Expression of the readthrough transcript CiDRE in alveolar macrophages boosts SARS-CoV-2 susceptibility and promotes COVID-19 severity.

Lung infection during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via the angiotensin-I-converting enzyme 2 (ACE2) receptor induces a cytokine storm. However, the precise mechanisms involved in severe COVID-19 pneumonia are unknown. Here, we showed that interleukin-10 (IL-10) induced the expression of ACE2 in normal alveolar macrophages, causing them to become vectors for SARS-CoV-2. The inhibition of this system in hamster models attenuated SARS-CoV-2 pathogenicity. Genome-wide association and quantitative trait locus analyses identified a IFNAR2-IL10RB readthrough transcript, COVID-19 infectivity-enhancing dual receptor (CiDRE), which was highly expressed in patients harboring COVID-19 risk variants at the IFNAR2 locus. We showed that CiDRE exerted synergistic effects via the IL-10-ACE2 axis in alveolar macrophages and functioned as a decoy receptor for type I interferons. Collectively, our data show that high IL-10 and CiDRE expression are potential risk factors for severe COVID-19. Thus, IL-10R and CiDRE inhibitors might be useful COVID-19 therapies.

Apple-shaped obesity: A risky soil for cytokine-accelerated severity in COVID-19.

Obesity has been recognized as one of the most significant risk factors for the deterioration and mortality associated with COVID-19, but the significance of obesity itself differs among ethnicity. Multifactored analysis of our single institute-based retrospective cohort revealed that high visceral adipose tissue (VAT) burden, but not other obesity-associated markers, was related to accelerated inflammatory responses and the mortality of Japanese COVID-19 patients. To elucidate the mechanisms how VAT-dominant obesity induces severe inflammation after severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection, we infected two different strains of obese mice, C57BL/6JHamSlc-ob/ob (ob/ob), C57BLKS/J-db/db (db/db), genetically impaired in the leptin ligand and receptor, respectively, and control C57BL/6 mice with mouse-adapted SARS-CoV-2. Here, we revealed that VAT-dominant ob/ob mice were extremely more vulnerable to SARS-CoV-2 due to excessive inflammatory responses when compared to SAT-dominant db/db mice. In fact, SARS-CoV-2 genome and proteins were more abundant in the lungs of ob/ob mice, engulfed in macrophages, resulting in increased cytokine production including interleukin (IL)-6. Both an anti-IL-6 receptor antibody treatment and the prevention of obesity by leptin replenishment improved the survival of SARS-CoV-2-infected ob/ob mice by reducing the viral protein burden and excessive immune responses. Our results have proposed unique insights and clues on how obesity increases the risk of cytokine storm and death in patients with COVID-19. Moreover, earlier administration of antiinflammatory therapeutics including anti-IL-6R antibody to VAT-dominant patients might improve clinical outcome and stratification of the treatment for COVID-19, at least in Japanese patients.

Anti-Inflammatory Effects of a Novel Nuclear Factor-κB Inhibitory Derivative Derived from Pyrazolo[3,4-d]Pyrimidine in Three Inflammation Models.

Nuclear factor-κB (NF-κB) plays a central role in inflammatory responses, and its physiologic functions are essential for cell survival and proliferation. Currently, drugs targeting NF-κB inhibition have not yet been applied in clinical practice. We investigated the physiologic effect of a novel NF-κB inhibitory compound, 1H-pyrazolo[3,4-d]pyrimidin-4-amine derivative (INH #1), on three inflammatory animal models. The pharmacokinetics were measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Acute hepatitis was induced by administrating lipopolysaccharide (LPS) and D-(+)-galactosamine hydrochloride followed by the analysis of survival time and inflammatory mediators. Collagen-induced arthritis (CIA) was induced by immunization with type II collagen (CII), and serum-transfer arthritis (STA) was caused by injecting K/BxN mice serum. Clinical and histologic scores were evaluated in both arthritis models. Immune cell subset analysis, CII-induced interferon-gamma (IFN-γ) production and proliferation, and measurement of anti-CII IgG antibodies were performed in the CIA model. In the acute hepatitis model, INH #1 suppressed tumor necrosis factor-α (TNF-α) production and prevented early death in a dose-dependent manner. INH #1 significantly attenuated arthritis scores and joint inflammation in both arthritis models. Additionally, in the CIA model, dendritic cells (DCs) in the regional lymph nodes were decreased in the treated mice and antigen-induced IFN-γ production and cell proliferation in splenocytes were inhibited, whereas the titers of anti-CII IgG antibodies were comparable regardless of the treatment. Here we revealed that INH #1 exerted anti-inflammatory effects in vivo via inhibition of inflammatory mediators and suppression of cellular immune responses. This compound could be a novel candidate for inhibition of NF-κB in certain inflammatory diseases. SIGNIFICANCE STATEMENT: A novel nuclear factor-κB (NF-κB) inhibitory compound, 1H-pyrazolo[3,4-d]pyrimidin-4-amine derivative (INH #1), which retains physiologically essential NF-κB bioactivity, suppressed inflammation in three different mouse models: the acute hepatitis model, the collagen-induced arthritis model, and the K/BxN serum-transfer arthritis model. These results suggest that this compound could be a novel and potent anti-inflammatory agent.

Diverse pro-inflammatory ability of mutated spike protein derived from variant strains of SARS-CoV-2.

The severity of COVID-19 has been reported to differ among SARS-CoV-2 mutant variants. The overactivation of macrophages is involved in severe COVID-19, yet the effects of SARS-CoV-2 mutations on macrophages remain poorly understood. To clarify the effects, we examined whether mutations of spike proteins (S-proteins) affect macrophage activation. CD14+ monocyte-derived macrophages were stimulated with the recombinant S-protein of the wild-type, Delta, and Omicron strains or live viral particles of individual strains. Regarding IL-6 and TNF-α, Delta or Omicron S-protein had stronger or weaker pro­inflammatory ability, respectively, than the wild-type. Similar trends were observed between S-proteins and viral particles. S-protein mutations could be related to the diversity in macrophage activation and severity rates in COVID-19 caused by various SARS-CoV-2 strains.

Chondroprotective effects of CDK4/6 inhibition via enhanced ubiquitin-dependent degradation of JUN in synovial fibroblasts.

OBJECTIVE: Targeting synovial fibroblasts (SF) using a cyclin-dependent kinase (CDK) 4/6 inhibitor (CDKI) could be a potent therapy for RA via inhibition of proliferation and MMP-3 production. This study was designed to elucidate the mechanism of chondroprotective effects on SFs by CDK 4/6 inhibition. METHODS: CDK4/6 activity was inhibited using CDKI treatment or enhanced by adenoviral gene transduction. Chondroprotective effects were evaluated using a collagen-induced arthritis model (CIA). Gene and protein expression were evaluated with quantitative PCR, ELISA and Western blotting. The binding of nuclear extracts to DNA was assessed with an electrophoresis mobility shift assay. RNA-Seq was performed to identify gene sets affected by CDKI treatment. RESULTS: CDKI attenuated cartilage destruction and MMP-3 production in CIA. In RASFs, CDKI impaired the binding of AP-1 components to DNA and inhibited the production of MMP-1 and MMP-3, which contain the AP-1 binding sequence in their promoter. CDK4/6 protected JUN from proteasome-dependent degradation by inhibiting ubiquitination. The RNA-Seq analysis identified CDKI-sensitive inflammatory genes, which were associated with the pathway of RA-associated genes, cytokine-cytokine receptor interaction and IL-17 signalling. Notably, the AP-1 motif was enriched in these genes. CONCLUSION: The mechanism of chondroprotective effects by CDK4/6 inhibition was achieved by the attenuation of AP-1 transcriptional activity via the impaired stability of JUN. Because the pharmacologic inhibition of CDK4/6 has been established as tolerable in cancer treatment, it could also be beneficial in patients with RA due to its chondroprotective and anti-inflammatory effects.

Factors associated with impaired physical function in elderly rheumatoid arthritis patients who had achieved low disease activity.

OBJECTIVE: We aimed to investigate factors associated with impaired physical function (defined as HAQ Disability Index [HAQ-DI] >0.5) of old-old (aged 75-84) patients with rheumatoid arthritis (RA). METHODS: Data from 15,185 RA patients in the National Database of Rheumatic Disease in Japan were extracted from 2017 to 2018. We enrolled 3,708 patients aged 55-84 in simplified disease activity index (SDAI) ≤11 and Steinbrocker stage I/II. Factors associated with HAQ-DI >0.5 were analyzed by multivariable logistic regression. RESULTS: About half of the old-old patients received methotrexate, which was lower than middle-aged (55-64) and young-old patients (65-74). The proportion of glucocorticoids in the old-old patients was highest among the three groups, and biological disease-modifying anti-rheumatic drugs were similarly used. The prevalence of HAQ-DI >0.5 was significantly higher in old-old patients with low disease activity than in those with remission. The same was true in the middle-aged and young-old patients. Multivariable analysis showed age, higher SDAI, glucocorticoid use, and methotrexate non-use were significantly associated with HAQ-DI >0.5 in the old-old patients. CONCLUSIONS: SDAI remission was an ideal goal for old-old patients in terms of physical function. Glucocorticoids and a low proportion of methotrexate use may influence the physical function of old-old patients.

Effectiveness and safety of treat-to-target strategy in elderly-onset rheumatoid arthritis: a 3-year prospective observational study.

OBJECTIVES: To evaluate 3-year outcomes of following a treat-to-target (T2T) strategy targeting low disease activity for patients with elderly-onset RA (EORA) and to confirm safety profile of T2T. METHODS: Treatment was adjusted to target low disease activity with conventional synthetic DMARDs, followed by biologic DMARDs (bDMARDs) in 197 MTX-naïve EORA patients (mean age 74.9 years) with moderate-to-high disease activity. Non-implementation of T2T was evaluated at week 12, 24, 36, 52, 76, 104 and 128. To evaluate risks of using MTX, bDMARDs and glucocorticoids, 2122 periods of 3 months each were analysed using Bayesian hierarchical logistic regression models. RESULTS: Of the patients, 84.7% received methotrexate, 34.0% glucocorticoids with DMARDs and 41.6% bDMARDs during the observation period. Sixty-nine of the 197 patients failed to adhere to T2T because of comorbidities or the patient's own decision: 33 failed once, 19 twice, 10 three times and 6 four times or more. Simplified disease activity index (SDAI) remission and HAQ Disability Index (HAQ-DI) ≤0.5 at 3 years were achieved in 57.8% and 70.3% of the 128 patients adhering to T2T, and 34.8% and 43.5% of the 69 patients who did not adhere to T2T, respectively, and these were significantly different. Eighty-nine serious adverse events (SAEs) of any type were reported in 61 patients. MTX, bDMARDs and glucocorticoid were not associated with SAEs when adjusted for mean SDAI during the observation period and comorbidities at baseline. CONCLUSION: T2T strategy for EORA by using MTX and bDMARDs was effective with an acceptable safety profile. Adhering to T2T led to better outcomes.

Induction of oligoclonal CD8 T cell responses against pulmonary metastatic cancer by a phospholipid-conjugated TLR7 agonist.

Recent advances in cancer immunotherapy have improved patient survival. However, only a minority of patients with pulmonary metastatic disease respond to treatment with checkpoint inhibitors. As an alternate approach, we have tested the ability of systemically administered 1V270, a toll-like receptor 7 (TLR7) agonist conjugated to a phospholipid, to inhibit lung metastases in two variant murine 4T1 breast cancer models, as well as in B16 melanoma, and Lewis lung carcinoma models. In the 4T1 breast cancer models, 1V270 therapy inhibited lung metastases if given up to a week after primary tumor initiation. The treatment protocol was facilitated by the minimal toxic effects exerted by the phospholipid TLR7 agonist compared with the unconjugated agonist. 1V270 exhibited a wide therapeutic window and minimal off-target receptor binding. The 1V270 therapy inhibited colonization by tumor cells in the lungs in an NK cell dependent manner. Additional experiments revealed that single administration of 1V270 led to tumor-specific CD8+ cell-dependent adaptive immune responses that suppressed late-stage metastatic tumor growth in the lungs. T cell receptor (TCR) repertoire analyses showed that 1V270 therapy induced oligoclonal T cells in the lungs and mediastinal lymph nodes. Different animals displayed commonly shared TCR clones following 1V270 therapy. Intranasal administration of 1V270 also suppressed lung metastasis and induced tumor-specific adaptive immune responses. These results indicate that systemic 1V270 therapy can induce tumor-specific cytotoxic T cell responses to pulmonary metastatic cancers and that TCR repertoire analyses can be used to monitor, and to predict, the response to therapy.

Evaluation of the alternative classification criteria of systemic lupus erythematosus established by Systemic Lupus International Collaborating Clinics (SLICC).

OBJECTIVE: To evaluate the performance of the 2012 Systemic Lupus International Collaborating Clinics criteria (SLICC-12) on classifying systemic lupus erythematosus (SLE) in an uncontrolled multi-centered study with real-life scenario of the patients in Japan. METHODS: This study comprised 495 patients with SLE or non-SLE rheumatic diseases and allied conditions from 12 institutes in Japan. Chart review of each patient was performed by the 27 expert rheumatologists and diagnosis of 487 cases reached to the consensus. Value of the SLICC-12 on SLE classification was analyzed comparing with the 1997 revised American College of Rheumatology SLE classification criteria (ACR-97) employing the expert-consented diagnoses. RESULTS: Compared to the ACR-97, the SLICC-12 had a higher sensitivity (ACR-97 vs. SLICC-12: 0.88 vs. 0.99, p < .01) and comparable specificity (0.85 vs. 0.80). The rate of misclassification (0.14 vs. 0.11) or the area under the receiver operating characteristic curves (0.863 vs. 0.894) was not statistically different. In the cases that diagnoses corresponded in high rates among experts, both criteria showed high accordance of SLE classification over 85% with the expert diagnoses. CONCLUSION: Although employment of SLICC-12 for the classification for SLE should be carefully considered, the SLICC-12 showed the higher sensitivity on classifying SLE in Japanese population.

Cell cycle regulation therapy combined with cytokine blockade enhances antiarthritic effects without increasing immune suppression.

OBJECTIVE: Biological disease-modifying antirheumatic drugs (DMARDs) that inhibit aberrant immune reactions in rheumatoid arthritis (RA) cannot induce complete remission in all patients. Combination therapies using two biological DMARDs have failed to exert additive effects and increased serious infections. We have found that cell cycle inhibition of synovial fibroblasts with cyclin-dependent kinase (CDK) inhibitors ameliorated the disease in animal models of RA without attenuating acquired immunity. The objective of this study was to determine whether a clinically well-tolerated selective CDK 4/6 inhibitor (CDKI), palbociclib, is effective and whether combination with cytokine blockers acts additively without enhancing immune suppression. METHODS: The effects of CDKI on haematopoiesis and physical and behavioural findings in mice were evaluated. Mice with collagen-induced arthritis (CIA) were treated with CDKI, etanercept or anti-interleukin (IL)-6 receptor antibody (MR16-1) alone or with a combination of CDKI with etanercept or MR16-1. Their clinical, histological and radiographic scores, serum anti-(type II collagen (CII)) antibody levels and proliferative responses of lymph node cells to CII were determined. RESULTS: Although CDKI induced marginal myelosuppression, it was well tolerated and ameliorated CIA dose-dependently. The combinations of low-dose CDKI and either tumour necrosis factor-α or IL-6 blocker enhanced the antiarthritic effects additively. The addition of CDKI to either cytokine blocker did not affect the levels of anti-CII antibodies and proliferative responses of lymphocytes to CII. CONCLUSIONS: A clinically well-tolerated CDK4/6 inhibitor exerted antiarthritic effects in this mouse model. By combining therapeutic agents targeting immune reaction and synovial proliferation, we have demonstrated for the first time that two molecular targeting treatments act additively and may not increase immune suppression.

Structural and functional outcomes of a therapeutic strategy targeting low disease activity in patients with elderly-onset rheumatoid arthritis: a prospective cohort study (CRANE).

OBJECTIVE: The aim of this study was to evaluate structural damage and physical disability in patients with elderly-onset RA (EORA) who were treated in clinical practice with a therapeutic strategy targeting low disease activity (LDA). METHODS: Data from 151 MTX-naive patients (mean age 74.9 years) with EORA from a prospective, monocentric registry were analysed. Treatment was adjusted every 3 months targeting LDA [28-joint DAS using ESR (DAS28-ESR) <3.2]. Treatment was initiated with non-biologic DMARDs (nbDMARDs), followed by TNF inhibitors (TNFis) or tocilizumab. The primary outcome was change from week 0 to week 52 in the modified total Sharp score (ΔmTSS). Secondary outcomes were derived from the HAQ Disability Index (HAQ-DI) and DAS28 at week 52. Predictors of clinically relevant radiographic progression [CRRP; ΔmTSS/year more than the smallest detectable change (2.1 points)] were examined using multivariate logistic regression models. RESULTS: Adherence to the treat-to-target strategy was observed in 83.4% of the 151 patients at week 24 and in 75.5% at week 52. At week 52, 67.6% of the patients were receiving a nbDMARD alone, 31.0% a TNFi with or without MTX and 1.4% tocilizumab. At week 52, structural remission (ΔmTSS/yr ≤0.5) was achieved in 49.7% of the patients, functional remission (HAQ-DI ≤0.5) in 63.4% and LDA in 51.0%. Clinical responses at weeks 12 and 24 were significant independent predictors of CRRP. Cumulative disease activity during the first 12 weeks predicted CRRP with a C-statistic of 0.888. CONCLUSION: Achieving structural remission, functional remission and LDA in clinical practice in EORA patients are realistic goals. Our results indicate significant benefits for a therapeutic strategy targeting LDA for EORA patients in clinical practice.

ß stimulator induces upregulation of miR-27a in the rat heart one hour after the injection.

MicroRNAs (miRs) are non-coding small RNA containing 18 to 22 nucleotides, that post-transcriptionally regulates mRNA expression. Chronic injection of ß stimulator is known to induce cardiac injury and change of miRs expression level in the heart with some pathological changes such as fibrosis, heart failure, myocardial infarction. We investigated the changes in the expression level of miRs in the rat heart one hour after isoproterenol (a ß stimulator) injection. Male Sprague-Dawley rats were assigned into three groups and received subcutaneous injection of normal sarin (NS) or 0.1 mg/kg isoproterenol (ISO-0.1) or 10 mg/kg isoproterenol (ISO-10). After one hour, we collected their heart and plasma. Total RNA was extracted from the left ventricle and used for deep miRNA sequencing. Based on the results of miRNA sequencing, we performed real-time polymerase chain reaction (RT-PCR) using 8 miR primers. Cardiac injury was evaluated by hematoxylin and eosin, and phosphotungstic acid-hematoxylin staining and measuring troponin-I levels in plasma. Troponin-I was significantly increased in ISO-0.1 and ISO-10 groups, but histological observation did not show any cardiac necrosis. miRNA sequencing identified 14 upregulated miRs and 12 downregulated miRs. Of the 26 miRs, RT-PCR confirmed miR-144-3p/5p and miR-451-5p were decreased, and that 5 miRs (miR-27a-5p, miR-30b-3p, miR-92a-1-5p, miR-132-5p, miR-582-3p) were upregulated. This study showed that ß stimulus causes downregulation of miR-144/451 cluster and increases expression of five 5 miRs in the heart, especially 6.5-fold upregulation of miR-27a-5p as early as one hour after isoproterenol injection. Therefore, these miRs might be good biomarkers for cardiac injury.

Variations in subtle cystic tumors of the atrioventricular node: Five autopsy cases.

Cystic tumor of the atrioventricular node (CTAVN) is the most common primary cardiac tumor cause of sudden death but is rarely found during forensic autopsy. We present five autopsy cases of sudden death from undiagnosed CTAVN. The tumors varied in their histological appearance, which may be related to their variation in clinical presentation. Some of the cases had been diagnosed with epilepsy before death; it seems that syncopal attacks caused by CTAVN may be misdiagnosed as epilepsy. When performing forensic autopsy, CTAVN should be considered in the differential diagnosis of sudden death. Careful examination of the cardiac conduction system is important in every sudden death case regardless of age.

Effectiveness and safety of treat-to-target strategy for methotrexate-naïve rheumatoid arthritis patients >75 years of age.

Objectives: To identify differences in effectiveness and safety of a treat-to-target (T2T) strategy comparing late-onset MTX-naïve RA patients (LORA) ≥75 or <75 years of age. Methods: Treatment was adjusted to target low disease activity with conventional synthetic DMARDs followed by biologic DMARDs (bDMARDs) in LORA ≥75 years (n = 98, mean age 80.0 years) and LORA <75 years (n = 99) with moderate-high disease activity. Achievement of Simplified Disease Activity Index (SDAI) remission at week 156 by non-responder imputation analysis was evaluated as a primary outcome. Results: LORA ≥75 years had more comorbidities than LORA <75 years, but SDAI and ACPA positivity were similar at baseline. Of the LORA ≥75 years, 70.4% started MTX and 34.1% and 37.1% received a bDMARD at week 52 and 156, respectively (very similar to the LORA <75 years). Glucocorticoid use was more frequent in the LORA ≥75 years than in the LORA <75 years. Comorbidities/adverse events more frequently contributed to the reasons for non-adherence to T2T in the LORA ≥75 than in the LORA <75. At week 156, 32.7% of the LORA ≥75 and 66.7% of the LORA <75 achieved SDAI remission (P < 0.001). The cumulative incidence of serious adverse events (SAEs) over 156 weeks was 42.8% in the LORA ≥75 and 22.1% in the LORA <75. Multivariable analysis indicated an increased risk of SDAI non-remission at week 156 in the LORA ≥75 [odds ratio 2.82 (95% CI 1.29. 6.14)] after adjusting for comorbidities at baseline, non-adherence to T2T and SAEs. Conclusions: It was more difficult to achieve remission in the LORA ≥75 patients than in the LORA <75 patients due to both poor treatment response and safety issues.

Clinical significance of complement as a biomarker of disease activity in 4 cases of IgG4-related disease with retroperitoneal fibrosis.

Hypocomplementaemia is frequently observed in IgG4-related diseases, however the clinical significance is unclear. We describe herein the clinical courses of 4 patients with IgG4-related disease with hypocomplementaemia. Our cases showed autoimmune pancreatitis, retroperitoneal fibrosis, Mikulicz's disease, interstitial lung disease, lymphadenopathy and mesenteric fibrosis around the aorta. A decrease in serum complement preceded deterioration of the disease and clinical improvement was observed in accordance with normalisation of serum complement. These clinical courses suggest that serum complement is a biomarker of the disease activity.

Mass-like Lesions Causing Compression of Large Vessels in Granulomatosis with Polyangiitis.

A 69-year-old woman with a history of otitis media with anti-neutrophil cytoplasmic antibody-associated vasculitis who had been receiving corticosteroid monotherapy presented with shortness of breath. The otitis media had been alleviated, but she had saddle nose. Chest enhanced computed tomography showed stenoses of the bronchi and large vessels surrounded by mass-like lesions in the mediastinum. These manifestations indicated an active state of granulomatosis with polyangiitis (GPA). After she was started on high-dose corticosteroids and intravenous cyclophosphamide, the mass-like lesions disappeared with improvements of the stenoses. Ameliorating mass-like lesions resulting from GPA requires therapeutic intervention using corticosteroids and immunosuppressants.

Leucine-rich alpha-2 glycoprotein as a potential biomarker for large vessel vasculitides.

Objectives: Serum levels of C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) have been used as useful biomarkers for reflecting the activity of large vessel vasculitides (LVV). However, a novel biomarker that could have a complementary role to these markers is still required. In this retrospective observational study, we investigated whether leucine-rich α-2 glycoprotein (LRG), a known biomarker in several inflammatory diseases, could be a novel biomarker for LVVs. Methods: 49 eligible patients with Takayasu arteritis (TAK) or giant cell arteritis (GCA) whose serum was preserved in our laboratory were enrolled. The concentrations of LRG were measured with an enzyme-linked immunosorbent assay. The clinical course was reviewed retrospectively from their medical records. The disease activity was determined according to the current consensus definition. Results: The serum LRG levels were higher in patients with active disease than those in remission, and decreased after the treatments. While LRG levels were positively correlated with both CRP and erythrocyte sedimentation rate, LRG exhibited inferior performance as an indicator of disease activity compared to CRP and ESR. Of 35 CRP-negative patients, 11 had positive LRG. Among the 11 patients, two had active disease. Conclusion: This preliminary study indicated that LRG could be a novel biomarker for LVV. Further large studies should be required to promise the significance of LRG in LVV.