Type 1 inflammatory endotype relates to low compliance, lung fibrosis, and severe complications in COVID-19.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is an acute respiratory disease; approximately 5% of patients developing severe COVID-19. It is known that cytokine release is associated with disease severity, but the relationship between the different clinical phenotypes and inflammatory endotypes is not well understood. OBJECTIVE: This study investigated the association between inflammatory biomarker-based endotypes and severe COVID-19 phenotypes. METHODS: Interleukin (IL) -6, C-reactive protein (CRP), C-X-C motif chemokine (CXCL) 9, IL-18, C-C motif chemokine (CCL) 3, CCL17, IL-10, and vascular endothelial growth factor (VEGF) were measured in 57 COVID-19 patients, and their association with clinical characteristics was examined using a cluster analysis. RESULTS: Significantly higher blood levels of the eight inflammatory markers were noted in patients who developed acute respiratory distress syndrome (ARDS) than in those who did not develop ARDS (non-ARDS). Using a cluster analysis, the patient groups were classified into four clusters, of which two had patients with high IL-6 and CRP levels. In the cluster with high levels of Type 1 (T1) inflammatory markers such as CXCL9 and IL-18, 85% of the patients had ARDS, 65% of the patients developed acute kidney injury (AKI), and 78% of the patients developed pulmonary fibrosis. CONCLUSIONS: In the cluster with high levels of T1 inflammatory markers, the patients frequently suffered from tissue damage, manifested as ARDS and AKI. Our findings identified distinct T1 inflammatory endotypes of COVID-19 and suggest the importance of controlling inflammation by monitoring T1 biomarkers and treating accordingly to limit the severity of the disease.

TNF-α induces expression of the circadian clock gene Bmal1 via dual calcium-dependent pathways in rheumatoid synovial cells.

Tumor necrosis factor (TNF)-α is responsible for expressions of several clock genes and affects joint symptoms of rheumatoid arthritis (RA) with diurnal fluctuation. We tried to determine the mechanism involved in over-expression of Bmal1, induced by TNF-α, in primary cultured rheumatoid synovial cells. Cells were incubated with intra-cellular Ca2+ chelator BAPTA-AM, calcineurin inhibitor FK506 and p300/CBP (CREB binding protein) inhibitor C646, respectively, or transfected with p300 and CBP small interfering RNA (siRNA) before stimulation with TNF-α. Oscillation phase and amplitude of Bmal1, transcriptional activator Rorα, transcriptional repressor Rev-erbα, and histone acetyltransferases (p300 and Cbp) were evaluated by quantitative real-time PCR. As results, TNF-α did not influence the oscillation phase of Rev-erbα, while enhanced those of Rorα, resulting in over-expression of Bmal1. When Ca2+ influx was inhibited by BAPTA-AM, TNF-α-mediated up-regulation of Rorα was cancelled, however, that of Bmal1 was still apparent. When we further explored another pathway between TNF-α and Bmal1, TNF-α suppressed the expression of Rev-erbα in the absence of Ca2+ influx, as well as those of p300 and Cbp genes. Finally, actions of TNF-α, in increasing Bmal1/Rorα and decreasing Rev-erbα, were cancelled by C646 treatment or silencing of both p300 and Cbp. In conclusion, we determined a novel role of TNF-α in inducing Bmal1 via dual calcium dependent pathways; Rorα was up-regulated in the presence of Ca2+ influx and Rev-erbα was down-regulated in the absence of that. Results proposed that inhibition of p300/CBP could be new therapeutic targets for RA.

Cellular immunity reflects the persistent symptoms among COVID-19 recovered patients in Japan.

Coronavirus disease (COVID-19) often causes persistent symptoms long after infection, referred to as "long COVID" or post-acute COVID-19 syndrome (PACS). This phenomenon has been studied primarily concerning B-cell immunity, while the involvement of T-cell immunity is still unclear. This retrospective study aimed to examine the relationship among the number of symptoms, cytokine levels, and the Enzyme-linked immunosorbent spot (ELISPOT) assay data in patients with COVID-19. To examine inflammatory conditions, plasma interleukin (IL)-6, IL-10, IL-18, chemokine ligand 9 (CXCL9), chemokine ligand 3 (CCL3), and vascular endothelial growth factor (VEGF) levels were analyzed using plasma obtained from COVID-19 recovery patients and healthy controls (HC). These levels were significantly higher in the COVID-19 group than those in the HC group. ELISPOT assays were performed to investigate the correlation between COVID-19 persistent symptoms and T-cell immunity. Cluster analysis of ELISPOT categorized COVID-19 recovery patients in the ELISPOT-high and -low groups, based on the values of S1, S2, and N. The number of persistent symptoms was significantly higher in the ELISPOT-low group than those in the ELISPOT-high group. Thus, T cell immunity is critical for the rapid elimination of COVID-19 persistent symptoms, and its measurement immediately after COVID-19 recovery might predict long-term COVID-19 or PACS.

Th1 cytokine endotype discriminates and predicts severe complications in COVID-19

Treatment of severe and critical cases of coronavirus disease 2019 (COVID-19) is still a top priority in public health. Previously, we reported distinct Th1 cytokines related to the pathophysiology of severe COVID-19 condition. In the present study, we investigated the association of Th1 and Th2 cytokine/chemokine endotypes with cell-mediated immunity via multiplex immunophenotyping, single-cell RNA-Seq analysis of peripheral blood mononuclear cells, and analysis of the clinical features of COVID-19 patients. Based on serum cytokine and systemic inflammatory markers, COVID-19 cases were classified into four clusters of increasing (I-IV) severity. Two prominent clusters were of interest and could be used as prognostic reference for a targeted treatment of severe COVID-19 cases. Cluster III reflected severe/critical pathology and was characterized by decreased in CCL17 levels and increase in IL-6, C-reactive protein CXCL9, IL-18, and IL-10 levels. The second cluster (Cluster II) showed mild to moderate pathology and was characterized by predominated CXCL9 and IL-18 levels, levels of IL-6 and CRP were relatively low. Cluster II patients received anti-inflammatory treatment in early-stage, which may have led prevent disease prognosis which is accompanied to IL-6 and CRP induction. In Cluster III, a decrease in the proportion of effector T cells with signs of T cell exhaustion was observed. This study highlights the mechanisms of endotype clustering based on specific inflammatory markers in related the clinical outcome of COVID-19.

Methotrexate upregulates circadian transcriptional factors PAR bZIP to induce apoptosis on rheumatoid arthritis synovial fibroblasts.

BACKGROUND: Effects of methotrexate (MTX) on the proliferation of rheumatoid arthritis (RA) synovial fibroblasts are incompletely understood. We explored actions of MTX in view of circadian transcriptions of synovial fibroblasts. METHODS: Under treatment with MTX, expression of core circadian clock genes, circadian transcriptional factor proline and acidic amino acid-rich basic leucine zipper (PAR bZIP), and proapoptotic molecule Bcl-2 interacting killer (Bik) was examined by real-time polymerase chain reaction. Protein expression of circadian clock gene PERIOD2 (PER2) and CYTOCHROME C was also examined by western blotting and ELISA. Promoter activities of Per2 and Bik were measured by Luciferase assay. Expression of PER2, BIK, and CYTOCHROME C and morphological changes of the nucleus were observed by fluorescent immunostaining. Synovial fibroblasts were transfected with Per2/Bik small interfering RNA, and successively treated with MTX to determine cell viabilities. Finally, synovial fibroblasts were treated with MTX according to the oscillation of Per2/Bik expression. RESULTS: MTX (10 nM) significantly decreased cell viabilities, but increased messenger RNA expression of Per2, Bik, and PAR ZIP including D site of the albumin promoter binding protein (Dbp), hepatic leukemia factor (Hlf), and thyrotroph embryonic factor (Tef). MTX also increased protein expression of PER2 and CYTOCHROME C, and promoter activities of Per2 and Bik via D-box. Under fluorescent observations, expression of PER2, BIK, and CYTOCHROME C was increased in apoptotic cells. Cytotoxicity of MTX was attenuated by silencing of Per2 and/or Bik, and revealed that MTX was significantly effective in situations where Per2/Bik expression was high. CONCLUSIONS: We present here novel unique action of MTX on synovial fibroblasts that upregulates PAR bZIP to transcribe Per2 and Bik, resulting in apoptosis induction. MTX is important in modulating circadian environments to understand a new aspect of pathogenesis of RA.

Circulating cell free DNA: a marker to predict the therapeutic response for biological DMARDs in rheumatoid arthritis.

AIM: To evaluate the correlation between circulating cell-free DNA (ccfDNA) in plasma and clinical disease activities in patients with rheumatoid arthritis (RA). METHOD: The study group included 30 patients with RA who started biological disease-modifying anti-rheumatic drugs (DMARDs) therapy. The concentration of ccfDNA in plasma was measured by quantitative real-time polymerase chain reaction at baseline to 24 weeks in every 4-week period from 30 patients and 21 healthy individuals. We also evaluated the correlation between ccfDNA and the clinical activity or the therapeutic response for biological DMARDs, using the simplified disease activity index (SDAI), Disease Activity Score of 28 joints (erythrocyte sedimentation rate) and the European League Against Rheumatism (EULAR) response criteria. Synovial fluid samples of knee joints were collected from 13 patients with RA and 12 with osteoarthritis (OA) to measure ccfDNA. RESULT: The concentration of ccfDNA in RA patients at baseline was higher than healthy controls (P = 0.016). After introducing biological DMARDs, ccfDNA was increased until 8 weeks from the baseline, and decreased after 12 weeks. The average of SDAI was improved in all patients enrolled. At 12 weeks after treatment, 15 patients were good responders to the EULAR response criteria, nine showed moderate response and six showed no response. ccfDNA in good responders was increased until 8 weeks, while those of moderate or no response were not (P = 0.042). In joint fluid of RA patients, ccfDNA was remarkably increased as compared to those from OA (P = 0.00011). CONCLUSION: After introducing biological DMARDs, increase of ccfDNA at 8 weeks was associated with improvement of disease activities. Compared with biomarkers reported, ccfDNA is able to predict the early therapeutic effects of biological DMARDs in RA patients.