Novel artesunate and isatin hybrid CT3-1 suppresses collagen-induced arthritis through abrogating dendritic cell chemotaxis-induced by CCR5.

BACKGROUND: Chemotaxis and trafficking of dendritic cells (DCs) induced by cytokine receptors are crucial steps in rheumatoid arthritis (RA) pathogenesis. C-C chemokine receptor type 5 (CCR5) plays a key role in DC movement and has been implicated in multitudinous inflammatory and immunology diseases. Thus, targeting CCR5 to suppress DC chemotaxis is considered as a potential strategy for the management of RA. METHODS: Herein, we first synthesized a new hybrid named CT3-1 which based on artesunate and isatin. Besides, we studied the regulating effectiveness of CT3-1 on bone marrow-derived DCs (BMDCs) and on collagen-induced arthritis (CIA) through RNA-seq analysis, cell function experiments in vitro and mice model in vivo. RESULTS: The results shown that CT3-1 mainly reduced CCR5 expression of immature BMDCs and importantly inhibited immature BMDC migration induced by CCR5 in vitro, with no or minor influence on other functions of DCs, such as phagocytosis and maturation. In the mouse model, CT3-1 relieved arthritis severity and inhibited CIA development. Furthermore, CT3-1 intervention decreased the expression of CCR5 in DCs and reduced the proportion of DCs in the peripheral blood of CIA mice. CONCLUSIONS: Our findings suggest that CCR5-induced chemotaxis and trafficking of immature DCs are important in RA. Targeting CCR5 and inhibiting immature DC chemotaxis may provide a novel choice for the treatment of RA and other similar autoimmune diseases. Moreover, we synthesized a new hybrid compound CT3-1 that could inhibit immature DC trafficking and effectively relieve RA by directly reducing the CCR5 expression of immature DCs.

Artemisitene suppresses rheumatoid arthritis progression via modulating METTL3-mediated N6-methyladenosine modification of ICAM2 mRNA in fibroblast-like synoviocytes.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoimmune disease. We previously revealed that the natural compound artemisitene (ATT) exhibits excellent broad anticancer activities without toxicity on normal tissues. Nevertheless, the effect of ATT on RA is undiscovered. Herein, we aim to study the effect and potential mechanism of ATT on RA management. METHODS: A collagen-induced arthritis (CIA) mouse model was employed to confirm the anti-RA potential of ATT. Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assays, cell cycle and apoptosis analysis, immunofluorescence, migration and invasion assays, quantitative real-time PCR (RT-qPCR), Western blot, RNA-sequencing (RNA-seq) analysis, plasmid construction and lentivirus infection, and methylated RNA immunoprecipitation and chromatin immunoprecipitation assays, were carried out to confirm the effect and potential mechanism of ATT on RA management. RESULTS: ATT relieved CIA in mice. ATT inhibited proliferation and induced apoptosis of RA-fibroblast-like synoviocytes (FLSs). ATT restrained RA-FLSs migration and invasion via suppressing epithelial-mesenchymal transition. RNA-sequencing analysis and bioinformatics analysis identified intercellular adhesion molecule 2 (ICAM2) as a promoter of RA progression in RA-FLSs. ATT inhibits RA progression by suppressing ICAM2/phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/p300 pathway in RA-FLSs. Moreover, ATT inhibited methyltransferase-like 3 (METTL3)-mediated N6-methyladenosine methylation of ICAM2 mRNA in RA-FLSs. Interestingly, p300 directly facilitated METTL3 transcription, which could be restrained by ATT in RA-FLSs. Importantly, METTL3, ICAM2 and p300 expressions in synovium tissues of RA patients were related to clinical characteristics and therapy response. CONCLUSIONS: We provided strong evidence that ATT has therapeutic potential for RA management by suppressing proliferation, migration and invasion, in addition to inducing apoptosis of RA-FLSs through modulating METTL3/ICAM2/PI3K/AKT/p300 feedback loop, supplying the fundamental basis for the clinical application of ATT in RA therapy. Moreover, METTL3, ICAM2 and p300 might serve as biomarkers for the therapy response of RA patients.

Efficacy and Safety of Low-Dose Interleukin 2 for Primary Sjögren Syndrome: A Randomized Clinical Trial.

Importance: Primary Sjögren syndrome (pSS) is a systemic autoimmune disease associated with dysregulated immune cells, with no efficient therapy. There is a need to study potential therapeutic approaches. Objective: To investigate the efficacy, safety, and immune response of low-dose interleukin 2 (LD-IL-2) in the treatment of pSS. Design, Setting, and Participants: A double-blind, placebo-controlled randomized clinical trial was conducted with a 2-group superiority design from June 2015 to August 2017. Sixty patients, aged 18 to 70 years, were recruited from Peking University People's Hospital. Efficacy analyses were based on the intention-to-treat (ITT) principle. Data were analyzed from December 2018 to March 2020. Interventions: Patients with pSS were treated with LD-IL-2 or placebo for 12 weeks and accompanied by 12 weeks of follow-up. Main Outcomes and Measures: The primary end point was defined as a 3-point or greater improvement on the European League Against Rheumatism Sjögren's Syndrome Disease Activity Index (ESSDAI) by week 24. The secondary end points included other clinical responses, safety, and changes of immune cell subsets at week 12 and 24. Results: Sixty patients with pSS were recruited, with 30 in the LD-IL-2 group (mean [SD] age, 47.6 [12.8] years; 30 [100%] women) and 30 in the placebo group (mean [SD] age, 51.0 [11.9] years; 30 [100%] women), and 57 completed the trial. More patients in the LD-IL-2 group (20 [66.7%]) achieved ESSDAI score reduction of at least 3 points than in the placebo group (8 [26.7%]) at week 24 (P = .004). There were greater resolutions of dryness, pain, and fatigue in the LD-IL-2 group than placebo group at week 12 (dryness: difference, -18.33 points; 95% CI, -28.46 to -8.21 points; P = .001; pain: difference, -10.33 points; 95% CI, -19.38 to -1.29 points; P = .03; fatigue: difference, -11.67 points; 95% CI, -20.65 to -2.68 points; P = .01). No severe adverse events were observed in either group. In addition, the LD-IL-2 group showed a significant decrease in infection compared with the placebo group (1 [3.3%] vs 9 [30.0%]; P = .006). Immunological analysis revealed that LD-IL-2 promoted an expansion of regulatory T cells and regulatory CD24highCD27+ B cells. Conclusions and Relevance: In this randomized clinical trial, LD-IL-2 was effective and well tolerated in patients with pSS, and it restored immune balance, with enhanced regulatory T cells and CD24highCD27+ B cells. Trial Registration: ClinicalTrials.gov Identifier: NCT02464319.

Shikonin attenuates rheumatoid arthritis by targeting SOCS1/JAK/STAT signaling pathway of fibroblast like synoviocytes.

BACKGROUND: Rheumatoid arthritis is a progressive and systemic autoimmune disease seriously compromises human health. Fibroblast like synoviocytes are the major effectors of proliferation and inflammation in rheumatoid arthritis synovial tissue. Shikonin has anti-inflammatory and immunomodulatory activities. But, its role on synovitis of rheumatoid arthritis is unknown. METHODS: The DBA/1 male mice were randomly divided into the following three groups (n = 6): (1) the normal control group of mice, (2) the CIA (collagen-induced arthritis) group in which mice suffered from arthritis induced by collagen, (3) the SKN (shikonin) group of mice which got arthritis and given intragastrically with shikonin 4 mg/kg per day continuously for 20 days,(4) the MTX (methotrexate) group of mice which got arthritis and orally administration with shikonin 0.5  mg/kg once two days continuously for 20 days. The therapeutic effect of shikonin on collagen induced arthritis mice was tested by arthritis incidence rate, arthritis score and inflammatory joint histopathology. The invasion, adhesion and migration of fibroblast like synoviocytes induced by tumor necrosis factor-α were applied to measure the anti-synovitis role of shikonin. The effect of shikonin on expression of interleukin-6, interleukin-1ß and tumor necrosis factor-α was measured by enzyme linked immunosorbent assay. The interaction between shikonin and suppressor of cytokine signaling 1 was verified by molecular docking. The signaling pathways activated by shikonin were measured by western blot. RESULTS: Shikonin decreased the arthritis score and arthritis incidence, and inhibited inflammation of inflamed joints in collagen induced arthritis mice. And shikonin reduced the number of vimentin+cells in collagen induced arthritis mice inflamed joints. Meanwhile, shikonin suppressed tumor necrosis factor-α-induced invasion, adhesion and migration of fibroblast like synoviocytes and reduced the expression of interleukin-6, interleukin-1ß and tumor necrosis factor-α. And we found that shikonin targeted suppressor of cytokine signaling 1. More interestingly, shikonin blocked the phosphorylation of Janus kinase 1/signal transducer andactivator of transcription 1/signal transducer andactivator of transcription 6 in synovial tissues and in fibroblast like synoviocytes. CONCLUSION: Shikonin represents a promising new anti-rheumatoid arthritis drug candidate that has anti-synovitis effect in collagen induced arthritis mice and inhibits tumor necrosis factor-α-induced fibroblast like synoviocytes by targeting suppressor of cytokine signaling 1/ Janus kinase/signal transducer andactivator of transcription signaling pathway. These findings demonstrate that shikonin has anti-synovitis effect and has great potential to be a new drug for the treatment of rheumatoid arthritis.

Role of IL-24 in NK cell activation and its clinical implication in systemic lupus erythematosus.

OBJECTIVES: Interleukin (IL)-24 has been considered as an inflammatory cytokine in autoimmune diseases. However, conflicting data exist and its biological function remains controversial. Additionally, little is known about its functional impact on natural killer (NK) cells. The aim of this study was to investigate the role of IL-24 in NK cell activation and its clinical implication in systemic lupus erythematosus (SLE). METHODS: Serum cohort consisting of 299 SLE patients, 214 RA patients, and 159 healthy controls (HCs) and plasma cohort consisting of 70 SLE patients, 82 RA patients, and 123 HCs were included in evaluating IL-24 concentrations. Impact of IL-24 on NK cell activation was assessed in two NK cell subsets, i.e., CD56dimCD16+ and CD56brightCD16- NK cells. Human NK-92 cell line was applied to evaluate functional potential of IL-24 on NK cell migration and invasion. RESULTS: Serum and plasma levels of IL-24 were comparable between patients with SLE or RA and HCs. While recombinant human (rh) IL-2 consistently induced an increased expression of CD69 on both CD56dimCD16+ and CD56brightCD16- cells derived from both healthy subjects and patients with SLE, IL-24 alone was insufficient to activate the CD56dim and CD56bright NK cells. Similarly, while the migratory NK-92 cell numbers were significantly increased with rhIL-2 stimulation, IL-24 alone was unable to enhance NK-92 cell migratory and invasive capacity. CONCLUSION: Our data indicate that there were no significant differences in serum and plasma concentrations of IL-24 between SLE patients and healthy controls. Recombinant IL-24 has no effect on NK cell activation and migration. Key points • This is the first study to investigate functional potential of IL-24 on NK cell activation. • Recombinant IL-24 lacks functional capacity on NK cell activation in either CD56dimCD16+ or CD56brightCD16- NK cell subsets derived from both healthy subjects and patients with SLE. • No significant differences in serum and plasma levels of IL-24 between SLE patients and healthy controls.

Interleukin 17E associates with haematologic involvement and autoantibody production in primary Sjögren's syndrome.

OBJECTIVES: Primary Sjögren's syndrome (pSS) is one of the most prevalent systemic autoimmune diseases characterised by inflammation and tissue damage of exocrine glands, especially salivary or lacrimal gland. IL-17 related immune response is pathogenic with proinflammatory feature in pSS. However, whether IL-17E, an IL-17 family member, is involved in pSS pathogenesis or not, has not been determined. METHODS: Serum levels of IL-17E and IL-17A as comparison in 107 patients with pSS and 42 healthy controls were determined with multiplex cytokine assays. EULAR Sjögren's syndrome disease activity index (ESSDAI) score was calculated. Laboratory parameters were measured by standard laboratory techniques. The inflammatory infiltration of minor labial gland biopsies was graded based on numbers of lymphocyte and quantified by Focus Score (FS). Expression of IL-17E and IL-17A in the biopsy was evaluated with immunohistochemistry. RESULTS: Significantly elevated IL-17E in pSS patients associated with ESSDAI, haematologic disorders and autoantibody production, including anti-nuclear antibodies (ANA), rheumatoid factor (RF) and anti-SSA antibodies were found. Histopathological features showed that expression of IL-17E was found in labial salivary gland and correlated with lymphocytic infiltration. CONCLUSIONS: IL-17E expression in pSS patients was increased and associated with haematologic disorders, autoantibody production and lymphocytic infiltration in salivary gland. This finding indicated that IL-17E is involved in pSS pathogenesis.

Searching for valuable differentially expressed miRNAs in postmenopausal osteoporosis by RNA sequencing.

AIM: Postmenopausal osteoporosis is a systemic and chronic bone disease in women. In order to understand the pathological mechanism of postmenopausal osteoporosis, we aimed to find the potential differentially expressed miRNAs in the disease. METHODS: Firstly, RNA sequencing was used to identify differentially expressed miRNAs, followed by the construction of the miRNA-target mRNA regulatory network. Then, Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes were used to analyze the biological function of target mRNAs. Finally, electronic validation of identified differentially expressed miRNAs and target mRNAs was performed. RESULTS: A total of 33 differentially expressed miRNAs (18 upregulated and 15 downregulated miRNAs) and 6820 miRNA-mRNA pairs were identified. Among which, seven miRNAs with high degree including hsa-miR-17-5p, hsa-miR-1-3p, hsa-miR-193b-3p, hsa-miR-125b-5p, hsa-miR-10b-5p, hsa-miR-100-5p and hsa-miR-30a-3p were obtained in the miRNA-mRNA regulatory network. TGF-beta was the most significantly enriched signaling pathway of target mRNAs. The electronic validation result of hsa-miR-1-3p, hsa-miR-193b-3p, hsa-miR-10b-5p, hsa-miR-100-5p, hsa-miR-133b, hsa-miR-708-5p, CRK, RAB5C, CCND1 and PCYOX1 was consisted with the RNA sequencing analysis. CONCLUSION: Dysfunctional miRNAs may play significant roles in postmenopausal osteoporosis.