IL-33 regulates age-dependency of long-term immune dysfunction induced by sepsis

Sepsis survival in adults is commonly followed by immunosuppression and increased susceptibility to secondary infections. However, the long-term immune consequences of pediatric sepsis are unknown. Here, we compared the frequency of Tregs, the activation of the IL-33/ILC2s axis in M2 macrophages, and the DNA methylation of epithelial lung cells from post-septic infant and adult mice. In contrast to adults, infant mice were resistant to secondary infection and did not show impairment in tumour controls upon melanoma challenge. Mechanistically, increased IL-33 levels, Tregs expansion, and activation of ILC2s and M2-macrophages were observed in post-septic adults but not infant mice. Impaired IL-33 production in post-septic infant mice was associated with increased DNA-methylation on lung epithelial cells. Notably, IL-33 treatment boosted the expansion of Tregs and induced immunosuppression in infant mice. Clinically, adults but not pediatric post-septic patients exhibited higher counts of Tregs and sera IL-33 levels. Hence, we describe a crucial and age-dependent role for IL-33 in post-sepsis immunosuppression.

Ginkgolic Acid Inhibits Coronavirus Strain 229E Infection of Human Epithelial Lung Cells

Since December 2019, the COVID-19 pandemic has affected more than 200 million individuals around the globe and caused millions of deaths. Although there are now multiple vaccines for SARS-CoV-2, their efficacy may be limited by current and future viral mutations. Therefore, effective antiviral compounds are an essential component to win the battle against the family of coronaviruses. Ginkgolic Acid (GA) is a pan-antiviral molecule with proven effective in vitro and in vivo activity. We previously demonstrated that GA inhibits Herpes Simplex Virus 1 (HSV-1) by disrupting viral structure, blocking fusion, and inhibiting viral protein synthesis. Additionally, we reported that GA displays broad-spectrum fusion inhibition encompassing all three classes of fusion proteins, including those of HIV, Ebola, influenza A, and Epstein Barr virus. Here, we report that GA exhibited potent antiviral activity against Human Coronavirus strain 229E (HCoV-229E) infection of human epithelial lung cells (MRC-5). GA significantly reduced progeny virus production, expression of viral proteins, and cytopathic effects (CPE). Furthermore, GA significantly inhibited HCoV-229E even when added post-infection. In light of our findings and the similarities of this family of viruses, GA holds promising potential as an effective antiviral treatment for SARS-CoV-2.

Pathological diagnosis of thoracic endometriosis

A 26-year-old woman, who underwent abdominal surgery because of pelvic endometriosis, suffered from upper abdominal pain, fever and dyspnoea 2 days postoperatively. Paralytic ileus and right-sided pneumothorax were revealed. Treatment with a chest drain was not successful and, thus, a video-assisted thoracoscopic surgery was performed, revealing endometriosis-like lesions. Basic histopathology did not confirm the visual diagnosis, but additional immunohistochemical staining for oestrogen and progesterone receptors showed positive reaction in epithelial lung cells, thus proved the diagnosis thoracic endometriosis. A resection of the apex of the right upper lobe and pleurodesis by talc poudrage was performed after which a mesh graft was applied on the diaphragm. After 5 years of follow-up, no recurrent pneumothorax occurred.

A suitable murine model for studying respiratory coronavirus infection and therapeutic countermeasures in BSL-2 laboratories

Several animal models are being used to explore important features of COVID-19, nevertheless none of them recapitulates all aspects of the disease in humans. The continuous refinement and development of other options of in vivo models are opportune, especially ones that are carried out at BSL-2 (Biosafety Level 2) laboratories. In this study, we investigated the suitability of the intranasal infection with the murine betacoronavirus MHV-3 to recapitulate multiple aspects of the pathogenesis of COVID-19 in C57BL/6J mice. We demonstrate that MHV-3 replicated in lungs 1 day after inoculation and triggered respiratory inflammation and dysfunction. This MHV-model of infection was further applied to highlight the critical role of TNF in cytokine-mediated coronavirus pathogenesis. Blocking TNF signaling by pharmacological and genetic strategies greatly increased the survival time and reduces lung injury of MHV-3-infected mice. In vitro studies showed that TNF blockage decreased SARS-CoV-2 replication in human epithelial lung cells and resulted in the lower release of IL-6 and IL-8 cytokines beyond TNF itself. Taken together, our results demonstrate that this model of MHV infection in mice is a useful BSL-2 screening platform for evaluating pathogenesis for human coronaviruses infections, such as COVID-19.

Immortalisation of primary human alveolar epithelial lung cells using a non-viral vector to study respiratory bioreactivity in vitro

To overcome the scarcity of primary human alveolar epithelial cells for lung research, and the limitations of current cell lines to recapitulate the phenotype, functional and molecular characteristics of the healthy human alveolar epithelium, we have developed a new method to immortalise primary human alveolar epithelial lung cells using a non-viral vector to transfect the telomerase catalytic subunit (hTERT) and the simian virus 40 large-tumour antigen (SV40). Twelve strains of immortalised cells (ICs) were generated and characterised using molecular, immunochemical and morphological techniques. Cell proliferation and sensitivity to polystyrene nanoparticles (PS) were evaluated. ICs expressed caveolin-1, podoplanin and receptor for advanced glycation end-products (RAGE), and most cells were negative for alkaline phosphatase staining, indicating characteristics of AT1-like cells. However, most strains also contained some cells that expressed pro-surfactant protein C, classically described to be expressed only by AT2 cells. Thus, the ICs mimic the cellular heterogeneity in the human alveolar epithelium. These ICs can be passaged, replicate rapidly and remain confluent beyond 15 days. ICs showed differential sensitivity to positive and negatively charged PS nanoparticles, illustrating their potential value as an in vitro model to study respiratory bioreactivity. These novel ICs offer a unique resource to study human alveolar epithelial biology.

FDA approved calcium channel blockers inhibit SARS-CoV-2 infectivity in epithelial lung cells

COVID-19 has infected more than 41 million people worldwide with over 1.1 million deaths and is caused by the severe acute respiratory syndrome coronavirus (CoV) 2 (SARS-CoV-2)(1). Currently there are no protective vaccinations available and the only antiviral therapy in active use in patients is remdesivir, which provides only limited benefit(2, 3). Hence, an urgent need for antiviral therapies against SARS-CoV-2 exists. SARS-CoV requires Ca2+ ions for host cell entry and based on the similarity between SARS-CoV and SARS-CoV-2 it is highly likely that the same requirements exist for both viruses(4, 5). Here, we tested whether FDA-approved calcium channel blocker (CCB) drugs can inhibit SARS-CoV-2 infection in cell culture. All the CCBs showed varying degrees of inhibition, with amlodipine and nifedipine strongly limiting SARS-CoV-2 entry and infection in epithelial lung cells at concentrations where cell toxicity was minimal. Further studies with pseudo-typed particles carrying the SARS CoV 2 Spike protein suggest that viral inhibition occurs at the level of viral host cell entry. Overall, our data suggest that CCBs have a high potential to treat SARS-CoV-2 infections and their current FDA approval would allow for a fast repurposing of these drugs.SignificanceCovid-19 infections are still increasing around the globe and a number of countries are currently facing a second wave of infection resulting in re-instated lockdowns and dramatic consequences for the public health systems. Vaccine developments are in progress but as of now there are no efficacious drugs on the market to fight the pandemic. Here, we present the first case of a FDA-approved class of drugs that inhibit SARS CoV 2 growth in vitro. Their FDA approval may reduce the time for repurposing, allowing bypass of time-and cost-intensive animal models and to test them directly in clinical trials for human application for treatment of Covid-19.

Engineering a dynamic model of the alveolar interface for the study of aerosol deposition

Nano and micro particles are widely used in industrial, household and medicinal applications. To understand the interaction between particles and epithelial cells, we developed a dynamic model of the alveolar interface. This system, named DALI (Dynamic Model for the ALveolar Interface), is a modular bioreactor composed of two chambers divided by a porous membrane where epithelial lung cells are seeded. The membrane is the support of the alveolar barrier that separates the two compartments of the alveolus: the air and blood side. The system integrates the following elements: i) Air/Liquid interface, thanks to the two chambers divided by the membrane: ii) Cell culture media flow, thanks to the presence of a peristaltic pump; iii) Lung breathing motion, thanks to an airflow that allows the stretching of the membrane; iv) Aerosol deposition system, to study the effects of drug efficacy or particle toxicity on the epithelial layer; v) Quartz Crystal Microbalance, to quantify the amount of aerosolized particles.

Interference with Pseudomonas aeruginosa Quorum Sensing and Virulence by the Mycobacterial Pseudomonas Quinolone Signal Dioxygenase AqdC in Combination with the N-Acylhomoserine Lactone Lactonase QsdA

ABSTRACT The nosocomial pathogen Pseudomonas aeruginosa regulates its virulence via a complex quorum sensing network, which, besides N-acylhomoserine lactones, includes the alkylquinolone signal molecules 2-heptyl-3-hydroxy-4(1H)-quinolone (Pseudomonas quinolone signal [PQS]) and 2-heptyl-4(1H)-quinolone (HHQ). Mycobacteroides abscessus subsp. abscessus, an emerging pathogen, is capable of degrading the PQS and also HHQ. Here, we show that although M. abscessus subsp. abscessus reduced PQS levels in coculture with P. aeruginosa PAO1, this did not suffice for quenching the production of the virulence factors pyocyanin, pyoverdine, and rhamnolipids. However, the levels of these virulence factors were reduced in cocultures of P. aeruginosa PAO1 with recombinant M. abscessus subsp. massiliense overexpressing the PQS dioxygenase gene aqdC of M. abscessus subsp. abscessus, corroborating the potential of AqdC as a quorum quenching enzyme. When added extracellularly to P. aeruginosa cultures, AqdC quenched alkylquinolone and pyocyanin production but induced an increase in elastase levels. When supplementing P. aeruginosa cultures with QsdA, an enzyme from Rhodococcus erythropolis which inactivates N-acylhomoserine lactone signals, rhamnolipid and elastase levels were quenched, but HHQ and pyocyanin synthesis was promoted. Thus, single quorum quenching enzymes, targeting individual circuits within a complex quorum sensing network, may also elicit undesirable regulatory effects. Supernatants of P. aeruginosa cultures grown in the presence of AqdC, QsdA, or both enzymes were less cytotoxic to human epithelial lung cells than supernatants of untreated cultures. Furthermore, the combination of both aqdC and qsdA in P. aeruginosa resulted in a decline of Caenorhabditis elegans mortality under P. aeruginosa exposure.