Molecular Endotypes of Idiopathic Pulmonary Fibrosis: A Latent Class Analysis of Two Multicenter Observational Cohorts.

Rationale: Idiopathic pulmonary fibrosis (IPF) causes irreversible fibrosis of the lung parenchyma. Although antifibrotic therapy can slow IPF progression, treatment response is variable. There exists a critical need to develop a precision medicine approach to IPF. Objectives: To identify and validate biologically driven molecular endotypes of IPF. Methods: Latent class analysis (LCA) was independently performed in prospectively recruited discovery (n = 875) and validation (n = 347) cohorts. Twenty-five plasma biomarkers associated with fibrogenesis served as class-defining variables. The association between molecular endotype and 4-year transplant-free survival was tested using multivariable Cox regression adjusted for baseline confounders. Endotype-dependent differential treatment response to future antifibrotic exposure was then assessed in a pooled cohort of patients naive to antifibrotic therapy at the time of biomarker measurement (n = 555). Measurements and Main Results: LCA independently identified two latent classes in both cohorts (P < 0.0001). WFDC2 (WAP four-disulfide core domain protein 2) was the most important determinant of class membership across cohorts. Membership in class 2 was characterized by higher biomarker concentrations and a higher risk of death or transplant (discovery, hazard ratio [HR], 2.02; 95% confidence interval [CI], 1.64-2.48; P < 0.001; validation, HR, 1.95; 95% CI, 1.34-2.82; P < 0.001). In pooled analysis, significant heterogeneity in treatment effect was observed between endotypes (P = 0.030 for interaction), with a favorable antifibrotic response in class 2 (HR, 0.64; 95% CI, 0.45-0.93; P = 0.018) but not in class 1 (HR, 1.19; 95% CI, 0.77-1.84; P = 0.422). Conclusions: In this multicohort study, we identified two novel molecular endotypes of IPF with divergent clinical outcomes and responses to antifibrotic therapy. Pending further validation, these endotypes could enable a precision medicine approach for future IPF clinical trials.

RSV enhances Staphylococcus aureus bacterial growth in the lung.

Patients coinfected with respiratory syncytial virus (RSV) and bacteria have longer hospital stays, higher risk of intensive care unit admission, and worse outcomes. We describe a model of RSV line 19F/methicillin-resistant Staphylococcus aureus (MRSA) USA300 coinfection that does not impair viral clearance, but prior RSV infection enhances USA300 MRSA bacterial growth in the lung. The increased bacterial burden post-RSV correlates with reduced accumulation of neutrophils and impaired bacterial killing by alveolar macrophages. Surprisingly, reduced neutrophil accumulation is likely not explained by reductions in phagocyte-recruiting chemokines or alterations in proinflammatory cytokine production compared with mice infected with S. aureus alone. Neutrophils from RSV-infected mice retain their ability to migrate toward chemokine signals, and neutrophils from the RSV-infected lung are better able to phagocytize and kill S. aureus ex vivo on a per cell basis. In contrast, while alveolar macrophages could ingest USA300 post-RSV, intracellular bacterial killing was impaired. The RSV/S. aureus coinfected lung promotes a state of overactivation in neutrophils, demonstrated by increased production of reactive oxygen species (ROS) that can drive formation of neutrophil extracellular traps (NETs), resulting in cell death. Mice with RSV/S. aureus coinfection had increased extracellular DNA and protein in bronchoalveolar lavage fluid and histological evidence confirmed NETosis in vivo. Taken together, these data highlight that prior RSV infection can prime the overactivation of neutrophils leading to cell death that impairs neutrophil accumulation in the lung. Additionally, alveolar macrophage killing of bacteria is impaired post-RSV. Together, these defects enhance USA300 MRSA bacterial growth in the lung post-RSV.

Early-life pulmonary viral infection leads to long-term functional and lower airway structural changes in the lungs.

Early-life respiratory virus infections have been correlated with enhanced development of childhood asthma. In particular, significant numbers of respiratory syncytial virus (RSV)-hospitalized infants go on to develop lung disease. It has been suggested that early-life viral infections may lead to altered lung development or repair that negatively impacts lung function later in life. Our data demonstrate that early-life RSV infection modifies lung structure, leading to decreased lung function. At 5 wk postneonatal RSV infection, significant defects are observed in baseline pulmonary function test (PFT) parameters consistent with decreased lung function as well as enlarged alveolar spaces. Lung function changes in the early-life RSV-infected group continue at 3 mo of age. The altered PFT and structural changes induced by early-life RSV were mitigated in TSLPR-/- mice that have previously been shown to have reduced immune cell accumulation associated with a persistent Th2 environment. Importantly, long-term effects were demonstrated using a secondary RSV infection 3 mo following the initial early-life RSV infection and led to significant additional defects in lung function, with severe mucus deposition within the airways, and consolidation of the alveolar spaces. These studies suggest that early-life respiratory viral infection leads to alterations in lung structure/repair that predispose to diminished lung function later in life.NEW & NOTEWORTHY These studies outline a novel finding that early-life respiratory virus infection can alter lung structure and function long-term. Importantly, the data also indicate that there are critical links between inflammatory responses and subsequent events that produce a more severe pathogenic response later in life. The findings provide additional data to support that early-life infections during lung development can alter the trajectory of airway function.

Proteomic Biomarkers of Survival in Idiopathic Pulmonary Fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) causes progressive lung scarring and high mortality. Reliable and accurate prognostic biomarkers are urgently needed. OBJECTIVE: To identify and validate circulating protein biomarkers of IPF survival. METHODS: High-throughput proteomic data were generated using prospectively collected plasma samples from patients with IPF from the Pulmonary Fibrosis Foundation Patient Registry (discovery cohort) and the Universities of California-Davis, Chicago, and Virginia (validation cohort). Proteins associated with three-year transplant-free survival (TFS) were identified using multivariable Cox proportional hazards regression. Those associated with TFS after adjustment for false discovery in the discovery cohort were advanced for testing in the validation cohort, with proteins maintaining TFS association with consistent effect direction considered validated. After combining cohorts, functional analyses were performed, and machine learning used to derive a proteomic signature of TFS. MAIN RESULTS: Of 2921 proteins tested in the discovery cohort (n=871), 231 were associated with differential TFS. Of these, 140 maintained TFS association with consistent effect direction in the validation cohort (n=355). After combining cohorts, validated proteins with strongest TFS association were latent-transforming growth factor beta-binding protein 2 (HR 2.43, 95% CI 2.09-2.82), collagen alpha-1(XXIV) chain (HR 2.21; 95% CI 1.86-2.39) and keratin 19 (HR 1.60; 95% CI 1.47-1.74). In decision curve analysis, a proteomic signature of TFS outperformed a similarly derived clinical prediction model. CONCLUSIONS: In largest proteomic investigation of IPF outcomes performed to date, we identified and validated 140 protein biomarkers of TFS. These results shed important light on potential drivers of IPF progression.

Fatal COVID-19 and Non-COVID-19 Acute Respiratory Distress Syndrome Is Associated with Incomplete Alveolar Type 1 Epithelial Cell Differentiation from the Transitional State without Fibrosis.

Acute respiratory distress syndrome (ARDS) due to coronavirus disease 2019 and other etiologies results from injury to the alveolar epithelial cell (AEC) barrier resulting in noncardiogenic pulmonary edema, which causes acute respiratory failure; recovery requires epithelial regeneration. During physiological regeneration in mice, type 2 AECs (AEC2s) proliferate, exit the cell cycle, transiently assume a transitional state, then differentiate into type 1 AECs (AEC1s); in humans, persistence of the transitional state is associated with pulmonary fibrosis. It is unknown whether transitional cells emerge and differentiate into AEC1s without fibrosis in human ARDS and why transitional cells differentiate into AEC1s during physiological regeneration but persist in fibrosis. We hypothesized that incomplete but ongoing AEC1 differentiation from transitional cells without fibrosis may underlie persistent barrier permeability and acute respiratory failure in ARDS. Immunostaining of postmortem ARDS lungs revealed abundant transitional cells without fibrosis. They were typically cuboidal or partially spread, sometimes flat, and occasionally expressed AEC1 markers. Immunostaining and/or single-cell RNA sequencing revealed that transitional cells in mouse models of physiological regeneration, ARDS, and fibrosis express markers of cell cycle exit but only in fibrosis express a specific senescence marker. Thus, in severe, fatal early ARDS, AEC1 differentiation from transitional cells is incomplete, underlying persistent barrier permeability and respiratory failure but ongoing without fibrosis; senescence of transitional cells may be associated with pulmonary fibrosis.

Myeloid- and Epithelial-derived Heparin-Binding Epidermal Growth Factor-like Growth Factor Promotes Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a poorly understood, progressive lethal lung disease with no known cure. In addition to alveolar epithelial cell (AEC) injury and excessive deposition of extracellular matrix proteins, chronic inflammation is a hallmark of IPF. Literature suggests that the persistent inflammation seen in IPF primarily consists of monocytes and macrophages. Recent work demonstrates that monocyte-derived alveolar macrophages (moAMs) drive lung fibrosis, but further characterization of critical moAM cell attributes is necessary. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is an important epidermal growth factor receptor ligand that has essential roles in angiogenesis, wound healing, keratinocyte migration, and epithelial-mesenchymal transition. Our past work has shown HB-EGF is a primary marker of profibrotic M2 macrophages, and this study seeks to characterize myeloid-derived HB-EGF and its primary mechanism of action in bleomycin-induced lung fibrosis using Hbegff/f;Lyz2Cre+ mice. Here, we show that patients with IPF and mice with pulmonary fibrosis have increased expression of HB-EGF and that lung macrophages and transitional AECs of mice with pulmonary fibrosis and humans all express HB-EGF. We also show that Hbegff/f;Lyz2Cre+ mice are protected from bleomycin-induced fibrosis and that this protection is likely multifactorial, caused by decreased CCL2-dependent monocyte migration, decreased fibroblast migration, and decreased contribution of HB-EGF from AEC sources when HB-EGF is removed under the Lyz2Cre promoter.

New Insights into Clinical and Mechanistic Heterogeneity of the Acute Respiratory Distress Syndrome: Summary of the Aspen Lung Conference 2021.

Clinical and molecular heterogeneity are common features of human disease. Understanding the basis for heterogeneity has led to major advances in therapy for many cancers and pulmonary diseases such as cystic fibrosis and asthma. Although heterogeneity of risk factors, disease severity, and outcomes in survivors are common features of the acute respiratory distress syndrome (ARDS), many challenges exist in understanding the clinical and molecular basis for disease heterogeneity and using heterogeneity to tailor therapy for individual patients. This report summarizes the proceedings of the 2021 Aspen Lung Conference, which was organized to review key issues related to understanding clinical and molecular heterogeneity in ARDS. The goals were to review new information about ARDS phenotypes, to explore multicellular and multisystem mechanisms responsible for heterogeneity, and to review how best to account for clinical and molecular heterogeneity in clinical trial design and assessment of outcomes. The report concludes with recommendations for future research to understand the clinical and basic mechanisms underlying heterogeneity in ARDS to advance the development of new treatments for this life-threatening critical illness.

Update on the Features and Measurements of Experimental Acute Lung Injury in Animals: An Official American Thoracic Society Workshop Report.

Advancements in methods, technology, and our understanding of the pathobiology of lung injury have created the need to update the definition of experimental acute lung injury (ALI). We queried 50 participants with expertise in ALI and acute respiratory distress syndrome using a Delphi method composed of a series of electronic surveys and a virtual workshop. We propose that ALI presents as a "multidimensional entity" characterized by four "domains" that reflect the key pathophysiologic features and underlying biology of human acute respiratory distress syndrome. These domains are 1) histological evidence of tissue injury, 2) alteration of the alveolar-capillary barrier, 3) presence of an inflammatory response, and 4) physiologic dysfunction. For each domain, we present "relevant measurements," defined as those proposed by at least 30% of respondents. We propose that experimental ALI encompasses a continuum of models ranging from those focusing on gaining specific mechanistic insights to those primarily concerned with preclinical testing of novel therapeutics or interventions. We suggest that mechanistic studies may justifiably focus on a single domain of lung injury, but models must document alterations of at least three of the four domains to qualify as "experimental ALI." Finally, we propose that a time criterion defining "acute" in ALI remains relevant, but the actual time may vary based on the specific model and the aspect of injury being modeled. The continuum concept of ALI increases the flexibility and applicability of the definition to multiple models while increasing the likelihood of translating preclinical findings to critically ill patients.

Persistent, Progressive Pulmonary Fibrosis and Epithelial Remodeling in Mice.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic interstitial lung disease with underlying mechanisms that have been primarily investigated in mice after intratracheal instillation of a single dose of bleomycin. However, the model has significant limitations, including transient fibrosis that spontaneously resolves and its failure to fully recapitulate the epithelial remodeling in the lungs of patients with IPF. Thus, there remains an unmet need for a preclinical model with features that more closely resemble the human disease. Repetitive intratracheal instillation of bleomycin has previously been shown to recapitulate some of these features, but the instillation procedure is complex, and the long-term consequences on epithelial remodeling and fibrosis persistence and progression remain poorly understood. Here, we developed a simplified repetitive bleomycin instillation strategy consisting of three bi-weekly instillations that leads to persistent and progressive pulmonary fibrosis. Lung histology demonstrates increased collagen deposition, fibroblast accumulation, loss of type I and type II alveolar epithelial cells within fibrotic areas, bronchiolization of the lung parenchyma with CCSP+ cells, remodeling of the distal lung into cysts reminiscent of simple honeycombing, and accumulation of hyperplastic transitional KRT8+ epithelial cells. Micro-computed tomographic imaging demonstrated significant traction bronchiectasis and subpleural fibrosis. Thus, the simplified repetitive bleomycin instillation strategy leads to progressive fibrosis and recapitulates the histological and radiographic characteristics of IPF. Compared with the single bleomycin instillation model, we suggest that the simplified repetitive instillation model may be better suited to address mechanistic questions about IPF pathogenesis and preclinical studies of antifibrotic drug candidates.

Long-term survivors of murine sepsis are predisposed to enhanced LPS-induced lung injury and proinflammatory immune reprogramming.

Millions of people who survive sepsis each year are rehospitalized and die due to late pulmonary complications. To prevent and treat these complications, biomarkers and molecular mediators must be identified. Persistent immune reprogramming in the form of immunoparalysis and impaired host defense is proposed to mediate late pulmonary complications after sepsis, particularly new pulmonary infections. However, immune reprogramming may also involve enhanced/primed responses to secondary stimuli, although their contribution to long-term sepsis complications remains understudied. We hypothesize that enhanced/primed immune responses in the lungs of sepsis survivors are associated with late pulmonary complications. To this end, we developed a murine sepsis model using cecal ligation and puncture (CLP) followed 3 wk later by administration of intranasal lipopolysaccharide to induce inflammatory lung injury. Mice surviving sepsis exhibit enhanced lung injury with increased alveolar permeability, neutrophil recruitment, and enhanced Ly6Chi monocyte Tnf expression. To determine the mediators of enhanced lung injury, we performed flow cytometry and RNA sequencing of lungs 3 wk after CLP, prior to lipopolysaccharide. Sepsis survivor mice showed expanded Ly6Chi monocytes populations and increased expression of many inflammatory genes. Of these, S100A8/A9 was also elevated in the circulation of human sepsis survivors for months after sepsis, validating our model and identifying S100A8/A9 as a potential biomarker and therapeutic target for long-term pulmonary complications after sepsis. These data provide new insight into the importance of enhanced/primed immune responses in survivors of sepsis and establish a foundation for additional investigation into the mechanisms mediating this response.

Efficient CD4Cre-Mediated Conditional KRas Expression in Alveolar Macrophages and Alveolar Epithelial Cells Causes Fatal Hyperproliferative Pneumonitis.

The CD4Cre transgenic model has been widely used for T cell-specific gene manipulation. We report unexpected highly efficient Cre-mediated recombination in alveolar macrophages (AMFs), bronchial epithelial cells (BECs), and alveolar epithelial cells (AECs) in this strain of mice. Different from CD4 T cells, AMFs, AECs, and BECs do not express detectable Cre protein, suggesting that Cre protein is either very transiently expressed in these cells or only expressed in their precursors. Mice carrying a conditional constitutively active KRas (caKRas) allele and the CD4Cre transgene contain not only hyperactivated T cells but also develop severe AMF accumulation, AEC and BEC hyperplasia, and adenomas in the lung, leading to early lethality correlated with caKRas expression in these cells. We propose that caKRas-CD4Cre mice represent, to our knowledge, a novel model of proliferative pneumonitis involving macrophages and epithelial cells and that the CD4Cre model may offer unique usefulness for studying gene functions simultaneously in multilineages in the lung. Our observations, additionally, suggest that caution in data interpretation is warranted when using the CD4Cre transgenic model for T cell-specific gene manipulation, particularly when lung pathophysiological status is being examined.

Diverse Injury Pathways Induce Alveolar Epithelial Cell CCL2/12, Which Promotes Lung Fibrosis.

Accumulating evidence suggests that fibrosis is a multicellular process with contributions from alveolar epithelial cells (AECs), recruited monocytes/macrophages, and fibroblasts. We have previously shown that AEC injury is sufficient to induce fibrosis, but the precise mechanism remains unclear. Several cell types, including AECs, can produce CCL2 and CCL12, which can promote fibrosis through CCR2 activation. CCR2 signaling is critical for the initiation and progression of pulmonary fibrosis, in part through recruitment of profibrotic bone marrow-derived monocytes. Attempts at inhibiting CCL2 in patients with fibrosis demonstrated a marked upregulation of CCL2 production and no therapeutic response. To better understand the mechanisms involved in CCL2/CCR2 signaling, we generated mice with conditional deletion of CCL12, a murine homolog of human CCL2. Surprisingly, we found that mice with complete deletion of CCL12 had markedly increased concentrations of other CCR2 ligands and were not protected from fibrosis after bleomycin injury. In contrast, mice with lung epithelial cell-specific deletion of CCL12 were protected from bleomycin-induced fibrosis and had expression of CCL2 and CCL7 similar to that of control mice treated with bleomycin. Deletion of CCL12 within AECs led to decreased recruitment of exudate macrophages. Finally, injury to murine and human primary AECs resulted in increased production of CCL2 and CCL12, in part through activation of the mTOR pathway. In conclusion, these data suggest that targeting CCL2 may be a viable antifibrotic strategy once the pathways involved in the production and function of CCL2 and other CCR2 ligands are better defined.

The ex vivo perfused human lung is resistant to injury by high-dose S. pneumoniae bacteremia.

Few patients with bacteremia from a nonpulmonary source develop acute respiratory distress syndrome (ARDS). However, the mechanisms that protect the lung from injury in bacteremia have not been identified. We simulated bacteremia by adding Streptococcus pneumoniae to the perfusate of the ex vivo perfused human lung model. In contrast to a pneumonia model in which bacteria were instilled into the distal air spaces of one lobe, injection of high doses of S. pneumoniae into the perfusate was not associated with alveolar epithelial injury as demonstrated by low protein permeability of the alveolar epithelium, intact alveolar fluid clearance, and the absence of alveolar edema. Unexpectedly, the ex vivo human lung rapidly cleared large quantities of S. pneumoniae even though the perfusate had very few intravascular phagocytes and lacked immunoglobulins or complement. The bacteria were cleared in part by the small number of neutrophils in the perfusate, alveolar macrophages in the airspaces, and probably by interstitial pathways. Together, these findings identify one mechanism by which the lung and the alveolar epithelium are protected from injury in bacteremia.

Protein tyrosine phosphatase-α amplifies transforming growth factor-ß-dependent profibrotic signaling in lung fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a progressive, often fatal, fibrosing lung disease for which treatment remains suboptimal. Fibrogenic cytokines, including transforming growth factor-ß (TGF-ß), are central to its pathogenesis. Protein tyrosine phosphatase-α (PTPα) has emerged as a key regulator of fibrogenic signaling in fibroblasts. We have reported that mice globally deficient in PTPα (Ptpra-/-) were protected from experimental pulmonary fibrosis, in part via alterations in TGF-ß signaling. The goal of this study was to determine the lung cell types and mechanisms by which PTPα controls fibrogenic pathways and whether these pathways are relevant to human disease. Immunohistochemical analysis of lungs from patients with IPF revealed that PTPα was highly expressed by mesenchymal cells in fibroblastic foci and by airway and alveolar epithelial cells. To determine whether PTPα promotes profibrotic signaling pathways in lung fibroblasts and/or epithelial cells, we generated mice with conditional (floxed) Ptpra alleles (Ptpraf/f). These mice were crossed with Dermo1-Cre or with Sftpc-CreERT2 mice to delete Ptpra in mesenchymal cells and alveolar type II cells, respectively. Dermo1-Cre/Ptpraf/f mice were protected from bleomycin-induced pulmonary fibrosis, whereas Sftpc-CreERT2/Ptpraf/f mice developed pulmonary fibrosis equivalent to controls. Both canonical and noncanonical TGF-ß signaling and downstream TGF-ß-induced fibrogenic responses were attenuated in isolated Ptpra-/- compared with wild-type fibroblasts. Furthermore, TGF-ß-induced tyrosine phosphorylation of TGF-ß type II receptor and of PTPα were attenuated in Ptpra-/- compared with wild-type fibroblasts. The phenotype of cells genetically deficient in PTPα was recapitulated with the use of a Src inhibitor. These findings suggest that PTPα amplifies profibrotic TGF-ß-dependent pathway signaling in lung fibroblasts.

Cellular senescence: friend or foe to respiratory viral infections?

Cellular senescence permanently arrests the replication of various cell types and contributes to age-associated diseases. In particular, cellular senescence may enhance chronic lung diseases including COPD and idiopathic pulmonary fibrosis. However, the role cellular senescence plays in the pathophysiology of acute inflammatory diseases, especially viral infections, is less well understood. There is evidence that cellular senescence prevents viral replication by increasing antiviral cytokines, but other evidence shows that senescence may enhance viral replication by downregulating antiviral signalling. Furthermore, cellular senescence leads to the secretion of inflammatory mediators, which may either promote host defence or exacerbate immune pathology during viral infections. In this Perspective article, we summarise how senescence contributes to physiology and disease, the role of senescence in chronic lung diseases, and how senescence impacts acute respiratory viral infections. Finally, we develop a potential framework for how senescence may contribute, both positively and negatively, to the pathophysiology of viral respiratory infections, including severe acute respiratory syndrome due to the coronavirus SARS-CoV-2.

Mechanisms of ATII-to-ATI Cell Differentiation during Lung Regeneration.

The alveolar epithelium consists of (ATI) and type II (ATII) cells. ATI cells cover the majority of the alveolar surface due to their thin, elongated shape and are largely responsible for barrier function and gas exchange. During lung injury, ATI cells are susceptible to injury, including cell death. Under some circumstances, ATII cells also die. To regenerate lost epithelial cells, ATII cells serve as progenitor cells. They proliferate to create new ATII cells and then differentiate into ATI cells [1,2,3]. Regeneration of ATI cells is critical to restore normal barrier and gas exchange function. Although the signaling pathways by which ATII cells proliferate have been explored [4,5,6,7,8,9,10,11,12], the mechanisms of ATII-to-ATI cell differentiation have not been well studied until recently. New studies have uncovered signaling pathways that mediate ATII-to-ATI differentiation. Bone morphogenetic protein (BMP) signaling inhibits ATII proliferation and promotes differentiation. Wnt/ß-catenin and ETS variant transcription factor 5 (Etv5) signaling promote proliferation and inhibit differentiation. Delta-like 1 homolog (Dlk1) leads to a precisely timed inhibition of Notch signaling in later stages of alveolar repair, activating differentiation. Yes-associated protein/Transcriptional coactivator with PDZ-binding motif (YAP/TAZ) signaling appears to promote both proliferation and differentiation. We recently identified a novel transitional cell state through which ATII cells pass as they differentiate into ATI cells, and this has been validated by others in various models of lung injury. This intermediate cell state is characterized by the activation of Transforming growth factor beta (TGFß) and other pathways, and some evidence suggests that TGFß signaling induces and maintains this state. While the abovementioned signaling pathways have all been shown to be involved in ATII-to-ATI cell differentiation during lung regeneration, there is much that remains to be understood. The up- and down-stream signaling events by which these pathways are activated and by which they induce ATI cell differentiation are unknown. In addition, it is still unknown how the various mechanistic steps from each pathway interact with one another to control differentiation. Based on these recent studies that identified major signaling pathways driving ATII-to-ATI differentiation during alveolar regeneration, additional studies can be devised to understand the interaction between these pathways as they work in a coordinated manner to regulate differentiation. Moreover, the knowledge from these studies may eventually be used to develop new clinical treatments that accelerate epithelial cell regeneration in individuals with excessive lung damage, such as patients with the Acute Respiratory Distress Syndrome (ARDS), pulmonary fibrosis, and emphysema.

Transitional human alveolar type II epithelial cells suppress extracellular matrix and growth factor gene expression in lung fibroblasts.

Epithelial-fibroblast interactions are thought to be very important in the adult lung in response to injury, but the specifics of these interactions are not well defined. We developed coculture systems to define the interactions of adult human alveolar epithelial cells with lung fibroblasts. Alveolar type II cells cultured on floating collagen gels reduced the expression of type 1 collagen (COL1A1) and α-smooth muscle actin (ACTA2) in fibroblasts. They also reduced fibroblast expression of hepatocyte growth factor (HGF), fibroblast growth factor 7 (FGF7, KGF), and FGF10. When type II cells were cultured at an air-liquid interface to maintain high levels of surfactant protein expression, this inhibitory activity was lost. When type II cells were cultured on collagen-coated tissue culture wells to reduce surfactant protein expression further and increase the expression of some type I cell markers, the epithelial cells suppressed transforming growth factor-ß (TGF-ß)-stimulated ACTA2 and connective tissue growth factor (CTGF) expression in lung fibroblasts. Our results suggest that transitional alveolar type II cells and likely type I cells but not fully differentiated type II cells inhibit matrix and growth factor expression in fibroblasts. These cells express markers of both type II cells and type I cells. This is probably a normal homeostatic mechanism to inhibit the fibrotic response in the resolution phase of wound healing. Defining how transitional type II cells convert activated fibroblasts into a quiescent state and inhibit the effects of TGF-ß may provide another approach to limiting the development of fibrosis after alveolar injury.