Vassallo, R.

Abstract Introduction The inhalation of aerosolized therapeutics is a promising strategy for the targeted delivery of drugs and biologics to the lungs. We have previously demonstrated the efficacy and feasibility of nebulized exosomes using a platelet-derived exosome product (PEP: Rion, Inc.), which significantly attenuated emphysema in a murine model (PMID: 38325750). However, the PEP aerosol’s physical properties—such as size, tissue deposition, anatomic location, and stability remain incompletely characterized. To address this, we utilized a cascade impactor to define aerosol particle size and deposition patterns and employed immunofluorescence staining to assess the regional and anatomic localization of nebulized PEP within the murine lung. Methods PEP particle size and tissue deposition were determined by passing the aerosol through a Marple cascade impactor (CI, DSI), which was connected to an inhalation tower (DSI). PEP aerosols were generated by a vibrating mesh nebulizer (Aerogen, Cat No AG-AL1100) at a controlled velocity and humidity and delivered to the CI at a flow rate of 2L/min. Aerosols were separated by size and allowed to deposit at the corresponding stages as they passed through the pinholes of the stage, by design. The stages were weighed after nebulization, and the percentage of aerosol deposition was calculated to simulate the human respiratory tree, according to the manufacturer’s specifications. In parallel, fluorescence DiR-labeled PEP was nebulized to spontaneously breathing mice, and confocal microscopy was used to determine intrapulmonary distribution. Results A total of 2.25 mL of PEP reconstituted in 0.9% normal saline (11.4 mg/mL final concentration) was nebulized for 35 minutes at a flow rate of 2 L/min, yielding 3.5 mg of total aerosol deposition, which is 13.6% of the total PEP mass (25.65mg) being nebulized, across the CI stages. Of this, 74% of the aerosolized particles with a particle size ≤ 1µm and deposited in Stages 7, 8 & F, corresponding to the deep regions of the lungs, specifically regions beyond the alveolar ducts and within the alveoli. In vivo 3D confocal imaging corroborated these findings, revealing abundant DiR-labeled PEP deep into the alveolar space within the peripheral regions of all lung lobes. Conclusion The study result demonstrates the feasibility and capability of delivering aerosolized PEP deeply into the alveolar regions of the lung at the given pressure, humidity, flow rate and nebulizer settings. This study establishes a methodological framework for characterizing and optimizing aerosolized exosome therapeutics for precise, region-specific delivery within the respiratory tract. This abstract is funded by: Department of Defense, Mayo Clinic Funding

Abstract Respiratory viral infections such as influenza and SARS-CoV-2 can lead to the development of chronic lung disease and impaired pulmonary function long after the recovery from acute illness. However, the mechanisms underlying these post-acute sequelae remain poorly understood. Using human lung samples from patients with post-acute sequelae of COVID-19 with pulmonary fibrosis (PASC-PF) and an aged mouse model of chronic lung disease following acute viral infection, we found that CD4+ T cells were enriched near the dysplastic Krt8Hi epithelial regions of the lung. Similarly, in aged mice, CD4+ T cells localized to Krt8Hi areas, and their spatial distribution correlated positively with the extent of epithelial dysplasia. Further analysis of bronchoalveolar lavage (BAL) fluid from COVID-19 convalescent individuals revealed a distinct population of FoxP3−PD-1HiCD4+ T cells highly expressing ICOS, CD69, and CD103 - markers characteristic of both T follicular helper (TFH) and tissue-resident memory (TRM) cells, which we have defined as tissue-resident helper (TRH) cells previously. Notably, the frequency of these TRH-like cells correlated positively with the extent of reticulate or consolidated regions on chest CT scans, indicative of lung fibrosis. Furthermore, the frequency of these cells was negatively associated with lung functional parameters including forced vital capacity (FVC). These findings suggest that PD-1HiCD4+ TRH-like cells expand in PASC and may contribute to chronic lung sequelae. Given that Bcl6 is required for TRH differentiation, we employed an aged, CD4-specific, inducible Bcl6-deficient (Bcl6iΔCD4) mouse model to evaluate the functional contribution of these cells to post viral lung fibrosis. Bcl6 deficiency led to a marked reduction in total and antigen-specific PD-1HiCD4+ TRH cells, as confirmed by flow cytometry and immunofluorescence. Strikingly, Bcl6 iΔCD4 mice displayed significantly decreased inflammatory and fibrotic lesions in the lung compared to wild-type controls. Moreover, Bcl6 iΔCD4 mice exhibited enhanced lung regeneration, evidenced by increased AT1 and AT2 epithelial cells and improved pulmonary function. Together, these findings identify Bcl6-dependent CD4+ TRH responses as key drivers of post-viral chronic lung sequelae. This study reveals a novel immunopathogenic mechanism linking persistent tissue helper memory T-cell responses to impaired lung repair and fibrosis, providing new insight into the development of post-viral lung disease and potential therapeutic targets for conditions such as respiratory long COVID. This abstract is funded by: American Lung Association, NIH

Abstract Introduction We previously demonstrated that nebulized platelet-derived exosome product (PEP) alleviates cigarette smoke (CS)-induced murine emphysema. To elucidate the underlying molecular mechanisms, we conducted single-nucleus RNA sequencing of lung tissue, which initially revealed a shift in the cell source for TNF signaling (a critical regulator of inflammation in COPD lungs) from innate lymphoid cells (ILC) to alveolar macrophages (aMac) after chronic CS exposure. In this study, we conducted a more in-depth analysis of cellular communication to determine the mechanism by which PEP modulates CS-induced TNF signaling in the lung microenvironment. Methods Single-cell transcriptomics and cell-cell communication analyses were performed between CS-exposed placebo control lungs vs. sham control lungs and CS-exposed PEP-treated vs. placebo control lungs using R packages, including Seurat, CellChat, Patchwork, Future, and NMF. A comparative analysis of ligand-receptor interactions across these datasets was conducted using CellChat to infer intercellular signaling networks. Results Single-nuclear RNA-seq analysis revealed that the Thy1-ITGAX_ITGB2 signaling pathway was significantly upregulated by CS, acting as the adhesive and activating bridge that facilitates the switch in TNF production from ILC to aMac. Thy1 is a GPI-anchored ligand expressed on ILCs, and ITGAX_ITGB2 is its cognate receptor on aMacs. Concurrently, Ptprc-Mrc1 signaling, an inhibitory pathway in ILC-aMac crosstalk, was downregulated, contributing to sustained aMac activation by CS. Furthermore, analysis of aMac-aMac interactions revealed upregulated GRN/Sort1 signaling, which promotes the internalization of progranulin, an anti-inflammatory glycoprotein that competes for TNF/TNFR signaling. Ptprc-Mrc1 signaling was also downregulated within the aMac autocrine/paracrine network, further perpetuating a pro-inflammatory state. In contrast, treatment with PEP downregulated Thy1-ITGAX_ITGB2 signaling and upregulated Lamc1-Cd44 signaling, a hallmark of aMac deactivation, thereby inducing a phenotypic switch in inflammatory macrophages toward a reparative M2 state, promoting the resolution of CS-induced chronic inflammation. Conclusions These data suggest that the transition of TNF production from ILCs to aMacs under chronic CS stress is driven by increased Thy1-ITGAX_ITGB2 interactions and decreased Ptprc-Mrc1 signaling between ILCs and aMacs crosstalk within the local tissue. Enhancing the crosstalk between Lamc1-Cd44 and Ptprc-Mrc1 may suppress TNF production and attenuate CS induced aMac activation. Targeting these pathways, as demonstrated by nebulized PEP treatment, holds therapeutic potential to deactivate inflamed aMacs and promote homeostasis in the inflamed lung. This abstract is funded by: Department of Defense, Mayo Clinic Funding

Abstract Rationale Interstitial lung disease (ILD) affects ∼50% of patients with systemic sclerosis (SSc) and is a leading cause of mortality. Visual assessment of ILD extent and morphologies, including ground-glass opacities (GGO) and reticular densities (RD), suffers from poor interobserver agreement, limiting prognostic consistency. CALIPER Quantitative CT (QCT) provides reproducible volumetric measures of parenchymal and vascular features. This study evaluated baseline QCT-derived lung features for association with transplant/mortality risk in SSc-ILD. Methods A retrospective cohort of SSc patients (≥18 years) seen at an academic center from 2007-2018 was identified using ICD codes, with ACR/EULAR 2013 SSc criteria confirmed by chart review. Inclusion required ILD on high-resolution CT (HRCT) ±2 years of SSc diagnosis, and PFTs ±6 months of HRCT. CALIPER quantified volumes of normal lung, GGO, RD, hyperlucency, honeycombing (HC), and pulmonary vascular-related structures (PVRS), which are expressed as percentages of total lung volume. Right heart catheterization confirmed pulmonary hypertension (PH) defined by 2022 ESC/ERS criteria (Eur Heart J, 2022). Cox hazards models assessed associations with combined mortality/transplant outcome, a priori adjusted for age, sex, diffuse SSc subtype, ILD duration, and PH (Column B), and further adjusted for baseline percent predicted FVC (ppFVC) and ppDLCO (Column C)(Table 1). Results Of 132 SSc-ILD patients, median SSc and ILD disease durations at HRCT were 1.4 (0.6, 2.4) and 0.0 (0.0, 0.5) years. Median feature percentages: total ILD 13.0% (IQR: 6.4, 21.0), GGO 10.2% (4.6, 16.9), RD 1.9% (0.8, 3.7), HC 0.03% (0.00, 0.09), PVRS 3.2% (2.3, 4.1). Median ppFVC was 81.1% (69.4, 92.6), and ppDLCO 60.0% (46.0, 75.7); 21 patients had PH. There were 2 transplants and 45 deaths. Every 5% increase in baseline total ILD and GGO associated with 22% (HR 1.22; 95% CI 1.12-1.33) and 24% (1.24; 1.12-1.36) higher risk of transplant/mortality on unadjusted models (Column A), respectively, remaining significant after a priori (CB) and full adjustment (CC)(Table 1). Every 1% increase in RD and PVRS conferred 19% (1.19; 1.08-1.31) and 33% (1.33; 1.08-1.63) higher risk of transplant/mortality in unadjusted models (CA); RD retained significance after a priori adjustment (1.17; 1.03-1.33), while PVRS trended non-significant in adjusted models. Conclusions In early SSc-ILD, QCT-derived GGO, RD, and PVRS provide robust prognostic value for mortality/transplant risk assessment, with small increases in baseline severity—at or below visual detection thresholds—associating with clinically meaningful risk increases. These findings support integrating baseline QCT biomarkers into risk stratification beyond clinical and PFT measures. This abstract is funded by: Rheumatology Research Foundation

Abstract Introduction: Prolonged mechanical ventilation can often be complicated with post-intubation tracheal stenosis (PITS). It usually manifests within few weeks following extubation. However, delayed presentations are rare and have disastrous outcomes if not identified. Early recognition of stridor and dyspnea post-extubation is crucial for timely intervention. We report a case of late presentation of PITS in a 67-year-old female patient with new-onset stridor after prolonged intubation. Case: A 67-year-old female with past medical history of coronary artery disease, type 1 diabetes, obstructive sleep apnea presented with a six-week history of progressive dyspnea on exertion and stridor. This occurred in the context of a three-week intubation following a pulseless electrical activity (PEA) arrest over five months prior. Immediately post-extubation, the patient had developed stridor without hypoxia or exertional symptoms and was subsequently discharged home without further investigation. On this presentation, she was afebrile and saturating 95% on room air with audible inspiratory stridor at bedside. Respiratory viral panel and autoimmune work up was negative. Computed tomography (CT) scan noted a significant circumferential narrowing in the proximal portion of the trachea measuring 2.5 cm in length with 0.5 cm in width with associated wall thickening. She was managed with 2.25% racemic epinephrine as needed and prednisone 10 mg daily with rescue continuous positive airway pressure (CPAP) until intervention could be safely performed after clopidogrel washout. On Day 5, she underwent rigid bronchoscopy with extensive CO2 laser vaporization (Fig. B) of scar release and paclitaxel-coated balloon dilation of the 3 mm narrowing (Fig. A) to 12 mm (Fig. C). Her stridor resolved with significant improvement in dyspnea and exercise tolerance. She was subsequently discharged on a steroid taper with outpatient interventional pulmonology follow up for repeat rigid bronchoscopy in 4 weeks to evaluate for restenosis. Discussion: Tracheostomy is often considered when extended period of intubation is anticipated as it can lead to increased risk of complications like PITS. It is important to have a high index of suspicion for tracheal stenosis in patients with a history of prolonged mechanical ventilation presenting with new-onset stridor, even when the symptoms are mild or intermittent. Early recognition and timely intervention can prevent the progression of stenosis and improve morbidity and clinical outcomes. Additionally, these patients tend to have an increased incidence of recurrence which requires close follow up for restenosis.