The Effect Of B Cell-specific TSC1 Knock Out On Osteoarthritis

1 BackgroundOsteoarthritis (OA) is one of the most common degenerative disorders characterized by cartilage damage, inflammation, synovial fibrosis, subchondral bone remodeling, and osteophyte formation, and is the single most important cause of disability in older adults. Currently, OA affects about half of the over 65 population with a greater percentage in women than in men after menopause. OA can broadly be classified in two different forms, primary and secondary. The primary or idiopathic OA, is a gene-dependent disease. Various studies have indicated a strong hereditary component in primary OA, probably due to its polygenic nature. Secondary OA, also called post-traumatic OA, frequently occurs sometime after a traumatic event. Secondary OA is exacerbated by inflammatory and repair processes that occur after the initial traumatic insult and after surgery. So far, although a lot of studies focus on OA, the mechanism of OA is still unclear. As the aging of population, the trend of age-related OA has increased year by year, especially in densely populated and aging Southern China. At present, the key scientific problems to be solved is gaining insight into the mechanism of OA and looking for effective prevention against this disease.OA was once considered to be a non-inflammatory condition; however, increasing evidence of inflammation in OA patients suggests that the immune system may play an important role in OA development and progression. Some research showed immune system fulfilled this function possibly through the secretion of inflammatory cytokines. Numerous studies have focused on identifying and describing the factors responsible for the development of the inflammatory processes involved in OA, and increasing numbers of reports have directed attention to the special role of the cytokine network in the pathogenesis of OA.Studies of human OA are limited by its multifactorial nature, and many studies have focused largely on changes in the articular cartilage. However, OA affects many organs, including the immune system, and inflammation of the synovial membrane is emerging as the main feature of OA, even in the early stages of the disease. Several soluble factors such as cytokines promote the progression of OA symptoms. Interleukin (IL)-1β, IL-6, and tumor necrosis factor a (TNFa) are the major pro-inflammatory cytokines responsible for the shift of cartilage homeostasis towards catabolism and degradation. IL-1β is considered to be a key cytokine in the pathogenesis of OA, by inducing inflammatory reactions and catabolic effects independently, as well as in combination with other mediators, with respect to the articular cartilage and other elements of the joints. TNFa is another important inflammatory cytokine in the pathophysiology of OA, and the effects of TNFa largely coincide with those of IL-1β, with marked synergism between them in relation tomany phenomena occurring during the course of OA. IL-6 is characterized by omnidirectional interactions in processes in the human body. It strongly activates the immune system and enhances the inflammatory response. IL-6 has similar effects to other cytokines on joint cartilage, and synergistically decreases the production of type II collagen and increases the production of matrix metalloproteinase (MMP) enzymes. Moreover, cellular infiltrates in the inflamed synovium in OA have been metalloproteinase (MMP) enzymes. Moreover, cellular infiltrates in the inflamed synovium in OA have been reported to contain activated B cells, as well as other cell types.Mammals rapamycin target protein (mTOR) is a relatively conservative serine/threonine protein kinase. mTOR is activated by nutrients (amino acids), growth factors (insulin, IGF, etc.) and cellular energy status (AMP/ATP ratio), and regulates several growth related process including protein synthesis, ribosome biogenesis, lipid synthesis, nutrient import, and autophagy. There are two kinds of mTOR complexes:mTORC1 and mTORC2. mTORC1 is sensitive to raprmycin, composed by mTOR, Raptor, mLST8 and PRAS40. mTORC2, containing mTOR, mLST8, Rictor, PRR5 and mSinl, is not sensitive to rapamycin. mTORCl has evolved to be a central signaling hub for coupling many cellular processes, including B-cell proliferation and activation in response to environmental inputs. Dysregulation of mTORC1 signaling is important in the pathology of cancer, diabetes, inflammatory diseases, and aging. Plasma cells were recently shown to be enriched in mice with B cell-specific activation of mTORCl by deletion of the gene for tuberous sclerosis complex 1 (TSC1), an mTORC1 upstream negative regulator. B cells are known to secrete inflammatory cytokines important for OA development, including IL-1β,IL-6, and TNF-a.Although inflammatory cytokines are known to play an important role in the progression of OA and B cells are a recognized source of these cytokines, the role of B-cell-derived cytokines in OA development remains ill-defined. In this study, we investigated the effects of B-cell-specific mTORC1 activation on plasma cells, inflammatory cytokine production, and severity of OA in TSC1 transgenic mice, to establish a link between mTORC1 activation in B cells and accelerated OA.In this study, we proposed that increasing activity of mTORC1 in B cell may be one of the pathogenesis of osteoarthritis, and research on that has not been reported. This research will provide a new mechanism for pathogenesis of osteoarthritis, and provide new strategies and targets for the treatment of osteoarthritis.2 ObjectiveThere are two points of objective in this study as follows:Firstly, control mice were treated with (DMM group) or without (SHAM group) DMM surgery to determine the effects of surgery. And then, we detected the changes of inflammatory cytokines in serum, spleen and synovial membrane after surgery. Secondly, we investigated the specific role of the immune system in OA pathophysiology in vivo by generating mice lacking TSC1 specifically in B cells, by crossing mice harboring a TSC1 conditional allele (TSClflox/flox) with mice expressing the Cre recombinase under the control of CD 19 regulatory elements. And we built the surgically induced osteoarthritis model by using DMM surgery.3 Materials and Methods1. CD19-Cre and TSC1-loxp mice were purchased from Jackson Laboratories. Using Cre-loxp system, we have successfully created mice with B lymphocyte-specific deletion of TSC1. The genotype of transgenic mice was identified by PCR and agarose gel electrophoresis, while effect of deletion was identified by western blot.2. Sections of the knee joint were stained with toluidine blue and Safranin-O/Fast Green according to the manufacturer’s recommendations. Slides were evaluated independently by two people in a blinded fashion. To determine the extent of cartilage deterioration after the deletion of TSC1 in B cell, three fields in joint sections of tibial and femoral articular cartilage were randomly selected, examined under high magnification, and evaluated using the modified Mankin scoring system.3. To determine the severity of degradation of articular cartilage after the deletion of TSC1 in B cell. Immunohistochemistry was performed using specific antibodies for MMP-13. And qPCR technology was used to examine the expression of type two and type ten collagen of articular cartilage.4. The impact of TSC1 deletion in B cell on inflammatory cytokines in serum. ELISA assay was used to determine the variation of inflammatory cytokines like IL-1β, IL-6 and TNFa of serum of mice.5. qPCR technology to examine the effects of TSC1 knockout on the expression of inflammatory cytokines like IL-1β, IL-6 and TNFa in synovial membrane and B cells from splenocytes.4 Results1. The mice model of B lymphocyte specificity knockout TSC1 was constructed successfully.We generated mice (KO mice) with a conditionally ablated TSC1 gene in B lymphocytes using a Cre expression cassette under the control of the CD 19 promoter. Western blotting showed that the expression of TSC1 was decreased in B cells-TSC1 KO mice, but not in control littermates. mTORC1 activity was monitored by assaying the level of phospho-S6(s235/236). The specific enrichment of phospho-S6(s235/236) in B lymphocytes was observed in B cells-TSC1 KO mice. In summary, the mice model of B lymphocyte specificity knockout TSC1 was constructed successfully.2. Histological changes were obvious in littermate control mice after DMM surgery induced OA.Littermate control mice (CON mice) were treated with (DMM group) or without (SHAM group) DMM surgery to determine the effects of surgery and changes in inflammatory cytokines after surgery. We evaluated cartilage changes by Safranin-O/Fast Green staining at 4 and 8 weeks after surgery using modified Mankin scores. Mankin score was significantly increased in the DMM compared with the SHAM group. Joint cartilage thickness, assessed by toluidine blue staining, showed severe abrasion and significantly reduced cartilage thickness in the DMM, compared with the SHAM group at 4 and 8 weeks after surgery. Gene expression levels of the inflammatory cytokines IL-1β, IL-6, and TNFa in the synovial membrane at 4 and 8 weeks after surgery showed similar trends, as assessed by qPCR, with significantly higher levels in the DMM group compared with the SHAM group. These results supported those of previous studies and indicated the suitability of DMM surgery for OA research, and showed that inflammatory cytokines in the synovial membrane were increased after DMM surgery.3. Inflammatory cytokine increased in serum and B cells in littermate control mice after DMM surgery induced OA.We comprehensively evaluated the progression of OA after DMM surgery by examining the expression of inflammatory cytokines in serum and B cells at 4 and 8 weeks after surgery. Serum IL-1β, IL-6, and TNFa protein levels were significantly higher in the DMM group at 4 and 8 weeks, according to ELISA. Gene expression levels of inflammatory cytokines in B cells were also significantly higher at 4 and 8 weeks after surgery in the DMM compared with the SHAM group, according to qPCR. Overall, these in vivo results suggest that inflammatory cytokines were enriched in B cells, serum, and synovial membranes after surgically induced OA, and are therefore likely to play a part in the progression of OA.4. Differences in expression of inflammatory cytokines between CD19-TSC1 transgenic and littermate control mice.We investigated the specific role of the immune system in OA pathophysiology in vivo by generating mice lacking TSC1 specifically in B cells, by crossing mice harboring a TSC1 conditional allele (TSC1flox/flox) with mice expressing the Cre recombinase under the control of CD 19 regulatory elements. We detected the gene expression levels of inflammatory cytokines in B cells by qPCR analysis and showed that IL-1β, IL-6, and TNFa were increased in KO mice compared with CON mice at 16 weeks postnatally. However, there was no significant difference in synovial membrane inflammatory cytokine levels between KO and CON mice, according to qPCR. These results suggested that KO mice may exhibited a more severe OA phenotype than CON mice after DMM surgery.5. KO mice exhibited accelerated OA phenotype.Based on the above assumptions, we subjected 8-week-old male CON and KO mice to a DMM model of OA and determined the OA phenotypes at 4 and 8 weeks post-surgery. As expected, histological analysis at 4 weeks post-surgery showed some loss of proteoglycans (loss of Safranin O staining) and roughening of the articular cartilage in knee joints in CON mice, but greater loss of proteoglycans and more destruction in some regions of the articular cartilage in KO mice. This phenotype became more pronounced at 8 weeks post-OA surgery, with significant and severe destruction of the articular cartilage associated with greater loss of proteoglycans in KO compared with CON mice. These results were confirmed by the increased modified Mankin score in KO compared with CON mice post-OA surgery. We also assessed cartilage thickness by toluidine blue staining and showed severe abrasion and significantly reduced cartilage thickness in KO compared with CON mice at 4 weeks following surgery. Moreover, cartilage thickness was also reduced in KO mice at 8 weeks after surgery. OA is known to be accompanied by cartilage degradation, which has been identified as the direct cause of articular cartilage reduction, and MMP-13 is known to be one of the major catabolic factors implicated in OA. We therefore determined MMP-13 expression by immunohistochemistry. The proportion of MMP-13-positive cells was significantly increased in KO compared with CON mice at 4 and 8 weeks after surgery-induced OA. Furthermore, qPCR analysis of synovial membrane and articular cartilage showed increased mRNA expression of type Ⅹ collagen and decreased mRNA expression of type Ⅱ collagen in KO compared with CON mice at both 4 and 8 weeks after surgery. In summary, these results indicated that KO mice exhibited a more severe OA phenotype than CON mice after DMM surgery, with significantly greater cartilage degradation.6. KO mice exhibited more severe inflammatory response after DMM surgery induced OA.KO mice subjected to DMM surgery exhibited an accelerated OA phenotype. We therefore observed the differences in serum inflammatory cytokines between KO and CON mice at 4 and 8 weeks after surgery using ELISA. Serum levels of IL-1β,IL-6, and TNFa were all increased in KO mice at 4 and 8 weeks after surgery. Furthermore, mRNA expression levels of these cytokines in the synovial membrane were also enhanced in KO compared with CON mice, as determined by qPCR. These results indicated that KO mice exhibited a more severe inflammatory response after surgically induced OA compared with CON mice, which may help to explain the accelerated OA phenotype observed in KO mice.5 Conclusions(1) We have successfully constructed the mice model of TSC1 specific knockout in B cells.(2) CON mice were treated with (DMM group) or without (SHAM group) DMM surgery to determine the effects of surgery.(3) CON mice were treated with (DMM group) or without (SHAM group) DMM surgery and in vivo results suggest that inflammatory cytokines were enriched in B cells, serum, and synovial membranes after surgically induced OA, and are therefore likely to play a part in the progression of OA.(4) We detected the gene expression levels of inflammatory cytokines in B cells by qPCR analysis and showed that IL-1β, IL-6, and TNFα were increased in KO mice compared with CON mice at 16 weeks postnatally. However, there was no significant difference in synovial membrane inflammatory cytokine levels between KO and CON mice, according to qPCR.(5) We subjected 8-week-old male CON and KO mice to a DMM model of OA and determined the OA phenotypes at 4 and 8 weeks post-surgery. And the results indicated that KO mice exhibited accelerated OA phenotype compared with CON mice.