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Background The aim of this study was to assess the anti-inflammatory effects of local cryotherapy in human non-septic knee arthritis. Methods In the phase I of the study, patients were randomized to receive either ice (30 min; N  = 16) or cold CO 2 (2 min; N  = 16) applied twice during 1 day at an 8-h interval on the arthritic knee. In phase II, 16 other ice-treated arthritic knees according to the same protocol were compared to the contralateral non-treated arthritic knees ( N  = 16). The synovial fluid was analyzed just before the first cold application, then 24 h later. IL-6, IL-1β, TNF-α, IL-17A, VEGF, NF-kB-p65 protein, and PG-E2 levels were measured in the synovial fluid and compared before/after the two cold applications. Results Forty-seven patients were included (17 gouts, 11 calcium pyrophosphate deposition diseases, 13 rheumatoid arthritides, 6 spondyloarthritides). Local ice cryotherapy significantly reduced the IL-6, IL-1β, VEGF, NF-kB-p65, and PG-E2 synovial levels, especially in the microcrystal-induced arthritis subgroup, while only phosphorylated NF-kB-p65 significantly decreased in rheumatoid arthritis and spondyloarthritis patients. Cold CO 2 only reduced the synovial VEGF levels. In the phase II of the study, the synovial PG-E2 was significantly reduced in ice-treated knees, while it significantly increased in the corresponding contralateral non-treated arthritic knees, with a significant inter-class effect size (mean difference − 1329 [− 2232; − 426] pg/mL; N  = 12). Conclusions These results suggest that local ice cryotherapy reduces IL-6, IL-1β, and VEGF synovial protein levels, mainly in microcrystal-induced arthritis, and potentially through NF-kB and PG-E2-dependent mechanisms. Trial registration Clinicaltrials.gov, NCT03850392 —registered February 20, 2019—retrospectively registered

Aim This study explored the systemic vascular effects of local cryotherapy with a focus on endothelial changes and arterial inflammation in the model of rat adjuvant-induced arthritis (AIA). Methods Cryotherapy was applied twice a day on hind paws of AIA rats from the onset of arthritis to the acute inflammatory phase. Endothelial activation was studied in the aorta by measuring the mRNA levels of chemokines (CXCL-1, MCP-1 (CCL-2), MIP-1α (CCL-3)) and adhesion molecules (ICAM-1, VCAM-1) by qRT-PCR. Endothelial dysfunction was measured in isolated aortic and mesenteric rings. Aortic inflammation was evaluated via the mRNA expression of pro-inflammatory cytokines (TNF-α, IL-6) by qRT-PCR and leucocyte infiltration analysis (flow cytometry). Plasma levels of TNF-α, IL-6, IL-1β, IL-17A, and osteoprotegerin (OPG) were measured using Multiplex/ELISA. Results AIA was associated with an increased aortic expression of CXCL-1 and ICAM-1 as well as an infiltration of leucocytes and increased mRNA expression of IL-6, IL-1β, and TNF-α. Local cryotherapy, which decreased arthritis score and structural damages, reduced aortic mRNA expression of CXCL-1, IL-6, IL-1β, and TNF-α, as well as aortic infiltration of leucocytes (T lymphocytes, monocytes/macrophages, neutrophils) and improved acetylcholine-induced vasorelaxation in the aorta and mesenteric arteries. Plasma levels of IL-17A and OPG were significantly reduced by cryotherapy, while the number of circulating leucocytes was not. IL-17A levels positively correlated with endothelial activation and dysfunction. Conclusion In the AIA model, local cryotherapy reduced systemic endothelial activation, immune cell infiltration, and endothelial dysfunction. Mechanistically, the reduction of circulating levels of IL-17A appears as the possible link between joint cooling and the remote vascular effects.

BackgroundMAIT (mucosal associated invariant T) cells are involved in mucosa defense against bacteria. This cellular subset is characterized by a semi-invariant αβ TCR and the expression of CD161. In patients with axial spondyloarthritis (ax SpA), previous works reported a decreased frequency of circulating MAIT cells compared to normal subjects. IL-17A is a relevant cytokine involved in SpA pathophysiology. MAIT cells are a cellular source of IL-17 and IL-17+ MAIT cells were found increased in ax SpA. We have previously reported an increased frequency of IFNγ+/IL-17+ MAIT cells as well as IL-22+ MAIT cells in ax SpA[1].ObjectivesIn this study, we aimed to complete our previous results by evaluating activation markers and chemokine receptor/integrin expression, especially those for gut homing, in MAIT cells.Methodspatients exhibited ax SpA (ASAS criteria) with a radiographic (r-ax SpA) or non-radiographic (nr-ax SpA) form. They all were under NSAIDs and biologic naïve. Healthy subjects were recruited as controls (HC). Circulating CD4+ and CD8+ T cells and MAIT cells were determined on blood samples by single platform flow cytometry (Cytoflex, Beckman Coulter). MAIT cells were identified by the co-expression of CD3, CD161 and TCRα7.2. In each subpopulation, we examined the expression of CD26, CD69, CCR9 and CD49d integrin.Results26 patients were included (11 r-ax SpA: 9 males [M]; mean age 54.1 ± 19.6 years; disease duration: 16.2 years; ASDAS score: 4.1; and 15 nr-ax SpA: 7 M; age: 36.4 ± 1.3; ASDAS: 5.6) and 27 HC (16 M; age: 43 ± 12.7). In patients with r-ax SpA, we observed an increased frequency of activated CD3+ CD69+ MAIT cells and CD3+ MAIT cells expressing the gut homing markers CCR9 and CD49d, as compared to HC (p <0.05). These higher frequencies were not observed in patients with nr-ax SpA. In addition, when examining the CD8+ MAIT population, similar higher frequencies of cells positive for CD69, CCR9 and CD49d were observed in patients with r-ax SpA compared to HC and patients with nr-ax SpA (p<0.01 and p<0.05, respectively).These modifications were specific for MAIT cells and were not observed in conventional CD4+ or CD8+ T lymphocytes.Table.HCr-ax SpAnr-ax SpA% CD69+ MAIT CD3+18.4 ± 9.928.3 ± 14.7 *19.7 ± 10.3% CD69+ MAIT CD8+19.1 ± 11.429.5 ± 15.6 *20.8 ± 12.0% CD49d+CCR9+ MAIT CD3+1.6 ± 1.72.5 ± 1.6 *1.9 ± 2.3% CD49d+CCR9+ MAIT CD8+1.4 ± 1.72.5 ± 1.8**1.7 ± 2ConclusionPatients with r-ax SpA are characterized by an increased frequency of activated MAIT cells that expressed homing receptors (chemokine and integrin) for the gut. These results confirm that MAIT cells are altered in ax SpA and highlight the relationships between ax SpA and gut inflammation, especially for the radiographic form.Figure 1.Reference[1]Toussirot E, Laheurte C, Gaugler B, Gabriel D, Saas P. Increased IL-22- and IL-17A-producing Mucosal-Associated Invariant T cells in the peripheral blood of patients with ankylosing spondylitis. Front Immunol. 2018; 9:1610.Acknowledgements:NIL.Disclosure of InterestsNone Declared.

Acute graft-versus-host disease (aGVHD) remains a major complication following allogeneic hematopoietic cell transplantation, limiting the success of this therapy. We previously reported that interleukin-22 (IL-22) participates to aGVHD development, but the underlying mechanisms of its contribution remain poorly understood. In this study, we analyzed the mechanism of the pathological function of IL-22 in intestinal aGVHD. Ex-vivo colon culture experiments indicated that IL-22 was able to induce Th1-like inflammation via signal transducer and activator of transcription factor-1 (STAT1) and CXCL10 induction in the presence of type I interferon (IFN). To evaluate a potential synergy between IL-22 and type I IFN in aGVHD, we transplanted recipient mice, either wild-type (WT) or type I IFN receptor deficient (IFNAR−/−), with bone marrow cells and WT or IL-22 deficient (IL-22−/−) T cells. We observed a decreased GVHD severity in IFNAR−/− recipient of IL-22−/− T cells, which was associated with a lower level of STAT1 activation and reduced CXCL10 expression in the large intestine. Finally, immunohistochemistry staining of STAT1 performed on gastrointestinal biopsies of 20 transplanted patients showed exacerbated STAT1 activation in gastrointestinal tissues of patients with aGVHD as compared with those without aGVHD. Thus, interfering with both IL-22 and type I IFN signaling may provide a novel approach to limit aGVHD.

Acute graft-versus-host disease (aGVHD) remains a major complication following allogeneic hematopoietic cell transplantation (allo-HCT), limiting the success of this therapy. Many proinflammatory cytokines secreted following the conditioning regimen have been linked to aGVHD initiation. Interleukin-22 (IL-22) is a cytokine related to IL-10 for its structure and is secreted by T helper type 17 (TH17) cells and innate immune cells. Given the paradoxical role of IL-22 in inflammation with both protective or proinflammatory functions, we investigated whether IL-22 could have a role in aGVHD pathophysiology in a mouse allo-HCT model. In this study, we show that IL-22 deficiency in donor T cells can decrease the severity of aGVHD, while limiting systemic and local inflammation in aGVHD target organs. In addition, we found that Foxp3+ regulatory T cells (Treg cells) were increased in recipient mice that received IL-22-deficient T cells, suggesting that Treg were involved in the reduced severity of GVHD. Finally, we found that the graft-versus-leukemia (GVL) effect mediated by donor T cells was preserved in the absence of IL-22. Overall, these data suggest that targeting of IL-22 may represent a valid approach towards decreasing aGVHD severity after allo-HCT while preserving the GVL effect.

The aim of this study was to assess the anti-inflammatory effects of local cryotherapy in human non-septic knee arthritis. In the phase I of the study, patients were randomized to receive either ice (30 min; N = 16) or cold CO.sub.2 (2 min; N = 16) applied twice during 1 day at an 8-h interval on the arthritic knee. In phase II, 16 other ice-treated arthritic knees according to the same protocol were compared to the contralateral non-treated arthritic knees (N = 16). The synovial fluid was analyzed just before the first cold application, then 24 h later. IL-6, IL-1[beta], TNF-[alpha], IL-17A, VEGF, NF-kB-p65 protein, and PG-E2 levels were measured in the synovial fluid and compared before/after the two cold applications. Forty-seven patients were included (17 gouts, 11 calcium pyrophosphate deposition diseases, 13 rheumatoid arthritides, 6 spondyloarthritides). Local ice cryotherapy significantly reduced the IL-6, IL-1[beta], VEGF, NF-kB-p65, and PG-E2 synovial levels, especially in the microcrystal-induced arthritis subgroup, while only phosphorylated NF-kB-p65 significantly decreased in rheumatoid arthritis and spondyloarthritis patients. Cold CO.sub.2 only reduced the synovial VEGF levels. In the phase II of the study, the synovial PG-E2 was significantly reduced in ice-treated knees, while it significantly increased in the corresponding contralateral non-treated arthritic knees, with a significant inter-class effect size (mean difference - 1329 [- 2232; - 426] pg/mL; N = 12). These results suggest that local ice cryotherapy reduces IL-6, IL-1[beta], and VEGF synovial protein levels, mainly in microcrystal-induced arthritis, and potentially through NF-kB and PG-E2-dependent mechanisms.

BackgroundAnkylosing spondylitis (AS) is the prototypic and most common form of spondyloarthritis (SpA). It was recently suggested that IL-23 could play a pivotal role in the pathophysiology of SpA. Indeed, there is a strong genetic association of SpA with polymorphisms of the IL-23 receptor (IL-23R) gene. In experimental models, IL-23 is produced during the misfolding phenomenon of HLA-B27 heavy chain in the endoplasmic reticulum. Furthermore, in animal models, IL-23 overexpression induced a SpA-like disease via IL-23+ resident T cells infiltrating entheseal structures and producing IL-17 and IL-22. In addition, there is an increased number of circulating cells producing IL-17 in patients with AS. Recent data also suggested that IL-17A is a potential target in the treatment of patients with AS.ObjectivesCells that expressed IL-23R as well as those producing IL-17 and/or IL-22 have been poorly characterized in AS and/or SpA. Thus, we aimed to better delineate the peripheral blood T cells that expressed IL-23R and the IL-17 and IL-22 producing cell subsets, notably conventional T cells as well as mucosal associated invariant T cells (MAIT) in patients with AS.Methods36 patients with AS (modified NY criteria; 27 M; age: 44.7 ± 2.6, disease duration: 12.7 ± 1.6; all under NSAIDs or traditional DMARDs) and 31 healthy controls (HC) (8 M; age: 46 ± 2.3) were evaluated. For each subject, peripheral blood T cell subsets were assessed using multi-color flow cytometry. Intracellular cytokine IL-17A, IL-22 and IFN-γ were evaluated after phorbol myristate acetate and ionomycine activation and permeabilization and analysed by flow cytometry. MAIT cells were identified by using TCRVα7.2 specific monoclonal antibodies. IL-22 serum levels were determined by ELISA using commercially available kit.Resultsthe number and percentage of CD3+ T cells, CD3+CD4+ and CD3+CD8+ T cells were comparable between patients and HC. Circulating IL-22 levels did not differ between patients and HC (22.6 ± 8.4 vs 15.1 ± 4.4 pg/ml, p=0.7). IFN-γ producing CD4+ and CD8+ T cells were decreased in AS compared to HC (4.6 ± 1.7 vs 11.4 ± 2.5% and 23.3 ± 1.9 vs 34.3 ± 2.3%, respectively, p=0.005 and p=0.002). CD8+ T cells expressing IL-23R were significantly increased in AS: 72.8 ± 1.5 vs 65.2 ± 2% (p=0.009). The percentage of MAIT T cells did not differ between patients and HC but we found an increased percentage of MAIT cells that produced IL-17 and IL-22: MAIT IFN-γ– IL-17+: 0.43 ± 0.1 vs 0.12 ± 0.008%, p=0.006; MAIT IFN-γ– IL-22+: 0.83 ± 0.1 vs 0.3 ± 0.1%, p=0.004. No correlation was found between the percentage of CD8+ IL-23R+ T cells and measurements of disease activity as well as between IL-17+ or IL-22+ MAIT cells and disease activity.ConclusionsThese preliminary results suggest that the peripheral blood of patients with AS is enriched in CD8+ IL-23R+ T cells as well as IL-17 and IL-22 producing MAIT cells. Since MAIT cells are involved in antibacterial immunity and bacterial agents are potentially involved in SpA, our preliminary data suggest that this T cell subset could potentially play a role in AS via IL-17 and IL22 production. This remains to be clarified by future and complementary studies.Disclosure of InterestNone declared

BackgroundMicroparticles (MPs) are small membrane-bound vesicles that arise from activated and dying cells. Although the majority of MPs in the blood originate from platelets, all cells appear to be able to release MPs. Many studies have raised the implication of these MP in various processes: inflammation, thrombosis, angiogenesis. Previous studies reported inconsistent results in inflammatory rheumatic diseases. Studies have shown the correlation between the circulating MPs, platelet MPs and lymphocyte MPs in patients with spondyloarthritis (SpA) compared to control patients. For rheumatoid arthritis (RA), platelet MPs levels were correlated with DAS28.ObjectivesThe aim of this study was to search a possible correlation between the disease activity and the pro-coagulant property of microparticles, potential indirect marker of inflammation.MethodsThe test used (STA Procoag PPL) is a standardised automated test. Results are expressed as coagulation times (in seconds). It is a functional test that provides information on the procoagulant potential of microparticles. The microparticles supply the phospholipids expressed on their membrane surface and the test provides calcium and factor Xa necessary to initiate coagulation: the shorter the coagulation time the greater the procoagulant activity of the phospholipids being studied, suggesting a higher number of MP.This is a prospective, single-centre study, including 39 patients with spondyloarthritis (ASAS criteria), 37 with rheumatoid arthritis (ACR criteria) and 26 control patients (healthy subjects, osteoarthritis). All patients underwent STA Procoag PPL test, and we collected medical data: disease activity (BASDAI, BASFI, DAS28vs and DAS28crp and HAQ), biological inflammation (VS, CRP), duration of disease, and current treatment.ResultsThe in vitro clotting time of serum of patients with spondyloarthritis and rheumatoid arthritis compared with controls was not significantly different (p=0.23 and p=0.44, respectively). Regarding the activity scores of inflammatory rheumatic disease: BASDAI and BASFI, DAS28esr, DAS28crp and HAQ for patients with RA, no correlation between these data and coagulation time was found; the same goes for biological inflammation (ESR, CRP), duration and type of treatment (Nonsteroidal anti-inflammatory drugs, DMARDs, biologics).Abstract AB0056 – Table 1STA PPLControls (n=26)SpA (n=39)pRA (n=37)p Mean±SD62,70±11,5866,48±12,77p=0,2365,25±13,54p=0,44 BASDAIr=0,0602 p=0,72DAS28 esrr=0230 p=0,17BASFIr=-0,125 p=0,45DAS28 crpr=0151 p=0,37ConclusionsIn this study, there is neither difference in values of procoagulant activity of MPs between inflammatory rheumatic diseases and control subjects, nor correlation with their activity scores or biological inflammation.Disclosure of InterestNone declared

BackgroundCryotherapy is widely used in rheumatic diseases, with a low level of evidence. Tissue mild hypothermia was reported to inhibit pivotal pro-inflammatory enzyme pathways such as COX-2/PG-E21 and NF-kB.2 ObjectivesWe hypothesised that local cryotherapy (LC) might reduce joint inflammation through PG-E2 and NF-kB repression.Methods47 patients suffering from non-septic knee arthritis were included (17 gouts, 11 calcium pyrophosphate dehydrate crystal deposition diseases, 6 spondyloarthritides, 13 rheumatoid arthritides), taking no concurrent anti-inflammatory drug/DMARD. They were first randomised to receive local ice (30 min-N=16) or cold pulsed CO2 (2 min – Cryo+ Cryonic-N=16) at 9 A.M and 5 P.M. Synovial fluid was collected just before the first cold application then 24 hours later (9 A.M). Synovial fluid IL-6, IL-1β, TNF-α, IL-17A, VEGF (Multiplex flow cytometry), NF-kB P65 (total/phosphorylated) and PG-E2 (ELISA) were measured and compared before and after 2 LC applications. Contralateral non-treated knees were then used as paired controls (in 16 other ice-treated patients).ResultsSynovial IL-6 significantly decreased after 2 LC applications (n=43 paired Wilcoxon:p=0.00015). This decrease was also significant in ice-treated subgroup (n=16 p<0.005) but not in CO2-treated patients (n=14;p=0.1). IL-1β and VEGF levels also decreased after treatment (n=44-p=0.02 and n=47-p=0.03) but not significantly in treatment subgroups. By contrast, IL-6 (n=13), IL-1β (n=15) and VEGF (n=15) didn’t change in contralateral non-treated knees. LC had no significant effect on IL-17A nor TNF-α synovial levels. LC significantly reduced synovial NF-kB (n=38-p=0.04) and NF-kB-P levels (n=38-p=0.004). These levels were also significantly decreased in ice-treated patients (n=26-p=0.03 and 0.003) but not in CO2-treated patients (n=13). We observed no effect on the NF-kB levels in contralateral non-treated knees (n=12). NF-kB variations correlated significantly with IL-6 (Pearson’s r=0.48-N=32 p<0.01) and VEGF (r=0.34-N=36-p=0.038) variations, and with the maximal skin temperature drop induced by LC (r=0.32-N=36-p=0.047). LC significantly reduced synovial PG-E2 (n=38-p=0.04), which was also significantly decreased in ice-treated patients (n=26-p=0.02) but not in CO2-treated patients (n=13). Conversely, we observed a significant increase in PG-E2 levels in contralateral non-treated knees (n=12-p=0.04), with a significant inter-class effect-size (weighted mean difference −1329 [−2232;−426] pg/mL-in 12 bi-arthritic patients).ConclusionsLocal ice cryotherapy applied twice during one day showed superior anti-inflammatory effects compared to local CO2, notably by reducing synovial IL-6 levels in arthritic knees. We also showed for the first time that this inhibitory effect on cytokine levels might be PG-E2 and NF-kB dependent, notably through significant inhibitory effects on the PG-E2 and (to a lesser extent) NF-kB pathways.References[1] Diestel. Cryobiology2008.[2] Yenari. Neurochem Int2006.Disclosure of InterestNone declared

TNF-related apoptosis-inducing ligand or Apo2L (Apo2L/TRAIL) is a promising anti-cancer drug owing to its ability to trigger apoptosis by binding to TRAIL-R1 or TRAIL-R2, two membrane-bound receptors that are often expressed by tumor cells. TRAIL can also bind non-functional receptors such as TRAIL-R4, but controversies still exist regarding their potential to inhibit TRAIL-induced apoptosis. We show here that TRAIL-R4, expressed either endogenously or ectopically, inhibits TRAIL-induced apoptosis. Interestingly, the combination of chemotherapeutic drugs with TRAIL restores tumor cell sensitivity to apoptosis in TRAIL-R4-expressing cells. This sensitization, which mainly occurs at the death-inducing signaling complex (DISC) level, through enhanced caspase-8 recruitment and activation, is compromised by c-FLIP expression and is independent of the mitochondria. Importantly, TRAIL-R4 expression prevents TRAIL-induced tumor regression in nude mice, but tumor regression induced by TRAIL can be restored with chemotherapy. Our results clearly support a negative regulatory function for TRAIL-R4 in controlling TRAIL signaling, and unveil the ability of TRAIL-R4 to cooperate with c-FLIP to inhibit TRAIL-induced cell death.