Inhibition of Toll-like receptor 4 breaks the inflammatory loop in autoimmune destructive arthritis

OBJECTIVE: Degeneration of extracellular matrix of cartilage leads to the production of molecules capable of activating the immune system via Toll-like receptor 4 (TLR-4). The objective of this study was to investigate the involvement of TLR-4 activation in the development and progression of autoimmune destructive arthritis. METHODS: A naturally occurring TLR-4 antagonist, highly purified lipopolysaccharide (LPS) from Bartonella quintana, was first characterized using mouse macrophages and human dendritic cells (DCs). Mice with collagen-induced arthritis (CIA) and mice with spontaneous arthritis caused by interleukin-1 receptor antagonist (IL-1Ra) gene deficiency were treated with TLR-4 antagonist. The clinical score for joint inflammation, histologic characteristics of arthritis, and local expression of IL-1 in joints were evaluated after treatment. RESULTS: The TLR-4 antagonist inhibited DC maturation induced by Escherichia coli LPS and cytokine production induced by both exogenous and endogenous TLR-4 ligands, while having no effect on these parameters by itself. Treatment of CIA using TLR-4 antagonist substantially suppressed both clinical and histologic characteristics of arthritis without influencing the adaptive anti-type II collagen immunity crucial for this model. Treatment with TLR-4 antagonist strongly reduced IL-1beta expression in articular chondrocytes and synovial tissue. Furthermore, such treatment inhibited IL-1-mediated autoimmune arthritis in IL-1Ra(-/-) mice and protected the mice against cartilage and bone pathology. CONCLUSION: In the present study, we demonstrate for the first time that inhibition of TLR-4 suppresses the severity of experimental arthritis and results in lower IL-1 expression in arthritic joints. Our data suggest that TLR-4 might be a novel target in the treatment of rheumatoid arthritis.     

Tumor necrosis factor α blockade treatment down‐modulates the increased systemic and local expression of toll‐like receptor 2 and toll‐like receptor 4 in spondylarthropathy

Objective

Abnormal host defense against pathogens has been implicated in the pathogenesis of spondylarthropathy (SpA), a disease characterized by abundant synovial infiltration with innate immune cells. Given the role of Toll‐like receptors (TLRs) in activation of innate inflammation and the occurrence of TLR‐dependent infections after tumor necrosis factor α (TNFα) blockade treatment, the present study was undertaken to analyze TLRs and their modulation by TNFα blockade in SpA.

Methods

Peripheral blood mononuclear cells (PBMCs) were obtained from SpA and rheumatoid arthritis (RA) patients during infliximab therapy, and from healthy controls. TLR‐2 and TLR‐4 expression and TNFα production upon lipopolysaccharide (LPS) stimulation were analyzed by flow cytometry on different monocyte subsets. Synovial biopsy specimens from 23 SpA patients before and after infliximab or etanercept treatment, from 15 RA patients, and from 18 osteoarthritis (OA) patients were analyzed by immunohistochemistry.

Results

Expression of TLR‐4, but not TLR‐2, was increased on PBMCs from patients with SpA, whereas both TLRs were increased in RA patients. TLR expression was particularly increased on the CD163+ macrophage subset. Infliximab reduced TLR‐2 and TLR‐4 expression on monocytes of SpA and RA patients, leading to lower levels than in controls and to impaired TNFα production upon LPS stimulation. In inflamed synovium, the expression of both TLRs and of CD163 was significantly higher in patients with SpA than in those with RA or OA. Paralleling the systemic effect, TLRs in synovium were down‐regulated following treatment with infliximab as well as etanercept, indicating a class effect of TNFα blockers.

Conclusion

Inflammation in SpA is characterized by increased TLR‐2 and TLR‐4 expression, which is sharply reduced by TNFα blockade. These findings suggest a potential role of innate immunity–mediated inflammation in SpA and provide an additional clue regarding the mechanism of action as well as the potential side effects of TNFα blockade.

     

Increased macrophage activation mediated through toll‐like receptors in rheumatoid arthritis

Objective

Macrophages are the major source of inflammation mediators that are important in the pathogenesis of rheumatoid arthritis (RA). This study was undertaken to analyze macrophages obtained from the joints of RA patients in order to characterize the expression of Toll‐like receptor 2 (TLR‐2) and TLR‐4 and the responses to TLR ligation.

Methods

Cells were isolated from the synovial fluid (SF) of RA patients or patients with other forms of inflammatory arthritis. Cell surface TLR‐2 and TLR‐4 expression and intracellular tumor necrosis factor α (TNFα) and interleukin‐8 (IL‐8) expression by CD14+ macrophages were determined by flow cytometry. Peptidoglycan (PG) and lipopolysaccharide (LPS) were used as ligands for TLR‐2 and TLR‐4, respectively.

Results

The expression of TLR‐2 and TLR‐4 was increased on CD14+ macrophages from the joints of RA patients compared with that on control in vitro–differentiated macrophages or control peripheral blood monocytes. Neither TLR‐2 expression nor TLR‐4 expression differed between RA and other forms of inflammatory arthritis. However, PG‐ and LPS‐induced TNFα expression and IL‐8 expression were greater with RA SF macrophages than with those obtained from the joints of patients with other forms of inflammatory arthritis or with control macrophages. PG‐induced TNFα expression and IL‐8 expression were highly correlated with TLR‐2 expression in normal macrophages, but not with that in macrophages obtained from joints of RA patients or patients with other forms of inflammatory arthritis.

Conclusion

TLR‐2 and TLR‐4 ligation resulted in increased activation of RA synovial macrophages compared with those from patients with other forms of inflammatory arthritis or compared with control macrophages. Factors other than the level of TLR‐2 and TLR‐4 expression contributed to the increased activation of RA SF macrophages. These observations support the notion of a potential role for activation through TLR‐2 and TLR‐4 in the inflammation and joint destruction of RA.

     

RNA released from necrotic synovial fluid cells activates rheumatoid arthritis synovial fibroblasts via toll‐like receptor 3

Objective

To assess the expression of Toll‐like receptor 3 (TLR‐3) protein in synovial tissues and cultured synovial fibroblasts obtained from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and to investigate the consequences of stimulation of cultured synovial fibroblasts with TLR‐3 ligands.

Methods

TLR‐3 expression in synovial tissues was determined by immunohistochemistry and immunofluorescence, and expression in cultured RA synovial fibroblasts (RASFs) was determined by fluorescence‐activated cell sorting and real‐time polymerase chain reaction techniques. TLR‐3 signaling was assessed by incubating RASFs with poly(I‐C), lipopolysaccharide, palmitoyl‐3‐cysteine‐serine‐lysine‐4, or necrotic synovial fluid cells from RA patients in the presence or absence of hydroxychloroquine or Benzonase. Subsequent determination of interferon‐β (IFNβ), CXCL10, CCL5, and interleukin‐6 (IL‐6) protein production in the culture supernatants was performed by enzyme‐linked immunosorbent assays.

Results

TLR‐3 protein expression was found to be higher in RA synovial tissues than in OA synovial tissues. TLR‐3 expression was localized predominantly in the synovial lining, with a majority of the TLR‐3–expressing cells coexpressing fibroblast markers. Stimulation of cultured RASFs with the TLR‐3 ligand poly(I‐C) resulted in the production of high levels of IFNβ, CXCL10, CCL5, and IL‐6 protein. Similarly, coincubation of RASFs with necrotic synovial fluid cells from patients with RA resulted in up‐regulation of these cytokines and chemokines in a TLR‐3–dependent manner.

Conclusion

Our findings demonstrate the expression of TLR‐3 in RA synovial tissue and the activation of RASFs in vitro by the TLR‐3 ligand poly(I‐C) as well as by necrotic RA synovial fluid cells, and indicate that RNA released from necrotic cells might act as an endogenous TLR‐3 ligand for the stimulation of proinflammatory gene expression in RASFs.

     

Significant association between toll‐like receptor gene polymorphisms and gallbladder cancer

Background: Toll‐like receptors (TLRs) are evolutionarily conserved cell surface receptors of innate immune system. Various polymorphisms in TLR genes have been identified and associated with susceptibility toward various malignancies such as prostate cancer, gastric cancer and colorectal cancer. The present study was undertaken to examine the potential association of two polymorphisms in TLR2 and TLR4 genes with gallbladder cancer (GBC) susceptibility. Methods: Genotypes and allelic frequencies of TLR2 and TLR4 gene polymorphisms were determined for 233 GBC patients and 257 cancer‐free controls randomly selected from the population, using polymerase chain reaction–restriction fragment length polymorphism. Odds ratio (OR) and 95% confidence interval (95% CI) were calculated in a multivariate logistic regression analysis for the association of TLR polymorphisms with GBC. Results: ‘del’ allele carriers of TLR2 (Δ22) polymorphism were associated with a 1.54‐fold increased risk for GBC (95% CI=1.02–2.24; P_trend=0.091). The TLR4 Ex4+936C >T polymorphism (g.14143C>T; rs4986791) was also found to be significantly associated with the overall higher risk of GBC under a dominant mode of inheritance (OR=1.96; 95% CI=1.11–2.26; P_trend=0.021). The false‐positive report probability (FPRP) approach advocated that these results were noteworthy (FPRP<0.5). Subgroup analysis showed that TLR4 Ex4+936C>T polymorphism was associated with an increased risk of GBC in females and GBC cases with gallstones (OR=2.85 and 2.22 respectively). Conclusion: In summary, low‐penetrance variants in TLR genes may alter the susceptibility towards gallbladder cancer.     

Expression of toll‐like receptor 2 on CD16+ blood monocytes and synovial tissue macrophages in rheumatoid arthritis

Objective

CD16 (IgG Fcγ receptor type IIIA [FcγRIIIA])–expressing CD14+ monocytes express high levels of Toll‐like receptor 2 (TLR‐2) and are able to efficiently produce proinflammatory cytokines such as tumor necrosis factor α (TNFα). To understand the role of CD16 and TLR‐2 in monocyte and macrophage activation in rheumatoid arthritis (RA), we investigated the expression of TLR‐2 on CD16+ blood monocytes and synovial tissue macrophages and the effect of CD16 and TLR‐2 activation on cytokine production.

Methods

The expression of CD14, CD16, TLR‐2, and TLR‐4 on blood monocytes was measured by flow cytometric analysis. CD16 and TLR‐2 expression in RA synovial tissue was detected by 2‐color immunofluorescence labeling. CD16+ mature monocytes were prepared by incubating blood monocytes in plastic plates for 24 hours. These adhered monocytes were stimulated with lipoteichoic acid (LTA), anti‐FcγRIII antibody, and Hsp60 for 5 hours, and culture supernatants were measured for various cytokines by immunoassay. The activation of NF‐κB was detected by electrophoretic mobility shift assay.

Results

The frequency of CD16+ cells in all blood monocytes was significantly increased in patients with RA compared with healthy controls. TLR‐2 was expressed at higher levels on CD16+ monocytes than on CD16− monocytes, while TLR‐4 was expressed similarly on both monocytes. In RA synovial tissue, CD16+/TLR‐2+ cells were distributed mainly in the lining layer. TLR‐2 expression on monocytes was enhanced by macrophage colony‐stimulating factor (M‐CSF) and interleukin‐10 (IL‐10), but was reduced by transforming growth factor β1, while CD16 expression was inducible by these cytokines. Adhered monocytes (∼50% CD16+) produced TNFα, IL‐1β, IL‐6, IL‐8, IL‐12 p40, IL‐1 receptor antagonist, and IL‐10 after LTA stimulation. This cytokine response was inhibited significantly by anti–TLR‐2 antibody and partly by anti–TLR‐4 antibody. Anti‐FcγRIII antibody stimulation markedly enhanced the LTA‐induced TNFα response. Hsp60 could stimulate TNFα production by adhered monocytes, which was inhibited similarly by anti–TLR‐2 antibody and anti–TLR‐4 antibody. NF‐κB activation in adhered monocytes was induced by LTA, but this NF‐κB activity was not augmented by anti‐FcγRIII antibody stimulation.

Conclusion

These results suggest that CD16+ monocytes and synovial tissue macrophages with high TLR‐2 expression may be induced by M‐CSF and IL‐10, and their production of TNFα could be simulated by endogenous TLR ligands such as Hsp60 and FcγRIIIA ligation by small immune complexes in RA joints.

     

Genetic variation in toll‐like receptor 9 and susceptibility to systemic lupus erythematosus

Objective

Autoantibodies produced by differentiated B cells play an important role in the pathogenesis of systemic lupus erythematosus (SLE). The Toll‐like receptor 9 (TLR‐9) gene has recently emerged as an important costimulatory molecule for both B cells and dendritic cells that respond to chromatin immune complexes. Genetic variation affecting the function of TLR‐9 may therefore increase or decrease the threshold of B cell or dendritic cell activation. This variability in activation threshold may, in turn, affect an individual's susceptibility to SLE. This study assessed the role of genetic variation within the TLR‐9 gene in susceptibility to SLE.

Methods

We genotyped 362 SLE‐affected subject/parent trios for 10 single‐nucleotide polymorphisms (SNPs) covering a 68,742‐bp genomic segment that contains the TLR‐9 gene and ∼60 kb of flanking sequence. We analyzed the data using the transmission disequilibrium test.

Results

There was no association of susceptibility to SLE with any of the 9 SNPs that generated usable data or the 8 haplotypes found at a frequency of >0.05 in this population. When analyzing the subset of 143 subjects with lupus nephritis, there was also no evidence of association between disease susceptibility and any SNP or haplotype.

Conclusion

These results indicate that there is no evidence that common (frequency higher than 5%) alleles of the TLR‐9 gene contribute significantly to the genetic risk involved in susceptibility to SLE or lupus nephritis.

     

SIGIRR/TIR‐8 is an inhibitor of toll‐like receptor signaling in primary human cells and regulates inflammation in models of rheumatoid arthritis

Objective

Single‐immunoglobulin interleukin‐1 receptor–related (SIGIRR), which is also known as Toll/interleukin‐1 receptor 8 (TIR‐8), is a member of the TIR domain–containing family of receptors and was first characterized as an inhibitor of interleukin‐1 receptor (IL‐1R) and Toll‐like receptor (TLR) signaling. In the Dextran sulfate sodium–induced colitis model, SIGIRR^−/− mice were shown to have increased inflammation and to be more susceptible to endotoxin challenge. Increasing evidence implicates TLR and IL‐1R signaling in the pathology of rheumatoid arthritis (RA). Therefore, the purpose of this study was to investigate the involvement of SIGIRR in regulating inflammation in disease‐relevant models.

Methods

Primary human monocyte‐derived macrophages and dendritic cells (DCs) were used to overexpress SIGIRR as well as to knock down endogenously expressed SIGIRR using small interfering RNAs. SIGIRR was also overexpressed in synovial cells derived from RA patients. To investigate the role of SIGIRR in vivo, zymosan‐induced arthritis (ZIA) and collagen antibody–induced arthritis (CAIA) were induced in SIGIRR‐knockout mice.

Results

SIGIRR overexpression inhibited TLR‐induced cytokine production in macrophages and DCs, while SIGIRR knockdown resulted in increased cytokine production following TLR stimulation. Moreover, SIGIRR overexpression inhibited the spontaneous release of cytokines by human RA synovial cells. The role of SIGIRR as an inhibitor of inflammation was confirmed in vivo, since SIGIRR^−/− mice developed a more severe disease in both the ZIA and CAIA models.

Conclusion

Our study is the first to show the expression pattern and function of SIGIRR in primary human cells. Furthermore, this investigation defines the role of SIGIRR in disease‐relevant cell types and demonstrates that SIGIRR is a potential therapeutic target for RA.

     

The expression of toll‐like receptors 3 and 7 in rheumatoid arthritis synovium is increased and costimulation of toll‐like receptors 3, 4, and 7/8 results in synergistic cytokine production by dendritic cells

Objective

To evaluate the expression of Toll‐like receptors (TLRs) 3 and 7 in synovium and to study potential differences in the maturation and cytokine production mediated by TLR‐2, TLR‐3, TLR‐4, and TLR‐7/8 by dendritic cells (DCs) from rheumatoid arthritis (RA) patients and DCs from healthy controls.

Methods

Synovial expression of TLR‐3 and TLR‐7 in RA was studied using immunohistochemistry. Monocyte‐derived DCs from RA patients and healthy controls were cultured for 6 days and subsequently stimulated for 48 hours via TLR‐mediated pathways (lipoteichoic acid, Pam₃Cys, and fibroblast‐stimulating lipopeptide 1 for TLR‐2, poly[I‐C] for TLR‐3, lipopolysaccharide and extra domain A for TLR‐4, and R848 for TLR‐7/8). Phenotypic DC maturation was measured using flow cytometry. The secretion of tumor necrosis factor α (TNFα), interleukin‐6 (IL‐6), IL‐10, and IL‐12 was measured using the Bio‐Plex system. Cell lines expressing TLR‐2 and TLR‐4 were used for the detection of TLR‐2 and TLR‐4 ligands in serum and synovial fluid from RA patients.

Results

TLR‐3 and TLR‐7 were highly expressed in RA synovium. All TLR ligands elicited phenotypic DC maturation equally between DCs from RA patients and those from healthy controls. TLR‐2– and TLR‐4–mediated stimulation of DCs from RA patients resulted in markedly higher production of inflammatory mediators (TNFα and IL‐6) compared with DCs from healthy controls. In contrast, upon stimulation of TLR‐3 and TLR‐7/8, the level of cytokine production was equal between DCs from RA patients and those from healthy controls. Remarkably, both TLR‐3 and TLR‐7/8 stimulation resulted in a skewed balance toward IL‐12. Intriguingly, the combined stimulation of TLR‐4 and TLR‐3–7/8 resulted in a marked synergy with respect to the production of inflammatory mediators. As a proof of concept, TLR‐4 ligands were increased in the serum and synovial fluid of RA patients.

Conclusion

TLRs are involved in the regulation of DC activation and cytokine production. We hypothesize that various TLR ligands in the joint trigger multiple TLRs simultaneously, favoring the breakthrough of tolerance in RA.