Activated PMN Exosomes: Pathogenic Entities Causing Matrix Destruction and Disease in the Lung.

Here, we describe a novel pathogenic entity, the activated PMN (polymorphonuclear leukocyte, i.e., neutrophil)-derived exosome. These CD63+/CD66b+ nanovesicles acquire surface-bound neutrophil elastase (NE) during PMN degranulation, NE being oriented in a configuration resistant to α1-antitrypsin (α1AT). These exosomes bind and degrade extracellular matrix (ECM) via the integrin Mac-1 and NE, respectively, causing the hallmarks of chronic obstructive pulmonary disease (COPD). Due to both ECM targeting and α1AT resistance, exosomal NE is far more potent than free NE. Importantly, such PMN-derived exosomes exist in clinical specimens from subjects with COPD but not healthy controls and are capable of transferring a COPD-like phenotype from humans to mice in an NE-driven manner. Similar findings were observed for another neutrophil-driven disease of ECM remodeling (bronchopulmonary dysplasia [BPD]). These findings reveal an unappreciated role for exosomes in the pathogenesis of disorders of ECM homeostasis such as COPD and BPD, providing a critical mechanism for proteolytic damage.

Tumor-Intrinsic Enhancer of Zeste Homolog 2 Controls Immune Cell Infiltration, Tumor Growth, and Lung Metastasis in a Triple-Negative Breast Cancer Model.

Triple-negative breast cancer (TNBC) is an aggressive and highly metastatic type of tumor. TNBC is often enriched in tumor-infiltrating neutrophils (TINs), which support cancer growth in part by counteracting tumor-infiltrating lymphocytes (TILs). Prior studies identified the enhancer of zeste homolog 2 (EZH2) as a pro-tumor methyltransferase in primary and metastatic TNBCs. We hypothesized that EZH2 inhibition in TNBC cells per se would exert antitumor activity by altering the tumor immune microenvironment. To test this hypothesis, we used CRISPR to generate EZH2 gene knockout (KO) and overexpressing (OE) lines from parent (wild-type-WT) 4T1 cells, an established murine TNBC model, resulting in EZH2 protein KO and OE, respectively. In vitro, EZH2 KO and OE cells showed early, transient changes in replicative capacity and invasiveness, and marked changes in surface marker profile and cytokine/chemokine secretion compared to WT cells. In vivo, EZH2 KO cells showed significantly reduced primary tumor growth and a 10-fold decrease in lung metastasis compared to WT cells, while EZH2 OE cells were unchanged. Compared to WT tumors, TIN:TIL ratios were greatly reduced in EZH2 KO tumors but unchanged in EZH2 OE tumors. Thus, EZH2 is key to 4T1 aggressiveness as its tumor-intrinsic knockout alters their in vitro secretome and in vivo primary tumor growth, TIN/TIL poise, and metastasis.

Immunomodulation in Cystic Fibrosis: Why and How?

Cystic fibrosis (CF) lung disease is characterized by unconventional mechanisms of inflammation, implicating a chronic immune response dominated by innate immune cells. Historically, therapeutic development has focused on the mutated cystic fibrosis transmembrane conductance regulator (CFTR), leading to the discovery of small molecules aiming at modulating and potentiating the presence and activity of CFTR at the plasma membrane. However, treatment burden sustained by CF patients, side effects of current medications, and recent advances in other therapeutic areas have highlighted the need to develop novel disease targeting of the inflammatory component driving CF lung damage. Furthermore, current issues with standard treatment emphasize the need for directed lung therapies that could minimize systemic side effects. Here, we summarize current treatment used to target immune cells in the lungs, and highlight potential benefits and caveats of novel therapeutic strategies.

Why Certain Repurposed Drugs Are Unlikely to Be Effective Antivirals to Treat SARS-CoV-2 Infections.

Most repurposed drugs have proved ineffective for treating COVID-19. We evaluated median effective and toxic concentrations (EC50, CC50) of 49 drugs, mostly from previous clinical trials, in Vero cells. Ratios of reported unbound peak plasma concentrations, (Cmax)/EC50, were used to predict the potential in vivo efficacy. The 20 drugs with the highest ratios were retested in human Calu-3 and Caco-2 cells, and their CC50 was determined in an expanded panel of cell lines. Many of the 20 drugs with the highest ratios were inactive in human Calu-3 and Caco-2 cells. Antivirals effective in controlled clinical trials had unbound Cmax/EC50 ≥ 6.8 in Calu-3 or Caco-2 cells. EC50 of nucleoside analogs were cell dependent. This approach and earlier availability of more relevant cultures could have reduced the number of unwarranted clinical trials.

Transcriptional reprogramming of infiltrating neutrophils drives lung pathology in severe COVID-19 despite low viral load.

Troubling disparities in COVID-19-associated mortality emerged early, with nearly 70% of deaths confined to Black/African American (AA) patients in some areas. However, targeted studies on this vulnerable population are scarce. Here, we applied multiomics single-cell analyses of immune profiles from matching airways and blood samples of Black/AA patients during acute SARS-CoV-2 infection. Transcriptional reprogramming of infiltrating IFITM2+/S100A12+ mature neutrophils, likely recruited via the IL-8/CXCR2 axis, leads to persistent and self-sustaining pulmonary neutrophilia with advanced features of acute respiratory distress syndrome (ARDS) despite low viral load in the airways. In addition, exacerbated neutrophil production of IL-8, IL-1ß, IL-6, and CCL3/4, along with elevated levels of neutrophil elastase and myeloperoxidase, were the hallmarks of transcriptionally active and pathogenic airway neutrophilia. Although our analysis was limited to Black/AA patients and was not designed as a comparative study across different ethnicities, we present an unprecedented in-depth analysis of the immunopathology that leads to acute respiratory distress syndrome in a well-defined patient population disproportionally affected by severe COVID-19.

Inactivation of SARS-CoV-2 and COVID-19 Patient Samples for Contemporary Immunology and Metabolomics Studies.

Due to the severity of COVID-19 disease, the U.S. Centers for Disease Control and Prevention and World Health Organization recommend that manipulation of active viral cultures of SARS-CoV-2 and respiratory secretions from COVID-19 patients be performed in biosafety level (BSL)3 laboratories. Therefore, it is imperative to develop viral inactivation procedures that permit samples to be transferred to lower containment levels (BSL2), while maintaining the fidelity of complex downstream assays to expedite the development of medical countermeasures. In this study, we demonstrate optimal conditions for complete viral inactivation following fixation of infected cells with commonly used reagents for flow cytometry, UVC inactivation in sera and respiratory secretions for protein and Ab detection, heat inactivation following cDNA amplification for droplet-based single-cell mRNA sequencing, and extraction with an organic solvent for metabolomic studies. Thus, we provide a suite of viral inactivation protocols for downstream contemporary assays that facilitate sample transfer to BSL2, providing a conceptual framework for rapid initiation of high-fidelity research as the COVID-19 pandemic continues.

Macrophage PD-1 associates with neutrophilia and reduced bacterial killing in early cystic fibrosis airway disease.

BACKGROUND: Macrophages are the major resident immune cells in human airways coordinating responses to infection and injury. In cystic fibrosis (CF), neutrophils are recruited to the airways shortly after birth, and actively exocytose damaging enzymes prior to chronic infection, suggesting a potential defect in macrophage immunomodulatory function. Signaling through the exhaustion marker programmed death protein 1 (PD-1) controls macrophage function in cancer, sepsis, and airway infection. Therefore, we sought to identify potential associations between macrophage PD-1 and markers of airway disease in children with CF. METHODS: Blood and bronchoalveolar lavage fluid (BALF) were collected from 45 children with CF aged 3 to 62 months and structural lung damage was quantified by computed tomography. The phenotype of airway leukocytes was assessed by flow cytometry, while the release of enzymes and immunomodulatory mediators by molecular assays. RESULTS: Airway macrophage PD-1 expression correlated positively with structural lung damage, neutrophilic inflammation, and infection. Interestingly, even in the absence of detectable infection, macrophage PD-1 expression was elevated and correlated with neutrophilic inflammation. In an in vitro model mimicking leukocyte recruitment into CF airways, soluble mediators derived from recruited neutrophils directly induced PD-1 expression on recruited monocytes/macrophages, suggesting a causal link between neutrophilic inflammation and macrophage PD-1 expression in CF. Finally, blockade of PD-1 in a short-term culture of CF BALF leukocytes resulted in improved pathogen clearance. CONCLUSION: Taken together, these findings suggest that in early CF lung disease, PD-1 upregulation associates with airway macrophage exhaustion, neutrophil takeover, infection, and structural damage.

Neutrophil Dysfunction in the Airways of Children with Acute Respiratory Failure Due to Lower Respiratory Tract Viral and Bacterial Coinfections.

Neutrophils are recruited to the airways of patients with acute respiratory distress syndrome (ARDS) where they acquire an activated pro-survival phenotype with an enhanced respiratory burst thought to contribute to ARDS pathophysiology. Our in vitro model enables blood neutrophil transepithelial migration into cell-free tracheal aspirate fluid from patients to recapitulate the primary airway neutrophil phenotype observed in vivo. Neutrophils transmigrated through our model toward airway fluid from children with lower respiratory viral infections coinfected with bacteria had elevated levels of neutrophil activation markers but paradoxically exhibited an inability to kill bacteria and a defective respiratory burst compared with children without bacterial coinfection. The airway fluid from children with bacterial coinfections had higher levels of neutrophil elastase activity, as well as myeloperoxidase levels compared to children without bacterial coinfection. Neutrophils transmigrated into the aspirate fluid from children with bacterial coinfection showed decreased respiratory burst and killing activity against H. influenzae and S. aureus compared to those transmigrated into the aspirate fluid from children without bacterial coinfection. Use of a novel transmigration model recapitulates this pathological phenotype in vitro that would otherwise be impossible in a patient, opening avenues for future mechanistic and therapeutic research.