interleukin-29 ( il-29 ) is the main cytokine among three members of interferon lambda family ( ifn-s ) , also known as type iii ifns , including ifn-1 , ifn-2 , and ifn-3 ( or il-29 , il-28a , and il-28b , resp . ) .
the activity of il-29 is determined in part by the distribution and expression of its specific il-28 receptor alpha chain ( il-28ra ) .
il-28ra is widely expressed by ranges of cells including epithelial cells , hepatocytes , fibroblasts [ 1 , 2 ] , and immune cells , such as plasmacytoid dcs , macrophages , th17 cells , and nk cells [ 36 ]
. the most studied biological role of ifn-s is its antiviral activity , but recent investigation starts to uncover a unique role of ifn-s in and beyond innate antiviral immunity [ 4 , 7 ] .
il-29 can induce antiproliferative and antitumor properties [ 8 , 9 ] , inhibit il-13 release in t cells , and stimulate il-6 , il-8 , and il-10 production in macrophages   and il-4 and il-13 in mast cells .
a recent study showed that il-29 is able to indirectly affect nk cells by regulating il-12 in macrophages .
moreover , our recent studies have demonstrated that il-29 levels are higher in peripheral blood mononuclear cells ( pbmcs ) and serum and synovium from rheumatoid arthritis ( ra ) patients when compared to healthy individuals .
recombinant il-29 enhances il-6 , il-8 , and matrix - metalloproteinases ( mmps ) production in ra fibroblasts and promotes ra inflammation .
il-29 has also been shown to contribute to other immune diseases , such as systemic lupus erythematosus ( sle ) [ 11 , 12 ] , asthma , and psoriasis .
however , the role of il-29 in inflammatory diseases remains largely unexplored and whether il-29 is involved in the pathogenesis of osteoarthritis ( oa ) is unclear .
oa is one of the most common disorders and has traditionally been classified as a noninflammatory arthritis ; however , increasing evidence shows that synovitis and the immune system are also active players in oa development and progression . for examples , oa synovial fibroblasts ( fls )
contribute to oa pathogenesis by producing inflammatory cytokines such as tumor necrosis factor- ( tnf- ) , il-1 , and chondrolytic mediators such as mmps ; fls - derived il-1 and tnf- induce cartilage degradation .
in addition , fls can mediate the innate immune response by responding to both inflammatory cytokines and toll - like receptor ( tlr ) ligands .
we have shown that il-29 enhanced ra inflammation and mediated tlr activation in ra fls [ 1 , 2 ] .
oa and ra share some similarities in pathogenesis , such as chronic inflammation , synovial hyperplasia , articular destruction , and abnormal immune response .
, we examined the potential role of il-29 as a proinflammatory cytokine in oa disease .
this study , for the first time , showed that il-29 was higher in blood and synovium from oa patients when compared with healthy controls ( hc ) .
recombinant il-29 increased the expression of proinflammatory cytokine in oa fls in vitro and promoted cartilage degradation in coculture of oa fls and cartilage explant ex vivo .
finally , our studies revealed that the effect of il-29 on oa fls was likely mediated by nf-b and mapk signaling pathway .
our present data support the hypothesis that il-29 may contribute to synovial inflammation and cartilage degradation in oa .
recombinant human il-29 and il-1 were purchased from peprotech ( rocky hill , nj , usa ) ; rabbit anti - human il-29/il-28r polyclonal antibody and mouse anti - human cd68 monoclonal antibody were purchased from abcam ( cambridge , ma , usa ) ; mouse anti - human fibroblast growth factor - basic ( fgf-2 ) monoclonal antibody was purchased from millipore ( billerica , ma , usa ) ; pe anti - human il-28r was purchased from biolegend ( san diego , ca , usa ) ; donkey anti - rabbit igg - r and goat anti - rabbit igg / tritc were purchased from santa cruz biotechnology ( santa cruz , ca , usa ) ; dylight488-conjugated affinipure donkey anti - mouse igg , peroxidase - conjugated sheep anti - rabbit secondary antibody , and peroxidase - conjugated sheep anti - mouse secondary antibody were purchased from jackson immunoresearch ( west grove , pa , usa ) ; human il-29 antibody was purchased from r&d systems ( minneapolis , mn , usa ) ; human il-29 enzyme - linked immunosorbent assay ( elisa ) reagent kits were purchased from adlitteram diagnostic laboratories ( san diego , ca , usa ) ; mmp-3 elisa kit was purchased from uscn life science inc .
( wuhan , china ) ; safranin o staining kit was purchased from sciencell research laboratories ( carlsbad , ca , usa ) ; primerscriptrt reagent kit was purchased from takara ( dalian , china ) ; power sybr green pcr master mix was purchased from applied biosystems ( carlsbad , ca , usa ) ; phospho - stat antibody sampler kit , phospho - c - jun n - terminal kinases ( jnk ) , phosphoextracellular signal - regulated kinases ( erk ) , phospho - p38 , phospho - akt , phospho - p65 , phospho - ib , and -actin antibody were purchased from cell signaling technology ( beverly , ma , usa ) ; tissue culture reagents including dulbecco 's modified eagle 's medium ( dmem ) and fetal bovine serum ( fbs ) were purchased from gibco ( carlsbad , ca , usa ) .
synovium and cartilage samples were harvested as surgical waste from 10 patients with end - stage symptomatic knee oa at the time of surgery for total knee replacement .
blood samples were collected from 30  oa patients and 30  hc . the general characteristics of both patients and controls subjects are summarized in tables 1 and 2 .
oa diagnosis was determined by clinician assessment according to the american college of rheumatology ( acr ) criteria .
usage of human tissues was approved by the ethical committee of the first affiliated hospital of nanjing medical university , and informed consent was obtained from all patients .
blood samples were collected from peripheral veins and were processed within 1  h to provide serum for elisa analysis and pbmc for rna extract .
knee synovial tissues ( 5  oa and 3  hc samples ) were used for immunohistology analysis , and the remaining 5  oa synovial samples were prepared for cell culture experiments .
synovial tissue was minced and digested with 1% collagenase ii at 37c for 4  h. oa fls were cultured in dmem medium supplemented with 10% fetal bovine serum ( fbs ) , 100  u / ml penicillin , and 100  g / ml streptomycin at 37c in a humidified atmosphere of 5% co2 in air .
purity of oa fls was determined by flow cytometry stained with antifibroblast marker ( fgf-2 ) .
cells were used for further experiments if > 95% cells were positive for fibroblast marker .
il-28r expression on cell surface was measured using flow cytometric analysis . in brief , oa fls ( 1  10 ) were harvested , blocked with 1% bovine serum albumin ( bsa ) for 30  min , and then stained with pe - conjugated il-28r for 25  min at room temperature in the dark . after washing 3 times with pbs , positive cells were determined using a bd facscalibur flow cytometer and cellquest software ( bd biosciences ) .
cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) assay following our published procedures .
briefly , oa fls ( 5  10 ) were incubated in a 96-well , flat - bottomed culture plate in a final volume of 200  l / well culture medium with il-29 ( 1 , 10 , and 100  ng / ml ) for 72  h in a humidified atmosphere ( 37c at 5% co2 ) .
next , 20  l of cell proliferation reagent wst-1 was added to each well and incubated for a further 4 hours .
after incubation , the absorbance was measured at 450  nm using an elisa plate reader ( biotek , winooski , vt , usa ) .
oa fls were stimulated with il-29 ( 1 , 10 , and 100  ng / ml ) in the presence or absence of il-29 blocking antibody ( 1000 , 2000 , and 3000  ng / ml ) for 48  h. after incubation , cells were collected for gene expression analysis by real - time pcr . in another set ,
oa fls were treated with 100  ng / ml il-29 for 0  min , 20  min , 40  min , and 60  min , respectively .
oa cartilages were obtained from knee joint tissue and submerged in a solution with betadine and pbs ( 1  :  3 ) for 10  min and then rinsed with pbs with no any trace of betadine .
after freeze - thaw three times at 80c to kill any live cell within the tissue , cartilages were stored at 20c and ready for use .
oa fls were cultured in 24-well plate until being 90% confluent and changed to fresh dmem medium .
then , the cells were treated with il-29 ( 1 , 10 , and 100  ng / ml ) and il-1 ( 20  ng / ml , positive control ) . thawed cartilage tissues were diced to similar size and placed to each well including oa fls and il-29 .
cartilage , oa fls , and il-29 or il-1 were coincubated in dmem medium at 37c in a humidified incubator ( 5% co2/air ) . after 72  h incubation
rna was transcribed into cdna in a 20  l mixture containing 1  g of total rna and primescript rt master mix ( takara ) according to the manufacturer 's instructions . quantitative real - time pcr was performed on applied biosystems 7900  ht instrument ( applied biosystems , ca , usa ) following our published procedures .
human il-29 and mmp-3 in serum or culture supernatants were measured by elisa according to the manufacturer 's instructions .
paraffin - embedded synovial tissues were sectioned , dewaxed , rehydrated , and stained following our published procedures .
each slide was evaluated by one of the authors under the microscopy ( nikon , japan ) .
tissue sections were scored for staining of the lining on a 0 to 5 scale as reported .
for each section , the number of positively stained cells was counted in 20 fields .
after sectioning and deparaffin , the slides were stained with the safranin o staining kit to examine the cartilage loss according to the manufacturer 's instructions . in brief , the slides were stained in 0.1% fast green solution for 10  min , rinsed in 1% acetic acid for 15  sec , and stained in 0.1% safranin o staining solution for 30  min .
after dehydration in ethanol and clearing in xylene , the slides were mounted and observed under microscope ( nikon , japan ) .
scored data were pooled , and the mean  sem was calculated . for cells immunofluorescence staining , oa fls were seeded on cover slips and stained following our published procedures . briefly , oa fls were fixed in 4% paraformaldehyde for 10  min and permeabilized with 0.3% triton x-100 in pbs for 5  min .
then , the cells were incubated with anti - human il-28r antibody overnight at 4c .
after washing , the cells were further incubated with goat anti - rabbit igg / tritc for 1  h in room temperature .
finally , the cells were washed and incubated with dapi staining solution for 1 - 2  min and analyzed by fluorescence microscopy ( nikon , japan ) .
il-28r was stained in red and nuclei were stained in blue . for tissue double immunofluorescence labeling
, sections were incubated with a mixture of primary antibodies ( rabbit anti - il-29 pab , mouse anti - cd68 , or fgf-2 mab ) at 4c overnight .
after washing , the slides were further incubated with a mixture of donkey anti - rabbit igg - r and dylight488-conjugated donkey anti - mouse igg for 1  h. finally , the slides were washed and incubated with dapi staining solution for 35  min .
images were acquired and processed digitally under the fluorescence microscopy ( olympus , japan ) .
the contained positive cells were stained in yellow and nuclei were stained in blue . for western blotting assay ,
cellular proteins were resolved by 8%10% sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) and were transferred to polyvinylidene fluoride membranes ( millipore , bedford , ma , usa ) .
nonspecific interactions were blocked with 5% skim milk for 2  h and were then probed with phospho - stat 1/2/3/4/5/6 , phospho - akt , phospho - p38 , phospho - erk , phospho - jnk , phospho - p65 , and phospho - ib .
the signals were visualized with super signal west dura chemiluminescent detection reagents following the manufacturer 's directions ( thermo fisher scientific inc . , rockford , usa ) , and protein bands were scanned and quantified with the gel - pro analyzer software ( bio - rad , ca , usa ) .
the relative quantification of target proteins was calculated by comparison of the bands density levels between samples with image j software .
all data were expressed as mean  sem and analyzed with graphpad prism 6 software ( graphpad software , la jolla , ca , usa ) .
differences between two groups were performed with student 's t - test for parametric data and mann - whitney u test for nonparametric data . a p value of < 0.05 was considered significant .
to investigate whether il-29 was involved in the pathogenesis of oa , we first examined the expression of il-29 mrna and its receptor il-28ra in pbmc .
real - time pcr analysis revealed that mrna expression of il-29 and il-28ra was significantly higher in oa pbmcs when compared to hc ( p = 0.0012 and 0.0017 , resp . ; figures 1(a ) and 1(b ) ) . in serum
, elisa analysis showed that il-29 was significantly higher in oa ( 25.26  3.34  pg / ml ) than in hc ( 5.61  0.71  pg / ml , p < 0.0001 ; figure 1(c ) ) .
next , we assessed the expression and localization of il-29 and il-28ra in synovial tissues from 5  oa patients and 3  hc with immunohistochemical analysis .
as shown in figure 2 , il-29 and il-28ra are strongly expressed in the lining layer of oa synovial tissues ( figure 2(a ) ) , and semiquantitative analysis indicated that il-29 and il-28ra were significantly increased in oa synovium compared to hc ( p = 0.0045 ) ( figure 2(b ) ) . because macrophages and fls are the major effector cells for inflammation in the infiltrated oa synovium , we next examined whether macrophages and fls can produce il-29 using double immunofluorescence staining .
the specific antibodies against cd68 and fgf-2 served as markers for macrophages and fibroblasts , respectively .
as shown in figure 2(c ) , il-29-positive cells were stained in red whereas cd68- or fgf-2-positive cells were stained in green , and positive cells for containing il-29 with cd68 or fgf-2 were shown in yellow . as expected , il-29 was strongly expressed in macrophages and fibroblasts in oa synovium , suggesting that these cells might be the important cellular sources of il-29 in oa synovium .
immunofluorescence staining showed that il-28ra was expressed in oa fls ( figures 3(a ) and 3(b ) ) , implying that oa fls may be the important target of il-29 .
il-29 at 1 , 10 , and 100  ng / ml did not affect the viability of oa fls after 72  h treatment ( figure 3(c ) ) .
as shown in figure 4 , il-29 induced a dose - dependent upregulation of il-1 , il-6 , il-8 , and mmp-3 mrna in oa fls following 48  h incubation .
furthermore , the action of il-29 on these cytokine expressions in oa fls was abolished by addition of il-29 blocking antibody at the concentration of 2000 and 3000  ng / ml .
il-29 can stimulate proinflammatory cytokines expression in oa fls as shown in figure 4 , and chronic inflammation is a major driver of ongoing cartilage damage and joint degeneration in oa pathogenesis .
therefore , we chose a coculture model of oa fls with ex vivo cartilage explant to examine the influence of il-29 on cartilage degradation .
after 72  h culture , safranin o / fast green staining of the cartilage in the coculture showed that glycosaminoglycan ( gag ) depletion ( mean  sem ) was significantly greater in il-29 ( 100  ng / ml ) ( 477.4  22.0  m ) or il-1 ( 20  ng / ml ) ( 717.4  20.9 
m ) treated conditions than that in the medium alone ( 154.4  7.1  m ) , indicated by the less intense red staining for safranin o and gag depletion deeper ( figures 5(a ) and 5(b ) ) . in oa
, cartilage damage is predominantly mediated by mmps and its specific inhibitors , the tissue inhibitors of metalloproteinases family ( timps ) , most notably by mmp-1 , mmp-3 , mmp-13 , and timp-1 .
we examined the ratio of mmp-1 , mmp-2 , mmp-3 , and mmp-13 to timp-1 in the oa fls after 72  h coculture and found that il-29 could significantly upregulate mmp-1/timp-1 , mmp-2/timp-1 , mmp-3/timp-1 , and mmp-13/timp-1 ratio at a dose - dependent manner in oa fls .
we also measured mmp-3 levels in supernatant at 72  h incubation in this experiment .
consistent with our finding in oa fls monocultures , there were higher levels of mmp-3 in oa fls / cartilage coculture system in response to il-29 at 100  ng / ml when compared to nontreatment control ( figure 5(c ) ) , similar to that stimulated by il-1 ( 20  ng / ml ) .
these data imply that il-29 promotes cartilage degradation by stimulating protease production in oa fls .
oa fls were stimulated with il-29 and the activation of downstream signal transduction pathways including canonical jak - stat and noncanonical mapk , akt , and nf-b pathways was evaluated ( figure 6 ) .
following stimulation of oa fls for 20  min , 40  min , and 60  min with il-29 ( 100  ng / ml ) , phosphorylation of stat 1 ( tyr701 ) and stat 5 ( tyr694 ) ; jnk , erk , and p38 ; and p65 and ib was significantly increased when compared to nontreatment controls . no change in phosphorylation of stats 2 ( tyr690 ) ,
3 ( tyr705 ) , and 6 ( tyr641 ) and akt was observed in response to il-29 .
phosphorylation of stat 3 ( ser727 ) and stat 4 ( tyr693 ) was not detectable in oa fls .
oa has traditionally been classified as a noninflammatory arthritis ; however , the accumulating evidences demonstrate that joint inflammation and synovitis play a critical role in the pathogenesis of oa .
our study for the first time showed that il-29 , a novel member of type iii interferon family , ( i ) was upregulated in blood and synovial tissues in patients with oa ; ( ii ) enhanced inflammation of oa fls and promoted cartilage degradation ; and ( iii ) activated mapk and nf-b signaling pathway . in the first case ,
we demonstrate an essential role of il-29 in inflammation in oa fls . we found that il-29 was abnormally elevated in pbmc , serum , synovial fluid , and synovium in oa patients . within the joint , il-29 and its receptor
were located at fls and macrophages , two main cell types in the inflamed synovium responsible for the inflammation and cartilage and bone damage in the joint .
abnormal il-29 was also found in ra , sle , gastric cancer , or other diseases . similar to ra , in this study , we did not find a significant correlation between il-29 in serum and oa disease activity ( data not shown ) .
the lack of a significant association may be due to the limited sample size and most of the patients having end - stage symptom of knee oa .
thus , further prospective studies including a larger sample size and oa patients in early and late disease are needed to determine whether il-29 can serve as a biomarker for systemic inflammation and oa disease activity .
we have reported that il-29 contributed to synovial inflammation by stimulating production of proinflammatory cytokines in ra fls .
the proinflammatory effects of il-29 have also been shown on pbmc by upregulation of ifn - inducible protein-10 ( ip-10 ) , monokine induced by ifn- ( mig ) , ifn--inducible t cell  chemoattractant ( i - tac / cxcl11 ) , and il-8 [ 11 , 18 ] and on t cells by downregulating th2 polarization and cytokine production [ 15 , 19 , 20 ] .
soluble inflammatory factors such as cytokines are central to most inflammatory processes , and several cytokines have been implicated in oa pathogenesis including il-6 and il-8 , tnf- , and il-1. indeed , our data showed that il-29 stimulated inflammatory cytokines il-1 , il-6 , and il-8 and production in oa fls , indicating that il-29 may contribute to oa inflammation .
recent epidemiological studies on large number of oa patients suggested that an inflammatory synovium / synovitis was linked to increased cartilage damage and pain .
fls , the major cellular constituent of the synovial membrane , are the main contributors to the inflammation and cartilage degrading mmp overproduction within the arthritic joint [ 11 , 12 , 22 ] .
proinflammatory cytokines derived from fls including il-6 and il-8 , tnf- , and il-1 could increase the production of mmps leading to cartilage damage .
our data also showed that mmp-3 mrna was significantly increased in fls upon il-29 treatment ; hence , we further address the interaction between il-29 , inflammation , mmp production , and cartilage degradation .
the second novel finding of this study is that il-29 plays a critical role in cartilage degradation in oa .
we used a coculture system to establish a direct link between il-29 and cartilage degradation . in this model ,
fls were cultured with oa cartilage explant to model inflammation - induced mmp production and cartilage degradation .
our data showed that il-29 enhanced mmps / timp-1 ratio in oa fls and then resulted in marked increased gag depletion in cartilage . taken together , our data suggest an important role for il-29 in regulating cartilage degradation by driving cytokine and mmp production in oa fls .
il-29 is thus capable of modulating one of the most important processes driving tissue degeneration in oa pathogenesis .
it has been suggested that il-29 elicits signal transduction via activation of the canonical jak - stat pathway and other alternative pathways including akt and the mitogen - activated protein kinase ( mapk ) in a panel of cells [ 2325 ] .
however , the ability of il-29 to trigger the pathways could be cell - type specific or altered in cells .
the present study is the first to report the signal transduction pathways in response to il-29 in oa fls . in these cells
phosphorylated stats form various homodimers and heterodimers , translocate to the nucleus , and induced il-29-specific biological activities , such as antiviral protection , antiproliferative response , and antitumor activities . and
the most prominent biological function of il-29 resides in their ability to induce antiviral state in cells ; thus , the canonical jak - stat signal pathway is the major pathway involved in il-29-induced antiviral action , whereas the effect of il-29 on proinflammatory cytokines production in oa fls may be one key point for studying oa pathogenesis .
it is therefore hypothesized that alternative signaling pathways could be more important in oa fls after il-29 stimulation .
indeed , il-29 induced strong nf-b and mapk ( p - jnk , p - erk , and p38 ) signal transduction in oa fls .
however , whether these pathways are involved in il-29 mediated effects on oa fls has to be investigated in the further study with the inhibitors of these pathways .
recent studies suggested that inhibitors of the above two pathways have received more attention in animal model studies and clinical trials in oa patients .
collectively , our present data combined with these reports suggested that il-29-mediated inflammation was the pivotal function of il-29 in oa pathogenesis and inhibition of il-29 would be a potential therapeutic target in oa disease .
in summary , this study provides the first evidence that il-29 is dysregulated in oa patients and may contribute to synovial inflammation and cartilage degeneration during oa pathogenesis by production of proinflammatory cytokine .
further studies of the immunoregulatory effects of il-29 and its underlying molecular mechanism are warranted to understand the pathological role of il-29 in oa disease .
 we have recently shown that il-29 was an important proinflammatory cytokine in pathogenesis of rheumatoid arthritis ( ra ) . 
 inflammation also contributes to the pathogenesis of osteoarthritis ( oa ) . 
 the aim of this study was to investigate the effect and mechanism of il-29 on cytokine production and cartilage degradation in oa . 
 the mrna levels of il-29 and its specific receptor il-28ra in peripheral blood mononuclear cells ( pbmcs ) were significantly increased in oa patients when compared to healthy controls ( hc ) . in the serum , 
 il-29 protein levels were higher in oa patients than those in hc . 
 immunohistochemistry revealed that both il-29 and il-28ra were dramatically elevated in oa synovium compared to hc ; synovial fibroblasts ( fls ) and macrophages were the main il-29-producing cells in oa synovium . 
 furthermore , recombinant il-29 augmented the mrna expression of il-1 , il-6 , il-8 , and matrix - metalloproteinase-3 ( mmp-3 ) in oa fls and increased cartilage degradation when ex vivo oa cartilage explant was coincubated with oa fls . 
 finally , in oa fls , il-29 dominantly activated mapk and nuclear factor-b ( nf-b ) , but not jak - stat and akt signaling pathway as examined by western blot . in conclusion , il-29 stimulates inflammation and cartilage degradation by oa fls , indicating that this cytokine is likely involved in the pathogenesis of oa . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions