HMGB1-LPS Complex Promotes The Transformaiton Of Synovial Fibroblasts To A Rheumatoid Arthritis Synovial Fibroblasts-like Phenotype

Object：To investigate the molecular mechanisms of HMGB1involved in theprogress of RA and induced the formation of RASFs，as well as the key signalingpathways.Materials and methods：In order to further study the effects of HMGB-LPS complexon the pathogenesis of rheumatoid arthritis and possible signaling pathway，we performedexperiments in vivo and in vitro to explore the role of HMGB1-LPS complex in thepathogenesis of rheumatoid arthritis. Experiment was divided into two parts：The first part： In current study, osteoarthritis synovial fibroblasts (OASFs) isolatedfrom tissue obtained during reconstructive surgery and cocultured with HMGB1-LPScomplex in vitro for5-8generations to induce the transformation of normal SFs to RA-likeSF (t-OASFs), RASFs as a positive control and OASFs as a negative control.To evaluatethe changes of cell cycle in t-OASFs, OASFs and RASFs,the cells were cultured withDMEM containing10%heat-activated FBS for24h, then media were replaced withDMEM containing10%heat-activated FBS and cultured for another24h. Then cells werecollected and stained with propidium iodide and the cell cycle and cell proliferation wereanalyzed by flow cytometry. To investigate the roles of HMGB1-LPS complex in theability of cells to resist invader and reduce apoptosis,the cells were exposed toapoptosis-inducing agent for8hours and then cell apoptosis was analyzed by flowcytometry, pro-survive protein(Bcl-2) and pro-apoptosis protein(Bax) were detected byWestern blotting.In order to investigate t-OASFs’ ability of inducing autophagy undercertain stimuli, Cells were grown on polylysine-treated coverslips in6-wells plate andcultured with DMEM containing10%heat-activated FBS for24h, then the media wasreplaced with PBS for4h to induce autophagy,the LC3II protein expression was observedwith a confocal microscopy and Western blotting. In order to evaluate the expression ofHMGB1-LPS complex associated receptors,chemokine receptors and adhesion moleculesin/on cells of three groups,the cells were stained with fluorescent-labeled antibodies andanalyzed by flow cytometry. To evaluate the expression of proinflammatory cytokines andMMPs of t-OASFs, OASFs and RASFs, the mRNA expression of TNF-α, IL-1β,IL-6MMP-3and MMP-13in cells were measured with Real-Time PCR, and the proteinexpression levels of TNF-α, IL-1β,IL-6MMP-3and MMP-13in the culture supernatantsof cells were detected with ELISA. To clarify the downstream signaling pathways ofHMGB1-LPS complex inducing transformation of SFs to RASFs, Multi target PathScan of protein assay kit used to screen the activated downstream signaling pathways of threegroups of cells.The second part:：According to the reference (Nat Med2009;15:1414-1420),t-OASFs induced by HMGB1-LPS complex, RASFs and OASFs were packaged withnormal cartilage,then implanted in SCID mice abdominal subcutaneous.After twomonths,the implanted cartilages were removed, fixed, embedded and sectioned.Then thecartilage slices were stained by HE, Alcian blue,Masson special staining andimmunohistochemistry analysis.The pathological score according to the staining resultswere compared among the three groups.Result:1、Compared with OASF cells,t-OASF cells in S/G2phase were increased and cellproliferation were accelerated.Cells were cultured with DMEM containing1%heat-activated FBS for24h, thenmedium was replaced with DMEM containing10%heat-activated FBS and cultured foranother24h. The cells were collected,stained with propidium iodide and then analyzed byflow cytometry. The results showed that there were16.83%in S phase and only0.07%inG2phase in OASF cells; t-OASF cells in S phase was30.82%, in G2phase was26.96%;RASF cells in S phase was9.17%, in G2phase cells was22.28%. Compared to OASFcells, more t-OASF cells had across the G1/G2checkpoint into S/G2phase, suggestingaccelerated proliferation occurred in t-OASF cells.2、The apoptosis induced by TNF-α of t-OASF cells were significantly decreased, andthe expression of pro-survive protein(BCL-2) was up-regulated, while pro-apoptosisprotein(Bax) was down-regulated.Reports in some literature, the RASFs can reduce the apoptosis sensitivity, therebyimproving survival. In order to evluate the apoptosis sensitivity of t-OASF cells, Cellswere exposed to TNF-α for8h and then the apoptosis was analyzed by flow cytometry.Theresults showed the proportion of apoptotic cells was6.57%, and that of necrotic cells was8.12%in OASF group. However，the proportion of apoptotic cells in t-OASF group andRASF group were3.16%and2.14%,that of necrotic cells were2.74%and1.08%respectively. Compared to OASF cells,the t-OASF cells were fewer apoptotic and necroticcells.The results of Western blotting also showed that pro-survive protein(BCL-2) wasup-regulated, while pro-apoptosis protein(Bax) was down-regulated in t-OASF cells andRASF cells.The experimental results of t-OASF group were similar with that of RASF group, suggesting the balance machanism of apoptosis-survival in the face of harmfulstimulation might be damaged,the survival rate of t-OASF cells was increased.3、 More autophagy punctate fluorescent spots were observed in t-OASF cellsThe studies confirmed that RASFs in hunger or rapamycin treatment, could increaseautophagy to evasion of apoptosis.In order to investigate the ability of inducing autophagyunder certain stimuli in t-OASF cells, Cells were grown on polylysine-treated coverslips in6-well plate and cultured with DMEM containing10%heat-activated FBS for24h to60%confluence, then the media were replaced with PBS for4hours to induce autophagy. TheLC3II expression was detected with a confocal microscopy and Western blotting. Byconfocal microscopy revealed the flourscence spots LC3Ⅱ in t-OASF cells were similarto RASF cell, significantly more than OASF cells.The Western blotting detection alsoconfirmed the the expression of LC3Ⅱin t-OASF cells and RASF cells were higher thanthat of in OASF cells.These results sugessted autophage incresed in t-OASF leading toreduction in apoptosis.4、HMGB1-LPS complex associated receptors were up-regulated in t-OASFThe HMGB1is combined by corresponding receptors of cell, mainly TLR2, TLR4and RAGE, then activated a series of downstream signaling pathways to achieve itsbiological activity. Our group preliminary studies have found that: the expression of TLR2and TLR4on RASFs surface were raised.In order to evaluate the expression ofHMGB1-LPS complex associated receptors on cells of three groups, cells were stainedwith fluorescent-labeled antibodies and analyzed by flow cytometry.The expressions ofTLR2,TLR4and RAGE in/on t-OASF cells(16.1%,26.1%and9.0%respectively) werehigher than OASF cells(6.7%,18.4%and2.6%respectively).Recently reseachers havefound that the TLR4on cell surface was sheding or turnover into inside after a long-termculture or a long term certain cytokine stimulation. RASFs cultured for five generations invitro, the cell surface TLR4can turnover into intracellular.Therefore,we adopted theintracellular flow cytometry staining analysis observed the intracellular TLR4expressionof RASF cells were19.8%and the surface were10.2%.the result confirm tha tthe cellsurface TLR4on RASF can turnover into intracellular.5、Chemokine ligands and adhesion molecules on t-OASF cells were up-regulatedRasied Chemokines ligands and adhesion molecules expression on RASF is one of thekey factors for lymphocyte infiltrate locality to induce inflammation andimmunopathological damage.To clear the expression of chemokines ligands and adhesion molecules on t-OASF cells and the difference with other group cells, cells were fixed andstained with fluorescent-labeled antibodies and analyzed by flow cytometry.The expressionof CCL2,CXCL12,ICAM and VCAM on t-OASF cells were41.6%,42.1%,78.4%and2.8%respectively, that were found higher than OASF cells(33.4%、21.4%、68.7%and2.0%respectively), similar to RASF cells (55.7%,28.8%,77.2%and5.7%respectively).The results might be indicated HMGB1-LPS complex could raise theexpression of chemokines ligands and adhesion molecules on cell surface and recruitlymphocytes,monocyte-macrophages and activated other immune cells into the local joints.6、The activation levels of NF-κB, SAPK/JNK and p38MAPK were significantincreased in t-OASF cellsInflammation related transcription factors,such as NF-κB, SAPK/JNK and MAPKare critical for up-regulate chemokines,MMPs, apoptosis and autophagy disorders.Weused a commercial pathway scan kit to evaluate the activation levels of inflammatoryassociated key intracellular signaling proteins in t-OASF, OASF and RASF cells. Theresults showed that the activation levels of NF-κB, SAPK/JNK and p38MAPK weresignificant increased in t-OASF cells compared to OASF cells. But the activation level ofSTAT3was significant decreased in t-OASF cells compared to OASF or RASF cells.7、The mRNA and protein expression levels of pro-inflammatory cytokines (TNF-α,IL-1β and IL-6) and MMPs were up-regulated in t-OASF cellsRASF cells were reported to express the high levels of pro-inflammatory cytokines,chemokines and MMPs, while the pro-inflammatory cytokines can also enhance RASFs onbone destruction.To evaluate the activation level of t-OASF, the cells were collected andthen measured the mRNA expression of pro-inflammatory cytokines and MMPs withReal-Time PCR, and cultured cell supernatant was collected to detect the protein level ofproinflammatory cytokines and MMPs by ELISA.The results revealed that the mRNAexpressions of TNF-α,IL-1β, IL-6,MMP-3and MMP-13in t-OASF cells weresignificantly higher than that of in OASF cells (P<0.05). The protein levels of TNF-α,IL-1β, IL-6, MMP-3and MMP-13in the supernatant of t-OASF cells were significantlyhigher than those of OASF cells (P<0.05). Compared with RASF cells, a majority ofTNF-α, IL-1β, IL-6, MMP-3and MMP-13mRNA and protein expression levels of t-OASFcells were observed no obvious difference. 8、The t-OASF cells could induce the degradation of human normal cartilage in aSCID mice modelA large number of matrix-degrading enzymes, such as MMPs could be produced byRASFs, causing the degradation and destruction of articular cartilage. To evaluate thepathogenicity of t-OASF cells to destroy cartilage, We implanted cartilage-spongecomplexes, containing t-OASF,RASF or OASF cells respectively, into SCID miceabdominal subcutaneous. After60days, the implants were removed for routinepathological examination, special staining and immunohistochemicalexamination.Pathological examination results showed the chondrocytes and cartilagematrix of cartilage implanted t-OASF cells had degradation and shrinkage significantlycompared with the cartilage implanted OASF cell.Alcian blue and masson’s trichromestaining showed the cartilage tissues implanted t-OASF cells were shallowed and thechondrocytes were degenerated obviously, indicating the collagen and acidmucopolysaccharide components in cartilage tissues were significantly reduced,and thebones were severely damaged.Immunohistochemistry detecting human-specific pro-matrixmetalloprotease-13(proMMP-13) showed that the expression of proMMP-13in cartilagetissues implanted t-OASF cells or RASF cells were increased significantly.Pathologicalscore according to the implanted cartilage pathological examination and the dyeing resultswere1.5in OASF group,3.8in t-OASF group and5.0in RASF group.The differencebetwween OASF group and t-OASF group were statistically significant(p<0.01).Conclusion：Our previous study found that HMGB1-LPS complex can induceapparent arthritis pathological changes in DBA/1mice,suggesting HMGB1-LPS complexcan mediate RASFs formation, resulting in RA pathology injury.In the present study,wedemonstrated that HMGB1-LPS complex might promote OASF cells transfomate toRASF-like cells(t-OASF) with a long-term stimulation.The cellls in S/G2phase wereincreased and cell proliferation was accelerated significantly.The autophagy was promotedand cell apoptosis was reduced. Moreover,the expressions of TLRs,RAGE in/on t-OASFcells were up-regulated,and the downstream signaling pathways,such as NF-κB,SAPK/JNK and p38MAPK were activated as well,leading to the upregulation ofproinflammatory cytokines (TNF-α,IL-1β and IL-6),chemokines receptors(CCL2,CXCL12),cell adhesion molecules (ICAM,VCAM) and MMPs(MMP3and MMP13)expressions.Finally,transformed RASF-like cells induced by HMGB1-LPS complexe werefurther confirmed with distinct pathogenicity,causing the degradation of normal cartilagetissue in SCID mice model.The results suggest that the exogenous PAMP (LPS) couldcombine with the endogenous DAMP (HMGB1)to form a compound,which migh recognize TLRs/RAGE receptors on the SFs, causing the activation of downstreamsignaling pathways, resulting in the increasement of pro-inflammatorycytokines,chemokines ligands and bone destructive enzymes,inducing the transformationof normal SFs to RASF-like cells (t-OASFs).These effects could promot the occurrenceand progression of RA eventually.