CLEC5A and TLR2 are critical in SARS-CoV-2-induced NET formation and lung inflammation

Coronavirus-induced disease 19 (COVID-19) infects more than three hundred and sixty million patients worldwide, and people with severe symptoms frequently die of acute respiratory distress syndrome (ARDS). Autopsy demonstrates the presence of thrombosis and microangiopathy in the small vessels and capillaries. Recent studies indicated that excessive neutrophil extracellular traps (NETs) contributed to immunothrombosis, thereby leading to extensive intravascular coagulopathy and multiple organ dysfunction. Thus, understanding the mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced NET formation would be helpful to reduce thrombosis and prevent ARDS. It has been shown that sera from individuals with COVID-19 triggered NET release in vitro, and spleen tyrosine kinase (Syk) inhibitor R406 inhibited NETosis caused by COVID-19 plasma. However, the serum components responsible for NET formation are still unknown. In this study, we found that virus-free extracellular vesicles (EVs) from COVID-19 patients (COVID-19 EVs) induced robust NET formation via Syk-coupled C-type lectin member 5A (CLEC5A). Blockade of CLEC5A inhibited COVID-19 EVs-induced NETosis, and simultaneous blockade of CLEC5A and TLR2 further suppressed SARS-CoV-2-induced NETosis in vitro. Moreover, thromboinflammation and lung fibrosis were attenuated dramatically in clec5a-/-/tlr2-/- mice. These results suggest that COVID-19 EVs play critical roles in SARS-CoV-2-induced immunothrombosis, and blockade of CLEC5A and TLR2 is a promising strategy to inhibit SARS-CoV-2-induced intravascular coagulopathy and reduce the risk of ARDS in COVID-19 patients.

Long non-coding RNA NEAT1 modulates hypoxia/reoxygenation-induced cardiomyocyte injury via targeting microRNA-520a.

In the present study, a hypoxia/reoxygenation (H/R) model of cardiomyocytes was established to investigate the effects of long non-coding RNA (LncRNA) Nuclear Enriched Abundant Transcript 1 (NEAT1) and microRNA (miR)-520a on H/R-induced cardiomyocyte apoptosis. Flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling staining were used to evaluate cell apoptosis. Luciferase activity assay was used to investigate whether miR-520a targets NEAT1. Results revealed that NEAT1 was significantly upregulated and miR-520a was downregulated in the ischemia/reperfusion myocardium and the cardiomyocytes that received H/R treatment. Further study demonstrated that knockdown of NEAT1 and overexpression of miR-520a serves a protective role against H/R-induced cardiomyocyte apoptosis. miR-520a directly targets NEAT1 and its expression level is negatively correlated with that of NEAT1. The findings suggested that NEAT1 and miR-520a may protect cardiomyocytes from apoptosis through regulating apoptotic proteins B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein, and altering cleaved caspase3 expression levels.

Localization of Vibrio vulnificus infection in dendritic cells and its effects on the cytoskeleton.

BACKGROUND: Vibrio vulnificus (Vv) is an estuarine bacterium that can cause primary septicemia as well as serious wound infections. However, little is known about the mechanisms by which Vv infects dendritic cells (DCs) and its effects on cytoskeleton. In this study, we aimed to investigate the invasion, internalization, and the organelles damage of the cultured dendritic cells (a DC 2.4 strain) during Vv infection. METHODS: The study model was the cultured DCs infected by a Vv 1.758 strain. Electron microscopy was used to observe the localization of bacteria at the different time points of infection, cell morphology, and the process of organelles changes. The cytoskeleton structure including the microfilaments and the microtubules rearrangement was examined under a fluorescence microscope. RESULTS: The Vv were pinocytosised into the DC cells through double-sides, and localized at 1 - 2 mm of the inner side membrane. It took 1.3, 1.9, and 3.4 hours to reach the infection ratio of 25%, 50%, and 75%, respectively. Using electron microscopy, the DCs had been observed to have developed chromatin aggregation within 4.0 hours, and significant cytoskeleton structure disruption was noted within 6.0 hours. CONCLUSION: The high lethality of Vv infection may be associated with the direct disruption of the DCs cytoskeleton structure.

[Association of activated CD4+ cell and HBeAg with liver damage and cirrhosis in patients with chronic hepatitis B].

OBJECTIVE: To study the association of the CD4+ cell counts and HBeAg with liver damage and cirrhosis in patients with chronic hepatitis B (CHB). METHOD: To measure the lymphocytes both CD4 and HLA-DR positive of 58 patients with CHB and 18 patients with HBsAg negative but HBcAb positive. The Platelets (PLT) were counted and the ALT and AST were measured, meanwhile, AST/PLT values were calculated. RESULTS: The CD4+ cell counts of the three types patients were lower than those of healthy controls significantly,and those of HBcAb positive patients without CHB were higher than those of HBeAg negative patients with CHB which were higher than those of HBeAg positive patients significantly (P < 0.05). And the ALT, AST and AST/PLT levels of both HBeAg negative and positive patients were much higher than the three indicators of both HBcAb positive patients without CHB and healthy controls significantly (P < 0.02), meanwhile, the three indicators of HBeAg negative patients were much lower than those of HBeAg positive ones. In addition, the CD4+ cell counts of the two types patients were negatively correlative with the three indicators(P < 0.05). CONCLUSION: Decreased CD4+ cell count can be used as the indicator to predicate the progress of liver damage in patients with CHB, and it is also useful for HBeAg to evaluate the development degree of liver damage and fibrosis in CHB patients.

[The clinical significance of interferon-gamma and its receptor in patients with schistosomiasis].

OBJECTIVE: To study the expression of interferon-gamma (IFN-gamma) and type I interferon-y receptor (IFN-gammaRI) in the chronic and advanced patients of schistosomiasis japonica, and to discuss its clinical relationship with hepatic fibrosis. Methods IFN-yR1 in the peripheral blood lymphocytes was detected by ELISA, and the hyaluronic acid (HA) , collagen type IV (C-IV), procollagen type III (PC III), laminin (LN) were detected by radio-immunoassay (RIA) in schistosomiasis patients. The level of IFN-y, IFN-gammaR1 and serum markers of hepatic fibrosis were observed, and the relationship with each other was analyzed by statistical method. RESULTS: There was no difference in the expression of IFN-gamma and IFN-gammaR1 between the patients with chronic schistosomiasis and the normal group (P > 0. 05 ), the IFN-gammaR1 in advanced cases without splenectomy was low (P <0. 05) , but IFN-y was high (P < 0. 01). The two indicators in the advanced schistosomiasis patients with splenectomy returned to normal. There was no corresponding relationship between the two indicators and HA, C-IV, PC III, LN with a r value of 0. 19, 0.20, 0. 14, and 0.21 respectively. CONCLUSION: There is a corresponding relationship between IFN-gamma and IFN-gammaR1; the expression of IFN-gammaR1 is related to the course of schistosomiasis, and the relationship with hepatic fibrosis needs further study.

[The value of reticulated platelet counts in diagnosing thrombocytopenic disorders].

OBJECTIVE: To evaluate the significance of reticulated platelets (RPs) in the diagnosis of thrombocytopenic disorders and the relationship between RPs and the proliferative degree of megakaryocyte (MK) in bone marrow. METHODS: With thiazole orange as a fluorescent dye, RPs were measured by analyzing the RNA content in platelets with flow cytometry and the percent and absolute counts of RPs were calculated. RESULTS: (1) The percent and absolute counts of the RPs in a normal group were (8.4 +/- 2.5)% and (16.8 +/- 6.8) x 10(9)/L respectively. (2) As compared with the normal group, the patients with idiopathic thrombocytopenic purpura (ITP) and hypersplenism had a significantly high percent and low absolute counts of RPs (P < 0.01). In patients with aplastic anemia, both the percent and absolute counts of RPs were at low levels (P < 0.05 and P < 0.01, respectively). There was no difference of RP percentage between the patients with acute leukemia or myelodysplastic syndromes and normal controls, but the absolute counts of RPs in the former was significantly lower than that in the latter. There was no difference between the percent and absolute counts of RPs among ITP patients with different proliferative degree of MK in bone marrow. (3) In all the diseases mentioned above, it was shown that RP percentage returned to normal in the effective cases after treatment, but no such change was found in the ineffective cases. CONCLUSIONS: Reticulated platelet counts contribute to the aetiology determination of the thrombocytopenia. It is also a valuable diagnostic method and a monitoring marker. There is no relationship between reticulated platelet counts and the counts of MK proliferation in bone marrow.