Interleukin-11 causes alveolar type 2 cell dysfunction and prevents alveolar regeneration

Following lung injury, alveolar regeneration is characterized by the transformation of alveolar type 2 (AT2) cells, via a transitional KRT8+ state, into alveolar type 1 (AT1) cells. In lung disease, dysfunctional intermediate cells accumulate, AT1 cells are diminished and fibrosis occurs. Using single cell RNA sequencing datasets of human interstitial lung disease, we found that interleukin-11 (IL11) is specifically expressed in aberrant KRT8 expressing KRT5-/KRT17+ and ba saloid cells. Stimulation of AT2 cells with IL11 or TGFβ1 caused EMT, induced KRT8+ and stalled AT1 differentiation, with TGFβ1 effects being IL11 dependent. In bleomycin injured mouse lung, IL11 was increased in AT2-derived KRT8+ cells and deletion of Il11ra1 in lineage labeled AT2 cells reduced KRT8+ expression, enhanced AT1 differentiation and promoted alveolar regeneration, which was replicated in therapeutic studies using anti-IL11. These data show that IL11 maintains AT2 cells in a dysfunctional transitional state, impairs AT1 differentiation and blocks


MAIN TEXT Introduction
The alveolar epithelium plays a pivotal role in lung homeostasis and protects the lung from inhaled environmental insults and pathogenic infections.In the alveolus, alveolar type 2 cells (AT2 cells) become activated after injury and proliferate and trans-differentiate into alveolar type 1 cells (AT1 cells) to restore alveolar structure and lung function (1,2).A number of human lung pathologies, including idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD) and post-infective lung damage, are characterized by failure of homeostatic AT2-to-AT1 transitions (3,4).
In mice, transitional cells, herein referred to as KRT8+ transitional cells, can be derived from either AT2 cells or airway stem cells, and possess the capacity to differentiate into mature AT1 cells (13,16).Importantly, KRT8+ transitional cells in the mouse exhibit transcriptional similarities to human disease-associated KRT5 -/KRT17 + / aberrant basaloid cells, including epithelial-mesenchymal transition (EMT), p53 and cell senescence pathways and expression of KRT8 itself (13,15).Furthermore, KRT8+ transitional cells are thought to contribute to fibrosis via expression of profibrotic and proinflammatory mediators.Recent studies have shown that elevated TGFβ signaling in AT2 cells and IRE1α activity in DATPS maintain the KRT8+ cell state following lung injury in mice (17)(18)(19).Similarly, the persistence of senescent AT2 cells promotes progressive pulmonary fibrosis (20).
Interleukin-11 (IL11), a member of the IL-6 family of cytokines, is upregulated in the airways following viral infections and has been associated with a range of respiratory disorders (21).We previously reported that IL11 was increased in the lungs and fibroblasts from patients with IPF, and its expression correlates with disease severity (22).A contemporaneous study found that IL11 was expressed in a range of cell types in fibrotic lungs of patients with Hermansky-Pudlak Syndrome (HPS) and also in SFTPC + cells in IPF (23).More recent pharmacologic studies using siRNA have validated the role of IL11 in lung fibrosis (24).
In the current study, we leveraged single cell RNA sequencing (scRNA-seq) datasets from patients with lung disease and analyzed IL11-lineage labeled cells in a mouse model to delineate the different lung cell types expressing IL11 in disease.We examined whether IL11 signaling plays a role in alveolar regeneration via its specific activity in AT2 cells using conditional Il11ra1 deletion in AT2 cells and lineage tracing in mice that were subjected to bleomycin lung injury and also in studies of AT2 cells in vitro.We also tested whether a  S1).Furthermore, the association between IL11 and the IL11 co-expression module was highly specific to disease (Fig 1I

IL11 is upregulated in activated AT2 cells, alveolar KRT8+ cells and stromal cells after lung injury in mice
To further characterize IL11 expressing cells in the injured lung, we used an IL11 EGFP reporter mouse (27).We performed single dose oropharyngeal injections of bleomycin (BLM), a drug that causes lung epithelial damage and fibrosis, and performed preliminary characterization of lung cells 10 days post-injury by flow cytometry.Using antibodies against a range of lung cell type markers: CD31 (endothelial cells), CD45 (hematopoietic cells), CD326 / EpCAM (epithelial cells), our analysis revealed that IL11 EGFP+ cells were rarely observed in the uninjured lung.However, following BLM injury, we found elevated proportions of IL11 EGFP+  Since the low detection/abundance of IL11 EGFP+ cells preclude further FACS-based analysis, we next focused on immunohistochemistry to determine the identities of IL11 expressing cells in the injured lung.To do this, we assessed the lungs of IL11 EGFP reporter mice at 7 or 21 days post-BLM injury by staining for GFP and counter-stained for SFTPC (AT2 marker), PDPN (AT1 marker), PDGFRA (pan-fibroblast marker) or CD45.Consistent with the flow cytometry analysis, IL11 EGFP+ cells were very rarely observed in the lungs of uninjured IL11 EGFP reporter mice.In contrast, in BLM injured lungs, IL11 EGFP  To investigate if IL11 is expressed by KRT8+ transitional cells, we performed immunostaining for GFP and KRT8 in lung sections from BLM-treated and uninjured IL11 EGFP reporter mice and excluded airway regions for quantification.In uninjured mice, KRT8 expression was limited to the airways whereas BLM-treatment resulted in the appearance of KRT8+ cells in the damaged alveolar regions (Fig 2E).There was overlap of IL11 EGFP expression in a proportion of KRT8 expressing cells in alveolar regions following BLM injury (Fig 2F).
To further test if IL11 expressing KRT8+ transitional cells are derived from AT2 cells during lung injury, we used Sftpc-CreER; R26-tdTomato (Sftpc-tdT) mice to trace AT2 cells and their descendants and monitored for the expression of IL11 specifically in this cell lineage after BLM injury.We exposed Sftpc-tdT mice to tamoxifen prior to BLM treatment and assessed the lungs 14 days post-injury (Fig 2G).We performed immunostaining using an anti-IL11 antibody, which showed consistent overlap with anti-GFP in injured IL11 EGFP lungs (Fig S5C), and counterstained for KRT8.This revealed the emergence of numerous IL11 + KRT8 + tdT + cells with spread out/elongated morphologies 14 days after BLM injury (Fig 2H).IL11 and KRT8 immunostaining were not observed in alveolar regions of uninjured Sftpc-tdT mice, as expected.These findings revealed that IL11 expressing KRT8+ transitional cells are derived from activated AT2 cells after lung injury.

IL11 induces cytopathic features in AT2 cells and delays AT2-to-AT1 differentiation
To investigate the functional importance of IL11 in alveolar epithelial cells, we performed 2 dimensional (2D) cultures of primary Human Pulmonary Alveolar Epithelial Cells (HPEpiC).By immunostaining, we first confirmed that HPEpiC expressed high levels of SFTPC and did not stain positive for AGER (Fig S6A).HPAEpiC expressed high levels of IL11RA and its coreceptor IL6ST (gp130) but lacked detectable IL6R expression (Fig S6A).
To test whether IL11 directly induces EMT in AT2 cells, we stimulated HPEpiC with IL11 (5 ng/ml, 24 h) and monitored for the expression of EMT related proteins (Collagen I, Fibronectin, SNAIL) along with KRT8 using immunostaining and immunofluorescence quantification (Fig 3A).In parallel, we treated HPEpiC with TGFβ1 (5 ng/ml; 24 h), a potent inducer of both EMT and KRT8 expression in AT2 cells (28)(29)(30), and simultaneously added a neutralizing IL11 antibody (X203) or an IgG control antibody to investigate the effect of IL11 signaling downstream of TGFβ stimulation (Fig 3A).This revealed that IL11 and TGFβ1 treatment led to upregulation of Collagen I, fibronectin, SNAIL and KRT8 expression, as compared to untreated cells (Fig 3B-C).By ELISA, we found that TGFβ1 stimulation significantly induced IL11 secretion by HPEpiC (Fig S6B).The effects of TGFβ1 on the expression of EMT related proteins and KRT8 were significantly blunted by X203 (Fig 3B-C).
In contrast to TGFβ1 treatment and consistent with data from other cell types (22,33), IL11 (5 ng/ml, 24 h) did not result in significant changes in global transcription levels in HPEpiC ( AT2 cells largely increase their cell area and spontaneously undergo differentiation towards AT1 cells when cultured under prolonged 2D culture conditions.Under these conditions, AT2 cells upregulate KRT8 during early differentiation, followed by a decline of KRT8 and the subsequent upregulation of mature AT1 markers (such as PDPN) during late differentiation (13,29).To test if IL11 stalls the transition of AT2 cells into mature AT1 cells, we isolated mouse AT2 cells from tamoxifen-exposed Sftpc-tdT mice by FACS sorting for tdT + cells and cultured these cells under 2D culture conditions followed by treatment with IL11 (5 ng/ml) from day 1 to day 5 (Fig 3E).By immunostaining and cell surface area analysis of tdT + cells, we found that numerous cells expressed PDPN and greatly increased their surface area after 5 days of culture in untreated cells (Fig 3F-G).In contrast, exposure to IL11 from days 1 to 5 stalled AT1 differentiation with cells expressing higher levels of KRT8, lower levels of PDPN and with reduced surface area, as compared to controls (Fig 3F-G).
Since prolonged TGFβ signaling impairs terminal AT1 maturation (17,29,34), we hypothesized that the maladaptive effects of TGFβ on AT1 maturation might be mediated, in part, by IL11.We tested for this by first priming AT2 cells with TGFβ1 for two consecutive days followed by subsequent treatment with TGFβ1 and X203 or IgG antibodies for an additional 2 days (Fig 3E).
Similar to the effects of sustained IL11 treatment, we found that cells treated with TGFβ1 followed by coincubation with IgG resulted in stalled AT1 differentiation, with cells that were less elongated and expressed higher levels of KRT8 as compared to controls (Fig 3F-G).On the other hand, coincubation with X203 partially-relieved the stalled AT1 differentiation phenotype and significantly increased cell surface area and PDPN expression as compared to IgG-treated cells (Fig 3F-G).These data show that IL11 promotes AT2 cell dysfunction by causing the accumulation of KRT8+ transitional cells and delaying the terminal differentiation of AT1 cells.

IL11 signaling in AT2 cells impairs AT2-to-AT1 cell differentiation during lung injury and promotes fibrosis
To test the hypothesis that IL11 signaling in AT2 cells promotes the accumulation of KRT8+ transitional cells and delays AT1 differentiation after lung injury in vivo, we generated AT2 cellspecific Il11ra1 deleted mice by crossing Sftpc-tdT mice and Il11ra1 fl/fl mice to create Sftpc-tdT; Il11ra1 fl/fl mice.This allowed the simultaneous deletion of Il11ra1 and the expression of tdT specifically in AT2 cells upon tamoxifen administration.Sftpc-tdT; Il11ra1 +/+ mice were used as controls.We injected tamoxifen 14 days prior to BLM injury and assessed the lungs of mice 12 days post-BLM treatment (Fig 4A).
To test the importance of IL11 signaling in AT2 cells for lung fibrogenesis, we used a separate cohort of Sftpc-CreER; Il11ra1 fl/fl mice.We administered tamoxifen 14   There was a non-significant trend of improved survival, body weights and decreased lung weights in AT2-specific Il11ra1-deleted mice by the end of the 21 day study period (Fig S7C-E).Serum surfactant protein D (SFTPD) levels, a marker of lung inflammation and epithelial injury (35) was elevated in BLM-injured control mice but was significantly reduced in injured mice with AT2 cell specific Il11ra1 deletion (Fig 4I).Consistent with the effects seen in Sftpc-tdT; Il11ra1 fl/fl mice, immunostaining for KRT8 revealed that KRT8 expressing cells were rarely observed in the alveolar compartment of AT2 cell-specific Il11ra1 deleted mice 21 days post-BLM injury, as compared to controls (Fig S7F).

Pharmacological blockade of IL11 promotes AT2-to-AT1 cell differentiation and alveolar regeneration after bleomycin-induced lung injury in vivo
We next investigated whether anti-IL11 antibodies could promote AT2-to-AT1 differentiation and enhance alveolar regeneration when administered after lung injury.To test this, we performed BLM-induced injury to tamoxifen-exposed Sftpc-CreER; R26-tdT mice followed by X203 or IgG control antibody administration starting from 4 days after injury and assessed the lungs on day 12 (Fig 5A).
As compared to uninjured lungs, we observed widespread architectural disruption in IgG treated mice, with a large increase in KRT8 + cells that adopted elongated morphologies, along with a decline in the number of tdT + cells (Fig 5B-C).Additionally, in IgG treated mice, we found an increase in non-lineage labeled KRT8 + cells (KRT8 + tdT -) that stained weakly for the AT1 marker PDPN (Fig 5B-D), likely reflecting an influx of airway/progenitor cells that have committed to alveolar fates in regions of severe lung injury (13,16,36,37).
As compared to IgG treated mice, BLM-induced parenchymal damage was attenuated by X203 treatment and this coincided with reduced numbers of KRT8 + cells and proportions of lineage labeled transitional cells (KRT8 + tdT + cells) and lineage negative cells (KRT8 + tdT -cells) (Fig 5B-E).Furthermore, X203 treatment restored tdT + cell numbers after injury to levels similar to those seen in uninjured lungs, which was associated with increased proliferation of surviving tdT + AT2 cells as determined by immunostaining for Ki67 (Fig 5B-C

Discussion
Severe respiratory diseases such as IPF and SARS-COV-2 pneumonia are associated with defects in alveolar epithelial repair and irreversible loss of alveolar epithelial cells, which ultimately leads to fibrosis and a decline in lung function.We previously discovered an important role for IL11 in lung fibrosis, mediated via its profibrotic activity in lung fibroblasts and IL11 expression was confirmed in diseased fibroblasts in the current study (22,25,38).Here, we show that IL11 is specifically upregulated in aberrant alveolar epithelial cells in human PF and its expression is associated with pathological pro-EMT and inflammatory gene signatures in diseased epithelial cells.In complementary studies of mice with severe lung injury, we found that IL11 is expressed by activated AT2 cells and KRT8+ transitional cells.
Due to the complex signaling milieu that occurs following severe lung injury, multiple pathways likely contribute to the emergence and maintenance of KRT8+ transitional cells, among which TGFβ, which shows IL11 dependency for its effects, is of particular importance (17,34).While inflammatory cytokines such as IL-1β and TNFα induce AT2 cell proliferation, IL-1β also primes a subset of Il1r1-expressing AT2 cells for differentiation into DATPS (14,39).Intriguingly, while IL6 is a therapeutic target in some forms of PF (26,40), we show that the cell types expressing IL6 in the fibrotic lung differ from those expressing IL11 and only IL11 expression is enriched in aberrant epithelial cells .
Our data identify an IL11-stimulated ERK-dependent signaling pathway that promotes and maintains AT2 cells in a KRT8+ state and induces EMT features in AT2 cells.Furthermore, we found that the anti-proliferative effects of TGFβ and its induction of EMT genes and KRT8 expression in AT2 cells was, in part, mediated by IL11 signaling.These findings may have implications for other airway/lung disorders such as Hermansky-Pudlak Syndrome-associated pulmonary fibrosis, severe asthma and severe viral pneumonitis, including SARS-COV-2 infection, where elevated IL11 levels are elevated and implicated in disease pathogenesis (11,23,(41)(42)(43).
Specialized lung mesenchymal cells form a supportive niche that maintains the progenitor properties of AT2 cells under homeostatic conditions (44,45).In disease, impaired alveolar repair may arise due to disruption of this supportive niche and the development instead of a profibrotic niche composed of pathological fibroblasts and dysfunctional alveolar epithelial cells (46).Given the elevated expression of IL11 in aberrant mesenchymal and epithelial cell types in PF and its role in both fibroblasts activation and AT2 cell dysfunction, we propose that IL11 may cause multiple aspects of pathobiology across cell types in the diseased niche.
There are limitations to our study.Although several recent studies have shown that IL11 is upregulated in the lungs and SFTPC + cells from patients with IPF (22)(23)(24), in situ studies of IL11 expression in diseased human lung tissue are required to further validate findings.We did not dissect the specific cell type expressing IL11 that impacts AT2-to-AT1 differentiation, although our earlier studies suggest a dominant role for IL11 secretion from fibroblasts for fibrosis phenotypes (25).In light of recent evidence highlighting the importance of distal airway secretory/basal cells in aberrant alveolar repair and fibrosis (46)(47)(48)(49), the effects of IL11 on the recruitment and differentiation of airway / progenitor cells towards KRT8+ and AT1 cells requires study.
In conclusion, we suggest that IL11 causes lung pathology in severe lung disease through at least two pathological processes.First, causing AT2 dysfunction and maintenance of a KRT8+ cell state, thus limiting terminal AT1 differentiation and impairing alveolar regeneration.And second, stimulating fibroblast-to-myofibroblast transformation that leads to lung fibrosis and inflammation (25).Hence, anti-IL11 therapeutics, which are advancing towards clinical trials in patients with PF, may promote alveolar regeneration and mitigate lung fibrosis that would differentiate anti-IL11 therapy from anti-fibrotics currently used in the clinic.

Materials and Methods
Computational analysis of scRNA-seq datasets of human pulmonary fibrosis.Processed human IPF scRNAseq dataset by Habermann et al and Adams et.al., were downloaded from GEO with the accession number GSE135893 and GSE136831 respectively.Cell-type annotations and Uniform Manifold Approximation and Projection (UMAP) coordinates provided by the authors were used in subsequent analyses.
Trajectory analysis.We re-classified alveolar epithelial cells in the Adams et.al., dataset with cell-type annotations defined by Habermann et.al., using Seurat's default label transfer pipeline.Quality of label transfer was evaluated by Jaccard Index (See Assessment of transcriptomic similarities between epithelial cell-types below).Transitional AT2, KRT5-/KRT17+, and AT1 cells were extracted from Habermann and Adams et al dataset for Slingshot trajectory analysis (Slingshot 1.4.0)(50), and the analysis was performed separately for each dataset.Briefly, Slingshot derives differentiation paths from a specified origin and calculates for each cell a pseudotime, which approximates differentiation progression of a cell toward the destination of the trajectory.In this analysis, transitional AT2 cells were specified as the origin, and two differentiation trajectories were derived, one to KRT5-/KRT17+ cells and the other to AT1 cells.Change in IL11 expression was evaluated along the two trajectories by fitting a Generalized Additive Model (GAM) with the expression of IL11 against pseudotime.
Assessment of transcriptomic similarities between epithelial cell-types.We examined transcriptional similarities of different epithelial clusters using the Jaccard index (a cluster here refers to cells of the same cell-type from a specific study, e.g., AT2 cells from Habermann et al dataset).First, we performed differentially expressed gene (DEGs) analysis in epithelial cells from the same study and for each cell-type retained upregulated DE genes with log2 fold change (log2FC) above the 85th percentile of the FC distribution and discarded genes with expression proportion in a cluster less than 40% compared to other cell-types.We refer to these genes as "markers'' of a cluster, and a Jaccard index value was derived for all possible cluster pairing (of all epithelial clusters pooling together both datasets).A Jaccard index between a cluster A and a cluster B was calculated by dividing the size of the intersection of their markers over the size of the union of their markers.
Network analysis.Cells assigned to the differentiation trajectory from transitional AT2 to KRT5-/KRT17+ cells by Slingshot analysis were selected for IL11 co-expression analysis, done individually in Habermann et.al., and Adams et.al., dataset.Briefly, spearman correlations were calculated between the expression of IL11 and genes expressed in the selected cells.Genes with spearman correlation with FDR < .2 were kept.In summary, 103 genes were found to be significantly correlated with IL11 in Adams et al dataset, 378 genes in Habermann et al dataset, and 32 genes in both datasets.Using the R package EnrichR (enrichR 2.1.0)(50,51), functional pathway enrichment analysis was performed on genes significantly correlated with IL11 (in individual datasets and combined) querying several annotation database including KEGG 2019 and MSigDB Hallmark 2020.Pathway terms with FDR < .1 were retained.De-novo network construction was performed on the 32 genes significantly correlated with IL11 in both datasets.Each node in the network represents a gene and each edge (connecting a pair of genes) the spearman correlation between the expression of the two genes in transitional AT2 and KRT5-/KRT17+ cells from Habermann et.al., dataset.Graphical representation of the network was constructed in Cytoscape (Cytoscape 3.8.2) (52), and genes overlapping with MSigDB Hallmark EMT process were colored.

Bleomycin model of lung injury.
The bleomycin model of lung fibrosis was performed as previously described (22).Briefly, female mice at 10-14 weeks of age were anesthetized by isoflurane inhalation and subsequently administered a single dose of bleomycin (Sigma-Aldrich) oropharyngeally at 0.75 U/kg body weight (for IL11 EGFP reporter mice) or 1.5 U/kg body weight (for all other mouse strains) in a volume of saline not exceeding 50 µl per mouse.Uninjured control mice received equal volumes of saline oropharyngeally.Mice were sacrificed at indicated time points post-bleomycin administration and lungs were collected for downstream analysis.
Mouse lung dissociation and FACS analysis.Mice lung dissociation was performed as previously described with slight adjustments (54).Briefly, lungs were perfused with cold sterile saline through the right ventricle.The lungs were then intratracheally inflated with 1.5 ml of Dispase 50 U/ml (Corning) followed by installation of 0.5 ml of 1% low melting agarose (BioRad) via the trachea.The lungs were excised and incubated on an orbital shaker for 45 min at room temperature.Each lobe was then minced into small pieces in DMEM (GIBCO) supplemented with 10% FBS (GIBCO) and 0.33 U/ml DNase I (Roche) and placed on the orbital shaker for an additional 10 minutes.The cells were then filtered through a 100 µm cell strainer and centrifuged at 400 g for 5 min at 4°C.The cell pellet was resuspended in ACK-buffer (GIBCO), incubated for 2 min on ice and then filtered through a 40 µm cell strainer.The cells were centrifuged at 400 g for 5 min at 4°C and resuspended in DPBS (GIBCO) supplemented with 5% FBS, and then stained with the following antibodies: EpCAM-BV785 (BioLegend #118245), CD45-APC (BioLegend, 103112), CD31-APC/Cy7 (BioLegend, 102534), I-A/I-E -AlexaFluor488 (MHC-II) (BioLegend, 107616) and 4', 6-diamidino-2-phenylindole (DAPI) (Life Technologies, 62248) was used to eliminate dead cells.The cells were then sorted on the BD FACS Aria III system (BD Bioscience) and the data was analyzed using FlowJo software (BD Bioscience).
RNA-seq.Total RNA was isolated from HPAEpiC with or without TGFβ1 or IL11 stimulation using RNeasy columns (Qiagen).RNA was quantified using Qubit™ RNA Broad Range Assay Kit (Life Technologies) and assessed for degradation based on RNA Quality Score (RQS) using the RNA Assay and DNA 5K/RNA/CZE HT Chip on a LabChip GX Touch HT Nucleic Acid Analyzer (PerkinElmer).TruSeq Stranded mRNA Library Prep kit (Illumina) was used to assess transcript abundance following standard instructions from the manufacturer.Briefly, poly(A)+ RNA was purified from 1ug of total RNA with RQS > 9, fragmented, and used for cDNA conversion, followed by 3' adenylation, adaptor ligation, and PCR amplification.The final libraries were quantified using Qubit™ DNA Broad Range Assay Kit (Life Technologies) according to the manufacturer's guide.The quality and average fragment size of the final libraries were determined using DNA 1K/12K/Hi Sensitivity Assay LabChip and DNA High Sensitivity Reagent Kit (PerkinElmer).Libraries with 16 unique dual indexes were pooled and sequenced on a NextSeq 500 benchtop sequencer (Illumina) using NextSeq 500 High Output v2 kit and 75-bp paired-end sequencing chemistry.
Colorimetric assays.Detection of secreted IL11 into the supernatant of HPAEpiC cultures was performed using the human IL-11 ELISA kit (R&D systems, D1100) according to manufacturers' instructions.Detection of SFTPD in mouse serum was performed using the mouse SP-D ELISA kit (ab240683) according to the manufacturer's instructions.Total lung hydroxyproline content of the right lobes of mice were measured using the Quickzyme Total Collagen assay kit (Quickzyme Biosciences) as previously described (22).Statistical analysis.Statistical analyses were performed using Graphpad Prism (v9).Analyses were performed using two-tailed Student's t-test, or one-way ANOVA as indicated in the figure legends.For comparisons between multiple treatment groups, P values were corrected for multiple testing using Sidak's test or Tukey's test.P values <0.05 were considered statistically significant.

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STAT5 signaling and p53 pathway (Fig 1G-H and Table and Fig S3D) suggesting a unique role of IL11 in dysfunctional alveolar epithelial cells in PF.
expression was notably upregulated in SFTPC + AT2 cells (Fig 2C-D), PDGFRA + fibroblasts and a subset of CD45 + hematopoietic cells (Fig S5A-B) within injured alveolar regions that were marked by areas of dense consolidation of nuclei DAPI staining.IL11 EGFP was localized to numerous SFTPC + cells adjacent to regions of tissue disruption with an elongated morphology suggestive of AT2-to-AT1 differentiation (Fig 2C).Immunostaining for an AT1 marker Podoplanin (PDPN) revealed that IL11 EGFP expression was very rarely detected in mature AT1 cells in injured or uninjured lungs (Fig 2C).

Fig. 2 .
Fig. 2. IL11 is expressed by KRT8+ transitional cells after bleomycin-induced lung injury in mice.(A) Schematic showing the induction of lung fibrosis via oropharyngeal injection of bleomycin (BLM) in IL11 EGFP reporter mice.(B) Representative flow cytometry analysis and proportions of EGFP + CD31 - CD45 -EpCAM + epithelial cells from uninjured or bleomycin (BLM)-challenged IL11 EGFP reporter mice.P value determined by two-tailed Student's t-test, n = 5 mice.Representative images and quantification of immunostaining for GFP and (C-D) SFTPC or PDPN 7 or 21 days post-BLM challenge.Data are represented as mean ± s.d.P value determined by one-way ANOVA (Tukey's test), n = 3 mice / group.(E) Representative images of immunostaining for GFP and KRT8 after 7 or 21 days post-BLM challenge.

Fig. 3 .
Fig. 3. IL11 induces cytopathic features in alveolar epithelial cells and delays AT2-to-AT1 differentiation in vitro.(A) Experimental design for the 2D culture of primary human pulmonary alveolar epithelial cells.(B) Representative image of immunostaining for KRT8, Fibronectin, Collagen I and SNAIL in human pulmonary alveolar epithelial cells treated with IL11 (5 ng/ml), TGFβ1 (5 ng/ml) in the presence Fig S6D), despite inducing the expression of EMT-related and KRT8 proteins (Fig 2F-G).Additionally, the effects of IL11 on the expression of KRT8 and EMT proteins Collagen I and fibronectin expression was blocked by the ERK inhibitor U0126 (Fig S6E-F), supportive of an important role for IL11-ERK post-transcriptional gene regulation in AT2 cells.In keeping with this, we observed numerous p-ERK + IL11 + tdT + cells within injured regions of lungs from BLMinjured Sftpc-tdT mice (Fig S6G) indicating the activation of IL11-ERK signaling in activated AT2 cells after lung injury that was not apparent in uninjured lungs (Fig S6G).
days prior to BLM-injury and assessed the lungs 21 days post-BLM treatment (Fig 4E).Tamoxifen-exposed Sftpc-CreER; Il11ra1 +/+ mice were used as controls.The deletion of Il11ra1 in AT2 cells from tamoxifen-exposed Sftpc-CreER; Il11ra1 fl/fl mice was verified by qPCR of FACS sorted CD31 - CD45 -EpCAM + MHCII + cells (Fig S7B).Histology assessment of lungs from BLM-injured Sftpc-CreER; Il11ra1 +/+ control mice indicated severe disruption to the lung architecture, increased collagen deposition and higher histopathological fibrosis scores, as compared to uninjured mice (Fig4F-G).These pathologies were significantly reduced in mice where Il11ra1 was deleted in AT2 cells (Fig4F-G).Lung hydroxyproline content was also significantly reduced in mice lacking Il11ra1 specifically in AT2 cells, as compared to controls (Fig4H).

Fig. 4 .
Fig. 4. IL11 signaling in AT2 cells disrupts AT2-to-AT1 differentiation after injury and is required for lung fibrosis.(A) Schematic showing the induction of lung injury in Sftpc-CreER; R26-tdT; Il11ra1 fl/fl or Il11ra1 +/+ mice via oropharyngeal injection of bleomycin (BLM).(B) Immunostaining of KRT8 and PDPN in the lungs of BLM-treated Sftpc-CreER; R26-tdTomato; Il11ra1-floxed or Il11ra1-WT mice.Scale bars: 100 µm.Yellow arrowheads indicate KRT8 + tdT + cells.(C-D) Proportion of KRT8 + tdT + or PDPN + tdT + to tdT + cells (n =3 mice/group).P values were determined by two-tailed Student's t-test.(E) Schematic showing the period of tamoxifen administration and the induction of lung fibrosis via oropharyngeal injection of BLM in Sftpc-CreER; Il11ra1 fl/fl or Il11ra1 +/+ mice.(F) Images of Masson's and Fig S8A).Consistent with the role of IL11-ERK signaling in promoting a dysfunctional KRT8 cell state in vitro, we found numerous p-ERK + KRT8 + cells in the lungs after BLM-injury in IgG-treated mice.The occurrence of p-ERK + KRT8 + cells was reduced in the lungs of X203-treated mice (Fig S8B).Finally, immunostaining for PDPN or AGER revealed that X203 treatment led to significantly enhanced differentiation of lineage labeled cells into AT1 cells (PDPN + tdT + or AGER + tdT + cells) as compared to IgG (Fig 5B and E and Fig S8C-D).

Fig S1 .
Fig S1.IL11 is elevated across mesenchymal and epithelial cell subsets in human pulmonary fibrosis.Violin plot of IL11 expression in individual cell types in scRNA-seq data from control and PF samples in (A-D) Habermann et.al. (GSE135893) and (E-I) Adams et.al. (GSE136831) datasets.Data are further grouped by PF or control in mesenchymal, epithelial, immune (myeloid or lymphoid) or endothelial cell types.

Fig. S2 .
Fig. S2.IL6 and IL11 expression in various epithelial cell types in the human lung.UMAP visualization of IL6 or IL11 expressing single cells in scRNA-seq data from control and PF samples in the (A-C) Habermann et.al. (GSE135893) and (D-F) Adams et.al. (GSE136831) dataset.IL6 or IL11 expressing cells are colored in dark blue in A, B, D, E and colored dots indicate different cell clusters in C and F.

Fig. S3 .
Fig. S3.IL11 is expressed by aberrant basaloid cells in human pulmonary fibrosis.(A) Heatmap showing the transcriptional similarities between selected epithelial cell types from Habermann et.al., (GSE135893) and Adams et.al. (GSE136831) datasets as assessed by the Jaccard index.(B) UMAP visualization of IL11 expressing single cells in the Adams et.al. dataset.IL11 expressing cells are colored in dark blue (left panel) and colored dots indicate cell type clustering (right panel).Cell labels were assigned using the classification from Habermann et.al. by Seurat's FindTransfer Algorithm (see Methods).Blue line indicates differentiation trajectory from transitional AT2 to AT1 cells; red line indicates differentiation trajectory from transitional AT2 to KRT5 -/KRT17 + .Data are composed of PF samples in the Adams et.al. dataset.(C) Expression of IL11 in the pseudotime trajectory from transitional AT2 to KRT5 - /KRT17 + versus from transitional AT2 to AT1 cells.Data are composed from PF samples in the Adams et.al. dataset.(D) Expression of IL11 co-expression module in transitional AT2, KRT5 -/KRT17 + and AT1 cells from control versus PF in combined Habermann et.al. and Adams et.al. datasets.

Fig. S4 .
Fig. S4.Flow cytometry gating of EGFP + cells from IL11 EGFP reporter mice lungs.(A) Representative gating for flow cytometry analysis of lung stromal and epithelial cells isolated from IL11 EGFP reporter mice based on antibody staining for CD31, CD45 or CD326 (EpCAM).Rightmost panel indicates the predetermined threshold for IL11 EGFP+ expression based on the EGFP signal in CD31 -CD45 -CD326 +

Fig. S5 .
Fig. S5.IL11 is upregulated in lung fibroblasts and CD45 + cells after bleomycin-induced lung injury in mice.Immunostaining for GFP and (A) CD45 or (B) PDGFRA in the lungs of IL11 EGFP reporter mice post-BLM injury.White arrowheads indicate marker positive IL11 EGFP+ cells.Scale bars: 50 µm.(C) Immunostaining of lungs from IL11 EGFP reporter mice post-BLM injury with anti-GFP and anti-IL11 antibodies, showing considerable overlap of GFP and IL11 signals.Scale bars: 100 µm.

Fig. S8 .
Fig. S8.Pharmacological inhibition of IL11 promotes AT2 cell proliferation and reduced p-ERK+ KRT8+ cells after bleomycin-induced lung injury.(A) Representative images of immunostaining for Ki67 and PDPN or (B) KRT8 and p-ERK in lung sections from BLM-injured Sftpc-CreER; R26-tdT mice treated with X203 or IgG antibodies.Scale bars: 50 µm in A and 100 µm in B. Yellow arrowheads indicate Ki67 + tdT + cells in panel A or p-ERK + KRT8 + tdT + cells in panel B. (C) Representative images of immunostaining for AGER and (D) the ratio of AGER + tdT + to tdT + cells in lung sections from BLM-injured Sftpc-CreER; R26-tdT mice treated with X203 or IgG antibodies.Scale bars: 100 µm.Data are represented as mean ± s.d.P values were determined by one way ANOVA (Tukey's test), n = 3 -6 mice / group.