RATIONALE Alveolar epithelial type 2 (AT2) cells from mouse and human lungs express the growth hormone releasing hormone receptor (GHRH-R) and require growth hormone for homeostasis. Inhibition of GHRH-R using an antagonist, MIA-602, reduces lung inflammation and fibrosis due to bleomycin in a murine model. MIA-602 also minimizes LPS-driven production of pro-inflammatory and pro-fibrotic mediators by AT2 cells derived from human pluripotent stem cells (iAT2 cells). GHRH-R and TLR4 (toll-like receptor 4) may share downstream pathways such as JAK2/STAT3 and NF-kB, both leading to inflammation through stimulation of cytokine and chemokine gene expression. To extend these observations, we have recently reported that MIA-602 modulates acute lung injury due to SARS-CoV-2 pneumonia in mice by inhibiting alveolar epithelial cell necroptosis thus limiting lung inflammation. METHODS We investigated how GHRH-R interacts with TLR4 to modulate AT2 cell driven lung inflammation and fibrosis. We used cultured human iAT2 cells treated with LPS (500 ng/mL for 24 h). Specific antibodies were then used to immunoprecipitate selected signaling proteins, followed by western blotting to identify precipitated complexes. Incubation with LPS increased expression of GHRH, the peptide ligand that normally binds to and activates GHRH-R. Importantly, GHRH-R antagonist MIA-602, was able to block activation of JAK2/STAT3 and NF-kB signaling after iAT2 cells were incubated with LPS. RESULTS Such evidence leads to the hypothesis that “crosstalk” occurs between GHRH-R and TLR4 during the inflammatory response of iAT2 cells to LPS. We explored evident functional relationships between GHRH-R and TLR4. Using specific antibodies for each receptor, pulldown assays revealed co-immunoprecipitation of phospho-STAT3 with GHRH-R as well as several adaptor proteins including TIR domain containing adaptor protein (TIRAP) and myeloid differentiation primary response 88 (MyD88) with TLR4. We conclusively identified G-protein coupled receptor kinase 4 (GRK4) as a joint target, which potentially binds GHRH-R, TLR4, phospho-STAT3 and TIRAP simultaneously. CONCLUSIONS In view of the kinase activities of GRK4 and the likelihood of GHRH-R, a G-protein coupled receptor, being a substrate of GRK4, we are currently investigating the role GRK4 may play in a complex consisting of GHRH-R, TLR4 and their direct downstream mediators. These studies provide foundational support for our observation that GRK4 can facilitate the inhibitory effects of GHRH-R antagonist, MIA-602, on inflammation mediated by AT2 cells through TLR-4. Such results indicate that anti-inflammatory actions of MIA-602 may be mediated by inhibition of the TLR4, which limits expression of the inflammatory phenotype in alveolar epithelial cells.

Electronic cigarettes (e-cigarettes) have emerged as potential harm reduction tools for conventional smokers, with proponents claiming reduced health risks. However, comprehensive evidence regarding their long-term cardiopulmonary effects remains limited. This scoping review aims to systematically map and compare the cardiopulmonary effects of e-cigarettes versus traditional cigarettes through the synthesis of clinical and preclinical evidence, focusing on chronic exposure outcomes and biological mechanisms. Through systematic searches in MEDLINE (n = 1,398), EMBASE (n = 1,300), and Web of Science (n = 642), we identified 30 relevant studies from 3,791 unique records examining cardiopulmonary endpoints with chronic exposure measurements. Although e-cigarettes generally demonstrated reduced toxicity compared with traditional cigarettes, they induced significant cardiopulmonary alterations. Key findings included increased inflammatory responses, oxidative stress, endothelial dysfunction, and altered immune responses. Both products affected cardiovascular function and tissue remodeling, although e-cigarettes typically showed reduced severity. Notably, the base components of e-cigarettes, propylene glycol/vegetable glycerin, independently contributed to observed effects. Current evidence suggests e-cigarettes may offer harm reduction potential compared with traditional cigarettes but are not harmless with respect to cardiopulmonary health. Long-term clinical data remain limited, highlighting the need for continued research on their chronic health outcomes.

Purpose: Growth hormone-releasing hormone (GHRH) is a 44-amino acid peptide that regulates growth hormone (GH) secretion. We hypothesized that GHRH receptor (GHRH-R) in alveolar type 2 (AT2) cells could modulate pro-inflammatory and possibly subsequent pro-fibrotic effects of lipopolysaccharide (LPS) or cytokines, such that AT2 cells could participate in lung inflammation and fibrosis. Methods: We used human alveolar type 2 (iAT2) epithelial cells derived from induced pluripotent stem cells (iPSC) to investigate how GHRH-R modulates gene and protein expression. We tested iAT2 cells' gene expression in response to LPS or cytokines, seeking whether these mechanisms caused endogenous production of pro-inflammatory molecules or mesenchymal markers. Quantitative real-time PCR (RT-PCR) and Western blotting were used to investigate differential expression of epithelial and mesenchymal markers. Result: Incubation of iAT2 cells with LPS increased expression of IL1-β and TNF-α in addition to mesenchymal genes, including ACTA2, FN1 and COL1A1. Alveolar epithelial cell gene expression due to LPS was significantly inhibited by GHRH-R peptide antagonist MIA-602. Incubation of iAT2 cells with cytokines like those in fibrotic lungs similarly increased expression of genes for IL1-β, TNF-α, TGFβ-1, Wnt5a, smooth muscle actin, fibronectin and collagen. Expression of mesenchymal proteins, such as N-cadherin and vimentin, were also elevated after prolonged exposure to cytokines, confirming epithelial production of pro-inflammatory molecules as an important mechanism that might lead to subsequent fibrosis. Conclusion: iAT2 cells clearly expressed the GHRH-R. Exposure to LPS or cytokines increased iAT2 cell production of pro-inflammatory factors. GHRH-R antagonist MIA-602 inhibited pro-inflammatory gene expression, implicating iAT2 cell GHRH-R signaling in lung inflammation and potentially in fibrosis.

COVID-19 pneumonia causes acute lung injury and acute respiratory distress syndrome (ALI/ARDS) characterized by early pulmonary endothelial and epithelial injuries with altered pulmonary diffusing capacity and obstructive or restrictive physiology. Growth hormone-releasing hormone receptor (GHRH-R) is expressed in the lung and heart. GHRH-R antagonist, MIA-602, has been reported to modulate immune responses to bleomycin lung injury and inflammation in granulomatous sarcoidosis. We hypothesized that MIA-602 would attenuate rVSV-SARS-CoV-2-induced pulmonary dysfunction and heart injury in a BSL-2 mouse model. Male and female K18-hACE2tg mice were inoculated with SARS-CoV-2/USA-WA1/2020, BSL-2-compliant recombinant VSV-eGFP-SARS-CoV-2-Spike (rVSV-SARS-CoV-2), or PBS, and lung viral load, weight loss, histopathology, and gene expression were compared. K18-hACE2tg mice infected with rVSV-SARS-CoV-2 were treated daily with subcutaneous MIA-602 or vehicle and conscious, unrestrained plethysmography performed on days 0, 3, and 5 (n = 7 to 8). Five days after infection mice were killed, and blood and tissues collected for histopathology and protein/gene expression. Both native SARS-CoV-2 and rVSV-SARS-CoV-2 presented similar patterns of weight loss, infectivity (~60%), and histopathologic changes. Daily treatment with MIA-602 conferred weight recovery, reduced lung perivascular inflammation/pneumonia, and decreased lung/heart ICAM-1 expression compared to vehicle. MIA-602 rescued altered respiratory rate, increased expiratory parameters (Te, PEF, EEP), and normalized airflow parameters (Penh and Rpef) compared to vehicle, consistent with decreased airway inflammation. RNASeq followed by protein analysis revealed heightened levels of inflammation and end-stage necroptosis markers, including ZBP1 and pMLKL induced by rVSV-SARS-CoV-2, that were normalized by MIA-602 treatment, consistent with an anti-inflammatory and pro-survival mechanism of action in this preclinical model of COVID-19 pneumonia.

Alveolar epithelial type 2 cells (AT2) respond to injury by proliferating and relining the alveolar surface as type 1 cells; they are also capable of developing basal and mesenchymal cell characteristics. Our aim was to determine whether AT2 cells could respond to cytokines by expressing pro-inflammatory or pro-fibrotic genes that could drive post-inflammatory pulmonary fibrosis. We used human AT2 cells derived from induced pluripotent stem cells (iAT2) as a model. iAT2 cells were exposed for 48h to cytokines like those found in fibrotic lung lavage fluid (TGF-B [300 pg/mL], IL-1B [10], TNF-a [100], IL-8 [1500], MCP1 [700], IL-33 [40], TSLP [100], IL-13 [2500] and IL-4 [160]). RNA and protein were then isolated to assess expression of genes related to inflammation and fibrosis. iAT2 cells expressed SP-C and SP-B proteins and contained lamellar bodies with tubular myelin. iAT2 cells expressed epithelial markers SFTPB, SFTPC and HOPX, in addition to KRT17, KRT19, CTGF and FN1, on single cell RNA-sequencing. After incubation with cytokines, cells expressed IL1-B, TNF-a, TGFB-1, a-smooth muscle actin, connective tissue growth factor, plasminogen activator inhibitor-1, fibronectin, type I collagen and tenascin C mRNA. After 14d incubation of iAT2 cells with cytokines, cells lost expression of SP-B, an epithelial marker, and expressed on western blotting N-cadherin and vimentin, markers of mesenchymal differentiation. Cytokeratin 5 protein, found in basal cells, was also expressed after cytokines. iAT2 cells respond to cytokines by expression of genes that produce inflammatory and fibrotic mediators, demonstrating the potential of AT2 cells to participate in lung inflammation and fibrosis.

Background: SARS-CoV-2 (COVID-19) transmits a multi-systemic disease that can lead to acute respiratory distress syndrome. Growth hormone-releasing hormone receptor (GHRH-R) and its splice variant are expressed in murine and human lung and heart. GHRH-R antagonist, MIA-602, has been shown to regulate inflammation in animal models and immune cell responses to bleomycin lung injury. Using a BSL2-compatible recombinant VSV-eGFP-SARS-CoV-2-S virus (rVSV-SARS-CoV-2-S) which mimics native SARS-CoV-2 infection in K18 hACE2tg mice, we tested our hypothesis that MIA-602 attenuates COVID-19-induced cardiopulmonary injury by reducing inflammation. Methods: Male and female K18-hACE2tg mice were infected with SARS-CoV-2/USA-WA1/2020, rVSV-SARS-CoV-2-S, or PBS and lung viral load, weight-loss and histopathology were compared (N=8). Mice infected with rVSV-SARS-CoV-2-S were subject to daily subcutaneous injections of 10 μg MIA-602 or vehicle (control) starting at 24h post-infection. Pulmonary function was measured via whole-body plethysmography on day 0, day 3, and day 5 (n=7). Five days after viral infection mice were sacrificed, and blood and tissues collected for histopathological analyses, H&E staining, RNA and protein work. Heart and lung tissues were used for RNASeq (n=3 per group). T-test or One-way ANOVA-test was used for statistical analysis. Results: SARS-CoV-2 and rVSV-SARS-CoV-2-S presented similar pathology for weight loss, infectivity (~60%) and histopathologic changes. Daily treatment with MIA-602 ameliorated weight loss, reduced lung inflammation, pneumonia and pulmonary dysfunction evidenced by rescued respiratory rate, expiratory parameters, and dysregulated airway parameters (p<.05). MIA-602 normalized the high expression of the inflammatory protein ICAM-1 in heart and lung (p<0.01), and master immune modulator Rag2 in lung (RNA:10-FC; Protein: 2-FC; p<.001). Conclusions: The results indicate a possible role for pulmonary Rag2 in protecting against pulmonary dysfunction and heart/lung inflammation by peptide GHRH-R antagonist MIA-602 in a novel animal model of COVID-19 pneumonia.

Background: COVID-19 causes severe pulmonary injury that can lead to acute respiratory distress syndrome. Growth hormone-releasing hormone receptor (GHRH-R) and its splice variant are expressed in murine and human lung and heart. GHRH-R antagonist, MIA-602, has been shown to modulate cellular immune responses to bleomycin lung injury and decrease inflammation in models of sarcoid granuloma. Using the BSL2-friendly rVSV-SARS-CoV-2-S of K18 hACE2tg mice to mimic native SARS-CoV-2 infection, we tested our hypothesis that MIA-602 attenuates cardiopulmonary injury in this COVID-19 model. Methods: Male and female K18 hACE2tg mice were inoculated with SARS-CoV-2 Washington (WA-1) native strain, recombinant VSV-SARS-CoV-2-Spike virus (rVSV-SARS-CoV-2-S), or PBS and lung viral load, weight loss and histopathology compared between groups (N=5-8). K18 h ACE2 tg mice infected with rVSV-SARS-CoV-2-S were subject to daily subcutaneous injections of 10 μg MIA-602 or vehicle starting at 24h post-infection. Pulmonary function was measured via whole-body plethysmography on day 0, day 3, and day 5 (n=7). Five days after viral infection mice were sacrificed; and blood and tissues collected for histopathological analyses, H&E staining and ICAM-1 immunohistochemistry. T-test or One-way ANOVA-test was used for statistical analysis. Results: Native SARS-CoV-2 and rVSV-SARS-CoV-2-S presented with similar patterns of weight loss, infectivity (~60%) and histopathologic changes. Daily treatment with MIA-602 ameliorated weight loss, reduced lung perivascular inflammation and pneumonia, and decreased lung/heart ICAM-1 expression compared to vehicle. MIA-602 rescued respiratory rate, increased expiratory parameters (Te, PEF, EEP) and mitigated dysregulated measures of airway obstruction (Penh and Rpef) compared to vehicle. Conclusions: rVSV-SARS-CoV-2-S is an accurate and safe alternative to native SARS-CoV-2 for preclinical studies. Daily treatment with the synthetic peptide GHRH-R antagonist MIA-602 attenuates pulmonary dysfunction and heart inflammation in this new preclinical mouse model of COVID-19 pneumonia. We hypothesize that the molecular mechanism involves anti-inflammatory actions of MIA-602.

Conjunctival epithelial cells, which express viral-entry receptors angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine type 2 (TMPRSS2), constitute the largest exposed epithelium of the ocular surface tissue and may represent a relevant viral-entry route. To address this question, we generated an organotypic air-liquid-interface model of conjunctival epithelium, composed of basal, suprabasal, and superficial epithelial cells, and fibroblasts, which could be maintained successfully up to day 75 of differentiation. Using single-cell RNA sequencing (RNA-seq), with complementary imaging and virological assays, we observed that while all conjunctival cell types were permissive to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome expression, a productive infection did not ensue. The early innate immune response to SARS-CoV-2 infection in conjunctival cells was characterised by a robust autocrine and paracrine NF-κB activity, without activation of antiviral interferon signalling. Collectively, these data enrich our understanding of SARS-CoV-2 infection at the human ocular surface, with potential implications for the design of preventive strategies and conjunctival transplantation.

Healthcare regulatory agencies have mandated a reduction in 30-day hospital readmission rates and have targeted COPD as a major contributor to 30-day readmissions. We aimed to develop and validate a simple tool deploying an artificial neural network (ANN) for early identification of COPD patients with high readmission risk. Using COPD patient data from eight hospitals within a large urban hospital system, four variables were identified, weighted and validated. These included the number of in-patient admissions in the previous 6 months, the number of medications administered on the first day, insurance status, and the Rothman Index on hospital day one. An ANN model was trained to provide a predictive algorithm and validated on an additional dataset from a separate time period. The model was implemented in a smartphone app (Re-Admit) incorporating four input risk factors, and a clinical care plan focused on high-risk readmission candidates was then implemented. Subsequent readmission data was analyzed to assess impact. The areas under the curve of receiver operating characteristics predicting readmission with ANN is 0.77, with sensitivity 0.75 and specificity 0.67 on the separate validation data. Readmission rates in the COPD high-risk subgroup after app and clinical intervention implementation saw a significant 48% decline. Our studies show the efficacy of ANN model on predicting readmission risks for COPD patients. The AI enabled Re-Admit smartphone app predicts readmission risk on day one of the patient's admission, allowing for early implementation of medical, hospital, and community resources to optimize and improve clinical care pathways.

MicroRNAs are non-coding RNAs that act to downregulate the expression of target genes by translational repression and degradation of messenger RNA molecules. Individual microRNAs have the ability to specifically target a wide array of gene transcripts, therefore allowing each microRNA to play key roles in multiple biological pathways. miR-324 is a microRNA predicted to target thousands of RNA transcripts and is expressed far more highly in the brain than in any other tissue, suggesting that it may play a role in one or multiple neurological pathways. Here we present data from the first global miR-324-null mice, in which increased excitability and interictal discharges were identified in vitro in the hippocampus. RNA sequencing was used to identify differentially expressed genes in miR-324-null mice which may contribute to this increased hippocampal excitability, and 3'UTR luciferase assays and western blotting revealed that two of these, Suox and Cd300lf, are novel direct targets of miR-324. Characterisation of microRNAs that produce an effect on neurological activity, such as miR-324, and identification of the pathways they regulate will allow a better understanding of the processes involved in normal neurological function and in turn may present novel pharmaceutical targets in treating neurological disease.

AbstractObjectivesGrowth hormone‐releasing hormone (GHRH) is a potent stimulator of growth hormone (GH) secretion from the pituitary gland. Although GHRH is essential for the growth of immune cells, the regulatory effects of its antagonist in granulomatous disease remain unknown.MethodsHere, we report expression of GHRH receptor (R) in human tissue with sarcoidosis granuloma and demonstrate the anti‐inflammatory effects of MIA602 (a GHRH antagonist) in two in vitro human granuloma models and an in vivo granuloma model using different methods including ELISA, immunohistochemistry, RNA‐seq analysis and flow cytometry.ResultsMIA602 decreases the levels of IL‐2, IL‐2R, IL‐7, IL‐12, IL‐17A and TNF‐α in an in vitro granuloma model. Further, we show that the anti‐inflammatory effect of MIA602 appears to be mediated by a reduction in CD45+CD68+ cells in granulomatous tissue and upregulation in PD‐1 expression in macrophages. Analysis of the expression of proteins involved in the mitochondrial stage of apoptosis showed that MIA602 increases the levels of caspase‐3, BCL‐xL/BAK dimer and MCl‐1/Bak dimer in the granuloma. These findings indicate that MIA602 may not induce apoptosis.ConclusionsOur findings further suggest that GHRH‐R is potentially a clinical target for the treatment of granulomatous disease and that MIA602 may be used as a novel therapeutic agent for sarcoidosis.

No study has compared the survival of patients with IPF with or without GERD on a large scale, so the impact of GERD on prognosis of IPF remains unclear. Our goal herein was to compare the two-year survival of patients with IPF with or without GERD using the Veterans Affairs (VA) national database. We completed a retrospective, cohort study using VA Informatics and Computing Infrastructure (VINCI) data. Using ICD-10 code J84.112, we identified 10,598 patients coded as IPF in VA outpatient settings between October 1, 2015 and December 24, 2019. We used codes ICD-9 530.81 and ICD-10 K21.9 to identify GERD. The Cox proportional model was used to compare survival of patients with IPF without GERD to patients with IPF who also had GERD, while adjusting for age and gender. Of 10,598 patients with IPF, 4,514 (42.7%) had GERD at the time of IPF diagnosis. Using the Cox proportional model adjusted for age and gender, the hazard ratio of 2-year death for patients with GERD compared to patients without GERD was 0.91 (95% CI: 0.85-0.98; p-value<0.01). The hazard ratio for 2-year death rate for males compared to females was 1.68 (95% CI: 1.29-2.20; p-value<0.01). Increased age was significantly associated with slightly higher risk of 2-year death (odds ratio: 1.02, 95% CI: 1.01-1.02; p-value<0.01). Although ICD codes may not identify all patients with GERD, this study shows importantly that patients with GERD and IPF do not have worse 2-year survival (in fact it appears better) compared to those without GERD, and thus suggests GERD is not responsible for worsened prognosis in patients with IPF. Supported by VA Research Service and South Florida VA Foundation for Research and Education.

Growth hormone releasing hormone (GHRH) is a potent stimulator of GH secretion from the pituitary gland. Although GHRH is essential for the growth of immune cells, the regulatory effects of its antagonist in granulomatous disease remains unknown. Here, we report expression of GHRH receptor (R) in human tissue with sarcoidosis granuloma and demonstrate the anti-inflammatory effects of MIA602 (a GHRH antagonist) in two in vitro human granuloma models and an in vivo granuloma model. MIA602 decreases levels of IL2, IL12, and IL17A in in vitro granuloma model.We show further that the anti-inflammatory effect of MIA602 appears to be mediated by reduction in CD45++CD68+ cells in granulomatous tissue and upregulation in PD-1 expression in macrophages.In analysis of expression of proteins involved in the mitochondrial stage of apoptosis, we show that MIA602 increases the levels of caspase 3, BCL-xL/BAK dimer, and MCl-1/Bak dimer in granuloma. These findings indicate that MIA602 may not induce apoptosis.The clinical relevance of our findings further suggest that HGRH-R is potentially a target for treatment of granulomatous disease and MIA602 possibly a novel therapeutic agent for sarcoidosis.### Competing Interest StatementThe authors have declared no competing interest.

Lung inflammation due to sarcoidosis is characterized by a complex cascade of immunopathologic events, including leukocyte recruitment and granuloma formation. α-melanocyte stimulating hormone (α-MSH) is a melanocortin signaling peptide with anti-inflammatory properties. We aimed to evaluate the effects of α-MSH in a novel in vitro sarcoidosis model. An in vitro sarcoidosis-like granuloma model was developed by challenging peripheral blood mononuclear cells (PBMCs) derived from patients with confirmed treatment-naïve sarcoidosis with microparticles generated from Mycobacterium abscessus cell walls. Unchallenged PBMCsand developed granulomas were treated daily with 10 μM α-MSH or saline as control. Cytokine concentrations in supernatants of culture and in cell extracts were measured using Illumina multiplex Elisa and western blot, respectively. Gene expression was analyzed using RNA-Seq and RT-PCR. Protein secretion and gene expression of IL-7, IL-7R, IFN-γ, MC1R, NF-κB, phosphorylated NF-κB (p-NF-κB), MARCO, and p-CREB were measured with western blot and RNAseq. A significant increase in IL-7, IL-7R, and IFN-γ protein expression was found in developed granulomas comparing to microparticle unchallenged PBMCs. IL-7, IL-7R, and IFN-γ protein expression was significantly reduced in developed granulomas after exposure to α-MSH compared with saline treated granulomas. Compared with microparticle unchallenged PBMCs, total NF-κB and p-NF-κB were significantly increased in developed granulomas, while expression of p-CREB was not changed. Treatment with α-MSH promoted a significantly higher concentration of p-CREB in granulomas. The anti-inflammatory effects of α-MSH were blocked by specific p-CREB inhibition. α-MSH has anti-inflammatory properties in this in vitro granuloma model, which is an effect mediated by induction of phosphorylation of CREB.