Phage Immunoprecipitation-Sequencing Reveals CDHR5 Autoantibodies in Select Patients With Interstitial Lung Disease.

OBJECTIVE: Interstitial lung diseases (ILDs) are a heterogeneous group of disorders that can develop in patients with connective tissue diseases. Establishing autoimmunity in ILD impacts prognosis and treatment. Patients with ILD are screened for autoimmunity by measuring antinuclear autoantibodies, rheumatoid factors, and other nonspecific tests. However, this approach may miss autoimmunity that manifests as autoantibodies to tissue antigens not previously defined in ILD. METHODS: We use Phage Immunoprecipitation-Sequencing (PhIP-Seq) to conduct an autoantibody discovery screen of patients with ILD and controls. We screened for novel autoantigen candidates using PhIP-Seq. We next developed a radio-labeled binding assay and validated the leading candidate in 398 patients with ILD recruited from two academic medical centers and 138 blood bank individuals that formed our reference cohort. RESULTS: PhIP-Seq identified 17 novel autoreactive targets, and machine learning classifiers derived from these targets discriminated ILD serum from controls. Among the 17 candidates, we validated CDHR5 and found CDHR5 autoantibodies in patients with rheumatologic disorders and importantly, patients not previously diagnosed with autoimmunity. Using survival and transplant free-survival data available from one of the two centers, patients with CDHR5 autoantibodies showed worse survival compared with other patients with connective tissue disease ILD. CONCLUSION: We used PhIP-Seq to define a novel CDHR5 autoantibody in a subset of select patients with ILD. Our data complement a recent study showing polymorphisms in the CDHR5-IRF7 gene locus strongly associated with titer of anticentromere antibodies in systemic sclerosis, creating a growing body of evidence suggesting a link between CDHR5 and autoimmunity.

Alveolar fibroblast lineage orchestrates lung inflammation and fibrosis.

Fibroblasts are present throughout the body and function to maintain tissue homeostasis. Recent studies have identified diverse fibroblast subsets in healthy and injured tissues1,2, but the origins and functional roles of injury-induced fibroblast lineages remain unclear. Here we show that lung-specialized alveolar fibroblasts take on multiple molecular states with distinct roles in facilitating responses to fibrotic lung injury. We generate a genetic tool that uniquely targets alveolar fibroblasts to demonstrate their role in providing niches for alveolar stem cells in homeostasis and show that loss of this niche leads to exaggerated responses to acute lung injury. Lineage tracing identifies alveolar fibroblasts as the dominant origin for multiple emergent fibroblast subsets sequentially driven by inflammatory and pro-fibrotic signals after injury. We identify similar, but not completely identical, fibroblast lineages in human pulmonary fibrosis. TGFß negatively regulates an inflammatory fibroblast subset that emerges early after injury and stimulates the differentiation into fibrotic fibroblasts to elicit intra-alveolar fibrosis. Blocking the induction of fibrotic fibroblasts in the alveolar fibroblast lineage abrogates fibrosis but exacerbates lung inflammation. These results demonstrate the multifaceted roles of the alveolar fibroblast lineage in maintaining normal alveolar homeostasis and orchestrating sequential responses to lung injury.

A fibroblast-dependent TGFß1/sFRP2 noncanonical Wnt signaling axis promotes epithelial metaplasia in idiopathic pulmonary fibrosis.

Reciprocal interactions between alveolar fibroblasts and epithelial cells are crucial for lung homeostasis, injury repair, and fibrogenesis, but underlying mechanisms remain unclear. To investigate, we administered the fibroblast-selective TGFß1 signaling inhibitor, epigallocatechin gallate (EGCG), to Interstitial Lung Disease (ILD) patients undergoing diagnostic lung biopsy and conducted single-cell RNA sequencing on spare tissue. Biopsies from untreated patients showed higher fibroblast TGFß1 signaling compared to non-disease donor or end-stage ILD tissues. In vivo, EGCG downregulated TGFß1 signaling and several pro-inflammatory and stress pathways in biopsy samples. Notably, EGCG reduced fibroblast secreted frizzle-like receptor protein 2 (sFRP2), an unrecognized TGFß1 fibroblast target gene induced near type II alveolar epithelial cells (AEC2s) in situ. Using AEC2-fibroblast coculture organoids and precision cut lung slices (PCLS) from non-diseased donors, we found TGFß1 signaling promotes a spread AEC2 KRT17+ basaloid state, whereupon sFRP2 then activates a mature Krt5+ basal cell program. Wnt-receptor Frizzled 5 (Fzd5) expression and downstream calcineurin signaling were required for sFRP2-induced nuclear NFATc3 accumulation and KRT5 expression. These findings highlight stage-specific TGFß1 signaling in ILD, the therapeutic potential of EGCG in reducing IPF-related transcriptional changes, and identify TGFß1-non-canonical Wnt pathway crosstalk via sFRP2 as a novel mechanism for dysfunctional epithelial signaling in Idiopathic Pulmonary Fibrosis/ILD.

Association between mosaic loss of chromosome Y and pulmonary fibrosis susceptibility and severity.

Background Pulmonary fibrosis (PF) is a rare lung disease with diverse pathogenesis and multiple interconnected underlying biological mechanisms. Mosaic loss of chromosome Y (mLOY) is one of the most common forms of acquired chromosome abnormality in men, which has been reported to be associated with increased risk of various chronic progressive diseases including fibrotic diseases. However, the exact role of mLOY in the development of PF remains elusive and to be elucidated. Methods: We adopted three complementary approaches to explore the role of mLOY in the pathogenesis of PF. We used copy number on chromosome Y to estimate mLOY comparing patients in PROFILE and gnomAD cohorts and between cases and control patients from the GE100KGP cohort. Correlation of mLOY with demographic and clinical variables was tested using patients from PROFILE cohort. Lung single-cell transcriptomic data were analysed to assess the cell types implicated in mLOY. We performed Mendelian randomisation to examine the causal relationship between mLOY, IPF, and telomere length. Results: The genetic analysis suggests that mLOY is found in PF from both case cohorts but when compared with an age matched population the effect is minimal (P = 0.0032). mLOY is related to age (P = 0.00021) and shorter telomere length (P = 0.0081) rather than PF severity or progression. Single-cell analysis indicates that mLOY appears to be found primarily in immune cells and appears to be related to presence and severity of fibrosis. Mendelian randomisation demonstrates that mLOY is not on the causal pathway for IPF, but partial evidence supports that telomere shortening is on the causal pathway for mLOY. Conclusion: Our study confirms the existence of mLOY in PF patients and suggests that mLOY is not a major driver of IPF. The combined evidence suggests a triangulation model where telomere shortening leads to both IPF and mLOY.

PAI-1 Deficiency Drives Pulmonary Vascular Smooth Muscle Remodeling and Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by vasoconstriction and remodeling of small pulmonary arteries (PAs). Central to the remodeling process is a switch of pulmonary vascular cells to a proliferative, apoptosis-resistant phenotype. Plasminogen activator inhibitor-1 (PAI-1) is the primary physiological inhibitor of urokinase-type and tissue-type plasminogen activators (uPA and tPA), but its role in PAH is unsettled. Here, we report that: (1) PAI-1 is deficient in remodeled small PAs and in early-passage PA smooth muscle and endothelial cells (PASMCs and PAECs) from subjects with PAH compared to controls; (2) PAI-1-/- mice spontaneously develop pulmonary vascular remodeling associated with up-regulation of mTORC1 signaling, pulmonary hypertension (PH), and right ventricle (RV) hypertrophy; and (3) pharmacological inhibition of uPA in human PAH PASMCs suppresses pro-proliferative mTORC1 and SMAD3 signaling, restores PAI-1 levels, reduces proliferation and induces apoptosis in vitro, and prevents the development of SU5416/hypoxia-induced PH and RV hypertrophy in vivo in mice. These data strongly suggest that down-regulation of PAI-1 in small PAs promotes vascular remodeling and PH due to unopposed activation of uPA and consequent up-regulation of mTOR and TGF-b signaling in PASMCs, and call for further studies to determine the potential benefits of targeting the PAI-1/uPA imbalance to attenuate and/or reverse pulmonary vascular remodeling and PH.

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. While antifibrotic therapy can slow IPF progression, treatment response is variable. There exists a critical need to develop a precision medicine approach to IPF. Objective: 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 naïve to antifibrotic therapy at time of biomarker measurement (n=555). Results: LCA independently identified two latent classes in both cohorts (p<0.0001). WAP four-disulfide core domain protein 2 (WFDC2) was the most important determinant of class membership across cohorts. Membership in Class 2 was characterized by higher biomarker concentrations and higher risk of death or transplantation (discovery: HR 2.02 [95% CI 1.64-2.48]; p<0.001; validation: HR 1.95 [1.34-2.82]; p<0.001). In pooled analysis, significant heterogeneity in treatment effect was observed between endotypes (pinteraction=0.030), with a favorable antifibrotic response in Class 2 (HR 0.64 [0.45-0.93]; p=0.018) but not in Class 1 (HR 1.19 [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 response to antifibrotics. Pending further validation, these endotypes could enable a precision medicine approach for future IPF clinical trials.

An in vivo screening platform identifies senolytic compounds that target p16INK4a+ fibroblasts in lung fibrosis.

The appearance of senescent cells in age-related diseases has spurred the search for compounds that can target senescent cells in tissues, termed senolytics. However, a major caveat with current senolytic screens is the use of cell lines as targets where senescence is induced in vitro, which does not necessarily reflect the identity and function of pathogenic senescent cells in vivo. Here, we developed a new pipeline leveraging a fluorescent murine reporter that allows for isolation and quantification of p16Ink4a+ cells in diseased tissues. By high-throughput screening in vitro, precision-cut lung slice (PCLS) screening ex vivo, and phenotypic screening in vivo, we identified a HSP90 inhibitor, XL888, as a potent senolytic in tissue fibrosis. XL888 treatment eliminated pathogenic p16Ink4a+ fibroblasts in a murine model of lung fibrosis and reduced fibrotic burden. Finally, XL888 preferentially targeted p16INK4a-hi human lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis (IPF), and reduced p16INK4a+ fibroblasts from IPF PCLS ex vivo. This study provides proof of concept for a platform where p16INK4a+ cells are directly isolated from diseased tissues to identify compounds with in vivo and ex vivo efficacy in mice and humans, respectively, and provides a senolytic screening platform for other age-related diseases.

Invasive mucinous adenocarcinoma of the lung with bronchorrhea - A marked reduction volume of sputum after SARS-CoV-2 infection.

Bronchorrhea is a watery sputum volume of at least 100 mL/day, which is commonly associated with lung malignancies. We report a 57-year-old woman was admitted to the hospital with a cough, profuse sputum. Chest CTs showed crazy paving pattern and lung nodules. Cell nests were visible on the Thinprep Cytologic Test. The case was considered an invasive mucinous adenocarcinoma of the lung combined with bronchorrhea. Significantly, the sputum volume declined rapidly and did not rise again when the patient was diagnosed with COVID-19 and treated with nirmatrelvir/ritonavir. This case is suggestive of studies related to regulatory mediators associated with bronchorrhea.

Genome-wide screens identify SEL1L as an intracellular rheostat controlling collagen turnover.

Accumulating evidence has implicated impaired extracellular matrix (ECM) clearance as a key factor in fibrotic disease. Despite decades of research elucidating the effectors of ECM clearance, relatively little is understood regarding the upstream regulation of this process. Collagen is the most abundant constituent of normal and fibrotic ECM in mammalian tissues. Its catabolism occurs through extracellular proteolysis and cell-mediated uptake of collagen fragments for intracellular degradation. Given the paucity of information regarding the regulation of this latter process, here we execute unbiased genome-wide screens to understand the molecular underpinnings of cell-mediated collagen clearance. Using this approach, we discover a mechanism through which collagen biosynthesis is sensed by cells internally and directly regulates clearance of extracellular collagen. The sensing mechanism appears to be dependent on endoplasmic reticulum-resident protein SEL1L and occurs via a noncanonical function of this protein. This pathway functions as a homeostatic negative feedback loop that limits collagen accumulation in tissues. In human fibrotic lung disease, the induction of this collagen clearance pathway by collagen synthesis is impaired, thereby contributing to the pathological accumulation of collagen in lung tissue. Thus, we describe cell-autonomous, rheostatic collagen clearance as an important pathway of tissue homeostasis.

Neutrophil extracellular traps linked to idiopathic pulmonary fibrosis severity and survival.

Background: Idiopathic pulmonary fibrosis (IPF) leads to progressive loss of lung function and mortality. Understanding mechanisms and markers of lung injury in IPF is paramount to improving outcomes for these patients. Despite the lack of systemic involvement in IPF, many analyses focus on identifying circulating prognostic markers. Using a proteomic discovery method followed by ELISA validation in multiple IPF lung compartments and cohorts we explored novel markers of IPF survival. Methods: In our discovery analysis, agnostic label-free quantitative proteomics differentiated lung tissue protein expression based on survival trajectory (n=10). Following selection of the candidate pathway (neutrophil extracellular trap (NET) formation), we subsequently validated the presence of NETs in the IPF lung microenvironment using fully quantitative assays of known NET remnants in separate IPF cohorts (n=156 and n=52) with bronchoalveolar lavage fluid. We then assessed the correlation of these markers with baseline pulmonary function and survival. Results: Discovery lung tissue proteomics identified NET formation as significantly associated with poor IPF survival. Using fully quantitative confirmatory tests for reproducibility we confirmed the presence of NET markers in IPF BALF and found significant correlations with worse pulmonary function in both cohorts (p<0.03 and p = 0.04 respectively). In the survival cohort, higher levels of NET markers predicted worse survival after adjusting for gender, age, and baseline physiologic severity (hazard ratio range: 1.79-2.19). Conclusions: NET markers were associated with disease severity and worse survival in IPF. These findings suggest NET formation contributes to lung injury and decreased survival in IPF and may represent a potential therapeutic target.

Gene expression meta-analysis reveals aging and cellular senescence signatures in scleroderma-associated interstitial lung disease.

Aging and cellular senescence are increasingly recognized as key contributors to pulmonary fibrosis. However, our understanding in the context of scleroderma-associated interstitial lung disease (SSc-ILD) is limited. To investigate, we leveraged previously established lung aging- and cell-specific senescence signatures to determine their presence and potential relevance to SSc-ILD. We performed a gene expression meta-analysis of lung tissues from 38 SSc-ILD and 18 healthy controls and found that markers (GDF15, COMP, and CDKN2A) and pathways (p53) of senescence were significantly increased in SSc-ILD. When probing the established aging and cellular senescence signatures, we found that epithelial and fibroblast senescence signatures had a 3.6- and 3.7-fold enrichment, respectively, in the lung tissue of SSc-ILD and that lung aging genes (CDKN2A, FRZB, PDE1A, and NAPI12) were increased in SSc-ILD. These signatures were also enriched in SSc skin and associated with degree of skin involvement (limited vs. diffuse cutaneous). To further support these findings, we examined telomere length (TL), a surrogate for aging, in the lung tissue and found that, independent of age, SSc-ILD had significantly shorter telomeres than controls in type II alveolar cells in the lung. TL in SSc-ILD was comparable to idiopathic pulmonary fibrosis, a disease of known aberrant aging. Taken together, this study provides novel insight into the possible mechanistic effects of accelerated aging and aberrant cellular senescence in SSc-ILD pathogenesis.

Short peripheral blood leukocyte telomere length in rheumatoid arthritis-interstitial lung disease.

Shortened telomere lengths (TLs) can be caused by single nucleotide polymorphisms and loss-of-function mutations in telomere-related genes (TRG), as well as ageing and lifestyle factors such as smoking. Our objective was to determine if shortened TL is associated with interstitial lung disease (ILD) in individuals with rheumatoid arthritis (RA). This is the largest study to demonstrate and replicate that shortened peripheral blood leukocytes-TL is associated with ILD in patients with RA compared with RA without ILD in a multinational cohort, and short PBL-TL was associated with baseline disease severity in RA-ILD as measured by forced vital capacity percent predicted.

Gene Expression Meta-Analysis Reveals Aging and Cellular Senescence Signatures in Scleroderma-associated Interstitial Lung Disease.

Aging and cellular senescence are increasingly recognized as key contributors to pulmonary fibrosis. However, our understanding in the context of scleroderma associated interstitial lung disease (SSc-ILD) is limited. To investigate, we leveraged previously established lung aging and cell-specific senescence signatures to determine their presence and potential relevance to SSc-ILD. We performed a gene expression meta-analysis of lung tissue from 38 SSc-ILD and 18 healthy controls and found markers (GDF15, COMP, CDKN2A) and pathways (p53) of senescence were significantly increased in SSc-ILD. When probing the established aging and cellular senescence signatures, we found epithelial and fibroblast senescence signatures had a 3.6-fold and 3.7-fold enrichment respectively in the lung tissue of SSc-ILD and that lung aging genes ( CDKN2A, FRZB, PDE1A, NAPI12) were increased in SSc-ILD. These signatures were also enriched in SSc skin and associated with degree of skin involvement (limited vs. diffuse cutaneous). To further support these findings, we examined telomere length (TL), a surrogate for aging, in lung tissue and found independent of age, SSc-ILD had significantly shorter telomeres than controls in type II alveolar cells in the lung. TL in SSc-ILD was comparable to idiopathic pulmonary fibrosis, a disease of known aberrant aging. Taken together, this study provides novel insight into the possible mechanistic effects of accelerated aging and aberrant cellular senescence in SSc-ILD pathogenesis.

PAI-1 Deficiency Drives Pulmonary Vascular Smooth Muscle Remodeling and Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive and potentially a rapidly fatal disease characterized by vasoconstriction and remodeling of small pulmonary arteries (PA) leading to increased pulmonary vascular resistance and right heart failure. Central to the remodeling process is a switch of the smooth muscle cells in small PAs (PASMC) to a proliferative, apoptosis-resistant phenotype. There is reason to suspect that the plasminogen activator system may play an important role in the remodeling program in PAH based on its roles in vascular post-injury restenosis, fibrosis, angiogenesis and tumorigenesis. Plasminogen activator inhibitor-1 (PAI-1) is the primary physiological inhibitor of the plasminogen activators - urokinase-type and tissue-type (uPA and tPA, respectively). Immunohisto- chemical and immunoblot analyses revealed that PAI-1 was deficient in smooth muscle areas of small remodeled PAs and early-passage PASMC from subjects with PAH compared to non-PAH controls. PAI1-/- male and female mice developed spontaneous pulmonary vascular remodeling and pulmonary hypertension (PH) as evidenced by significant increase in PA medial thickness, systolic right ventricular pressure, and right ventricular hypertrophy. Lastly, the uPA inhibitors upamostat (WX-671) and amiloride analog BB2-30F down-regulated mTORC1 and SMAD3, restored PAI-1 levels, reduced proliferation, and induced apoptosis in human PAH PASMC. We examined the effect of inhibition of uPA catalytic activity by BB2-30F on the development of SU5416/Hypoxia (SuHx)-induced PH in mice. Vehicletreated SuHx-exposed mice had up-regulated mTORC1 in small PAs, developed pulmonary vascular remodeling and PH, as evidenced by significant increase of PA MT, sRVP, RV hypertrophy, and a significant decrease in the pulmonary artery acceleration time/pulmonary ejection time (PAAT/PET) ratio compared to age- and sex-matched normoxia controls, whereas BB2-30F-treated group was protected from all these pathological changes. Taken together, our data strongly suggest that PAI-1 down- regulation in PASMC from human PAH lungs promotes PASMC hyper-proliferation, remodeling, and spontaneous PH due to unopposed uPA activation. Further studies are needed to determine the potential benefits of targeting the PAI-1/uPA imbalance to attenuate the progression and/or reverse pulmonary vascular remodeling and PH.

Short airway telomeres are associated with primary graft dysfunction and chronic lung allograft dysfunction.

Primary graft dysfunction (PGD) is a major risk factor for chronic lung allograft dysfunction (CLAD) following lung transplantation, but the mechanisms linking these pathologies are poorly understood. We hypothesized that the replicative stress induced by PGD would lead to erosion of telomeres, and that this telomere dysfunction could potentiate CLAD. In a longitudinal cohort of 72 lung transplant recipients with >6 years median follow-up time, we assessed tissue telomere length, PGD grade, and freedom from CLAD. Epithelial telomere length and fibrosis-associated gene expression were assessed on endobronchial biopsies taken at 2 to 4 weeks post-transplant by TeloFISH assay and nanoString digital RNA counting. Negative-binomial mixed-effects and Cox-proportional hazards models accounted for TeloFISH staining batch effects and subject characteristics including donor age. Increasing grade of PGD severity was associated with shorter airway epithelial telomere lengths (p = 0.01). Transcriptomic analysis of fibrosis-associated genes showed alteration in fibrotic pathways in airway tissue recovering from PGD, while telomere dysfunction was associated with inflammation and impaired remodeling. Shorter tissue telomere length was in turn associated with increased CLAD risk, with a hazard ratio of 1.89 (95% CI 1.16-3.06) per standard deviation decrease in airway telomere length, after adjusting for subject characteristics. PGD may accelerate telomere dysfunction, potentiating immune responses and dysregulated repair. Epithelial cell telomere dysfunction may represent one of several mechanisms linking PGD to CLAD.

Telomere length and immunosuppression in non-idiopathic pulmonary fibrosis interstitial lung disease.

BACKGROUND: Studies suggest a harmful pharmacogenomic interaction exists between short leukocyte telomere length (LTL) and immunosuppressants in idiopathic pulmonary fibrosis (IPF). It remains unknown if a similar interaction exists in non-IPF interstitial lung disease (ILD). METHODS: A retrospective, multicentre cohort analysis was performed in fibrotic hypersensitivity pneumonitis (fHP), unclassifiable ILD (uILD) and connective tissue disease (CTD)-ILD patients from five centres. LTL was measured by quantitative PCR for discovery and replication cohorts and expressed as age-adjusted percentiles of normal. Inverse probability of treatment weights based on propensity scores were used to assess the association between mycophenolate or azathioprine exposure and age-adjusted LTL on 2-year transplant-free survival using weighted Cox proportional hazards regression incorporating time-dependent immunosuppressant exposure. RESULTS: The discovery and replication cohorts included 613 and 325 patients, respectively. In total, 40% of patients were exposed to immunosuppression and 22% had LTL <10th percentile of normal. fHP and uILD patients with LTL <10th percentile experienced reduced survival when exposed to either mycophenolate or azathioprine in the discovery cohort (mortality hazard ratio (HR) 4.97, 95% CI 2.26-10.92; p<0.001) and replication cohort (mortality HR 4.90, 95% CI 1.74-13.77; p=0.003). Immunosuppressant exposure was not associated with differential survival in patients with LTL ≥10th percentile. There was a significant interaction between LTL <10th percentile and immunosuppressant exposure (discovery pinteraction=0.013; replication pinteraction=0.011). Low event rate and prevalence of LTL <10th percentile precluded subgroup analyses for CTD-ILD. CONCLUSION: Similar to IPF, fHP and uILD patients with age-adjusted LTL <10th percentile may experience reduced survival when exposed to immunosuppression.

A fibroblast-dependent TGFß1/sFRP2 noncanonical Wnt signaling axis underlies epithelial metaplasia in idiopathic pulmonary fibrosis.

Reciprocal interactions between alveolar fibroblasts and epithelial cells are crucial for lung homeostasis, injury repair, and fibrogenesis, but underlying mechanisms remain unclear. To investigate this, we administered the fibroblast-selective TGFß1 signaling inhibitor, epigallocatechin gallate (EGCG), to Interstitial Lung Disease (ILD) patients undergoing diagnostic lung biopsy and conducted single-cell RNA sequencing on spare tissue. Unexposed biopsy samples showed higher fibroblast TGFß1 signaling compared to non-disease donor or end-stage ILD tissues. In vivo, EGCG significantly downregulated TGFß1 signaling and several pro-inflammatory and stress pathways in biopsy samples. Notably, EGCG reduced fibroblast secreted Frizzle-like Receptor Protein 2 (sFRP2), an unrecognized TGFß1 fibroblast target gene induced near type II alveolar epithelial cells (AEC2s). In human AEC2-fibroblast coculture organoids, sFRP2 was essential for AEC2 trans-differentiation to basal cells. Precision cut lung slices (PCLS) from normal donors demonstrated that TGFß1 promoted KRT17 expression and AEC2 morphological change, while sFRP2 was necessary for KRT5 expression in AEC2-derived basaloid cells. Wnt-receptor Frizzled 5 (Fzd5) expression and downstream calcineurin-related signaling in AEC2s were required for sFRP2-induced KRT5 expression. These findings highlight stage-specific TGFß1 signaling in ILD, the therapeutic potential of EGCG in reducing IPF-related transcriptional changes, and identify the TGFß1-non-canonical Wnt pathway crosstalk via sFRP2 as a novel mechanism for dysfunctional epithelial signaling in Idiopathic Pulmonary Fibrosis/ILD.

Targeted alveolar regeneration with Frizzled-specific agonists.

Wnt ligands oligomerize Frizzled (Fzd) and Lrp5/6 receptors to control the specification and activity of stem cells in many species. How Wnt signaling is selectively activated in different stem cell populations, often within the same organ, is not understood. In lung alveoli, we show that distinct Wnt receptors are expressed by epithelial (Fzd5/6), endothelial (Fzd4), and stromal (Fzd1) cells. Fzd5 is uniquely required for alveolar epithelial stem cell activity, whereas fibroblasts utilize distinct Fzd receptors. Using an expanded repertoire of Fzd-Lrp agonists, we could activate canonical Wnt signaling in alveolar epithelial stem cells via either Fzd5 or, unexpectedly, non-canonical Fzd6. A Fzd5 agonist (Fzd5ag) or Fzd6ag stimulated alveolar epithelial stem cell activity and promoted survival in mice after lung injury, but only Fzd6ag promoted an alveolar fate in airway-derived progenitors. Therefore, we identify a potential strategy for promoting regeneration without exacerbating fibrosis during lung injury.