osteoarthritis ( oa ) is considered to be a multifactorial disease with risk factors such as inflammation , aging , menopause , obesity , genetic background , and joint instability . clinical manifestations of oa may include joint pain , tenderness , stiffness , creaking , locking of joints , and local inflammation . currently , no curative treatment for oa has been developed , and the major therapeutic strategies for oa are based on conservative treatments and surgical intervention ( joint replacement ) . for the conservative treatments , most guidelines include recommendations for muscle exercise with medications to increase joint stability and to relieve joint inflammation and pain [ 2 , 3 ] . for example , the 2014 oarsi guidelines for nonsurgical management of knee osteoarthritis indicated that land - based exercise ( such as t'ai chi ) , water - based exercise , and strength training are all recommended to improve pain and physical function in knee oa . for the pharmacological interventions , the guideline indicated that both oral nonselective and cox2-selective nsaids ( nonsteroidal anti - inflammatory drugs ) as well as topical nsaids are conditionally recommended to improve the symptoms in knee oa . in addition to their effects on joint inflammation and pain relief , recent animal research has shown that some nsaids have anticatabolic and antiapoptotic effects on articular cartilage [ 5 , 6 ] . these findings suggest that control of joint inflammation may be an important therapeutic target to avoid oa progression and improve oa symptoms . in this study , we examined the anti - inflammatory and anticatabolic effects of follistatin on a carrageenan - induced mouse arthritis model . follistatin is an extracellular decoy receptor for the proinflammatory cytokine , activin ( inhibin ) . it is a glycosylated polypeptide structurally unrelated to activins and was originally isolated from ovarian fluid , where it mimics the action of inhibin on follicle stimulating hormone ( fsh ) secretion . since binding affinity of follistatin to activin is high , follistatin is considered to neutralize the actions of activins almost irreversibly . activin was originally isolated from gonadal fluids based on the ability to induce fsh secretion from the pituitary gland . activins are homo- or heterodimeric cysteine knot proteins belonging to the tgf superfamily . to date , 4 genes have been identified in the activin family ( inhibina , b , c , and e ) and 3 dimeric proteins ( activin a ( a/a ) , activin b ( b/b ) , and activin ab ( a/b ) ) have been studied in detail . subsequent analyses indicated that , in addition to being integral to reproductive physiology , activins are involved in many physiological processes such as embryonic development , tissue homeostasis , and tissue repair [ 9 , 10 ] . furthermore , recent reports indicated that the release of activins is involved in the acute inflammatory processes and immune responses in various clinical contexts such as sepsis , liver fibrosis , acute lung injury , asthma , wound healing , ischemia - reperfusion injury , and rheumatoid arthritis [ 11 , 12 ] . in acute systemic inflammatory situations , jones et al . reported that administration of 50  g of lipopolysaccharide ( lps ) into the jugular vein of corriedale ewes increased plasma activin a levels within one hour after lps injection [ 13 , 14 ] . the surge of activins after lps injection occurred at almost the same time or just prior to the surge of key proinflammatory cytokines such as tnf and il6 . importantly , recent reports confirmed the inhibitory effects of follistatin on activin action during inflammatory processes in various mouse models . reported that administration of follistatin neutralized the action of activin and alleviated symptoms in a mouse colitis model . hardy et al . suggested that the balance between activin a and follistatin is a determinant of severity of allergic inflammation or tissue phenotypic shift in asthma . jones et al . showed that treatment with follistatin altered the expression profiles of proinflammatory cytokines and increased survival after administration of a lethal dose of lps . their data strongly suggest the crucial roles of activin in the inflammatory response and that follistatin has significant therapeutic potential to reduce the severity of inflammatory diseases . since expression of both activins and follistatin is observed in the joint tissues [ 12 , 18 ] , we hypothesized that follistatin may function as an anti - inflammatory cytokine in the joint . to test this hypothesis , we examined the effect of recombinant mouse follistatin protein in the carrageenan - induced arthritis model . in this study , we showed that administration of follistatin in arthritic mice significantly alleviates synovial inflammation and inhibits proteoglycan loss from articular cartilage . our data suggest the involvement of activin signaling in the process of joint inflammation and that follistatin may have therapeutic potential to alleviate oa symptoms . animal care and all experiments were conducted in accordance with the guideline of the animal committee of tokyo medical and dental university . mice were housed under a 12-hour light - dark cycle and allowed food and water ad libitum . ( minneapolis , mn ) . rat anti - mouse monoclonal f4/80 antibody was purchased from abd serotec ( oxford , uk ) . thirty - three male mice ( 12-week - old ) were registered in this study . mice were randomly divided into three groups : carrageenan group ( ca ) , carrageenan + follistatin ( ca + flt ) group ( n = 14/group ) , and follistatin group ( flt , n = 5 , figure 1(a ) ) . a small skin incision was created to expose the knee joint ( figure 1(b ) ) . first , 19 mice ( 14 of ca + flt and 5 of flt ) were injected with follistatin ( 25  ng in 5  l of saline ) in the left knee joint ( figure 1(a ) ) . needles were inserted through the lateral infrapatellar area toward the intercondylar space of the femur in a deep knee - flexed position ( figure 1(c ) ) . after 30  min , all the mice in ca and ca + flt groups were injected with -carrageenan solution ( 30  g in 5  l saline ) into the left knee joint through the lateral margin of the patellar tendon to induce joint inflammation . ca + flt groups and 5 in flt group were sacrificed at day 3 , and the remaining mice in each group ( n = 7 ) were sacrificed at day 14 after injection ( figure 1(a ) ) . left knee joints were dissected , fixed in 4% pfa ( paraformaldehyde ; sigma - aldrich , mo ) in the fixed angle ( 30 degrees ) , decalcified in 20% edta ( ethylenediaminetetraacetic acid in pbs , ph 7.4 ) , and embedded in paraffin . five m thick sagittal sections of medial weight - bearing regions were prepared , and sections of equivalent position from each mouse were selected by the shape of the growth plate and further analyzed in this study . each section was stained with safranin - o / fast green or hematoxylin and eosin ( h&e ) for the histological evaluation of cartilage degeneration and joint inflammation . we analyzed two other sections 100  m away ( on both the medial and lateral sides ) from the analyzed sections of each mouse and observed similar results ( data not shown ) . to evaluate the early stage of articular cartilage degeneration , which is indicated by the loss of proteoglycan from articular cartilage , we employed a semiquantitative scoring system described by coles et al . with minor modification as described in ( modified coles score ( coles et al . , arthritis & rheumatology , 2010 ) ) . histological scores of articular cartilage from femurs and tibiae were evaluated and total scores were compared between the two groups at each time point . 
 modified coles score ( coles et al . , arthritis & rheumatology , 2010 ) 
 articular cartilage structure is as follows : 
 0 indicates normal ;  1 indicates some surface irregularities , no fibrillation ;  2 indicates severe surface irregularities and undulation ;  3 indicates fibrillation , clefts , or cartilage loss into superficial zone ;  4 indicates fibrillation , clefts , or cartilage loss into middle zone . surface layer morphology is as follows : 
 0 indicates smooth ;  1 indicates some small irregularities ;  2 indicates moderately roughened or enlarged ;  3 indicates severely enlarged , cellular infiltrate . pericellular loss of safranin - o staining is as follows : 
 0 indicates no pericellular loss of staining ;  1 indicates proteoglycan loss observed in less than 30% of articular surface ;  2 indicates proteoglycan loss observed in between 30% and 60% of articular surface ;  3 indicates proteoglycan loss observed in more than 60% of articular surface . articular cartilage structure is as follows : 
 0 indicates normal ;  1 indicates some surface irregularities , no fibrillation ;  2 indicates severe surface irregularities and undulation ;  3 indicates fibrillation , clefts , or cartilage loss into superficial zone ;  4 indicates fibrillation , clefts , or cartilage loss into middle zone . 1 indicates some surface irregularities , no fibrillation ; 2 indicates severe surface irregularities and undulation ; 3 indicates fibrillation , clefts , or cartilage loss into superficial zone ; 4 indicates fibrillation , clefts , or cartilage loss into middle zone . surface layer morphology is as follows : 
 0 indicates smooth ;  1 indicates some small irregularities ;  2 indicates moderately roughened or enlarged ;  3 indicates severely enlarged , cellular infiltrate . 1 indicates some small irregularities ; 2 indicates moderately roughened or enlarged ; 3 indicates severely enlarged , cellular infiltrate . pericellular loss of safranin - o staining is as follows : 
 0 indicates no pericellular loss of staining ;  1 indicates proteoglycan loss observed in less than 30% of articular surface ;  2 indicates proteoglycan loss observed in between 30% and 60% of articular surface ;  3 indicates proteoglycan loss observed in more than 60% of articular surface . 0 indicates no pericellular loss of staining ; 1 indicates proteoglycan loss observed in less than 30% of articular surface ; 2 indicates proteoglycan loss observed in between 30% and 60% of articular surface ; 3 indicates proteoglycan loss observed in more than 60% of articular surface . the severity of synovial inflammation was evaluated according to the synovitis scoring system described by blom et al . the synovitis scores were indicated as the total of four different areas in the knee joint as denoted in figure 2(c ) ( upper panel ) . synovitis score ( blom et al . , osteoarthritis and cartilge , 2004 ) 
 0 indicates no changes compared to normal joints ;  1 indicates thickening of the synovial lining and some influx of inflammatory cells ;  2 indicates thickening of the synovial lining and intermediate influx of inflammatory cells ;  3 indicates profound thickening of the synovial lining ( more than four cell layers ) and maximal observed influx of inflammatory cells . 0 indicates no changes compared to normal joints ; 1 indicates thickening of the synovial lining and some influx of inflammatory cells ; 2 indicates thickening of the synovial lining and intermediate influx of inflammatory cells ; 3 indicates profound thickening of the synovial lining ( more than four cell layers ) and maximal observed influx of inflammatory cells . briefly , sections were deparaffinized , rinsed with pbs , and then incubated in 3% h2o2 in methanol for 30  min at room temperature to deactivate endogenous peroxidase . after blocking with normal rabbit serum ( vector laboratories , ca ) for 30  min , sections were incubated with rat anti - mouse monoclonal f4/80 antibody ( 1  :  2000 dilution ) at 4c overnight . after extensive washing with pbs 0.1% tween 20 , signals were visualized by the vectastain abc kit ( vector laboratories , ca ) . the degree of f4/80-positive macrophage infiltration into synovial membrane , which represents the severity of synovitis , was scored as described by willis et al . the scores were indicated as the total of four different areas of the synovial membrane ( superficial layer and deep layer of suprapatellar or infrapatellar region ) as described in figure 2(f ) . f4/80 score ( willis et al . , joi , 2012 ) 
 0 indicates no staining ;  1 indicates minimal to few faintly positive cells ;  2 indicates scattered single positive cells ;  3 indicates clusters of two or more positive cells ;  4 indicates larger clusters of positive cells , multifocal to coalescing . 0 indicates no staining ; 1 indicates minimal to few faintly positive cells ; 2 indicates scattered single positive cells ; 3 indicates clusters of two or more positive cells ; 4 indicates larger clusters of positive cells , multifocal to coalescing . statistical analyses were performed using mann - whitney u test and p values less than 0.05 were considered significant . since it was reported that intra - articular injection of carrageenan initiates a localized synovial inflammatory response that causes articular cartilage degeneration , we examined the anti - inflammatory effects of follistatin at day 3 after carrageenan injection . as shown in figure 2(a ) , single intra - articular injection of carrageenan induced synovial hyperplasia and increased cellularity in the synovial membrane at day 3 . blom 's synovitis score indicated that carrageenan significantly enhanced synovitis ( figure 2(c ) , middle panel ) . interestingly , preinjection of follistatin significantly reduced infiltration of inflammatory cells into the synovial membrane and alleviated synovitis , although fibrotic changes in the synovial membrane were still observed ( figure 2(a ) ) . these acute inflammatory responses induced by carrageenan injection were almost quenched at 14 days after carrageenan challenge ( figures 2(b ) and 2(c ) lower panels ) . to further elucidate the physiological roles of follistatin in alleviation of synovial inflammation , we analyzed macrophage infiltration after carrageenan challenge . as shown in figures 2(d ) and 2(e ) , significant numbers of f4/80-positive macrophages migrated into both superficial and deep regions of synovial membrane at day 3 after carrageenan challenge . in contrast , preincubation with follistatin greatly reduced macrophage accumulation in both regions of the synovial membrane . these effects were specific to the 3rd day after carrageenan challenge as we did not observe any significant alteration in macrophage infiltration between control , ca , and ca + flt groups at day 14 ( figures 2(d)2(f ) ) . as reported previously , single intra - articular injection of carrageenan reduced proteoglycan content in both femur and tibia articular cartilage at day 3 , indicated by the reduced dyeability of safranin - o ( figure 3(a ) ) in comparison to control and ca groups . modified coles score indicated that intra - articular injection of carrageenan significantly induced articular cartilage degeneration at this time point ( figure 3(b ) ) . however , we did not observe any significant alterations in the articular surface structures such as fissure formation , increased cellularity , and hypertrophic differentiation of articular chondrocytes at this stage ( figure 3(a ) ) . interestingly , preinjection of follistatin preserved safranin - o dyeability and improved the modified coles score almost to the same level as that of control ( figures 3(a ) and 3(b ) ) . follistatin itself does not seem to have effects on proteoglycan metabolism since no apparent histological alteration was observed between the control and flt groups ( figure 3(a ) ) . at day 14 , after carrageenan challenge , safranin - o dyeability was reversed to the control levels in the ca group and we did not observe any significant alteration in the articular surface structure between control , ca , and ca + flt groups ( figures 4(a ) and 4(b ) ) . in this study , we showed that follistatin significantly alleviated carrageenan - induced synovial inflammation and articular cartilage degeneration . detailed histological analyses indicated that preinjection of follistatin significantly reduced macrophage infiltration into the synovial membrane and protected articular cartilage from proteoglycan erosion at day 3 after carrageenan injection . our data suggest the potential for follistatin as a novel anti - inflammatory and anticatabolic drug for oa treatment . carrageenan is a sulphated mucopolysaccharide extracted from the seaweeds chondrus spp . and gigartina spp . , it is known for its remarkable capacity to stimulate local inflammation dominated by intense macrophage aggregation and by fibroblastic proliferation [ 25 , 26 ] . molecular action of carrageenan is mediated by members of the family of innate immune receptors , such as tlr2 and tlr4 ( toll - like receptors 2 and 4 ) . tlr2 and tlr4 are expressed in a broad range of mammalian cells including epithelial / endothelial cells and immune - related cells such as monocytes , macrophages , and t - cells [ 27 , 28 ] . upon recognition of their cognate ligands , tlrs form homo- or heterodimers and activate intracellular signals through recruitment of different combinations of tir - domain - containing adaptor proteins . the major components of these adaptor molecules include myd88 ( myeloid differentiation factor 88 ) and mal / tirap ( myd88-adaptor - like ) . the interaction between myd88 and iraks ( il1 receptor associated kinase ) induces the formation of macromolecular complexes that ultimately impinge on tak1 ( tgf activated kinase 1 ) and lead to activation of nfb , which has pivotal roles in the induction of various cytokines and chemokines such as il1 , il6 , and il8 in various cell types . carrageenan - induced arthritis is a well - established experimental animal model to investigate inflammation - mediated articular cartilage degradation in rodents and rabbits . it is reported that single intra - articular injection of carrageenan initiates a localized synovial inflammatory response , which is indicated by synovial hyperplasia and macrophage accumulation , and decreases both the proteoglycan content and the rate of de novo proteoglycan synthesis in the articular cartilage . although it is not fully elucidated , the tlr - interleukin axis is likely to be involved in the process of joint inflammation and cartilage degeneration induced by carrageenan , since synovial cells also express both tlr2 and tlr4 and their expression levels are increased in ra ( rheumatoid arthritis ) synovium . activin levels are reported to surge in the very early stage during the inflammatory response , which is almost the same time or slightly prior to the induction of proinflammatory cytokines tnf and il6 [ 13 , 14 ] . although there is no evidence that proves the direct involvement of activins on the upregulation of proinflammatory cytokines and chemokines induced by tlr signals , we consider that it is likely because previous studies reported that activin a release after lps exposure was significantly suppressed in mice lacking myd88 and that follistatin significantly inhibited the surge of serums tnf , il1 , and il6 after lps challenge in mice . we speculate that our data correlates with these findings since our synovitis score was significantly improved in the ca + flt group at day 3 when compared to that in the ca group ( figure 2(c ) ) . interestingly , synovial fibrosis was still observed in the ca + flt group ( figure 2(a ) ) while f4/80-positive macrophage infiltration into the synovial membrane was significantly inhibited at day 3 ( figure 2(d ) ) . synovial fibrosis is a major contributor to joint stiffness in oa , and tgf is considered to play central roles in the onset and persistence of this process . reported that the tlr4-myd88-nfb dependent pathway enhances tgf signaling through downregulation of bambi ( bmp and activin membrane - bound inhibitor ) , a pseudoreceptor for tgf/bmp family molecules . it is still unclear if activin signal negatively contributes to the process of synovial fibrosis . our data suggest that follistatin may specifically interfere with the tlr4-myd88-nfb dependent pathway of proinflammatory cytokine expressions but may not be involved in the tlr - bambi - tgf pathway . in this study , we also showed that follistatin significantly inhibited proteoglycan erosion induced by carrageenan . accumulating data indicate that cartilage matrix degrading enzymes such as adamtss and mmps are regulated by proinflammatory cytokines such as il1. tian et al . reported that adamts4 expression in nucleus pulposus ( np ) cells was enhanced by il1 through mapk and nfb activation . kataoka et al . reported that adamts4 expression in synoviocytes was enhanced by il1 and this upregulation was reversed by synthetic inhibitors for p38mapk ( sb239063 ) and jnk ( sp600125 ) . reported that il1 induced mmp1 , mmp2 , mmp9 , mmp13 , and adamts5 in human chondrocytes . our data suggest that chondroprotective effects of follistatin may also be through the downregulation of tlr - myd88-nfb - il1 signaling pathways . molecular mechanisms by which activins modulate tlr - myd88-nfb signal pathways are still unclear . several recent publications reported negative cross talk between tgf/bmp and tlr - myd88-nfb signaling pathways . reported that k48-linked polyubiquitination and degradation of myd88 were induced by tgf , which was mediated by smad6-specific recruitment of smurf e3 ligases . huang et al . reported that tlr4 dependent nfb activation inhibited bmp2-induced phosphorylation of smad1/5/8 . based on our study , we speculate that signals mediated by activins may positively regulate tlr - myd88-nfb dependent proinflammatory cytokine expression in the joint . further studies are required to elucidate the molecular mechanisms by which activins / follistatin signals regulate joint inflammation and articular cartilage metabolism . in summary , we demonstrated that follistatin significantly alleviated synovial inflammation and articular cartilage degeneration induced by carrageenan . we expect that follistatin or other extracellular activin blocking reagents such as actriia - fc and actriib - fc can be utilized as potent inhibitors for inflammation - mediated articular cartilage degeneration .
activins are proinflammatory cytokines which belong to the tgf superfamily . 
 follistatin is an extracellular decoy receptor for activins . 
 since both activins and follistatin are expressed in articular cartilage , we hypothesized that activin - follistatin signaling participates in the process of joint inflammation and cartilage degeneration . to test this hypothesis , we examined the effects of follistatin in a carrageenan - induced mouse arthritis model . 
 synovitis induced by intra - articular injection of carrageenan was significantly alleviated by preinjection with follistatin . 
 macrophage infiltration into the synovial membrane was significantly reduced in the presence of follistatin . 
 in addition , follistatin inhibited proteoglycan erosion induced by carrageenan in articular cartilage . 
 these data indicate that activin - follistatin signaling is involved in joint inflammation and cartilage homeostasis . 
 our data suggest that follistatin can be a new therapeutic target for inflammation - induced articular cartilage degeneration .