Neutrophil-Derived Proteases in Lung Inflammation: Old Players and New Prospects.

Neutrophil-derived proteases are critical to the pathology of many inflammatory lung diseases, both chronic and acute. These abundant enzymes play roles in key neutrophil functions, such as neutrophil extracellular trap formation and reactive oxygen species release. They may also be released, inducing tissue damage and loss of tissue function. Historically, the neutrophil serine proteases (NSPs) have been the main subject of neutrophil protease research. Despite highly promising cell-based and animal model work, clinical trials involving the inhibition of NSPs have shown mixed results in lung disease patients. As such, the cutting edge of neutrophil-derived protease research has shifted to proteases that have had little-to-no research in neutrophils to date. These include the cysteine and serine cathepsins, the metzincins and the calpains, among others. This review aims to outline the previous work carried out on NSPs, including the shortcomings of some of the inhibitor-orientated clinical trials. Our growing understanding of other proteases involved in neutrophil function and neutrophilic lung inflammation will then be discussed. Additionally, the potential of targeting these more obscure neutrophil proteases will be highlighted, as they may represent new targets for inhibitor-based treatments of neutrophil-mediated lung inflammation.

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 (ARDS). Interleukin-10 (IL-10) plays a crucial role in regulating the balance between antimicrobial immunity and immunopathology. In the current study, we have investigated the role of IL-10 in acute lung injury (ALI) induced by influenza A virus (IAV) and methicillin-resistant Staphylococcus aureus (MRSA) coinfection. This unique coinfection model resembles acute pneumonia patients undergoing appropriate antibiotic therapies. Using global IL-10 and IL-10 receptor (IL-10R) gene-deficient mice, as well as in vivo neutralizing antibodies, here 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 hyper-production and acute mortality. Importantly, this anti-inflammatory 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 ARDS pathogenesis by counteracting the IFN-γ response.

Immune predisposition drives susceptibility to pneumococcal pneumonia after mild influenza A virus infection in mice.

Introduction: A frequent sequela of influenza A virus (IAV) infection is secondary bacterial pneumonia. Therefore, it is clinically important to understand the genetic predisposition to IAV and bacterial coinfection. Methods: BALB/c and C57BL/6 (B6) mice were infected with high or low-pathogenic IAV and Streptococcus pneumoniae (SPn). The contribution of cellular and molecular immune factors to the resistance/susceptibility of BALB/c and B6 mice were dissected in nonlethal and lethal IAV/SPn coinfection models. Results: Low-virulent IAV X31 (H3N2) rendered B6 mice extremely susceptible to SPn superinfection, while BALB/c mice remained unaffected. X31 infection alone barely induces IFN-γresponse in two strains of mice; however, SPn superinfection significantly enhances IFN-γ production in the susceptible B6 mice. As a result, IFN-γ signaling inhibits neutrophil recruitment and bacterial clearance, leading to lethal X31/SPn coinfection in B6 mice. Conversely, the diminished IFN-γ and competent neutrophil responses enable BALB/c mice highly resistant to X31/SPn coinfection. Discussion: The results establish that type 1 immune predisposition plays a key role in lethal susceptibility of B6 mice to pneumococcal pneumonia after mild IAV infection.

Valaciclovir for Epstein-Barr Virus Suppression in Moderate-to-Severe COPD: A Randomized Double-Blind Placebo-Controlled Trial.

BACKGROUND: Epstein-Barr virus (EBV) frequently is measured at high levels in COPD using sputum quantitative polymerase chain reaction, whereas airway immunohistochemistry analysis has shown EBV detection to be common in severe disease. RESEARCH QUESTION: Is valaciclovir safe and effective for EBV suppression in COPD? STUDY DESIGN AND METHODS: The Epstein-Barr Virus Suppression in COPD (EViSCO) trial was a randomized double-blind placebo-controlled trial conducted at the Mater Hospital Belfast, Northern Ireland. Eligible patients had stable moderate-to-severe COPD and sputum EBV (measured using quantitative polymerase chain reaction) and were assigned randomly (1:1) to valaciclovir (1 g tid) or matching placebo for 8 weeks. The primary efficacy outcome was sputum EBV suppression (defined as ≥ 90% sputum viral load reduction) at week 8. The primary safety outcome was the incidence of serious adverse reactions. Secondary outcome measures were FEV1 and drug tolerability. Exploratory outcomes included changes in quality of life, sputum cell counts, and cytokines. RESULTS: From November 2, 2018, through March 12, 2020, 84 patients were assigned randomly (n = 43 to valaciclovir). Eighty-one patients completed trial follow-up and were included in the intention-to-treat analysis of the primary outcome. A greater number of participants in the valaciclovir group achieved EBV suppression (n = 36 [87.8%] vs n = 17 [42.5%]; P < .001). Valaciclovir was associated with a significant reduction in sputum EBV titer compared with placebo (-90,404 copies/mL [interquartile range, -298,000 to -15,200 copies/mL] vs -3,940 copies/mL [interquartile range, -114,400 to 50,150 copies/mL]; P = .002). A statistically nonsignificant 24-mL numerical FEV1 increase was shown in the valaciclovir group (difference, -44 mL [95% CI, -150 to 62 mL]; P = .41). However, a reduction in sputum white cell count was noted in the valaciclovir group compared with the placebo group (difference, 2.89 [95% CI, 1.5 × 106-7.4 × 106]; P = .003). INTERPRETATION: Valaciclovir is safe and effective for EBV suppression in COPD and may attenuate the sputum inflammatory cell infiltrate. The findings from the current study provide support for a larger trial to evaluate long-term clinical outcomes. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT03699904; URL: www. CLINICALTRIALS: gov.

IFN-γ transforms the transcriptomic landscape and triggers myeloid cell hyperresponsiveness to cause lethal lung injury.

Acute Respiratory Distress Syndrome (ARDS) is an inflammatory disease that is associated with high mortality but no specific treatment. Our understanding of initial events that trigger ARDS pathogenesis is limited. We have developed a mouse model of inflammatory lung injury by influenza and methicillin-resistant Staphylococcus aureus (MRSA) coinfection plus daily antibiotic therapy. Using this pneumonic ARDS model, here we show that IFN-γ receptor signaling drives inflammatory cytokine storm and lung tissue damage. By single-cell RNA sequencing (scRNA-seq) analysis, we demonstrate that IFN-γ signaling induces a transcriptional shift in airway immune cells, particularly by upregulating macrophage and monocyte expression of genes associated with inflammatory diseases. Further evidence from conditional knockout mouse models reveals that IFN-γ receptor signaling in myeloid cells, particularly CD11c+ mononuclear phagocytes, directly promotes TNF-α hyperproduction and inflammatory lung damage. Collectively, the findings from this study, ranging from cell-intrinsic gene expression to overall disease outcome, demonstrate that influenza-induced IFN-γ triggers myeloid cell hyperresponsiveness to MRSA, thereby leading to excessive inflammatory response and lethal lung damage during coinfection.

Type I IFN Signaling Protects Mice from Lethal SARS-CoV-2 Neuroinvasion.

Multiple organ damage is common in patients with severe COVID-19, even though the underlying pathogenic mechanisms remain unclear. Acute viral infection typically activates type I IFN (IFN-I) signaling. The antiviral role of IFN-I is well characterized in vitro. However, our understanding of how IFN-I regulates host immune response to SARS-CoV-2 infection in vivo is incomplete. Using a human ACE2-transgenic mouse model, we show in the present study that IFN-I receptor signaling is essential for protection against the acute lethality of SARS-CoV-2 in mice. Interestingly, although IFN-I signaling limits viral replication in the lung, the primary infection site, it is dispensable for efficient viral clearance at the adaptive phase of SARS-CoV-2 infection. Conversely, we found that in the absence of IFN-I receptor signaling, the extreme animal lethality is consistent with heightened infectious virus and prominent pathological manifestations in the brain. Taken together, our results in this study demonstrate that IFN-I receptor signaling is required for restricting virus neuroinvasion, thereby mitigating COVID-19 severity.

EchidnaCSI: Engaging the public in research and conservation of the short-beaked echidna.

The short-beaked echidna is an iconic Australian animal and the most-widespread native mammal, inhabiting diverse environments. The cryptic nature of echidnas has limited research into their ecology in most areas; however, from the well-researched and endangered Kangaroo Island echidna population, we understand that the threats include habitat loss, roads, and invasive species. To obtain more information about echidnas Australia-wide, we established the Echidna Conservation Science Initiative (EchidnaCSI) citizen science project. EchidnaCSI calls on members of the public to submit photographs of wild echidnas and learn to identify and collect echidna scats for molecular analysis. To facilitate participation, we developed a smartphone application as well as ongoing social and traditional media activities and community events. In 3 y, more than 9,000 members of the public have downloaded the EchidnaCSI app, collecting 400 scats and submitting over 8,000 sightings of echidnas from across Australia. A subset of submitted scat samples were subjected to DNA extraction and PCR, which validated the approach of using citizen science for scat collection and viability for molecular analysis. To assess the impact of the project through public participation, we surveyed our participants (n = 944) to understand their demographics and motivations for engagement. Survey results also revealed that EchidnaCSI served as a gateway into citizen science more generally for many participants. EchidnaCSI demonstrates the potential for using citizen science approaches to collect high-quality data and material from a cryptic species over a very large geographic area and the considerable engagement value of citizen science research.

Cathepsin S Contributes to Lung Inflammation in Acute Respiratory Distress Syndrome.

Rationale: Although the cysteine protease cathepsin S has been implicated in the pathogenesis of several inflammatory lung diseases, its role has not been examined in the context of acute respiratory distress syndrome, a condition that still lacks specific and effective pharmacological treatments. Objectives: To characterize the status of cathepsin S in acute lung inflammation and examine the role of cathepsin S in disease pathogenesis. Methods: Human and mouse model BAL fluid samples were analyzed for the presence and activity of cathepsin S and its endogenous inhibitors. Recombinant cathepsin S was instilled directly into the lungs of mice. The effects of cathepsin S knockout and pharmacological inhibition were examined in two models of acute lung injury. Protease-activated receptor-1 antagonism was used to test a possible mechanism for cathepsin S-mediated inflammation. Measurements and Main Results: Pulmonary cathepsin S concentrations and activity were elevated in acute respiratory distress syndrome, a phenotype possibly exacerbated by the loss of the endogenous antiprotease cystatin SN. Direct cathepsin S instillation into the lungs induced key pathologies of acute respiratory distress syndrome, including neutrophilia and alveolar leakage. Conversely, in murine models of acute lung injury, genetic knockdown and prophylactic or therapeutic inhibition of cathepsin S reduced neutrophil recruitment and protein leakage. Cathepsin S may partly mediate its pathogenic effects via protease-activated receptor-1, because antagonism of this receptor abrogated cathepsin S-induced airway inflammation. Conclusions: Cathepsin S contributes to acute lung injury and may represent a novel therapeutic target for acute respiratory distress syndrome.

The relationship between plasma cystatin C, mortality and acute respiratory distress syndrome subphenotype in the HARP-2 trial.

Objective: To evaluate the performance of cystatin C as a prognostic and predictive marker in a trial of patients with acute respiratory distress syndrome (ARDS). Design, patients and setting: A retrospective analysis was performed on plasma samples from patients included in the HARP-2 (hydroxymethylglutaryl-CoA reductase inhibition with simvastatin in acute lung injury to reduce pulmonary dysfunction) trial - a multicentre, phase 2b trial carried out in general intensive care units across 40 hospitals in the United Kingdom and Ireland. Cystatin C concentrations in plasma obtained from 466 patients with ARDS (before they were randomly assigned in the trial) were quantified by ELISA (enzyme-linked immunosorbent assay). Results: In a univariate analysis, plasma cystatin C concentrations were significantly higher in patients with ARDS who did not survive past 28 days (odds ratio [OR], 1.39 [95% CI, 1.12-1.72]; P = 0.002). In a multivariate model adjusted for selected covariates, cystatin C concentrations remained higher among patients with ARDS who did not survive, although this did not reach statistical significance (OR, 1.28 [95% CI, 0.96-1.71]; P = 0.090). Cystatin C concentration was also significantly associated with hyperinflammatory ARDS (OR, 2.64 [95% CI, 1.83-3.89]; P < 0.001). In multivariate models adjusted for both cystatin C concentration and ARDS subphenotype, hyperinflammatory ARDS was prognostic for mortality (OR, 2.06 [95% CI, 1.16-3.64]; P = 0.013) but cystatin C concentration was not (OR, 1.16 [95% CI, 0.85-1.57]; P = 0.346). In a multivariate analysis, hyperinflammatory ARDS was predictive of a beneficial effect of simvastatin on mortality (OR, 2.05 [95% CI, 1.16-3.62]; P = 0.014) but cystatin C concentration was not (OR, 1.10 [95% CI, 0.77-1.56]; P = 0.614). Conclusion: The association between cystatin C concentration and mortality in ARDS may be dependent on inflammatory subphenotype. Cystatin C concentration does not appear to add to existing prognostic or predictive approaches.

Proteases, Mucus, and Mucosal Immunity in Chronic Lung Disease.

Dysregulated protease activity has long been implicated in the pathogenesis of chronic lung diseases and especially in conditions that display mucus obstruction, such as chronic obstructive pulmonary disease, cystic fibrosis, and non-cystic fibrosis bronchiectasis. However, our appreciation of the roles of proteases in various aspects of such diseases continues to grow. Patients with muco-obstructive lung disease experience progressive spirals of inflammation, mucostasis, airway infection and lung function decline. Some therapies exist for the treatment of these symptoms, but they are unable to halt disease progression and patients may benefit from novel adjunct therapies. In this review, we highlight how proteases act as multifunctional enzymes that are vital for normal airway homeostasis but, when their activity becomes immoderate, also directly contribute to airway dysfunction, and impair the processes that could resolve disease. We focus on how proteases regulate the state of mucus at the airway surface, impair mucociliary clearance and ultimately, promote mucostasis. We discuss how, in parallel, proteases are able to promote an inflammatory environment in the airways by mediating proinflammatory signalling, compromising host defence mechanisms and perpetuating their own proteolytic activity causing structural lung damage. Finally, we discuss some possible reasons for the clinical inefficacy of protease inhibitors to date and propose that, especially in a combination therapy approach, proteases represent attractive therapeutic targets for muco-obstructive lung diseases.

The Impact of Aging in Acute Respiratory Distress Syndrome: A Clinical and Mechanistic Overview.

Acute respiratory distress syndrome (ARDS) is associated with increased morbidity and mortality in the elderly population (≥65 years of age). Additionally, age is widely reported as a risk factor for the development of ARDS. However, the underlying pathophysiological mechanisms behind the increased risk of developing, and increased severity of, ARDS in the elderly population are not fully understood. This is compounded by the significant heterogeneity observed in patients with ARDS. With an aging population worldwide, a better understanding of these mechanisms could facilitate the development of therapies to improve outcomes in this population. In this review, the current clinical evidence of age as a risk factor and prognostic indicator in ARDS and the potential underlying mechanisms that may contribute to these factors are outlined. In addition, research on age-dependent treatment options and biomarkers, as well as future prospects for targeting these underlying mechanisms, are discussed.

Airway Inflammation and Host Responses in the Era of CFTR Modulators.

The arrival of cystic fibrosis transmembrane conductance regulator (CFTR) modulators as a new class of treatment for cystic fibrosis (CF) in 2012 represented a pivotal advance in disease management, as these small molecules directly target the upstream underlying protein defect. Further advancements in the development and scope of these genotype-specific therapies have been transformative for an increasing number of people with CF (PWCF). Despite clear improvements in CFTR function and clinical endpoints such as lung function, body mass index (BMI), and frequency of pulmonary exacerbations, current evidence suggests that CFTR modulators do not prevent continued decline in lung function, halt disease progression, or ameliorate pathogenic organisms in those with established lung disease. Furthermore, it remains unknown whether their restorative effects extend to dysfunctional CFTR expressed in phagocytes and other immune cells, which could modulate airway inflammation. In this review, we explore the effects of CFTR modulators on airway inflammation, infection, and their influence on the impaired pulmonary host defences associated with CF lung disease. We also consider the role of inflammation-directed therapies in light of the widespread clinical use of CFTR modulators and identify key areas for future research.

Giant breast tumors: surgical management of phyllodes tumors, potential for reconstructive surgery and a review of literature.

BACKGROUND: Phyllodes tumors are biphasic fibroepithelial neoplasms of the breast. While the surgical management of these relatively uncommon tumors has been addressed in the literature, few reports have commented on the surgical approach to tumors greater than ten centimeters in diameter - the giant phyllodes tumor. CASE PRESENTATION: We report two cases of giant breast tumors and discuss the techniques utilized for pre-operative diagnosis, tumor removal, and breast reconstruction. A review of the literature on the surgical management of phyllodes tumors was performed. CONCLUSION: Management of the giant phyllodes tumor presents the surgeon with unique challenges. The majority of these tumors can be managed by simple mastectomy. Axillary lymph node metastasis is rare, and dissection should be limited to patients with pathologic evidence of tumor in the lymph nodes.

Body mass, age and sexual maturity in short-beaked echidnas, Tachyglossus aculeatus.

During the course of this 12 year field study body masses of 11 hatchling echidnas (Tachyglossus aculeatus multiaculeatus) and 25 pouch young between the ages of 5 and 60 days were recorded. Body mass increased from 0.3 to approximately 50 g in the first half of pouch life. It then quadrupled before young were placed in a burrow at 45 to 55 days of age. There was a positive correlation between the body mass of the female and that of her young at weaning. From 33 subadult echidnas located, tagged and radio tracked during this study, body masses of 10 were monitored to sexual maturity, i.e. when first encountered in a courtship train. Minimum age of sexual maturity ranged between 5 and 12 years. As subadults, there was no difference between mean body masses of males and females. At sexual maturity, mean body mass of females was significantly higher. No correlation was found between age at sexual maturity and body mass nor was there a significant difference in age of males and females at sexual maturity.