Microglial MERTK eliminates phosphatidylserine‐displaying inhibitory post‐synapses

Glia contribute to synapse elimination through phagocytosis in the central nervous system. Despite the important roles of this process in development and neurological disorders, the identity and regulation of the "eat‐me" signal that initiates glia‐mediated phagocytosis of synapses has remained incompletely understood. Here, we generated conditional knockout mice with neuronal‐specific deletion of the flippase chaperone Cdc50a, to induce stable exposure of phosphatidylserine, a well‐known "eat‐me" signal for apoptotic cells, on the neuronal outer membrane. Surprisingly, acute Cdc50a deletion in mature neurons causes preferential phosphatidylserine exposure in neuronal somas and specific loss of inhibitory post‐synapses without effects on other synapses, resulting in abnormal excitability and seizures. Ablation of microglia or the deletion of microglial phagocytic receptor Mertk prevents the loss of inhibitory post‐synapses and the seizure phenotype, indicating that microglial phagocytosis is responsible for inhibitory post‐synapse elimination. Moreover, we found that phosphatidylserine is used for microglia‐mediated pruning of inhibitory post‐synapses in normal brains, suggesting that phosphatidylserine serves as a general "eat‐me" signal for inhibitory post‐synapse elimination.     

Stem Cell‐based therapies for COVID‐19‐related acute respiratory distress syndrome

As the number of confirmed cases and resulting death toll of the COVID‐19 pandemic continue to increase around the globe ‐ especially with the emergence of new mutations of the SARS‐CoV‐2 virus in addition to the known alpha, beta, gamma, delta and omicron variants ‐ tremendous efforts continue to be dedicated to the development of interventive therapeutics to mitigate infective symptoms or post‐viral sequelae in individuals for which vaccines are not accessible, viable or effective in the prevention of illness. Many of these investigations aim to target the associated acute respiratory distress syndrome, or ARDS, which induces damage to lung epithelia and other physiologic systems and is associated with progression in severe cases. Recently, stem cell‐based therapies have demonstrated preliminary efficacy against ARDS based on a number of preclinical and preliminary human safety studies, and based on promising outcomes are now being evaluated in phase II clinical trials for ARDS. A number of candidate stem cell therapies have been found to exhibit low immunogenicity, coupled with inherent tropism to injury sites. In recent studies, these have demonstrated the ability to modulate suppression of pro‐inflammatory cytokine signals such as those characterizing COVID‐19‐associated ARDS. Present translational studies are aiming to optimize the safety, efficacy and delivery to fully validate stem cell‐based strategies targeting COVID‐19 associated ARDS for viable clinical application.     

Kinetics and persistence of cellular and humoral immune responses to SARS‐CoV‐2 vaccine in healthcare workers with or without prior COVID‐19

SARS‐CoV‐2 vaccines are highly efficient against severe forms of the disease, hospitalization and death. Nevertheless, insufficient protection against several circulating viral variants might suggest waning immunity and the need for an additional vaccine dose. We conducted a longitudinal study on the kinetics and persistence of immune responses in healthcare workers vaccinated with two doses of BNT162b2 mRNA vaccine with or without prior SARS‐CoV‐2 infection. No new infections were diagnosed during follow‐up. At 6 months, post‐vaccination or post‐infection, despite a downward trend in the level of anti‐S IgG antibodies, the neutralizing activity does not decrease significantly, remaining higher than 75% (85.14% for subjects with natural infection, 88.82% for vaccinated after prior infection and 78.37% for vaccinated only). In a live‐virus neutralization assay, the highest neutralization titres were present at baseline and at 6 months follow‐up in persons vaccinated after prior infection. Anti‐S IgA levels showed a significant descending trend in vaccinated subjects (p < 0.05) after 14 weeks. Cellular immune responses are present even in vaccinated participants with declining antibody levels (index ratio 1.1–3) or low neutralizing activity (30%–40%) at 6 months, although with lower T‐cell stimulation index (p = 0.046) and IFN‐γ secretion (p = 0.0007) compared to those with preserved humoral responses.     

20‐HETE synthesis inhibition attenuates traumatic brain injury–induced mitochondrial dysfunction and neuronal apoptosis via the SIRT1/PGC‐1α pathway: A translational study

Objectives20‐hydroxyeicosatetraenoic acid (20‐HETE) is a metabolite of arachidonic acid catalysed by cytochrome P450 enzymes and plays an important role in cell death and proliferation. We hypothesized that 20‐HETE synthesis inhibition may have protective effects in traumatic brain injury (TBI) and investigated possible underlying molecular mechanisms.Materials and methodsNeurologic deficits, and lesion volume, reactive oxygen species (ROS) levels and cell death as assessed using immunofluorescence staining, transmission electron microscopy and Western blotting were used to determine post‐TBI effects of HET0016, an inhibitor of 20‐HETE synthesis, and their underlying mechanisms.ResultsThe level of 20‐HETE was found to be increased significantly after TBI in mice. 20‐HETE synthesis inhibition reduced neuronal apoptosis, ROS production and damage to mitochondrial structures after TBI. Mechanistically, HET0016 decreased the Drp1 level and increased the expression of Mfn1 and Mfn2 after TBI, indicating a reversal of the abnormal post‐TBI mitochondrial dynamics. HET0016 also promoted the restoration of SIRT1 and PGC‐1α in vivo, and a SIRT1 activator (SRT1720) reversed the downregulation of SIRT1 and PGC‐1α and the abnormal mitochondrial dynamics induced by 20‐HETE in vitro. Furthermore, plasma 20‐HETE levels were found to be higher in TBI patients with unfavourable neurological outcomes and were correlated with the GOS score.ConclusionsThe inhibition of 20‐HETE synthesis represents a novel strategy to mitigate TBI‐induced mitochondrial dysfunction and neuronal apoptosis by regulating the SIRT1/PGC‐1α pathway.     

An organoid‐derived bronchioalveolar model for SARS‐CoV‐2 infection of human alveolar type II‐like cells

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) causes coronavirus disease 2019 (COVID‐19), which may result in acute respiratory distress syndrome (ARDS), multiorgan failure, and death. The alveolar epithelium is a major target of the virus, but representative models to study virus host interactions in more detail are currently lacking. Here, we describe a human 2D air–liquid interface culture system which was characterized by confocal and electron microscopy and single‐cell mRNA expression analysis. In this model, alveolar cells, but also basal cells and rare neuroendocrine cells, are grown from 3D self‐renewing fetal lung bud tip organoids. These cultures were readily infected by SARS‐CoV‐2 with mainly surfactant protein C‐positive alveolar type II‐like cells being targeted. Consequently, significant viral titers were detected and mRNA expression analysis revealed induction of type I/III interferon response program. Treatment of these cultures with a low dose of interferon lambda 1 reduced viral replication. Hence, these cultures represent an experimental model for SARS‐CoV‐2 infection and can be applied for drug screens.     

DNA asymmetry promotes SUMO modification of the single‐stranded DNA‐binding protein RPA

Repair of DNA double‐stranded breaks by homologous recombination (HR) is dependent on DNA end resection and on post‐translational modification of repair factors. In budding yeast, single‐stranded DNA is coated by replication protein A (RPA) following DNA end resection, and DNA–RPA complexes are then SUMO‐modified by the E3 ligase Siz2 to promote repair. Here, we show using enzymatic assays that DNA duplexes containing 3'' single‐stranded DNA overhangs increase the rate of RPA SUMO modification by Siz2. The SAP domain of Siz2 binds DNA duplexes and makes a key contribution to this process as highlighted by models and a crystal structure of Siz2 and by assays performed using protein mutants. Enzymatic assays performed using DNA that can accommodate multiple RPA proteins suggest a model in which the SUMO‐RPA signal is amplified by successive rounds of Siz2‐dependent SUMO modification of RPA and dissociation of SUMO‐RPA at the junction between single‐ and double‐stranded DNA. Our results provide insights on how DNA architecture scaffolds a substrate and E3 ligase to promote SUMO modification in the context of DNA repair.     

Interleukin‐37 improves T‐cell‐mediated immunity and chimeric antigen receptor T‐cell therapy in aged backgrounds

Aging‐associated declines in innate and adaptive immune responses are well documented and pose a risk for the growing aging population, which is predicted to comprise greater than 40 percent of the world''s population by 2050. Efforts have been made to improve immunity in aged populations; however, safe and effective protocols to accomplish this goal have not been universally established. Aging‐associated chronic inflammation is postulated to compromise immunity in aged mice and humans. Interleukin‐37 (IL‐37) is a potent anti‐inflammatory cytokine, and we present data demonstrating that IL‐37 gene expression levels in human monocytes significantly decline with age. Furthermore, we demonstrate that transgenic expression of interleukin‐37 (IL‐37) in aged mice reduces or prevents aging‐associated chronic inflammation, splenomegaly, and accumulation of myeloid cells (macrophages and dendritic cells) in the bone marrow and spleen. Additionally, we show that IL‐37 expression decreases the surface expression of programmed cell death protein 1 (PD‐1) and augments cytokine production from aged T‐cells. Improved T‐cell function coincided with a youthful restoration of Pdcd1, Lat, and Stat4 gene expression levels in CD4⁺ T‐cells and Lat in CD8⁺ T‐cells when aged mice were treated with recombinant IL‐37 (rIL‐37) but not control immunoglobin (Control Ig). Importantly, IL‐37‐mediated rejuvenation of aged endogenous T‐cells was also observed in aged chimeric antigen receptor (CAR) T‐cells, where improved function significantly extended the survival of mice transplanted with leukemia cells. Collectively, these data demonstrate the potency of IL‐37 in boosting the function of aged T‐cells and highlight its therapeutic potential to overcome aging‐associated immunosenescence.     

On‐site genetic analysis for species identification using lab‐on‐a‐chip

This paper presents a microfluidic device capable of performing genetic analysis on dung samples to identify White Rhinoceros (Ceratotherium simum). The development of a microfluidic device, which can be used in the field, offers a portable and cost‐effective solution for DNA analysis and species identification to aid conservation efforts. Optimization of the DNA extraction processes produced equivalent yields compared to conventional kit‐based methods within just 5 minutes. The use of a color‐changing loop‐mediated isothermal amplification reaction for simultaneous detection of the cytochrome B sequence of C. simum enabled positive results to be obtained within as little as 30 minutes. Field testing was performed at Knowsley Safari to demonstrate real‐world applicability of the microfluidic device for testing of biological samples.     

New insights into the central sympathetic hyperactivity post‐myocardial infarction: Roles of METTL3‐mediated m6A methylation

Ventricular arrhythmias (VAs) triggers by sympathetic nerve hyperactivity contribute to sudden cardiac death in myocardial infarction (MI) patients. Microglia‐mediated inflammation in the paraventricular nucleus (PVN) is involved in sympathetic hyperactivity after MI. N6‐methyladenosine (m⁶A), the most prevalent mRNA and epigenetic modification, is critical for mediating cell inflammation. We aimed to explore whether METTL3‐mediated m⁶A modification is involved in microglia‐mediated sympathetic hyperactivity after MI in the PVN. MI model was established by left coronary artery ligation. METTL3‐mediated m⁶A modification was markedly increased in the PVN at 3 days after MI, and METTL3 was primarily located in microglia by immunofluorescence. RNA‐seq, MeRIP‐seq, MeRIP‐qPCR, immunohistochemistry, ELISA, heart rate variability measurements, renal sympathetic nerve activity recording and programmed electrical stimulation confirmed that the elevated toll‐like receptor 4 (TLR4) expression by m⁶A modification on TLR4 mRNA 3''‐UTR region combined with activated NF‐κB signalling led to the overwhelming production of pro‐inflammatory cytokines IL‐1β and TNF‐α in the PVN, thus inducing the sympathetic hyperactivity and increasing the incidence of VAs post‐MI. Targeting METTL3 attenuated the inflammatory response and sympathetic hyperactivity and reduced the incidence of VAs post‐MI.     

TMPRSS2 expression dictates the entry route used by SARS‐CoV‐2 to infect host cells

SARS‐CoV‐2 is a newly emerged coronavirus that caused the global COVID‐19 outbreak in early 2020. COVID‐19 is primarily associated with lung injury, but many other clinical symptoms such as loss of smell and taste demonstrated broad tissue tropism of the virus. Early SARS‐CoV‐2–host cell interactions and entry mechanisms remain poorly understood. Investigating SARS‐CoV‐2 infection in tissue culture, we found that the protease TMPRSS2 determines the entry pathway used by the virus. In the presence of TMPRSS2, the proteolytic process of SARS‐CoV‐2 was completed at the plasma membrane, and the virus rapidly entered the cells within 10 min in a pH‐independent manner. When target cells lacked TMPRSS2 expression, the virus was endocytosed and sorted into endolysosomes, from which SARS‐CoV‐2 entered the cytosol via acid‐activated cathepsin L protease 40–60 min post‐infection. Overexpression of TMPRSS2 in non‐TMPRSS2 expressing cells abolished the dependence of infection on the cathepsin L pathway and restored sensitivity to the TMPRSS2 inhibitors. Together, our results indicate that SARS‐CoV‐2 infects cells through distinct, mutually exclusive entry routes and highlight the importance of TMPRSS2 for SARS‐CoV‐2 sorting into either pathway.     

17β‐Oestradiol facilitates M2 macrophage skewing and ameliorates arrhythmias in ovariectomized female infarcted rats

Epidemiological studies have suggested a lower incidence of arrhythmia‐induced sudden cardiac death in women than in men. 17β‐oestradiol (E2) has been reported to have a post‐myocardial infarction antiarrhythmic effect, although the mechanisms have yet to be elucidated. We investigated whether E2‐mediated antioxidation regulates macrophage polarization and affects cardiac sympathetic reinnervation in rats after MI. Ovariectomized Wistar rats were randomly assigned to placebo pellets, E2 treatment, or E2 treatment +3‐morpholinosydnonimine (a peroxynitrite generator) and followed for 4 weeks. The infarct sizes were similar among the infarcted groups. At Day 3 after infarction, post‐infarction was associated with increased superoxide levels, which were inhibited by administering E2. E2 significantly increased myocardial IL‐10 levels and the percentage of regulatory M2 macrophages compared with the ovariectomized infarcted alone group as assessed by immunohistochemical staining, Western blot and RT‐PCR. Nerve growth factor colocalized with both M1 and M2 macrophages at the magnitude significantly higher in M1 compared with M2. At Day 28 after infarction, E2 was associated with attenuated myocardial norepinephrine levels and sympathetic hyperinnervation. These effects of E2 were functionally translated in inhibiting fatal arrhythmias. The beneficial effect of E2 on macrophage polarization and sympathetic hyperinnervation was abolished by 3‐morpholinosydnonimine. Our results indicated that E2 polarized macrophages into the M2 phenotype by inhibiting the superoxide pathway, leading to attenuated nerve growth factor‐induced sympathetic hyperinnervation after myocardial infarction.     

T‐cell infiltration,contribution and regulation in the central nervous system post‐traumatic injury

T cells participate in the repair process and immune response in the CNS post‐traumatic injury and play both a beneficial and harmful role. Together with nerve cells and other immune cells, they form a microenvironment in the CNS post‐traumatic injury. The repair of traumatic CNS injury is a long‐term process. T cells contribute to the repair of the injury site to influence the recovery. Recently, with the advance of new techniques, such as mass spectrometry‐based flow cytometry, modern live‐cell imaging, etc, research focusing on T cells is becoming one of the valuable directions for the future therapy of traumatic CNS injury. In this review, we summarized the infiltration, contribution and regulation of T cells in post‐traumatic injury, discussed the clinical significance and predicted the future research direction.     

MicroRNA‐411‐3p inhibits bleomycin‐induced skin fibrosis by regulating transforming growth factor‐β/Smad ubiquitin regulatory factor‐2 signalling

Skin fibrosis, which is characterized by fibroblast proliferation and increased extracellular matrix, has no effective treatment. An increasing number of studies have shown that microRNAs (miRNAs/miRs) participate in the mechanism of skin fibrosis, such as in limited cutaneous systemic sclerosis and pathological scarring. The objective of the present study was to determine the role of miR‐411‐3p in bleomycin (BLM)‐induced skin fibrosis and skin fibroblast transformation. Using Western blot analysis and real‐time quantitative polymerase chain reaction assess the expression levels of miR‐411‐3p, collagen (COLI) and transforming growth factor (TGF)‐β/Smad ubiquitin regulatory factor (Smurf)‐2/Smad signalling factors both in vitro and in vivo with or without BLM. To explore the regulatory relationship between miR‐411‐3p and Smurf2, we used the luciferase reporter assay. Furthermore, miR‐411‐3p overexpression was identified in vitro and in vivo via transfection with Lipofectamine 2000 reagent and injection. Finally, we tested the dermal layer of the skin using haematoxylin and eosin and Van Gieson''s staining. We found that miR‐411‐3p expression was decreased in bleomycin (BLM)‐induced skin fibrosis and fibroblasts. However, BLM accelerated transforming growth factor (TGF)‐β signalling and collagen production. Overexpression of miR‐411‐3p inhibited the expression of collagen, F‐actin and the TGF‐β/Smad signalling pathway factors in BLM‐induced skin fibrosis and fibroblasts. In addition, miR‐411‐3p inhibited the target Smad ubiquitin regulatory factor (Smurf)‐2. Furthermore, Smurf2 was silenced, which attenuated the expression of collagen via suppression of the TGF‐β/Smad signalling pathway. We demonstrated that miR‐411‐3p exerts antifibrotic effects by inhibiting the TGF‐β/Smad signalling pathway via targeting of Smurf2 in skin fibrosis.     

Attenuation of SARS‐CoV‐2 replication and associated inflammation by concomitant targeting of viral and host cap 2'‐O‐ribose methyltransferases

The SARS‐CoV‐2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2''‐O‐ribose cap needed for viral immune escape. We find that the host cap 2''‐O‐ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS‐CoV‐2 replication. Using in silico target‐based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti‐SARS‐CoV‐2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co‐substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID‐19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection‐induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID‐19.