Fibroblast-derived extracellular vesicles contain SFRP1 and mediate pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a lethal chronic lung disease characterized by aberrant intercellular communication, extracellular matrix deposition, and destruction of functional lung tissue. While extracellular vesicles (EVs) accumulate in the IPF lung, their cargo and biological effects remain unclear. We interrogated the proteome of EV and non-EV fractions during pulmonary fibrosis and characterized their contribution to fibrosis. EVs accumulated 14 days after bleomycin challenge, correlating with decreased lung function and initiated fibrogenesis in healthy precision-cut lung slices. Label-free proteomics of bronchoalveolar lavage fluid EVs (BALF-EVs) collected from mice challenged with bleomycin or control identified 107 proteins enriched in fibrotic vesicles. Multiomic analysis revealed fibroblasts as a major cellular source of BALF-EV cargo, which was enriched in secreted frizzled related protein 1 (SFRP1). Sfrp1 deficiency inhibited the activity of fibroblast-derived EVs to potentiate lung fibrosis in vivo. SFRP1 led to increased transitional cell markers, such as keratin 8, and WNT/ß-catenin signaling in primary alveolar type 2 cells. SFRP1 was expressed within the IPF lung and localized at the surface of EVs from patient-derived fibroblasts and BALF. Our work reveals altered EV protein cargo in fibrotic EVs promoting fibrogenesis and identifies fibroblast-derived vesicular SFRP1 as a fibrotic mediator and potential therapeutic target for IPF.

Antibiotic use during influenza infection augments lung eosinophils that impair immunity against secondary bacterial pneumonia.

A leading cause of mortality after influenza infection is the development of a secondary bacterial pneumonia. In the absence of a bacterial superinfection, prescribing antibacterial therapies is not indicated but has become a common clinical practice for those presenting with a respiratory viral illness. In a murine model, we found that antibiotic use during influenza infection impaired the lung innate immunologic defenses toward a secondary challenge with methicillin-resistant Staphylococcus aureus (MRSA). Antibiotics augment lung eosinophils, which have inhibitory effects on macrophage function through the release of major basic protein. Moreover, we demonstrated antibiotic treatment during influenza infection causes a fungal dysbiosis that drive lung eosinophilia and impair MRSA clearance. Finally, we evaluated three cohorts of hospitalized patients and found eosinophils positively correlated with antibiotic use, systemic inflammation, and worsened outcomes. Altogether, our work demonstrates a detrimental effect of antibiotic treatment during influenza infection that has harmful immunologic consequences via recruitment of eosinophils to the lungs thereby increasing the risk of developing a secondary bacterial infection.

An aberrant immune-epithelial progenitor niche drives viral lung sequelae.

The long-term physiological consequences of respiratory viral infections, particularly in the aftermath of the COVID-19 pandemic-termed post-acute sequelae of SARS-CoV-2 (PASC)-are rapidly evolving into a major public health concern1-3. While the cellular and molecular aetiologies of these sequelae are poorly defined, increasing evidence implicates abnormal immune responses3-6 and/or impaired organ recovery7-9 after infection. However, the precise mechanisms that link these processes in the context of PASC remain unclear. Here, with insights from three cohorts of patients with respiratory PASC, we established a mouse model of post-viral lung disease and identified an aberrant immune-epithelial progenitor niche unique to fibroproliferation in respiratory PASC. Using spatial transcriptomics and imaging, we found a central role for lung-resident CD8+ T cell-macrophage interactions in impairing alveolar regeneration and driving fibrotic sequelae after acute viral pneumonia. Specifically, IFNγ and TNF derived from CD8+ T cells stimulated local macrophages to chronically release IL-1ß, resulting in the long-term maintenance of dysplastic epithelial progenitors and lung fibrosis. Notably, therapeutic neutralization of IFNγ + TNF or IL-1ß markedly improved alveolar regeneration and pulmonary function. In contrast to other approaches, which require early intervention10, we highlight therapeutic strategies to rescue fibrotic disease after the resolution of acute disease, addressing a current unmet need in the clinical management of PASC and post-viral disease.

Caveolin scaffolding domain (CSD) peptide LTI-2355 modulates the phagocytic and synthetic activity of lung derived myeloid cells in Idiopathic Pulmonary Fibrosis (IPF) and Post-acute sequelae of COVID-fibrosis (PASC-F).

Rationale: The role of the innate immune system in Idiopathic Pulmonary Fibrosis (IPF) remains poorly understood. However, a functional myeloid compartment is required to remove dying cells and cellular debris, and to mediate innate immune responses against pathogens. Aberrant macrophage activity has been described in patients with Post-acute sequelae of COVID fibrosis (PASC-F). Therefore, we examined the functional and synthetic properties of myeloid cells isolated from normal donor lung and lung explant tissue from both IPF and PASC-F patients and explored the effect of LTI-2355, a Caveolin Scaffolding Domain (CSD) peptide, on these cells. Methods & Results: CD45 + myeloid cells isolated from lung explant tissue from IPF and PASC-F patients exhibited an impaired capacity to clear autologous dead cells and cellular debris. Uptake of pathogen-coated bioparticles was impaired in myeloid cells from both fibrotic patient groups independent of type of pathogen highlighting a cell intrinsic functional impairment. LTI-2355 improved the phagocytic activity of both IPF and PASC-F myeloid cells, and this improvement was paired with decreased pro-inflammatory and pro-fibrotic synthetic activity. LTI-2355 was also shown to primarily target CD206-expressing IPF and PASC-F myeloid cells. Conclusions: Primary myeloid cells from IPF and PASC-F patients exhibit dysfunctional phagocytic and synthetic properties that are reversed by LTI-2355. Thus, these studies highlight an additional mechanism of action of a CSD peptide in the treatment of IPF and progressive fibrotic lung disease.

Interstitial Macrophages Mediate Efferocytosis of Alveolar Epithelium during Influenza Infection.

Efferocytosis is a process whereby apoptotic cells are cleared to maintain tissue homeostasis. In the lungs, efferocytosis has been implicated in several acute and chronic inflammatory diseases. A long-standing method to study efferocytosis in vivo is to instill apoptotic cells into the lungs to evaluate macrophage uptake. However, this approach provides nonphysiologic levels of cells to the airspaces, where there is preferential access to the alveolar macrophages. To circumvent this limitation, we developed a new method to study efferocytosis of damaged alveolar type 2 (AT2) epithelial cells in vivo. A reporter mouse that expresses TdTomato in AT2 epithelial cells was injured with influenza (strain PR8) to induce apoptosis of AT2 cells. We were able to identify macrophages that acquire red fluorescence after influenza injury, indicating efferocytosis of AT2 cells. Furthermore, evaluation of macrophage populations led to the surprising finding that lung interstitial macrophages were the primary efferocyte in vivo. In summary, we present a novel finding that the interstitial macrophage, not the alveolar macrophage, primarily mediates clearance of AT2 cells in the lungs after influenza infection. Our method of studying efferocytosis provides a more physiologic approach in evaluating the spatiotemporal dynamics of apoptotic cell clearance in vivo and opens new avenues to study the mechanisms by which efferocytosis regulates inflammation.

Proximal immune-epithelial progenitor interactions drive chronic tissue sequelae post COVID-19.

The long-term physiological consequences of SARS-CoV-2, termed Post-Acute Sequelae of COVID-19 (PASC), are rapidly evolving into a major public health concern. The underlying cellular and molecular etiology remain poorly defined but growing evidence links PASC to abnormal immune responses and/or poor organ recovery post-infection. Yet, the precise mechanisms driving non-resolving inflammation and impaired tissue repair in the context of PASC remain unclear. With insights from three independent clinical cohorts of PASC patients with abnormal lung function and/or viral infection-mediated pulmonary fibrosis, we established a clinically relevant mouse model of post-viral lung sequelae to investigate the pathophysiology of respiratory PASC. By employing a combination of spatial transcriptomics and imaging, we identified dysregulated proximal interactions between immune cells and epithelial progenitors unique to the fibroproliferation in respiratory PASC but not acute COVID-19 or idiopathic pulmonary fibrosis (IPF). Specifically, we found a central role for lung-resident CD8+ T cell-macrophage interactions in maintaining Krt8hi transitional and ectopic Krt5+ basal cell progenitors, thus impairing alveolar regeneration and driving fibrotic sequelae after acute viral pneumonia. Mechanistically, CD8+ T cell derived IFN-γ and TNF stimulated lung macrophages to chronically release IL-1ß, resulting in the abnormal accumulation of dysplastic epithelial progenitors and fibrosis. Notably, therapeutic neutralization of IFN-γ and TNF, or IL-1ß after the resolution of acute infection resulted in markedly improved alveolar regeneration and restoration of pulmonary function. Together, our findings implicate a dysregulated immune-epithelial progenitor niche in driving respiratory PASC. Moreover, in contrast to other approaches requiring early intervention, we highlight therapeutic strategies to rescue fibrotic disease in the aftermath of respiratory viral infections, addressing the current unmet need in the clinical management of PASC and post-viral disease.

Proximal immune-epithelial progenitor interactions drive chronic tissue sequelae post COVID-19.

The long-term physiological consequences of SARS-CoV-2, termed Post-Acute Sequelae of COVID-19 (PASC), are rapidly evolving into a major public health concern. The underlying cellular and molecular etiology remain poorly defined but growing evidence links PASC to abnormal immune responses and/or poor organ recovery post-infection. Yet, the precise mechanisms driving non-resolving inflammation and impaired tissue repair in the context of PASC remain unclear. With insights from three independent clinical cohorts of PASC patients with abnormal lung function and/or viral infection-mediated pulmonary fibrosis, we established a clinically relevant mouse model of post-viral lung sequelae to investigate the pathophysiology of respiratory PASC. By employing a combination of spatial transcriptomics and imaging, we identified dysregulated proximal interactions between immune cells and epithelial progenitors unique to the fibroproliferation in respiratory PASC but not acute COVID-19 or idiopathic pulmonary fibrosis (IPF). Specifically, we found a central role for lung-resident CD8+ T cell-macrophage interactions in maintaining Krt8hi transitional and ectopic Krt5+ basal cell progenitors, thus impairing alveolar regeneration and driving fibrotic sequelae after acute viral pneumonia. Mechanistically, CD8+ T cell derived IFN-γ and TNF stimulated lung macrophages to chronically release IL-1ß, resulting in the abnormal accumulation of dysplastic epithelial progenitors and fibrosis. Notably, therapeutic neutralization of IFN-γ and TNF, or IL-1ß after the resolution of acute infection resulted in markedly improved alveolar regeneration and restoration of pulmonary function. Together, our findings implicate a dysregulated immune-epithelial progenitor niche in driving respiratory PASC. Moreover, in contrast to other approaches requiring early intervention, we highlight therapeutic strategies to rescue fibrotic disease in the aftermath of respiratory viral infections, addressing the current unmet need in the clinical management of PASC and post-viral disease.

Senescence of alveolar epithelial progenitor cells: a critical driver of lung fibrosis.

Pulmonary fibrosis comprises a range of chronic interstitial lung diseases (ILDs) that impose a significant burden on patients and public health. Among these, idiopathic pulmonary fibrosis (IPF), a disease of aging, is the most common and most severe form of ILD and is treated largely by lung transplantation. The lack of effective treatments to stop or reverse lung fibrosis-in fact, fibrosis in most organs-has sparked the need to understand causative mechanisms with the goal of identifying critical points for potential therapeutic intervention. Findings from many groups have indicated that repeated injury to the alveolar epithelium-where gas exchange occurs-leads to stem cell exhaustion and impaired alveolar repair that, in turn, triggers the onset and progression of fibrosis. Cellular senescence of alveolar epithelial progenitors is a critical cause of stemness failure. Hence, senescence impairs repair and thus contributes significantly to fibrosis. In this review, we discuss recent evidence indicating that senescence of epithelial progenitor cells impairs alveolar homeostasis and repair creating a profibrotic environment. Moreover, we discuss the impact of senescent alveolar epithelial progenitors, alveolar type 2 (AT2) cells, and AT2-derived transitional epithelial cells in fibrosis. Emerging evidence indicates that transitional epithelial cells are prone to senescence and, hence, are a new player involved in senescence-associated lung fibrosis. Understanding the complex interplay of cell types and cellular regulatory factors contributing to alveolar epithelial progenitor senescence will be crucial to developing targeted therapies to mitigate their downstream profibrotic sequelae and to promote normal alveolar repair.NEW & NOTEWORTHY With an aging population, lung fibrotic diseases are becoming a global health burden. Dysfunctional repair of the alveolar epithelium is a key causative process that initiates lung fibrosis. Normal alveolar regeneration relies on functional progenitor cells; however, the senescence of these cells, which increases with age, hinders their ability to contribute to repair. Here, we discuss studies on the control and consequence of progenitor cell senescence in fibrosis and opportunities for research.

A wireless patch for the monitoring of C-reactive protein in sweat.

The quantification of protein biomarkers in blood at picomolar-level sensitivity requires labour-intensive incubation and washing steps. Sensing proteins in sweat, which would allow for point-of-care monitoring, is hindered by the typically large interpersonal and intrapersonal variations in its composition. Here we report the design and performance of a wearable and wireless patch for the real-time electrochemical detection of the inflammatory biomarker C-reactive (CRP) protein in sweat. The device integrates iontophoretic sweat extraction, microfluidic channels for sweat sampling and for reagent routing and replacement, and a graphene-based sensor array for quantifying CRP (via an electrode functionalized with anti-CRP capture antibodies-conjugated gold nanoparticles), ionic strength, pH and temperature for the real-time calibration of the CRP sensor. In patients with chronic obstructive pulmonary disease, with active or past infections or who had heart failure, the elevated concentrations of CRP measured via the patch correlated well with the protein's levels in serum. Wearable biosensors for the real-time sensitive analysis of inflammatory proteins in sweat may facilitate the management of chronic diseases.

Understanding Interstitial Lung Diseases Associated with Connective Tissue Disease (CTD-ILD): Genetics, Cellular Pathophysiology, and Biologic Drivers.

Connective tissue disease-associated interstitial lung disease (CTD-ILD) is a collection of systemic autoimmune disorders resulting in lung interstitial abnormalities or lung fibrosis. CTD-ILD pathogenesis is not well characterized because of disease heterogeneity and lack of pre-clinical models. Some common risk factors are inter-related with idiopathic pulmonary fibrosis, an extensively studied fibrotic lung disease, which includes genetic abnormalities and environmental risk factors. The primary pathogenic mechanism is that these risk factors promote alveolar type II cell dysfunction triggering many downstream profibrotic pathways, including inflammatory cascades, leading to lung fibroblast proliferation and activation, causing abnormal lung remodeling and repairs that result in interstitial pathology and lung fibrosis. In CTD-ILD, dysregulation of regulator pathways in inflammation is a primary culprit. However, confirmatory studies are required. Understanding these pathogenetic mechanisms is necessary for developing and tailoring more targeted therapy and provides newly discovered disease biomarkers for early diagnosis, clinical monitoring, and disease prognostication. This review highlights the central CTD-ILD pathogenesis and biological drivers that facilitate the discovery of disease biomarkers.

Basal Cell-derived WNT7A Promotes Fibrogenesis at the Fibrotic Niche in Idiopathic Pulmonary Fibrosis.

Loss of epithelial integrity, bronchiolarization, and fibroblast activation are key characteristics of idiopathic pulmonary fibrosis (IPF). Prolonged accumulation of basal-like cells in IPF may impact the fibrotic niche to promote fibrogenesis. To investigate their role in IPF, basal cells were isolated from IPF explant and healthy donor lung tissues. Single-cell RNA sequencing was used to assess differentially expressed genes in basal cells. Basal cell and niche interaction was demonstrated with the sLP-mCherry niche labeling system. Luminex assays were used to assess cytokines secreted by basal cells. The role of basal cells in fibroblast activation was studied. Three-dimensional organoid culture assays were used to interrogate basal cell effects on AEC2 (type 2 alveolar epithelial cell) renewal capacity. Perturbation was used to investigate WNT7A function in vitro and in a repetitive bleomycin model in vivo. We found that WNT7A is highly and specifically expressed in basal-like cells. Proteins secreted by basal cells can be captured by neighboring fibroblasts and AEC2s. Basal cells or basal cell-conditioned media activate fibroblasts through WNT7A. Basal cell-derived WNT7A inhibits AEC2 progenitor cell renewal in three-dimensional organoid cultures. Neutralizing antibodies against WNT7A or a small molecule inhibitor of Frizzled signaling abolished basal cell-induced fibroblast activation and attenuated lung fibrosis in mice. In summary, basal cells and basal cell-derived WNT7A are key components of the fibrotic niche, providing a unique non-stem cell function of basal cells in IPF progression and a novel targeting strategy for IPF.

Translational Studies Reveal the Divergent Effects of Simtuzumab Targeting LOXL2 in Idiopathic Pulmonary Fibrosis.

The composition of extracellular matrix (ECM) is altered during pathologic scarring in damaged organs including the lung. One major change in the ECM involves the cross-linking of collagen, which promotes fibroblast to myofibroblast differentiation. We examined the role of lysyl oxidase (LOX)-like 2 in lung progenitors and fibroblasts cultured from normal or IPF lung samples and in a humanized mouse model of IPF using a monoclonal antibody (Simtuzumab). Primary lung fibroblasts from normal donor lungs and IPF lung explants were examined for expression of LOXL2. Targeting LOXL2 with Simtuzumab on normal and IPF fibroblasts was examined both in vitro and in vivo for synthetic, functional, and profibrotic properties. LOXL2 was increased at transcript and protein level in IPF compared with normal lung samples. In a dose-dependent manner, Simtuzumab enhanced differentiation of fibroblasts into myofibroblasts. Inhibition of LOXL2 also enhanced fibroblast invasion and accelerated the outgrowth of fibroblasts from dissociated human lung cell preparations. Finally, preventative or delayed delivery of Simtuzumab enhanced lung fibrosis in a humanized mouse model of pulmonary fibrosis. Consistent with its failure in a Phase 2 clinical trial, Simtuzumab exhibited no therapeutic efficacy in translational in vitro and in vivo assays.

Evidence of premature lymphocyte aging in people with low anti-spike antibody levels after BNT162b2 vaccination.

SARS-CoV-2 vaccines have unquestionably blunted the overall impact of the COVID-19 pandemic, but host factors such as age, sex, obesity, and other co-morbidities can affect vaccine efficacy. We identified individuals in a relatively healthy population of healthcare workers (CORALE study cohort) who had unexpectedly low peak anti-spike receptor binding domain (S-RBD) antibody levels after receiving the BNT162b2 vaccine. Compared to matched controls, "low responders" had fewer spike-specific antibody-producing B cells after the second and third/booster doses. Moreover, their spike-specific T cell receptor (TCR) repertoire had less depth and their CD4+ and CD8+T cell responses to spike peptide stimulation were less robust. Single cell transcriptomic evaluation of peripheral blood mononuclear cells revealed activation of aging pathways in low responder B and CD4+T cells that could underlie their attenuated anti-S-RBD antibody production. Premature lymphocyte aging may therefore contribute to a less effective humoral response and could reduce vaccination efficacy.

Extracellular Vesicles, Inflammation, and Cardiovascular Disease.

Cardiovascular disease is a leading cause of death worldwide. The underlying mechanisms of most cardiovascular disorders involve innate and adaptive immune responses, and extracellular vesicles are implicated in both. In this review, we describe the mechanistic role of extracellular vesicles at the intersection of inflammatory processes and cardiovascular disease. Our discussion focuses on atherosclerosis, myocardial ischemia and ischemic heart disease, heart failure, aortic aneurysms, and valvular pathology.

Current Therapeutics for COVID-19, What We Know about the Molecular Mechanism and Efficacy of Treatments for This Novel Virus.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has caused significant morbidity and mortality worldwide. Though previous coronaviruses have caused substantial epidemics in recent years, effective therapies remained limited at the start of the Coronavirus disease 19 (COVID-19) pandemic. The emergence and rapid spread throughout the globe of the novel SARS-CoV-2 virus necessitated a rapid development of therapeutics. Given the multitude of therapies that have emerged over the last two years and the evolution of data surrounding the efficacy of these therapies, we aim to provide an update on the major clinical trials that influenced clinical utilization of various COVID-19 therapeutics. This review focuses on currently used therapies in the United States and discusses the molecular mechanisms by which these therapies target the SARS-CoV-2 virus or the COVID-19 disease process. PubMed and EMBASE were used to find trials assessing the efficacy of various COVID-19 therapies. The keywords SARS-CoV-2, COVID-19, and the names of the various therapies included in this review were searched in different combinations to find large-scale randomized controlled trials performed since the onset of the COVID-19 pandemic. Multiple therapeutic options are currently approved for the treatment of SARS-CoV-2 and prevention of severe disease in high-risk individuals in both in the inpatient and outpatient settings. In severe disease, a combination of antiviral and immunomodulatory treatments is currently recommended for treatment. Additionally, anti-viral agents have shown promise in preventing severe disease and hospitalization for those in the outpatient setting. More recently, current therapeutic approaches are directed toward early treatment with monoclonal antibodies directed against the SARS-CoV-2 virus. Despite this, no treatment to date serves as a definitive cure and vaccines against the SARS-CoV-2 virus remain our best defense to prevent further morbidity and mortality.

Cellular Senescence: Pathogenic Mechanisms in Lung Fibrosis.

Pulmonary fibrosis is a chronic and fatal lung disease that significantly impacts the aging population globally. To date, anti-fibrotic, immunosuppressive, and other adjunct therapy demonstrate limited efficacies. Advancing our understanding of the pathogenic mechanisms of lung fibrosis will provide a future path for the cure. Cellular senescence has gained substantial interest in recent decades due to the increased incidence of fibroproliferative lung diseases in the older age group. Furthermore, the pathologic state of cellular senescence that includes maladaptive tissue repair, decreased regeneration, and chronic inflammation resembles key features of progressive lung fibrosis. This review describes regulatory pathways of cellular senescence and discusses the current knowledge on the senescence of critical cellular players of lung fibrosis, including epithelial cells (alveolar type 2 cells, basal cells, etc.), fibroblasts, and immune cells, their phenotypic changes, and the cellular and molecular mechanisms by which these cells contribute to the pathogenesis of pulmonary fibrosis. A few challenges in the field include establishing appropriate in vivo experimental models and identifying senescence-targeted signaling molecules and specific therapies to target senescent cells, known collectively as "senolytic" or "senotherapeutic" agents.

Categorization of lung mesenchymal cells in development and fibrosis.

Pulmonary mesenchymal cells are critical players in both the mouse and human during lung development and disease states. They are increasingly recognized as highly heterogeneous, but there is no consensus on subpopulations or discriminative markers for each subtype. We completed scRNA-seq analysis of mesenchymal cells from the embryonic, postnatal, adult and aged fibrotic lungs of mice and humans. We consistently identified and delineated the transcriptome of lipofibroblasts, myofibroblasts, smooth muscle cells, pericytes, mesothelial cells, and a novel population characterized by Ebf1 expression. Subtype selective transcription factors and putative divergence of the clusters during development were described. Comparative analysis revealed orthologous subpopulations with conserved transcriptomic signatures in murine and human lung mesenchymal cells. All mesenchymal subpopulations contributed to matrix gene expression in fibrosis. This analysis would enhance our understanding of mesenchymal cell heterogeneity in lung development, homeostasis and fibrotic disease conditions.