Citations

AbstractEnterocutaneous fistulas (ECFs) profoundly impact patients’ quality of life, contributing to high morbidity rates and increased mortality due to ineffective treatment options. To address this challenge, ECFGel, a multifunctional, tissue adhesive injectable hydrogel, designed to occlude, sterilize, and promote healing of ECF tracts, is developed. ECFGel is formulated using gelatin and oxidized dextran (O‐Dex) as base components, which form chemical crosslinks within the hydrogel and with surrounding biological tissues, ensuring tissue adhesiveness. A choline and geranate‐based ionic liquid (IL) is incorporated to provide dual functionality, potent antimicrobial activity, and mechanical enhancement. By optimizing IL concentration, ECFGel achieves rapid gelation, enhanced mechanical strength, and improved elastic recoverability. Additionally, iohexol (IOH) is added for radiopacity, enabling real‐time imaging and further strengthening the hydrogel's mechanical properties. ECFGel demonstrates antiswelling properties, biodegradability, and effective tract occlusion in porcine soft tissues. It shows strong antimicrobial activity against highly resistant, patient‐derived pathogens isolated from clinical ECF cases. In a porcine perianal fistula model, ECFGel enables rapid occlusion and complete healing, promoting tissue maturation, reducing bacterial load, and increasing markers of cell proliferation and vascularization compared to untreated controls. These promising results highlight ECFGel's potential as a new therapeutic option for treating infected ECFs.

Abstract Celiac disease (CeD) is an immune-mediated condition driven by dietary gluten resulting in small intestinal mucosal inflammation and injury, along with myriads of symptoms. The only treatment is a lifelong gluten-free diet (GFD) and although most patients improve, the restriction can lead to nutrient deficiencies including fiber. While duodenal microbiota is altered in CeD, its fiber metabolic capacity is unknown. Here we show that both active and GFD-treated CeD patients had impaired microbial fiber metabolism in the small intestine which associates with depletion of the carbolytic taxa Prevotella spp. Colonization of germ-free mice with Prevotella spp increased small intestinal short chain fatty acids (SCFA). In gluten-sensitized mice expressing the celiac risk gene, HLA-DQ8, an inulin-supplemented diet facilitated microbial carbolytic function, SCFAs production and accelerated mucosal healing in the small intestine during GFD. The results support clinical investigations of dietary fiber supplementation and microbial carbolytic function to enhance responses to GFD in CeD.

Abstract Rationale: Primary ciliary dyskinesia (PCD) is a heterogeneous, rare genetic disorder characterized by dysfunctional motile cilia. PCD pulmonary manifestations include impaired airway mucociliary clearance, mucus obstruction, recurrent infection, and bronchiectasis (BE). Notably, thoracic imaging studies indicate that alveolar atelectasis is more prevalent in children with PCD than in age-matched children with cystic fibrosis (CF). However, morphologic and molecular analyses of the site of initiation and progression of PCD airway disease and relationships to PCD alveolar disease have not been reported. In this study, we analyzed region-specific morphologic and molecular characterizations of airway epithelia and alveolar regions in PCD lungs excised at transplantation to address these questions. Methods: Nine PCD lungs and seven non-diseased control lungs were included in the morphometric studies. Bronchi and proximal and distal bronchioles were defined using unique morphologic structures, and distal airway secretory cell (DASC) markers were used to differentiate between proximal and distal bronchioles. Morphologic characteristics of airway epithelia and luminal mucus were quantitated by histology, immunohistochemistry, and RNA in situ hybridization. Spatial transcriptomic technologies (GeoMx) characterized region-specific transcript expression in three airway epithelial regions (bronchi, proximal bronchioles, and distal bronchioles), intraluminal mucus plugs, and alveolar regions of PCD compared to control lungs. Finally, pulmonary function data from a cohort of PCD subjects were analyzed to investigate correlations between alveolar morphologic findings and alveolar gas exchange function, i.e. diffusing capacity for carbon monoxide (DLCO). Results: PCD bronchi and proximal bronchioles exhibited ectasia, characterized by increased MUC5AC and MUC5B-producing cells, mucus plugs containing MUC5AC and MUC5B mucins infiltrated by neutrophils, and epithelial gene signatures associated with ectasia. In contrast, PCD distal bronchioles were not ectatic and exhibited MUC5B-dominant DASCs, mucus plugs containing selectively MUC5B mucin infiltrated by mixture of neutrophils and macrophages, and gene signatures suggestive of impaired ciliogenesis. Molecular analysis revealed upregulation of pathways associated with muco-inflammation, antimicrobial defense, and the innate immune system routinely in PCD airway epithelia. IL-1β gene signatures were observed in both PCD airway and alveolar epithelia, whereas interferon-stimulated gene signatures were restricted to airway epithelia. Transcriptomic and morphologic analyses suggested that PCD alveolar disease was initiated by distal bronchiolar obstruction, associated with chronic microatelectasis, progressive loss of alveolar epithelial type 2 (AT2) cells, and eventual non-resolving fibrosis. A significant percentage (28%) of people with PCD exhibited reduced DLCO. Conclusions: PCD airway disease is manifest by distinct profiles of proximal vs distal bronchiolar disease associated with microatelectatic and fibrotic alveolar disease.

Abstract RATIONALE: Alveolar Type 2 (AT2) cells play a critical role in maintaining alveolar function and facilitating repair processes in the lung. Despite recent advancement on transcriptomic mapping of cell identities and AT fate selection, the mechanisms governing AT2 fate plasticity remain elusive. Notably, in AT2-derived pulmonary diseases such as COPD and IPF, disruptions in AT2 differentiation are observed, suggesting alterations may exist in the regulation of fate plasticity. Therefore, a refined model describing the regulation of AT2 fate plasticity is needed, along with an assessment of the model's conservation across murine and human AT2s. METHODS: We performed scRNA-sequencing, ATAC-seq, and bioinformatic analysis on samples across murine lung development and validating findings by immunostaining of embryonic lungs. We conducted organotypic cultures as well as genetically engineered mouse model experiments to assess changes in AT2 fate plasticity in. Further, we established a Mayo Clinic patient lung cell biorepository from patient tissue derived AT2s. In parallel, we performed long read scRNA-sequencing for isoform discovery, organotypic culture and immunostaining in human AT2 organoids, to characterize the presence of similar regulatory mechanisms. RESULTS: Throughout the late embryonic and early perinatal period, we found a window of fate plasticity exists in AT2s whereby they retain the capacity to give rise to AT1s, and that its being closed over time by the bZIP transcription factor C/EBPα, as its genetic deletion in AT2s results in regaining fate plasticity. By ATAC-seq and ChIP-seq, we found C/EBPα binds closely to the Dlk1 locus, a Notch signaling regulator implicated in AT2-to-AT1 conversion that is upregulated following Cebpa deletion. Following adult lung injury, we found C/EBPα downregulation is required for re-accessing AT2 fate plasticity and is likely mediated by the dominant negative C/EBP family member CHOP. Finally, we observe Cebpa loss also activates a “defender” AT2 state enriched in interferon stimulated antipathogenic gene expression that is distinct from its reparative state and propose AT2s can toggle between these states following infection to robustly protect and repair alveoli.Further, we validated the presence of C/EBPα and absence of Dlk1 in human AT2s isolated from healthy adult patients, which persists following in vitro expansion. We conclude that AT2 cells window of fate plasticity is dictated by a molecular circuit involving C/EBPα suppressing and Dlk1. Future works aim to remove C/EBPα in human AT2s to reopen their fate plasticity for cell therapy.

Abstract RATIONALE: The alveolar epithelium is primarily comprised of two cell types – the flat alveolar type I (AT1) cell responsible for gas exchange, and the cuboidal AT2 cell responsible for surfactant secretion, alveolar defense and acts as facultative stem cells in injury repair. Both cell types emerge from a population of bipotent distal progenitors (DPs). While the FGF signaling involved in directing AT2 fate has been identified, the mechanism by which AT2 differentiation is precisely timed is not well understood. In embryonic stem cells, it has been demonstrated that differentiation is regulated by a biophysical parameter, cell membrane tension (CMT), that acts upstream of FGF signaling by suppressing receptor endocytosis. Given this, we sought to investigate whether and how CMT plays a role in timing the alveolar epithelial differentiation, particularly focusing on how CMT might regulate FGF signaling. METHODS: DPs were isolated from E15.5 mice and plated on top of or embedded in Matrigel. DPs were treated with small molecules, to rise CMT (CHIR22901 and Methyl Beta cyclodextrin; MBCD), inhibiting either endocytosis (Dynasore) or pathways downstream of FGF signaling (FR180204, Akt VIII, KRpep-2d, NSC 74859). CMT is measured both with optical trap technology and FlipTR dye, which uses fluorescence lifetime imaging microscopy. Tissues and cells were immunostained and imaged using markers for AT1 and AT2 cells, Fgfr2, and the endocytosis marker EEA1. The transgenic mice for this project are in a C57B6 strain background and include a pan-lung epithelial Cre (TgNkx2.1-Cre) bred to Cre-dependent ERK reporter (Rosa26SAE). RESULTS: We found that CMT reduces immediately prior to alveolar differentiation, and that increasing CMT with CHIR and MBCD blocks differentiation of DPs in culture. As CMT reduces, we observe an increase in endocytosis and internalization of Fgfr2 in both cultured cells and in vivo at E17.5. Further, scRNAseq analysis showed an upregulation of ERK target genes and we have validated higher ERK activity in nAT2s in the reporter line. Further, we validated that CMT regulates FGF signaling, as decrease in CMT results in more Fgfr2 endocytosis that likely activates ERK and Akt signaling. Our small molecule inhibition data suggests that ERK and Akt signaling are required for AT2 fate selection and we propose that they are the downstream targets of FGF signaling that are activated following lowered CMT and increased Fgfr2 endocytosis. Our findings are of fundamental importance for understanding how cell mechanics regulates biochemical signaling and fate selection during lung development.

Abstract Non-tubercular mycobacteria (NTM) present a serious challenge in managing lung transplant candidates and recipients. Pretransplant NTM isolation has been associated with post-transplant NTM infection and significantly increased mortality risk. Strategies for managing pre-transplant NTM isolation vary widely in clinical practice. We present a 35-year-old lung transplant candidate from a tuberculosis-endemic country with severe pulmonary hypertension and Eisenmenger Syndrome due to unrepaired atrial septal defect, who underwent bilateral lung transplantation. Two years prior to transplant, she was successfully treated for pulmonary tuberculosis with six months of an unknown regimen in her home country. After moving to the U.S., she received M. tuberculosis (MTB) clearance from her state tuberculosis board, with three negative sputum samples six months prior to transplant. During her transplant evaluation, multi-drug-resistant M. fortuitum complex was isolated from one mycobacterial sputum culture. There was no clinical or radiographic evidence of active lung infection, and no targeted therapies were added. Standard peri-transplant antibiotics (vancomycin, cefepime, and caspofungin) were given. The patient received basilixamab for induction immunosuppression, and tacrolimus, mycophenolate mofetil, and prednisone for maintenance immunosuppression. Explant pathology showed necrotizing granuloma in the right upper lobe with acid-fast bacilli (AFB). The differential diagnosis included non-viable MTB, viable MTB despite prior clearance, or M. fortuitum. Three bronchoscopy washings were obtained in the first week post-transplant and returned negative for MTB by PCR, AFB stain, and culture. In collaboration with the Mycobacteriology laboratory, broad-range PCR testing was performed on the formalin-fixed paraffin-embedded samples from the explanted right lung, which was positive for MTB complex DNA. In total, the patient completed six sputum samples within the year pre-transplant and six respiratory samples within a month post-transplant, all of which returned negative for MTB by PCR, AFB stain, and culture. Blood flow cytometric (FC) immunoprofiling with mycobacterial antigens and controls was conducted (Escalante et al. 2015). The patient's FC antigen-specific activated T-cell profiling was consistent with a reduced risk of latent TB infection (FC CD4+ CD25+CD134+ T-cells (RD1-peptides-nil) = 0.19%). T-cell reactivity to MTB300 and PPD antigens suggested prior MTB, NTM, and/or BCG vaccination exposures. She progressed well without any preventive therapy for LTBI. FC immunoprofiling one year after transplantation showed similar results. She continues to have good pulmonary function and quality of life in her second-year post-transplant without evidence of NTM or MTB infection. Escalante P, et al. Combinatorial Immunoprofiling in Latent Tuberculosis Infection. Am J Respir Crit Care Med. 2015;192(5):605-17. PMCID: PMC4595688.

Abstract Rationale: Tandem exposure to cannabinoids and nicotine during pregnancy poses significant risks to fetal development and health including low birth weight, preterm birth, and perinatal death. However, the effect of co-exposure with the most prominent cannabinoids found in Cannabis sativa (Trans-Δ-9-tetrahydrocannabinol (THC) and cannabidiol (CBD)) alongside nicotine on fetal lung development is not well understood. This study aims to investigate the combinatorial impact of CBD, THC, and nicotine, on human lung development through investigating cellular senescence and the potential role of senolytics in alleviating these effects.Methods: Human fetal lung explants from 10 to 16 weeks gestation were cultured on air-liquid interface and treated with various concentrations of nicotine (1µM), CBD, and THC (5µM, or 2.5µM each when used in combination), for 72 hours. To assess the effect of senolytics, the explants were also treated for 48 hours with a combination of CBD, THC, and nicotine. Subsequently, senolytics, Dasatinib (D, 250nM) and Quercetin (Q, 375nM), were added or not for an additional 24 hours. After the 72-hour treatment, explants were collected and either fixed for immunofluorescence staining (IF) analyses or used for RNA extraction and quantitative reverse transcription PCR (qRT-PCR). Additionally, the supernatants were used to evaluate the expression of the senescence-associated secretory phenotype (SASP), conducted by Eve Technologies Corporation-Canada. This study used Luminex xMAP technology for multiplexed quantification of 96 Human cytokines, chemokines, and growth factors.Results: Our results demonstrated that co-exposure to CBD, THC, and nicotine increases the expression of the senescence markers CDKN1A (0.1163±0.02384, p=0.0094, n=8) and CDKN2A (0.005793±0.002157, p=0.0516, n=8) encoding P21 and P16 respectively. Moreover, the IF staining showed increased expression of the DNA damage marker ɣH2A.X following treatment with CBD+THC+nicotine (7.375±2.154% vs 2.245±0.8329%, p=0.0604, n=6). Furthermore, the lung explants treated with cannabinoids and nicotine released cytokines and chemokines involved in immune responses and inflammation among others IL-6, IL-8, M-CSF, MCP-3, MIP-1β, TNFα, MIP-3β, and TSLP (p<0.05). The addition of D+Q reduced SASP secretion and showed a decreasing trend in expression of the senescence genes CDKN1A and CDKN2A compared to the triple treatment.Conclusion: Taken together, our results showed that the combinatorial exposure to CBD, THC, and nicotine increases senescence, DNA damage, and the senescence-associated secretory phenotypes (SASP) secretion. The use of senolytics (D+Q) may prove to be a promising therapeutic option, alleviating several of the negative effects induced by the different substances.

Abstract RATIONALE: Susceptibility to severe COVID-19 and long COVID may be influenced by pre-existing lung structure. ECG-gated, non-contrast cardiac CT scans, originally acquired for assessing coronary artery calcium, are available in multiple longitudinal cohort studies and have been previously validated against lung CT scans for densitometry; however, protocol variation across cohorts may hinder direct comparison. To address this, we developed a quality control (QC) rubric to grade cardiac CT scans for standardized quantitative lung measures across cohorts. METHODS: C4R assessed SARS-CoV-2 infection and outcomes in 14 NHLBI cohorts, of which 10 (six community-based and four longitudinal case-control studies) included CT scans (12,459 participants with lung and 13,752 with cardiac CTs). Logic rules were applied to categorize scans (Table): Grade “A-B” scans had 2-3.5mm slice thickness, at least 20 axial slices, no lung cropping in axial view and lung and airway volumes greater than the cohort-specific 1st percentile. “C-D” scans had one lung obscured by cropping around the heart, so quantitative measures were restricted to the lung in complete view. “F-H” scans were deemed unusable, suffering from low slice count (<20), severe image cropping, low lung or airway volumes (< 1st%ile). Visual inspection was performed on a sample from each category to ensure accuracy. Percent of lung voxels in low attenuation areas < -950 HU (LAA950), high attenuation areas (-600 to -250 HU, HAA) and 15th percentile HU value (perc15) were measured. Within-participant variability was quantified using the intraclass correlation coefficient (ICC). RESULTS: We performed QC on cardiac CT scans from an initial sample of 6,275 participants from three C4R cohorts (ARIC, CARDIA, MASALA), imaged with GE or Siemens scanners. Of these, 5,125 (81.7%) were graded “A-B”, 496 (7.9%) were “C-D” and 654 (10.4%) were “F-H”. The percentage of “F-H” scans varied widely across cohorts [<1% to 26%] due to image cropping. Among the 5,621 cardiac CT scans graded A-D, the mean±SD for LAA950, HAA and perc15 respectively were 1.1±2.1%, 7.2±4.7% and -887±33 HU. Repeated imaging was available for 107 participants (time interval 5.1±0.7y). For paired scans in grades A-B (n=101/107), the within-participant correlation for LAA950, HAA and perc15 were 0.91, 0.38 and 0.74 respectively. CONCLUSION: Initial application of QC to cardiac CT scans identified scans with high reproducibility, with the aim to provide valid quantitative lung metrics on over 20,000 highly characterized participants in C4R.

Abstract Rationale Extracellular matrix (ECM) accumulation results from an imbalance between ECM deposition and degradation, due to persistently increased ECM synthesis or reduced resorption. Fibroblasts, as key regulators of ECM homeostasis in both health and disease, are essential targets to rebalance ECM turnover. Recent findings indicate that fibroblasts are regulated by immune cells, which can influence ECM deposition. Specifically, myeloperoxidase (MPO), an enzyme produced by neutrophils, has been shown to enhance fibroblast-driven ECM accumulation in various diseases. In patients with radiographic Interstitial Lung Disease (ILD), elevated MPO levels have been correlated with reduced lung capacity (Cantin et al., 1987), while recent studies report increased MPO levels in the bronchoalveolar lavage fluid of ILD patients (Ngo et al., EJROR 2024). Additionally, MPO and related immune dysregulation proteins are found in fibrotic alveoli in Idiopathic Pulmonary Fibrosis (IPF) (Herrera et al., JCI Insight 2022). These findings suggest an association between MPO and lung fibrosis, yet it remains uncertain whether MPO directly influences ECM accumulation in fibrotic progression. Methods Patient tissue and serum samples were collected with consent, and MPO levels were quantified using ELISA and immunofluorescence techniques. Biochemical assays were performed using MPO isolated from human leukocytes, recombinant cathepsin K, and hydrogen peroxide to investigate whether MPO influences cathepsin K activity. Precision cut lung slices (PCLS) were prepared from untreated C57BL/6 mice and treated with transforming growth factor-beta (TGFβ) and/or MPO for four days, with cathepsin K activity assessed using fluorescence assays. Additionally, MPO-knockout and wild-type mice received intratracheal bleomycin, and after 14 days, cathepsin K activity was quantified via in vivo imaging. Collagen content, as an indicator of fibrosis, was measured by quantifying hydroxyproline in harvested lung tissue. Results/Discussion/Conclusion Increased MPO levels were observed in immunofluorescent-stained samples (57%; 4/7) and serum samples (30%; 3/10) from IPF patients. Biochemical assays demonstrated that physiological levels of MPO (0.2 µg/ml) combined with H2O2 reduced recombinant cathepsin K activity by 85%. In PCLS treated with MPO, cathepsin K activity decreased by 50% with or without TGFβ. After bleomycin treatment, wild-type mice showed reduced cathepsin K activity, while MPO-knockout mice maintained activity levels. Both survival and weight loss were similar between wild-type and MPO-knockout mice; however, collagen content was significantly higher in wild-type mice, whereas MPO-knockout mice were protected from bleomycin-induced collagen accumulation. These findings suggest that MPO contributes to ECM accumulation in lung fibrosis, potentially through inhibition of collagenolytic enzymes such as cathepsin K.

Abstract Rationale: The 7th World Symposium on Pulmonary Hypertension highlighted the importance of patient-reported outcomes in clinical assessment of pulmonary arterial hypertension (PAH). Patient-reported outcomes, such as health-related quality of life (HRQOL), have been associated with morbidity and mortality in PAH, but the added value of HRQOL measures to validated risk prediction models remains unknown. Methods: Adults with RHC-proven PAH from the Redefining Pulmonary Hypertension through Pulmonary Vascular Disease Phenomics (PVDOMICS) Study, a multi-center observational study, were included in this study. The Pulmonary Hypertension Association Registry (PHAR) was used as a validation cohort. HRQOL was measured using the emPHasis-10 score, where higher scores reflect worse HRQOL. Noninvasive risk assessment was measured by REVEAL Lite 2 risk score, categorized into 3-strata. Cox proportional hazard models were used to model survival evaluating the interaction between risk strata and HRQOL scores, adjusting for age, sex, and incident diagnosis. Harrell's C-statistics were calculated to compare discriminative performance. Results: From the PVDOMICS and PHAR cohorts, 265 and 2671 individuals with PAH and complete emPHasis-10 and REVEAL Lite 2 scores were included. Both cohorts had similar demographics, with an average age of 52±13 and 56±16 years, 75% and 72% female, 81% and 75% White, in the PVDOMICS and PHAR cohorts respectively. Over 87% of PVDOMICS were prevalent cases of PAH, while in the PHAR half were incident. Most individuals (62%) in PVDOMICS were REVEAL Lite 2 low risk, with 16% intermediate and 22% high risk; by contrast, in PHAR 33% were low, 29% intermediate, and 38% high risk. REVEAL Lite 2 scores demonstrated weak to moderate correlation with emPHasis-10 scores in both cohorts (PVDOMICS r=0.48 p<0.001; PHAR r=0.33 p<0.001). Notably, the full range of emPHasis-10 scores were observed in the low-risk individuals across both cohorts. In the PVDOMICS adjusted Cox models, higher emPHasis-10 scores predicted poorer survival (p=0.047) particularly among low-risk individuals (interaction versus high-risk group p=0.08). Similar adjusted models in PHAR demonstrated emPHasis-10 to be a confounder (p=0.001) without significant interaction by risk strata (low vs high-risk interaction p=0.94), but the greatest change in median survival time was observed in the low-risk group (Figure 1). Addition of emPHasis-10 to REVEAL Lite 2 scores improved discrimination in predicting survival (PVDOMICS: C-statistic 0.71 vs 0.73; PHAR: C-statistic 0.67 vs 0.69). Conclusions: Across two multicenter cohorts of incident and prevalent PAH, HRQOL as measured by the emPHasis-10 score improved discrimination for survival, particularly among individuals with lower risk scores.