Pulmonary Fibrosis Stakeholder Summit: A Joint NHLBI, Three Lakes Foundation, and Pulmonary Fibrosis Foundation Workshop Report.

Despite progress in elucidation of disease mechanisms, identification of risk factors, biomarker discovery, and the approval of two medications to slow lung function decline in idiopathic pulmonary fibrosis and one medication to slow lung function decline in progressive pulmonary fibrosis, pulmonary fibrosis remains a disease with a high morbidity and mortality. In recognition of the need to catalyze ongoing advances and collaboration in the field of pulmonary fibrosis, the NHLBI, the Three Lakes Foundation, and the Pulmonary Fibrosis Foundation hosted the Pulmonary Fibrosis Stakeholder Summit on November 8-9, 2022. This workshop was held virtually and was organized into three topic areas: 1) novel models and research tools to better study pulmonary fibrosis and uncover new therapies, 2) early disease risk factors and methods to improve diagnosis, and 3) innovative approaches toward clinical trial design for pulmonary fibrosis. In this workshop report, we summarize the content of the presentations and discussions, enumerating research opportunities for advancing our understanding of the pathogenesis, treatment, and outcomes of pulmonary fibrosis.

Disruption of proteostasis causes IRE1 mediated reprogramming of alveolar epithelial cells.

Disruption of alveolar type 2 cell (AEC2) protein quality control has been implicated in chronic lung diseases, including pulmonary fibrosis (PF). We previously reported the in vivo modeling of a clinical surfactant protein C (SP-C) mutation that led to AEC2 endoplasmic reticulum (ER) stress and spontaneous lung fibrosis, providing proof of concept for disruption to proteostasis as a proximal driver of PF. Using two clinical SP-C mutation models, we have now discovered that AEC2s experiencing significant ER stress lose quintessential AEC2 features and develop a reprogrammed cell state that heretofore has been seen only as a response to lung injury. Using single-cell RNA sequencing in vivo and organoid-based modeling, we show that this state arises de novo from intrinsic AEC2 dysfunction. The cell-autonomous AEC2 reprogramming can be attenuated through inhibition of inositol-requiring enzyme 1 (IRE1α) signaling as the use of an IRE1α inhibitor reduced the development of the reprogrammed cell state and also diminished AEC2-driven recruitment of granulocytes, alveolitis, and lung injury. These findings identify AEC2 proteostasis, and specifically IRE1α signaling through its major product XBP-1, as a driver of a key AEC2 phenotypic change that has been identified in lung fibrosis.

Activation of Alveolar Epithelial ER Stress by ß-Coronavirus Infection Disrupt Surfactant Homeostasis in Mice: Implications for Covid-19 Respiratory Failure.

COVID-19 syndrome is characterized by acute lung injury, hypoxemic respiratory failure, and high mortality. Alveolar Type 2 (AT2) cells are essential for gas exchange, repair, and regeneration of distal lung epithelium. We have shown that the causative agent, SARS-CoV-2 and other ß-coronavirus genus members induce an ER stress response in vitro, however the consequences for host AT2 function in vivo are less understood. To study this, two murine models of coronavirus infection were employed- mouse hepatitis virus-1 (MHV-1) in A/J mice and a mouse adapted SARS-CoV-2 strain. MHV-1 infected mice exhibited dose-dependent weight loss with histological evidence of distal lung injury accompanied by elevated bronchoalveolar lavage fluid (BALF) cell counts and total protein. AT2 cells showed evidence of both viral infection and increased BIP/GRP78 expression, consistent with activation of the unfolded protein response (UPR). The AT2 UPR included increased IRE1α signaling and a biphasic response in PERK signaling accompanied marked reductions in AT2 and BALF surfactant protein (SP-B, SP-C) content, increases in surfactant surface tension, and emergence of a re-programmed epithelial cell population (Krt8+, Cldn4+). The loss of a homeostatic AT2 endophenotype was attenuated by treatment with the IRE1α inhibitor OPK711. As proof-of-concept, C57BL6 mice infected with mouse-adapted SARS-CoV-2 demonstrated similar lung injury and evidence of disrupted surfactant homeostasis. We conclude that lung injury from ß-coronavirus infection results from an aberrant host response activating multiple AT2 UPR pathways, altering surfactant metabolism/function, and changing AT2 endophenotypes offering a mechanistic link between SARS-CoV-2 infection, AT2 cell biology, and acute respiratory failure.

Stem Cells, Cell Therapies and Bioengineering in Lung Biology and Diseases 2023.

Repair and regeneration of a diseased lung using stem cells or bioengineered tissues is an exciting therapeutic approach for a variety of lung diseases and critical illnesses. Over the past decade increasing evidence from preclinical models suggests that cells, which are not normally resident in the lung can be utilized to modulate immune responses after injury, but there have been challenges in translating these promising findings to the clinic. In parallel, there has been a surge in bioengineering studies investigating the use of artificial and acellular lung matrices as scaffolds for three-dimensional lung or airway regeneration, with some recent attempts of transplantation in large animal models. The combination of these studies with those involving stem cells, induced pluripotent stem cell derivatives, and/or cell therapies is a promising and rapidly developing research area. These studies have been further paralleled by significant increases in our understanding of the molecular and cellular events by which endogenous lung stem and/or progenitor cells arise during lung development and participate in normal and pathologic remodeling after lung injury. For the 2023 Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases Conference, scientific symposia were chosen to reflect the most cutting-edge advances in these fields. Sessions focused on the integration of "-omics" technologies with function, the influence of immune cells on regeneration, and the role of the extracellular matrix in regeneration. The necessity for basic science studies to enhance fundamental understanding of lung regeneration and to design innovative translational studies was reinforced throughout the conference.

Chronic Expression of a Clinical SFTPC Mutation Causes Murine Lung Fibrosis with Idiopathic Pulmonary Fibrosis Features.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic interstitial lung disease. A barrier to developing more effective therapies for IPF is the dearth of preclinical models that recapitulate the early pathobiology of this disease. Intratracheal bleomycin, the conventional preclinical murine model of IPF, fails to reproduce the intrinsic dysfunction to the alveolar epithelial type 2 cell (AEC2) that is believed to be a proximal event in the pathogenesis of IPF. Murine fibrosis models based on SFTPC (Surfactant Protein C gene) mutations identified in patients with interstitial lung disease cause activation of the AEC2 unfolded protein response and endoplasmic reticulum stress-an AEC2 dysfunction phenotype observed in IPF. Although these models achieve spontaneous fibrosis, they do so with precedent lung injury and thus are challenged to phenocopy the general clinical course of patients with IPF-gradual progressive fibrosis and loss of lung function. Here, we report a refinement of a murine Sftpc mutation model to recapitulate the clinical course, physiological impairment, parenchymal cellular composition, and biomarkers associated with IPF. This platform provides the field with an innovative model to understand IPF pathogenesis and index preclinical therapeutic candidates.

Identification and Prognosis of Patients With Interstitial Pneumonia With Autoimmune Features.

BACKGROUND/OBJECTIVE: Patients classified as interstitial pneumonia with autoimmune features (IPAF) have interstitial lung disease (ILD) and features of autoimmunity but do not fulfill criteria for connective tissue diseases (CTDs). Our goal was to identify patients classifiable as IPAF, CTD-ILD, and idiopathic pulmonary fibrosis (IPF) from a preexisting pulmonary cohort and evaluate the prognosis of patients with IPAF. METHODS: We reviewed the medical records of 456 patients from a single-center pulmonary ILD cohort whose diagnoses were previously established by a multidisciplinary panel that did not include rheumatologists. We reclassified patients as IPAF, CTD-ILD, or IPF. We compared transplant-free survival using Kaplan-Meier methods and identified prognostic factors using Cox models. RESULTS: We identified 60 patients with IPAF, 113 with CTD-ILD, and 126 with IPF. Transplant-free survival of IPAF was not statistically significantly different from that of CTD-ILD or IPF. Among IPAF patients, male sex (hazard ratio, 4.58 [1.77-11.87]) was independently associated with worse transplant-free survival. During follow-up, only 10% of IPAF patients were diagnosed with CTD-ILD, most commonly antisynthetase syndrome. CONCLUSION: Despite similar clinical characteristics, most patients with IPAF did not progress to CTD-ILD; those who did often developed antisynthetase syndrome, highlighting the critical importance of comprehensive myositis autoantibody testing in this population. As in other types of ILD, male sex may portend a worse prognosis in IPAF. The routine engagement of rheumatologists in the multidisciplinary evaluation of ILD will help ensure the accurate classification of these patients and help clarify prognostic factors.

Heterogeneity in Human Induced Pluripotent Stem Cell-derived Alveolar Epithelial Type II Cells Revealed with ABCA3/SFTPC Reporters.

Alveolar epithelial type 2 cells (AEC2s), the facultative progenitors of lung alveoli, are typically identified through the use of the canonical markers, SFTPC and ABCA3. Self-renewing AEC2-like cells have been generated from human induced pluripotent stem cells (iPSCs) through the use of knock-in SFTPC fluorochrome reporters. However, developmentally, SFTPC expression onset begins in the fetal distal lung bud tip and thus is not specific to mature AEC2s. Furthermore, SFTPC reporters appear to identify only those iPSC-derived AEC2s (iAEC2s) expressing the highest SFTPC levels. Here, we generate an ABCA3 knock-in GFP fusion reporter (ABCA3:GFP) that enables the purification of iAEC2s while allowing visualization of lamellar bodies, organelles associated with AEC2 maturation. Using an SFTPCtdTomato and ABCA3:GFP bifluorescent line for in vitro distal lung-directed differentiation, we observe later onset of ABCA3:GFP expression and broader identification of the subsequently emerging iAEC2 population based on ABCA3:GFP expression compared with SFTPCtdTomato expression. Comparing ABCA3:GFP/SFTPCtdTomato double-positive with ABCA3:GFP single-positive (SP) cells by RNA sequencing and functional studies reveals iAEC2 cellular heterogeneity with both populations functionally processing surfactant proteins but the SP cells exhibiting faster growth kinetics, increased clonogenicity, increased expression of progenitor markers, lower levels of SFTPC expression, and lower levels of AEC2 maturation markers. Over time, we observe that each population (double-positive and SP) gives rise to the other and each can serve as the parents of indefinitely self-renewing iAEC2 progeny. Our results indicate that iAEC2s are a heterogeneous population of cells with differing proliferation versus maturation properties, the majority of which can be tracked and purified using the ABCA3:GFP reporter or surrogate cell surface proteins, such as SLC34A2 and CPM.

The common ABCA3E292V variant disrupts AT2 cell quality control and increases susceptibility to lung injury and aberrant remodeling.

ATP-binding cassette class A3 (ABCA3) is a lipid transporter that plays a critical role in pulmonary surfactant function. The substitution of valine for glutamic acid at codon 292 (E292V) produces a hypomorphic variant that accounts for a significant portion of ABCA3 mutations associated with lung disorders spanning from neonatal respiratory distress syndrome and childhood interstitial lung disease to diffuse parenchymal lung disease (DPLD) in adults including pulmonary fibrosis. The mechanisms by which this and similar ABCA3 mutations disrupt alveolar type 2 (AT2) cell homeostasis and cause DPLD are largely unclear. The present study, informed by a patient homozygous for the E292V variant, used an in vitro and a preclinical murine model to evaluate the mechanisms by which E292V expression promotes aberrant lung injury and parenchymal remodeling. Cell lines stably expressing enhanced green fluorescent protein (EGFP)-tagged ABCA3 isoforms show a functional deficiency of the ABCA3E292V variant as a lipid transporter. AT2 cells isolated from mice constitutively homozygous for ABCA3E292V demonstrate the presence of small electron-dense lamellar bodies, time-dependent alterations in macroautophagy, and induction of apoptosis. These changes in AT2 cell homeostasis are accompanied by a spontaneous lung phenotype consisting of both age-dependent inflammation and fibrillary collagen deposition in alveolar septa. Older ABCA3E292V mice exhibit increased vulnerability to exogenous lung injury by bleomycin. Collectively, these findings support the hypothesis that the ABCA3E292V variant is a susceptibility factor for lung injury through effects on surfactant deficiency and impaired AT2 cell autophagy.

Integrated plasma proteomics and lung transcriptomics reveal novel biomarkers in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease with a significant unmet medical need. Development of transformational therapies for IPF is challenging in part to due to lack of robust predictive biomarkers of prognosis and treatment response. Importantly, circulating biomarkers of IPF are limited and none are in clinical use. METHODS: We previously reported dysregulated pathways and new disease biomarkers in advanced IPF through RNA sequencing of lung tissues from a cohort of transplant-stage IPF patients (n = 36) in comparison to normal healthy donors (n = 19) and patients with acute lung injury (n = 11). Here we performed proteomic profiling of matching plasma samples from these cohorts through the Somascan-1300 SomaLogics platform. RESULTS: Comparative analyses of lung transcriptomic and plasma proteomic signatures identified a set of 34 differentially expressed analytes (fold change (FC) ≥ ± 1.5, false discovery ratio (FDR) ≤ 0.1) in IPF samples compared to healthy controls. IPF samples showed strong enrichment of chemotaxis, tumor infiltration and mast cell migration pathways and downregulated extracellular matrix (ECM) degradation. Mucosal (CCL25 and CCL28) and Th2 (CCL17 and CCL22) chemokines were markedly upregulated in IPF and highly correlated within the subjects. The mast cell maturation chemokine, CXCL12, was also upregulated in IPF plasma (fold change 1.92, FDR 0.006) and significantly correlated (Pearson r = - 0.38, p = 0.022) to lung function (%predicted FVC), with a concomitant increase in the mast cell Tryptase, TPSB2. Markers of collagen III and VI degradation (C3M and C6M) were significantly downregulated (C3M p < 0.001 and C6M p < 0.0001 IPF vs control) and correlated, Pearson r = 0.77) in advanced IPF consistent with altered ECM homeostasis. CONCLUSIONS: Our study identifies a panel of tissue and circulating biomarkers with clinical utility in IPF that can be validated in future studies across larger cohorts.

Epithelial Expression of an Interstitial Lung Disease-Associated Mutation in Surfactant Protein-C Modulates Recruitment and Activation of Key Myeloid Cell Populations in Mice.

Patients with idiopathic pulmonary fibrosis (IPF) often experience precipitous deteriorations, termed "acute exacerbations" (AE), marked by diffuse alveolitis and altered gas exchange, resulting in a significant loss of lung function or mortality. The missense isoleucine to threonine substitution at position 73 (I73T) in the alveolar type 2 cell-restricted surfactant protein-C (SP-C) gene (SFTPC) has been linked to clinical IPF. To better understand the sequence of events that impact AE-IPF, we leveraged a murine model of inducible SP-CI73T (SP-CI73T/I73TFlp+/- ) expression. Following administration of tamoxifen to 8-12-wk-old mice, an upregulation of SftpcI73T initiated a diffuse lung injury marked by increases in bronchoalveolar lavage fluid (BALF) protein and histochemical evidence of CD45+ and CD11b+ cell infiltrates. Flow cytometry of collagenase-digested lung cells revealed a transient, early reduction in SiglecFhiCD11blowCD64hiCD11chi macrophages, countered by the sequential accumulation of SiglecFloCD11b+CD64-CD11c-CCR2+Ly6C+ immature macrophages (3 d), Ly6G+ neutrophils (7 d), and SiglecFhiCD11bhiCD11clo eosinophils (2 wk). By mRNA analysis, BALF cells demonstrated a time-dependent phenotypic shift from a proinflammatory (3 d) to an anti-inflammatory/profibrotic activation state, along with serial elaboration of monocyte and eosinophil recruitment factors. The i.v. administration of clodronate effectively reduced total BALF cell numbers, CCR2+ immature macrophages, and eosinophil influx while improving survival. In contrast, resident macrophage depletion from the intratracheal delivery of clodronate liposomes enhanced SftpcI73T -induced mortality. These results using SftpcI73T mice provide a detailed ontogeny for AE-IPF driven by alveolar epithelial dysfunction that induces a polycellular inflammation initiated by the early influx of proinflammatory CCR2+Ly6Chi immature macrophages.

Grp78 Loss in Epithelial Progenitors Reveals an Age-linked Role for Endoplasmic Reticulum Stress in Pulmonary Fibrosis.

Rationale: Alveolar epithelial cell (AEC) injury and dysregulated repair are implicated in the pathogenesis of pulmonary fibrosis. Endoplasmic reticulum (ER) stress in AEC has been observed in idiopathic pulmonary fibrosis (IPF), a disease of aging.Objectives: To investigate a causal role for ER stress in the pathogenesis of pulmonary fibrosis (PF) and therapeutic potential of ER stress inhibition in PF.Methods: The role of ER stress in AEC dysfunction and fibrosis was studied in mice with tamoxifen (Tmx)-inducible deletion of ER chaperone Grp78, a key regulator of ER homeostasis, in alveolar type II (AT2) cells, progenitors of distal lung epithelium, and in IPF lung slice cultures.Measurements and Main Results:Grp78 deletion caused weight loss, mortality, lung inflammation, and spatially heterogeneous fibrosis characterized by fibroblastic foci, hyperplastic AT2 cells, and increased susceptibility of old and male mice, all features of IPF. Fibrosis was more persistent in more severely injured Grp78 knockout (KO) mice. Grp78 KO AT2 cells showed evidence of ER stress, apoptosis, senescence, impaired progenitor capacity, and activation of TGF-ß (transforming growth factor-ß)/SMAD signaling. Glucose-regulated protein 78 is reduced in AT2 cells from old mice and patients with IPF, and ER stress inhibitor tauroursodeoxycholic acid ameliorates ER stress and fibrosis in Grp78 KO mouse and IPF lung slice cultures.Conclusions: These results support a causal role for ER stress and resulting epithelial dysfunction in PF and suggest ER stress as a potential mechanism linking aging to IPF. Modulation of ER stress and chaperone function may offer a promising therapeutic approach for pulmonary fibrosis.

Linking Fibrotic Remodeling and Ultrastructural Alterations of Alveolar Epithelial Cells after Deletion of Nedd4-2.

Our previous study showed that in adult mice, conditional Nedd4-2-deficiency in club and alveolar epithelial type II (AE2) cells results in impaired mucociliary clearance, accumulation of Muc5b and progressive, terminal pulmonary fibrosis within 16 weeks. In the present study, we investigated ultrastructural alterations of the alveolar epithelium in relation to interstitial remodeling in alveolar septa as a function of disease progression. Two, eight and twelve weeks after induction of Nedd4-2 knockout, lungs were fixed and subjected to design-based stereological investigation at the light and electron microscopic level. Quantitative data did not show any abnormalities until 8 weeks compared to controls. At 12 weeks, however, volume of septal wall tissue increased while volume of acinar airspace and alveolar surface area significantly decreased. Volume and surface area of alveolar epithelial type I cells were reduced, which could not be compensated by a corresponding increase of AE2 cells. The volume of collagen fibrils in septal walls increased and was linked with an increase in blood-gas barrier thickness. A high correlation between parameters reflecting interstitial remodeling and abnormal AE2 cell ultrastructure could be established. Taken together, abnormal regeneration of the alveolar epithelium is correlated with interstitial septal wall remodeling.

Congenital Deletion of Nedd4-2 in Lung Epithelial Cells Causes Progressive Alveolitis and Pulmonary Fibrosis in Neonatal Mice.

Recent studies found that expression of NEDD4-2 is reduced in lung tissue from patients with idiopathic pulmonary fibrosis (IPF) and that the conditional deletion of Nedd4-2 in lung epithelial cells causes IPF-like disease in adult mice via multiple defects, including dysregulation of the epithelial Na+ channel (ENaC), TGFß signaling and the biosynthesis of surfactant protein-C proprotein (proSP-C). However, knowledge of the impact of congenital deletion of Nedd4-2 on the lung phenotype remains limited. In this study, we therefore determined the effects of congenital deletion of Nedd4-2 in the lung epithelial cells of neonatal doxycycline-induced triple transgenic Nedd4-2fl/fl/CCSP-rtTA2S-M2/LC1 mice, with a focus on clinical phenotype, survival, lung morphology, inflammation markers in BAL, mucin expression, ENaC function and proSP-C trafficking. We found that the congenital deletion of Nedd4-2 caused a rapidly progressive lung disease in neonatal mice that shares key features with interstitial lung diseases in children (chILD), including hypoxemia, growth failure, sterile pneumonitis, fibrotic lung remodeling and high mortality. The congenital deletion of Nedd4-2 in lung epithelial cells caused increased expression of Muc5b and mucus plugging of distal airways, increased ENaC activity and proSP-C mistrafficking. This model of congenital deletion of Nedd4-2 may support studies of the pathogenesis and preclinical development of therapies for chILD.

Surfactant protein-D modulation of pulmonary macrophage phenotype is controlled by S-nitrosylation.

Surfactant protein-D (SP-D) is a regulator of pulmonary innate immunity whose oligomeric state can be altered through S-nitrosylation to regulate its signaling function in macrophages. Here, we examined how nitrosylation of SP-D alters the phenotypic response of macrophages to stimuli both in vivo and in vitro. Bronchoalveolar lavage (BAL) from C57BL6/J and SP-D-overexpressing (SP-D OE) mice was incubated with RAW264.7 cells ± LPS. LPS induces the expression of the inflammatory genes Il1b and Nos2, which is reduced 10-fold by SP-D OE-BAL. S-nitrosylation of the SP-D OE-BAL (SNO-SP-D OE-BAL) abrogated this inhibition. SNO-SP-D OE-BAL alone induced Il1b and Nos2 expression. PCR array analysis of macrophages incubated with SP-D OE-BAL (±LPS) shows increased expression of repair genes, Ccl20, Cxcl1, and Vcam1, that was accentuated by LPS. LPS increases inflammatory gene expression, Il1a, Nos2, Tnf, and Ptgs2, which was accentuated by SNO-SP-D OE-BAL but inhibited by SP-D OE-BAL. The transcription factor NF-κB was identified as a target for SNO-SP-D by IPA, which was confirmed by Trans-AM ELISA in vitro. In vivo, SP-D overexpression increases the burden of infection in a Pneumocystis model while increasing cellular recruitment. Expression of iNOS and the production of NO metabolites were significantly reduced in SP-D OE mice relative to C57BL6/J. Inflammatory gene expression was increased in infected C57BL6/J mice but decreased in SP-D OE. SP-D oligomeric structure was disrupted in C57BL6/J infected mice but unaltered within SP-D OE. Thus SP-D modulates macrophage phenotype and the balance of multimeric to trimeric SP-D is critical to this regulation.

Protein kinase R-like endoplasmatic reticulum kinase is a mediator of stretch in ventilator-induced lung injury.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a severe form of lung injury characterized by damage to the epithelial barrier with subsequent pulmonary edema and hypoxic respiratory failure. ARDS is a significant medical problem in intensive care units with associated high care costs. There are many potential causes of ARDS; however, alveolar injury associated with mechanical ventilation, termed ventilator-induced lung injury (VILI), remains a well-recognized contributor. It is thus critical to understand the mechanism of VILI. Based on our published preliminary data, we hypothesized that the endoplasmic reticulum (ER) stress response molecule Protein Kinase R-like Endoplasmic Reticulum Kinase (PERK) plays a role in transmitting mechanosensory signals the alveolar epithelium. METHODS: ER stress signal responses to mechanical stretch were studied in ex-vivo ventilated pig lungs. To explore the effect of PERK inhibition on VILI, we ventilated live rats and compared lung injury parameters to non-ventilated controls. The effect of stretch-induced epithelial ER Ca2+ signaling on PERK was studied in stretched alveolar epithelial monolayers. To confirm the activation of PERK in human disease, ER stress signaling was compared between ARDS and non-ARDS lungs. RESULTS: Our studies revealed increased PERK-specific ER stress signaling in response to overstretch. PERK inhibition resulted in dose-dependent improvement of alveolar inflammation and permeability. Our data indicate that stretch-induced epithelial ER Ca2+ release is an activator of PERK. Experiments with human lung tissue confirmed PERK activation by ARDS. CONCLUSION: Our study provides evidences that PERK is a mediator stretch signals in the alveolar epithelium.

When Is an Alveolar Type 2 Cell an Alveolar Type 2 Cell? A Conundrum for Lung Stem Cell Biology and Regenerative Medicine.

Generating mature, differentiated, adult lung cells from pluripotent cells, such as induced pluripotent stem cells and embryonic stem cells, offers the hope of both generating disease-specific in vitro models and creating definitive and personalized therapies for a host of debilitating lung parenchymal and airway diseases. With the goal of advancing lung-regenerative medicine, several groups have developed and reported on protocols using defined media, coculture with mesenchymal components, or sequential treatments mimicking lung development, to obtain distal lung epithelial cells from stem cell precursors. However, there remains significant controversy about the degree of differentiation of these cells compared with their primary counterparts, coupled with a lack of consistency or uniformity in assessing the resultant phenotypes. Given the inevitable, exponential expansion of these approaches and the probable, but yet-to-emerge second and higher generation techniques to create such assets, we were prompted to pose the question, what makes a lung epithelial cell a lung epithelial cell? More specifically for this Perspective, we also posed the question, what are the minimum features that constitute an alveolar type (AT) 2 epithelial cell? In addressing this, we summarize a body of work spanning nearly five decades, amassed by a series of "lung epithelial cell biology pioneers," which carefully describes well characterized molecular, functional, and morphological features critical for discriminately assessing an AT2 phenotype. Armed with this, we propose a series of core criteria to assist the field in confirming that cells obtained following a differentiation protocol are indeed mature and functional AT2 epithelial cells.

Impaired Lysosomal Integral Membrane Protein 2-dependent Peroxiredoxin 6 Delivery to Lamellar Bodies Accounts for Altered Alveolar Phospholipid Content in Adaptor Protein-3-deficient pearl Mice.

The Hermansky Pudlak syndromes (HPS) constitute a family of disorders characterized by oculocutaneous albinism and bleeding diathesis, often associated with lethal lung fibrosis. HPS results from mutations in genes of membrane trafficking complexes that facilitate delivery of cargo to lysosome-related organelles. Among the affected lysosome-related organelles are lamellar bodies (LB) within alveolar type 2 cells (AT2) in which surfactant components are assembled, modified, and stored. AT2 from HPS patients and mouse models of HPS exhibit enlarged LB with increased phospholipid content, but the mechanism underlying these defects is unknown. We now show that AT2 in the pearl mouse model of HPS type 2 lacking the adaptor protein 3 complex (AP-3) fails to accumulate the soluble enzyme peroxiredoxin 6 (PRDX6) in LB. This defect reflects impaired AP-3-dependent trafficking of PRDX6 to LB, because pearl mouse AT2 cells harbor a normal total PRDX6 content. AP-3-dependent targeting of PRDX6 to LB requires the transmembrane protein LIMP-2/SCARB2, a known AP-3-dependent cargo protein that functions as a carrier for lysosomal proteins in other cell types. Depletion of LB PRDX6 in AP-3- or LIMP-2/SCARB2-deficient mice correlates with phospholipid accumulation in lamellar bodies and with defective intraluminal degradation of LB disaturated phosphatidylcholine. Furthermore, AP-3-dependent LB targeting is facilitated by protein/protein interaction between LIMP-2/SCARB2 and PRDX6 in vitro and in vivo Our data provide the first evidence for an AP-3-dependent cargo protein required for the maturation of LB in AT2 and suggest that the loss of PRDX6 activity contributes to the pathogenic changes in LB phospholipid homeostasis found HPS2 patients.

The biology of the ABCA3 lipid transporter in lung health and disease.

The lipid transporter, ATP-binding cassette class A3 (ABCA3), is a highly conserved multi-membrane-spanning protein that plays a critical role in the regulation of pulmonary surfactant homeostasis. Mutations in ABCA3 have been increasingly recognized as one of the causes of inherited pulmonary diseases. These monogenic disorders produce familial lung abnormalities with pathological presentations ranging from neonatal surfactant-deficiency-induced respiratory failure to childhood or adult diffuse parenchymal lung diseases for which specific treatment modalities remain limited. More than 200 ABCA3 mutations have been reported to date with approximately three quarters of patients presenting as compound heterozygotes. Recent advances in our understanding of the molecular basis underlying normal ABCA3 biosynthesis and processing and of the mechanisms of alveolar epithelial cell dysregulation caused by the expression of its mutant forms are beginning to emerge. These insights and the role of environmental factors and modifier genes are discussed in the context of the considerable variability in disease presentation observed in patients with identical ABCA3 gene mutations. Moreover, the opportunities afforded by an enhanced understanding of ABCA3 biology for targeted therapeutic strategies are addressed.

Neutrophils promote alveolar epithelial regeneration by enhancing type II pneumocyte proliferation in a model of acid-induced acute lung injury.

Alveolar epithelial regeneration is essential for resolution of the acute respiratory distress syndrome (ARDS). Although neutrophils have traditionally been considered mediators of epithelial damage, recent studies suggest they promote type II pneumocyte (AT2) proliferation, which is essential for regenerating alveolar epithelium. These studies did not, however, evaluate this relationship in an in vivo model of alveolar epithelial repair following injury. To determine whether neutrophils influence alveolar epithelial repair in vivo, we developed a unilateral acid injury model that creates a severe yet survivable injury with features similar to ARDS. Mice that received injections of the neutrophil-depleting Ly6G antibody had impaired AT2 proliferation 24 and 72 h after acid instillation, which was associated with decreased reepithelialization and increased alveolar protein concentration 72 h after injury. As neutrophil depletion itself may alter the cytokine response, we questioned the contribution of neutrophils to alveolar epithelial repair in neutropenic granulocyte-colony stimulating factor (G-CSF)-/- mice. We found that the loss of G-CSF recapitulated the neutrophil response of Ly6G-treated mice and was associated with defective alveolar epithelial repair, similar to neutrophil-depleted mice, and was reversed by administration of exogenous G-CSF. To approach the mechanisms, we employed an unbiased protein analysis of bronchoalveolar lavage fluid from neutrophil-depleted and neutrophil-replete mice 12 h after inducing lung injury. Pathway analysis identified significant differences in multiple signaling pathways that may explain the differences in epithelial repair. These data emphasize an important link between the innate immune response and tissue repair in which neutrophils promote alveolar epithelial regeneration.