Activation of cytotoxic lymphocytes through CD6 enhances killing of cancer cells.

Immune checkpoint inhibitors (ICIs) have demonstrated efficacy and improved survival in a growing number of cancers. Despite their success, ICIs are associated with immune-related adverse events that can interfere with their use. Therefore, safer approaches are needed. CD6, expressed by T-lymphocytes and human NK cells, engages in cell-cell interactions by binding to its ligands CD166 (ALCAM) and CD318 (CDCP1). CD6 is a target protein for regulating immune responses and is required for the development of several mouse models of autoimmunity. Interestingly, CD6 is exclusively expressed on immune cells while CD318 is strongly expressed on most cancers. Here we demonstrate that disrupting the CD6-CD318 axis with UMCD6, an anti-CD6 monoclonal antibody, prolongs survival of mice in xenograft mouse models of human breast and prostate cancer, treated with infusions of human lymphocytes. Analysis of tumor-infiltrating immune cells showed that augmentation of lymphocyte cytotoxicity by UMCD6 is due to effects of this antibody on NK, NKT and CD8 + T cells. In particular, tumor-infiltrating cytotoxic lymphocytes from UMCD6-treated mice expressed higher levels of perforin and were found in higher proportions than those from IgG-treated mice. Moreover, RNA-seq analysis of human NK-92 cells treated with UMCD6 revealed that UMCD6 up-regulates the NKG2D-DAP10 receptor complex, important in NK cell activation, as well as its downstream target PI3K. Our results now describe the phenotypic changes that occur on immune cells upon treatment with UMCD6 and further confirm that the CD6-CD318 axis can regulate the activation state of cytotoxic lymphocytes and their positioning within the tumor microenvironment.

Mammalian Glycosylation Patterns Protect Citrullinated Chemokine MCP-1/CCL2 from Partial Degradation.

Monocyte chemoattractant protein-1 (MCP-1/CCL2) is a potent chemotactic agent for monocytes, primarily produced by macrophages and endothelial cells. Significantly elevated levels of MCP-1/CCL2 were found in synovial fluids of patients with rheumatoid arthritis (RA), compared to osteoarthritis or other arthritis patients. Several studies suggested an important role for MCP-1 in the massive inflammation at the damaged joint, in part due to its chemotactic and angiogenic effects. It is a known fact that the post-translational modifications (PTMs) of proteins have a significant impact on their properties. In mammals, arginine residues within proteins can be converted into citrulline by peptidylarginine deiminase (PAD) enzymes. Anti-citrullinated protein antibodies (ACPA), recognizing these PTMs, have become a hallmark for rheumatoid arthritis (RA) and other autoimmune diseases and are important in diagnostics and prognosis. In previous studies, we found that citrullination converts the neutrophil attracting chemokine neutrophil-activating peptide 78 (ENA-78) into a potent macrophage chemoattractant. Here we report that both commercially available and recombinant bacterially produced MCP-1/CCL2 are rapidly (partially) degraded upon in vitro citrullination. However, properly glycosylated MCP-1/CCL2 produced by mammalian cells is protected against degradation during efficient citrullination. Site-directed mutagenesis of the potential glycosylation site at the asparagine-14 residue within human MCP-1 revealed lower expression levels in mammalian expression systems. The glycosylation-mediated recombinant chemokine stabilization allows the production of citrullinated MCP-1/CCL2, which can be effectively used to calibrate crucial assays, such as modified ELISAs.

CCL25 and CCR9 is a unique pathway that potentiates pannus formation by remodeling RA macrophages into mature osteoclasts.

This study elucidates the mechanism of CCL25 and CCR9 in rheumatoid arthritis (RA). RA synovial fluid (SF) expresses elevated levels of CCL25 compared to OA SF and plasma from RA and normal. CCL25 was released into RA SF by fibroblasts (FLS) and macrophages (MΦs) stimulated with IL-1ß and IL-6. CCR9 is also presented on IL-1ß and IL-6 activated RA FLS and differentiated MΦs. Conversely, in RA PBMCs neither CCL25 nor CCR9 are impacted by 3-month longitudinal TNF inhibitor therapy. CCL25 amplifies RA FLS and monocyte infiltration via p38 and ERK phosphorylation. CCL25-stimulated RA FLS secrete potentiated levels of IL-8 which is disrupted by p38 and ERK inhibitors. CCL25 polarizes RA monocytes into nontraditional M1 MΦs that produce IL-8 and CCL2. Activation of p38 and ERK cascades are also responsible for the CCL25-induced M1 MΦ development. Unexpectedly, CCL25 was unable to polarize RA PBMCs into effector Th1/Th17 cells. Consistently, lymphokine like RANKL was uninvolved in CCL25-induced osteoclastogenesis; however, this manifestation was regulated by osteoclastic factors such as RANK, cathepsin K (CTSK), and TNF-α. In short, we reveal that CCL25/CCR9 manipulates RA FLS and MΦ migration and inflammatory phenotype in addition to osteoclast formation via p38 and ERK activation.

Inhibition of EZH2 prevents fibrosis and restores normal angiogenesis in scleroderma.

Scleroderma (SSc) is a complex disease that involves activation of the immune system, vascular complications, and tissue fibrosis. The histone methyltransferase enhancer of zeste homolog 2 (EZH2) mediates trimethylation of lysine 27 of histone 3 (H3K27me3), which acts as a repressive epigenetic mark. Both EZH2 and H3K27me3 were elevated in SSc dermal fibroblasts and endothelial cells compared with healthy controls. EZH2 inhibitor DZNep halted fibrosis both in vitro and in vivo. In SSc fibroblasts, DZNep dose-dependently reduced the expression of profibrotic genes and inhibited migratory activity of SSc fibroblasts. We show that epigenetic dysregulation and overexpression of LRRC16A explains EZH2-mediated fibroblast migration in SSc. In endothelial cells, inhibition of EZH2 restored normal angiogenesis in SSc via activating the Notch pathway, specifically by up-regulating the Notch ligand DLL4. Our results demonstrate that overexpression of EZH2 in SSc fibroblasts and endothelial cells is profibrotic and antiangiogenic. Targeting EZH2 or EZH2-regulated genes might be of therapeutic potential in SSc.

Scleroderma dermal microvascular endothelial cells exhibit defective response to pro-angiogenic chemokines.

OBJECTIVES: Angiogenesis plays a critical role in SSc (scleroderma). The aim of this study was to examine the expression of growth-regulated protein-γ (Gro-γ/CXCL3), granulocyte chemotactic protein 2 (GCP-2/CXCL6) and their receptor CXCR2 in endothelial cells (ECs) isolated from SSc skin and determine whether these cells mount an angiogenic response towards pro-angiogenic chemokines. The downstream signalling pathways as well as the pro-angiogenic transcription factor inhibitor of DNA-binding protein 1 (Id-1) were also examined. METHODS: Skin biopsies were obtained from patients with dcSSc. ECs were isolated via magnetic positive selection. Angiogenesis was measured by EC chemotaxis assay. RESULTS: Gro-γ/CXCL3 and GCP-2/CXCL6 were minimally expressed in both skin types but elevated in SSc serum. Pro-angiogenic chemokine mRNA was greater in SSc ECs than in normal ECs. SSc ECs did not migrate to vascular endothelial growth factor (VEGF), Gro-γ/CXCL3, GCP-2/CXCL6 or CXCL16. The signalling pathways stimulated by these chemokines were also dysregulated. Id-1 mRNA in SSc ECs was lower compared with normal ECs, and overexpression of Id-1 in SSc ECs increased their ability to migrate towards VEGF and CXCL16. CONCLUSION: Our results show that SSc ECs are unable to respond to pro-angiogenic chemokines despite their increased expression in serum and ECs. This might be due to the differences in the signalling pathways activated by these chemokines in normal vs SSc ECs. In addition, the lower expression of Id-1 also decreases the angiogenic response. The inability of pro-angiogenic chemokines to promote EC migration provides an additional mechanism for the impaired angiogenesis that characterizes SSc.

Ligation of TLR5 promotes myeloid cell infiltration and differentiation into mature osteoclasts in rheumatoid arthritis and experimental arthritis.

Our aim was to examine the impact of TLR5 ligation in rheumatoid arthritis (RA) and experimental arthritis pathology. Studies were conducted to investigate the role of TLR5 ligation on RA and mouse myeloid cell chemotaxis or osteoclast formation, and in addition, to uncover the significance of TNF-α function in TLR5-mediated pathogenesis. Next, the in vivo mechanism of action was determined in collagen-induced arthritis (CIA) and local joint TLR5 ligation models. Last, to evaluate the importance of TLR5 function in RA, we used anti-TLR5 Ab therapy in CIA mice. We show that TLR5 agonist, flagellin, can promote monocyte infiltration and osteoclast maturation directly through myeloid TLR5 ligation and indirectly via TNF-α production from RA and mouse cells. These two identified TLR5 functions are potentiated by TNF-α, because inhibition of both pathways can more strongly impair RA synovial fluid-driven monocyte migration and osteoclast differentiation compared with each factor alone. In preclinical studies, flagellin postonset treatment in CIA and local TLR5 ligation in vivo provoke homing and osteoclastic development of myeloid cells, which are associated with the TNF-α cascade. Conversely, CIA joint inflammation and bone erosion are alleviated when TLR5 function is blocked. We found that TLR5 and TNF-α pathways are interconnected, because TNF-α is produced by TLR5 ligation in RA myeloid cells, and anti-TNF-α therapy can markedly suppress TLR5 expression in RA monocytes. Our novel findings demonstrate that a direct and an indirect mechanism are involved in TLR5-driven RA inflammation and bone destruction.

Evidence that CXCL16 is a potent mediator of angiogenesis and is involved in endothelial progenitor cell chemotaxis : studies in mice with K/BxN serum-induced arthritis.

OBJECTIVE: To examine the possibility that CXCL16 recruits endothelial cells (ECs) to developing neovasculature in rheumatoid arthritis (RA) synovium. METHODS: We utilized the RA synovial tissue SCID mouse chimera system to examine human microvascular EC (HMVEC) and human endothelial progenitor cell (EPC) recruitment into engrafted human synovium that was injected intragraft with CXCL16-immunodepleted RA synovial fluid (SF). CXCR6-deficient and wild-type (WT) C57BL/6 mice were primed to develop K/BxN serum-induced arthritis and evaluated for angiogenesis. HMVECs and EPCs from human cord blood were also examined for CXCR6 expression, by immunofluorescence and assessment of CXCL16 signaling activity. RESULTS: CXCR6 was prominently expressed on human EPCs and HMVECs, and its expression on HMVECs could be up-regulated by interleukin-1ß. SCID mice injected with CXCL16-depleted RA SF exhibited a significant reduction in EPC recruitment. In experiments using the K/BxN serum-induced inflammatory arthritis model, CXCR6(-/-) mice showed profound reductions in hemoglobin levels, which correlated with reductions in monocyte and T cell recruitment to arthritic joint tissue compared to that observed in WT mice. Additionally, HMVECs and EPCs responded to CXCL16 stimulation, but exhibited unique signal transduction pathways and homing properties. CONCLUSION: These results indicate that CXCL16 and its receptor CXCR6 may be a central ligand/receptor pair that is closely associated with EPC recruitment and blood vessel formation in the RA joint.

Metabolic reprogramming by Syntenin-1 directs RA FLS and endothelial cell-mediated inflammation and angiogenesis.

A novel rheumatoid arthritis (RA) synovial fluid protein, Syntenin-1, and its receptor, Syndecan-1 (SDC-1), are colocalized on RA synovial tissue endothelial cells and fibroblast-like synoviocytes (FLS). Syntenin-1 exacerbates the inflammatory landscape of endothelial cells and RA FLS by upregulating transcription of IRF1/5/7/9, IL-1ß, IL-6, and CCL2 through SDC-1 ligation and HIF1α, or mTOR activation. Mechanistically, Syntenin-1 orchestrates RA FLS and endothelial cell invasion via SDC-1 and/or mTOR signaling. In Syntenin-1 reprogrammed endothelial cells, the dynamic expression of metabolic intermediates coincides with escalated glycolysis along with unchanged oxidative factors, AMPK, PGC-1α, citrate, and inactive oxidative phosphorylation. Conversely, RA FLS rewired by Syntenin-1 displayed a modest glycolytic-ATP accompanied by a robust mitochondrial-ATP capacity. The enriched mitochondrial-ATP detected in Syntenin-1 reprogrammed RA FLS was coupled with mitochondrial fusion and fission recapitulated by escalated Mitofusin-2 and DRP1 expression. We found that VEGFR1/2 and Notch1 networks are responsible for the crosstalk between Syntenin-1 rewired endothelial cells and RA FLS, which are also represented in RA explants. Similar to RA explants, morphological and transcriptome studies authenticated the importance of VEGFR1/2, Notch1, RAPTOR, and HIF1α pathways in Syntenin-1 arthritic mice and their obstruction in SDC-1 deficient animals. Consistently, dysregulation of SDC-1, mTOR, and HIF1α negated Syntenin-1 inflammatory phenotype in RA explants, while inhibition of HIF1α impaired synovial angiogenic imprint amplified by Syntenin-1. In conclusion, since the current therapies are ineffective on Syntenin-1 and SDC-1 expression in RA synovial tissue and blood, targeting this pathway and its interconnected metabolic intermediates may provide a novel therapeutic strategy.

Pharmacological inhibition of TAK1 prevents and induces regression of experimental organ fibrosis.

Multiorgan fibrosis in systemic sclerosis (SSc) accounts for substantial mortality and lacks effective therapies. Lying at the crossroad of TGF-ß and TLR signaling, TGF-ß-activated kinase 1 (TAK1) might have a pathogenic role in SSc. We therefore sought to evaluate the TAK1 signaling axis in patients with SSc and to investigate pharmacological TAK1 blockade using a potentially novel drug-like selective TAK1 inhibitor, HS-276. Inhibiting TAK1 abrogated TGF-ß1 stimulation of collagen synthesis and myofibroblasts differentiation in healthy skin fibroblasts, and it ameliorated constitutive activation of SSc skin fibroblasts. Moreover, treatment with HS-276 prevented dermal and pulmonary fibrosis and reduced the expression of profibrotic mediators in bleomycin-treated mice. Importantly, initiating HS-276 treatment even after fibrosis was already established prevented its progression in affected organs. Together, these findings implicate TAK1 in the pathogenesis of SSc and identify targeted TAK1 inhibition using a small molecule as a potential strategy for the treatment of SSc and other fibrotic diseases.

Activation of Cytotoxic Lymphocytes Through CD6 Enhances Killing of Cancer Cells.

Immune checkpoint inhibitors (ICIs) have demonstrated efficacy and improved survival in a growing number of cancers. Despite their success, ICIs are associated with immune-related adverse events that can interfere with their use. Therefore, safer approaches are needed. CD6, expressed by T-lymphocytes and human NK cells, engages in cell-cell interactions by binding to its ligands CD166 (ALCAM) and CD318 (CDCP1). CD6 is a target protein for regulating immune responses and is required for the development of several mouse models of autoimmunity. Interestingly, CD6 is exclusively expressed on immune cells while CD318 is strongly expressed on most cancers. Here we demonstrate that disrupting the CD6-CD318 axis with UMCD6, an anti-CD6 monoclonal antibody, prolongs survival of mice in xenograft models of human breast and prostate cancer, treated with infusions of human lymphocytes. Analysis of tumor-infiltrating immune cells showed that augmentation of lymphocyte cytotoxicity by UMCD6 is due to effects of this antibody on NK, NKT and CD8+ T cells. Tumor-infiltrating cytotoxic lymphocytes were found in higher proportions and were activated in UMCD6-treated mice compared to controls. Similar changes in gene expression were observed by RNA-seq analysis of NK cells treated with UMCD6. Particularly, UMCD6 up-regulated the NKG2D-DAP10 complex and activated PI3K. Thus, the CD6-CD318 axis can regulate the activation state of cytotoxic lymphocytes and their positioning within the tumor microenvironment.

Inhibition of bromodomain extraterminal histone readers alleviates skin fibrosis in experimental models of scleroderma.

Binding of the bromodomain and extraterminal domain proteins (BETs) to acetylated histone residues is critical for gene transcription. We sought to determine the antifibrotic efficacy and potential mechanisms of BET inhibition in systemic sclerosis (SSc). Blockade of BETs was done using a pan-BET inhibitor, JQ1; BRD2 inhibitor, BIC1; or BRD4 inhibitors AZD5153 or ARV825. BET inhibition, specifically BRD4 blockade, showed antifibrotic effects in an animal model of SSc and in patient-derived diffuse cutaneous SSc (dcSSc) fibroblasts. Transcriptome analysis of JQ1-treated dcSSc fibroblasts revealed differentially expressed genes related to extracellular matrix, cell cycle, and calcium (Ca2+) signaling. The antifibrotic effect of BRD4 inhibition was mediated at least in part by downregulation of Ca2+/calmodulin-dependent protein kinase II α and reduction of intracellular Ca2+ concentrations. On the basis of these results, we propose targeting Ca2+ pathways or BRD4 as potentially novel therapeutic approaches for progressive tissue fibrosis.

Soluble CD13 induces inflammatory arthritis by activating the bradykinin receptor B1.

CD13, an ectoenzyme on myeloid and stromal cells, also circulates as a shed, soluble protein (sCD13) with powerful chemoattractant, angiogenic, and arthritogenic properties, which require engagement of a G protein-coupled receptor (GPCR). Here we identify the GPCR that mediates sCD13 arthritogenic actions as the bradykinin receptor B1 (B1R). Immunofluorescence and immunoblotting verified high expression of B1R in rheumatoid arthritis (RA) synovial tissue and fibroblast-like synoviocytes (FLSs), and demonstrated binding of sCD13 to B1R. Chemotaxis, and phosphorylation of Erk1/2, induced by sCD13, were inhibited by B1R antagonists. In ex vivo RA synovial tissue organ cultures, a B1R antagonist reduced secretion of inflammatory cytokines. Several mouse arthritis models, including serum transfer, antigen-induced, and local innate immune stimulation arthritis models, were attenuated in Cd13-/- and B1R-/- mice and were alleviated by B1R antagonism. These results establish a CD13/B1R axis in the pathogenesis of inflammatory arthritis and identify B1R as a compelling therapeutic target in RA and potentially other inflammatory diseases.

Expression and function of CXCL16 in a novel model of gout.

OBJECTIVE: To better define the activity of soluble CXCL16 in the recruitment of polymorphonuclear neutrophils (PMNs) in vivo, utilizing a novel animal model of gout involving engraftment of SCID mice with normal human synovial tissue (ST) injected intragraft with gouty human synovial fluid (SF). METHODS: For in vitro studies, a modified Boyden chemotaxis system was used to identify CXCL16 as an active recruitment factor for PMNs in gouty SF. Migration of PMNs could be reduced by neutralization of CXCL16 activity in gouty SF. For in vivo analyses, fluorescent dye-tagged PMNs were injected intravenously into SCID mice while, simultaneously, diluted gouty SF containing CXCL16, or depleted of CXCL16 by antibody blocking, was administered intragraft. In addition, the receptor for CXCL16, CXCR6, was inhibited by incubating PMNs with a neutralizing anti-CXCR6 antibody prior to injection into the mouse chimeras. Recruitment of PMNs to the gouty SF-injected normal human ST was then examined in this SCID mouse chimera system. RESULTS: CXCL16 concentrations were highly elevated in gouty SF, and PMNs were observed to migrate in response to CXCL16 in vitro. Normal human ST-SCID mouse chimeras injected intragraft with gouty SF that had been depleted of CXCL16 during PMN transfer showed a significant reduction of 50% in PMN recruitment to engrafted tissue as compared with that after administration of sham-depleted gouty SF. Similar findings were achieved when PMNs were incubated with a neutralizing anti-CXCR6 antibody before injection into chimeras. CONCLUSION: Overall, the results of this study outline the effectiveness of the human-SCID mouse chimera system as a viable animal model of gout, serving to identify the primary function of CXCL16 as a significant mediator of in vivo recruitment of PMNs to gouty SF.

IRAK4 inhibition: a promising strategy for treating RA joint inflammation and bone erosion.

Flares of joint inflammation and resistance to currently available biologic therapeutics in rheumatoid arthritis (RA) patients could reflect activation of innate immune mechanisms. Herein, we show that a TLR7 GU-rich endogenous ligand, miR-Let7b, potentiates synovitis by amplifying RA monocyte and fibroblast (FLS) trafficking. miR-Let7b ligation to TLR7 in macrophages (MΦs) and FLSs expanded the synovial inflammatory response. Moreover, secretion of M1 monokines triggered by miR-Let7b enhanced Th1/Th17 cell differentiation. We showed that IRAK4 inhibitor (i) therapy attenuated RA disease activity by blocking TLR7-induced M1 MΦ or FLS activation, as well as monokine-modulated Th1/Th17 cell polarization. IRAK4i therapy also disrupted RA osteoclastogenesis, which was amplified by miR-Let7b ligation to joint myeloid TLR7. Hence, the effectiveness of IRAK4i was compared with that of a TNF inhibitor (i) or anti-IL-6R treatment in collagen-induced arthritis (CIA) and miR-Let7b-mediated arthritis. We found that TNF or IL-6R blocking therapies mitigated CIA by reducing the infiltration of joint F480+iNOS+ MΦs, the expression of certain monokines, and Th1 cell differentiation. Unexpectedly, these biologic therapies were unable to alleviate miR-Let7b-induced arthritis. The superior efficacy of IRAK4i over anti-TNF or anti-IL-6R therapy in miR-Let7b-induced arthritis or CIA was due to the ability of IRAK4i therapy to restrain the migration of joint F480+iNOS+ MΦs, vimentin+ fibroblasts, and CD3+ T cells, in addition to negating the expression of a wide range of monokines, including IL-12, MIP2, and IRF5 and Th1/Th17 lymphokines. In conclusion, IRAK4i therapy may provide a promising strategy for RA therapy by disconnecting critical links between inflammatory joint cells.

Interleukin-34 Reprograms Glycolytic and Osteoclastic Rheumatoid Arthritis Macrophages via Syndecan 1 and Macrophage Colony-Stimulating Factor Receptor.

OBJECTIVE: In rheumatoid arthritis (RA), elevated serum interleukin-34 (IL-34) levels are linked with increased disease severity. IL-34 binds to 2 receptors, macrophage colony-stimulating factor receptor (M-CSFR) and syndecan 1, which are coexpressed in RA macrophages. Expression of both IL-34 and syndecan 1 is strikingly elevated in the RA synovium, yet their mechanisms of action remain undefined. This study was undertaken to investigate the mechanism of action of IL-34 in RA. METHODS: To characterize the significance of IL-34 in immunometabolism, its mechanism of action was elucidated in joint macrophages, fibroblasts, and T effector cells using RA and preclinical models. RESULTS: Intriguingly, syndecan 1 activated IL-34-induced M-CSFR phosphorylation and reprogrammed RA naive cells into distinctive CD14+CD86+GLUT1+ M34 macrophages that expressed elevated levels of IL-1ß, CXCL8, and CCL2. In murine M34 macrophages, the inflammatory phenotype was accompanied by potentiated glycolytic activity, exhibited by transcriptional up-regulation of GLUT1, c-Myc, and hypoxia-inducible factor 1α (HIF-1α) and amplified pyruvate and l-lactate secretion. Local expression of IL-34 provoked arthritis by expanding the glycolytic F4/80-positive, inducible nitric oxide synthase (iNOS)-positive macrophage population, which in turn attracted fibroblasts and polarized Th1/Th17 cells. The cross-talk between murine M34 macrophages and Th1/Th17 cells broadened the inflammatory and metabolic phenotypes, resulting in the expansion of IL-34 pathogenicity. Consequently, IL-34-instigated joint inflammation was alleviated in RAG-/- mice compared to wild-type mice. Syndecan 1 deficiency attenuated IL-34-induced arthritis by interfering with joint glycolytic M34 macrophage and osteoclast remodeling. Similarly, inhibition of glycolysis by 2-deoxy-d-glucose reversed the joint swelling and metabolic rewiring triggered by IL-34 via HIF-1α and c-Myc induction. CONCLUSION: IL-34 is a novel endogenous factor that remodels hypermetabolic M34 macrophages and facilitates their cross-regulation with T effector cells to advance inflammatory bone destruction in RA.

CD6 is a target for cancer immunotherapy.

Limitations of checkpoint inhibitor cancer immunotherapy include induction of autoimmune syndromes and resistance of many cancers. Since CD318, a novel CD6 ligand, is associated with the aggressiveness and metastatic potential of human cancers, we tested the effect of an anti-CD6 monoclonal antibody, UMCD6, on killing of cancer cells by human lymphocytes. UMCD6 augmented killing of breast, lung, and prostate cancer cells through direct effects on both CD8+ T cells and NK cells, increasing cancer cell death and lowering cancer cell survival in vitro more robustly than monoclonal antibody checkpoint inhibitors that interrupt the programmed cell death 1 (PD-1)/PD-1 ligand 1 (PD-L1) axis. UMCD6 also augmented in vivo killing by human peripheral blood lymphocytes of a human breast cancer line xenotransplanted into immunodeficient mice. Mechanistically, UMCD6 upregulated the expression of the activating receptor NKG2D and downregulated expression of the inhibitory receptor NKG2A on both NK cells and CD8+ T cells, with concurrent increases in perforin and granzyme B production. The combined capability of an anti-CD6 monoclonal antibody to control autoimmunity through effects on CD4+ lymphocyte differentiation while enhancing killing of cancer cells through distinct effects on CD8+ and NK cells opens a potential new approach to cancer immunotherapy that would suppress rather than instigate autoimmunity.

Interleukin 18 induces angiogenesis in vitro and in vivo via Src and Jnk kinases.

BACKGROUND: Interleukin 18 (IL-18) is a novel mediator of angiogenesis in rheumatoid arthritis (RA). OBJECTIVE: To examine the role of IL-18 in RA angiogenesis and the signalling mechanisms involved. METHODS: Human dermal microvascular endothelial cell (HMVEC) chemotaxis, capillary morphogenesis assays and Matrigel plug angiogenesis assays were performed in vivo using IL-18 with or without signalling inhibitors. A novel model of angiogenesis was devised using dye-tagged HMVECs to study their homing into RA and normal (NL) synovial tissues (STs) engrafted in severe combined immunodeficient (SCID) mice. RESULTS: IL-18-mediated angiogenesis depended on Src and Jnk, as the inhibitors of Src and Jnk blocked IL-18-induced HMVEC chemotaxis, tube formation and angiogenesis in Matrigel plugs. However, inhibitors of Janus kinase 2, p38, MEK, phosphatidylinositol-3-kinase and neutralising antibodies to vascular endothelial growth factor or stromal derived factor-1α did not alter IL-18-induced HMVEC migration. These results were confirmed with Jnk or Src sense or antisense oligodeoxynucleotides. Moreover, IL-18 induced phosphorylation of Src and Jnk in HMVECs. As proof of principle, IL-18 null mice had a significantly decreased angiogenesis compared with wild-type mice in Matrigel plug angiogenesis assays in vivo. IL-18 markedly enhanced mature HMVEC homing to human RA ST compared with NL ST in SCID mice, confirming the role of IL-18-induced angiogenesis in RA ST in vivo. CONCLUSION: Targeting IL-18 or its signalling intermediates may prove to be a potentially novel therapeutic strategy for angiogenesis-dependent diseases, such as RA.

Attenuation of Murine Collagen-Induced Arthritis by Targeting CD6.

OBJECTIVE: CD6 is an important regulator of T cell function that interacts with the ligands CD166 and CD318. To further clarify the significance of CD6 in rheumatoid arthritis (RA), we examined the effects of targeting CD6 in the mouse model of collagen-induced arthritis (CIA), using CD6-knockout (CD6-KO) mice and CD6-humanized mice that express human CD6 in lieu of mouse CD6 on their T cells. METHODS: We immunized wild-type (WT) and CD6 gene-KO mice with a collagen emulsion to induce CIA. For treatment studies using CD6-humanized mice, mice were immunized similarly and a mouse anti-human CD6 IgG (UMCD6) or control IgG was injected on days 7, 14, and 21. Joint tissues were evaluated for tissue damage, leukocyte infiltration, and local inflammatory cytokine production. Collagen-specific Th1, Th9, and Th17 responses and serum levels of collagen-specific IgG subclasses were also evaluated in WT and CD6-KO mice with CIA. RESULTS: The absence of CD6 reduced 1) collagen-specific Th9 and Th17, but not Th1 responses, 2) the levels of many proinflammatory joint cytokines, and 3) serum levels of collagen-reactive total IgG and IgG1, but not IgG2a and IgG3. Joint homogenate hemoglobin content was significantly reduced in CD6-KO mice with CIA compared to WT mice with CIA (P < 0.05) (reduced angiogenesis). Moreover, treating CD6-humanized mice with mouse anti-human CD6 monoclonal antibody was similarly effective in reducing joint inflammation in CIA. CONCLUSION: Taken together, these data suggest that interaction of CD6 with its ligands is important for the perpetuation of CIA and other inflammatory arthritides that are T cell driven.

A unique role for galectin-9 in angiogenesis and inflammatory arthritis.

BACKGROUND: Galectin-9 (Gal-9) is a mammalian lectin secreted by endothelial cells that is highly expressed in rheumatoid arthritis synovial tissues and synovial fluid. Roles have been proposed for galectins in the regulation of inflammation and angiogenesis. Therefore, we examined the contribution of Gal-9 to angiogenesis and inflammation in arthritis. METHODS: To determine the role of Gal-9 in angiogenesis, we performed human dermal microvascular endothelial cell (HMVEC) chemotaxis, Matrigel tube formation, and mouse Matrigel plug angiogenesis assays. We also examined the role of signaling molecules in Gal-9-induced angiogenesis by using signaling inhibitors and small interfering RNA (siRNA). We performed monocyte (MN) migration assays in a modified Boyden chamber and assessed the arthritogenicity of Gal-9 by injecting Gal-9 into mouse knees. RESULTS: Gal-9 significantly increased HMVEC migration, which was decreased by inhibitors of extracellular signal-regulating kinases 1/2 (Erk1/2), p38, Janus kinase (Jnk), and phosphatidylinositol 3-kinase. Gal-9 HMVEC-induced tube formation was reduced by Erk1/2, p38, and Jnk inhibitors, and this was confirmed by siRNA knockdown. In mouse Matrigel plug assays, plugs containing Gal-9 induced significantly higher angiogenesis, which was attenuated by a Jnk inhibitor. Gal-9 also induced MN migration, and there was a marked increase in MN ingress when C57BL/6 mouse knees were injected with Gal-9 compared with the control, pointing to a proinflammatory role for Gal-9. CONCLUSIONS: Gal-9 mediates angiogenesis, increases MN migration in vitro, and induces acute inflammatory arthritis in mice, suggesting a novel role for Gal-9 in angiogenesis, joint inflammation, and possibly other inflammatory diseases.

DEK-targeting DNA aptamers as therapeutics for inflammatory arthritis.

Novel therapeutics are required for improving the management of chronic inflammatory diseases. Aptamers are single-stranded RNA or DNA molecules that have recently shown utility in a clinical setting, as they can specifically neutralize biomedically relevant proteins, particularly cell surface and extracellular proteins. The nuclear chromatin protein DEK is a secreted chemoattractant that is abundant in the synovia of patients with juvenile idiopathic arthritis (JIA). Here, we show that DEK is crucial to the development of arthritis in mouse models, thus making it an appropriate target for aptamer-based therapy. Genetic depletion of DEK or treatment with DEK-targeted aptamers significantly reduces joint inflammation in vivo and greatly impairs the ability of neutrophils to form neutrophil extracellular traps (NETs). DEK is detected in spontaneously forming NETs from JIA patient synovial neutrophils, and DEK-targeted aptamers reduce NET formation. DEK is thus key to joint inflammation, and anti-DEK aptamers hold promise for the treatment of JIA and other types of arthritis.