematoxylin - eosin ( h&e ) , safranin o , and toluidine blue to detect proteoglycans . for immunohistochemistry
, the sections were performed using the vectastain abc kit ( vector laboratories , burlingame , ca , usa ) .
tissues were stained anti - receptor activator of nf-b ( rank ) , anti - receptor activator of nf-b ligand ( rankl ) , anti - tnf- , anti - tnf receptor - associated factor 6 ( traf6 ) , anti - il-17 , and anti - il-6 ( all from santa cruz biotechnology inc . ) and anti - phosphorylated(p ) stat3 ( y705 ) , anti - pstat3 ( s727 ) , and anti - pampk anti - pmtor ( all from cell signaling , danvers , ma ) .
the tissue was stained using pe - conjugated anti - cd4 , fitc - conjugated anti - forkhead box p3 ( foxp3 ) , apc - conjugated anti - cd25 , fitc - conjugated anti - il-17 , fitc - conjugated anti - pstat3 ( y705 ) , and fitc - conjugated anti - pstat3 ( s727 ) ( all from ebiosciences , san diego , ca , usa ) .
stained sections were analyzed using a zeiss microscope ( lsm 510 meta ; carl zeiss , oberkochen , germany ) .
anti - igg , igg1 , and igg2a were measured by mouse igg , igg1 , and igg2a elisa quantitation kits ( bethyl laboratories , montgomery , tx ) . to analyze intracellular cytokines ,
splenocytes were stained with percp - conjugated anti - cd4 , apc - conjugated anti - cd25 , fitc - conjugated anti - il-17 , and pe - conjugated anti - foxp3 ( ebiosciences ) , followed by fixation and permeabilization with a foxp3 staining buffer kit ( bd bioscience ) according to the manufacturer 's instructions .
four  hours before the staining , the cells were stimulated with phorbol myristate acetate ( 25  ng / ml ) and ionomycin ( 250  ng / ml ) ( all from sigma - aldrich ) and golgistop ( bd bioscience ) .
all data was analyzed using flowjo software ( tree star , ashland , or , usa ) .
the mrna expression levels were estimated using a lightcycler 2.0 instrument ( roche diagnostic , mannheim , germany ) with the version 4.0 software .
all reactions were performed with the lightcycler faststart dna master sybr green i ( takara , shiga , japan ) , following the manufacturer 's instructions .
the primers sequences are shown in table 1 . to purify splenic cd4 + t cells ,
the splenocytes were incubated with cd4-coated magnetic beads and isolated using magnetic activated cell sorting ( macs ) separation columns ( miltenyi biotec ) .
isolated cd4 + t cells were stimulated with th17 cell - polarizing conditions for 3 days : plate - bound anti - cd3 ( 0.5  g / ml ) , anti - cd28 ( 1  g / ml ) ( both from bd pharmingen , ca , usa ) ; anti - interferon ( ifn)- ( 2  g / ml ) , anti - il-4 ( 2  g / ml ) , transforming growth factor ( tgf)- ( 2  ng / ml ) , and il-6 ( 20  ng / ml ) ( all from r&d systems , minneapolis , mn , usa ) .
osteoclasts were stimulated in the presence of macrophage colony - stimulating factor ( m - csf ) ( 10  ng / ml ) ( r&d systems ) and receptor activator of nuclear factor kappa - b ligand ( rankl ) ( 50  ng / ml ) ( peprotech , london , uk ) and absence or presence of metformin 1  mm .
the protein was separated by sds - page and transferred on nitrocellulose membranes ( amersham pharmacia biotech , piscataway , nj , usa ) . western blot was performed by snap i.d .
the hybridized bands were detected by enhanced chemiluminescence ( ecl ) detection kit ( thermo scientific brand of thermo fisher scientific , inc . ) .
the antibodies were as follows : anti - ampk , anti - pampk , anti - mtor , anti - pmtor , anti - pstat3 y705 , anti - pstat3 s727 , and anti - stat3 ( all from cell signaling ) ; traf6 ( santa cruz ) ; and -actin ( sigma ) .
comparing numerical data between groups was performed with 2-way anova and nonparametric mann - whitney tests .
differences in the mean values of various groups were analyzed by using anova with a post hoc test .
to investigate the antiarthritic effect of metformin , mice with cia were orally fed with either metformin or saline three times a week from day 7 after first immunization .
metformin - treated cia mice showed significantly reduced severity ( figure 1(a ) , left ) and incidence of clinical arthritis ( figure 1(a ) , right ) .
histological and cartilage scores measured based on inflammatory cell infiltration and cartilage damage were also significantly lower in metformin - treated mice ( figure 1(b ) ) .
il-17 , il-6 , and tnf positive cells decreased in metformin - treated cia mice ( figure 1(c ) ) .
total igg , igg1 , and igg2a antibodies significantly decreased in metformin - treated cia mice when compared to saline - treated controls ( figure 1(d ) ) .
the numbers of cd4+il-17 + t cells and cd4 + pstat3 + ( both at s727 and y705 ) t cells were reduced in metformin - treated cia mice more than in the controls . in contrast , the numbers of cd4 + cd25 + foxp3 + treg cells and cd4 + pstat5 + t cells were higher in the metformin - treated group ( figure 2 ) .
next , spleen cells isolated from each group were analyzed for the expression of il-17 and foxp3 using flow cytometry .
the results showed that cd4+il-17+th17 cells were lower in the metformin - treated cia , whereas cd4+cd25 + foxp3+treg cells were increased in metformin group ( figure 3(a ) ) . as well , mrna expressions of the th17-related cytokines and transcription factors were significantly reduced in the splenocytes from metformin - treated cia mice and there was greater expression of foxp3 ( figure 3(b ) ) .
as the proportion of th17 cell was decreased in metformin - treated cia mice in vivo , the effect of metformin on th17 cell and treg cell differentiation in vitro was examined .
cd4 + t cells isolated from healthy c57bl/6 mice were cultured in th17 cell - polarizing condition in the presence or absence of metformin .
the differentiation of th17 cells was inhibited with metformin as there was a simultaneous increase in treg differentiation ( figure 3(d ) ) .
in addition , metformin inhibited the expression levels of the th17-associated genes , il-17 , ahr , runx1 , and rort , as it increased mrna levels of foxp3 ( figure 3(e ) ) .
treatment with metformin increased the expression of pampk in il-6 10  ng / ml stimulated cd4 t cell ( figure 3(f ) ) ) .
these results indicate that metformin suppresses th17 cell differentiation while enhancing treg differentiation and ampk activation in a proinflammatory condition ( figure 6 ) .
metformin - treated cia mice showed reduced bone erosion and cartilage destruction in the arthritic joints ( figure 1(b ) ) .
to determine effect of metformin on osteoclastogenesis , we were immunochemical staining for rank and rankl in joint tissues .
as expected , the expression of rank and rankl decreased in the inflamed joint tissue of metformin - treated cia ( figure 4(a ) ) .
the preosteoclasts derived from the control cia mice and metformin - treated cia mice were stimulated with m - csf and rankl .
the results showed that the osteoclasts from metformin - treated cia mice were less capable of differentiating into osteoclasts ( figure 4(b ) ) .
the expression levels of various osteoclastogenic markers , such as trap , mmp-9 , integrin 3 , calcitonin receptor , and cathepsin k , were decreased , also .
the reduction in osteoclastic activity was observed following metformin - treated mice ( figure 4(c ) ) .
to verify the observation that metformin reduced the expression of osteoclastogenic markers ex vivo , we investigated whether metformin could inhibit osteoclast differentiation in vitro .
the results showed that metformin treatment inhibited osteoclast differentiation as determined by the trap staining assay ( figure 5(a ) ) .
we also measured osteoclastogenic markers mrna expression levels in control osteoclasts and metformin - treated ones .
the mrna expression of the osteoclast - related markers , trap , cathepsin k , mmp-9 , calcitonin receptor , carbonic ii , and integrin 3 , decreased by meformin treatment .
we also found that metformin suppressed the expression of hif-1 mrna whereas it induced expression of ampk mrna ( figure 5(b ) ) .
as the pharmacologic effect of metformin is largely dependent on ampk , the activation of ampk , mtor , and stat3 during osteoclastogenesis was addressed .
western blot analysis demonstrated that expressions of traf6 , pmtor , and pstat3 ( y705 and s727 ) were reduced with metformin treatment .
consistently , immunohistochemical staining of arthritic joints revealed that expressions of these molecules were significantly lower in metformin - treated cia mice ( figure 5 ) .
collectively , these results suggest that metformin suppresses osteoclastogenesis via inhibition of mtor and stat3 , which is mediated by metformin - activated ampk .
in the present study , we demonstrated that metformin suppressed cia via reciprocal regulation of th17 cell and treg cell .
metformin - activated ampk seems to be involved in this regulation through the inhibition of mtor and its downstream molecules , hif-1 and stat3 .
as th17 receives the spotlight in the pathogenesis of ra , a number of medications that target th17 have been developed .
however , tocilizumab , an il-6 receptor blocking antibody , is currently being used , whereas tofacitinib , a jak inhibitor , is under clinical trials .
the molecules shown to inhibit stat3 or rort have demonstrated promising results in animal models .
recently , metabolism has been raised as an important check point in regulating th17/treg balance .
therefore , the role of ampk , the master metabolic sensor , in t cell fate was investigated .
kang et al . reported that metformin - activated ampk inhibited mtor - stat3 pathway contributing to a reduction in the population of th17 cells .
furthermore , our results revealed that metformin not only suppressed th17 cells but also simultaneously enhanced treg cells .
this might be explained as the metabolic check point determines the differentiation of th17 cell or treg cell .
for instance , th17 cells are highly glycolytic while treg cells have a high lipid oxidation rate .
the high glycolytic activity of th17 cell is dependent on mtor , which is inhibited by ampk .
mtor enhances the glycolytic pathway promoting th17 cell differentiation whereas ampk inhibits mtor and promotes mitochondrial oxidative metabolism to enhance treg cell .
furthermore , the high glycolytic activity of th17 cells is attributed to hif-1 , a downstream molecule of mtor pathway .
in addition , hif-1 promotes the nuclear translocation of rort and degrades foxp3 , resulting in a decrease in treg cell .
it is widely known that pstat3 competes for the same binding locus as pstat5 in il-17 promoter to enhance il-17 .
therefore , activating ampk and inhibiting mtor are an efficient way to regulate th17 and treg concurrently .
our results revealed that metformin suppressed th17 cell while increasing treg cell in cia mice .
though confocal microscopy showed an increase in stat5 phosphorylation , it seems to result from reduced pstat3 and consequently increase the number of treg rather than the direct effect of metformin on stat5 phosphorylation .
undeniably , metformin did not affect il-2 induced stat5 phosphorylation levels in vitro ( data not shown ) .
it was reported that metformin ameliorated experimental autoimmune encephalitis   and collagen antibody induced arthritis , both involving th17 cells .
our results confirmed that metformin - activated ampk could suppress cia via reciprocal regulation of th17 cell and treg cell .
therefore , future investigation of whether metformin can be used as a therapeutic in th17-driven chronic inflammatory diseases is promising .
the number of trap+ multinucleated cells was lower in the joints of metformin - treated mice .
this may partially result from decreased expression of inflammatory cytokines that promote osteoclastogenesis via the upregulation of rankl in the arthritic joint .
metformin stimulates osteoprotegerin and reduces rankl expression in osteoblasts , and ampk inhibits rank signaling , which might also contribute to reduced osteoclastogenesis . to investigate the direct effect on osteoclastogenesis
, preosteoclasts were cultured with m - csf and rankl in the presence or absence of metformin , decreasing osteoclastogenesis in the metformin - treated cells .
our results also revealed that the suppression was associated with an increase in ampk phosphorylation and subsequent decrease in mtor and stat3 phosphorylation .
the decreased expression of hif-1 in metformin - treated osteoclasts verifies the recent observation that mtor - hif-1 pathway was critical in osteoclastogenesis . to date , there is no prevention in the development of rheumatoid arthritis .
however , proper early treatment can prevent progressive damage of the joints . in this study ,
the effect of metformin on joint destruction and regulation of immune cells in occurrence of rheumatoid arthritis was evaluated .
collectively , these results suggest a new role of metformin . metformin has been shown to ameliorate autoimmune arthritis by regulating the th17/treg balance and inhibiting osteoclastogenesis through ampk . in cases where secondary diabetes mellitus develops due to excessive corticosteroid use in ra patients , metformin can be the optimal therapeutic option due to its glucose lowering and anti - inflammatory properties .
the present study demonstrated that metformin had an anti - inflammatory effect on cia due to the inhibition of th17 cell differentiation and the upregulation of treg cell differentiation along with the suppression of osteoclast differentiation .
our data suggest that metformin might be a potential candidate for therapeutic modulation of experimental animal model with rheumatoid arthritis .
 metformin is widely used to suppress certain functions of the cells found in diseases including diabetes and obesity . in this study , 
 the effects of metformin on downregulating il-17-producing t ( th17 ) cells , activating and upregulating regulatory t ( treg ) cells , suppressing osteoclastogenesis , and clinically scoring collagen - induced arthritis ( cia ) were investigated . to evaluate the effect of metformin on cia , mice were orally fed with either metformin or saline as control three times a week for nine weeks . 
 histological analysis of the joints was performed using immunohistochemistry and th17 cells and treg cells of the spleen tissue were examined by confocal microscopy staining . 
 metformin mitigated the severity of cia , reduced serum immunoglobulin concentrations , and reciprocally regulated th17/treg axis . also , metformin treatment of normal cells cultured in th17 conditions decreased the number of th17 cells and increased the number of treg cells . 
 metformin decreased gene expression and osteoclastogenic activity in cia and normal mice . 
 these results indicate that metformin had immunomodulatory actions influencing anti - inflammatory action on cia through the inhibition of th17 cell differentiation and the upregulation of treg cell differentiation along with the suppression of osteoclast differentiation . 
 our results suggest that metformin may be a potential therapeutic for rheumatoid arthritis . 