Gasdermin E-mediated keratinocyte pyroptosis participates in the pathogenesis of psoriasis by promoting skin inflammation.

BACKGROUND: Psoriasis is a common chronic inflammatory disease with an unclear aetiology. Keratinocytes in psoriasis are susceptible to exogenous triggers that induce inflammatory cell death. OBJECTIVES: To investigate whether gasdermin E (GSDME)-mediated pyroptosis in keratinocytes contributes to the pathogenesis of psoriasis. METHODS: Skin samples from patients with psoriasis and from healthy controls were collected to evaluate the expression of GSDME, cleaved caspase-3 and inflammatory factors. We then analysed the data series GSE41662 to further compare the expression of GSDME between lesional and nonlesional skin samples in those with psoriasis. In vivo, a caspase-3 inhibitor and GSDME-deficient mice (Gsdme-/-) were used to block caspase-3/GSDME activation in an imiquimod-induced psoriasis model. Skin inflammation, disease severity and pyroptosis-related proteins were analysed. In vitro, tumour necrosis factor (TNF)-α-induced caspase-3/GSDME-mediated pyroptosis in the HACAT cell line was explored. RESULTS: Our analysis of the GSE41662 data series found that GSDME was upregulated in psoriasis lesions vs. normal skin. High levels of inflammatory cytokines such as interleukin (IL)-1ß, IL-6 and TNF-α were also found in psoriasis lesions. In mice in the Gsdme-/- and caspase-3 inhibitor groups, the severity of skin inflammation was attenuated and GSDME and cleaved caspase-3 levels decreased after imiquimod treatment. Similarly, IL-1ß, IL-6 and TNF-α expression was decreased in the Gsdme-/- and caspase-3 inhibitor groups. In vitro, TNF-α induced HACAT cell pyroptosis through caspase-3/GSDME pathway activation, which was suppressed by blocking caspase-3 or silencing Gsdme. CONCLUSIONS: Our study provides a novel explanation of TNF-α/caspase-3/GSDME-mediated keratinocyte pyroptosis in the initiation and -acceleration of skin inflammation and the progression of psoriasis.

Psoriasis is chronic and autoinflammatory common skin disease that affects 2­3% of the world's population. The disease is characterized by persistent inflammation in various body systems, including the skin and joints. However, the exact cause of the disease is unclear. In this study from China, we found that in people with psoriasis a protein called 'gasdermin E' (or 'GSDME') is increased in a type of skin cell called keratinocytes. In psoriasis, these keratinocytes are susceptible to a type of cell death called 'pyroptosis'. We aimed to find out whether pyroptosis caused by GSDME in keratinocytes contributes to the development of psoriasis. To do this, we looked at samples of skin from people with psoriasis and compared these to samples from healthy controls (those without psoriasis). Firstly, we investigated the levels of GSDME, another protein called caspase-3 and other inflammatory factors in the skin lesions from patients with psoriasis. Secondly, we analysed previously published data from 24 patients with psoriasis. Finally, we carried out a range of experiments to confirm our findings. We found that keratinocyte pyroptosis was mediated by the messenger proteins TNF-α/caspase-3, and that GSDME played a key role in the initiation and acceleration of skin inflammation and the progression of psoriasis. Targeting the GSDME pathway may be a novel strategy in treating psoriasis.

Protection of MCC950 against Alzheimer's disease via inhibiting neuronal pyroptosis in SAMP8 mice.

Neuronal dysfunction and loss are thought to be one of the causes of cognitive impairment in Alzheimer's disease (AD), but the specific mechanism of neuronal loss in the pathogenesis of AD remains controversial. This study explored the role of NLRP3 inflammasome-induced neuronal pyroptosis in neuronal loss of AD, and pioneered the use of NLRP3 inhibitor MCC950 to intervene in the treatment of senescence-accelerated mouse prone 8 (SAMP8) mice. In vitro, human primary neurons (HPNs) pretreated with MCC950 were stimulated with amyloid-ß1-42 (Aß1-42), and it was found that MCC950 significantly reduced the neurotoxicity of Aß1-42 by inhibiting neuronal pyroptosis. In vivo, SAMP8 mice were randomly divided into vehicle-treated group and MCC950-treated group, and it was found that MCC950 also played a positive role in treatment. The intervention of MCC950 improved the spatial memory ability and brain histological morphology of SAMP8 mice, and reduced the deposition of amyloid-ß in the brain. Furthermore, MCC950 was found to inhibit the overexpressions of NLRP3, caspase-1, and GSDMD, which were the response factors of pyroptosis in SAMP8 mouse neurons, by immunofluorescence staining. In this study, we found that neuronal pyroptosis induced by the NLRP3/caspase-1/GSDMD axis was an important factor in neuronal loss of AD, and revealed that MCC950 might be a potential AD therapeutic agent.

Blocking Matrix Metalloproteinase-9 Abrogates Collagen-Induced Arthritis via Inhibiting Dendritic Cell Migration.

Trafficking of dendritic cells (DCs) to lymph nodes (LNs) to present Ags is a crucial step in the pathogenesis of rheumatoid arthritis (RA). Matrix metalloproteinase-9 (MMP-9) is the key molecule for DC migration. Thus, blocking MMP-9 to inhibit DC migration may be a novel strategy to treat RA. In this study, we used anti-MMP-9 Ab to treat collagen-induced arthritis (CIA) in DBA/1J mice and demonstrated that anti-MMP-9 Ab treatment significantly suppressed the development of CIA via the modulation of DC trafficking. In anti-MMP-9 Ab-treated CIA mice, the number of DCs in draining LNs was obviously decreased. In vitro, anti-MMP-9 Ab and MMP-9 inhibitor restrained the migration of mature bone marrow-derived DCs in Matrigel in response to CCR7 ligand CCL21. In addition, blocking MMP-9 decreased T and B cell numbers in LNs of CIA mice but had no direct influence on the T cell response to collagen II by CD4+ T cells purified from LNs or spleen. Besides, anti-MMP-9 Ab did not impact on the expression of MHC class II, CD40, CD80, CD86, and chemokine receptors (CCR5 and CCR7) of DCs both in vivo and in vitro. Furthermore, we discovered the number of MMP-9-/- DCs trafficking from footpads to popliteal LNs was dramatically reduced as compared with wild type DCs in both MMP-9-/- mice and wild type mice. Taken together, these results indicated that DC-derived MMP-9 is the crucial factor for DC migration, and blocking MMP-9 to inhibit DC migration may constitute a novel strategy of future therapy for RA and other similar autoimmune diseases.

Inflammatory or Neuropathic Pain: Characteristics and Their Relationships with Disease Activity and Functional Status in Axial Spondyloarthritis Patients.

OBJECTIVE: To assess inflammatory back pain (IBP) and neuropathic pain (NP) in patients with axial spondyloarthritis (axSpA) and explore their relationships with disease activity and functional status. DESIGN: Descriptive, cross-sectional study. SETTING AND SUBJECTS: A convenience sample of 132 patients with axSpA was recruited from a university-affiliated hospital in China. METHODS: The presence of IBP or NP, pain intensity and quality, pain interference, disease activity, and functional status were assessed. Univariate analyses and binary logistic regressions were used to identify factors related to IBP or NP. RESULTS: Fifty patients (37.9%) had IBP, and 22 (16.7%) had NP. Scores of pain intensity and pain interference, disease activity, and functional limitation were significantly higher in patients with IBP or NP than those without either (P < 0.05). The quality of IBP or NP was mainly presented as sensory descriptors, and patients with IBP had a higher level of "tiring-exhausting" in affective descriptors (P < 0.05). There was a significant correlation between IBP and disease activity (odds ratio [OR] = 1.813, 95% confidence interval [CI] = 1.196-2.750). NP was related to functional limitation (OR = 1.544, 95% CI = 1.098-2.171). CONCLUSIONS: Though pain quality (sensory and affective pain descriptors) show differently in patients with IBP or NP, the axSpA patients with IBP or NP experience more severe pain intensity and pain interference, higher disease activity, and greater functional limitation. The presence of IBP or NP could reflect higher disease activity or greater functional limitation in patients with axSpA.

Associations Between Pain Patterns and Self-Reported Clinical Outcomes in Patients with Knee Osteoarthritis.

OBJECTIVES: To study pain locations and patterns in knee osteoarthritis (OA) and determine associations between knee pain patterns and pain characteristics, functional status, and psychological status. DESIGN: A cross-sectional study. SETTING AND SUBJECTS: A convenience sample of 203 patients with knee OA was recruited from two university-affiliated hospitals from July 2016 to July 2017. METHODS: Pain locations were recorded with Photographic Knee Pain Map. Pain characteristics, functional dysfunction, pain catastrophizing, and pain self-efficacy were assessed, and demographic and radiographic data were also collected. Self-reported clinical outcomes across common pain patterns were compared. RESULTS: The medial joint line area (72.9%) was the most prevalent pain zone. The common pain patterns were diffuse (34.5%), isolated medial (22.7%), isolated anterior (9.9%), and medial-lateral pain (9.9%). Scores of intermittent pain (mean difference [MD] = 9.669, 95% confidence interval [CI] = 2.815 to 16.523) and knee-related function in daily living (MD = -10.353, 95% CI= -17.358 to -3.349) were worse in diffuse pain compared with isolated medial pain. Knee-related symptom scores were higher with isolated medial (MD = 13.146, 95% CI = 6.725 to 19.566), isolated anterior (MD = 15.102, 95% CI = 6.525 to 23.679), and medial-lateral (MD = 15.281, 95% CI = 6.704 to 23.857) pain relative to diffuse pain. Diffuse pain, after adjusting for confounding factors, was associated with more severe knee pain at rest, higher scores of constant and intermittent pain, lower knee-related function scores, higher pain catastrophizing, and lower pain self-efficacy scores (P < 0.05). CONCLUSIONS: The most common pattern is diffuse pain, which is associated with worse knee pain intensity at rest, constant and intermittent pain experiences, and worse physical and psychological status, compared with more localized patterns.

Not only anti-inflammation, etanercept abrogates collagen-induced arthritis by inhibiting dendritic cell migration and maturation.

The application of tumor necrosis factor inhibitors (TNFi) is a major breakthrough in the treatment of rheumatoid arthritis (RA). While the anti-inflammatory nature of TNFi is thought to contribute to the therapeutic effects, recent data show that the pharmacology of TNF-α blockade is probably more complex than previously thought. This study investigates whether etanercept (ETN), one of the TNF antagonists, suppresses arthritis development through modulation of dendritic cell (DC) functions. Bone marrow-derived DCs (BMDCs) were stimulated with lipopolysaccharide (LPS) and treated with ETN for 24 hrs. DC functions, including maturation and migration, were determined. DCs from the lymph nodes (LNs) of ETN-treated collagen-induced arthritis (CIA) mice were analyzed for phenotypes and subsets. ETN efficiently inhibited the phenotypic maturation both in vitro and in vivo. ETN treatment delayed the onset and reduced the severity of arthritis in CIA mice. Moreover, ETN treatment strongly down regulated the number of both myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in LNs, possibly due to the depressive effect on the expression of CXCR4 on DCs in peripheral blood. The impaired DC migration to local LNs by ETN down regulated the number of T cells and B cells, and changed the LN cellular composition. The data show that TNF-α blockade has profound effects on DC maturation and migration, which may contribute to its immune regulatory effects in RA patients.

FTY720 Abrogates Collagen-Induced Arthritis by Hindering Dendritic Cell Migration to Local Lymph Nodes.

Because dendritic cells (DCs) play critical roles in the pathogenesis of rheumatoid arthritis, modulation of their functions could serve as a novel therapy. In this study, we demonstrated that FTY720 treatment significantly suppressed the incidence and severity of collagen-induced arthritis (CIA) in DBA/1J mice via the modulation of DC functions. In FTY720-treated CIA mice, a decrease in the number of DCs in local draining lymph nodes (LNs) was observed. In vitro, FTY720 inhibited the trafficking of LPS-stimulated bone marrow-derived DCs (BMDCs). Decreased secretion of CCL19 and downregulation of CCR7 on DCs may explain the mechanisms underlying the impairment of DC migration induced by FTY720. In a DC-induced mouse arthritis model, FTY720 treatment also suppressed the incidence and severity of arthritis, which was correlated with a decrease in the migration of injected BMDCs to draining LNs. Although lower levels of costimulatory molecules (CD40, CD80, and CD86) and I-A(q) expressed on LN DCs were observed in FTY720-treated mice, in vitro analysis showed no effect of FTY720 on LPS-stimulated BMDC maturation. Furthermore, LN cells from FTY720-treated CIA mice displayed diminished production of proinflammatory cytokines in response to collagen II and Con A stimulation. In addition, the ratio of Th1/Th2 in the draining LNs of mice with DC-induced arthritis was decreased upon FTY720 treatment. This finding was consistent with the fact that FTY720 suppressed IL-12p70 production in cultured BMDCs. Taken together, these results indicate that inhibition of DC migration by FTY720 may provide a novel approach in treating autoimmune diseases such as rheumatoid arthritis.

Apigenin, a potent suppressor of dendritic cell maturation and migration, protects against collagen-induced arthritis.

This study aimed to investigate whether apigenin (API) suppresses arthritis development through the modulation of dendritic cell functions. Bone marrow-derived dendritic cells (BMDCs) were stimulated in vitro with lipopolysaccharide (LPS) and treated with API for 24 hrs; DC functions, including phenotype expressions, cytokine secretion, phagocytosis and chemotaxis, were then investigated. The effects of API on collagen-induced arthritis (CIA) were examined in vivo, and purified DCs from the lymph nodes (LNs) of API-treated CIA mice were analysed for phenotypes and subsets. In in vitro, API efficiently restrained the phenotypic and functional maturation of LPS-stimulated BMDCs while maintaining phagocytotic capabilities. Moreover, API inhibited the chemotactic responses of LPS-stimulated BMDCs, which may be related to the depressive effect on chemokine receptor 4 (CXCR4). In in vivo, API treatment delayed the onset and reduced the severity of arthritis in CIA mice, and diminished secretion of pro-inflammatory cytokines in the serum and supernatants from the LN cells of the CIA mice. Similar to the in vitro findings, the API-treated mice exhibited reduced expression of co-stimulatory molecules and major histocompatibility complex II on DCs. Furthermore, API treatment strongly down-regulated the number of Langerhans cells, but not plasmacytoid DCs (pDCs) in LNs, which may be related to the depressive effect of API on the expression of CXCR4 on DCs of peripheral blood. These data provide new insight into the mechanism of action of API on arthritis and indicate that the inhibition of maturation and migration of DCs by API may contribute to its immunosuppressive effects.

Sniping the scout: Targeting the key molecules in dendritic cell functions for treatment of autoimmune diseases.

Dendritic cells (DCs) are a power tool for manipulating immune system. They play important roles in the induction of immunity as well as inducing intrathymic and peripheral tolerance. After generated from stem cells in the bone marrow, DCs traffic to the peripheral tissues, where they capture and process antigens, express lymphocyte co-stimulators, migrate to the secondary lymph organs and present the processed antigen to naive T cells to either activate or tolerize them. These processes are modulated subtly and influenced by various factors. Aberrant regulation of the processes may cause autoimmunity. Investigation into the biology of DCs and the molecules and mechanisms that regulate them helps us understanding the pathogenesis of autoimmune diseases and reveals numerous steps for pharmacological manipulation. In this review, we made a sketch line of the critical events of DC biology that are potential pharmacologic targets for the treatment of autoimmune diseases.

[Clinical applications of apparent diffusion coefficient value for evaluation of hip joint of ankylosing spondylitis active lesions].

OBJECTIVE: To evaluate the apparent diffusion coefficient (ADC) value in differential diagnosis of ankylosing spondylitis active lesions. METHOD: A total of 68 patients with AS involving hip joint who were diagnosed in Third Affiliated Hospital of Southern Medical University form March 2010 to December 2014 were evaluated by MRI. The ADC values of the hip joint lesions were measured and compared in 38 cases of ankylosing spondylitis active lesions (active group) and 30 cases of ankylosing spondylitis in stable period (stable group), all cases were confirmed by clinical diagnosis. RESULTS: There are 238 lesions were detected demonstrating hyperintens in DWI and ADC picture. The mean ADC values of active group and stable group were (1.00 ± 0.13) × 10⁻³ mm²/s and (0.65 ± 0.12) × 10⁻³ mm²/s (t=-11.85, P=0.00). The correlation coefficient between ADC and BASDAI score is 0.831 (P<0.01). ROC curve showed that the threshold ADC value differentiating active group and stable group was 0.81 × 10⁻³ mm²/s, with a sensitivity of 94.7%and specificity of 90%. CONCLUSION: The ADC value of hip joint lesion of AS patient can help to determine the nature of the ankylosing spondylitis.

[The relevance of ADC value, T1intensive rate and the clinical activities in ankylosing spondylitis sacroiliitis].

OBJECTIVE: To evaluate the value of judging for the activity in AS by diffusion-weighted sequences (DWI) and enhance sequences MRI(DCE-MRI), to explore the correlation of Apparent diffusion coefficient(ADC) values, intensive rate and the clinical activity index in sacroiliitis (SIJ) of ankylosing spondylitis (AS). METHODS: 56 patients prospectively choiced and diagnosed were divided into two groupsas, active group (n = 32) and chronic group (n = 24) by rheumatologist according to BASDAI and laboratory parameters. Twenty healthy adults were as control group. The values of ADC and intensive rate of all sacroiliac joints (SIJs) were measured on MRI. BASDAI score were got by Bath ankylosing spondylitis disease activity index. ESR, CPR and were got by laboratory. Statisticaly to analysis whether the parameters were significantly different amang AS active, chronic, and the control group. To assess the correlation of the values of ADC, intensive rate and BASDAI score, ESR, CPR and in SIJ. RESULTS: the values of ADC and intensive rate were significantly different among AS active, chronic and the control group. There were the significant correlation between the values of ADC, intensive rate and BASDAI score. CONCLUSION: Diffusion-weighted sequences and Contrast-enhanced sequences is superior to other methods in judging the activity in AS.combined with clinical activity index, the accuracy can significantly be improved to explore whether the activities of AS are.

Cytokine signatures of human whole blood for monitoring immunosuppression.

How to evaluate status of the immune system is extremely critical for clinical immunosuppressive treatment. In this study, we tested the secretion of cytokines in undiluted whole blood samples stimulated with Phorbol 12-myristate 13-acetate (PMA) and ionomycin (IONO), and compared the effects of dexamethasone (DEX), cyclosporine A (CsA) or mycophenolic acid (MPA), either alone or in combination, on cytokine profiles. The results showed that both DEX and CsA dose-dependently inhibited the production of eleven cytokines: interleukin (IL)-2, IL-4, IL-5, IL-6, IL-13, IL-17, interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF). Unexpectedly, MPA showed no obvious influences except for the mild inhibition on GM-CSF production. In combination treatment, cytokine profiles reflect not only the synergistic effects among drugs, but also the specific effect of the individual drug. Thus, the effects of different immunosuppressants could be reflected through their specific cytokine signatures, which can be applied to maximize immunosuppressive effects, while to minimize risk of infections and help physicians to reasonably apply immunosuppressants.

Anti-Toxoplasma gondii antibodies as a risk factor for the prevalence and severity of systemic lupus erythematosus.

BACKGROUND: Systemic lupus erythematosus (SLE) is a complex systemic autoimmune disease characterized by the presence of numerous autoantibodies. The interaction of infectious agents (viruses, bacteria and parasites) and a genetically susceptible host may be a key mechanism for SLE. Toxoplasma gondii is a widespread intracellular parasite that has been implicated in the pathogenesis of autoimmune diseases. However, the relationship between T. gondii infection and the increased risk of SLE in Chinese populations remains unclear. METHODS: The seroprevalence of T. gondii infection was assessed in 1771 serum samples collected from Chinese individuals (908 healthy controls and 863 SLE patients) from different regions of China using an enzyme-linked immunosorbent assay. Serum autoantibodies and clinical information were obtained and analysed. RESULTS: Our observations revealed a higher prevalence of anti-T. gondii antibodies (ATxA) immunoglobulin G (IgG) in serum samples from SLE patients (144/863, 16.7%) than in those from the healthy controls (53/917, 5.8%; P < 0.0001), indicating a 2.48-fold increased risk of SLE in the ATxA-IgG+ population, after adjustment for age and sex (95% confidence interval [CI] 1.70-3.62, P < 0.0001). ATxA-IgG+ SLE patients also showed a 1.75-fold higher risk of developing moderate and severe lupus symptoms (95% CI 1.14-2.70, P = 0.011) compared to ATxA-IgG- patients. Relative to ATxA-IgG- patients, ATxA-IgG+ patients were more likely to develop specific clinical symptoms, including discoid rash, oral ulcer, myalgia and alopecia. Seven antibodies, namely anti-ribosomal RNA protein (rRNP), anti-double stranded DNA (dsDNA), anti-cell membrane DNA (cmDNA), anti-scleroderma-70 (Scl-70), anti-cardiolipin (CL), anti-beta2-glycoprotein-I (B2GPI) and rheumatoid factor (RF), occurred more frequently in ATxA-IgG+ patients. When combined with anti-dsDNA and RF/anti-rRNP/anti-cmDNA/ESR, ATxA-IgG significantly increased the risk for severe lupus. CONCLUSIONS: Our results suggest that ATxA-IgG may be a significant risk factor for SLE prevalence and severity in Chinese populations.

Downregulation of Sox8 mediates monosodium urate crystal-induced autophagic impairment of cartilage in gout arthritis.

The deposition of monosodium urate (MSU) crystals in arthritic joints of gout seriously damages cartilage. This study aimed to investigate whether MSU crystal-induced cartilage impairment was related to autophagic signaling. mRNAs of cartilage from MSU-induced gouty arthritis rat model were sequenced. MSU crystal-treated human chondrocytes were used to evaluate the function of Sox8. The recombinant Sox8 lentiviral vector (lenti-Sox8) was applied to upregulate the expression of Sox8. Transfection of the mRFP-GFP-LC3 plasmid was evaluated by confocal microscopy. The autophagic vacuoles were stained with monodansylcadaverine and examined by flow cytometry. The morphology of autophagosomes was observed by transmission electron microscopy. The ratio of LC3-II/I in the presence or absence of bafilomycin A1 and the expression levels of Beclin1, Sox8, p-PI3K, PI3K, p-AKT, AKT, p-mTOR, and mTOR were detected by Western blot. In vivo, the effect of Sox8 on cartilage of acute gouty model rats was evaluated by safranin-O/fast green staining and Western blot. The expression of Sox8 was significantly downregulated both in vivo and in vitro. In chondrocytes, MSU crystals reduced the expression of Sox8, inhibited the PI3K/AKT/mTOR signaling pathway, and increased the level of autophagy. Overexpression of Sox8 notably inhibited MSU crystal-induced autophagy by rescuing the phosphorylation levels in the PI3K/AKT/mTOR signaling pathway. In vivo, overexpression of Sox8 remarkably alleviated cartilage damage in acute gouty model rats. These results indicate that downregulation of Sox8 plays an important role in MSU-induced chondrocyte autophagy by modulating PI3K/AKT/mTOR signaling, and overexpression of Sox8 may serve as a novel therapy to prevent the impairment of cartilage in gout arthritis.

Polydatin protects against gouty nephropathy by inhibiting renal tubular cell pyroptosis.

OBJECTIVE: To investigate the protective effect and mechanism of polydatin (PD) against gouty nephropathy (GN) in mice. METHODS: Twenty-four mice were randomly divided into three groups: the control group (no treatment), the GN group (300 mg/kg hypoxanthine + 150 mg/kg potassium oxonate), and the GN + PD group (300 mg/kg hypoxanthine + 150 mg/kg potassium oxonate + 50 mg/kg PD). Histological changes in the kidneys and the levels of uric acid (UA), blood urea nitrogen (BUN), and serum creatinine (SCr) in the sera were measured. In addition, the expression of gasdermin D (GSDMD) protein in renal tubular epithelial cells, and the expression of NOD-like receptor protein 3 (NLRP3), GSDMD, and caspase-1 proteins in the kidney tissues were determined by immunohistochemistry, immunofluorescence, and Western blot. RESULTS: In vitro, PD inhibited the expression of NLRP3, caspase-1, and GSDMD and protected the renal tubular epithelial cells from pyroptosis. In vivo, PD treatment significantly ameliorated the pathological changes in kidney tissue, and reversed the decrease of serum UA and BUN in GN model mice. The expression of NLRP3, GSDMD, and caspase-1 proteins was also decreased in the PD-treated GN mice. CONCLUSION: The results suggest that PD has a protective effect on mice with GN, which may be related to the downregulation of NLRP3, GSDMD, and caspase-1 proteins and the inhibition of renal tubular epithelial cells pyroptosis.

Effects and mechanisms of Celastrol on the formation of neutrophil extracellular traps (NETs).

Background: To investigate the effect and mechanism of Celastrol on the formation of neutrophil extracellular traps (NETs), and to provide a theoretical basis for the clinical application of Tripterygium wilfordii. Methods: First, we isolated neutrophils from the peripheral blood of healthy volunteers, and then observed the effect of Celastrol on Phorbol Myristate Acetate (PMA)-induced neutrophil release of NETs. The level of NETs was detected by using the membrane-impermeable nucleic acid dye, SytoxGreens. In addition, the levels of reactive oxygen species (ROS) were also examined to determine whether Celastrol affects ROS production during PMA-induced NETs. Results: Celastrol produced significant cytotoxicity at a concentration of 5 µM (213.2±75.07), and the effect of stimulant PMA (25 nM) treatment was not statistically different (197.3±25.15) (P=0.9167). Celastrol (1.25, 0.625, and 0.3125 µM) did not exhibit cytotoxicity when treating neutrophils. Compared with the PMA (25 nM) + Celastrol (1.25, 0.625, and 0.3125 µM) group and the PMA (25 nM) monotherapy group, SytoxGreen showed a statistically significant reduction in fluorescence at 528 µM under 485 µM light excitation. Also, under the co-localization marker of Hochest and SytoxGreen double staining, we observed that the release of NETs in the PMA-treated group was higher than that in the control group. The PMA-induced neutrophil release of NETs was markedly reduced compared to the PMA-treated group. The NET release was substantially decreased under double staining with the Hochest and SytoxGreen co-localization markers. The fluorescence intensity of the Celastrol plus PMA group was significantly lower than that of the PMA treatment group alone, indicating a decrease in the level of intracellular ROS. Interestingly, the level of ROS in the treatment group who received Celastrol alone was lower than that in the control group, indicating that Tripterygium wilfordii could inhibit the spontaneous production of ROS by neutrophils in the absence of stimulation. Conclusions: The molecular mechanism of Celastrol involves inhibition of PMA-stimulated neutrophil NETs formation in vitro, which is possibly related to the reduction of ROS levels. This indicates that Celastrol, the main component in Tripterygium wilfordii, can inhibit the formation of NETs, which provides a theoretical basis for the study of NETs-related diseases.

Single-Cell Analysis of Patients with Axial Spondyloarthritis After Anti-TNFα Treatment: Experimental Data and Review of the Literature.

Axial spondyloarthritis (Ax-SpA) is a chronic inflammatory disease that predominantly affects the axial joints and is most common in young men. However, the precise immune cell subset involved in Ax-SpA remains unclear. Our study characterized the periphery immune landscape of Ax-SpA patients before and after anti-TNFα treatment using single-cell transcriptomics and proteomics sequencing and elucidated the effects of anti-TNFα treatment at the single-cell level. First, we found that peripheral granulocytes and monocytes significantly increased in Ax-SpA patients. Second, we identified a more functional subtype of regulatory T cells, which was present in synovial fluid and increased in patients after treatment. Third, we identified a cluster of inflammatory monocyte subset with stronger inflammatory and chemotactic characteristics. A potential interaction between classical monocytes and granulocytes via the CXCL8/2-CXCR1/2 signaling pathway was observed, which decreased after treatment. Together, these results defined the complex expression profiles and advanced our understanding of the immune atlas in Ax-SpA patients before and after anti-TNFα treatment.

Efficacy and Safety of CMAB008 Compared with Innovator Infliximab in Patients with Moderate-to-Severe Rheumatoid Arthritis Receiving Concomitant Methotrexate: A Randomized, Double-blind, Multi-center, Phase III Non-inferiority Study.

OBJECTIVES: The aim of this work is to verify the non-inferior efficacy and safety of CMAB008 compared with innovator infliximab in rheumatoid arthritis patients combined with methotrexate. METHODS: We conducted a randomized, double-blinded, parallel, positive control design, multicenter study, with a stable dose of methotrexate. Patients were enrolled randomly with a ratio of 1:1 to receive intravenously CMAB008 3 mg/kg or innovator infliximab 3 mg/kg at weeks 0, 2, 6, 14, 22 and 30. The primary efficacy endpoint was American College of Rheumatology 20% improvement criteria (ACR20) response rate at week 30. The non-inferiority was established if the lower limit of the one-sided 97.5% confidence interval (CI) for the difference was more than - 15% and the equivalence was established if the two-sided 95% CI was within ± 15% in an exploratory equivalence analysis. The secondary endpoints included other efficacy assessment parameters, as well as immunogenicity, safety, and pharmacokinetics. RESULTS: In the full analysis population (FAS), 110 (57.6%) of 191 patients in the CMAB008 group and 120 (62.2%) of 193 patients in the innovator infliximab group reached the primary outcome of ACR20 at week 30. The differences of the rates were - 4.6% and the lower limit of one-sided 97.5% confidence interval was - 14.29%, not less than the lower limit of the non-inferiority margin (- 15%); so CMAB008 was non-inferior to innovator infliximab. Further, CMAB008 was equivalent to innovator infliximab both in FAS (difference - 4.6%, 95% CI - 14.29% to 5.12%) and PPS (difference - 3.3%, 95% CI - 13.18% to 6.62%). The efficacy, safety, immunogenicity, and pharmacokinetics are highly similar between CMAB008 and innovator infliximab. CONCLUSIONS: Non-inferior efficacy of CMAB008 to innovator infliximab is illustrated with similar early and lasting therapeutic effects, and the equivalence is further demonstrated. CMAB008 is well tolerated and has semblable safety compared with the innovator infliximab. TRIAL REGISTRATION NUMBER: NCT03478111.

Association between clinical characteristics and pain relief in patients with axial spondyloarthritis treated with non-steroidal anti-inflammatory drugs.

Objectives: The aim of this study was to evaluate pain relief in axial spondyloarthritis (axSpA) patients treated with non-steroidal anti-inflammatory drugs (NSAIDs) and to investigate the relation of the demographic, clinical, and psychological characteristics with pain relief. Patients and methods: Between February 2017 and December 2019, a total of 94 patients (61 males, 33 females; mean age: 28.3±8.1 years; range, 14 to 54 years) who were diagnosed with axSpA and treated with NSAIDs were included. The patients were assessed at baseline and at three months. A reduction of 30% in the Numeric Rating Scale (NRS) indicates a clinically meaningful improvement. The patients were divided into the relief group (≥30% improvement in NRS) and non-relief group (<30% improvement). Potential influential factors for pain relief such as neuropathic pain (NP), disease activity, function, pain catastrophizing, and pain self-efficacy were assessed. The relationship between patients' characteristics and pain relief was analyzed. Results: Seventy-two (76.6%) patients achieved pain relief. These patients had significantly higher baseline erythrocyte sedimentation rate, C-reactive protein, and lower percentage of NP. There was no significant difference between the two groups in function, pain catastrophizing, and pain self-efficacy. Multiple logistic regression analysis revealed that patients with NP were less likely to achieve pain relief (odds ratio [OR]: 3.531, 95% confidence interval [CI]: 1.155-10.789; p=0.027). Conclusion: Pain relief is still insufficient in some axSpA patients, despite the administration of NSAIDs. The presence of NP is significantly associated with poor pain relief. Alternative medications instead of NSAIDs are needed to achieve optimal pain relief, when NP is diagnosed.

Attenuation of Rheumatoid Arthritis Through the Inhibition of Tumor Necrosis Factor-Induced Caspase 3/Gasdermin E-Mediated Pyroptosis.

OBJECTIVE: To determine the role of gasdermin E (GSDME)-mediated pyroptosis in the pathogenesis and progression of rheumatoid arthritis (RA), and to explore the potential of GSDME as a therapeutic target in RA. METHODS: The expression and activation of caspase 3 and GSDME in the synovium, macrophages, and monocytes of RA patients were determined by immunohistochemistry, immunofluorescence, and Western blot analysis. The correlation of activated GSDME with RA disease activity was evaluated. The pyroptotic ability of monocytes from RA patients was tested, and the effect of tumor necrosis factor (TNF) on caspase 3/GSDME-mediated pyroptosis of monocytes and macrophages was investigated. In addition, collagen-induced arthritis (CIA) was induced in mice lacking Gsdme, and the incidence and severity of arthritis were assessed. RESULTS: Compared to cells from healthy controls, monocytes and synovial macrophages from RA patients showed increased expression of activated caspase 3, GSDME, and the N-terminal fragment of GSDME (GSDME-N). The expression of GSDME-N in monocytes from RA patients correlated positively with disease activity. Monocytes from RA patients with higher GSDME levels were more susceptible to pyroptosis. Furthermore, TNF induced pyroptosis in monocytes and macrophages by activating the caspase 3/GSDME pathway. The use of a caspase 3 inhibitor and silencing of GSDME significantly blocked TNF-induced pyroptosis. Gsdme deficiency effectively alleviated arthritis in a mouse model of CIA. CONCLUSION: These results support the notion of a pathogenic role of GSDME in RA and provide an alternative mechanism for RA pathogenesis involving TNF, which activates GSDME-mediated pyroptosis of monocytes and macrophages in RA. In addition, targeting GSDME might be a potential therapeutic approach for RA.