A zebrafish model of COVID-19-associated cytokine storm syndrome reveals differential proinflammatory activities of Spike proteins of SARS-CoV-2 variants of concern.

The sudden and unexpected appearance of the COVID-19 pandemic turned the whole world upside down in a very short time. One of the main challenges faced has been to understand COVID-19 patient heterogeneity, as a minority develop life-threatening hyperinflammation, the so-called cytokine storm syndrome (CSS). Using the unique advantages of the zebrafish model we report here the proinflammatory role of Spike (S) proteins from different SARS-CoV-2 variants of concern after injection into the hindbrain ventricle, a cavity filled with cerebrospinal fluid to which immune cells can be easily recruited and that mimics the alveolar environment of the human lung. We found that wild type/Wuhan variant S1 (S1WT) protein promoted neutrophil and macrophage recruitment, local and systemic hyperinflammation, emergency myelopoiesis, and hemorrhages. In addition, S1γ protein was more proinflammatory and S1δ was less proinflammatory than S1WT and, strikingly, Sβ promoted delayed and long-lasting inflammation. Pharmacological inhibition of the canonical inflammasome robustly alleviated S1 protein-induced inflammation and emergency myelopoiesis. In contrast, genetic inhibition of angiotensin-converting enzyme 2 strengthened the proinflammatory activity of S1, and the administration of angiopoietin (1-7) fully rescued S1-induced hyperinflammation and hemorrhages. These results shed light into the mechanisms orchestrating the COVID-19-associated CSS and the host immune response to different SARS-CoV-2 S protein variants.

Role of the Redox State of Human Peroxiredoxin-5 on Its TLR4-Activating DAMP Function

Human peroxiredoxin-5 (PRDX5) is a unique redox-sensitive protein that plays a dual role in brain ischemia-reperfusion injury. While intracellular PRDX5 has been reported to act as a neuroprotective antioxidative enzyme by scavenging peroxides, once released extracellularly from necrotic brain cells, the protein aggravates neural cell death by inducing expression of proinflammatory cytokines in macrophages through activation of Toll-like receptor (TLR) 2 (TLR2) and 4 (TLR4). Although recent evidence showed that PRDX5 was able to interact directly with TLR4, little is known regarding the role of the cysteine redox state of PRDX5 on its DAMP function. To gain insights into the role of PRDX5 redox-active cysteine residues in the TLR4-dependent proinflammatory activity of the protein, we used a recombinant human PRDX5 in the disulfide (oxidized) form and a mutant version lacking the peroxidatic cysteine, as well as chemically reduced and hyperoxidized PRDX5 proteins. We first analyzed the oxidation state and oligomerization profile by Western blot, mass spectrometry, and SEC-MALS. Using ELISA, we demonstrate that the disulfide bridge between the enzymatic cysteines is required to allow improved TLR4-dependent IL-8 secretion. Moreover, single-molecule force spectroscopy experiments revealed that TLR4 alone is not sufficient to discriminate the different PRDX5 redox forms. Finally, flow cytometry binding assays show that disulfide PRDX5 has a higher propensity to bind to the surface of living TLR4-expressing cells than the mutant protein. Taken together, these results demonstrate the importance of the redox state of PRDX5 cysteine residues on TLR4-induced inflammation.

Exosomes produced by melanoma cells significantly influence the biological properties of normal and cancer-associated fibroblasts

The incidence of cutaneous malignant melanoma is increasing worldwide. While the treatment of initial stages of the disease is simple, the advanced disease frequently remains fatal despite novel therapeutic options . This requires identification of novel therapeutic targets in melanoma. Similarly to other types of tumours, the cancer microenvironment plays a prominent role and determines the biological properties of melanoma. Importantly, melanoma cell-produced exosomes represent an important tool of intercellular communication within this cancer ecosystem. We have focused on potential differences in the activity of exosomes produced by melanoma cells towards melanoma-associated fibroblasts and normal dermal fibroblasts. Cancer-associated fibroblasts were activated by the melanoma cell-produced exosomes significantly more than their normal counterparts, as assessed by increased transcription of genes for inflammation-supporting cytokines and chemokines, namely IL-6 or IL-8. We have observed that the response is dependent on the duration of the stimulus via exosomes and also on the quantity of exosomes. Our study demonstrates that melanoma-produced exosomes significantly stimulate the tumour-promoting proinflammatory activity of cancer-associated fibroblasts. This may represent a potential new target of oncologic therapy .

Miconazole Suppresses 27-Hydroxycholesterol-induced Inflammation by Regulating Activation of Monocytic Cells to a Proinflammatory Phenotype

Miconazole is effective in treating inflammatory skin conditions and has well-established antifungal effects. To elucidate the underlying mechanisms mediating its additional beneficial effects, we assessed whether miconazole influences the inflammation induced by 27-hydroxycholesterol (27OHChol), an oxygenated cholesterol derivative with high proinflammatory activity, using THP-1 monocytic cells. Miconazole dose-dependently inhibited the expression of proinflammatory markers, including CCL2 and CCR5 ligands such as CCL3 and CCL4, and impaired the migration of monocytic cells and CCR5-positive T cells. In the presence of 27OHChol, miconazole decreased CD14 surface levels and considerably weakened the lipopolysaccharide response. Furthermore, miconazole blocked the release of soluble CD14 and impaired the transcription of the matrix metalloproteinase-9 gene and secretion of its active gene product. Additionally, it downregulated the expression of ORP3 and restored the endocytic function of THP-1 cells. Collectively, these findings indicate that miconazole regulates the 27OHChol-induced expression of proinflammatory molecules in monocytic cells, thereby suppressing inflammation in an oxysterol-rich milieu.

SARS-CoV-2 spike protein induces abnormal inflammatory blood clots neutralized by fibrin immunotherapy

Blood clots are a central feature of coronavirus disease-2019 (COVID-19) and can culminate in pulmonary embolism, stroke, and sudden death. However, it is not known how abnormal blood clots form in COVID-19 or why they occur even in asymptomatic and convalescent patients. Here we report that the Spike protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to the blood coagulation factor fibrinogen and induces structurally abnormal blood clots with heightened proinflammatory activity. SARS-CoV-2 Spike virions enhanced fibrin-mediated microglia activation and induced fibrinogen-dependent lung pathology. COVID-19 patients had fibrin autoantibodies that persisted long after acute infection. Monoclonal antibody 5B8, targeting the cryptic inflammatory fibrin epitope, inhibited thromboinflammation. Our results reveal a procoagulant role for the SARS-CoV-2 Spike and propose fibrin-targeting interventions as a treatment for thromboinflammation in COVID-19.

Stress Induces Release of Extracellular Vesicles by Trypanosoma cruzi Trypomastigotes

All extracellular forms of Trypanosoma cruzi, the causative agent of Chagas disease, release extracellular vesicles (EVs) containing major surface molecules of the parasite. EV release depends on several mechanisms (internal and external). However, most of the environmental conditions affecting this phenomenon are still unknown. In this work, we evaluated EV release under different stress conditions and their ability to be internalized by the parasites. In addition, we investigated whether the release conditions would affect their immunomodulatory properties in preactivated bone marrow-derived macrophages (BMDM). Sodium azide and methyl-cyclo-β-dextrin (CDB) reduced EV release, indicating that this phenomenon relies on membrane organization. EV release was increased at low temperatures (4°C) and acidic conditions (pH 5.0). Under this pH, trypomastigotes differentiated into amastigotes. EVs are rapidly liberated and reabsorbed by the trypomastigotes in a concentration-dependent manner. Nitrosative stress caused by sodium nitrite in acid medium or S-nitrosoglutathione also stimulated the secretion of EVs. EVs released under all stress conditions also maintained their proinflammatory activity and increased the expression of iNOS, Arg 1, IL-12, and IL-23 genes in IFN-γ and LPS preactivated BMDM. In conclusion, our results suggest a budding mechanism of release, dependent on the membrane structure and parasite integrity. Stress conditions did not affect functional properties of EVs during interaction with host cells. EV release variations under stress conditions may be a physiological response against environmental changes.

B-Cell Compartmental Features and Molecular Basis for Therapy in Autoimmune Disease

Background and ObjectivesTo assess the molecular landscape of B-cell subpopulations across different compartments in patients with neuromyelitis optica spectrum disorder (NMOSD).MethodsWe performed B-cell transcriptomic profiles via single-cell RNA sequencing across CSF, blood, and bone marrow in patients with NMOSD.ResultsAcross the tissue types tested, 4 major subpopulations of B cells with distinct signatures were identified: naive B cells, memory B cells, age-associated B cells, and antibody-secreting cells (ASCs). NMOSD B cells show proinflammatory activity and increased expression of chemokine receptor genes (CXCR3 and CXCR4). Circulating B cells display an increase of antigen presentation markers (CD40 and CD83), as well as activation signatures (FOS, CD69, and JUN). In contrast, the bone marrow B-cell population contains a large ASC fraction with increased oxidative and metabolic activity reflected by COX genes and ATP synthase genes. Typically, NMOSD B cells become hyperresponsive to type I interferon, which facilitates B-cell maturation and anti–aquaporin-4 autoantibody production. The pool of ASCs in blood and CSF were significantly elevated in NMOSD. Both CD19− and CD19+ ASCs could be ablated by tocilizumab, but not rituximab treatment in NMOSD.DiscussionB cells are compartmentally fine tuned toward autoreactivity in NMOSD and become hyperreactive to type I interferon. Inhibition of type I interferon pathway may provide a new therapeutic avenue for NMOSD.

Microglial Calhm2 regulates neuroinflammation and contributes to Alzheimer’s disease pathology

Alzheimer’s disease (AD) is the most common neurodegenerative disease in the world. Neuronal calcium dysfunction and microglial-mediated neuroinflammation are closely associated with the development of AD. However, it remains unknown whether calcium dysfunction contributes to microglial activation and, in turn, AD pathology in vivo. In this study, we demonstrated that the expression of calcium homeostasis modulator family protein 2 (Calhm2) is increased in an AD mouse model. In 5×FAD mice carrying five familial AD gene mutations, both conventional knockout of Calhm2 and conditional microglial knockout of Calhm2 significantly reduced amyloid β deposition, neuroinflammation, and cognitive impairments. Mechanistically, knockout of Calhm2 inhibited microglial proinflammatory activity but increased phagocytic activity, leading to restoration of the balance between inflammation and phagocytosis. In addition, knockout of Calhm2 reduced acute LPS-induced neuroinflammation. These results highlight an important role for Calhm2 in microglial activation and provide a potential therapeutic target for diseases related to microglia-mediated neuroinflammation.

AB0043 EFFECT OF ANTI-INFLAMMATORY MEDICATION ON INTERACTION OF SYNOVIAL FIBROBLASTS WITH MACROPHAGES IN RHEUMATOID ARTHRITIS

Background:One of the key mechanisms in the pathogenesis of rheumatoid arthritis (RA) is the interaction of macrophages and synovial fibroblasts during joint inflammation. Increased synergistic proinflammatory activity of both cell types leads to the release of high levels of proinflammatory cytokines, especially of interleukin-6 (IL-6), and of matrix degrading enzymes. If this mechanism is uncontrolled, progressive destruction of articular cartilage and bone will take place.In active disease, immediate anti-inflammatory treatment with glucocorticoids is usually replaced by disease-modifying anti-rheumatic drugs (DMARDS), especially by methotrexate (MTX) and biologics such as TNF-α- or IL-6-inhibitors. This led to great improvements in prognosis and outcome for RA patients. However, about 40% of patients experience no remission or suffer from side effects of medication. To optimize established substances and to develop new treatment strategies, it is necessary to understand the mechanisms underlying the limited therapeutic effects.Objectives:Evaluation of the effect of prednisolone, MTX, adalimumab, tocilizumab on IL-6 secretion by RA synovial fibroblasts (RASF) and macrophages.Methods:RA synovium was used for RASF isolation. Peripheral blood mononuclear cells (PBMCs) were isolated from blood of healthy donors and RA patients by using Ficoll© medium followed by density gradient centrifugation. Mononuclear cells were seeded on six well plates (6x10^6/well) and incubated for one week. Then they were stimulated with Interferon-у (20 ng/ml) and LPS (50 ng/ml) for 48h to initiate differentiation into proinflammatory M1 macrophages. The M1 macrophages were co-cultured with RASF (100.000/well) and different treatments added (prednisolone: 10, 25, 50, 75, 100 nM, 1 µM; adalimumab: 100, 500 µg/ml; tocilizumab: 1, 5 µg/ml; MTX: 0,5, 1, 5, 10, 100 nM, 1µM). After 24h culture supernatants were collected and IL-6- and TNFα-ELISAs were performed.Results:IL-6 concentrations of untreated controls were comparable, regardless whether M1 macrophages from healthy donors or RA-patients were used for co-culture. Prednisolone reduced co-culture-induced IL-6 up to 56% (p<0.001) in co-culture of RASF and M1 macrophages of healthy donors and up to 60% (p<0.001) in co-culture of RASF and RA M1 macrophages. Adalimumab reduced IL-6 up to 28% (p<0.05) in M1 of healthy donors and up to 45% (p<0.01) in RA M1 macrophage co-cultures. A minor reduction by 10-20% of IL-6 was observed with tocilizumab and no significant effect could be achieved after treatment with MTX.Conclusion:Prednisolone and adalimumab clearly decrease but do not eliminate proinflammatory synergistic activity of RASF and M1 macrophages. These results confirm the clinical observation, that there is a large number of RA-patients that independent of anti-inflammatory treatment still suffer from low-level joint inflammation.The synergistic proinflammatory activity of M1 macrophages and RASF seems to be a complex and multifactorial mechanism that is difficult to eliminate by a single treatment substance. Since it is one of the key mechanisms in RA pathogenesis, there is a critical need to investigate how therapy effects could be optimized. This study confirmed RASFs as one of the leading effector cells of increased synergistic proinflammatory activity, thus underlining their promising role as a treatment target in rheumatoid arthritis.Disclosure of Interests:None declared