Abstract

   Currently, there is no pharmacological treatment targeting defective
   tissue repair in chronic disease. Here, we used a
   transcriptomics-guided drug target discovery strategy using gene
   signatures of smoking-associated chronic obstructive pulmonary disease
   (COPD) and from mice chronically exposed to cigarette smoke,
   identifying druggable targets expressed in alveolar epithelial
   progenitors, of which we screened the function in lung organoids. We
   found several drug targets with regenerative potential, of which EP and
   IP prostanoid receptor ligands had the most profound therapeutic
   potential in restoring cigarette smoke–induced defects in alveolar
   epithelial progenitors in vitro and in vivo. Mechanistically, we found,
   using single-cell RNA sequencing analysis, that circadian clock and
   cell cycle/apoptosis signaling pathways were differentially expressed
   in alveolar epithelial progenitor cells in patients with COPD and in a
   relevant model of COPD, which was prevented by prostaglandin E2 or
   prostacyclin mimetics. We conclude that specific targeting of EP and IP
   receptors offers therapeutic potential for injury to repair in COPD.
     __________________________________________________________________

   EP and IP receptors offer novel therapeutic potential for lung repair
   in COPD.

INTRODUCTION

   One of the main challenges in pharmacology today is the generation of
   drugs with regenerative potential, with the ability to restore tissue
   repair in chronic disease. Regenerative medicine has thus far mainly
   focused on transplantation, tissue engineering approaches, stem or
   progenitor cell therapy, or a combination of these ([54]1). A
   regenerative pharmacological approach will have considerable additional
   potential because it can be applied on a relatively large scale.
   Furthermore, it can be used to halt the disease process in an early
   stage, resulting in real disease-modifying treatment. In addition,
   pharmacological targeting may aid or support other regenerative
   strategies.

   There is a need for regenerative pharmacology in respiratory,
   cardiovascular, and neurological diseases as well as many other disease
   areas. In respiratory medicine, chronic obstructive pulmonary disease
   (COPD) is one of the most common lung diseases with a need for
   regenerative therapies. The disease is characterized by airflow
   limitation that is not fully reversible and which deteriorates
   progressively. The main difficulty underlying COPD pathogenesis is
   increased tissue destruction in combination with abnormal tissue repair
   in susceptible individuals. As current therapies do not modify the
   course of the disease, developing new therapeutic strategies aiming at
   regeneration of tissue is necessary.

   In affected individuals, there is an increase in alveolar air space
   associated with destruction of alveolar epithelial cells along with
   reduced capacity of epithelial progenitors to restore this defect. In
   the distal lung, alveolar type II cells and alveolar epithelial
   progenitors harbor stem cell capacity and function to maintain alveolar
   epithelium ([55]2). These cells reside in a local tissue
   microenvironment called the niche, which is composed of supporting
   cells such as fibroblasts and alveolar macrophages. The niche controls
   adequate activation of the progenitor cell ([56]1–[57]3) by means of
   secreted factors such as Wingless-related integration sites (WNTs),
   fibroblast growth factors (FGFs), retinoic acid, and many other factors
   that control stemness, proliferation, and differentiation ([58]3).

   As in many chronic diseases associated with aging, this local lung
   microenvironment is insufficiently supportive for lung repair in COPD
   ([59]1, [60]4, [61]5). For example, studies have indicated that an
   imbalance of canonical and noncanonical WNT signaling results in
   impaired alveolar regeneration in COPD ([62]4, [63]6). Moreover,
   lymphotoxin-β (LTβ), released from CD8^+ T cells in COPD, can
   negatively interfere with repair. LTβ induces noncanonical nuclear
   factor κB signaling, thereby repressing functional Wnt/β-catenin
   signaling in the lung ([64]5). A recent study showed longitudinal
   changes in lung tissue gene expression associated with repair to
   underlie disease progression ([65]7). Accordingly, the challenge of
   successful regenerative pharmacology in COPD needs to take into
   consideration the specific hostile microenvironment and the abnormal
   repair process that stands in the way of adequate regeneration in COPD.

   In the present study, we hypothesized that a transcriptomics-guided
   drug target discovery strategy based on gene signatures differentially
   expressed in COPD and in response to cigarette smoke (CS) may be used
   to identify druggable gene targets that are specifically involved in
   defective lung repair in COPD. Our results demonstrate that such a
   strategy coupled to functional studies in organoids yields receptor
   ligands of which EP and IP prostanoid receptors show the most
   significant potential in counteracting the negative effects of CS on
   alveolar progenitor cell function.

RESULTS

Transcriptomics-guided screening to identify drug targets

   We set out to identify drug targets that may help restore defective
   lung repair. To achieve this, we used a transcriptomics-guided target
   discovery strategy (described in [66]Fig. 1A) based on gene signatures
   of COPD lung tissues ([67]8) and of a model of CS exposure ([68]9) to
   identify differentially regulated druggable genes. We found reactome
   pathways related to inflammation such as neutrophil degranulation and
   innate immune system to be enriched in both datasets ([69]Fig. 1, C and
   D) and pathways related senescence, apoptosis, and extracellular matrix
   regulation to be enriched in COPD, which was also reported in a recent
   study ([70]7) using longitudinal samples. We identified 38 individual
   target genes that were concordantly up-regulated and 30 individual
   target genes concordantly down-regulated. These genes were filtered
   through the “Drug-gene interactions and potential druggability in the
   Drug Gene Interaction Database” ([71]www.dgidb.org), which rendered 25
   druggable up-regulated genes and 16 druggable down-regulated genes
   ([72]Fig. 1B). Genes were further filtered for expression in lung
   epithelial cells or fibroblasts by consulting the human Lung Cell Atlas
   ([73]https://asthma.cellgeni.sanger.ac.uk/) and LungMAP
   ([74]https://lungmap.net/), which yielded 15 druggable target genes.

Fig. 1. Overview of the transcriptomics-guided drug discovery strategy.

   [75]Fig. 1.
   [76]Open in a new tab

   (A) Schematic outline of the drug screening strategy. (B) Heatmap shows
   the gene expression pattern of the druggable genes ([77]www.dgidb.org)
   identified both in CS-exposed mice and patient with COPD databases. (C)
   Reactome pathway enrichment analysis of genes differentially expressed
   from patients with COPD ([78]8) using gene set enrichment analysis
   (GSEA); the top 50 pathways enriched are presented. TCF, T cell factor;
   GPCR, G protein–coupled receptor; ESR, estrogen receptor; MHC, major
   histocompatibility complex; RUNX1, runt-related transcription factor 1.
   (D) Reactome pathway enrichment analysis of differentially genes
   differentially expressed from CS-exposed mice ([79]9) using GSEA
   ([80]www.gsea-msigdb.org/gsea/msigdb/annotate.jsp).

   To assess the potential relevance of signaling functionally associated
   with the 15 genes of interest, we set up an in vitro organoid model to
   perform specific drug screening tests. An adult lung epithelial
   organoid model can recapitulate the in vivo functionality and genetic
   signature of alveolar epithelium and follow the initial progenitor cell
   division, subsequent growth, and differentiation of adult lung
   epithelial cells in a single assay ([81]6, [82]10). Moreover, the
   organoid assay allows us to model the interactions between CS extract
   (CSE) exposure and the alveolar progenitor cell in the most direct
   possible way. The majority (~70%) of organoids express mature alveolar
   markers such as pro-SPC (surfactant protein C). Approximately 10%
   express airway markers [acetylated tubulin (ACT)], and ~10% expressed a
   mixed phenotype (pro-SPC^+/ACT^+) ([83]10). Fibroblasts do not
   integrate into the organoids, which consist entirely of epithelial
   structures. Thus, we cocultured mouse CD31^−/CD45^−/Epcam^+ (epithelial
   cellular adhesion molecule^+) lung epithelial cells with murine CCL-206
   lung fibroblasts or human CD31^−/CD45^−/Epcam^+ lung epithelial cells
   with human MRC5 (Medical Research Council cell strain 5) lung
   fibroblasts in organoids in Matrigel and exposed these in vitro to
   different concentrations (1.25, 2.5, and 5%) of CSE ([84]Fig. 2, A and
   C). The number and size of organoids established by coculturing human
   lung tissue–derived CD31^−/CD45^−/EpCAM^+ cells and MRC5 fibroblasts
   were significantly decreased by CSE in a concentration-dependent manner
   at day 14 ([85]Fig. 2B). The total number of murine organoids
   quantified at day 14 of treatment with different concentrations of CSE
   yielded similar results and was decreased in a CSE dose–dependent
   manner as well ([86]Fig. 2E). To specifically analyze the impact of CSE
   on organoids derived from alveolar epithelial progenitors, we
   morphologically subclassified organoids into airway- and alveolar-type
   ([87]10) organoids ([88]Fig. 2D), which revealed that alveolar organoid
   numbers were more susceptible to CSE exposure than airway organoids
   ([89]Fig. 2E). Immunofluorescence studies confirmed that the number of
   ACT (ciliated cell marker, airway-type organoids) and pro-SPC^+ (type
   II cell marker, alveolar-type organoids) organoids was significantly
   decreased by 5% CSE ([90]Fig. 2F). The size of both organoid types was
   decreased at day 14 ([91]Fig. 2G).

Fig. 2. CS exposure represses adult epithelial lung organoid formation.

   [92]Fig. 2.
   [93]Open in a new tab

   (A) Schematic of in vitro human experimental design. (B) Quantification
   of total amount of human organoids and the quantification of average
   human organoid diameters after treatment with CSE (0, 1, 2.5, and 5%).
   N = 7 experiments (two healthy and five COPD donors), n > 150 organoids
   per group. (C) Schematic of in vitro murine experimental design. (D)
   Representative images of murine lung organoids. Left: Light microscopy.
   Right: Immunofluorescence (IF) of organoids. Green, pro-SPC (SPC); red,
   ACT; blue, 4′,6-diamidino-2-phenylindole (DAPI). White arrowheads,
   airway-type organoid; yellow arrowheads, alveolar-type organoid. Scale
   bars, 100 μm. (E) Quantification of the normalized number of total
   organoids, airway, and alveolar-type organoids on day 14 obtained after
   treatment with different concentrations of CSE (0, 1.25, 2.5, and 5%).
   (F) Quantification of normalized ACT^+ and pro-SPC^+ organoids obtained
   after treatment with 0, 1.25, 2.5, and 5% CSE. (G) Quantification of
   average organoid diameter after treatment with 0, 1.25, 2.5, and 5% CSE
   measured on day 14. N = 5 experiments, n > 380 organoids per group. (H
   and I) Overview of drug screening using the in vitro murine lung
   organoid model. Comparison of the normalized number of airway and
   alveolar-type organoids treated with the different drugs of interest in
   the absence (H) or presence (I) of 5% CSE. Red bars, PTGIR; yellow
   bars, PTGES2; blue bars, antagonists. Data are presented as means ± SEM
   in number quantification. Data are presented as scatter plots with
   medians in size quantification. VEH, vehicle; ACSS2, acetate-dependent
   acetyl CoA synthetase 2; CHRM3, muscarinic M3 receptor; CPS1, carbamoyl
   phosphate synthetase 1; CTSC, cathepsin C; CXCL1, C-X-C motif chemokine
   ligand 1; DMKN, dermokine; FGF17, fibroblast growth factor 17; IBSP,
   integrin binding sialoprotein; LEPR, leptin receptor; PTGIR,
   prostaglandin I receptor; PTGES2, prostaglandin E synthase 2; RAB8B,
   Ras-related protein Rab-8B; SLC1A4, solute carrier family 1 member 4;
   SLC16A3, solute carrier family 16 member 3; TNNIK3, TNNI3 interacting
   kinase. For all panels, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P
   < 0.0001.

   We next aimed to use this lung organoid model to evaluate the efficacy
   of existing COPD therapeutics. Increasing evidence ([94]11, [95]12) has
   linked phosphodiesterase 4 (PDE4) inhibition to the therapeutic
   management of respiratory diseases, and roflumilast has been used as an
   oral medication in patients with COPD with a prior history of
   hospitalization for an acute exacerbation (GOLD 2021). This led us to
   explore whether the classic PDE4 inhibitor, rolipram, was able to
   rescue the CS-induced reduction in organoid formation by alveolar
   progenitors. Thus, organoids were subjected in vitro to different
   concentrations (1 and 10 μM) of rolipram in the presence and absence of
   5% CSE for up to 14 days (fig. S1A). Rolipram (10 μM) alone
   significantly increased the total number of organoids at day 7 and the
   pro-SPC^+ organoids at day 14 (fig. S1, B and C) but had no beneficial
   effects on organoid numbers when combined with CSE exposures. Treatment
   with rolipram (1 μM) either alone or in combination with CSE induced
   significantly increase alveolar organoid size (fig. S1D). The
   combination of rolipram and olodaterol tended to restore the defective
   organoid formation under the exposure of 5% CSE (fig. S1, K to M).

   Budesonide is an inhaled corticosteroid used in COPD management
   ([96]13–[97]16). Therefore, we examined its effect also in our in vitro
   (fig. S1E) and in vivo (fig. S1H) CS/organoid models. Budesonide (1,
   10, and 100 nM) in combination with CSE exposure further reduced the
   number and the size of both airway- and alveolar-type organoids as
   compared to CSE exposure alone (fig. S1, F and G). In vivo exposure to
   budesonide together with CS increased the number of airway but not
   alveolar organoids (fig. S1I), without affecting the organoid size
   (fig. S1J). Together, these data show that in vitro exposure to CSE
   functionally represses human alveolar epithelial progenitor organoid
   formation, resulting in reduced growth and differentiation, which can
   be mimicked using murine alveolar epithelial progenitors. Validating
   the limitations of current pharmacology, PDE4 inhibitors and
   corticosteroids do not prevent or reduce the detrimental effects of CS
   on organoid formation.

   The assay was used subsequently to screen for the functionality of the
   targets in restoring organoid growth. Genes down-regulated in response
   to CS and COPD were targeted with activating ligands or with ligands
   mimicking an active state of the protein, whereas genes up-regulated in
   response to CS and COPD were targeted using antagonists, with the
   exception of ACSS2 and CHRM3 for which we included both an agonist and
   an antagonist. In addition, we included an agonist of PTGIR because of
   its previously suggested beneficial effects in COPD ([98]17). The
   effects of the drugs targeting the 15 selected genes alone (compared to
   vehicle; [99]Fig. 2H) and in the presence of 5% CSE exposure ([100]Fig.
   2I) on the number of organoids were determined. Specific information of
   all drug effects on organoid number and size are summarized in figs. S2
   and S3. The compound activating ACSS2 [acetyl coenzyme A (CoA)
   synthetase short-chain family member 2] increased the number of
   airway-type organoids and the size of alveolar organoids in combination
   with CSE (figs. S2 and S3), whereas the ACSS2 inhibitor had the
   opposite effects. Atropine [muscarinic M3 receptor (CHRM3) antagonist],
   IBSP (integrin binding sialoprotein), and LEPR (leptin receptor) tended
   to increase the number and size of alveolar organoids in response to
   CSE as well (figs. S2 and S3). However, considering the overall
   magnitude of alveolar-type organoids, particularly, 16,16-dimethyl
   prostaglandin E2 (PGE[2]) (stable analog of PGE[2], the enzymatic
   product of target gene PTGES) and iloprost [prostacyclin (PGI[2])
   analog, ligand for target gene PTGIR] were identified as the by far
   most promising targets with regards to their capacity in restoring the
   CSE-induced repression of organoid formation ([101]Fig. 2, H and I, and
   fig. S5).

PGE[2] and PGI[2] significantly prevent alveolar epithelial dysfunction

   The PTGES2 and PTGIR genes encode membrane-associated PGE synthase and
   the PGI[2] receptor, respectively. PGE[2] acts on four receptor
   subtypes, being PTGER1 to PTGER4, whereas PGI[2] acts primarily on
   PTGIR. We assessed their expression in human lung tissue of healthy
   smokers and patients with COPD ([102]GSE76925) and found maintained
   expression of all receptors in disease with some small differences in
   expression, most notably a reduced expression of PTGER2 and increased
   expression of PTGIR ([103]Fig. 3A). Single-cell RNA sequencing
   (scRNA-seq) data from human lung tissue shows similar expression of all
   five receptors in alveolar epithelial cells and in fibroblasts
   ([104]www.copdcellatlas.com) ([105]18). scRNA-seq of mouse lung tissue
   shows that expression of Ptger2 and Ptger4 were the highest compared to
   that of Ptger1 and Ptger3 in mesenchymal cells ([106]Fig. 3, B and C).
   The expression of PTGES and PTGES2, the enzymes responsible for PGE[2]
   synthesis, was relatively ubiquitous in human and mouse lung tissue,
   whereas PTGIS, the enzyme responsible for PGI[2] synthesis, was the
   highest in mesenchymal cell types including fibroblasts.

Fig. 3. 16,16-dimethyl PGE[2], iloprost, and selective EP2 and EP4 analogs
restore lung organoid formation in response to CS(E).

   [107]Fig. 3.
   [108]Open in a new tab

   (A) The relative gene expression of PTGER1, PTGER2, PTGER3, PTGER4, and
   PTGIR in healthy smokers (N = 40) and patients with COPD (N = 111)
   downloaded from the National Center for Biotechnology Information
   (NCBI) Gene Expression Omnibus (GEO) database ([109]GSE76925). (B and
   C) Data are extracted from the NCBI GEO database ([110]GSE151674). (B)
   The expression of Ptges, Ptges2, Ptgis, Ptger1, Ptger2, Ptger3, Ptger4,
   and Ptgir in epithelial cells using scRNA-seq analysis of mouse lung
   tissue. (C) The expression of Ptges, Ptges2, Ptgis, Ptger1, Ptger2,
   Ptger3, Ptger4, and Ptgir in mesenchymal cells using scRNA-seq analysis
   of mouse lung tissue. SMCs, smooth muscle cells. (D) Quantification of
   normalized number of alveolar-type organoids treated with vehicle
   control or 5% CSE ± PGE[2] agonist (16,16-dimethyl PGE[2])/iloprost.
   DMSO, dimethyl sulfoxide. (E) Quantification of normalized number of
   SPC^+ organoids treated with vehicle control or 5% CSE ± PGE[2] agonist
   (16,16-dimethyl PGE[2]) or iloprost. (F) Quantification of normalized
   number of alveolar-type of organoids treated with vehicle control or 5%
   CSE ± selective EP2 or EP4 agonist. Data are presented as means ± SEM.
   *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns, not
   significant.

   To further characterize the effects of PGE[2] and PGI[2] on defective
   alveolar epithelial progenitors, we examined them in vitro ([111]Fig.
   3, D to F) and in vivo ([112]Fig. 4) CS(E). The PGE[2] analog
   16,16-dimethyl PGE[2] and the PGI[2] analog iloprost both increased the
   number of alveolar-type organoids even in the presence of 5% CSE
   ([113]Fig. 3D) and significantly increased the number of SPC^+
   organoids under conditions of CSE exposure ([114]Fig. 3E). To address
   the relative roles of the two G[s]-coupled PGE[2] receptors, EP2 and
   EP4, we evaluated the selective agonists [(R)-butaprost and
   5-[(3S)-3-hydroxy-4-phenyl-1-buten-1-yl]1-[6-(2H-tetrazol-5R-yl)hexyl]-
   2-pyrrolidinone; CAY10598] of these receptors. Focus was on these
   G[s]-coupled receptors, as we found that cholera toxin, a well-known
   inducer of constitutive adenylyl cyclase activity and adenosine
   3′,5′-monophosphate (cAMP) signaling, increased organoid formation both
   with and without the exposure to CSE (fig. S4A). The EP2-selective
   butaprost had no effect on organoid number ([115]Fig. 3F) but increased
   the alveolar size in the absence and presence of 5% CSE (fig. S5C). The
   EP4-selective agonist increased the number of alveolar-type organoids
   significantly and prevented the number reduction resulting from 5% CSE
   exposure ([116]Fig. 3F). The EP4 agonist also increased the size of
   both types of organoids in the absence and presence of 5% CSE (fig.
   S5C). To explore whether the duration of drug exposure affected
   organoid formation, we treated the organoids in vitro with PGE[2] or
   PGI[2] analog for three different time windows during organoid
   development as illustrated in fig. S5 (D to G). These time windows were
   identified previously ([117]10) and mark the initial division phase
   (days 0 to 2), proliferation (days 2 to 7), and differentiation phase
   (days 7 to 14). We observed no effect on the number of organoids for
   any of the short-term drug treatments, suggesting that continuous
   treatment with or iloprost during all phases of organoid formation is
   required.

Fig. 4. Administration (in vivo and in vitro) of misoprostol and iloprost to
CS-exposed mice restored lung organoid formation.

   [118]Fig. 4.
   [119]Open in a new tab

   (A) Schematic of in vivo CS exposure experimental setup. (B and C)
   Number of airway- and alveolar-type organoids quantified on day 14 of
   coculturing CCL-206 fibroblasts and Epcam^+ cells (isolated from
   air-exposed/CS-exposed mice). N = 11 experiments. (D) Schematic of
   experimental design. Organoids were generated from air- or CS-exposed
   mice treated in vivo with misoprostol [intraperitoneal (i.p.)] or
   iloprost (intraperitoneal); all organoids were treated with normal
   organoid medium. (E and F) Number of alveolar-type and pro-SPC^+
   organoids quantified on day 14 from coculture of CCL-206 fibroblasts
   and Epcam^+ cells [isolated from air-exposed (control) and CS-exposed
   mice treated intraperitoneally with misoprostol or iloprost]. (G)
   Schematic of experimental design. Organoids were generated from mice
   exposed to air or CS. Misoprostol and iloprost were added in vitro to
   the organoid medium for treatment. (H and I) Number of alveolar-type
   and SPC^+ organoids quantified on day 14 from coculture of CCL206
   fibroblasts and Epcam^+ cells (isolated from air- and CS-exposed mice)
   treated with misoprostol/iloprost in vitro. (J) Schematic of
   experimental design. (K and L) Number of alveolar-type and SPC^+
   organoids quantified on day 14 from coculture of CCL-206 fibroblasts
   and Epcam^+ cells (isolated from air- and CS-exposed mice for 4 months)
   treated with misoprostol/iloprost in vitro. Data are presented as means
   ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

   To examine the effects of PGE[2] and PGI[2] in vivo, we exposed mice to
   air (vehicle control), CS, CS + misoprostol (PGE[2] analog), or CS +
   iloprost as shown in [120]Fig. 4. For the in vivo studies, we diverted
   to misoprostol as PGE[2] analog, as this is a well-tolerated, safe
   analog of PGE[2] that is also used clinically. To assess the impact of
   in vivo CS exposure to the apical epithelial side only and to
   investigate the immediate impact of CS on the alveolar epithelial
   progenitors, we exposed mice to air or CS for 1 week, subsequently
   isolated CD31^−/CD45^−/Epcam^+ cells, and cocultured these with CCL-206
   fibroblasts in vitro for 14 days ([121]Fig. 4A). The number of alveolar
   organoids was significantly decreased after in vivo CS exposure,
   indicating that a relatively short exposure to CS in vivo is sufficient
   to capture early changes in progenitor cell function ([122]Fig. 4C). CS
   exposure did not change the yield of CD31^−/CD45^−/Epcam^+ cells,
   whereas exposing mice to misoprostol or iloprost increased the yield of
   Epcam^+ cells (fig. S6A). The organoid assay revealed that in vivo
   (intraperitoneal) treatment with either misoprostol or iloprost
   significantly increased the number of alveolar-type and SPC^+ organoids
   ([123]Fig. 4, E and F) ex vivo. Next, to investigate whether in vitro
   drug treatment would have similar effects on damage caused by in vivo
   CS exposure, we isolated Epcam^+ cells from either air- or CS-exposed
   mice and subjected these to in vitro misoprostol or iloprost treatment
   in the organoid assay for 14 days ([124]Fig. 4G). In vitro misoprostol
   increased the number of alveolar-type organoids in cultures derived
   from CS-exposed mice ([125]Fig. 4H). Only in vitro misoprostol
   increased the size of alveolar organoids derived from CS-exposed
   animals (fig. S6C). To validate these findings in a chronic CS model,
   we isolated CD31^−/CD45^−/ Epcam^+ cells from mice exposed to CS for 4
   months and subjected these cells in vitro to misoprostol or iloprost
   treatment in the organoid assay for 14 days. In vitro misoprostol and
   iloprost increased the number of alveolar-type organoids in cultures
   derived from chronically CS-exposed mice to the same extent as what we
   observed for the 1-week exposure model ([126]Fig. 4K). Misoprostol also
   increased the number of SPC^+-differentiated organoids ([127]Fig. 4L).
   Together, our data show that PGE[2] and PGI[2] analogs protect alveolar
   epithelial progenitor function from the effects of CS exposure. In
   addition, EP4 rather than EP2 seems to mediate the protective effects
   of PGE[2].

Distinct genetic signatures in regulation of defective alveolar epithelial
repair

   To unravel the transcriptional changes leading to impaired lung
   organoid formation after exposure to CS and the mechanisms underlying
   the beneficial effects of PGE[2] and PGI[2] treatment, we performed
   RNA-seq ([128]Fig. 5A) on Epcam^+ cells isolated from mice exposed to
   air (control), CS, CS + misoprostol (intraperitoneal), or CS + iloprost
   (intraperitoneal) directly after the isolation procedure (i.e., before
   inclusion in the organoid assay). Principal component analysis (PCA)
   revealed that the CS-exposed group is transcriptionally distinct from
   the control group ([129]Fig. 5B) and that the CS + misoprostol and CS +
   iloprost groups are transcriptionally different from the CS-exposed
   group. The top differentially expressed genes from these three
   comparisons, including both up- and down-regulated genes, are shown in
   the volcano plot ([130]Fig. 5C) and summarized in the Supplementary
   Materials. Reactome pathway analysis was used to identify molecular
   pathways overrepresented in CS/misoprostol/iloprost-modulated genes in
   alveolar epithelial cells. Within the top 20 enriched pathways
   ([131]Fig. 5D and the Supplementary Materials), genes associated with
   cell cycle, mitotic prometaphase, DNA replication/synthesis, and RHO
   guanosine triphosphatases (GTPases) activating formins signaling
   pathways were up-regulated by CS exposure compared to air (control)
   exposure; however, these were down-regulated by treatment with
   misoprostol or iloprost. Notably, genes associated with the circadian
   clock signaling pathway were down-regulated by CS exposure but restored
   by treatment with misoprostol or iloprost. Moreover, signaling by FGF
   receptor 1 (FGFR1), FGFR3, and FGFR4 were down-regulated in response to
   CS exposure, whereas the same signaling pathways were up-regulated by
   misoprostol but not iloprost treatment. These findings suggest that the
   repair mechanisms of misoprostol and iloprost in response to CS include
   cell cycle and circadian clock signaling and that the distorted FGFR
   signaling resulting from CS was corrected only by misoprostol. In
   addition, the nuclear receptor transcription pathway, growth hormone
   receptor signaling, mitogen-activated protein kinase (MAPK), WNT, and
   cell-cell communication signaling pathways appear to be down-regulated
   in response to CS, but not in either the misoprostol or iloprost
   treatment group. Overall, the RNA-seq analysis demonstrates both common
   and distinct transcriptomic mechanisms of misoprostol and iloprost
   treatment in response to CS exposure in alveolar epithelial
   progenitors.

Fig. 5. Transcriptomic signatures in response to CS with(out) misoprostol and
iloprost.

   [132]Fig. 5.
   [133]Open in a new tab

   (A) Schematic experimental design for RNA-seq. (B) PCA plots
   demonstrate the clusters between different comparisons: air versus CS,
   CS versus CS + misoprostol, and CS versus CS + iloprost. (C) Volcano
   plots displaying the differentially expressed genes with log[2] fold
   change (FC) of at least one with their corresponding P values. The
   entire list of differently expressed genes is provided in the
   Supplementary Materials. (D) The top five significantly up- and
   down-regulated reactome pathways enrichment form differentially
   expressed genes within the comparisons of air versus CS exposure, CS
   exposure versus CS + misoprostol, and CS exposure versus CS + iloprost.
   The top 20 significantly enriched pathways are shown in table S3. RORA,
   RAR-related orphan receptor alpha; BMAL, brain and muscle ARNT-like 1;
   CLOCK, circadian locomotor output cycles kaput; NPAS2, neuronal PAS
   Domain Protein 2; RAF, rapidly accelerated fibrosarcoma.

Circadian clock signaling and alveolar epithelial repair

   Having demonstrated that the circadian clock signaling pathway was
   down-regulated by CS exposure and restored by in vivo treatment of
   misoprostol or iloprost, we next set out to explore this finding in
   more detail. We assessed the mRNA expression of core circadian clock
   genes in the lung tissue from patients with COPD ([134]GSE76925) and
   found that the expression of CLOCK, CRY1, CRY2, RORA, and PER2 were all
   significantly decreased in COPD ([135]Fig. 6A). We next assessed these
   same transcriptional nine core circadian clock genes in the mouse lung
   epithelial cells we obtained after in vivo CS exposure and
   misoprostol/iloprost treatment (described in [136]Fig. 5A). The gene
   expression of these clock genes was mostly down-regulated in CS-exposed
   alveolar epithelial progenitors ([137]Fig. 6B). Misoprostol and
   iloprost increased the expression of Per2, Per3, and Nr1d2 in
   particular ([138]Fig. 6B). scRNA-seq of mouse lung tissue showed that
   these nine core circadian clock genes were mainly expressed in alveolar
   epithelial cells and fibroblasts ([139]Fig. 6, C and D, and fig. S7).
   In particular, the expression of Arntl, Clock, Cry2, Rora, Per3, and
   Nr1d1 were highly expressed in alveolar epithelial cells ([140]Fig.
   6C), whereas Clock, Rora, and Nr1d1 were highly expressed in the
   fibroblasts ([141]Fig. 6D). Among epithelial cell types, the core
   circadian clock gene Rora was mainly expressed in alveolar epithelial
   type II cells; among mesenchymal cell types, it was mainly expressed in
   leucine-rich repeat-containing G protein-coupled receptor 5^+/6^+
   (Lgr5^+/Lgr6^+) fibroblasts ([142]Fig. 6, C and D, and fig. S7).

Fig. 6. Circadian clock signaling in healthy and diseased lung tissue.

   [143]Fig. 6.
   [144]Open in a new tab

   (A) The relative genes expression of ARNTL (BMAL1), CLOCK, CRY1, CRY2,
   RORA, PER2, PER3, NR1D1, and NR1D2 in healthy smokers (N = 40) and
   patients with COPD (N = 111) downloaded from the NCBI GEO database
   ([145]GSE76925). (B) The expression of Arntl (Bmal1), Clock, Cry1,
   Cry2, Rora, Per2, Per3, Nr1d1, and Nr1d2 genes in air-, CS-, CS +
   misoprostol–, and CS + iloprost–exposed epithelial cells. FPKM,
   fragments per kilobase of transcript per million mapped reads. (C and
   D) The expression of Arntl (Bmal1), Clock, Cry1, Cry2, Rora, Per2,
   Per3, Nr1d1, and Nr1d2 in epithelial cells and mesenchymal cells using
   scRNA-seq of mouse lung tissue ([146]GSE151674). (E) Immunofluorescence
   staining of pro-SPC (yellow), NR1D1 (green), and PER3 (green) on lung
   sections acquired from wild-type mice. Scale bars, 50 μm. (F) Schematic
   of experimental design. Scale bars, 200 μm. (G) Representative images
   of lung organoids treated with vehicle control or 5% CSE ± GSK4112 (10
   μM)/SR8278 (10 μM). (H) Quantification of normalized number of
   alveolar-type of organoids treated with vehicle control or 5% CSE ±
   GSK4112 (10 μM)/SR8278 (10 μM). Data are presented as means ± SEM. *P <
   0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

   The nuclear heme receptor Rev-erbα, encoded by NR1D1, plays an
   essential role in clock-dependent lung physiology and anti-inflammatory
   response ([147]19–[148]21). In line with these findings, the
   immunofluorescence staining of mouse lung tissue showed that NR1D1
   (nuclear receptor subfamily 1, group D, member 1) and PER3 (period
   circadian clock 3) were expressed at the protein level in alveolar
   epithelial cells and PER3 was particularly enriched in alveolar
   epithelial type II cells ([149]Fig. 6E). Next, to functionally evaluate
   the role of Rev-erbα in defective lung repair, we used an agonist and
   antagonist of Rev-erbα ([150]Fig. 6, F and G). The Rev-erbα agonist
   GSK4112 (10 μM) increased the number of alveolar-type organoids, both
   compared to vehicle and in the presence of 5% CSE ([151]Fig. 6H).
   Conversely, the Rev-erbα antagonist SR8278 (10 μM) significantly
   prevented organoid formation ([152]Fig. 6H), both in the absence and in
   the presence of 5% CSE.

DISCUSSION

   COPD results from repeated microinjuries to the epithelium often caused
   by tobacco smoking. In susceptible individuals, this results in tissue
   remodeling in the conducting airways but destruction of the respiratory
   bronchioles and alveoli ([153]22). Such a disparity was also found in
   our organoid assay, which revealed higher numbers of airway organoids
   but lower numbers of alveolar organoids in response to in vivo CS
   exposure. No clinically approved pharmacological treatment prevents or
   reverses the tissue destruction in the distal lung. The results of our
   study are in line with this contention and demonstrate that the PDE4
   inhibitor rolipram and the corticosteroid budesonide had no, or only
   very limited, beneficial effects on impaired organoid growth and
   differentiation in response to CSE in vitro or in response to CS
   exposure in vivo. If anything, budesonide appeared to restrict
   progenitor cell growth, which is a concern, given the wide use of
   corticosteroids in the management of COPD. These data underscore the
   need for novel drug targets.

   Consequently, we set out to search for new potential drug targets for
   lung repair in COPD and identify EP and IP receptor agonists as two
   such potential targets using a transcriptomics-guided drug discovery
   strategy. Both EP and IP receptor agonists were able to promote
   epithelial repair responses after exposure to CS(E). Whereas PGE[2] and
   PGI[2] showed the most profound changes, other methods including ACSS2
   agonism, LEPR agonism, and IBSP agonism yielded smaller effects. ACSS2
   supports acetyl-CoA synthesis from acetate in the cytosol ([154]23,
   [155]24) and thereby plays an important role in lipid metabolism and in
   the regulation of histone acetylation in the nucleus during gene
   transcription. IBSP is a member of the small integrin-binding ligand
   N-linked glycoprotein family ([156]25, [157]26), which is associated
   with bone metastases of lung cancer ([158]27). LEPR is an adipocytokine
   that not only has a central role in regulating food intake and energy
   expenditure ([159]28) but also has been linked to lung function decline
   in a population in COPD ([160]29). Nonetheless, among all the candidate
   targets, PGE[2] and PGI[2] analogs emerged as the most promising
   compounds among all drugs in the current study. PGs (prostaglandins)
   are lipid mediators synthesized from arachidonic acid via the
   cyclooxygenase pathway and include PGD2, PGI[2], PGH2, PGE[2], and
   PGF2α ([161]30). PGI[2] signals via IP receptors to induce cAMP
   signaling, similar to EP4 receptors. We show EP4 receptors to have
   similar expression as in non-COPD controls, whereas IP receptors are
   expressed at higher levels in patients with COPD, indicating that the
   expression of both receptors is maintained in disease.

   We show that PGE[2] agonists are beneficial in reducing CS-induced
   damage to alveolar epithelial progenitors. However, PGE[2] has been
   reported as an unstable molecule with an extremely short half-life;
   therefore, targeting its receptors with specific more stable analogs
   may be a better alternative. PGE2 is the most widely produced PG in the
   human body, and it signals via four specific G protein–coupled
   receptors (EP1 to EP4) ([162]31, [163]32). The interactions between
   PGE[2] and EP receptors depend on tissue and cell type, specific
   receptor expression, and differences in binding affinities, leading to
   unique patterns of EP receptor activation ([164]33). PGE[2] can
   stimulate cAMP production through EP2 and EP4 receptors, whereas EP3
   activation results in decreased cAMP synthesis and EP1 stimulation is
   coupled to G[q] activation and (enhanced) Ca^2+ signaling ([165]30,
   [166]33, [167]34). EP1 and EP3 receptors can mediate
   bronchoconstriction indirectly through activation of neural pathways
   ([168]35), as a consequence nonselective PGE[2] analogs are unsuitable
   as pulmonary drugs. Therefore, we selected analogs of EP2 and EP4 to
   mimic effect of PGE[2] in our organoid assay and demonstrated that EP4
   agonism showed beneficial effects against impaired organoid formation
   in response to CS(E) exposure. Targeting EP4 receptors is worthwhile
   investigating in more detail in the future, as the effects may surpass
   epithelial repair only. Additional beneficial effects of EP4 agonism in
   COPD may include bronchoprotection (in humans; EP2 in mice) ([169]36)
   and inhibition of inflammation ([170]37). Expression of the EP4
   receptor is maintained in COPD and in smokers. Smoking and aging are
   associated with increased expression of PGE[2] in the human lung
   ([171]38, [172]39). Whereas this may serve as an endogenous protective
   mechanism, PGE[2] has multiple effects, and not all of these are
   protective to the pathophysiology of COPD. We propose that selective
   EP4 agonism could unify several functional features that support the
   treatment of COPD, making this an intriguing pharmacological target.

   Iloprost, a stable PGI[2] analog ([173]30, [174]40–[175]44), has been
   shown to have anti-inflammatory effects and protects against
   bleomycin-induced pulmonary fibrosis in mice ([176]41) and is also
   clinically used for the treatment of pulmonary hypertension ([177]40).
   Although a recent study ([178]43) showed that iloprost improved
   clinical outcomes in patients with COPD with poor lung oxygenation, its
   impact on alveolar repair is unknown. Here, we show that iloprost
   prevents the repressed organoid formation resulting from CS(E)
   exposure. Whereas IP and EP4 receptors are both linked to cAMP
   generation, rolipram and olodaterol had no effect on organoid growth
   and only very limited effect when applied in combination. This apparent
   discrepancy might be explained by cAMP compartmentalization. This
   phenomenon explains why and how distinct G[s] protein–coupled receptors
   or drugs activating the cAMP pathway can have distinct effects on cAMP
   generation and function ([179]45). An alternative explanation is that
   EP4 receptor expression is higher than that of the β[2]-adrenergic
   receptor. As corticosteroids have been shown to inhibit the release of
   arachidonic acid metabolites including PGI[2] ([180]46) and PGE[2]
   ([181]17), we speculate that the negative effect of budesonide on the
   progenitor cell growth may be due to the restriction of PG synthesis.

   Analogs of PGE[2] and PGI[2] have previously been demonstrated to
   attenuate other features of COPD in mice. For example, iloprost
   attenuated inflammation, airway hyperresponsiveness, and airway
   remodeling in a mouse model of lipopolysaccharide and elastase exposure
   ([182]45). Systemically administered EP2 agonists have beneficial
   effects on angiogenesis in a mouse model of emphysema ([183]45).
   Moreover, PGE[2] represses fibroblast activation, fibroblast to
   myofibroblast differentiation, and migratory capacity ([184]47). PGE[2]
   has been reported ([185]48) as a key regulator released from alveolar
   epithelial cells regulating the barrier function of the lung
   microvasculature. However, we did not see supportive effects of
   misoprostol or iloprost using a scratch wound assay on human pulmonary
   endothelial cells exposed to CS (fig. S8), indicating that the
   beneficial effects of PGE[2] and PGI[2] on repair may be specific for
   the alveolar epithelial cell. In addition, studies have shown PGE[2] to
   be anti-inflammatory by attenuating macrophages activation and
   proliferation ([186]39, [187]49), While beneficial, the
   anti-inflammatory activity of PGE[2] and PGI[2] analogs could also
   predispose to pulmonary infections, which is a risk that needs to be
   considered moving forward. Here, we identified the potential
   therapeutic effect of PGE[2] and PGI[2] analogs on defective alveolar
   progenitors in response to CS that appears to be primarily related to
   the disturbance of circadian clock signaling rather than its
   anti-inflammatory response. Given the diverse and occasionally opposite
   roles of PGE[2] and PGI[2] in the pathophysiology of COPD, the main
   challenge will be to balance broad spectrum with specificity, for
   example, using subtype-selective drugs or drugs targeted to specific
   lung cell types.

   By generating transcriptomic signatures of epithelial progenitors
   derived from mice exposed in vivo to air, CS, CS + misoprostol, or CS +
   iloprost, we uncovered dynamic molecular signaling pathways in response
   to CS exposure. We identified circadian clock signaling as being
   significantly repressed in the alveolar epithelial progenitors derived
   from mice exposed to CS, which could be improved by either misoprostol
   or iloprost treatment. Circadian rhythms ([188]50–[189]53) are normally
   generated and regulated by clock genes, including BMAL1 (ARNTL1) and
   CLOCK encoding activators, period (PER1 to PER3) and cryptochrome genes
   (CRY1 and CRY2) that encode repressors, and the nuclear receptors
   Rev-erb (NR1D1 and NR1D2) and RORA, which constitute secondary
   regulatory loops. These core clock genes not only activate or repress a
   cell-autonomous clock but also regulate the clock-controlled genes
   ([190]54), thus interacting with other molecular signaling pathways.
   Previously, it has been demonstrated that clock signaling is
   down-regulated in CS-exposed mice, linked to an impairment of
   antioxidant defense mechanisms ([191]55), and Rev-erbα has been shown
   as an key regulator of inflammatory response in lung injury models
   ([192]19–[193]21, [194]56). Here, we show that the Rev-erbα agonist
   prevents the impaired organoid formation resulted from CSE exposure,
   suggesting that PGE[2]/PGI[2]/Rev-erbα signaling pathway plays an
   important role in defective lung repair.

   Furthermore, we found that CS exposure up-regulated pathways associated
   with cell cycle activity in alveolar epithelial progenitors, which
   could be counteracted by in vivo misoprostol or iloprost treatment. The
   cell cycle ([195]57–[196]61) is driven by a set of tightly regulated
   molecular events controlling DNA replication and mitosis with four
   phases, and each individual cell may require different triggers to
   decide whether to enter proliferation or apoptosis. To further assess
   alveolar epithelial progenitors under which cell cycle/apoptotic status
   in response to CS exposure and additional PGE[2]/PGI[2] treatments may
   be the next step to investigate in the future. A link between circadian
   clock and cell cycle signaling pathways has been proposed ([197]54,
   [198]60, [199]62). The molecular control of the biological clock is
   dependent on cAMP signaling, and cAMP activators are known to entrain
   the biological clock ([200]63), explaining the link between PGE[2] and
   PGI[2] activations and restoration of the defective clock signaling in
   combination with CS exposure. Hence, it is of great interest to
   determine in more molecular detail how these two oscillatory systems
   communicate in regulating PGE[2]/PGI[2]-mediated lung repair in future
   studies.

   In conclusion, in this study, we demonstrate the protective effects of
   several drug candidates, including PGE[2] and PGI[2] analogs, against
   in vivo and in vitro CS(E)-induced damage of alveolar epithelial
   progenitors. Furthermore, using transcriptome analysis, we show that CS
   induces a wide range of transcriptional changes, including alterations
   of circadian clock and cell cycle signaling pathways, which can be
   counteracted by either misoprostol (PGE[2]) or iloprost (PGI[2])
   treatment. While the focus of our studies was on COPD, similar
   beneficial effects may be operative in diseases affected by similar
   defects in alveolar progenitors such as acute respiratory distress
   syndrome and pulmonary fibrosis. Overall, these data provide promising
   therapeutic strategies to specifically address defective lung repair in
   respiratory diseases, in particular, targeting EP4 and IP receptors.

MATERIALS AND METHODS

Animals

   Mouse experiments for organoid study were performed at the Central
   Animal Facility of the University Medical Center Groningen (UMCG) in
   accordance with the national guidelines and upon approval of the
   experimental procedures by CDP and the Institutional Animal Care and
   Use Committee of the University of Groningen (license AVD105002015303).
   C57BL/6J (555) and BALB/cByJ (Jax strain) mice (both genders, 8 to 12
   weeks of age) were maintained under a 12-hour light/dark cycles and
   were allowed food and water ad libitum. Animals for circadian clock
   studies were exposed to CS and/or administrated with compounds at the
   same time of the day for all mice in all groups. Animals were
   euthanized at the same time of the day. Animals for the chronic CS
   model were adult (female, 8 to 10 weeks of age at the start of the
   study) C57BL/6N mice, which were obtained from the Charles River
   Laboratories (Sulzfeld, Germany) and used for both the scRNA-seq
   analysis and organoid assay. These experiments were performed at the
   Helmholtz Zentrum München and approved by the ethics committee for
   animal welfare of the local government for the administrative region of
   Upper Bavaria (Regierungspräsidium Oberbayern) and were conducted under
   strict governmental and international guidelines in accordance with
   European Union Directive 2010/63/EU.

Human material

   The human lung tissue was obtained from lung transplant donors
   according to the Eurotransplant guidelines including the absence of
   primary lung diseases such as asthma and COPD and no more than 20 pack
   years of smoking history ([201]64). Human lung tissue specimens were
   obtained with full informed consent from patients undergoing lung
   volume reduction surgery or lung transplantation at UMCG. The study
   protocol was consistent with the Research Code of the UMCG and national
   ethical and professional guidelines. All tissue samples were anonymized
   before use. Gene expression in human lung published datasets was
   obtained by downloading a series matrix files from the National Center
   for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO)
   database for [202]GSE76925 ([203]65) and gene expression normalized to
   healthy smokers.

In vivo CS exposure

   Mice (n = 4 to 11 per group, 10 to 12 weeks old) were exposed (whole
   body) to 3R4F research cigarettes (Tobacco Research Institute,
   University of Kentucky, Lexington, KY) for four consecutive days (two
   sessions per day, 8 hours between each exposure) to establish an acute
   smoke-induced inflammation model, as described previously ([204]9). In
   the CS group, mice were exposed to one cigarette in the morning and
   three in the afternoon on day 1. From days 2 to 4, mice were exposed to
   five cigarettes each session. All cigarettes were smoked without a
   filter in 5 min at a rate of 5 liter/hour in a ratio with 60 liter/hour
   air using a peristaltic pump (45 rpm; Watson Marlow 323 E/D, Rotterdam,
   the Netherlands). In the control group, mice were exposed to fresh air
   using similar exposure chambers as the CS group.

   In some studies, budesonide was nebulized (0.1 mM; 15 min per mouse per
   exposure) to wild-type mice (n = 6) before each CS exposure. In
   separate studies, intraperitoneal injections of 50 μg of misoprostol or
   50 μg of iloprost were given to wild-type mice (n = 6 to 8) 30 min
   before each CS exposure. On day 5, mice were euthanized, and the lungs
   were immediately used for establishing organoid cultures or stored at
   −80°C for further experimental uses.

   For the scRNA-seq analysis and the organoid assay setup in the chronic
   model, CS was generated from 3R4F research cigarettes, with the filters
   removed to expose to the murine lungs. Mice were whole body exposed to
   active 100% mainstream CS of 500 mg/m^3 total particulate matter for 50
   min twice per day for 4 m in a manner mimicking natural human smoking
   habits as previously described ([205]65).

Fibroblast culture

   Mouse fibroblasts, CCL-206 [Mlg (CCL-206); American Type Culture
   Collection (ATCC), Wesel, Germany] were cultured in Dulbecco’s modified
   Eagle’s medium (DMEM)/F12 medium supplemented with 10% (v/v) fetal
   bovine serum (FBS), penicillin/streptomycin (100 U/ml), 2 mM
   l-glutamine, and 1% amphotericin B in a humidified atmosphere under 5%
   CO[2]/95% air at 37°C, as previously described ([206]6, [207]10,
   [208]66). For organoid experiments, fibroblasts were
   proliferation-inactivated by incubation in mitomycin C (10 μg/ml;
   M4287, Sigma-Aldrich) for 2 hours, followed by three washes with
   phosphate-buffered saline (PBS), after which the cells were trypsinized
   before introduction into the organoid cocultures. Human lung
   fibroblasts MRC5 (CCL-171, ATCC, Wesel, Germany) were cultured in Ham’s
   F12 medium supplemented with the same additives as the murine
   fibroblasts’ medium.

Cigarette smoke extract

   The smoke of two 3R4F research cigarettes was pumped into 25 ml of warm
   fibroblasts culture medium to produce 100% CSE ([209]12). All
   cigarettes were without a filter, and smoke was passed through the
   medium using a peristaltic pump (45 rpm; Watson Marlow 323 E/D,
   Rotterdam, the Netherlands). CSE was freshly prepared before each set
   of experiments.

Murine primary alveolar epithelial isolation

   The primary alveolar epithelial cells, brief in Epcam^+ cells
   (CD31^−/CD45^−/CD326^+) were isolated on the basis of the protocols
   published previously from our group ([210]67). The isolation of Epcam^+
   cells generated from the chronic CS mice was slightly different. Murine
   lungs harvested from mice exposed with CS (4 months) were stored in
   MACS tissue storage solution (130-100-008) at 4°C overnight (due to
   shipment). Lungs were chopped into small pieces and incubated in the
   enzyme mix containing dispase (#354235, Corning) and deoxyribonuclease
   I (A3778, Analytics-Shop) for 1 hour at 37°C. The rest procedure is the
   same as the ordinary epithelial isolation.

Organoid culture

   The adult alveolar epithelial progenitor cell–derived organoid assay
   provides a platform to study the response of alveolar epithelial cells
   to various conditions rapidly and easily where complex in vivo analysis
   is less feasible ([211]68) and typical two-dimensional cell culture
   cannot recapitulate the cell-cell interactions. Moreover, organoids
   represent a powerful tool to model various pathological states of
   environmental or genetic origin that can be easily manipulated in vitro
   for validation of therapeutic approaches ([212]69). The organoid
   culture system is based on previously published protocols from our
   group ([213]6, [214]10, [215]66). Briefly, epithelial cells
   (CD31^−/CD45^−/CD326^+) were freshly isolated from murine or human lung
   tissue and cocultured with murine CCL-206 or human MRC5 fibroblasts,
   respectively, in Matrigel (Corning Life Sciences B.V., Amsterdam, The
   Netherlands). EpCAM^+ (CD31^−/CD45^−/CD326^+) cells were isolated from
   mouse lung tissue (without the trachea) using the QuadroMACS Separator
   and antibody-bound magnetic beads (Miltenyi Biotec, Leiden, The
   Netherlands). EpCAM^+ cells and fibroblasts were mixed 1:1 (20,000
   cells each) and suspended in 100 μl of Matrigel prediluted 1:1 (v/v)
   with DMEM supplemented with 10% FBS. This mixture of cells was added to
   a 24-well Falcon cell culture insert (Corning, USA) within a 24-well
   plate containing 400 μl of organoid media [DMEM/F-12 supplemented with
   5% FBS, 1% penicillin/streptomycin, 1% glutamine, 1% amphotericin B,
   0.025 per mil (‰) epidermal growth factor, 1%
   insulin-transferrin-selenium, and 1.75‰ bovine pituitary extract]
   underneath the insert in each well. Adult human donor tissue was
   isolated from histologically normal regions of lung tissue specimens
   obtained at UMCG (Groningen, The Netherlands) from n = 7 patients (two
   non-COPD and five patients with COPD). Human lung tissues were
   incubated and homogenized overnight in an enzyme mixture at 4°C; the
   EpCAM^+ isolation process was similar to that described above for
   murine lung tissue. Organoids were cultured in a humidified atmosphere
   under 5% CO[2]/95% air at 37°C, and medium in the wells was refreshed
   every 2 to 3 days. To quantify the number of organoids, light
   microscopy at ×20 magnification was used, and organoids were counted
   manually. The diameter of the organoids (organoid size) was measured
   using NIS-Elements software with a light microscope. The number
   parameter represents the ability of alveolar epithelial progenitors to
   form organoids, whereas the size parameter may result from swelling or
   proliferation. Subsequent phenotyping identifies effects on alveolar
   type II cell differentiation.

   For in vitro organoid experiments, organoids were continuously treated
   with control, 1.25 (1% for human organoids), 2.5, or 5% CSE; and
   organoid culture medium was refreshed every other day. All information
   about the pharmacological compounds used in this study is provided in
   the table S1.

Immunofluorescence staining

   The immunofluorescence staining assay for organoids was performed as
   described previously by our group with minor modifications ([216]6,
   [217]10, [218]66). Organoids were fixed in acetone diluted 1:1 (v/v)
   with methanol for 15 min at −20°C. After fixation, 1 ml of PBS with
   0.02% sodium azide was added to the well underneath the insert.
   Organoids were kept at 4°C for 1 week after fixation. Bovine serum
   albumin (BSA) medium was added on top of the insert for blocking at
   room temperature (RT) for 2 hours. Afterward, primary antibody
   incubation was performed in PBS buffer with 0.1% BSA and 0.1% Triton
   X-100 overnight at 4°C. The next day, the organoids were washed three
   times with PBS for 30 min, and secondary antibody incubation was
   performed for 2 hours at RT. After washing with PBS for 15 min, the
   organoids on the insert membrane were transferred to a glass slide with
   two drops of mounting medium containing 4 the organoids on the insert
   (DAPI) (104139, Abcam, Cambridge, UK), and a coverslip was applied.

   The immunofluorescence staining assay for the lung slices was performed
   as described previously with minor modifications ([219]6). The frozen
   murine lung sections (5 μm) were dried for 30 min and then were fixed
   in 4% paraformaldehyde/PBS solution at RT for 15 min. After rinsing
   three times with PBS, the lung sections were blocked within PBS buffer
   containing 5% donkey serum and 0.3% Triton X-100 at RT for 60 min. The
   primary antibody incubation [Rev-erbAα/NR1D1 antibody, NBP2-75645,
   Novus Biologicals (1:100 dilution); anti-PER3 antibody, ab224594, Abcam
   (1:100 dilution); and anti–pro-SPC antibody, AB3786, Sigma-Aldrich
   (1:100 dilution)] was performed in the PBS buffer containing 1% BSA and
   0.3% Triton X-100 at 4°C overnight. After rinsing three times with PBS,
   the lung sections were incubated with secondary antibodies (donkey
   anti-rabbit immunoglobulin G Alexa Fluor 488, A21206, Thermo Fisher
   Scientific; 1:1000 dilution) at RT for 2 hours. The sections were
   rinsed twice with PBS and once with ultrapure water and then were
   transferred to a glass slide, and then the coverslip was applied.

   The slides were kept at 4°C. Confocal images were acquired using a
   Leica SP8 microscope or a Leica DM4000B microscope. Images were
   obtained and analyzed with LAS X (Leica) software (open resource, Leica
   Microsystems GmbH, Wetzlar, Germany).

RNA extraction and RNA-seq analysis

   The Epcam^+ cells isolated from mice exposed to air, CS, CS +
   misoprostol, or CS + iloprost were used to extract total RNA for
   RNA-seq using NucleoSpin RNA kit (740955, Macherey-Nagel, Germany)
   according to the manufacturer’s instructions. RNA concentrations and
   qualities were analyzed using NanoDrop spectrophotometer. An Illumina
   NovaSeq 6000 sequencer was used for the RNA-seq data analysis by
   GenomeScan ([220]www.genomescan.nl). The procedure included data
   quality control, adapter trimming, alignment of short reads, and
   feature counting. Library preparation was checked by calculating the
   ribosomal (and globin) content. Checks for possible sample and barcode
   contamination were performed, and a set of standard quality metrics for
   the raw dataset was determined using quality control tools (FastQC
   v0.34 and FastQA). Before alignment, the reads were trimmed for adapter
   sequences using Trimmomatic v0.30. To align the reads of each sample,
   the ensemble mouse reference GRCm38 (patch 6) was used. Analyses
   following the RNA-seq studies were performed using the BioJupies
   platform ([221]https://amp.pharm.mssm.edu/biojupies/) ([222]70) and
   using the DeSeq2 package in R. Gene expression in murine lung published
   datasets was obtained by downloading a series of matrix files from the
   NCBI GEO database ([223]GSE151674).

Immunohistochemistry

   Immunohistochemistry was performed on the murine lung tissue harvested
   from mice exposed by air/CS/CS + misoprostol (intraperitoneal)/CS +
   iloprost (intraperitoneal) based on the optimized protocol described
   previously ([224]71). Briefly, paraffin-embedded frozen murine lung
   tissue was cut with a Microm HM 340E microtome. Five-micrometer
   transverse cross sections were used for analysis. Tissue sections were
   first dried 30 min with hair blower and then were rehydrated in PBS.
   Tissue sections were stained with 3,3′-diamino-benzidine (DAB)
   (Sigma-Aldrich) NaCN and H[2]O[2] for 7 min. After rising within PBS,
   sections were counterstained with hematoxylin and imbedded in Kaiser
   gelatin. Sections were stained with Mayer’s hematoxylin for 10 min,
   rinsed in running tap water for 10 min, and then were stained with
   eosin for 3 min. After a short rinsing step (UP water), the sections
   were quickly dehydrated in 96% ethanol and 100% ethanol and xylene. The
   section was mounted with mounting medium.

Statistics analysis

   All data are presented as means ± SEM unless indicated otherwise.
   Unless stated otherwise, all data were assessed for statistical
   significance using two-tailed Student’s t test or one-way analysis of
   variance (ANOVA). The P value indicating statistically significant
   differences between the mean/median values are defined as follows: *P <
   0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Statistical
   analyses were performed with GraphPad Prism 8 software.

Acknowledgments