Bone marrow mononuclear cells for joint therapy: The role of macrophages in inflammation resolution and tissue repair

Osteoarthritis (OA) is the most prevalent joint disease causing major disability and medical expenditures. Synovitis is a central feature of OA and is primarily driven by macrophages. Synovial macrophages not only drive inflammation but also its resolution, through a coordinated, simultaneous expression of pro- and anti-inflammatory mechanisms that are essential to counteract damage and recover homeostasis. Current OA therapies are largely based on anti-inflammatory principles and therefore block pro-inflammatory mechanisms such as prostaglandin E₂ and Nuclear factor-kappa B signaling pathways. However, such mechanisms are also innately required for mounting a pro-resolving response, and their blockage often results in chronic low-grade inflammation. Following minor injury, macrophages shield the damaged area and drive tissue repair. If the damage is more extensive, macrophages incite inflammation recruiting more macrophages from the bone marrow to maximize tissue repair and ultimately resolve inflammation. However, sustained damage and inflammation often overwhelms pro-resolving mechanisms of synovial macrophages leading to the chronic inflammation and related tissue degeneration observed in OA. Recently, experimental and clinical studies have shown that joint injection with autologous bone marrow mononuclear cells replenishes inflamed joints with macrophage and hematopoietic progenitors, enhancing mechanisms of inflammation resolution, providing remarkable and long-lasting effects. Besides creating an ideal environment for resolution with high concentrations of interleukin-10 and anabolic growth factors, macrophage progenitors also have a direct role in tissue repair. Macrophages constitute a large part of the early granulation tissue, and further transdifferentiate from myeloid into a mesenchymal phenotype. These cells, characterized as fibrocytes, are essential for repairing osteochondral defects. Ongoing “omics” studies focused on identifying key drivers of macrophage-mediated resolution of joint inflammation and those required for efficient osteochondral repair, have the potential to uncover ways for developing engineered macrophages or off-the-shelf pro-resolving therapies that can benefit patients suffering from many types of arthropaties, not only OA.     

Plasma proteins present in osteoarthritic synovial fluid can stimulate cytokine production via Toll-like receptor 4

Introduction

Osteoarthritis (OA) is a degenerative disease characterized by cartilage breakdown in the synovial joints. The presence of low-grade inflammation in OA joints is receiving increasing attention, with synovitis shown to be present even in the early stages of the disease. How the synovial inflammation arises is unclear, but proteins in the synovial fluid of affected joints could conceivably contribute. We therefore surveyed the proteins present in OA synovial fluid and assessed their immunostimulatory properties.

Methods

We used mass spectrometry to survey the proteins present in the synovial fluid of patients with knee OA. We used a multiplex bead-based immunoassay to measure levels of inflammatory cytokines in serum and synovial fluid from patients with knee OA and from patients with rheumatoid arthritis (RA), as well as in sera from healthy individuals. Significant differences in cytokine levels between groups were determined by significance analysis of microarrays, and relations were determined by unsupervised hierarchic clustering. To assess the immunostimulatory properties of a subset of the identified proteins, we tested the proteins' ability to induce the production of inflammatory cytokines by macrophages. For proteins found to be stimulatory, the macrophage stimulation assays were repeated by using Toll-like receptor 4 (TLR4)-deficient macrophages.

Results

We identified 108 proteins in OA synovial fluid, including plasma proteins, serine protease inhibitors, proteins indicative of cartilage turnover, and proteins involved in inflammation and immunity. Multiplex cytokine analysis revealed that levels of several inflammatory cytokines were significantly higher in OA sera than in normal sera, and levels of inflammatory cytokines in synovial fluid and serum were, as expected, higher in RA samples than in OA samples. As much as 36% of the proteins identified in OA synovial fluid were plasma proteins. Testing a subset of these plasma proteins in macrophage stimulation assays, we found that Gc-globulin, α₁-microglobulin, and α₂-macroglobulin can signal via TLR4 to induce macrophage production of inflammatory cytokines implicated in OA.

Conclusions

Our findings suggest that plasma proteins present in OA synovial fluid, whether through exudation from plasma or production by synovial tissues, could contribute to low-grade inflammation in OA by functioning as so-called damage-associated molecular patterns in the synovial joint.     

Cells of the synovium in rheumatoid arthritis. Osteoclasts

Osteoclasts are multinucleated cells of hematopoietic origin and are the primary bone resorbing cells. Numerous osteoclasts are found within the synovial tissue at sites adjacent to bone, creating resorption pits and local bone destruction. They are equipped with specific enzymes and a proton pump that enable them to degrade bone matrix and solubilize calcium, respectively. The synovial tissue of inflamed joints has a particularly high potential to accumulate osteoclasts because it harbors monocytes/macrophages, which function as osteoclast precursors, as well as cells that provide the specific molecular signals that drive osteoclast formation. Osteoclasts thus represent a link between joint inflammation and structural damage since they resorb mineralized tissue adjacent to the joint and destroy the joint architecture.     

Interleukin-1β signaling in osteoarthritis – chondrocytes in focus

Osteoarthritis (OA) can be regarded as a chronic, painful and degenerative disease that affects all tissues of a joint and one of the major endpoints being loss of articular cartilage. In most cases, OA is associated with a variable degree of synovial inflammation. A variety of different cell types including chondrocytes, synovial fibroblasts, adipocytes, osteoblasts and osteoclasts as well as stem and immune cells are involved in catabolic and inflammatory processes but also in attempts to counteract the cartilage loss. At the molecular level, these changes are regulated by a complex network of proteolytic enzymes, chemokines and cytokines (for review: [1]). Here, interleukin-1 signaling (IL-1) plays a central role and its effects on the different cell types involved in OA are discussed in this review with a special focus on the chondrocyte.     

Melatonin abolished proinflammatory factor expression and antagonized osteoarthritis progression in vivo

Progressive structural changes in osteoarthritis (OA) involve synovial inflammation and angiogenesis, as well as activation of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin (IL)-8, and the angiogenic factor vascular endothelial growth factor (VEGF). The endogenous hormone melatonin (N-acetyl-5-methoxytryptamine) is involved in antioxidative and anti-inflammatory activities, but how it antagonizes OA progression via its specific receptors is unclear. Here, we demonstrate that the MT₁ melatonin receptor, but not the MT₂ receptor, is highly expressed in normal tissue and only minimally in OA tissue. By targeting the MT₁ receptor, melatonin reversed OA-induced pathology and effectively reduced levels of TNF-α, IL-8, and VEGF expression in OA synovial fibroblasts and synovium from rats with severe OA. Interestingly, we found that the anabolic activities of melatonin involved the MT₁ receptor, which upregulated microRNA-185a through the PI3K/Akt and ERK signaling pathways in OA synovial fibroblasts. Our investigation confirms the role of the MT₁ receptor in melatonin-induced anti-catabolic effects in OA disease.Subject terms: miRNAs, Chronic inflammation     

The loss of sympathetic nerve fibers in the synovial tissue of patients with rheumatoid arthritis is accompanied by increased norepinephrine release from synovial macrophages.

Our objective was to investigate sympathetic and sensory nerve fibers in synovial tissue in rheumatoid arthritis (RA) and osteoarthritis (OA) in relation to histological inflammation and synovial cytokine and norepinephrine (NE) secretion. Immunohistochemistry was used to detect nerve fibers and inflammatory parameters. A superfusion technique of synovial tissue pieces was used to investigate cytokine and NE secretion. In RA, we detected 0.2 +/- 0.04 tyrosine hydroxylase-positive (TH-positive=sympathetic) nerve fibers/mm2 as compared to 4.4 +/- 0. 8 nerve fibers/mm2 in OA (P<0.001). In RA, there was a negative correlation between the number of TH-positive nerve fibers and inflammation index (RRank=-0.705, P=0.002) and synovial IL-6 secretion (RRank=-0.630, P=0.009), which was not found in OA. Substance P-positive (=sensory) nerve fibers were increased in RA as compared to OA (3.5+/-0.2 vs. 2.3+/-0.3/mm2, P=0.009). Despite lower numbers of sympathetic nerve fibers in RA than in OA, NE release was similar at baseline (RA vs. OA: 152+/-36 vs. 106+/-21 pg/ml, n.s.). Basal synovial NE secretions correlate with the number of TH-positive CD 163+ synovial macrophages (RA: RRank=0.622, P=0.031; OA: RRank=0.299, n.s.), and synovial macrophages have been shown to produce NE in vitro. Whereas sympathetic innervation is reduced, sensory innervation is increased in the synovium from patients with longstanding RA when compared to the synovium from OA patients. The differential patterns of innervation are dependent on the severity of the inflammation. However, NE secretion from the synovial tissue is maintained by synovial macrophages. This demonstrates a loss of the influence of the sympathetic nervous system on the inflammation, accompanied by an up-regulation of the sensory inputs into the joint, which may contribute to the maintenance of the disease.     

B cell clonal expansion and somatic hypermutation of Ig variable heavy chain genes in the synovial membrane of patients with osteoarthritis

Inflammatory mediators have been explored as possible factors in the initiation and/or progression of osteoarthritis (OA). This study shows that synovial infiltration by B lymphocytes is present in almost half of the knee OA cases. The degree of B lymphocyte infiltration is associated with more pronounced synovial inflammation and with the presence of plasma cells and lymphoid follicles in more severe cases. To examine whether these B cells are merely bystanders or could be involved in the pathogenesis of OA, we analyzed the Ig H chain variable region (V(H)) genes of B cells recovered from the synovial membrane of five OA patients with marked B cell infiltration. Sequence analysis of CDR3 regions of rearranged VDJ genes revealed clonal or oligoclonal B cell expansions in all cases. Expanded B cell clones in four of five OA patients showed clustered somatic mutations, occurring mainly in the CDRs and with a high replacement-to-silent ratio (>2.9), indicating that these cells are postgerminal center B cells that had been positively selected through their Ag receptor. These data demonstrate the presence in inflamed knee OA synovium of clonally expanded, Ag-driven B cells that may contribute to the development or progression of the disease.     

Plasma proteins take their toll on the joint in osteoarthritis

In their recent study, Sohn and colleagues identify multiple plasma proteins in the synovial fluid of patients with osteoarthritis (OA) and demonstrate the capacity of several of the proteins to activate macrophages via the innate immune receptor Toll-like receptor-4 (TLR-4). The authors speculate that the plasma proteins transit into the synovial compartment at sites of tissue damage where the endothelial barrier may be compromised, thus bringing them into contact with the articular surface and cells within the synovium. These results demonstrate a novel mechanism by which synovial inflammation can be initiated in patients with OA and how this process may contribute to the pathogenesis of OA joint pathology.     

A critical role for allograft inflammatory factor-1 in the pathogenesis of rheumatoid arthritis

Rheumatoid arthritis (RA) is characterized by massive synovial proliferation, angiogenesis, subintimal infiltration of inflammatory cells and the production of cytokines such as TNF-alpha and IL-6. Allograft inflammatory factor-1 (AIF-1) has been identified in chronic rejection of rat cardiac allografts as well as tissue inflammation in various autoimmune diseases. AIF-1 is thought to play an important role in chronic immune inflammatory processes, especially those involving macrophages. In the current work, we examined the expression of AIF-1 in synovial tissues and measured AIF-1 in synovial fluid (SF) derived from patients with either RA or osteoarthritis (OA). We also examined the proliferation of synovial cells and induction of IL-6 following AIF-1 stimulation. Immunohistochemical staining showed that AIF-1 was strongly expressed in infiltrating mononuclear cells and synovial fibroblasts in RA compared with OA. Western blot analysis and semiquantitative RT-PCR analysis demonstrated that synovial expression of AIF-1 in RA was significantly greater than the expression in OA. AIF-1 induced the proliferation of cultured synovial cells in a dose-dependent manner and increased the IL-6 production of synovial fibroblasts and PBMC. The levels of AIF-1 protein were higher in synovial fluid from patients with RA compared with patients with OA (p < 0.05). Furthermore, the concentration of AIF-1 significantly correlated with the IL-6 concentration (r = 0.618, p < 0.01). These findings suggest that AIF-1 is closely associated with the pathogenesis of RA and is a novel member of the cytokine network involved in the immunological processes underlying RA.     

Stromal Transcriptional Profiles Reveal Hierarchies of Anatomical Site,Serum Response and Disease and Identify Disease Specific Pathways

Synovial fibroblasts in persistent inflammatory arthritis have been suggested to have parallels with cancer growth and wound healing, both of which involve a stereotypical serum response programme. We tested the hypothesis that a serum response programme can be used to classify diseased tissues, and investigated the serum response programme in fibroblasts from multiple anatomical sites and two diseases. To test our hypothesis we utilized a bioinformatics approach to explore a publicly available microarray dataset including rheumatoid arthritis (RA), osteoarthritis (OA) and normal synovial tissue, then extended those findings in a new microarray dataset representing matched synovial, bone marrow and skin fibroblasts cultured from RA and OA patients undergoing arthroplasty. The classical fibroblast serum response programme discretely classified RA, OA and normal synovial tissues. Analysis of low and high serum treated fibroblast microarray data revealed a hierarchy of control, with anatomical site the most powerful classifier followed by response to serum and then disease. In contrast to skin and bone marrow fibroblasts, exposure of synovial fibroblasts to serum led to convergence of RA and OA expression profiles. Pathway analysis revealed three inter-linked gene networks characterising OA synovial fibroblasts: Cell remodelling through insulin-like growth factors, differentiation and angiogenesis through _3 integrin, and regulation of apoptosis through CD44. We have demonstrated that Fibroblast serum response signatures define disease at the tissue level, and that an OA specific, serum dependent repression of genes involved in cell adhesion, extracellular matrix remodelling and apoptosis is a critical discriminator between cultured OA and RA synovial fibroblasts.     

Interleukin-34 as a promising clinical biomarker and therapeutic target for inflammatory arthritis

Interleukin-34 (IL-34), recently identified as a novel inflammatory cytokine and the second ligand for colony-stimulating factor-1 receptor, is known to play regulatory roles in the development, maintenance, and function of mononuclear phagocyte lineage cells – especially osteoclasts. Regarding its primary effect on osteoclasts, IL-34 has been shown to stimulate formation and activation of osteoclasts, which in turn magnifies osteoclasts-resorbing activity. In addition to its role in osteoclastogenesis, IL-34 has been implicated in inflammation of synovium via augmenting production of inflammatory mediators, in which altered IL-34 expression is regulated by pro-inflammatory cytokines responsible for cartilage degradation. Indeed, IL-34 has been documented to be highly expressed in inflamed synovium of rheumatoid arthritis (RA) and knee osteoarthritis (OA) patients, which are recognized as inflammatory arthritis. Furthermore, a number of clinical studies demonstrated that IL-34 levels were significantly increased in the circulation and synovial fluid of patients with RA and knee OA. Its levels were also found to be positively associated with disease severity – especially radiographic severity of both RA and knee OA patients. Interestingly, emerging evidence has accumulated that functional blockage of IL-34 with specific antibody can alleviate the severity of inflammatory arthritis. It is therefore reasonable to speculate that IL-34 may be developed as a potential biomarker and a new therapeutic candidate for inflammatory arthritis. To date, there are numerous studies showing IL-34 involvement and association with many aspects of inflammatory arthritis. Herein, this review aimed to summarize the recent findings regarding regulatory role of IL-34 in synovial inflammation-mediated cartilage destruction and update the current comprehensive knowledge on usefulness of IL-34-based treatment in inflammatory arthritis – particularly RA and knee OA.     

Physiology and pathophysiology of nitrosative and oxidative stress in osteoarthritic joint destruction

Osteoarthritis (OA) is one of the most common chronic diseases, with increasing importance due to increased life expectancy. On a cellular level, the pathophysiology of joint function impairment and ultimate destruction associated with OA remains poorly understood. Free radicals are highly reactive molecules involved in both normal intracellular signal transduction and degenerative cellular processes. An imbalance between the free radical burden and cellular scavenging mechanisms, defined as oxidative stress, has been identified as a relevant factor in OA pathogenesis. This literature review elucidates the involvement of nitrosative and oxidative stress in cellular ageing in joints, cell senescence, and apoptosis. Free radical exposure is known to promote cellular senescence and apoptosis, and the involvement of radical oxygen species (ROS) in inflammation, fibrosis control, and pain nociception has been proven. A relatively novel approach to OA pathophysiology considers the joint to be a dynamic system consisting of 3, continuously interacting compartments, cartilage, synovial tissue, and subchondral bone. Current knowledge concerning free radical involvement in paracrine signalling in OA is reviewed. The interrelationship between oxidative imbalances and OA pathophysiology may provide a novel approach to the comprehension, and therefore modification, of OA disease progression and symptom control.     

滑膜细胞在骨关节炎中的研究进展

骨关节炎(osteoarthritis,OA)作为最常见的退行性关节疾病,其主要临床特点是软骨的破坏降解,进而导致关节功能丧失,严重影响患者的生活质量.越来越多的证据表明,除了软骨组织,OA的病理改变还涉及滑膜、骨以及软骨下骨在内的多个组织系统.其中,滑膜作为组织系统的重要组成部分,其病变在OA中的作用日益突出.滑膜细胞分为A型滑膜巨噬细胞和B型滑膜成纤维细胞,在OA中发挥着不同但又密切联系的作用.本文综述不同类型滑膜细胞在OA中的作用,为进一步认识OA发病机制及治疗方法提供科学的理论依据.     

Impaired macrophage autophagy increases the immune response in obese mice by promoting proinflammatory macrophage polarization

Recent evidence that excessive lipid accumulation can decrease cellular levels of autophagy and that autophagy regulates immune responsiveness suggested that impaired macrophage autophagy may promote the increased innate immune activation that underlies obesity. Primary bone marrow-derived macrophages (BMDM) and peritoneal macrophages from high-fat diet (HFD)-fed mice had decreased levels of autophagic flux indicating a generalized impairment of macrophage autophagy in obese mice. To assess the effects of decreased macrophage autophagy on inflammation, mice with a Lyz2-Cre-mediated knockout of Atg5 in macrophages were fed a HFD and treated with low-dose lipopolysaccharide (LPS). Knockout mice developed systemic and hepatic inflammation with HFD feeding and LPS. This effect was liver specific as knockout mice did not have increased adipose tissue inflammation. The mechanism by which the loss of autophagy promoted inflammation was through the regulation of macrophage polarization. BMDM and Kupffer cells from knockout mice exhibited abnormalities in polarization with both increased proinflammatory M1 and decreased anti-inflammatory M2 polarization as determined by measures of genes and proteins. The heightened hepatic inflammatory response in HFD-fed, LPS-treated knockout mice led to liver injury without affecting steatosis. These findings demonstrate that autophagy has a critical regulatory function in macrophage polarization that downregulates inflammation. Defects in macrophage autophagy may underlie inflammatory disease states such as the decrease in macrophage autophagy with obesity that leads to hepatic inflammation and the progression to liver injury.     

Pentraxin 3 regulated by miR-224-5p modulates macrophage reprogramming and exacerbates osteoarthritis associated synovitis by targeting CD32

Emerging evidence has shown an imbalance in M1/M2 macrophage polarization to play an essential role in osteoarthritis (OA) progression. However, the underlying mechanistic basis for this polarization is unknown. RNA sequencing of OA M1-polarized macrophages found highly expressed levels of pentraxin 3 (PTX3), suggesting a role for PTX3 in OA occurrence and development. Herein, PTX3 was found to be increased in the synovium and articular cartilage of OA patients and OA mice. Intra-articular injection of PTX3 aggravated, while PTX3 neutralization reversed synovitis and cartilage degeneration. No metabolic disorder or proteoglycan loss were observed in cartilage explants when treated with PTX3 alone. However, cartilage explants exhibited an OA phenotype when treated with culture supernatants of macrophages stimulated with PTX3, suggesting that PTX3 did not have a direct effect on chondrocytes. Therefore, the OA anti-chondrogenic effects of PTX3 are primarily mediated through macrophages. Mechanistically, PTX3 was upregulated by miR-224-5p deficiency, which activated the p65/NF-κB pathway to promote M1 macrophage polarization by targeting CD32. CD32 was expressed by macrophages, that when stimulated with PTX3, secreted abundant pro-inflammation cytokines that induced severe articular cartilage damage. The paracrine interaction between macrophages and chondrocytes produced a feedback loop that enhanced synovitis and cartilage damage. The findings of this study identified a functional pathway important to OA development. Blockade of this pathway and PTX3 may prevent and treat OA.Subject terms: Osteoarthritis, Extracellular signalling molecules     

Gene expression,protein profiling,and chemotactic activity of infrapatellar fat pad mesenchymal stem cells in pathologies of the knee joint

The infrapatellar fat pad (IPFP) is a periarticular adipose knee tissue. This tissue contains a large number of mesenchymal stem cells (MSCs). In the present work, we wanted to study the IPFP MSCs and their relationship and differences in two groups, anterior cruciate ligament (ACL) ruptures knees and ostheoarthrosis (OA). The IPFP of 42 patients with OA or ACL rupture were analyzed. Isolation, primary culture, and a genetic and proteomic study of MSCs from IPFP were performed. Gene expression of IL-6, tumor necrosis factor (TNF), IL-8, HSPA1A (Hsp70), CXCL10, RANTES, MMP1, MMP3, TIMP1, and BMP7 was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). We analyzed MSCs from from 12 diferents patients in two cellular pools (6 from AO disease and 6 from ALC rupture to form two cell pool), for the iTRAQ Proteomic Assay. The conditional media were used in quantitative analysis of MSC soluble factors by Luminex and for de migration assay. A higher gene expression of IL-6, TNF, CXCL10, RANTES, and MMP1 and OPG in MSCs from OA versus ACL (p < 0.05) was observed. Conversely HSPA1A, TIMP1, and RANKL showed a significant lower expression in OA-MSCs (p < 0.05). In the secretome analysis, adipsin and visfantin levels in the supernatants from OA-MSCs were lower (p < 0.05) respect to ACL-MSCs. Also, the monocytic cells migrated two-folds in the presence of conditioned media from OA-MSCs patients versus patients with ACL-MSC. The infrapatellar pad should be considered as an adipose tissue capable of producing and excreting inflammatory mediators directly in the knee joint, influencing the development and progression of knee joint pathologies.     

CXCL12 chemokine up-regulates bone resorption and MMP-9 release by human osteoclasts: CXCL12 levels are increased in synovial and bone tissue of rheumatoid arthritis patients

Chemokines are involved in a number of inflammatory pathologies and some of them show a pivotal role in the modulation of osteoclast development. Therefore, we evaluated the role of CXCL12 chemokine on osteoclast differentiation and function and we analyzed its expression on synovial and bone tissue biopsies from rheumatoid arthritis (RA) patients. Osteoclasts were obtained by 7 days in vitro differentiation with RANKL and M-CSF of CD11b positive cells in the presence or absence of CXCL12. The total number of osteoclast was analyzed by Tartrate-resistant acid phosphatase (TRAP)-staining and bone-resorbing activity was assessed by pit assay. MMP-9 and TIMP-1 release was evaluated by ELISA assay. CXCL12 expression on biopsies from RA patients was analyzed by immunohistochemistry. Osteoclasts obtained in the presence of CXCL12 at 10 nM concentration displayed a highly significant increase in bone-resorbing activity as measured by pit resorption assay, while the total number of mature osteoclasts was not affected. The increased resorption is associated with overexpression of MMP-9. Immunostaining for CXCL12 on synovial and bone tissue biopsies from both rheumatoid arthritis (RA) and osteoarthritis (OA) samples revealed a strong increase in the expression levels under inflammatory conditions. CXCL12 chemokine showed a clear activating role on mature osteoclast by inducing bone-resorbing activity and specific MMP-9 enzymatic release. Moreover, since bone and synovial biopsies from RA patients showed an elevated CXCL12 expression, these findings may provide useful tools for achieving a full elucidation of the complex network that regulates osteoclast function in course of inflammatory diseases.