Circulating tRNA-derived fragments are decreased in patients with rheumatoid arthritis and increased in patients with psoriatic arthritis.

INTRODUCTION: tRNA-derived fragments (tRFs) play an important role in immune responses. To clarify the role of tRFs in autoimmunity we studied circulating tRF-levels in patients with rheumatoid arthritis (RA) and psoriatic arthritis (PsA), and in a murine model for arthritis. MATERIAL AND METHODS: Circulating tRF-levels were quantified by miR-Q RT-qPCR. tRNA processing and modification enzyme expression was analysed by RT-qPCR and public transcriptomics data. RESULTS: Significant reduction (up to 3-fold on average) of tRF-levels derived from tRNA-Gly-GCC,CCC, tRNA-Glu-CTC and tRNA-Val-CAC,AAC was observed in RA patients, whereas tRNA-Glu-CTC and tRNA-Val-CAC,AAC tRFs were found at significantly higher levels (up to 3-fold on average) in PsA patients, compared to healthy controls. Also in arthritic IL1Ra-KO mice reduced levels of tRNA-Glu-CTC fragments were seen. The expression of NSUN2, a methyltransferase catalysing tRNA methylation, was increased in RA-peripheral blood mononuclear cells (PBMCs) compared to PsA, but this is not consistently supported by public transcriptomics data. DISCUSSION: The observed changes of specific tRF-levels may be involved in the immune responses in RA and PsA and may be applicable as new biomarkers. CONCLUSION: Circulating tRF-levels are decreased in RA and increased in PsA and this may, at least in part, be mediated by methylation changes.

Separating friend from foe: Inhibition of TGF-ß-induced detrimental SMAD1/5/9 phosphorylation while maintaining protective SMAD2/3 signaling in OA chondrocytes.

OBJECTIVE: Transforming growth factor-ß (TGF-ß) signaling via SMAD2/3 is crucial to control cartilage homeostasis. However, TGF-ß can also have detrimental effects by signaling via SMAD1/5/9 and thereby contribute to diseases like osteoarthritis (OA). In this study, we aimed to block TGF-ß-induced SMAD1/5/9 signaling in primary human OA chondrocytes, while maintaining functional SMAD2/3 signaling. DESIGN: Human OA chondrocytes were pre-incubated with different concentrations of ALK4/5/7 kinase inhibitor SB-505124 before stimulation with TGF-ß. Changes in SMAD C-terminal phosphorylation were analyzed using Western blot and response genes were measured with quantitative Polymerase Chain Reaction. To further explore the consequences of our ability to separate pathways, we investigated TGF-ß-induced chondrocyte hypertrophy. RESULTS: Pre-incubation with 0.5 µM SB-505124, maintained ±50% of C-terminal SMAD2/3 phosphorylation and induction of JUNB and SERPINE1, but blocked SMAD1/5/9-C phosphorylation and expression of ID1 and ID3. Furthermore, TGF-ß, in levels comparable to those in the synovial fluid of OA patients, resulted in regulation of hypertrophic and dedifferentiation markers in OA chondrocytes; i.e. an increase in COL10, RUNX2, COL1A1, and VEGF and a decrease in ACAN expression. Interestingly, in a subgroup of OA chondrocyte donors, blocking only SMAD1/5/9 caused stronger inhibition on TGF-ß-induced RUNX2 than blocking both SMAD pathways. CONCLUSION: Our findings indicate that using low dose of SB-505124 we maintained functional SMAD2/3 signaling that blocks RUNX2 expression in a subgroup of OA patients. We are the first to show that SMAD2/3 and SMAD1/5/9 pathways can be separately modulated using low and high doses of SB-505124 and thereby split TGF-ß's detrimental from protective function in chondrocytes.

Synergy between 15-lipoxygenase and secreted PLA2 promotes inflammation by formation of TLR4 agonists from extracellular vesicles.

Damage-associated endogenous molecules induce innate immune response, thus making sterile inflammation medically relevant. Stress-derived extracellular vesicles (stressEVs) released during oxidative stress conditions were previously found to activate Toll-like receptor 4 (TLR4), resulting in expression of a different pattern of immune response proteins in comparison to lipopolysaccharide (LPS), underlying the differences between pathogen-induced and sterile inflammation. Here we report that synergistic activities of 15-lipoxygenase (15-LO) and secreted phospholipase A2 (sPLA2) are needed for the formation of TLR4 agonists, which were identified as lysophospholipids (lysoPLs) with oxidized unsaturated acyl chain. Hydroxy, hydroperoxy, and keto products of 2-arachidonoyl-lysoPI oxidation by 15-LO were identified by mass spectrometry (MS), and they activated the same gene pattern as stressEVs. Extracellular PLA2 activity was detected in the synovial fluid from rheumatoid arthritis and gout patients. Furthermore, injection of sPLA2 promoted K/BxN serum-induced arthritis in mice, whereby ankle swelling was partially TLR4 dependent. Results confirm the role of oxidized lysoPL of stressEVs in sterile inflammation that promotes chronic diseases. Both 15-LO and sPLA2 enzymes are induced during inflammation, which opens the opportunity for therapy without compromising innate immunity against pathogens.

Extracellular vesicles regulate purinergic signaling and epithelial sodium channel expression in renal collecting duct cells.

Purinergic signaling regulates several renal physiological and pathophysiological processes. Extracellular vesicles (EVs) are nanoparticles released by most cell types, which, in non-renal tissues, modulate purinergic signaling. The aim of this study was to investigate the effect of EVs from renal proximal tubule (HK2) and collecting duct cells (HCD) on intra- and intersegment modulation of extracellular ATP levels, the underlying molecular mechanisms, and the impact on the expression of the alpha subunit of the epithelial sodium channel (αENaC). HK2 cells were exposed to HK2 EVs, while HCD cells were exposed to HK2 and HCD EVs. Extracellular ATP levels and αENaC expression were measured by chemiluminescence and qRT-PCR, respectively. ATPases in EV populations were identified by mass spectrometry. The effect of aldosterone was assessed using EVs from aldosterone-treated cells and urinary EVs (uEVs) from primary aldosteronism (PA) patients. HK2 EVs downregulated ectonucleoside-triphosphate-diphosphohydrolase-1 (ENTPD1) expression, increased extracellular ATP and downregulated αENaC expression in HCD cells. ENTPD1 downregulation could be attributed to increased miR-205-3p and miR-505 levels. Conversely, HCD EVs decreased extracellular ATP levels and upregulated αENaC expression in HCD cells, probably due to enrichment of 14-3-3 isoforms with ATPase activity. Pretreatment of donor cells with aldosterone or exposure to uEVs from PA patients enhanced the effects on extracellular ATP and αENaC expression. We demonstrated inter- and intrasegment modulation of renal purinergic signaling by EVs. Our findings postulate EVs as carriers of information along the renal tubules, whereby processes affecting EV release and/or cargo may impact on purinergically regulated processes.

Comparing Approaches to Normalize, Quantify, and Characterize Urinary Extracellular Vesicles.

BACKGROUND: Urinary extracellular vesicles (uEVs) are a promising source for biomarker discovery, but optimal approaches for normalization, quantification, and characterization in spot urines are unclear. METHODS: Urine samples were analyzed in a water-loading study, from healthy subjects and patients with kidney disease. Urine particles were quantified in whole urine using nanoparticle tracking analysis (NTA), time-resolved fluorescence immunoassay (TR-FIA), and EVQuant, a novel method quantifying particles via gel immobilization. RESULTS: Urine particle and creatinine concentrations were highly correlated in the water-loading study (R2 0.96) and in random spot urines from healthy subjects (R2 0.47-0.95) and patients (R2 0.41-0.81). Water loading reduced aquaporin-2 but increased Tamm-Horsfall protein (THP) and particle detection by NTA. This finding was attributed to hypotonicity increasing uEV size (more EVs reach the NTA size detection limit) and reducing THP polymerization. Adding THP to urine also significantly increased particle count by NTA. In both fluorescence NTA and EVQuant, adding 0.01% SDS maintained uEV integrity and increased aquaporin-2 detection. Comparison of intracellular- and extracellular-epitope antibodies suggested the presence of reverse topology uEVs. The exosome markers CD9 and CD63 colocalized and immunoprecipitated selectively with distal nephron markers. Conclusions uEV concentration is highly correlated with urine creatinine, potentially replacing the need for uEV quantification to normalize spot urines. Additional findings relevant for future uEV studies in whole urine include the interference of THP with NTA, excretion of larger uEVs in dilute urine, the ability to use detergent to increase intracellular-epitope recognition in uEVs, and CD9 or CD63 capture of nephron segment-specific EVs.

Systemic overexpression of interleukin-22 induces the negative immune-regulator SOCS3 and potently reduces experimental arthritis in mice.

OBJECTIVE: High levels of IL-22 are present in serum and synovial fluid of patients with RA. As both pro- and anti-inflammatory roles for IL-22 have been described in studies using animal models of RA, its exact function in arthritis remains poorly defined. With this study we aimed to further unravel the mechanism by which IL-22 exerts its effects and to decipher its therapeutic potential by overexpression of IL-22 either locally or systemically during experimental arthritis. METHODS: CIA was induced in DBA-1 mice by immunization and booster injection with type II collagen (col II). Before arthritis onset, IL-22 was overexpressed either locally by intra-articular injection or systemically by i.v. injection using an adenoviral vector and clinical arthritis was scored for a period of 10 days. Subsequently, joints were isolated for histological analysis of arthritis severity and mRNA and protein expression of various inflammatory mediators was determined in the synovium, spleen and serum. RESULTS: Local IL-22 overexpression did not alter arthritis pathology, whereas systemic overexpression of IL-22 potently reduced disease incidence, severity and pathology during CIA. Mice systemically overexpressing IL-22 showed strongly reduced serum cytokine levels of TNF-α and macrophage inflammatory protein 1α that correlated significantly with the enhanced expression of the negative immune regulator SOCS3 in the spleen. CONCLUSION: With this study, we revealed clear anti-inflammatory effects of systemic IL-22 overexpression during CIA. Additionally, we are the first to show that the protective effect of systemic IL-22 during experimental arthritis is likely orchestrated via upregulation of the negative regulator SOCS3.

Therapeutic Potential for Regulation of the Nuclear Factor Kappa-B Transcription Factor p65 to Prevent Cellular Senescence and Activation of Pro-Inflammatory in Mesenchymal Stem Cells.

Mesenchymal stem cells have an important potential in the treatment of age-related diseases. In the last years, small extracellular vesicles derived from these stem cells have been proposed as cell-free therapies. Cellular senescence and proinflammatory activation are involved in the loss of therapeutic capacity and in the phenomenon called inflamm-aging. The regulators of these two biological processes in mesenchymal stem cells are not well-known. In this study, we found that p65 is activated during cellular senescence and inflammatory activation in human umbilical cord-derived mesenchymal stem cell. To demonstrate the central role of p65 in these two processes, we used small-molecular inhibitors of p65, such as JSH-23, MG-132 and curcumin. We found that the inhibition of p65 prevents the cellular senescence phenotype in human umbilical cord-derived mesenchymal stem cells. Besides, p65 inhibition produced the inactivation of proinflammatory molecules as components of a senescence-associated secretory phenotype (SASP) (interleukin-6 and interleukin-8 (IL-6 and IL-8)). Additionally, we found that the inhibition of p65 prevents the transmission of paracrine senescence between mesenchymal stem cells and the proinflammatory message through small extracellular vesicles. Our work highlights the important role of p65 and its inhibition to restore the loss of functionality of small extracellular vesicles from senescent mesenchymal stem cells and their inflamm-aging signature.

Osteoarthritis-Related Inflammation Blocks TGF-ß's Protective Effect on Chondrocyte Hypertrophy via (de)Phosphorylation of the SMAD2/3 Linker Region.

Osteoarthritis (OA) is a degenerative joint disease characterized by irreversible cartilage damage, inflammation and altered chondrocyte phenotype. Transforming growth factor-ß (TGF-ß) signaling via SMAD2/3 is crucial for blocking hypertrophy. The post-translational modifications of these SMAD proteins in the linker domain regulate their function and these can be triggered by inflammation through the activation of kinases or phosphatases. Therefore, we investigated if OA-related inflammation affects TGF-ß signaling via SMAD2/3 linker-modifications in chondrocytes. We found that both Interleukin (IL)-1ß and OA-synovium conditioned medium negated SMAD2/3 transcriptional activity in chondrocytes. This inhibition of TGF-ß signaling was enhanced if SMAD3 could not be phosphorylated on Ser213 in the linker region and the inhibition by IL-1ß was less if the SMAD3 linker could not be phosphorylated at Ser204. Our study shows evidence that inflammation inhibits SMAD2/3 signaling in chondrocytes via SMAD linker (de)-phosphorylation. The involvement of linker region modifications may represent a new therapeutic target for OA.

A roadmap to target interleukin-6 in osteoarthritis.

Joint inflammation is present in the majority of OA patients and pro-inflammatory mediators, such as IL-6, are actively involved in disease progression. Increased levels of IL-6 in serum or synovial fluid from OA patients correlate with disease incidence and severity, with IL-6 playing a pivotal role in the development of cartilage pathology, e.g. via induction of matrix-degrading enzymes. However, IL-6 also increases expression of anti-catabolic factors, suggesting a protective role. Until now, this dual role of IL-6 is incompletely understood and may be caused by differential effects of IL-6 classic vs trans-signalling. Here, we review current evidence regarding the role of IL-6 classic- and trans-signalling in local joint pathology of cartilage, synovium and bone. Furthermore, we discuss targeting of IL-6 in experimental OA models and provide future perspective for OA treatment by evaluating currently available IL-6 targeting strategies.

Commensal microbiota influence systemic autoimmune responses.

Antinuclear antibodies are a hallmark feature of generalized autoimmune diseases, including systemic lupus erythematosus and systemic sclerosis. However, the processes underlying the loss of tolerance against nuclear self-constituents remain largely unresolved. Using mice deficient in lymphotoxin and Hox11, we report that approximately 25% of mice lacking secondary lymphoid organs spontaneously develop specific antinuclear antibodies. Interestingly, we find this phenotype is not caused by a defect in central tolerance. Rather, cell-specific deletion and in vivo lymphotoxin blockade link these systemic autoimmune responses to the formation of gut-associated lymphoid tissue in the neonatal period of life. We further demonstrate antinuclear antibody production is influenced by the presence of commensal gut flora, in particular increased colonization with segmented filamentous bacteria, and IL-17 receptor signaling. Together, these data indicate that neonatal colonization of gut microbiota influences generalized autoimmunity in adult life.

The level of synovial AXL expression determines the outcome of inflammatory arthritis, possibly depending on the upstream role of TGF-ß1.

OBJECTIVE: To investigate the role of AXL, a member of the anti-inflammatory TYRO3, AXL MER (TAM) receptor family, in arthritis. METHODS: KRN serum transfer arthritis was induced in Axl-/- and wild-type mice. Knee and ankle joints were scored macro- and microscopically. Synovial gene and protein expression of Axl was determined in naïve and TGF-ß1-overexpressing joints. AXL expression was determined in M1-like or M2-like macrophages and RA synovium. Human macrophages, fibroblasts and synovial micromasses were stimulated with TGF-ß1 or the AXL inhibitor R428. RESULTS: Ankle joints of Axl-/- mice showed exacerbated arthritis pathology, whereas no effect of Axl gene deletion was observed on gonarthritis pathology. To explain this spatial difference, we examined the synovium of naïve mice. In contrast to the knee, the ankle synovial cells prominently expressed AXL. Moreover, the M2-like macrophage phenotype was the dominant cell type in the naïve ankle joint. Human M2-like macrophages expressed higher levels of AXL and blocking AXL increased their inflammatory response. In the murine ankle synovium, gene expression of Tgfb1 was increased and Tgb1 correlated with Axl. Moreover, TGFB1 and AXL expression also correlated in human RA synovium. In human macrophages and synovial micromasses, TGF-ß1 enhanced AXL expression. Moreover, TGF-ß1 overexpression in naïve murine knee joints induced synovial AXL expression. CONCLUSION: Differences in synovial AXL expression are in accordance with the observation that AXL dampens arthritis in ankle, but not in knee joints. We provide evidence that the local differences in AXL expression could be due to TGF-ß1, and suggest similar pathways operate in RA synovium.

High LDL-C levels attenuate onset of inflammation and cartilage destruction in antigen-induced arthritis.

OBJECTIVES: In this study, we used hypercholesterolaemic apolipoprotein E-deficient (Apoe-/-) mice to investigate LDL/oxLDL effect on synovial inflammation and cartilage destruction during antigen-induced arthritis (AIA). Further, as macrophage FcγRs are crucial to immune complex-mediated AIA, we investigated in vitro the effects of high cholesterol levels on the expression of FcγRs on macrophages. METHODS: AIA was induced by intra-articular injection of mBSA into knee joints of immunised Apoe-/- and wild type (WT) control mice. Joint swelling was measured by uptake of 99mTc pertechnetate (99mTc). Joint inflammation and cartilage destruction were assessed by histology. Anti-mBSA IgGs were measured by ELISA and specific T-cell response by lymphocyte stimulation test. Upon oxLDL stimulation of WT macrophages, protein levels of FcγRs were measured by flow cytometry. RESULTS: Local induction of AIA resulted in less joint swelling, synovial infiltrate and exudate in the joint cavity in Apoe-/- mice compared to WT controls, even though both their humoral and adaptive immune response were comparable. Whereas Apoe deficiency alone did not affect macrophage expression of FcγRs, oxLDL sharply reduced the protein levels of activating FcγRs, crucial in mediating cartilage damage. In agreement with the reduced inflammation in Apoe-/- mice, we observed decreased MMP activity and destruction in the articular cartilage. CONCLUSIONS: Taken together, our findings suggest that high levels of LDL/oxLDL during inflammation, dampen the initiation and chronicity of joint inflammation and cartilage destruction in AIA by regulating macrophage FcγR expression.

Milk-Derived Nanoparticle Fraction Promotes the Formation of Small Osteoclasts But Reduces Bone Resorption.

The general consensus is that milk promotes bone growth and density because is a source of calcium and contains components that enhance intestinal calcium uptake or directly affect bone metabolism. In this study, we investigated the effect of bovine-derived milk 100,000 g pellet (P100), which contains nanoparticles (<220 nm) including extracellular vesicles, on osteoclast differentiation and bone resorption. Bone marrow-derived osteoclast precursor cells were differentiated into osteoclasts by M-CSF and RANKL (control) and in the presence of milk P100. Milk P100 treatment until day 4 increased the number of TRAP-positive mononuclear cells and small (≤5 nuclei) osteoclasts. The number of large (≥6 nuclei) osteoclasts remained the same. These alterations were associated with increased expression of TRAP, NFATc1, and c-Fos. Cells seeded in a calcium-phosphate coated plate or bone slices showed reduced resorption area when exposed to milk P100 during the differentiation phase and even after osteoclast formation. Interestingly, milk P100 treatment enhanced Cathepsin K expression but reduced Carbonic Anhydrase 2 gene expression. Moreover, intracellular acid production was also decreased by milk P100 treatment. Oral delivery of milk P100 to female DBA1/J mice for 7 weeks did not alter bone area; however, increased osteoclast number and area in tibia without changes in serum RANKL and CTX-I levels. We showed for the first time the effect of milk P100 on osteoclast differentiation both in vitro and in vivo and found that milk P100 increased the formation of small osteoclasts but this does not lead to more bone resorption probably due to reduced acid secretion. J. Cell. Physiol. 232: 225-233, 2017. © 2016 Wiley Periodicals, Inc.

IL37 dampens the IL1ß-induced catabolic status of human OA chondrocytes.

Objective: A crucial feature of OA is cartilage degradation. This process is mediated by pro-inflammatory cytokines, among other factors, via induction of matrix-degrading enzymes. Interleukin 37 (IL37) is an anti-inflammatory cytokine and is efficient in blocking the production of pro-inflammatory cytokines during innate immune responses. We hypothesize that IL37 is therapeutic in treating the inflammatory cytokine cascade in human OA chondrocytes and can act as a counter-regulatory cytokine to reduce cartilage degradation in OA. Methods: Human OA cartilage was obtained from patients undergoing total knee or hip arthroplasty. Immunohistochemistry was applied to study IL37 protein expression in cartilage biopsies from OA patients. Induction of IL37 expression by IL1ß, OA synovium-conditioned medium and TNFα was investigated in human OA chondrocytes. Adenoviral overexpression of IL37 followed by IL1ß stimulation was performed to investigate the anti-inflammatory potential of IL37. Results: IL37 expression was detected in cartilage biopsies of OA patients and induced by IL1ß. After IL1ß stimulation, increased IL1ß, IL6 and IL8 expression was observed in OA chondrocytes. Elevated IL37 levels diminished the IL1ß-induced IL1ß , IL6 and IL8 gene levels and IL1ß and IL8 protein levels. In addition to the reduction in pro-inflammatory cytokine expression, IL37 reduced MMP1 , MMP3 , MMP13 and disintegrin and metalloproteinase with thrombospondin motifs 5 gene levels and MMP3 and MMP13 protein levels. Conclusion: IL37 is induced by IL1ß, and IL37 itself reduced IL1ß, IL6 and IL8 production, indicating that IL37 is able to induce a counter-regulatory anti-inflammatory feedback loop in chondrocytes. In addition, IL37 dampens catabolic enzyme expression. This supports IL37 as a potential therapeutic target in OA.

Transcriptional profiling distinguishes inner and outer annulus fibrosus from nucleus pulposus in the bovine intervertebral disc.

BACKGROUND: Cells in the intervertebral disc have unique phenotypes and marker genes that separate the nucleus pulposus (NP), annulus fibrosus (AF) and articular cartilage (AC) have been identified. Recently, it was shown that phenotypic marker genes exhibit variable expression in humans. In this study, the bovine tail was used to determine the ability of marker genes to distinguish the outer and inner AF from NP tissue and isolated cells. METHODS: Bovine tail intervertebral discs from 13 donors were dissected and correct isolation of tissue was confirmed. mRNA was isolated directly from tissue or passage 0 monolayer cells and used for gene expression measurements (qPCR). Conventional marker genes (bAcan, bCol1a1, bCol2a1) and novel marker genes (bAdamts17, bBrachyury/T, bCD24, bCol5a1, bCol12a1, bFoxf1, bKrt19, bPax1, bSfrp2) were evaluated. RESULTS: As expected bAcan, bCol2a1 and bCol1a1 distinguished outer AF from NP tissue, while inner AF and NP could not be discriminated. The NP markers bT, bCd24 and bKrt19 were significantly higher expressed in NP than inner and outer AF tissue. bFoxF1 and bPax1 only distinguished IVD tissues from AC. The AF markers bAdamts17, bCol5a1, bCol12a1 and bSfrp2 were higher expressed in the outer AF compared with inner AF and NP tissue. Monolayer culturing strongly decreased bAcan, bCol2a1, bCD24 and bCol5a1 expression, while bCol1a1, bT, bKrt19 and bSfrp2 were not affected. CONCLUSION: The IVD phenotypic marker genes bT, bKrt19, bSfrp2 and bCol12a1 convincingly distinguished NP from outer AF in situ and in vitro.

Induction of Canonical Wnt Signaling by Synovial Overexpression of Selected Wnts Leads to Protease Activity and Early Osteoarthritis-Like Cartilage Damage.

Proteins from the Wnt signaling pathway are very important for joint development. Curiously, osteoarthritis (OA) is thought to be a recapitulation of developmental processes. Various members of the Wnt signaling pathway are overexpressed in the synovium during experimental OA. Here, we investigated the potency of specific Wnt proteins, when expressed in the synovium, to induce OA pathology. We overexpressed Wnt5a, Wnt8a, Wnt16, and WISP1 in the synovium using adenoviral vectors. We determined whether overexpression resulted in OA pathology by histology, and we measured whether Wnt signaling led to increased protease activity in the joint. Synovial overexpression of Wnt8a and Wnt16 led to canonical Wnt signaling in the cartilage, whereas overexpression of Wnt5a did not. Canonical Wnt signaling increased protease activity and induced cartilage damage shortly after overexpression. Specific blocking of the canonical Wnt signaling pathway with Dickkopf-1 reduced the Wnt-signaling-induced cartilage damage. By contrast, the noncanonical signaling Wnt5a did not cause cartilage lesions. Overexpression of WISP1, a downstream protein of canonical Wnt signaling, resulted in increased cartilage damage. In conclusion, our data show that canonical Wnts and WISP1, which we found overexpressed in the synovium during experimental OA, may conduce to OA pathology.

Periodontal pathogens directly promote autoimmune experimental arthritis by inducing a TLR2- and IL-1-driven Th17 response.

Increasing epidemiologic evidence supports a link between periodontitis and rheumatoid arthritis. The actual involvement of periodontitis in the pathogenesis of rheumatoid arthritis and the underlying mechanisms remain, however, poorly understood. We investigated the influence of concomitant periodontitis on clinical and histopathologic characteristics of T cell-mediated experimental arthritis and evaluated modulation of type II collagen (CII)-reactive Th cell phenotype as a potential mechanism. Repeated oral inoculations of periodontal pathogens Porphyromonas gingivalis and Prevotella nigrescens induced periodontitis in mice, as evidenced by alveolar bone resorption. Interestingly, concurrent periodontitis induced by both bacteria significantly aggravated the severity of collagen-induced arthritis. Exacerbation of arthritis was characterized by increased arthritic bone erosion, whereas cartilage damage remained unaffected. Both P. gingivalis and P. nigrescens skewed the CII-specific T cell response in lymph nodes draining arthritic joints toward the Th17 phenotype without affecting Th1. Importantly, the levels of IL-17 induced by periodontal pathogens in CII-specific T cells directly correlated with the intensity of arthritic bone erosion, suggesting relevance in pathology. Furthermore, IL-17 production was significantly correlated with periodontal disease-induced IL-6 in lymph node cell cultures. The effects of the two bacteria diverged in that P. nigrescens, in contrast to P. gingivalis, suppressed the joint-protective type 2 cytokines, including IL-4. Further in vitro studies showed that the Th17 induction strongly depended on TLR2 expression on APCs and was highly promoted by IL-1. Our data provide evidence of the involvement of periodontitis in the pathogenesis of T cell-driven arthritis through induction of Ag-specific Th17 response.

Disease-regulated local IL-10 gene therapy diminishes synovitis and cartilage proteoglycan depletion in experimental arthritis.

OBJECTIVES: Rheumatoid arthritis is a chronic destructive autoimmune disease, but the course is unpredictable in individual patients. An attractive treatment would provide a disease-regulated therapy that offers personalised drug delivery. Therefore, we expressed the anti-inflammatory interleukin-10 (IL-10) gene under the control of inflammation-dependent promoters in a mouse model of arthritis. METHODS: Proximal promoters of S100a8, Cxcl1, Mmp13, Saa3, IL-1b and Tsg6 were selected by whole-genome expression analysis of inflamed synovial tissues from arthritic mice. Mice were injected intraarticularly in knee joints with lentiviral vectors expressing a luciferase reporter or the therapeutic protein IL-10 under control of the Saa3 or Mmp13 promoter. After 4 days, arthritis was induced by intraarticular injection of streptococcal cell walls (SCW). At different time points after arthritis induction, in vivo bioluminescent imaging was performed and knee joints were dissected for histological and RNA analysis. RESULTS: The disease-regulated promoter-luciferase reporter constructs showed different activation profiles during the course of the disease. The Saa3 and Mmp13 promoters were significantly induced at day 1 or day 4 after arthritis induction respectively and selected for further research. Overexpression of IL-10 using these two disease-inducible promoters resulted in less synovitis and markedly diminished cartilage proteoglycan depletion and in upregulation of IL-1Ra and SOCS3 gene expression. CONCLUSIONS: Our study shows that promoters of genes that are expressed locally during arthritis can be candidates for disease-regulated overexpression of biologics into arthritic joints, as shown for IL-10 in SCW arthritis. The disease-inducible approach might be promising for future tailor-made local gene therapy in arthritis.

In vivo molecular imaging of cathepsin and matrix metalloproteinase activity discriminates between arthritic and osteoarthritic processes in mice.

Rheumatoid arthritis (RA) and osteoarthritis (OA) are serologically and clinically distinctive, but at the local level, both diseases have many molecular pathways in common. In vivo molecular imaging can unravel the local pathologic processes involved in both diseases. In this study, we investigated matrix metalloproteinase (MMP) and cathepsin activity during cartilage destruction, in an RA and an OA mouse model, using biophotonic imaging of substrate-based probes. Mice with collagen-induced arthritis (CIA) or destabilization of the medial meniscus (DMM) were imaged using near-infrared fluorescent probes, activated by several cathepsins or MMPs. Fluorescence signal intensity was compared to synovial gene expression, histology, and cartilage staining of a neoepitope of aggrecan cleaved by MMPs with the amino acids DIPEN. Increased cathepsin and MMP activity was seen during CIA, whereas the DMM model only showed increased MMP activity. DIPEN expression was seen only during CIA. A possible explanation can be differences in gene expressions; MMP3 and -13, known to produce DIPEN neoepitopes, were upregulated in the CIA model, whereas MMP12, known to be involved in elastin degradation and chemokine inhibition, was upregulated in the DMM model. Thus, molecular imaging showed no cathepsin activity at the time of cartilage damage in the DMM model, whereas both cathepsins and MMPs are active in the CIA model during disease progression.

Toll-like receptor 2 controls acute immune complex-driven arthritis in mice by regulating the inhibitory Fcγ receptor IIB.

OBJECTIVE: Previous studies have demonstrated a protective role of Toll-like receptor 2 (TLR-2) and a proinflammatory function of TLR-4 during chronic T cell-driven arthritis. The involvement of TLRs in T cell-independent arthritic processes, however, remains unclear. This study was undertaken to determine the functional significance of TLR-2 and TLR-4 in T cell-independent immune complex-driven arthritis. METHODS: Serum-transfer arthritis was induced in wild-type and TLR-deficient mice by intraperitoneal injections of arthritogenic K/BxN mouse serum. Arthritis was assessed macroscopically and by histologic analysis. The influence of TLR-2 on macrophage cytokine profile, Fcγ receptor (FcγR) expression, and response to immune complexes was determined. RESULTS: While TLR-4, unexpectedly, did not play any significant role, TLR-2 deficiency accelerated the onset and markedly increased the severity of acute immune complex-driven arthritis in mice. TLR-2 deficiency resulted in a substantial increase in joint inflammation, bone erosion, and cartilage pathology, indicating a protective function of TLR-2 in passive FcγR-driven disease. Ex vivo study of the macrophage inflammatory phenotype revealed increased production of tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) despite similar levels of IL-10, along with a significant increase in FcγR-specific response, in TLR-2-/- mouse macrophages early in the disease. Although distinct FcγR messenger RNA expression was not affected, cell surface protein expression of the inhibitory FcγRIIB in TLR-2-/- naive primary macrophages was specifically diminished, resulting in a higher proinflammatory response. Accordingly, TLR-2 stimulation specifically up-regulated FcγRIIB, but not the activating FcγR, on macrophages. CONCLUSION: TLR-2 regulates acute immune complex-driven arthritis by controlling macrophage FcγR response. Our findings indicate that the protective role of TLR-2 is extended beyond its previously described role in promoting Treg cells during T cell-mediated arthritis.