Notch ligands are biomarkers of anti-TNF response in RA patients.

Notch and its ligands play a critical role in rheumatoid arthritis (RA) pathogenesis. Hence, studies were conducted to delineate the functional significance of the Notch pathway in RA synovial tissue (ST) cells and the influence of RA therapies on their expression. Morphological studies reveal that JAG1, DLL4, and Notch1 are highly enriched in RA ST lining and sublining CD68+CD14+ MΦs. JAG1 and DLL4 transcription is jointly upregulated in RA MΦs reprogrammed by TLR4/5 ligation and TNF, whereas Syntenin-1 exposure expands JAG1, DLL4, and Notch1 expression levels in these cells. Single-cell RNA-seq data exhibit that JAG1 and Notch3 are overexpressed on all fibroblast-like synoviocyte (FLS) subpopulations, in parallel, JAG2, DLL1, and Notch1 expression levels are modest on RA FLS and are predominately potentiated by TLR4 ligation. Intriguingly, JAG1, DLL1/4, and Notch1/3 are presented on RA endothelial cells, and their expression is mutually reconfigured by TLR4/5 ligation in the endothelium. Synovial JAG1/JAG2/DLL1 or Notch1/3 transcriptomes were unchanged in patients who received disease-modifying anti-rheumatic drugs (DMARDs) or IL-6R Ab therapy regardless of disease activity score. Uniquely, RA MΦs and endothelial cells rewired by IL-6 displayed DLL4 transcriptional upregulation, and IL-6R antibody treatment disrupted RA ST DLL4 transcription in good responders compared to non-responders or moderate responders. Nevertheless, the JAG1/JAG2/DLL1/DLL4 transcriptome was diminished in anti-TNF good responders with myeloid pathotype and was unaltered in the fibroid pathotype except for DLL4. Taken together, our findings suggest that RA myeloid Notch ligands can serve as markers for anti-TNF responsiveness and trans-activate Notch receptors expressed on RA FLS and/or endothelial cells.

Syntenin-1-mediated arthritogenicity is advanced by reprogramming RA metabolic macrophages and Th1 cells.

OBJECTIVES: Syntenin-1, a novel endogenous ligand, was discovered to be enriched in rheumatoid arthritis (RA) specimens compared with osteoarthritis synovial fluid and normal synovial tissue (ST). However, the cellular origin, immunoregulation and molecular mechanism of syntenin-1 are undescribed in RA. METHODS: RA patient myeloid and lymphoid cells, as well as preclinical models, were used to investigate the impact of syntenin-1/syndecan-1 on the inflammatory and metabolic landscape. RESULTS: Syntenin-1 and syndecan-1 (SDC-1) co-localise on RA ST macrophages (MΦs) and endothelial cells. Intriguingly, blood syntenin-1 and ST SDC-1 transcriptome are linked to cyclic citrullinated peptide, erythrocyte sedimentation rate, ST thickness and bone erosion. Metabolic CD14+CD86+GLUT1+MΦs reprogrammed by syntenin-1 exhibit a wide range of proinflammatory interferon transcription factors, monokines and glycolytic factors, along with reduced oxidative intermediates that are downregulated by blockade of SDC-1, glucose uptake and/or mTOR signalling. Inversely, IL-5R and PDZ1 inhibition are ineffective on RA MΦs-reprogrammed by syntenin-1. In syntenin-1-induced arthritis, F4/80+iNOS+RAPTOR+MΦs represent glycolytic RA MΦs, by amplifying the inflammatory and glycolytic networks. Those networks are abrogated in SDC-1-/- animals, while joint prorepair monokines are unaffected and the oxidative metabolites are moderately replenished. In RA cells and/or preclinical model, syntenin-1-induced arthritogenicity is dependent on mTOR-activated MΦ remodelling and its ability to cross-regulate Th1 cells via IL-12 and IL-18 induction. Moreover, RA and joint myeloid cells exposed to Syntenin-1 are primed to transform into osteoclasts via SDC-1 ligation and RANK, CTSK and NFATc1 transcriptional upregulation. CONCLUSION: The syntenin-1/SDC-1 pathway plays a critical role in the inflammatory and metabolic landscape of RA through glycolytic MΦ and Th1 cell cross-regulation (graphical abstract).

Inhibition of IRAK4 dysregulates SARS-CoV-2 spike protein-induced macrophage inflammatory and glycolytic reprogramming.

Escalated innate immunity plays a critical role in SARS-CoV-2 pathology; however, the molecular mechanism is incompletely understood. Thus, we aim to characterize the molecular mechanism by which SARS-CoV-2 Spike protein advances human macrophage (MÏ´) inflammatory and glycolytic phenotypes and uncover novel therapeutic strategies. We found that human MÏ´s exposed to Spike protein activate IRAK4 phosphorylation. Blockade of IRAK4 in Spike protein-stimulated MÏ´s nullifies signaling of IRAK4, AKT, and baseline p38 without affecting ERK and NF-κB activation. Intriguingly, IRAK4 inhibitor (IRAK4i) rescues the SARS-CoV-2-induced cytotoxic effect in ACE2+HEK 293 cells. Moreover, the inflammatory reprogramming of MÏ´s by Spike protein was blunted by IRAK4i through IRF5 and IRF7, along with the reduction of monokines, IL-6, IL-8, TNFα, and CCL2. Notably, in Spike protein-stimulated MÏ´s, suppression of the inflammatory markers by IRAK4i was coupled with the rebalancing of oxidative phosphorylation over metabolic activity. This metabolic adaptation promoted by IRAK4i in Spike protein-activated MÏ´s was shown to be in part through constraining PFKBF3, HIF1α, cMYC, LDHA, lactate expression, and reversal of citrate and succinate buildup. IRAK4 knockdown could comparably impair Spike protein-enhanced inflammatory and metabolic imprints in human MÏ´s as those treated with ACE2, TLR2, and TLR7 siRNA. Extending these results, in murine models, where human SARS-CoV-2 Spike protein was not recognized by mouse ACE2, TLRs were responsible for the inflammatory and glycolytic responses instigated by Spike protein and were dysregulated by IRAK4i therapy. In conclusion, IRAK4i may be a promising strategy for severe COVID-19 patients by counter-regulating ACE2 and TLR-mediated MÏ´ hyperactivation. IRAK4i therapy counteracts MÏ´ inflammatory and glycolytic reprogramming triggered by Spike protein. This study illustrates that SARS-CoV-2 Spike protein activates IRAK4 signaling via ACE2 as well as TLR2 and TLR7 sensing in human MÏ´s. Remarkably, IRAK4i treatment can dysregulate both ACE-dependent and independent (via TLR sensing) SARS-CoV-2 Spike protein-activated inflammatory and metabolic imprints.

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.

Metabolic reprogramming of macrophages instigates CCL21-induced arthritis.

This study was designed to delineate the functional significance of CCL21 in metabolic reprogramming in experimental arthritis and differentiated rheumatoid arthritis (RA) macrophages (MΦs). To characterize the influence of CCL21 on immunometabolism, its mechanism of action was elucidated by dysregulating glucose uptake in preclinical arthritis and RA MΦs. In CCL21 arthritic joints, the glycolytic intermediates hypoxia-inducible factor 1α (HIF1α), cMYC and GLUT1 were overexpressed compared with oxidative regulators estrogen-related receptor γ and peroxisome proliferator-activated receptor gamma coactivator 1 (PGC1)-α. Interestingly, 2-deoxy-D-glucose (2-DG) therapy mitigated CCL21-induced arthritis by restraining the number of joint F4/80+ iNOS+ MΦs without impacting F4/80+ Arginase+ MΦs. Similar to the preclinical findings, blockade of glycolysis negated CCL21-polarized CD14+ CD86+ GLUT+ MΦ frequency; however, CD14+ CD206+ GLUT+ MΦs were not implicated in this process. In CCL21-induced arthritis and differentiated RA MΦs, the inflammatory imprint was uniquely intercepted by 2-DG via interleukin-6 (IL-6) downregulation. Despite the more expansive inflammatory response of CCL21 in the arthritic joints relative to the differentiated RA MΦs, 2-DG was ineffective in joint tumor necrosis factor-α, IL-1ß, CCL2 and CCL5 enrichment. By contrast, disruption of glycolysis markedly impaired CCL21-induced HIF1α and cMYC signaling in arthritic mice. Notably, in RA MΦs, glycolysis interception was directed toward dysregulating CCL21-enhanced HIF1α transcription. Nonetheless, in concurrence with the diminished IL-6 levels, CCL21 differentiation of CD14+ CD86+ GLUT1+ MΦs was reversed by glycolysis and HIIF1α inhibition. Moreover, in the CCL21 experimental arthritis or differentiated RA MΦs, the malfunctioning metabolic machinery was accompanied by impaired oxidative phosphorylation because of reduced PGC1α or peroxisome proliferator-activated receptor-γ expression. CCL21 reconfigures naïve myeloid cells into glycolytic RA CD14+ CD86+ GLUT+ IL-6high HIF1αhigh MΦs. Therefore, inhibiting the CCL21/CCR7 pathway may provide a promising therapeutic strategy.

Metabolic regulation of RA macrophages is distinct from RA fibroblasts and blockade of glycolysis alleviates inflammatory phenotype in both cell types.

Recent studies have shown the significance of metabolic reprogramming in immune and stromal cell function. Yet, the metabolic reconfiguration of RA macrophages (MΦs) is incompletely understood during active disease and in crosstalk with other cell types in experimental arthritis. This study elucidates a distinct regulation of glycolysis and oxidative phosphorylation in RA MΦs compared to fibroblast (FLS), although PPP (Pentose Phosphate pathway) is similarly reconfigured in both cell types. 2-DG treatment showed a more robust impact on impairing the RA M1 MΦ-mediated inflammatory phenotype than IACS-010759 (IACS, complexli), by reversing ERK, AKT and STAT1 signaling, IRF8/3 transcription and CCL2 or CCL5 secretion. This broader inhibitory effect of 2-DG therapy on RA M1 MΦs was linked to dysregulation of glycolysis (GLUT1, PFKFB3, LDHA, lactate) and oxidative PPP (NADP conversion to NADPH), while both compounds were ineffective on oxidative phosphorylation. Distinctly, in RA FLS, 2-DG and IACS therapies constrained LPS/IFNγ-induced AKT and JNK signaling, IRF5/7 and fibrokine expression. Disruption of RA FLS metabolic rewiring by 2-DG or IACS therapy was accompanied by a reduction of glycolysis (HIF1α, PFKFB3) and suppression of citrate or succinate buildup. We found that 2-DG therapy mitigated CIA pathology by intercepting joint F480+iNOS+MΦ, Vimentin+ fibroblast and CD3+T cell trafficking along with downregulation of IRFs and glycolytic intermediates. Surprisingly, IACS treatment was inconsequential on CIA swelling, cell infiltration, M1 and Th1/Th17 cytokines (IFN-γ/IL-17) and joint glycolytic mediators. Collectively, our results indicate that blockade of glycolysis is more effective than inhibition of complex 1 in CIA, in part due to its effectiveness on the MΦ inflammatory phenotype.

CCL21/CCR7 signaling in macrophages promotes joint inflammation and Th17-mediated osteoclast formation in rheumatoid arthritis.

In rheumatoid arthritis (RA), synovial tissue abundantly expresses CCL21, a chemokine strongly associated with RA susceptibility. In this study, we aimed to characterize the functional significance of CCL21/CCR7 signaling in different phases of RA pathogenesis. We determined that CCR7 is a hallmark of RA M1 synovial fluid (SF) macrophages, and its expression in RA monocytes and in vitro differentiated macrophages is closely associated with disease activity score (DAS28). In early stages of RA, monocytes infiltrate the synovial tissue. However, blockade of SF CCL21 or CCR7 prevents RA SF-mediated monocyte migration. CCR7 expression in the newly migrated macrophages can be accentuated by LPS and IFNγ and suppressed by IL-4 treatment. We also uncovered that CCL21 stimulation increases the number of M1-polarized macrophages (CD14+CD86+), resulting in elevated transcription of IL-6 and IL-23. These CCL21-induced M1 cytokines differentiate naïve T cells to Th17 cells, without affecting Th1 cell polarization. In the erosive stages of disease, CCL21 potentiates RA osteoclastogenesis through M1-driven Th17 polarization. Disruption of this intricate crosstalk, by blocking IL-6, IL-23, or IL-17 function, impairs the osteoclastogenic capacity of CCL21. Consistent with our in vitro findings, we establish that arthritis mediated by CCL21 expands the joint inflammation to bone erosion by connecting the differentiation of M1 macrophages with Th17 cells. Disease progression is further exacerbated by CCL21-induced neovascularization. We conclude that CCL21 is an attractive novel target for RA therapy, as blockade of its function may abrogate erosive arthritis modulated by M1 macrophages and Th17 cell crosstalk.

Inhibition of Human Cytomegalovirus Entry into Host Cells Through a Pleiotropic Small Molecule.

Human cytomegalovirus (HCMV) infections are wide-spread among the general population with manifestations ranging from asymptomatic to severe developmental disabilities in newborns and life-threatening illnesses in individuals with a compromised immune system. Nearly all current drugs suffer from one or more limitations, which emphasizes the critical need to develop new approaches and new molecules. We reasoned that a 'poly-pharmacy' approach relying on simultaneous binding to multiple receptors involved in HCMV entry into host cells could pave the way to a more effective therapeutic outcome. This work presents the study of a synthetic, small molecule displaying pleiotropicity of interactions as a competitive antagonist of viral or cell surface receptors including heparan sulfate proteoglycans and heparan sulfate-binding proteins, which play important roles in HCMV entry and spread. Sulfated pentagalloylglucoside (SPGG), a functional mimetic of heparan sulfate, inhibits HCMV entry into human foreskin fibroblasts and neuroepithelioma cells with high potency. At the same time, SPGG exhibits no toxicity at levels as high as 50-fold more than its inhibition potency. Interestingly, cell-ELISA assays showed downregulation in HCMV immediate-early gene 1 and 2 (IE 1&2) expression in presence of SPGG further supporting inhibition of viral entry. Finally, HCMV foci were observed to decrease significantly in the presence of SPGG suggesting impact on viral spread too. Overall, this work offers the first evidence that pleiotropicity, such as demonstrated by SPGG, may offer a new poly-therapeutic approach toward effective inhibition of HCMV.

IL-11 facilitates a novel connection between RA joint fibroblasts and endothelial cells.

IL-11 has been detected in inflamed joints; however, its role in the pathogenesis of arthritis is not yet clear. Studies were conducted to characterize the expression and functional significance of IL-11 and IL-11Rα in rheumatoid arthritis (RA). IL-11 levels were elevated in RA synovial fluid (SF) compared to osteoarthritis (OA) SF and plasma from RA, OA and normal individuals (NLs). Morphologic studies established that IL-11 was detected in lining fibroblasts and macrophages in addition to sublining endothelial cells and macrophages at higher levels in RA compared to NL synovial tissues. Since IL-11Rα was exclusively expressed in RA fibroblasts and endothelial cells, macrophages were not involved in IL-11 effector function. Ligation of IL-11 to IL-11Rα strongly provoked fibroblast infiltration into RA joint, while cell proliferation was unaffected by this process. Secretion of IL-8 and VEGF from IL-11 activated RA fibroblasts was responsible for the indirect effect of IL-11 on endothelial cell transmigration and tube formation. Moreover, IL-11 blockade impaired RA SF capacity to elicit endothelial cell transmigration and tube formation. We conclude that IL-11 binding to endothelial IL-11Rα can directly induce RA angiogenesis. In addition, secretion of proangiogenic factors from migrating fibroblasts potentiated by IL-11 can indirectly contribute to RA neovascularization.

Differential impact of obesity on the pathogenesis of RA or preclinical models is contingent on the disease status.

OBJECTIVE: Studies were performed to uncover the significance of obesity in rheumatoid arthritis (RA) and preclinical models. METHODS: Preclinical arthritis models were used to examine the impact of obesity on disease onset and remission. Conditioned media from RA adipose tissues were used to investigate the mechanism contributing to joint neutrophil influx and M1 macrophage differentiation observed in early and remission phases of arthritis. RESULTS: We report that mice fed with high fat diet (HFD) have an earlier onset of collagen-induced arthritis (CIA) compared with mice on regular diet. However, the differences in CIA joint swelling between the two diet groups are lost once disease is established. We found that early arthritis triggered by obesity is due to elevated joint MIP2/interleukin-8 levels detected in CIA as well as in the RA and mouse adipose tissues and the effect of this chemokine on neutrophil recruitment. Although active disease progression is similarly affected in both diet groups, arthritis resolution is accelerated in lean mice while joint inflammation is sustained in obese mice. We document that HFD can prolong toll-like receptor (TLR)4-induced arthritis by increasing joint monocyte migration and further remodelling the recruited cells into M1 macrophages. Consistently, we show that adipose condition media can transform RA and wild-type naïve myeloid cells into M1 macrophages; however, this function is impaired by TLR4 blockade or deficiency. CONCLUSIONS: We conclude that despite established disease being unaffected by obesity, the early and the resolution phases of RA are impacted by obesity through different mechanisms.

A role for 3-O-sulfated heparan sulfate in promoting human cytomegalovirus infection in human iris cells.

Human cytomegalovirus (HCMV) has emerged as a clinically opportunistic pathogen that targets multiple types of ocular cells and tissues, including the iris region of the uveal tract during anterior uveitis. In this report, we used primary cultures of human iris stroma (HIS) cells derived from human eye donors to investigate HCMV entry. The following lines of evidence suggested the role of 3-O-sulfated heparan sulfate (3-OS HS) during HCMV-mediated entry and cell-to-cell fusion in HIS cells. First, 3-O-sulfotransferase-3 (3-OST-3) expression in HIS cells promoted HCMV internalization, while pretreatment of HIS cells with heparinase enzyme or with anti-3-OS HS (G2) peptide significantly reduced the HCMV-mediated formation of plaques/foci. Second, coculture of the HCMV-infected HIS cells with CHO-K1 cells expressing 3-OS HS significantly enhanced cell fusion. Finally, a similar trend of enhanced fusion was observed with cells expressing HCMV glycoproteins (gB, gO, and gH-gL) cocultured with 3-OS HS cells. Taken together, these results highlight the role of 3-OS HS during HCMV plaque formation and cell-to-cell fusion and identify a novel target for future therapeutic interventions.

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.

The pathogenic role of angiogenesis in rheumatoid arthritis.

Angiogenesis is the formation of new capillaries from pre-existing vasculature, which plays a critical role in the pathogenesis of several inflammatory autoimmune diseases such as rheumatoid arthritis (RA), spondyloarthropathies, psoriasis, systemic lupus erythematosus, systemic sclerosis, and atherosclerosis. In RA, excessive migration of circulating leukocytes into the inflamed joint necessitates formation of new blood vessels to provide nutrients and oxygen to the hypertrophic joint. The dominance of the pro-angiogenic factors over the endogenous angiostatic mediators triggers angiogenesis. In this review article, we highlight the underlying mechanisms by which cells present in the RA synovial tissue are modulated to secrete pro-angiogenic factors. We focus on the significance of pro-angiogenic factors such as growth factors, hypoxia-inducible factors, cytokines, chemokines, matrix metalloproteinases, and adhesion molecules on RA pathogenesis. As pro-angiogenic factors are primarily produced from RA synovial tissue macrophages and fibroblasts, we emphasize the key role of RA synovial tissue lining layer in maintaining synovitis through neovascularization. Lastly, we summarize the specific approaches utilized to target angiogenesis. We conclude that the formation of new blood vessels plays an indispensable role in RA progression. However, since the function of several pro-angiogenic mediators is cross regulated, discovering novel approaches to target multiple cascades or selecting an upstream cascade that impairs the activity of a number of pro-angiogenic factors may provide a promising strategy for RA therapy.

Characterising the expression and function of CCL28 and its corresponding receptor, CCR10, in RA pathogenesis.

OBJECTIVE: This study was conducted to determine the expression pattern, regulation and function of CCL28 and CCR10 in rheumatoid arthritis (RA) pathogenesis. METHODS: Expression of CCL28 and CCR10 was assessed in RA compared with other arthritis synovial tissues (STs) or fluids (SFs) by histology or ELISA. The factors modulating CCL28 and CCR10 expression were identified in RA myeloid and endothelial cells by ELISA, FACS and Western blotting. The mechanism by which CCL28 ligation promotes RA angiogenesis was examined in control and CCR10-knockdown endothelial cell chemotaxis and capillary formation. RESULTS: CCL28 and/or CCR10 expression levels were accentuated in STs and SFs of patients with joint disease compared with normal controls and they were predominately coexpressed in RA myeloid and endothelial cells. We show that protein expression of CCL28 and CCR10 was modulated by tumour necrosis factor (TNF)-α and toll-like receptor 4 ligation in RA monocytes and endothelial cells and by interleukin (IL)-6 stimulation in RA macrophages. Neutralisation of CCL28 in RA SF or blockade of CCR10 on human endothelial progenitor cells (EPCs) significantly reduced SF-induced endothelial migration and capillary formation, demonstrating that ligation of joint CCL28 to endothelial CCR10+ cells is involved in RA angiogenesis. We discovered that angiogenesis driven by ligation of CCL28 to CCR10 is linked to the extracellular signal regulated kinase (ERK) cascade, as CCR10-knockdown cells exhibit dysfunctional CCL28-induced ERK signalling, chemotaxis and capillary formation. CONCLUSIONS: The overexpression of CCL28 and CCR10 in RA ST and their contribution to EPC migration into RA joints support the CCL28/CCR10 cascade as a potential therapeutic target for RA.

The novel role of IL-7 ligation to IL-7 receptor in myeloid cells of rheumatoid arthritis and collagen-induced arthritis.

Although the role of IL-7 and IL-7R has been implicated in the pathogenesis of rheumatoid arthritis (RA), the majority of the studies have focused on the effect of IL-7/IL-7R in T cell development and function. Our novel data, however, document that patients with RA and greater disease activity have higher levels of IL-7, IL-7R, and TNF-α in RA monocytes, suggesting a feedback regulation between IL-7/IL-7R and TNF-α cascades in myeloid cells that is linked to chronic disease progression. Investigations into the involved mechanism showed that IL-7 is a novel and potent chemoattractant that attracts IL-7R(+) monocytes through activation of the PI3K/AKT1 and ERK pathways at similar concentrations of IL-7 detected in RA synovial fluid. To determine whether ligation of IL-7 to IL-7R is a potential target for RA treatment and to identify their mechanism of action, collagen-induced arthritis (CIA) was therapeutically treated with anti-IL-7 Ab or IgG control. Anti-IL-7 Ab treatment significantly reduces CIA monocyte recruitment and osteoclast differentiation as well as potent joint monocyte chemoattractants and bone erosion markers, suggesting that both direct and indirect pathways might contribute to the observed effect. We also demonstrate that reduction in joint MIP-2 levels is responsible for suppressed vascularization detected in mice treated with anti-IL-7 Ab compared with the control group. To our knowledge, we show for the first time that expression of IL-7/IL-7R in myeloid cells is strongly correlated with RA disease activity and that ligation of IL-7 to IL-7R contributes to monocyte homing, differentiation of osteoclasts, and vascularization in the CIA effector phase.

Susceptibility of human iris stromal cells to herpes simplex virus 1 entry.

Ocular herpes simplex virus 1 (HSV-1) infection can lead to multiple complications, including iritis, an inflammation of the iris. Here, we use human iris stroma cells as a novel in vitro model to demonstrate HSV-1 entry and the inflammatory mediators that can damage the iris. The upregulated cytokines observed in this study provide a new understanding of the intrinsic immune mechanisms that can contribute to the onset of iritis.

Angiogenesis in rheumatoid arthritis is fostered directly by toll-like receptor 5 ligation and indirectly through interleukin-17 induction.

OBJECTIVE: To examine the impact of Toll-like receptor 5 (TLR-5) on endothelial cell function in rheumatoid arthritis (RA) and vascularization in collagen-induced arthritis (CIA). METHODS: Endothelial cell migration and tube formation assays were used to demonstrate the direct role of TLR-5 ligation in angiogenesis. Mice with CIA were treated with the TLR-5 agonist flagellin to document the effect of TLR-5 ligation in RA pathology. Vascularization in CIA was determined by immunohistochemical analysis and determination of cytokine levels in ankle joints. Spleen Th17 cells and joint interleukin-17 (IL-17) were quantified by fluorescence-activated cell sorting analysis and enzyme-linked immunosorbent assay. The development of Th17 cells induced by TLR-5 ligation was validated in RA peripheral blood mononuclear cells. RESULTS: Ligation of TLR-5 to endogenous ligands expressed in RA synovial fluid contributed to endothelial cell infiltration and tube formation. Furthermore, treatment with flagellin after the onset of CIA exacerbated joint inflammation; in contrast, inflammation in control mice remained at a plateau phase. We showed that TLR-5-enhanced disease severity was attributable to Th17 cell differentiation and joint vascularization in CIA. Examination of the underlying mechanism using RA peripheral blood mononuclear cells documented that ligation of TLR-5 in myeloid cells and production of Th17-promoting cytokines were necessary for Th17 cell polarization. Additionally, we demonstrated that blockade of the IL-17 cascade markedly reduced endothelial cell migration activated by flagellin-conditioned medium, suggesting that TLR-5 ligation can mediate RA angiogenesis either directly by attracting endothelial cells or indirectly by fostering Th17 cell development. CONCLUSION: Our data demonstrate a novel role for TLR-5 in RA angiogenesis; thus, TLR-5 may be a promising new target for RA treatment.

TLR5, a novel and unidentified inflammatory mediator in rheumatoid arthritis that correlates with disease activity score and joint TNF-α levels.

The innate immune system plays an important role in rheumatoid arthritis (RA) pathogenesis. Previous studies support the role of TLR2 and 4 in RA and experimental arthritis models; however, the regulation and pathogenic effect of TLR5 is undefined in RA. In this study, we show that TLR5 is elevated in RA and osteoarthritis ST lining and sublining macrophages and endothelial cells compared with normal individuals. Furthermore, expression of TLR5 is elevated in RA synovial fluid macrophages and RA peripheral blood monocytes compared with RA and normal peripheral blood in vitro-differentiated macrophages. We also found that TLR5 on RA monocytes is an important modulator of TNF-α in RA synovial fluid and that TLR5 expression on these cells strongly correlates with RA disease activity and TNF-α levels. Interestingly, TNF-α has a feedback regulation with TLR5 expression in RA monocytes, whereas expression of this receptor is regulated by IL-17 and IL-8 in RA macrophages and fibroblasts. We show that RA monocytes and macrophages are more responsive to TLR5 ligation compared with fibroblasts despite the proinflammatory response being mediated through the same signaling pathways in macrophages and fibroblasts. In conclusion, we document the potential role of TLR5 ligation in modulating transcription of TNF-α from RA synovial fluid and the strong correlation of TLR5 and TNF-α with each other and with disease activity score in RA monocytes. Our results suggest that expression of TLR5 may be a predictor for RA disease progression and that targeting TLR5 may suppress RA.

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.

Ligation of TLR7 by rheumatoid arthritis synovial fluid single strand RNA induces transcription of TNFα in monocytes.

OBJECTIVE: The aim of the study was to characterise the expression, regulation and pathogenic role of toll-like receptor 7 (TLR7) and TLR8 in rheumatoid arthritis (RA). METHODS: Expression of TLR7 and TLR8 was demonstrated in RA, osteoarthritis (OA) and normal (NL) synovial tissues (STs) employing immunohistochemistry. The authors next examined the mechanism by which TLR7 and TLR8 ligation mediates proinflammatory response by Western blot analysis and ELISA. Expression of TLR7 and TLR8 in RA monocytes was correlated to disease activity score (DAS28) and tumour necrosis factor α (TNFα) levels. Further, the effect of TLR7 ligation in RA monocytes was determined on synovial fluid (SF)-mediated TNFα transcription. RESULTS: TLR7/8 are predominately expressed in RA ST lining and sublining macrophages. The authors show that NF-κB and/or PI3K pathways are essential for TLR7/8 induction of proinflammatory factors in RA peripheral blood (PB)-differentiated macrophages. Expression of TLR7 in RA monocytes shows a strong correlation with DAS28 and TNFα levels. By contrast, expression of TLR8 in these cells does not correlate with DAS28, TLR7 or TNFα levels. The authors further demonstrate that RNA from RA SF, but not RA or NL plasma, could modulate TNFα transcription from RA monocytes that can be downregulated by antagonising TLR7 ligation or degradation of single stand (ss) RNA. Thus, ssRNA present in RA SF may function as a potential endogenous ligand for TLR7. CONCLUSIONS: These results suggest that expression of TLR7, but not TLR8, may be a predictor for RA disease activity and anti-TNFα responsiveness, and targeting TLR7 may suppress chronic progression of RA.