Adhesion G protein-coupled receptors (aGPCRs) constitute a structurally and functionally distinct group within the superfamily of GPCRs. In 2015, the International Union of Pharmacology invited the Adhesion GPCR Consortium to publish a comprehensive review about aGPCRs and establish a unified nomenclature. Since then, substantial progress has been made in delineating the biological roles, molecular architecture, biochemical properties, expression profiles, ligand repertoire, and activation and signaling strategies of aGPCRs. Commensurate with these advances, their relevance to human pathophysiology has become increasingly apparent. In a coordinated effort, the Adhesion GPCR Consortium has reviewed recent progress in this field and provides a comprehensive assessment of the current understanding of aGPCR biology, including a focus on human and mammalian aGPCRs, their evolutionary origins, methodological approaches, and model systems for their investigation, as well as emerging approaches for their therapeutic targeting. SIGNIFICANCE STATEMENT: Adhesion G protein-coupled receptors are versatile cell-surface proteins that integrate structural, biochemical, and physiological functions, with major roles in health and disease. This review summarizes current knowledge of their molecular features, functions in diverse model systems, and emerging opportunities for therapeutic targeting, providing a comprehensive resource that connects basic biology with translational applications across multiple scientific disciplines.

Idiopathic pulmonary fibrosis (IPF) is a progressive, fatal lung disease that develops in response to chronic epithelial injury. Unlike injury-induced homeostatic lung repair during which fibroblasts undergo apoptosis and clearance, the lungs of IPF patients continue to accumulate apoptosis-resistant, pro-fibrotic, extracellular matrix-producing fibroblasts. Here, we show that prevention of PDGFRα+ fibroblast apoptosis by conditional BCL-2 expression leads to the emergence and persistence of senescent, pro-fibrotic fibroblasts along with enduring, pathologic fibrotic lung remodeling. Additionally, spatial transcriptomic studies of human IPF lungs confirmed the presence of senescent, BCL-2 expressing α-smooth muscle actin+ myofibroblasts in fibrotic regions. Of translational significance, selective BCL-2 inhibition with ABT-199 in fibrotic mice re-engaged the apoptotic pathway in fibroblasts, reduced senescence, and promoted fibrosis resolution and lung regeneration. Our findings suggest that sustained BCL-2 expression in fibroblasts prevents homeostatic lung repair, drives persistent fibrosis and is a therapeutically relevant target to reverse persistent pulmonary fibrosis.

The mechanisms by which e-cigarette vaping (EV) affects lung health remain unclear. Clinical data from clusters of EV-associated lung injury indicate that EV damages distal lung parenchyma and increases vulnerability to second-hit injury, including respiratory viral infections. Using human lung endothelial and epithelial cells and precision-cut lung slices, we investigated the mechanisms underlying distal lung cell injury and repair triggered by brief (24-hour) EV exposure. Using RNA sequencing of lung tissue from Golden Syrian hamsters, we evaluated the persistence of lung stress responses (10 days after 5 days of EV exposure and determined the impact of EV on host defense against influenza A virus (IAV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. EV disrupted the barrier function of human distal lung cells through JNK stress-response signaling, triggered autophagy with impaired autophagolysosomal degradation, suppressed mTOR signaling and cell proliferation, and culminated in apoptosis. Analysis of transcriptional responses in EV-exposed hamster lungs revealed persistent activation of pathways involving JNK signaling, autophagy, barrier dysfunction, tissue remodeling, and impaired Th1 immunity. EV pre-exposure increased the viral burden of SARS-CoV-2, downregulated antiviral genes (Ifit1, Isg15, Nfkbia), and altered Stat1 and Irf7 immune signaling, while amplifying oxidative stress and IL-12 signaling. These findings show that short-term EV exposure triggered stress-induced distal lung cell injury with persistent changes in antiviral immunity and molecular pathways associated with tissue remodeling. When sustained, as with habitual EV use, these alterations may increase susceptibility to respiratory viral infections and contribute to the development of chronic lung disease.

Sonic Hedgehog (SHH) signaling functions in temporal- and context-dependent manners to pattern diverse tissues during embryogenesis. The signal transducer Smoothened (SMO) is activated by sterols, oxysterols, and arachidonic acid (AA) through binding pockets in its extracellular cysteine-rich domain (CRD) and 7-transmembrane (7TM) bundle. In vitro analyses suggest SMO signaling is allosterically enhanced by combinatorial ligand binding to these pockets but in vivo evidence of SMO allostery is lacking. Herein, we map an AA binding pocket at the top of the 7TM bundle and show that its disruption attenuates SHH and sterol-stimulated SMO induction. A knockin mouse model of compromised AA binding reveals that homozygous mutant mice are cyanotic, exhibit high perinatal lethality, and show congenital heart disease. Surviving mutants demonstrate pulmonary maldevelopment and fail to thrive. Neurodevelopment is unaltered in these mice, suggesting that context-dependent allosteric regulation of SMO signaling allows for precise tuning of pathway activity during cardiopulmonary development.

Heterotrimeric Gi proteins are crucial modulators of G protein-coupled receptor signaling, with Gαi2 ubiquitously expressed and implicated in diverse physiological processes. Previous reports described partial lethality in Gnai2-deficient mice, but the timing and mechanism of death remained unclear. Here, we demonstrate that impaired neonatal respiratory adaptation contributes to mortality in Gnai2-deficient neonates. Despite normal Mendelian distribution at birth and no overt malformations, at least 20% of the expected Gnai2-deficient neonates died within minutes after birth, displaying abnormal breathing, cyanosis, and features resembling neonatal respiratory distress syndrome (RDS). Histological and ultrastructural analyses revealed reduced alveolar surface area, thickened septa, increased mesenchymal tissue, and impaired surfactant ultrastructure, despite unaltered alveolar surfactant phospholipid levels. These findings suggest that Gαi2 modulates the structural deployment and functional organization of surfactant within alveoli, although the incomplete phenotype and survival of some neonates indicate a regulatory rather than indispensable role of Gαi2. Our data underscore the complexity of neonatal respiratory adaptation and highlight potential systemic and intercellular mechanisms underlying alveolar stabilization.

IntroductionEarly-life dysbiosis is associated with increased risk of asthma development but the underlying mechanisms remain unclear. Although eosinophils have been reported in the developing lung, their contributions to alveolar morphogenesis and lung mechanics have not been functionally interrogated.MethodsMaternal exposure to antibiotics (ABX) was used to induce early-life offspring dysbiosis, and the effects on lung function and development was assessed. Similar measurements were made in mice lacking eosinophils due to genetic modification, or administration of IL-5 blocking agents.ResultsABX exposure between Embryonic Day 15 (E15) and post-natal day 28 (PN28), increased allergen-induced, and baseline airway hyperreactivity (AHR). Similar observations were made when maternal ABX exposure was limited to PN10 to PN20. Complete characterization of baseline lung mechanics demonstrated downward-shifted pulmonary PV loops, increased small airway resistance, decreased compliance, and reduced inspiratory capacity at weaning and 14 months of age. Consistent with observation of small airway dysfunction, offspring of ABX-exposed dams demonstrated significantly smaller alveoli at multiple stages of lung development. Examination of recruitment to developing lungs demonstrated an exaggerated recruitment of eosinophils at key developmental periods (PN14) in offspring of ABX-exposed dams. Mice with fewer eosinophils (through genetic knockout, or treatment with anti-IL-5) display altered patterns of lung mechanics opposite to that seen in offspring of ABX-exposed dams.DiscussionThese data underscore an underappreciated role of eosinophils in homeostatic lung development and suggest that early life modulation of pulmonary eosinophil activity has long-term effects on susceptibility to the development of chronic lung diseases such as asthma.

Rationale: The increasing use of e-cigarette vaping (EV) and the evidence that EV contributes to lung inflammation, emphasize the need for better understanding of mechanisms by which EV injures the lung. Building on our observation that EV exposure triggers autophagy in lung cells and activates autophagy signaling pathways governed by JUND in hamster lungs, we hypothesize that JNK activation is required for EV-induced autophagy. Methods: Cultured small airway epithelial cells (hSAEC1-KT) were treated with a broad JNK inhibitor (SP600125, 50 mM, 1h; Selleckchem) prior to EV exposure (Juul, Virgina Tobacco, 5 mg/mL nicotine; 7.5 mM; 2, 4, or 24 hours). Autophagy markers such as the lipidated form of LC3B (Microtubule-associated protein 1A/1B-light chain 3B) or LC3BII, and the autophagy adaptor protein 62/SQSTM1 (sequestosome 1) or p62; as well as JNK phosphorylation were measured by western blot and immunofluorescence. Results: EV decreased levels of ribosomal S6k phosphorylation within 2 hours, and this effect was sustained for 24 hours. EV increased LC3BII levels 2-fold at 4 hours and ∼5-fold at 24 hours. EV also increased p62 by∼1.5-fold at 4 hours and ∼2-fold at 24 hours. EV increased JNK phosphorylation by 2-fold at 24 hours. Inhibition of JNK did not affect levels of phosphorylated ribosomal S6k, but significantly decreased LC3BII (p=0.008) and p62 (p=0.01) at 24hrs, to similar levels as in control unexposed cells. Conclusion: Following inhibition of mTOR activity, EV triggered autophagy with progressive accumulation of p62/SQTM1, denoting incomplete fusion/digestion of autophagosomes. JNK activation by EV contributes to impaired autophagy completion, by yet unknown mechanisms. JNK inhibition may ameliorate lung injury repair following EV exposure, prompting further studies into which specific JNK isoform is involved in this process. Funding: RO1HL144396, Wollowick Chair in COPD Research.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a progressive disease with limited treatment options and unknown etiology. The ATP Binding Cassette Transporter ABCG1 is an intracellular sterol transporter required for pulmonary surfactant homeostasis. Targeted disruptions in lipid homeostasis result in perturbations to pulmonary surfactant and spontaneous lung pathologies. Mice deficient in ABCG1 develop age-dependent accumulation of lipid-filled foam cell alveolar macrophages (AM). Patients with IPF have evidence of increased neutral lipid content evident on Oil-red-O staining of AM in lung tissue. We hypothesize that aberrant cholesterol metabolism plays a role in IPF. Methods: We performed lipidomic analysis on lung tissue and AM from Abcg1-null mice. We generated animals with macrophage-specific deletion of ABCG1 and performed bone marrow transplants in WT and Abcg1-null mice. Results: Spontaneous fibrosis developed in Abcg1-deficient animals by 12 months, while lipid levels rose as early as 6 months and preceding the onset of fibrosis. Mice with macrophage-specific deletion of ABCG1 also developed fibrosis by 12 months. Transplantation of WT mice with Abcg1-null macrophages was sufficient to promote fibrosis development whereas transplantation of Abcg1-null mice with WT macrophages prevented fibrosis. Conclusions: Levels of AM pathologic lipids increase before the onset of fibrosis, suggesting AM foam cell lipid accumulation drives the progression of fibrosis. Furthermore, transplantation of lipid-filled macrophages into WT mice and mice with macrophage-specific loss of cholesterol handling resulted in fibrosis development, demonstrating aberrant macrophage cholesterol handling was sufficient to promote fibrosis.

Idiopathic pulmonary fibrosis (IPF) is etiologically complex, with well-documented genetic and nongenetic origins. In this Review, we speculate that the development of IPF requires two hits: the first establishes a vulnerable bronchoalveolar epithelium, and the second triggers mechanisms that reprogram distal epithelia to initiate and perpetuate a profibrotic phenotype. While vulnerability of the bronchoalveolar epithelia is most often driven by common or rare genetic variants, subsequent injury of the bronchoalveolar epithelia results in persistent changes in cell biology that disrupt tissue homeostasis and activate fibroblasts. The dynamic biology of IPF can best be contextualized etiologically and temporally, including stages of vulnerability, early disease, and persistent and progressive lung fibrosis. These dimensions of IPF highlight critical mechanisms that adversely disrupt epithelial function, activate fibroblasts, and lead to lung remodeling. Together with better recognition of early disease, this conceptual approach should lead to the development of novel therapeutics directed at the etiologic and temporal drivers of lung fibrosis that will ultimately transform the care of patients with IPF from palliative to curative.

Rationale: Acute lung injury (ALI) is characterized by the loss and damage of type I alveolar epithelial cells (ATI). Repair depends on alveolar type II (ATII) cells, key progenitor cells of the lung, to proliferate and transdifferentiate into new ATI cells. The signals driving ATII proliferation and transdifferentiation are not well characterized. Our data suggest alveolar macrophages (AMs) play a critical role. AMs are subdivided into two categories resident (RAMs) and recruited (RecAMs). RecAMs are derived from circulating monocytes that migrate into the lung in response to ALI. In vivo studies from our lab have shown that RecAMs are needed for repair and upregulate HIF-1α and its secreted downstream effectors in response to ALI. Thus, we hypothesize that HIF-1α is critical in RecAM dependent repair. Methods: To assess the effect of macrophages on ATII proliferation, we isolated AMs from bronchoalveolar lavage of Csf1rCre x HIF-1αfl/fl mice. AMs were harvested from naïve or LPS treated (80 ug intratracheally) mice after day 6. The macrophages were cultured for 24 hours in ATII cell base media, and conditioned media was collected to use as growth medium for ATII cells. To isolate ATII cells, murine lungs were digested with Dispase and negatively selected using anti-CD45, CD32, and CD16 monoclonal antibodies. The isolated ATII cells were plated onto transwell inserts coated with 50:50 ratio of rat tail collagen and Matrigel, cultured in macrophage-conditioned medium for 24 hours, then liberated and assessed via flow cytometry for proliferation marker Ki67. Results: ATII cells were isolated from the murine lung at 85% purity, as assessed by flow cytometry. Cultured ATII cells showed increased Ki67 positivity when treated with both naïve and LPS macrophage-conditioned media compared with ATII base media. Conclusion: In this study, we demonstrate the ability to isolate ATII cells for in vitro culture and show that macrophage-conditioned media augments ATII proliferation. Our data suggest that alveolar macrophages secrete factors that stimulate ATII cell proliferation. Future directions include utilizing this model to assess the effect of purified macrophage subsets (RAMs and RecAMs) and HIF1a KO macrophages in stimulating ATII repair, as well as utilizing neutralizing antibodies to identify the responsible growth factor in the macrophage-conditioned media.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive condition marked by significant morbidity. While the pathology of the disease is well understood, its underlying cause remains unidentified.Cellular lipids and lipid mediators play roles in inflammation and in the development of several pathologies. Lipid homeostasis is important in the lung as lipid makes up 90% of pulmonary surfactant. Loss of cholesterol handling via transporter ABCG1 has been reported as a key to fibrosis development in mice. We hypothesise that aberrant cholesterol metabolism plays a role in the development of IPF. Methods: IPF surgical lung biopsy specimens were assessed for subpleural fat and foamy macrophages. Human IPF and control tissue (n=10) were analysed on the Sciex Lipidyzer Platform for targeted quantitative measurement of >1,100 lipid species across 13 classes. We exposed human fibroblasts to cholesteryl esters (CE), measuring expression of pro-fibrotic markers and associated lipid handling and fibrosis genes. Results: There was an increase in total pulmonary lipid content (p=0.0317) in IPF compared to control lung tissue. Patients with IPF have evidence of increased neutral lipid contentevident in the sub-pleural space with evidence of foamy alveolar macrophages and lipid-filled fibroblasts on IPF histology specimens compared to non-IPF controls. Human IPF lung fibroblasts showed a marked increase in fibrotic marker expression when exposed to saturated CE 16:0 (Col1a1=1.5-fold change, p=0.0079; α-SMA=2.1-fold change, p=0.0125), whereas exposure to unsaturated CE 20:4 resulted in decreased fibrotic marker expression. Conclusions: Abnormal lipid deposition was observed in IPF tissue. Saturated CE caused production of pro-fibrotic cytokines in human fibroblasts. Nonetheless, more research is necessary to elucidate why unsaturated CE exerts protective effect in human IPF fibroblasts.

Pulmonary fibrosis (PF) can be idiopathic or driven by a specific insult, genetic susceptibility, or disease process. Inflammation plays a role in the pathophysiology, the extent of which remains a longstanding topic of debate. More recently, there has been increasing interest in a potential inciting role for aberrant lipid metabolism. Lipids are essential for the structure and function of all cell membranes, but specifically in the lung for surfactant composition, intra- and intercellular lipid mediators, and lipofibroblasts. Clinically, there is evidence of increased lipid deposition in the subpleural space and at a whole-lung tissue level in PF. There is evidence of increased parenchymal lipid deposition and abnormal mediastinal fat shape on chest computed tomography. A protective role for cholesterol-lowering drugs, including statins and ezetimibe, has been described in PF. At a cellular level, fatty acid, phospholipid, and glucose metabolism are disordered, as is the production of lipid mediators. Here we put forward the argument that there is substantive clinical and biological evidence to support a role for aberrant lipid metabolism and lipid mediators in the pathogenesis of PF.

Pulmonary alveolar proteinosis (PAP) is a life-threatening, rare lung syndrome for which there is no cure and no approved therapies. PAP is a disease of lipid accumulation characterized by alveolar macrophage foam cell formation. While much is known about the clinical presentation, there is a paucity of information regarding temporal changes in lipids throughout the course of disease. Our objectives were to define the detailed lipid composition of alveolar macrophages in PAP patients at the time of diagnosis and during treatment. We performed comprehensive mass spectrometry to profile the lipid signature of alveolar macrophages obtained from three independent mouse models of PAP and from PAP and nonPAP patients. Additionally, we quantified changes in macrophage-associated lipids during clinical treatment of PAP patients. We found remarkable variations in lipid composition in PAP patients, which were consistent with data from three independent mouse models. Detailed lipidomic analysis revealed that the overall alveolar macrophage lipid burden inversely correlated with clinical improvement and response to therapy in PAP patients. Specifically, as PAP patients experienced clinical improvement, there was a notable decrease in the total lipid content of alveolar macrophages. This crucial observation suggests that the levels of these macrophage-associated lipids can be utilized to assess the efficacy of treatment. These findings provide valuable insights into the dysregulated lipid metabolism associated with PAP, offering the potential for lipid profiling to serve as a means of monitoring therapeutic interventions in PAP patients.

Introduction: There is increasing interest in the role of lipids in processes that modulate fibrosis. Specifically, some inherited surfactant dysfunction disorders result in pulmonary fibrosis. Surfactant consists of phospholipids and smaller quantities of neutral lipids such as cholesterol ester (CE). Tissue lipidomics on mouse bleomycin models demonstrates increased total lipid compared to control. Aims & objectives: To perform lipidomic analysis on human IPF lung tissue and assess the impact of specific lipids on human fibroblasts. Methods: Lung tissue from human IPF and control patients (n=10) were analysed for targeted quantitative measurement of >1,100 lipid species. Primary cell culture of human IPF and control fibroblasts was performed and cells were exposed to lipid species identified as increased on IPF tissue lipidomic analysis. Expression of alpha-smooth muscle actin (aSMA) and collagen 1 (Col1a1) were measured to assess for myofibroblast-like transition following lipid exposure. Results: There was an increase in pulmonary lipid content in patients with IPF compared to control (23.16nmol/mg vs. 18.66nmol/mg, p=0.0317). Specific increases were noted in 16:0, 20:4, and 22:6-containing lipids within both CE and PC species. Dose-related increases in aSMA and Col1a1 protein and RNA expression occur in human fibroblasts exposed to PC-16.0 and decreased expression when exposed to PC-20.4, CE 16.0, and CE 20.4. Conclusions: Abnormal lipid deposition is seen in IPF on lipidomic analysis. Specific lipid subgroups appear to impact fibroblast biology as seen by changes in aSMA and Col1a1 expression. Further investigation is still warranted to identify the specific action of lipids in fibrosis.