No abstract

BackgroundTreatment effect of tumor necrosis factor α (TNFα) inhibitors is still low in patients with rheumatoid arthritis (RA), around 50%-70%. Thus more drugs by targeting proliferation of synovial fibroblasts (FLS) and TNFα induced inflammatory cytokine production are needed. Repurposed use of drugs that have been used in clinic is a quick and cost-effective way to find new drugs.ObjectivesThis study aims to screen drugs that could inhibit the proliferation and inflammation induced by TNFα in FLS from FDA approved on market drug library, then to assess the treatment effect of the identified drugs on collagen-induced arthritis (CIA) mouse model.MethodsCCK8 assay was performed to screen the drugs that could inhibit FLS proliferation, followed by qRT-PCR and ELISA to select the drugs that could suppress TNFα induced inflammatory cytokine production. Then, treatment effects of the identified drugs were assessed in CIA mouse model.ResultsThe first and second round drug librabry screening aimed to select out the drugs that could inhibit the proliferation of FLS. Results showed, from 1815 drugs, 372 drugs were indentified at the initial screening (Figure 1A) and 121 drugs were identified from the second screening (Figure 1B). The third round screening was performed to screen the drugs that could inhibit TNFα-induced inflammation, and results showed that a total of 77 drugs could inhibit mRNA expression levels of both IL-6 and IL-8 by over 50%, and 64 drugs could suppress secretion levels of both IL-6 and IL-8 for more than 20% (Figure 1C-F). Then a total of 14 drugs that were not anti-cancer drugs were used to further confirm the inhibitory effect on the TNFα induced inflammation, and results showed only Agomelatine (AOM), Cinacalcet (CCC), Amlodipine (ALM), Simvastatin (SV), and Pindolol (PDL) could suppress mRNA expression and secretion levels of IL-6, IL-8 (Figure 1G-J).As highly proliferated FLS and inflammatory cytokines play a critical role in the pathology of rheumatoid arthritis (RA), we tested the treatment effect of AOM, CCC, ALM, SV, and PDL in the CIA mose model. Results showed ALM (5mg/kg), CCC (10mg/kg), and PDL (10mg/kg) had a trend to reduce the clinical score but did not reach statistical meaning while SV (10mg/kg) did not show any treatment effect. Whereas, AOM (10mg/kg) could effectively alleviate the swelling of the the joint, which was as effective as the positive control drug methotrexate (MTX) (Figure 1K). ALM, AOM, as well as MTX could reduce the pathological score of inflammation and bone erosion at both the knee and the anke while PDL could only reduce the inflammation and relieve bone erosion at the knee(Figure 1L-N).ConclusionAgomelatine indentified from the FDA approved drug library could inhibit FLS proliferation and TNFα induced inflammation, and was therapeutic against CIA mouse model.Figure 1.Agomelatine identified from the FDA-approved drug library is therapeutic against collagen induced arthritis. (A) The first round screening by CCK8 assay. The drugs could inhibit more than 20% absorbance at 450nm, under the red lines, were the target ones, n=372. (B) The second round screening. The drugs could inhibit more than 10% absorbance at 450nm, under the red lines, were the target ones, n=121. (C-F) The third round screening. (C-D) mRNA expression levels of IL-6 and IL-8. (E-F) the secretion levels of IL-6 and IL-8. (G-J) The confirmation of drugs’ inhibitory effect on the inflammation cytokine production. Red asterisk indicated the conparison between TNFα group and DMSO group and blue pound signs suggested the conparison between drugs and TNFα group. (G-H) the mRNA expression levels of IL-6 and IL-8. (I-J) the secretion levels of IL-6 and IL-8. CIA mouse model was established to test the treatment effect of SV (10mg/kg), PDL (10mg/kg), ALM (5mg/kg), CCC (10mg/kg), and AOM (10mg/kg), and MTX (2mg/kg) was set as the positive control. (K) clinical score of joint swelling (L) H&E staining of knee joint (M) H&E staining of ankle joint (N) pathological scoring of H&E staining for joint inflammation and bone erosion.AcknowledgementsThis study is supported by Sichuan Science and Technology Program (2021JDRC0045 and 2021YFS0164), Post-doctoral Research and Development Fund of West China Hospital of Sichuan University (2019HXBH090), and National Natural Science Foundation of China (No. 82201985).Disclosure of InterestsNone Declared.

BackgroundAnkylosing Spondylitis (AS) and psoriatic arthritis (PsA) are chronic inflammatory diseases of undetermined etiology. Both entities exhibit features of autoimmunity (exhibited by clonal expansion of T cell populations) and autoinflammation, as noted by the aberrant activity of innate and innate-like cells [1].ObjectivesIn this study, we screen for genetic variants associated with known autoinflammatory disease in patients with AS, PsA, and compare it to rheumatoid arthritis (RA) and psoriasis (Ps).MethodsThe UK genome biobank identified exomes of AS, PsA, RA, and Ps patients. Autoinflammatory genes were selected from the blueprint autoinflammatory syndrome panel [2]. Twenty thousand nine hundred five missense mutations were identified from the 47 autoinflammatory genes in the blueprint panel and screened in these four cohorts. Mutational burden, mutation number, and mutation rate in these 47 genes were calculated.ResultsExonic mutations of 1264 in AS, 886 in PsA, 5361 in RA and 5567 in Ps patients from the UK biobank were enrolled and analyzed in this study. The number of autoinflammatory variants identified was 937 in AS, 780 in PsA, 2081 in RA and 2015 in Ps. The average mutation rate in 47 genes is around 0.26 for these four diseases. The autoinflammatory panel’s average mutation burden was not significantly different between these diseases (116 in AS and PsA, 118 in RA and Ps). However, when we accounted for the number of individuals screened, AS and PsA had a higher mean number of autoinflammatory variants than RA and Ps. Specifically, AS and PsA patients had a mean number of 0.741 and 0.880 variants per individual, respectively, compared to 0.388 and 0.362 for RA and Ps, respectively.ConclusionThis study strengths the role of autoinflammation in AS and PsA as we report an overabundance of autoinflammatory variants in AS and PsA compared to RA and psoriasis. Consideration of autoinflammatory etiology may impact current diagnostic and therapeutic approaches.References[1]Mauro D, Thomas R, Guggino G, Lories R, Brown MA, Ciccia F. Ankylosing spondylitis: an autoimmune or autoinflammatory disease? Nat Rev Rheumatol. 2021 Jul;17(7):387-404. doi: 10.1038/s41584-021-00625-y. Epub 2021 Jun 10. PMID: 34113018.[2]https://blueprintgenetics.com/tests/panels/immunology/autoinflammatory-syndrome-panel/Table 1.Autoinflammatory variants in AS, PsA, RA and PsDiseaseNo. of PatientsNo. of Autoinflammatory VariantsAverage No. of Autoinflammatory Variants per patientAS12649370.741PsA8867800.880RA536120810.388Ps556720150.362Acknowledgements:NIL.Disclosure of InterestsQuan Li: None declared, Amanda Dohey: None declared, Dianne Codner: None declared, Proton Rahman Speakers bureau: Abbott, AbbVie, Amgen, Bristol-Myers Squibb, Celgene, Eli Lilly, Janssen, Novartis, and Pfizer, Consultant of: Abbott, AbbVie, Amgen, Bristol-Myers Squibb, Celgene, Eli Lilly, Janssen, Novartis, and Pfizer, Grant/research support from: Janssen and Novartis.

BackgroundInterstitial pulmonary fibrosis is the deadliest manifestation of rheumatoid arthritis (RA), and its pathogenesis is complicated. As a new drug for controlling RA, clinical studies have found that iguratimod (IGU) has certain advantages in improving lung function and shows great potential for pulmonary fibrosis therapy[1]. However, the specific mechanism of IGU in treating pulmonary fibrosis is not clear.ObjectivesThis study was designed to observe and investigate the therapeutic effects of IGU on bleomycin-induced pulmonary fibrosis in mice and further investigate its underlying mechanism, aiming to provide a further theoretical basis for clinical rational drug use.MethodsA mouse model of pulmonary fibrosis was induced by intratracheal injection of bleomycin (BLM)[2]. Model mice were randomly assigned to receive Sodium carboxymethyl cellulose (CMC) or different concentrations of IGU. HE staining and immunohistochemical staining were performed to observe the therapeutic effects of IGU on mouse fibrosis. TGF-β induced A549 EMT cell model was utilized to investigate the effects of IGU on EMT in vitro[3]. NLRP3 inflammasome was activated by the co-stimulation of TGF-β+LPS+ATP (TLA) to evaluate the effects of IGU in vitro[4].ResultsWe found that IGU resulted in favorable therapeutic outcomes by affecting the inflammation infiltration (Figure 1A) and collagen deposition (Figure 1B). Immunohistochemical results showed that IGU downregulated the levels of α-SM and NLRP3. Additionally, the markers of BLM-mediated EMT (Figure 1C) phenotype and NLRP3-activated (Figure 1D) phenotype in the lung were also attenuated after IGU administration. In vitro experiments, the results confirmed its anti-EMT effects of reducing the expression of interstitial proteins Vimentin and α-SMA and restoring the content of epithelial protein E-cadherin. Besides, IGU could inhibit NLRP3 activation by downregulating NLRP3 related marker proteins, reducing the secretion of IL-1β, and attenuating caspase-1 activity. We then found that IGU anti-EMT and anti-NLRP3 effect was accompanied by decreased ROS production.Figure 1.IGU ameliorates BLM-induced pulmonary inflammation and pulmonary fibrosis. (A) Representative lung micrographs of lung tissues stained with HE. Magnification, × 200. (B) Representative photographs of lung tissues stained with Masson’s trichrome staining. Magnification, × 200. (C) Representative results of western blot for E-cadherin, Vimentin, α-SMA expression in lung tissues. GAPDH was used as the endogenous control. (D) Representative results of western blot for NLRP3, caspase-1, cleaved-caspase-1 p20, IL-1β expression in lung tissues. GAPDH was used as the endogenous control.ConclusionIGU can inhibit the EMT process and NLRP3 inflammasome activation, reduce ROS production to ameliorate pulmonary fibrosis, which may provide new insights into the further application of IGU in Interstitial pulmonary fibrosis.References[1]Shu P, et al. Eur Rev Med Pharmacol Sci 2021, 25(4):4687 v Med[2]Philipp K, et al. Eur Respir J 2020, 55(6):1901105.[3]Justin C. H, et al. Matrix Biol 2018, 71-72:112-127.[4]Anita A P, et al. Front Pharmacol 2020, 11:1201.Disclosure of InterestsNone declared.