Neutrophilia and NETopathy as Key Pathologic Drivers of Progressive Lung Impairment in Patients With COVID-19

There is an urgent need for new therapeutic strategies to contain the spread of the novel coronavirus disease 2019 (COVID-19) and to curtail its most severe complications. Severely ill patients experience pathologic manifestations of acute respiratory distress syndrome (ARDS), and clinical reports demonstrate striking neutrophilia, elevated levels of multiple cytokines, and an exaggerated inflammatory response in fatal COVID-19. Mechanical respirator devices are the most widely applied therapy for ARDS in COVID-19, yet mechanical ventilation achieves strikingly poor survival. Many patients, who recover, experience impaired cognition or physical disability. In this review, we argue the need to develop therapies aimed at inhibiting neutrophil recruitment, activation, degranulation, and neutrophil extracellular trap (NET) release. Moreover, we suggest that currently available pharmacologic approaches should be tested as treatments for ARDS in COVID-19. In our view, targeting host-mediated immunopathology holds promise to alleviate progressive pathologic complications of ARDS and reduce morbidities and mortalities in severely ill patients with COVID-19

Regeneration of Alveolar Type I and II Cells from Scgb1a1-Expressing Cells following Severe Pulmonary Damage Induced by Bleomycin and Influenza

The lung comprises an extensive surface of epithelia constantly exposed to environmental insults. Maintaining the integrity of the alveolar epithelia is critical for lung function and gaseous exchange. However, following severe pulmonary damage, what progenitor cells give rise to alveolar type I and II cells during the regeneration of alveolar epithelia has not been fully determined. In this study, we have investigated this issue by using transgenic mice in which Scgb1a1-expressing cells and their progeny can be genetically labeled with EGFP. We show that following severe alveolar damage induced either by bleomycin or by infection with influenza virus, the majority of the newly generated alveolar type II cells in the damaged parenchyma were labeled with EGFP. A large proportion of EGFP-expressing type I cells were also observed among the type II cells. These findings strongly suggest that Scgb1a1-expressing cells, most likely Clara cells, are a major cell type that gives rise to alveolar type I and II cells during the regeneration of alveolar epithelia in response to severe pulmonary damage in mice..Singapore. National Research FoundationSingapore–MIT Alliance for Research and Technology (Infectious Disease-IRG research programme

Neutrophils infected with highly virulent influenza H3N2 virus exhibit augmented early cell death and rapid induction of type I interferon signaling pathways

AbstractWe developed a model of influenza virus infection of neutrophils by inducing differentiation of the MPRO promyelocytic cell line. After 5days of differentiation, about 20–30% of mature neutrophils could be detected. Only a fraction of neutrophils were infected by highly virulent influenza (HVI) virus, but were unable to support active viral replication compared with MDCK cells. HVI infection of neutrophils augmented early and late apoptosis as indicated by annexin V and TUNEL assays. Comparison between the global transcriptomic responses of neutrophils to HVI and low virulent influenza (LVI) revealed that the IFN regulatory factor and IFN signaling pathways were the most significantly overrepresented pathways, with activation of related genes in HVI as early as 3h. Relatively consistent results were obtained by real-time RT-PCR of selected genes associated with the type I IFN pathway. Early after HVI infection, comparatively enhanced expression of apoptosis-related genes was also elicited

VAPC, an human endogenous inhibitor for hepatitis C virus (HCV) infection, is intrinsically unstructured but forms a "fuzzy complex" with HCV NS5B

10.1371/journal.pone.0040341PLoS ONE77

Streptococcus Pneumoniae Secretes Hydrogen Peroxide Leading to DNA Damage and Apoptosis in Lung Cells

Streptococcus pneumoniae is a leading cause of pneumonia and one of the most common causes of death globally. The impact of S. pneumoniae on host molecular processes that lead to detrimental pulmonary consequences is not fully understood. Here, we show that S. pneumoniae induces toxic DNA double-strand breaks (DSBs) in human alveolar epithelial cells, as indicated by ataxia telangiectasia mutated kinase (ATM)-dependent phosphorylation of histone H2AX and colocalization with p53-binding protein (53BP1). Furthermore, results show that DNA damage occurs in a bacterial contact-independent fashion and that Streptococcus pyruvate oxidase (SpxB), which enables synthesis of H[subscript 2]O[subscript 2], plays a critical role in inducing DSBs. The extent of DNA damage correlates with the extent of apoptosis, and DNA damage precedes apoptosis, which is consistent with the time required for execution of apoptosis. Furthermore, addition of catalase, which neutralizes H[subscript 2]O[subscript 2], greatly suppresses S. pneumoniae-induced DNA damage and apoptosis. Importantly, S. pneumoniae induces DSBs in the lungs of animals with acute pneumonia, and H[subscript 2]O[subscript 2] production by S. pneumoniae in vivo contributes to its genotoxicity and virulence. One of the major DSBs repair pathways is nonhomologous end joining for which Ku70/80 is essential for repair. We find that deficiency of Ku80 causes an increase in the levels of DSBs and apoptosis, underscoring the importance of DNA repair in preventing S. pneumoniae-induced genotoxicity. Taken together, this study shows that S. pneumoniae-induced damage to the host cell genome exacerbates its toxicity and pathogenesis, making DNA repair a potentially important susceptibility factor in people who suffer from pneumonia

A Co-culture Model of PBMC and Stem Cell Derived Human Nasal Epithelium Reveals Rapid Activation of NK and Innate T Cells Upon Influenza A Virus Infection of the Nasal Epithelium

Background: We established an in vitro co-culture model involving H3N2-infection of human nasal epithelium with peripheral blood mononuclear cells (PBMC) to investigate their cross-talk during early H3N2 infection. Methods: Nasal epithelium was differentiated from human nasal epithelial stem/progenitor cells and cultured wtih fresh human PBMC. PBMC and supernatants were harvested after 24 and 48 h of co-culture with H3N2-infected nasal epithelium. We used flow cytometry and Luminex to characterize PBMC subpopulations, their activation and secretion of cytokine and chemokines. Results: H3N2 infection of the nasal epithelium associated with significant increase in interferons (IFN-alpha, IFN-gamma, IL-29), pro-inflammatory cytokines (TNF-alpha, BDNF, IL-3) and viral-associated chemokines (IP-10, MCP-3, I-TAC, MIG), detectable already after 24 h. This translates into rapid activation of monocytes, NK-cells and innate T-cells (MAIT and gamma delta T cells), evident with CD38+ and/or CD69+ upregulation. Conclusions: This system may contribute to in vitro mechanistic immunological studies bridging systemic models and possibly enable the development of targeted immunomodulatory therapies.Peer reviewe

Impact of BMI and waist circumference on epigenome-wide DNA methylation and identification of epigenetic biomarkers in blood: an EWAS in multi-ethnic Asian individuals

Background The prevalence of obesity and its related chronic diseases have been increasing especially in Asian countries. Obesity-related genetic variants have been identified, but these explain little of the variation in BMI. Recent studies reported associations between DNA methylation and obesity, mostly in non-Asian populations. Methods We performed an epigenome-wide association study (EWAS) on general adiposity (body mass index, BMI) and abdominal adiposity (waist circumference, WC) in 409 multi-ethnic Asian individuals and replicated BMI and waist-associated DNA methylation CpGs identified in other populations. The cross-lagged panel model and Mendelian randomization were used to assess the temporal relationship between methylation and BMI. The temporal relationship between the identified CpGs and inflammation and metabolic markers was also examined. Results EWAS identified 116 DNA methylation CpGs independently associated with BMI and eight independently associated with WC at false discovery rate P-FDR < 0.05 in 409 Asian samples. We replicated 110 BMI-associated CpGs previously reported in Europeans and identified six novel BMI-associated CpGs and two novel WC-associated CpGs. We observed high consistency in association direction of effect compared to studies in other populations. Causal relationship analyses indicated that BMI was more likely to be the cause of DNA methylation alteration, rather than the consequence. The causal analyses using BMI-associated methylation risk score also suggested that higher levels of the inflammation marker IL-6 were likely the consequence of methylation change. Conclusion Our study provides evidence of an association between obesity and DNA methylation in multi-ethnic Asians and suggests that obesity can drive methylation change. The results also suggested possible causal influence that obesity-related methylation changes might have on inflammation and lipoprotein levels

Successful Genetic Transfection of the Colonic Protistan Parasite Blastocystis for Reliable Expression of Ectopic Genes

The microbial parasite Blastocystis colonizes the large intestines of numerous animal species and increasing evidence has linked Blastocystis infection to enteric diseases with signs and symptoms including abdominal pain, constipation, diarrhea, nausea, vomiting, and flatulence. It has also recently been reported to be an important member of the host intestinal microbiota. Despite significant advances in our understanding of Blastocystis cell biology and host-parasite interactions, a genetic modification tool is absent. In this study, we successfully established a robust gene delivery protocol for Blastocystis subtype 7 (ST7) and ectopic protein expression was further tested using a high sensitivity nano-luciferase (Nluc) reporter system, with promoter regions from several genes. Among them, a strong promoter encompassing a region upstream of the legumain 5? UTR was identified. Using this promoter combined with the legumain 3? UTR, which contains a conserved, precise polyadenylation signal, a robust transient transfection technique was established for the first time in Blastocystis. This system was validated by ectopic expression of proteins harbouring specific localization signals. The establishment of a robust, reproducible gene modification system for Blastocystis is a significant advance for Blastocystis research both in vitro and in vivo. This technique will spearhead further research to understand the parasite’s biology, its role in health and disease, along with novel ways to combat the parasite

Bench to Bed Evidences for Pharmacokinetic and Pharmacodynamic Interactions Involving Oseltamivir and Chinese Medicine

Oseltamivir (OA), an ethyl ester prodrug of oseltamivir carboxylate (OC), is clinically used as a potent and selective inhibitor of neuraminidase. Chinese medicines have been advocated to combine with conventional drug for avian influenza. The current study aims to investigate the potential pharmacokinetic and pharmacodynamic interactions of a Chinese medicine formula, namely, Yin Qiao San and Sang Ju Yin (CMF1), commonly used for anti-influenza in combination with OA in both rat and human, and to reveal the underlined mechanisms. It was found that although Cmax, AUC and urinary recovery of OC, as well as metabolic ratio (AUCOC/AUCOA), were significantly decreased in a dose-dependent manner following combination use of CMF1 and OA in rat studies (P<0.01), such coadministration in 14 healthy volunteers only resulted in a trend of minor decrease in the related parameters. Further mechanistic studies found that although CMF1 could reduce absorption and metabolism of OA, it appears to enhance viral inhibition of OA (P<0.01). In summary, although there was potential interaction between OA and CMF1 found in rat studies, its clinical impact was expected to be minimal. The coadministration of OA and CMF1 at the clinical recommended dosages is, therefore, considered to be safe

A connection between star formation activity and cosmic rays in the starburst galaxy M 82

Although Galactic cosmic rays (protons and nuclei) are widely believed to be dominantly accelerated by the winds and supernovae of massive stars, definitive evidence of this origin remains elusive nearly a century after their discovery [1]. The active regions of starburst galaxies have exceptionally high rates of star formation, and their large size, more than 50 times the diameter of similar Galactic regions, uniquely enables reliable calorimetric measurements of their potentially high cosmic-ray density [2]. The cosmic rays produced in the formation, life, and death of their massive stars are expected to eventually produce diffuse gamma-ray emission via their interactions with interstellar gas and radiation. M 82, the prototype small starburst galaxy, is predicted to be the brightest starburst galaxy in gamma rays [3, 4]. Here we report the detection of >700 GeV gamma rays from M 82. From these data we determine a cosmic-ray density of 250 eV cm-3 in the starburst core of M 82, or about 500 times the average Galactic density. This result strongly supports that cosmic-ray acceleration is tied to star formation activity, and that supernovae and massive-star winds are the dominant accelerators.Comment: 18 pages, 4 figures; published in Nature; Version is prior to Nature's in-house style editing (differences are minimal