The aim of the study was to characterize the expression, regulation and pathogenic role of TLR7 and TLR8 in rheumatoid arthritis (RA).
Expression of TLR7 and TLR8 was demonstrated in RA, osteoarthritis (OA) and normal (NL) synovial tissues (ST) employing immunohistochemistry. We 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 TNF-α levels. Further the effect of TLR7 ligation in RA monocytes was determined on synovial fluid (SF) mediated TNF-α transcription.
TLR7/TLR8 are predominately expressed in RA ST lining and sublining macrophages. We show that NF-κB and/or PI3K pathways are essential for TLR7/TLR8 induction of proinflammatory factors in RA peripheral blood (PB) differentiated macrophages. Expression of TLR7 in RA monocytes shows a strong correlation with DAS28 and TNF-α levels. In contrast, expression of TLR8 in these cells does not correlate with DAS28, TLR7 or TNF-α levels. We further demonstrate that RNA from RA SF but not RA or NL plasma could modulate TNF-α transcription from RA monocytes that can be downregulated by antagonizing TLR7 ligation or degradation of single stand (ss) RNA. Thus, ssRNA present in RA SF may function as a potential endogenous ligand for TLR7.
These results suggest that expression of TLR7 but not TLR8 may be a predictor for RA disease activity and anti-TNF-α responsiveness, and targeting TLR7 may suppress chronic progression of RA.
RA synovial tissue; RA monocytes/macrophages; TLR7; TLR8 and ssRNA
IL-17-induced joint inflammation is associated with increased angiogenesis. However, the mechanism by which IL-17 mediates angiogenesis is undefined. Therefore, the pathologic role of CXCL1 and CXCL5 was investigated in arthritis mediated by local expression of IL-17, employing a neutralizing antibody to each chemokine. Next, endothelial chemotaxis was utilized to examine whether endothelial migration was differentially mediated by CXCL1 and CXCL5. Our results demonstrate that IL-17-mediated disease activity was not affected by anti-CXCL1 treatment alone. In contrast, mice receiving anti-CXCL5 demonstrated significantly reduced clinical signs of arthritis, compared to the mice treated with IgG control. Consistently, while inflammation, synovial lining thickness, bone erosion and vascularization were markedly reduced in both the anti-CXCL5 and combination anti-CXCL1 and 5 treatment groups, mice receiving anti-CXCL1 antibody had clinical scores similar to the control group. In contrast to joint FGF2 and VEGF levels, TNF-α was significantly reduced in mice receiving anti-CXCL5 or combination of anti-CXCL1 and 5 therapies compared to the control group. We found that, like IL-17, CXCL1-induced endothelial migration is mediated through activation of PI3K. In contrast, activation of NF-κB pathway was essential for endothelial chemotaxis induced by CXCL5. Although CXCL1 and CXCL5 can differentially mediate endothelial trafficking, blockade of CXCR2 can inhibit endothelial chemotaxis mediated by either of these chemokines. These results suggest that blockade of CXCL5 can modulate IL-17-induced inflammation in part by reducing joint blood vessel formation through a non-overlapping IL-17 mechanism.
IL-17-induced arthritis; CXCL1; CXCL5; Angiogenesis
These studies were performed to determine the role of CCL21 and its corresponding receptor CCR7 in the pathogenesis of Rheumatoid Arthritis (RA).
Histological studies were performed to compare the expression of CCR7 and CCL21 in RA synovial tissues. Next the role of CCL21 and/or CCR7 in angiogenesis was examined employing in vitro chemotaxis, tube formation and in vivo matrigel plug assays. Finally the mechanism by which CCL21 mediates angiogenesis was determined by Western blot analysis, endothelial chemotaxis and tube formation.
In this study, we document that CCR7 and CCL21 colocolize in VWF+ cells where their expression is elevated in RA synovial tissue. Hence the ability to induce angiogenesis was examined for CCR7 ligands, CCL19 and CCL21. CCL21, but not CCL19, at concentrations present in the RA joint, induces human microvascular endothelial cell (HMVEC) migration that is mediated through CCR7 ligation. Further, suppression of the PI3K pathway markedly reduces CCL21-induced HMVEC chemotaxis and tube formation, however suppression of ERK and JNK pathways has no effect on these processes. Neutralization of either CCL21 in RA synovial fluids or CCR7 on HMVECs significantly reduces the induction of HMVEC migration and/or tube formation by RA synovial fluid. We further demonstrate that CCL21 is angiogenic, by showing its ability to promote blood vessel growth in matrigel plugs in vivo at concentrations present in RA joint.
These observations identify a novel function for CCL21 as an angiogenic mediator in RA, supporting CCL21/CCR7 as a therapeutic target in RA.
CCL21; CCR7; RA synovial fluid; angiogenesis and migration
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 (OA) synovial tissue lining and sublining macrophages and endothelial cells compared to normal individuals. Further, expression of TLR5 is elevated in RA synovial fluid macrophages and RA peripheral blood (PB) monocytes compared to RA and normal PB 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 feed back regulation with TLR5 expression in RA monocytes, while 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 to 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.
RA monocytes; TLR5; TNF-α; DAS28; RA fibroblasts and RA differentiated macrophages
Electronic health records (EHR) can allow for the generation of large cohorts of individuals with given diseases for clinical and genomic research. A rate-limiting step is the development of electronic phenotype selection algorithms to find such cohorts. This study evaluated the portability of a published phenotype algorithm to identify rheumatoid arthritis (RA) patients from EHR records at three institutions with different EHR systems.
Materials and Methods
Physicians reviewed charts from three institutions to identify patients with RA. Each institution compiled attributes from various sources in the EHR, including codified data and clinical narratives, which were searched using one of two natural language processing (NLP) systems. The performance of the published model was compared with locally retrained models.
Applying the previously published model from Partners Healthcare to datasets from Northwestern and Vanderbilt Universities, the area under the receiver operating characteristic curve was found to be 92% for Northwestern and 95% for Vanderbilt, compared with 97% at Partners. Retraining the model improved the average sensitivity at a specificity of 97% to 72% from the original 65%. Both the original logistic regression models and locally retrained models were superior to simple billing code count thresholds.
These results show that a previously published algorithm for RA is portable to two external hospitals using different EHR systems, different NLP systems, and different target NLP vocabularies. Retraining the algorithm primarily increased the sensitivity at each site.
Electronic phenotype algorithms allow rapid identification of case populations in multiple sites with little retraining.
Automated learning; biomedical informatics; discovery and text and data mining methods; electronic health record; genetic; improving the education and skills training of health professionals; infection control; knowledge representations; linking the genotype and phenotype; medical informatics; natural language processing; other methods of information extraction; phenotype algorithms DNA databank machine learning; phenotype identification; phenotyping; rheumatoid arthritis; rheumatology; translational research – application of biological knowledge to clinical care
The aim of the study was to characterize the expression of IL-7 and IL-7R in rheumatoid arthritis (RA) synovial tissue and to examine their regulation and pathogenic role in macrophages, endothelial cells and RA synovial tissue fibroblasts.
Expression of IL-7 and IL-7R was demonstrated in RA and normal synovial tissues employing immunohistochemistry. Expression and regulation of IL-7 and IL-7R was determined in RA peripheral blood in vitro differentiated macrophages, RA synovial tissue fibroblasts and human microvascular endothelial cells (HMVECs) by real-time RT-PCR and/or flow cytometry. Next, IL-7 activated macrophages, RA fibroblasts and endothelial cells were examined for production of proangiogenic factors employing ELISA.
IL-7 and IL-7R were coexpressed on RA synovial tissue lining and sublining macrophages and endothelial cells. Consistently, expression of IL-7 and its receptor were significantly elevated in RA synovial fluid and peripheral blood macrophages as well as RA fibroblasts compared to normal cells. TLR4 ligation and stimulation with TNF-α modulated expression of IL-7 and IL-7R on RA macrophages and HMVECs. However, in RA fibroblasts only expression of IL-7R was increased by LPS and TNF-α activation. IL-7 also mediated RA pathogenesis by inducing production of potent proangiogenic factors from macrophages and endothelial cells.
We identify, for the first time, regulators of IL-7 and IL-7R expression in RA fibroblasts, RA peripheral blood in vitro differentiated macrophages and endothelial cells and we document a novel role of IL-7 in RA angiogenesis.
IL-7; IL-7R; RA synovial tissue fibroblast; macrophages and proangiogenic factors
To determine the mechanism of action of IL-27 against rheumatoid arthritis (RA).
Adenovirus containing IL-27 transcript was constructed and was locally delivered into the ankles of collagen-induced arthritis (CIA) mice. Progression of arthritis was assessed in treated versus untreated mice by measuring ankle circumference and through histological analysis. IL-17 and its downstream targets as well as cytokines promoting TH-17 cell differentiation were quantified by ELISA in CIA ankles locally expressing adenoviral IL-27 versus controls. Ankles from both treatment groups were immunostained for neutrophil and monocyte migration. Last, vascularization was quantified by histology and by determining ankle hemoglobin levels.
Our results demonstrate that ectopic expression of IL-27 in CIA ameliorates inflammation, lining hypertrophy, and bone erosion compared to the control group. Serum and joint IL-17 levels were significantly reduced in the IL-27 treatment group compared to controls. Two of the main cytokines which induce TH-17 cell differentiation and IL-17 downstream target molecules were greatly downregulated in CIA ankles receiving forced expression of IL-27. The control mice had higher levels of vascularization and monocyte trafficking compared to those mice ectopically expressing IL-27.
Our results suggest that increased IL-27 levels relieve arthritis in CIA ankles. This amelioration of arthritis involves a reduction in CIA serum and joint IL-17 levels and results in decreased IL-17-mediated monocyte recruitment and angiogenesis. Hence, IL-27 may be a therapeutic target in RA.
IL-27; collagen induced arthritis; IL-17; IL-1 and IL-6
The aim was to characterize the expression of CCL19 and CCL21 in rheumatoid arthritis (RA) synovial tissue and to examine their regulation and pathogenic role in macrophages and RA synovial tissue fibroblasts.
Expression of CCL19 and CCL21 was demonstrated in RA and normal (NL) synovial tissues employing immunohistochemistry. CCL19 and CCL21 levels were quantified in fluids from osteoarthritis (OA), juvenile idiopathic arthritis (JIA), psoriatic arthritis (PsA) and RA using ELISA. Regulation of CCL19 and CCL21 expression was determined in RA peripheral blood in vitro differentiated macrophages as well as RA synovial tissue fibroblasts by real-time RT-PCR. CCL19 and CCL21 activated peripheral blood in vitro differentiated macrophages and RA synovial tissue fibroblasts were examined for proangiogenic factor production employing ELISA.
CCL19 and CCL21 were elevated in RA synovial tissue compared to NL controls. Levels of CCL19 and CCL21 were greatly increased in RA and PsA synovial fluid versus OA synovial fluid. In RA macrophages and fibroblasts, expression of CCL19 was increased by LPS, TNF-α and IL-1β stimulation. However, CCL21 expression was modulated by IL-1β in RA fibroblasts as well as TNF-α and RA synovial fluid in RA macrophages. CCL19 and CCL21 activation induced VEGF and Ang-1 production from RA synovial tissue fibroblasts and secretion of IL-8 and Ang-1 from macrophages.
We identify, for the first time, regulators of CCL19 and CCL21 in RA fibroblasts and RA peripheral blood in vitro differentiated macrophages and we document a novel role of CCL19/21 in RA angiogenesis.
CCL19; CCL21; RA synovial tissue fibroblast; macrophages and proangiogenic factors
Rheumatoid arthritis (RA) is a chronic inflammatory disease which is in part mediated by proinflammatory factors produced by RA synovial tissue fibroblasts and macrophages, resulting in monocyte migration from the blood to the synovial tissue. In order to characterize the potential role of IL-17 in monocyte migration, RA synovial fibroblasts and macrophages were activated with IL-17 and examined for the expression of monocyte chemokines. The two potentially important monocyte chemoattractants identified were CCL20/MIP-3α and CCL2/MCP-1, which were significantly induced in RA synovial fibroblasts and macrophages. However, in vivo, only CCL2/MCP-1 was detectable following adenovirus (Ad)-IL-17 injection. We found that IL-17 induction of CCL2/MCP-1 was mediated by PI3K, ERK, and JNK pathways in RA synovial tissue fibroblasts and PI3K and ERK pathways in macrophages. Further, we show that neutralization of CCL2/MCP-1 significantly reduced IL-17-mediated monocyte recruitment into the peritoneal cavity. We demonstrate that local expression of IL-17 in ankle joints was associated with significantly increased monocyte migration and CCL2/MCP-1 levels. Interestingly, we show that RA synovial fluids immunoneutralized for both IL-17 and CCL2/MCP-1 have similar monocyte chemotaxis activity as those immunoneutralized for each factor alone. In short, CCL2/MCP-1 produced from cell types present in the RA joint as well as in experimental arthritis may be in part responsible for IL-17-induced monocyte migration, hence these results suggest that CCL2/MCP-1 is a downstream target of IL-17 that may be important in RA.
IL-17; CCL2/MCP-1; macrophages; synovial tissue fibroblasts; monocytes; rheumatoid arthritis
Angiogenesis is an early and a critical event in the pathogenesis of Rheumatoid arthritis (RA). Neovascularization is dependent on endothelial cell activation, migration and proliferation, and inhibition of angiogenesis may provide a novel therapeutic approach in RA. In this study, we document a novel role of IL-17 in mediating angiogenesis. Local expression of IL-17 in mouse ankles increases vascularity. We further demonstrate that IL-17 is angiogenic, by showing its ability to promote blood vessel growth in matrigel plugs in vivo. Additionally, IL-17, in concentrations present in the RA joint, induces human microvascular endothelial cell (HMVEC) migration mediated through the PI3K/AKT1 pathway. Further, suppression of the PI3K pathway markedly reduces IL-17-induced tube formation. We also show that both IL-17-induced HMVEC chemotaxis and tube formation are mediated primarily through IL-17 receptor (R) C. Neutralization of either IL-17 in RA synovial fluids or IL-17RC on HMVECs significantly reduces the induction of HMVEC migration by RA synovial fluid. Finally, RA synovial fluid immunoneutralized with IL-17 and VEGF does not reduce HMVEC migration beyond the effect detected with each factor alone. These observations identify a novel function for IL-17 as an angiogenic mediator in RA, supporting IL-17 as a therapeutic target in RA.
IL-17; angiogenesis; HMVECs; migration; rheumatoid arthritis
Macrophages are important mediators of chronic inflammation and are prominent in the synovial lining and sublining of patients with rheumatoid arthritis (RA). Recently, we demonstrated increased toll like receptor (TLR) 2 and 4 expression and increased response to microbial TLR2 and TLR4 ligands in macrophages from the joints of RA. The current study characterized the expression of the 96-kDa heat shock glycoprotein (gp96) in the joints of RA and its role as an endogenous TLR ligand to promote innate immunity in RA. Gp96 was increased in RA compared with osteoarthritis and arthritis-free control synovial tissues. The expression of gp96 strongly correlated with inflammation and synovial lining thickness. Gp96 was increased in synovial fluid from the joints of RA compared with disease controls. Recombinant gp96 was a potent activator of macrophages, and the activation was mediated primarily through TLR2 signaling. The cellular response to gp96 was significantly stronger with RA synovial macrophages compared to peripheral blood monocytes from RA or healthy controls. The transcription of TLR2, TNFα and IL-8, but not TLR4, was significantly induced by gp96, and the induction was significantly greater in purified RA synovial macrophages. The expression of TLR2, but not TLR4, on synovial fluid macrophages strongly correlated with the level of gp96 in the synovial fluid. The present study documents the potential role of gp96 as an endogenous TLR2 ligand in RA and provides insight into the mechanism by which gp96 promotes the chronic inflammation of RA, identifying gp96 as a potential new therapeutic target.
gp96; grp94; macrophages; TLR2; TLR4; Rheumatoid Arthritis
The aim of this study was to quantify the number of T-helper (TH)-17 cells present in rheumatoid arthritis (RA) synovial fluid (SF) and to determine the level of interleukin (IL)-17 cytokine in RA, osteoarthritis (OA) and normal synovial tissue, as well as to examine SF macrophages for the presence of IL-23, IL-27 and interferon (IFN)-γ.
Peripheral blood (PB) mononuclear cells from normal and RA donors and mononuclear cells from RA SF were examined either without stimulation or after pretreatment with IL-23 followed by stimulation with phorbol myristate acetate (PMA) plus ionomycin (P/I). The abundance of TH-17 cells in RA SF was determined by flow cytometry. IL-17 levels were quantified in synovial tissue from RA, OA and normal individuals by ELISA and IL-23 was identified in SFs by ELISA. RA SF and control in vitro differentiated macrophages were either untreated or treated with the toll-like receptor (TLR) 2 ligand peptidoglycan, and then IL-23, IL-27 and IFN-γ mRNA levels were quantified by real-time polymerase chain reaction (RT-PCR).
Treatment with P/I alone or combined with IL-23 significantly increased the number of TH-17 cells in normal, RA PB and RA SF. With or without P/I plus IL-23, the percentage of TH-17 cells was higher in RA SF compared with normal and RA PB. IL-17 levels were comparable in OA and normal synovial tissues, and these values were significantly increased in RA synovial tissue. Although IL-17 was readily detected in RA SFs, IL-23 was rarely identified in RA SF. However, IL-23 mRNA was significantly increased in RA SF macrophages compared with control macrophages, with or without TLR2 ligation. IL-27 mRNA was also significantly higher in RA SF compared with control macrophages, but there was no difference in IL-27 levels between RA and control macrophages after TLR2 ligation. IFN-γ mRNA was also detectable in RA SF macrophages but not control macrophages and the increase of IFN-γ mRNA following TLR2 ligation was greater in RA SF macrophages compared with control macrophages.
These observations support a role for TH-17 cells in RA. Our observations do not strongly support a role for IL-23 in the generation of TH-17 cells in the RA joint, however, they suggest strategies that enhance IL-27 or IFN-γ might modulate the presence of TH-17 cells in RA.