Disruption of pulmonary microvascular endothelial barrier by dysregulated claudin-8 and claudin-4: uncovered mechanisms in porcine reproductive and respiratory syndrome virus infection.

The pulmonary endothelium is a dynamic and metabolically active monolayer of endothelial cells. Dysfunction of the pulmonary endothelial barrier plays a crucial role in the acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), frequently observed in the context of viral pneumonia. Dysregulation of tight junction proteins can lead to the disruption of the endothelial barrier and subsequent leakage. Here, the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) served as an ideal model for studying ALI and ARDS. The alveolar lavage fluid of pigs infected with HP-PRRSV, and the supernatant of HP-PRRSV infected pulmonary alveolar macrophages were respectively collected to treat the pulmonary microvascular endothelial cells (PMVECs) in Transwell culture system to explore the mechanism of pulmonary microvascular endothelial barrier leakage caused by viral infection. Cytokine screening, addition and blocking experiments revealed that proinflammatory cytokines IL-1ß and TNF-α, secreted by HP-PRRSV-infected macrophages, disrupt the pulmonary microvascular endothelial barrier by downregulating claudin-8 and upregulating claudin-4 synergistically. Additionally, three transcription factors interleukin enhancer binding factor 2 (ILF2), general transcription factor III C subunit 2 (GTF3C2), and thyroid hormone receptor-associated protein 3 (THRAP3), were identified to accumulate in the nucleus of PMVECs, regulating the transcription of claudin-8 and claudin-4. Meanwhile, the upregulation of ssc-miR-185 was found to suppress claudin-8 expression via post-transcriptional inhibition. This study not only reveals the molecular mechanisms by which HP-PRRSV infection causes endothelial barrier leakage in acute lung injury, but also provides novel insights into the function and regulation of tight junctions in vascular homeostasis.

IL-10 Counteracts IFN-γ to Alleviate Acute Lung Injury in a Viral-Bacterial Superinfection Model.

Immune activation is essential for lung control of viral and bacterial infection, but an overwhelming inflammatory response often leads to the onset of acute respiratory distress syndrome. IL-10 plays a crucial role in regulating the balance between antimicrobial immunity and immunopathology. In the present study, we investigated the role of IL-10 in acute lung injury induced by influenza A virus and methicillin-resistant Staphylococcus aureus coinfection. This unique coinfection model resembles patients with acute pneumonia undergoing appropriate antibiotic therapies. Using global IL-10 and IL-10 receptor gene-deficient mice, as well as in vivo neutralizing antibodies, we show that IL-10 deficiency promotes IFN-γ-dominant cytokine responses and triggers acute animal death. Interestingly, this extreme susceptibility is fully preventable by IFN-γ neutralization during coinfection. Further studies using mice with Il10ra deletion in selective myeloid subsets reveal that IL-10 primarily acts on mononuclear phagocytes to prevent IFN-γ/TNF-α hyperproduction and acute mortality. Importantly, this antiinflammatory IL-10 signaling is independent of its inhibitory effect on antiviral and antibacterial defense. Collectively, our results demonstrate a key mechanism of IL-10 in preventing hypercytokinemia and acute respiratory distress syndrome pathogenesis by counteracting the IFN-γ response.

Pulmonary mesenchymal stem cells are engaged in distinct steps of host response to respiratory syncytial virus infection.

Lung-resident (LR) mesenchymal stem and stromal cells (MSCs) are key elements of the alveolar niche and fundamental regulators of homeostasis and regeneration. We interrogated their function during virus-induced lung injury using the highly prevalent respiratory syncytial virus (RSV) which causes severe outcomes in infants. We applied complementary approaches with primary pediatric LR-MSCs and a state-of-the-art model of human RSV infection in lamb. Remarkably, RSV-infection of pediatric LR-MSCs led to a robust activation, characterized by a strong antiviral and pro-inflammatory phenotype combined with mediators related to T cell function. In line with this, following in vivo infection, RSV invades and activates LR-MSCs, resulting in the expansion of the pulmonary MSC pool. Moreover, the global transcriptional response of LR-MSCs appears to follow RSV disease, switching from an early antiviral signature to repair mechanisms including differentiation, tissue remodeling, and angiogenesis. These findings demonstrate the involvement of LR-MSCs during virus-mediated acute lung injury and may have therapeutic implications.

Alcohol Increases Lung Angiotensin-Converting Enzyme 2 Expression and Exacerbates Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein Subunit 1-Induced Acute Lung Injury in K18-hACE2 Transgenic Mice.

During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, alcohol consumption increased markedly. Nearly one in four adults reported drinking more alcohol to cope with stress. Chronic alcohol abuse is now recognized as a factor complicating the course of acute respiratory distress syndrome and increasing mortality. To investigate the mechanisms behind this interaction, a combined acute respiratory distress syndrome and chronic alcohol abuse mouse model was developed by intratracheally instilling the subunit 1 (S1) of SARS-CoV-2 spike protein (S1SP) in K18-human angiotensin-converting enzyme 2 (ACE2) transgenic mice that express the human ACE2 receptor for SARS-CoV-2 and were kept on an ethanol diet. Seventy-two hours after S1SP instillation, mice on an ethanol diet showed a strong decrease in body weight, a dramatic increase in white blood cell content of bronchoalveolar lavage fluid, and an augmented cytokine storm, compared with S1SP-treated mice on a control diet. Histologic examination of lung tissue showed abnormal recruitment of immune cells in the alveolar space, abnormal parenchymal architecture, and worsening Ashcroft score in S1SP- and alcohol-treated animals. Along with the activation of proinflammatory biomarkers [NF-κB, STAT3, NLR family pyrin domain-containing protein 3 (NLRP3) inflammasome], lung tissue homogenates from mice on an alcohol diet showed overexpression of ACE2 compared with mice on a control diet. This model could be useful for the development of therapeutic approaches against alcohol-exacerbated coronavirus disease 2019.

Current and Past Infections of HBV Do Not Increase Mortality in Patients With COVID-19.

BACKGROUND AND AIMS: We compared risk of acute liver injury and mortality in patients with COVID-19 and current, past, and no HBV infection. APPROACH AND RESULTS: This was a territory-wide retrospective cohort study in Hong Kong. Patients with COVID-19 between January 23, 2020, and January 1, 2021, were identified. Patients with hepatitis C or no HBsAg results were excluded. The primary outcome was mortality. Acute liver injury was defined as alanine aminotransferase or aspartate aminotransferase ≥2 × upper limit of normal (ULN; i.e., 80 U/L), with total bilirubin ≥2 × ULN (i.e., 2.2 mg/dL) and/or international normalized ratio ≥1.7. Of 5,639 patients included, 353 (6.3%) and 359 (6.4%) had current and past HBV infection, respectively. Compared to patients without known HBV exposure, current HBV-infected patients were older and more likely to have cirrhosis. Past HBV-infected patients were the oldest, and more had diabetes and cardiovascular disease. At a median follow-up of 14 (9-20) days, 138 (2.4%) patients died; acute liver injury occurred in 58 (1.2%), 8 (2.3%), and 11 (3.1%) patients with no, current, and past HBV infection, respectively. Acute liver injury (adjusted HR [aHR], 2.45; 95% CI, 1.52-3.96; P < 0.001), but not current (aHR, 1.29; 95% CI, 0.61-2.70; P = 0.507) or past (aHR, 0.90; 95% CI, 0.56-1.46; P = 0.681) HBV infection, was associated with mortality. Use of corticosteroid, antifungal, ribavirin, or lopinavir-ritonavir (adjusted OR [aOR], 2.55-5.63), but not current (aOR, 1.93; 95% CI, 0.88-4.24; P = 0.102) or past (aOR, 1.25; 95% CI, 0.62-2.55; P = 0.533) HBV infection, was associated with acute liver injury. CONCLUSION: Current or past HBV infections were not associated with more liver injury and mortality in COVID-19.

Recombinant ACE2 protein protects against acute lung injury induced by SARS-CoV-2 spike RBD protein.

BACKGROUND: SARS-CoV-2 infection leads to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Both clinical data and animal experiments suggest that the renin-angiotensin system (RAS) is involved in the pathogenesis of SARS-CoV-2-induced ALI. Angiotensin-converting enzyme 2 (ACE2) is the functional receptor for SARS-CoV-2 and a crucial negative regulator of RAS. Recombinant ACE2 protein (rACE2) has been demonstrated to play protective role against SARS-CoV and avian influenza-induced ALI, and more relevant, rACE2 inhibits SARS-CoV-2 proliferation in vitro. However, whether rACE2 protects against SARS-CoV-2-induced ALI in animal models and the underlying mechanisms have yet to be elucidated. METHODS AND RESULTS: Here, we demonstrated that the SARS-CoV-2 spike receptor-binding domain (RBD) protein aggravated lipopolysaccharide (LPS)-induced ALI in mice. SARS-CoV-2 spike RBD protein directly binds and downregulated ACE2, leading to an elevation in angiotensin (Ang) II. AngII further increased the NOX1/2 through AT1R, subsequently causing oxidative stress and uncontrolled inflammation and eventually resulting in ALI/ARDS. Importantly, rACE2 remarkably reversed SARS-CoV-2 spike RBD protein-induced ALI by directly binding SARS-CoV-2 spike RBD protein, cleaving AngI or cleaving AngII. CONCLUSION: This study is the first to prove that rACE2 plays a protective role against SARS-CoV-2 spike RBD protein-aggravated LPS-induced ALI in an animal model and illustrate the mechanism by which the ACE2-AngII-AT1R-NOX1/2 axis might contribute to SARS-CoV-2-induced ALI.

Human convalescent plasma protects K18-hACE2 mice against severe respiratory disease.

SARS-CoV-2 is the causative agent of COVID-19 and human infections have resulted in a global health emergency. Small animal models that reproduce key elements of SARS-CoV-2 human infections are needed to rigorously screen candidate drugs to mitigate severe disease and prevent the spread of SARS-CoV-2. We and others have reported that transgenic mice expressing the human angiotensin-converting enzyme 2 (hACE2) viral receptor under the control of the Keratin 18 (K18) promoter develop severe and lethal respiratory disease subsequent to SARS-CoV-2 intranasal challenge. Here we report that some infected mice that survive challenge have residual pulmonary damages and persistent brain infection on day 28 post-infection despite the presence of anti-SARS-COV-2 neutralizing antibodies. Because of the hypersensitivity of K18-hACE2 mice to SARS-CoV-2 and the propensity of virus to infect the brain, we sought to determine if anti-infective biologics could protect against disease in this model system. We demonstrate that anti-SARS-CoV-2 human convalescent plasma protects K18-hACE2 against severe disease. All control mice succumbed to disease by day 7; however, all treated mice survived infection without observable signs of disease. In marked contrast to control mice, viral antigen and lesions were reduced or absent from lungs and absent in brains of antibody-treated mice. Our findings support the use of K18-hACE2 mice for protective efficacy studies of anti-SARS-CoV-2 medical countermeasures (MCMs). They also support the use of this system to study SARS-CoV-2 persistence and host recovery.

A brief review of interplay between vitamin D and angiotensin-converting enzyme 2: Implications for a potential treatment for COVID-19.

The novel coronavirus disease 2019 (COVID-19) is rapidly expanding and causing many deaths all over the world with the World Health Organization (WHO) declaring a pandemic in March 2020. Current therapeutic options are limited and there is no registered and/or definite treatment or vaccine for this disease or the causative infection, severe acute respiratory coronavirus 2 syndrome (SARS-CoV-2). Angiotensin-converting enzyme 2 (ACE2), a part of the renin-angiotensin system (RAS), serves as the major entry point into cells for SARS-CoV-2 which attaches to human ACE2, thereby reducing the expression of ACE2 and causing lung injury and pneumonia. Vitamin D, a fat-soluble-vitamin, is a negative endocrine RAS modulator and inhibits renin expression and generation. It can induce ACE2/Ang-(1-7)/MasR axis activity and inhibits renin and the ACE/Ang II/AT1R axis, thereby increasing expression and concentration of ACE2, MasR and Ang-(1-7) and having a potential protective role against acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Therefore, targeting the unbalanced RAS and ACE2 down-regulation with vitamin D in SARS-CoV-2 infection is a potential therapeutic approach to combat COVID-19 and induced ARDS.

A novel cell-based assay for dynamically detecting neutrophil extracellular traps-induced lung epithelial injuries.

Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS) are common lung disorders characterized by alveolar-capillary barrier disruption and dyspnea, which can cause substantial morbidity and mortality. Currently, a cluster of acute respiratory illnesses, known as novel coronavirus (2019-nCoV)-infected pneumonia (NCIP), which allegedly originally occurred in Wuhan, China, has increased rapidly worldwide. The critically ill patients with ARDS have high mortality in subjects with comorbidities. Previously, the excessive recruitment and activation of neutrophils (polymorphonuclear leukocytes [PMNs]), accompanied by neutrophil extracellular traps (NETs) formation were reported being implicated in the pathogenesis of ALI/ARDS. However, the direct visualization of lung epithelial injuries caused by NETs, and the qualitative and quantitative evaluations of this damage are still lacking. Additionally, those already reported methods are limited for their neglect of the pathological role exerted by NETs and focusing only on the morphological features of NETosis. Therefore, we established a cell-based assay for detecting NETs during lung epithelial cells-neutrophils co-culture using the xCELLigence system, a recognized real-time, dynamic, label-free, sensitive, and high-throughput apparatus. Our results demonstrated that lung epithelial injuries, reflected by declines in cell index (CI) values, could be induced by lipopolysaccharide (LPS)-activated PMNs, or NETs in a time and dose-dependent manner. NETs generation was verified to be the major contributor to the cytotoxicity of activated PMNs; protein components of NETs were the prevailing cytotoxic mediators. Moreover, this cell-based assay identified that PMNs from severe pneumonia patients had a high NETs formative potential. Additionally, acetylsalicylic acid (ASA) and acetaminophen (APAP) were discovered alleviating NETs formation. Thus, this study not only presents a new methodology for detecting the pathophysiologic role of NETs but also lays down a foundation for exploring therapeutic interventions in an effort to cure ALI/ARDS in the clinical setting of severe pneumonia, including the emerging of NCIP.

Study on Laboratory Indicators Related to Lung Injury of Corona Virus Disease 2019.

BACKGROUND: Since December 2019, a series of pneumonia cases caused by COVID-19 emerged in Wuhan, Hubei Province, China. People are generally susceptible to COVID-19 because people lack immunity to this new virus. With the spread of this epidemic disease from Wuhan, a national outbreak soon appeared, and now many countries have this disease. Unfortunately, no effective drug for COVID-19 treatment has been found so far. METHODS: We designed a retrospective study based on patients admitted to The Affiliated Infectious Hospital of Soochow University from January 22, 2020, to February 25, 2020, with diagnosed COVID-19. We analyzed correlations between RT-PCR negative time and laboratory indicators, then divided all cases into 2 groups according to oxygenation index, data of RT-PCR negative time and related laboratory indicators of the two groups were com-pared. RESULTS: We collected 84 confirmed patients whose RT-PCR had turned negative, including 23 patients with the lowest oxygenation index ≤ 300 mmHg and 61 patients had > 300 mmHg. There was a positive correlation between the RT-PCR negative time and age, WBC count, LDH, SCr. There were statistically significant differences in fever numbers, WBC count, lymphocyte count, CRP, ALT, AST, albumin, LDH, SCr, D-dimer, and fibrinogen between the two groups based on the oxygenation index. CONCLUSIONS: Age, WBC count, LDH, and SCr may be related to the duration of COVID-19 disease. Fever, WBC count, lymphocyte count, CRP, ALT, AST, albumin, LDH, SCr, D-dimer, and fibrinogen are related to the severity of acute lung injury.

The Outcomes of Patients Incidentally Confirmed with Covid-19 After Cardiac Surgery.

BACKGROUND: The aim of this study was to investigate the clinical outcomes of cardiac surgery in patients who were incidentally diagnosed with Covid-19 in the postoperative period. PATIENTS AND METHODS: We performed 826 open cardiac surgeries in five tertiary centers. Most of the surgeries were elective coronary artery bypass grafting (CABG) (93.8%). A preoperative RT-PCR test and transcutaneous oxygen saturation were routinely investigated prior to surgery. We also investigated whether the patients already received Covid-19 treatment or had any contact with a Covid-19 patient in the last two weeks. We analyzed high sensitive C-reactive protein (hs-CRP), d-dimer, and fibrinogen, which plays a main role in the activation of procoagulant state after surgeries. RESULTS: Acute lung injury related to Covid-19 activation was observed in 48 out of 826 patients (5.8%). The median age of 48 patients was 63.9±12.4 years. Euro-Score and body mass index (BMI) were 6.1±1.1 and 29.2±4.1kg/m², respectively. RT-PCR test results were positive in 29 patients (60.4%). We performed thoracic computed tomography (CT) in all patients with or without positive RT-PCR test results. Thoracic CT images showed that there was a different degree of ARDS (mild, moderate, and serious). The median time of extracorporeal circulation (ECC) was 93.2±14.6 min. in on-pump surgery (IQR, 68-155 min.). Common symptoms included dyspnea (N = 22; 45.8%) and fever (N = 12; 25%). Eleven patients needed readmission to ICU. Compared with non-admitted to ICU patients, ICU patients were higher comorbidities and severe laboratory abnormalities (eg, high blood d-dimer and fibrinogen). We also detected significantly low oxygen saturation, hypercapnia, and severe acidosis in readmitted patients. Radiologic investigations showed that there were severe ARDS with bilateral pneumonic infiltration resistant to medical treatment in 6 out of 11 patients who died (54.5%). CONCLUSION: Diffuse pneumonic infiltration related to Covid-19 may develop in asymptomatic cardiac surgery patients with negative RT-PCR test results. Immunologic disorders resulting from ECC, physiologic distress, and anesthesia may activate Covid-19 during the incubation period. We need randomized clinical trials to explain Covid-19 activation in the latent period of the virus, and clinical outcomes in cardiac surgery.

The Disruption of the Endothelial Barrier Contributes to Acute Lung Injury Induced by Coxsackievirus A2 Infection in Mice.

Sporadic occurrences and outbreaks of hand, foot, and mouth disease (HFMD) caused by Coxsackievirus A2 (CVA2) have frequently reported worldwide recently, which pose a great challenge to public health. Epidemiological studies have suggested that the main cause of death in critical patients is pulmonary edema. However, the pathogenesis of this underlying comorbidity remains unclear. In this study, we utilized the 5-day-old BALB/c mouse model of lethal CVA2 infection to evaluate lung damage. We found that the permeability of lung microvascular was significantly increased after CVA2 infection. We also observed the direct infection and apoptosis of lung endothelial cells as well as the destruction of tight junctions between endothelial cells. CVA2 infection led to the degradation of tight junction proteins (e.g., ZO-1, claudin-5, and occludin). The gene transcription levels of von Willebrand factor (vWF), endothelin (ET), thrombomodulin (THBD), granular membrane protein 140 (GMP140), and intercellular cell adhesion molecule-1 (ICAM-1) related to endothelial dysfunction were all significantly increased. Additionally, CVA2 infection induced the increased expression of inflammatory cytokines (IL-6, IL-1ß, and MCP-1) and the activation of p38 mitogen-activated protein kinase (MAPK). In conclusion, the disruption of the endothelial barrier contributes to acute lung injury induced by CVA2 infection; targeting p38-MAPK signaling may provide a therapeutic approach for pulmonary edema in critical infections of HFMD.

ACE2 and Innate Immunity in the Regulation of SARS-CoV-2-Induced Acute Lung Injury: A Review.

Despite the protracted battle against coronavirus acute respiratory infection (COVID-19) and the rapid evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), no specific and effective drugs have to date been reported. Angiotensin-converting enzyme 2 (ACE2) is a zinc metalloproteinase and a critical modulator of the renin-angiotensin system (RAS). In addition, ACE2 has anti-inflammatory and antifibrosis functions. ACE has become widely known in the past decade as it has been identified as the primary receptor for SARS-CoV and SARS-CoV-2, being closely associated with their infection. SARS-CoV-2 primarily targets the lung, which induces a cytokine storm by infecting alveolar cells, resulting in tissue damage and eventually severe acute respiratory syndrome. In the lung, innate immunity acts as a critical line of defense against pathogens, including SARS-CoV-2. This review aims to summarize the regulation of ACE2, and lung host cells resist SARS-CoV-2 invasion by activating innate immunity response. Finally, we discuss ACE2 as a therapeutic target, providing reference and enlightenment for the clinical treatment of COVID-19.

The Effects of Insulin-Like Growth Factor I and BTP-2 on Acute Lung Injury.

Acute lung injury (ALI) afflicts approximately 200,000 patients annually and has a 40% mortality rate. The COVID-19 pandemic has massively increased the rate of ALI incidence. The pathogenesis of ALI involves tissue damage from invading microbes and, in severe cases, the overexpression of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß). This study aimed to develop a therapy to normalize the excess production of inflammatory cytokines and promote tissue repair in the lipopolysaccharide (LPS)-induced ALI. Based on our previous studies, we tested the insulin-like growth factor I (IGF-I) and BTP-2 therapies. IGF-I was selected, because we and others have shown that elevated inflammatory cytokines suppress the expression of growth hormone receptors in the liver, leading to a decrease in the circulating IGF-I. IGF-I is a growth factor that increases vascular protection, enhances tissue repair, and decreases pro-inflammatory cytokines. It is also required to produce anti-inflammatory 1,25-dihydroxyvitamin D. BTP-2, an inhibitor of cytosolic calcium, was used to suppress the LPS-induced increase in cytosolic calcium, which otherwise leads to an increase in proinflammatory cytokines. We showed that LPS increased the expression of the primary inflammatory mediators such as toll like receptor-4 (TLR-4), IL-1ß, interleukin-17 (IL-17), TNF-α, and interferon-γ (IFN-γ), which were normalized by the IGF-I + BTP-2 dual therapy in the lungs, along with improved vascular gene expression markers. The histologic lung injury score was markedly elevated by LPS and reduced to normal by the combination therapy. In conclusion, the LPS-induced increases in inflammatory cytokines, vascular injuries, and lung injuries were all improved by IGF-I + BTP-2 combination therapy.

Poly(ADP-Ribose) Polymerase Inhibition in Acute Lung Injury. A Reemerging Concept.

PARP1, the major isoform of a family of ADP-ribosylating enzymes, has been implicated in the regulation of various biological processes including DNA repair, gene transcription, and cell death. The concept that PARP1 becomes activated in acute lung injury (ALI) and that pharmacological inhibition or genetic deletion of this enzyme can provide therapeutic benefits emerged over 20 years ago. The current article provides an overview of the cellular mechanisms involved in the pathogenetic roles of PARP1 in ALI and provides an overview of the preclinical data supporting the efficacy of PARP (poly[ADP-ribose] polymerase) inhibitors. In recent years, several ultrapotent PARP inhibitors have been approved for clinical use (for the therapy of various oncological diseases): these newly-approved PARP inhibitors were recently reported to show efficacy in animal models of ALI. These observations offer the possibility of therapeutic repurposing of these inhibitors for patients with ALI. The current article lays out a potential roadmap for such repurposing efforts. In addition, the article also overviews the scientific basis of potentially applying PARP inhibitors for the experimental therapy of viral ALI, such as coronavirus disease (COVID-19)-associated ALI.

Expression pattern of the interferon regulatory factor family members in influenza virus induced local and systemic inflammatory responses.

Type I interferon is considered to be a key cytokine in influenza virus-induced acute lung injury (ALI), in which IRF3 and IRF7 play particularly important roles. However, whether all nine members of IRF family are involved in influenza virus-induced immune response is currently unknown. In this study, we found that all members of IRF family responded to influenza virus. The IRF family expression profile seems to be related to the pathogenicity of the particular influenza virus strain. The influenza virus mainly relies on endosomal TLR signals and the positive feedback loop of IFN-I to cause either direct or indirect different expression of all IRF family members locally or systemically. Interestingly, IRF6 was somewhat different from other IRF family members during influenza virus infection. Overall, the expression profile of the IRF family may be a valuable reference for the prevention and treatment of influenza complications, which encourage further, more in-depth research.

Clinical phenotypes of SARS-CoV-2: implications for clinicians and researchers.

Patients with COVID-19 present a broad spectrum of clinical presentation. Whereas hypoxaemia is the marker of severity, different strategies of management should be customised to five specific individual phenotypes. Many intubated patients present with phenotype 4, characterised by pulmonary hypoxic vasoconstriction, being associated with severe hypoxaemia with "normal" (>40 mL·cmH2O-1) lung compliance and likely representing pulmonary microvascular thrombosis. Phenotype 5 is often associated with high plasma procalcitonin and has low pulmonary compliance, Which is a result of co-infection or acute lung injury after noninvasive ventilation. Identifying these clinical phenotypes and applying a personalised approach would benefit the optimisation of therapies and improve outcomes.

Absence of ß6 Integrin Reduces Influenza Disease Severity in Highly Susceptible Obese Mice.

Obese individuals are considered a high-risk group for developing severe influenza virus infection. While the exact mechanisms for increased disease severity remain under investigation, obese-mouse models suggest that increased acute lung injury (ALI), potentially due to enhanced viral spread and decreased wound repair, is likely involved. We previously demonstrated that upregulation of the lung epithelial cell ß6 integrin during influenza virus infection was involved in disease severity. Knocking out ß6 (ß6 KO) resulted in improved survival. Of interest, obese mice have increased lung ß6 integrin levels at homeostasis. Thus, we hypothesized that the protective effect seen in ß6 KO mice would extend to the highly susceptible obese-mouse model. In the current study, we show that crossing ß6 KO mice with genetically obese (ob/ob) mice (OBKO) resulted in reduced ALI and impaired viral spread, like their lean counterparts. Mechanistically, OBKO alveolar macrophages and epithelial cells had increased type I interferon (IFN) signaling, potentially through upregulated type I IFN receptor expression, which was important for the enhanced protection during infection. Taken together, our results indicate that the absence of an epithelial integrin can beneficially alter the pulmonary microenvironment by increasing protective type I IFN responses even in a highly susceptible obese-mouse model. These studies increase our understanding of influenza virus pathogenesis in high-risk populations and may lead to the development of novel therapies.IMPORTANCE Obesity is a risk factor for developing severe influenza virus infection. However, the reasons for this are unknown. We found that the lungs of obese mice have increased expression of the epithelial integrin ß6, a host factor associated with increased disease severity. Knocking out integrin ß6 in obese mice favorably altered the pulmonary environment by increasing type I IFN signaling, resulting in decreased viral spread, reduced lung injury, and increased survival. This study furthers our understanding of influenza virus pathogenesis in the high-risk obese population and may potentially lead to the development of novel therapies for influenza virus infection.

Bone marrow-derived mesenchymal stem cells attenuate pulmonary inflammation and lung damage caused by highly pathogenic avian influenza A/H5N1 virus in BALB/c mice.

BACKGROUND: The highly pathogenic avian influenza A/H5N1 virus is one of the causative agents of acute lung injury (ALI) with high mortality rate. Studies on therapeutic administration of bone marrow-derived mesenchymal stem cells (MSCs) in ALI caused by the viral infection have been limited in number and have shown conflicting results. The aim of the present investigation is to evaluate the therapeutic potential of MSC administration in A/H5N1-caused ALI, using a mouse model. METHODS: MSCs were prepared from the bone marrow of 9 to 12 week-old BALB/c mice. An H5N1 virus of A/turkey/East Java/Av154/2013 was intranasally inoculated into BALB/c mice. On days 2, 4, and 6 after virus inoculation, MSCs were intravenously administered into the mice. To evaluate effects of the treatment, we examined for lung alveolar protein as an indicator for lung injury, PaO2/FiO2 ratio for lung functioning, and lung histopathology. Expressions of NF-κB, RAGE (transmembrane receptor for damage associated molecular patterns), TNFα, IL-1ß, Sftpc (alveolar cell type II marker), and Aqp5+ (alveolar cell type I marker) were examined by immunohistochemistry. In addition, body weight, virus growth in lung and brain, and duration of survival were measured. RESULTS: The administration of MSCs lowered the level of lung damage in the virus-infected mice, as shown by measuring lung alveolar protein, PaO2/FiO2 ratio, and histopathological score. In the MSC-treated group, the expressions of NF-κB, RAGE, TNFα, and IL-1ß were significantly suppressed in comparison with a mock-treated group, while those of Sftpc and Aqp5+ were enhanced. Body weight, virus growth, and survival period were not significantly different between the groups. CONCLUSION: The administration of MSCs prevented further lung injury and inflammation, and enhanced alveolar cell type II and I regeneration, while it did not significantly affect viral proliferation and mouse morbidity and mortality. The results suggested that MSC administration was a promissing strategy for treatment of acute lung injuries caused by the highly pathogenic avian influenza A/H5N1 virus, although further optimization and combination use of anti-viral drugs will be obviously required to achieve the goal of reducing mortality.

ß-sitosterol ameliorates influenza A virus-induced proinflammatory response and acute lung injury in mice by disrupting the cross-talk between RIG-I and IFN/STAT signaling.

ß-Sitosterol (24-ethyl-5-cholestene-3-ol) is a common phytosterol Chinese medical plants that has been shown to possess antioxidant and anti-inflammatory activity. In this study we investigated the effects of ß-sitosterol on influenza virus-induced inflammation and acute lung injury and the molecular mechanisms. We demonstrate that ß-sitosterol (150-450 µg/mL) dose-dependently suppresses inflammatory response through NF-κB and p38 mitogen-activated protein kinase (MAPK) signaling in influenza A virus (IAV)-infected cells, which was accompanied by decreased induction of interferons (IFNs) (including Type I and III IFN). Furthermore, we revealed that the anti-inflammatory effect of ß-sitosterol resulted from its inhibitory effect on retinoic acid-inducible gene I (RIG-I) signaling, led to decreased STAT1 signaling, thus affecting the transcriptional activity of ISGF3 (interferon-stimulated gene factor 3) complexes and resulting in abrogation of the IAV-induced proinflammatory amplification effect in IFN-sensitized cells. Moreover, ß-sitosterol treatment attenuated RIG-I-mediated apoptotic injury of alveolar epithelial cells (AEC) via downregulation of pro-apoptotic factors. In a mouse model of influenza, pre-administration of ß-sitosterol (50, 200 mg·kg-1·d-1, i.g., for 2 days) dose-dependently ameliorated IAV-mediated recruitment of pathogenic cytotoxic T cells and immune dysregulation. In addition, pre-administration of ß-sitosterol protected mice from lethal IAV infection. Our data suggest that ß-sitosterol blocks the immune response mediated by RIG-I signaling and deleterious IFN production, providing a potential benefit for the treatment of influenza.