*Review* **Emerging Roles of Airway Epithelial Cells in Idiopathic Pulmonary Fibrosis**

**Ashesh Chakraborty, Michal Mastalerz, Meshal Ansari , Herbert B. Schiller and Claudia A. Staab-Weijnitz \***

Member of the German Center for Lung Research (DZL), Institute of Lung Health and Immunity and Comprehensive Pneumology Center with the CPC-M BioArchive, Helmholtz Zentrum München GmbH, 81377 Munich, Germany; ashesh.chakraborty@helmholtz-muenchen.de (A.C.); michal.mastalerz@helmholtz-muenchen.de (M.M.); meshal.ansari@helmholtz-muenchen.de (M.A.);

herbert.schiller@helmholtz-muenchen.de (H.B.S.) **\*** Correspondence: staab-weijnitz@helmholtz-muenchen.de; Tel.: +49-(0)89-3187-4681

**Abstract:** Idiopathic pulmonary fibrosis (IPF) is a fatal disease with incompletely understood aetiology and limited treatment options. Traditionally, IPF was believed to be mainly caused by repetitive injuries to the alveolar epithelium. Several recent lines of evidence, however, suggest that IPF equally involves an aberrant airway epithelial response, which contributes significantly to disease development and progression. In this review, based on recent clinical, high-resolution imaging, genetic, and single-cell RNA sequencing data, we summarize alterations in airway structure, function, and cell type composition in IPF. We furthermore give a comprehensive overview on the genetic and mechanistic evidence pointing towards an essential role of airway epithelial cells in IPF pathogenesis and describe potentially implicated aberrant epithelial signalling pathways and regulation mechanisms in this context. The collected evidence argues for the investigation of possible therapeutic avenues targeting these processes, which thus represent important future directions of research.

**Keywords:** basal cells; bronchial epithelium; airway epithelium; lung fibrosis; MUC5B; single cell RNA sequencing; epithelial populations; IPF

#### **1. Introduction: An Emerging Role of the Airway Epithelium in IPF Aetiology**

Idiopathic pulmonary fibrosis (IPF) is characterized by excessive deposition of extracellular matrix (ECM) within the alveolar compartment of the lung, leading to impairment of gas exchange, increased stiffness and, ultimately, loss of lung function. Despite approval of the two first effective antifibrotic drugs more than six years ago [1,2] and intensive sustained efforts in clinical drug development, IPF remains associated with high mortality rates. Current therapeutic options do not halt disease progression and prevalence of IPF appears to be rising worldwide [3].

The aetiology of IPF is incompletely understood. Traditionally, IPF was believed to be mainly caused by repetitive injuries to the alveolar epithelium. A growing body of evidence, however, based on genome-wide association studies (GWAS), molecular profiling of patient samples, high-resolution micro-CT imaging, and single cell RNA-Sequencing (scRNA-Seq), suggests that IPF equally involves an aberrant response of the bronchial and bronchiolar epithelium, which contributes significantly to disease development and progression. In this review, we summarize known alterations in airway structure, function, and cell type composition in IPF. We furthermore give a comprehensive overview on the genetic and mechanistic evidence pointing towards an essential role of the airway epithelium in IPF pathogenesis. Potential mechanisms of aberrant airway epithelial regeneration and, finally, possible therapeutic avenues targeting these processes are discussed.

**Citation:** Chakraborty, A.; Mastalerz, M.; Ansari, M.; Schiller, H.B.; Staab-Weijnitz, C.A. Emerging Roles of Airway Epithelial Cells in Idiopathic Pulmonary Fibrosis. *Cells* **2022**, *11*, 1050. https://doi.org/ 10.3390/cells11061050

Academic Editors: Malgorzata Wygrecka and Elie El Agha

Received: 1 February 2022 Accepted: 17 March 2022 Published: 19 March 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **2. General Airway Structure**

The lung is structurally and functionally categorized into two regions, the conducting zone and the respiratory zone. The conducting airways consist of the trachea, the bronchi, and the conducting bronchioles, whereas the respiratory zone contains the areas of gas exchange, the terminal (respiratory) bronchioles and the alveoli (Figure 1A). The conducting airways are lined with a pseudostratified epithelium composed primarily of basal, club, goblet and ciliated cells, which play an essential role in the first-line defence against inhaled toxins, particles, and pathogens. Structure and cell type composition of the conducting airway epithelium gradually changes with increasing airway generations from a pseudostratified appearance with mainly ciliated next to secretory and basal cells over a simple columnar to a simple cuboidal epithelium, which harbours fewer ciliated cells and more secretory cells, particularly club cells. In contrast, the alveolar epithelium in the respiratory airways is lined with alveolar type 1 (AT1) and type 2 (AT2) cells, which, together with endothelial cells below and the interjacent basement membrane, make up the blood-air barrier for O2/CO<sup>2</sup> exchange [4,5] (Figure 1A). Basal cells are established as the main human progenitor cells for all cell types in the pseudostratified epithelium lining the conducting airways [6] while AT2 cells give rise to AT1 cells in the alveoli [7]. More recently, in the murine lung, the bronchoalveolar duct junction at the transition between bronchioles and alveoli has been described to harbour additional multipotent stem cells, the so-called bronchoalveolar stem cells (BASCs). These can give rise to club and ciliated cells on the one hand and AT1 and AT2 cells on the other hand, in particular in response to injury [8]. Whether such a population exists in the human lung, however, is unclear to date.

**Figure 1.** Schematic overview of airways in healthy lung and idiopathic pulmonary fibrosis (IPF). (**A**) Airways in the healthy lung, depicting normal cell type distribution in the proximal and distal

airways as well as in the bronchioalveolar duct junction. (**B**) Airways in the IPF lung, depicting dilated bronchioles, impaired mucociliary clearance and the thickened basement membrane in the distal airways, two types of honeycomb cysts (HC, mucociliary, basaloid), and accumulation of extracellular matrix (ECM) in the alveolar region. AT1, alveolar cell type 1; AT2, alveolar cell type II; ECM, extracellular matrix; SMC, smooth muscle cell. Figure was created with biorender.com.

#### **3. Changes in Airway Morphology in IPF**

#### *3.1. Airway Dilation*

In recent years, multiple evidence has emerged that strongly argues for considerable changes in airway morphology and physiology in IPF, which contribute to disease progression. For instance, clinical CT findings in IPF patients as well as experimental micro-CT imaging of explanted IPF lungs demonstrate that proximal and distal airways are dilated [9–12], which may explain why FEV1/FVC ratios for IPF patients are higher than expected [13,14]. This is in agreement with aerosol-derived airway morphometry and capnographic measurements, which equally show increased airway volumes in IPF patients [15,16]. While changes in conducting airway volumes seem independent of disease severity [16], they appear to facilitate the distinction between stable and progressive disease, hence bear prognostic value [9]. The underlying mechanisms for airway dilation in IPF are not fully understood. Traditionally, traction bronchiectasis and bronchiolectasis, caused by increased collagen deposition and contraction of the peripheral fibrotic areas, have been thought to "pull open" the bronchi and bronchioles, respectively [17,18]. This concept is supported by the observation that the quantity of fibroblast foci correlates with traction bronchiectasis in high-resolution CT (HRCT) scans [19]. However, considering the comparatively distant location of fibrotic areas relative to the affected airways in IPF, and recent findings on emerging proliferative epithelial cell type populations in IPF (discussed below in Section 4), it has been suggested that the HRCT pattern of traction bronchiectasis in IPF is rather caused by bronchiolar proliferation than by mechanical traction alone [20].

#### *3.2. Increased Airway Wall Thickness (AWT)*

Recent studies report increases in airway wall thickness (AWT). Verleden et al. performed clinical CT and micro-CT of IPF explant and donor lungs, in combination with matched histological examinations. The authors observed that, due to increased AWT, more small airways are visible in CT scans of IPF specimens [21], a finding which was very recently confirmed by Ikezoe et al. [12] (Figure 2A). Additionally, a retrospective analysis of clinical chest CT images by Miller et al. suggested that lungs of IPF patients display significant increases in AWT, notably already in early disease stages [22]. Here, the authors performed so-called Pi10 measurements, which rely on a series of experimental determinations of total airway and luminal airway areas at different luminal perimeters. For each patient, the airway wall areas are calculated by subtraction of the luminal airway from the total airway area and the square root of these values is plotted against the perimeter. Regression analysis allows for the determination of the airway wall thickness of a hypothetical airway with an internal perimeter of 10 mm, the Pi10, a measure, which can then be directly compared between patients and disease cohorts. Interestingly, and explicitly mentioned by the authors as a limitation of their study, the way Pi10 is determined implies that changes in the internal luminal area, e.g., altered mucus layers, may have impacted the findings. As altered mucociliary clearance and increased MUC5B expression indeed are important features of IPF airways (discussed in Section 5), this raises the question whether Pi10 measurements are affected by increased levels of airway MUC5B, for example.

accessed 8/03/2022) α α **Figure 2.** Airway epithelial abnormalities in IPF. (**A**) Comparison of airway features in control and IPF lungs as monitored by computed tomography (CT, adapted from Ikezoe et al. [12] with permission of the American Thoracic Society). Computed tomography (CT) scans from lungs of a control subject (upper row) and a case of IPF (lower row). The panels show from left to right: (1) Axial midslice multidetector computed tomography (MDCT) scans indicating where a random tissue sample was obtained for microCT (red circles); (2) reconstructed airway tree for the same scan from the lateral perspective; (3) midslice microCT scans of the tissue sample circled in red; (4) small airway tree segmentations obtained from the microCT scans visualized in three dimensions, identifying terminal bronchioles (TB, white arrowheads) and transitional bronchioles (asterisks); (5) representative crosssectional image of the terminal bronchiole (TB) highlighted by the yellow arrowhead. This figure panel is adapted from Ikezoe et al. [12] with permission of the American Thoracic Society. Copyright © 2022 American Thoracic Society. All rights reserved. The American Journal of Respiratory and Critical Care Medicine is an official journal of the American Thoracic Society. Readers are encouraged to read the entire article for the correct context at https://www.atsjournals.org/doi/10.1164/rccm.20 2103-0585OC (last accessed 8 March 2022). The authors, editors, and The American Thoracic Society are not responsible for errors or omissions in adaptations. (**B**) Immunofluorescent stainings of serial lung sections of a representative control subject (upper row) and a case of IPF (lower row) with mouse isotype control antibody (mIgG1) and antibodies directed towards keratin 5 (KRT5), keratin 14 (KRT14), club cell-specific protein 10 (CC10), α-smooth muscle actin (α-SMA) as a marker for smooth muscle cells and myofibroblasts, and type I collagen (Coll I). Scale bar 100 µm.

#### *3.3. Bronchiolar Abnormalities*

Bronchiolar lesions involving abnormal bronchiolar proliferation and migration are typical features of IPF and represent regions of injury and active regeneration [23–25]. While the observed increase in bronchiolar proliferation has been interpreted to result in an increased number of bronchioles in IPF [14,23], recent evidence based on micro-CT imaging and histology suggests it more likely leads to dilation and distortion of the small airways [10–12,21] (Figure 2A). In contrast, the number of terminal bronchioles is even reduced in IPF [10,12,21]. Importantly, the latter observation was made in areas of mild fibrosis and the number of terminal bronchioles did not further decline in areas with more severe fibrosis, indicating that loss of terminal bronchioles is an early event in IPF [21]. In addition, it was demonstrated in two very recent independent studies that loss of terminal bronchioles correlates with honeycomb formation and that conducting airways directly lead into honeycomb cysts [10,12]. In agreement, early studies have demonstrated that peripheral cystic air spaces are ventilated, but represent physiological dead-space because they are not perfused [26]. This supports the concept that small airways are the origin of honeycomb cysts, abnormal peripheral airway spaces that will be discussed in more detail in the following.

#### *3.4. Honeycomb Formation and Bronchiolization*

In thoracic radiology, the term "honeycombing" refers to clustered cystic airspaces which typically are located in the subpleural region of the lung [27]. While clinical HRCT only detects honeycomb cysts with a diameter of about 1 mm and bigger, smaller honeycomb cysts are usually observed in histology [28]. Typical microscopic honeycomb cysts in IPF are small, subpleural, and localized in vicinity to fibrotic areas. Figure 2B (lower row, IPF) shows a collapsed honeycomb cyst characterized by KRT5<sup>+</sup> KRT14<sup>+</sup> CC10<sup>−</sup> cells in close proximity to fibroblast foci. On a cellular level, these honeycomb cysts are characterized by p63<sup>+</sup> KRT5<sup>+</sup> airway epithelial-like cell types replacing the normal alveolar epithelium, a process termed bronchiolization [29]. Some honeycomb cysts appear to be composed of stratified layers of hyperplastic p63<sup>+</sup> KRT5<sup>+</sup> KRT14<sup>+</sup> cells [25] (e.g., Figure 2B), while others display a pseudostratified mucociliary epithelium, containing ciliated, p63<sup>+</sup> KRT5<sup>+</sup> basal, and goblet cells expressing *MUC5B* as the main mucin component [25,30,31]. Whether honeycomb cysts derive from the small airways or from the alveolar epithelium as a result of ectopic bronchiolar differentiation is still controversially discussed. Considering the current knowledge about epithelial progenitor cells in the distal lung, bronchiolization could be a result of AT2 cells committing to an aberrant differentiation program [7], or derive from migrating basal cells [6] or BASCs [8,32] originating from the small airway or of bronchoalveolar duct junction, respectively. BASCs, at least in the mouse, can give rise to AT2 and club cells upon injury [32], but there is, to the best of our knowledge, no evidence that they can give rise to p63<sup>+</sup> KRT5<sup>+</sup> basal cell-like populations, which most frequently line bronchiolized areas in the IPF lung [23,25,30,33]. This, in contrast, has been unambiguously demonstrated for airway stem cells in distal lung regeneration after injury: After influenza infection of mice, for example, p63<sup>+</sup> cells emerge in the bronchioles and form extra-bronchiolar parenchymal clusters of p63<sup>+</sup> Krt5<sup>+</sup> basal cells, despite of little *TP63*-expression in normal murine bronchioles [34,35]. Lineage tracing experiments performed in independent laboratories have demonstrated that these cells derive from a rare population of SOX2<sup>+</sup> p63<sup>+</sup> Krt5+/<sup>−</sup> progenitor cells, but not from alveolar epithelial cells or BASCs [34,36,37]. Hence, studies in mouse models of lung injury have argued against an alveolar origin of bronchiolized areas in IPF and rather suggested that bronchiolization may originate from the airways.

However, it is important to mention in this context, that studies in human organoid culture systems have provided compelling evidence that, in contrast to mouse AT2 cells, human AT2 cells can give rise to Krt5<sup>+</sup> basal cells. This differentiation capacity into KRT5<sup>+</sup> basal-like cells was strictly dependent on adult human lung mesenchymal cells (AHLM) as feeder cells. The resulting Krt5<sup>+</sup> basal cells expressed canonical basal cell markers (*SOX2*, *TP63*) in addition to genes typically associated with aberrant basal epithelial populations in IPF [38]. Interestingly. scRNA-Seq analysis of AHLM further revealed that during organoid culture mesenchymal subpopulations emerge that resemble such enriched in IPF lung tissue [38]. Collectively, these findings indicate that pathological mesenchymal cells in IPF generate a niche that is supportive of aberrant differentiation of human AT2 cells into KRT5<sup>+</sup> basal cells. Whether this is what happens in IPF, too, remains elusive, but it is plausible that aberrant basal cells in IPF derive from both airway and alveolar epithelial cells.

In summary, airways are drastically altered in IPF, with changes that (1) include macroscopic morphological changes visible by clinical and experimental CT imaging (airway dilation, increased airway wall thickness, honeycomb cysts), (2) manifest in physiological parameters like increased dead-space ventilation and higher FEV1/FVC ratios, and (3) involve repopulation of the injured alveolar region with basal-like epithelial cells, which may be both airway- and alveolar-derived (Figures 1B and 2). On a cellular level, recent scRNA-Seq analyses of IPF lungs have provided even more weight to the importance of airway-like cells in IPF and will be discussed in the following chapter.

#### **4. Recent Insights from Single Cell RNA-Sequencing (scRNA-Seq) Studies**

Since the advent of single-cell RNA sequencing (scRNA-Seq), several studies in the past five years have revolutionized the concept of epithelial cell populations in IPF. In the earliest study, Xu et al. isolated Epcam+/HTII-280<sup>+</sup> cells from peripheral regions of control and IPF lung and subjected that cell population to scRNA-Seq. Initially, they found that the yield of Epcam+/HTII-280<sup>+</sup> cells, classically reflecting AT2 cells, drastically decreased in IPF lungs. However, more interestingly, in IPF, Epcam+/HTII-280<sup>+</sup> subpopulations emerged which expressed transcripts typically associated with conducting airways and extracellular matrix-expressing cells, at the expense of genes typically associated with AT2 function [39]. Overall, the authors identified four subpopulations of Epcam+/HTII-280<sup>+</sup> cells in IPF including (1) normal AT2 cells, (2) cells which expressed Goblet cellspecific markers, (3) cells which expressed basal cell-specific markers, and (4) indeterminate cells, which expressed multi-lineage markers including such for AT2, AT1, conducting airway cells and mesenchymal cells, and could thus not unambiguously be assigned to one cell type. Remarkably, the latter often co-expressed *SOX2* and *SOX9*, genes that typically define proximal airway progenitor and distal airway progenitor cells in the adult lung, respectively, thus indicating a loss of proximal-distal patterning in the IPF lung. Notably, SOX2+/SOX9<sup>+</sup> progenitor cells otherwise only emerge in human lung development during the pseudoglandular stage in the distal epithelium but are already lost in the canalicular stage [40]. In addition, a more recent study suggests that surfactant processing is lost in these newly emerging epithelial cell populations, adding an important functional outcome of these changes [41]. Hence, in summary, in IPF a drastic loss of normal AT2 cells is paralleled by an increase of conducting airway characteristics in peripheral alveolar epithelial cells and an activation of aberrant differentiation programs or possibly reactivation of early lung developmental programs.

While the study above analyzed sorted Epcam+/HTII-280<sup>+</sup> cells, isolated from a limited number of control and IPF lungs (*n* = 3), four more recent studies analyzed single cell suspensions from more specimens, without prior experimental enrichment for epithelial cells [42–45]. For visualization of the most important and consistent findings regarding epithelial cell populations in IPF/interstitial lung disease (ILD), we generated an integrative data set comprising all four studies (Figure 3A–C) using the Scanpy package (v1.8.0) [46]. To address potential batch effects, the integration was performed as described in Mayr et al. [43]. Briefly, the publicly available raw count matrices were re-processed data set wise with the same procedure. To mitigate effects of background mRNA contamination, the matrices were corrected by using the function adjustCounts() from the R library SoupX [47]. The expression matrices were normalized with scran's size factor based approach [48], log transformed via scanpy's pp.log1p() and finally scaled to unit variance

and zero mean before concatenating them. A shared set of variable genes was selected by calculating gene variability patient-wise (flavor = "cell\_ranger", n\_top\_genes = 4000) and excluding known cell cycle genes. The intersection of the variable genes across all data cohorts was used as input for principal component analysis (1311 genes). After subsetting to the epithelial cell populations, the BBKNN method [49] was used to generate a batch balanced data manifold (Munich: ILD = 7, controls *n* = 12; Chicago: ILD *n* = 9, controls *n* = 8; Nashville: ILD *n* = 20, controls *n* = 10; and New Haven: ILD = 32, controls *n* = 22). Cell type identities from the original publication were retained and harmonized across studies. All four studies consistently confirmed the concept of an emerging diverse repertoire of epithelial cell types in ILD including IPF, most strikingly an increase in cells with features of conducting airways at the expense of classical alveolar epithelial cells (Figure 3D).

**Figure 3.** Single cell RNA-Sequencing has revealed drastic changes in epithelial cell populations in ILD. (**A**) Uniform Manifold Approximation and Projection (UMAP)-based dimension reduction of single cell transcriptomic data to delineate epithelial cell types, labelled by cell type. (**B**) Same UMAP visualization labelled by ILD cohort. Data used for visualization was derived from in total four datasets [42–45] of control and interstitial lung disease (ILD) samples: New Haven [45], Nashville [44], Chicago [42], and Munich [43]. (**C**) Same UMAP visualization labelled by disease. (**D**) Relative frequencies of epithelial cell populations demonstrate a consistent increase in conducting airway cell populations in ILD at the expense of alveolar type 1 (AT1) and 2 (AT2) cells. ab., aberrant.

In more detail, up to 10 distinct clusters of epithelial cells were defined in these studies. While all identified most classical epithelial cell types, i.e., AT1, AT2, basal, ciliated, and secretory cells by similar expression signatures (Figure 4), there are some differences in subcategorization of the described cell type clusters. For instance, while Habermann et al. [44] distinguished between ciliated cells and differentiating ciliated cells, such a distinction was not made in the other studies [42,45]. Furthermore, categorization of secretory cells differs significantly between these reports. Reyfman et al. categorized club cells based on *SCGB1A1* (also termed *CC10* or *CCSP*) expression and did not report goblet cells but *MUC5B*-expressing cells within their cluster of club cells [42]. Adams et al. distinguished between club and goblet cells, but in their report *SCGB1A1* expression is a characteristic of both cell types and club and goblet cells are differentiated from each other by *SCGB3A2* and *MUC5B* expression, respectively [45]. Published and unpublished results from our lab have shown that *SCGB1A1* is expressed by a subpopulation of MUC5AC<sup>+</sup> goblet cells, too [50]; so indeed, *SCGB1A1* should rather be considered a more general marker for secretory cells

than specifically for club cells. Possibly reflecting similar considerations, Habermann et al. refrained from the attempt to distinguish between club and goblet cells and instead defined several secretory cell type clusters based on expression of *SCGB1A1, SCGB3A2,* and *MUC5B* and combinations thereof. Collectively, these studies show that, at least based on single cell transcript analysis, there is a continuum of secretory cells with overlapping gene expression patterns, which are not easily sorted into club and goblet cells without information on cell shape, spatial distribution within the bronchial tree, and protein expression patterns. Therefore, here, we also refer to those as secretory cells, without further distinction into goblet and club cells (Figures 4 and 5). Independent of secretory cell subcategorization, all studies consistently demonstrate an increase in secretory cells including MUC5B<sup>+</sup> cells. This was equally observed in an independent scRNA-Seq study where the authors refer to SCGBB1A1<sup>+</sup> MUC5B<sup>+</sup> cells as club cells, which, as explained above, may not be entirely accurate due to the ambiguity of SCGBB1A1 as a marker in that context. Still, also this study clearly demonstrates an increase of secretory cells in IPF relative to the healthy lung [51]. Furthermore, beyond quantitative alterations in epithelial cell populations, all IPF/ILD airway subpopulations displayed many significantly upregulated genes in their expression signatures when compared to their healthy counterparts (Figure 5B).

**Figure 4.** Cell type-specific markers for epithelial cell populations in ILD derived from scRNA-Seq data. Using the data set described in Figure 3, the top 5 specific markers for the described epithelial populations are plotted, (**A**) ranked by adjusted *p*-value or (**B**) ranked by log fold changes of relevant cell type vs. all other epithelial cell types. pct., percentage; avg. expr., average expression; ab., aberrant.

Basal cells appear to be particularly important in the context of IPF aetiology and progression for several reasons. For instance, a basal cell signature detected in the bronchioalveolar lavage transcriptome in IPF patients was predictive of mortality, strongly suggesting that basal cells play a central role in IPF progression [31]. Basal cell numbers are drastically increased in ILD (Figure 3D) and novel basal cell subpopulations and characteristics have already been demonstrated before the scRNA-Seq era. In 2015, Jonsdottir et al. reported that p63<sup>+</sup> KRT14<sup>+</sup> cells overlay fibroblastic foci in IPF (see also Figure 2B) and displayed characteristics of epithelial-to-mesenchymal transition (EMT) [52]. Shortly after, using immunofluorescence studies, Smirnova et al. quantified KRT5<sup>+</sup> and KRT14<sup>+</sup> basal cell population in healthy and IPF lungs and equally observed a drastic increase of basal cell populations in the distal IPF lung and proposed KRT14<sup>+</sup> as a marker for an aberrantly differentiating progenitor cell pool [25]. The above-mentioned scRNA-Seq studies confirm

these findings, showing that *KRT14* is overexpressed in basal cells in ILD, and also a marker of aberrant basaloid cells, which will be described below [42,44,45].

last accessed 22/12/2021 **Figure 5.** Epithelial cell populations show distinct expression changes in ILD. Using the data set described in Figure 3, differential gene expression analysis was performed with diffxpy (https: //github.com/theislab/diffxpy, last accessed 22 December 2021) while accounting for number of transcripts per cell and patient cohort. The top 50 deregulated genes in specific subpopulations of epithelial cells are given, ranked by log2 fold change. (**A**) Top 50 genes induced in aberrant basaloid cells relative to gene expression of all other healthy epithelial cell types. (**B**) Top 50 genes increased in ILD in other airway epithelial cell populations. pct., percentage; avg. expr., average expression; ab., aberrant.

A recent scRNA-Seq study focussed on changes in basal cell plasticity in IPF and defined basal cell heterogeneity in the normal and IPF lung in greater detail [53]. According to this study, basal cells in the healthy lung can be subdivided in at least four subpopulations, namely classical multipotent basal cells (MPB), proliferating basal cells (PB), secretory-primed basal cells (SPB), and activated basal cells (AB). Based on scRNA-Seq data, surface marker screening, as well as bronchosphere assays, the authors established CD66 as a surface marker for SPBs and demonstrated an increase of CD66<sup>+</sup> KRT5<sup>+</sup> SPBs in IPF. With the importance of MUC5B and thus secretory airway cells in disease aetiology, these observations put forward modulation of basal cell priming as a novel therapeutic strategy in IPF [53].

− Interestingly, Habermann et al. as well as Adams et al. identified a novel epithelial cell population with features of basal cells, which exclusively emerged in pulmonary fibrosis, namely KRT5−/KRT17<sup>+</sup> epithelial cells [44], or aberrant basaloid cells [45]. These cells are comparably rare (Figure 3D) and characterized by expression of basal cell markers like *TP63*, *KRT17*, *LAMB3*, and *LAMC2* (but not *KRT5*, see Figure 4), in combination with mesenchymal markers like *COL1A1*, *VIM, TNC*, and *FN1,* and markers of senescence like *CDKN1A* (Figure 5A) [44,45]. Expression of *SOX9* and other markers of a distal differentiation program suggested that these cells also display characteristics of alveolar epithelial cells. Furthermore, these cells also showed the highest expression levels of *MMP7*, encoding

matrix metallopeptidase 7, the probably best-validated peripheral blood biomarker for IPF (Figure 5A). Using RNA in situ hybridization, KRT17+/COL1A1<sup>+</sup> basaloid cells were shown to cover fibrotic foci in IPF lungs but were not detected in non-fibrotic controls [44]. Given that these cells display characteristics of conducting and respiratory airways, the cellular origin is not clear. ScRNA-Seq-based pseudo-time analysis has raised the possibility that both transitional AT2 and *SCGB3A2*-expressing secretory cells may act as precursors for aberrant basaloid cells [43,44], a hypothesis which still requires experimental validation. Notably, studies in mouse models of lung fibrosis and injury have identified similar converging differentiation pathways, namely from club cells on the one hand and AT2 cells on the other to a population called Krt8<sup>+</sup> alveolar differentiation intermediate (ADI) cells. This cell population is highly similar to the aberrant basaloid cells in IPF [54], but of transient character in bleomycin-induced lung fibrosis: Krt8<sup>+</sup> ADI cells peak in the fibrotic phase and gradually disappear during resolution of fibrosis. Importantly, lineage tracing using Sox2- and Sftpc-Cre drivers has confirmed the dual, conducting airway and alveolar, origin of Krt8<sup>+</sup> ADI cells. Collectively, this supports a model where an intermediate cell type, transiently emerging during a normal repair process, accumulates and persists in IPF.

In summary, scRNA-Seq studies have consistently demonstrated drastic changes in epithelial subpopulations in ILD, which strongly argue for an essential role of airway epithelial cells in disease development and progression. These include: (1) A dramatic decrease of normal alveolar cell types of the respiratory zone and their replacement by diverse conducting airway cell populations (Figure 3D). (2) The emergence of a novel ILD-specific cell type reminiscent of an intermediate cell involved in normal alveolar repair, which probably derives from both proximal and distal precursors and persists in lung fibrosis (Figures 3D and 4). (3) Considerable changes in overall gene expression patterns in epithelial cell types (Figure 5).

#### **5. Changes in Airway Function**

#### *5.1. Mucociliary Clearance*

The discovery of the *MUC5B* polymorphism (see below, Section 6) has drawn a lot of attention to dysregulated mucociliary clearance as a major aetiological mechanism in IPF [29]. IPF is characterized by increased expression of *MUC5B* in the distal airways and honeycomb cysts. Increased expression is often driven by the minor allele (T) of the risk single nucleotide polymorphism (SNP) rs35705950, which is overrepresented in IPF patients. Consequently, the mucin MUC5B accumulates in airways of the distal lung where even mucous plugs can be observed within microscopic honeycomb cysts [55]. From other lung diseases, most prominently cystic fibrosis, it is very well known that overproduction of mucus impairs mucociliary clearance, leads to accumulation of particles and pathogens in the airways and increases the risk for chronic injury and inflammation. Indicating that this likely applies to lung fibrosis as well, *MUC5B* overexpression in distal airways has been shown to significantly impair mucociliary clearance and aggravate lung fibrosis in the mouse model of bleomycin-induced lung injury [56]. Importantly, in the same model, mucolytic treatment led to clearance of inflammatory cells from the lungs and counteracted the production of fibrillar collagen, providing proof-of-concept that restoring impaired mucociliary clearance may be beneficial in prevention and treatment of pulmonary fibrosis [56].

A potential key role of impaired mucociliary clearance for lung fibrogenesis is further emphasized by an independent study, where the issue of mucociliary clearance was approached from a very different angle. The E3 ubiquitin-protein ligase NEDD4-2 targets the epithelial Na<sup>+</sup> channel (ENaC, encoded by *SCNN1A*) for intracellular degradation and thus plays a key role in limiting the levels of active ENaC at the cell surface. ENaC in turn is a critical regulator of epithelial surface hydration and consequently affects mucus properties. Overexpression of *SCNN1A* and activation of ENaC increases transepithelial transport of salt and water leading to dehydration of the apical epithelial mucous layer and thus impaired mucociliary clearance [57]. NEDD4-2 levels are decreased in IPF airways. With NEDD4-2 representing an antagonist of ENaC, conditional deletion of NEDD4-2 from airway epithelial cells in mice, as expected, increased ENaC activity and significantly impaired mucociliary clearance. A striking long-term consequence of this NEDD4-2 deficiency in murine airways, however, was the development of patchy lung fibrosis, bronchiolar remodelling, and increased MUC5B production in the peripheral airways, all features strongly reminiscent of IPF and actually reflecting IPF pathology more accurately than the most commonly used bleomycin-induced mouse model of lung fibrosis [58]. Collectively, these findings strongly indicate that mucociliary dysfunction is a major aetiological factor in IPF and, even though the minor risk allele within the *MUC5B* promoter will probably remain the most important cause, may have multiple origins including, e.g., dysregulation of epithelial surface hydration properties by NEDD4-2/ENaC.

#### *5.2. Epithelial Barrier Dysfunction in IPF Pathogenesis*

The bronchial epithelial barrier plays an important role in protecting the airways against environmental insults not only via mucociliary clearance and production of antimicrobial substances to eliminate inhaled pathogens, but also by tight junctions that maintain the cell–cell contact and regulate paracellular permeability [59]. Even if this has not been comprehensively assessed, some reports suggest that epithelial barrier function is altered during IPF pathogenesis. Zou et al., for instance, have demonstrated by immunohistochemistry (IHC) stainings for several tight junction proteins, that specifically levels of claudin-2 were elevated in IPF bronchiolar regions [60]. Others have found that levels of protein kinase D (PKD), a negative regulator of airway barrier integrity [61], were increased in IPF bronchiolar epithelium relative to normal lung tissue sections [62].

#### *5.3. Other Changes in Airway Function*

In a study designed to investigate the pathogenesis of cough in IPF, authors found increased levels of nerve growth factor and brain-derived neurotrophic factor in induced sputa of IPF patients compared to healthy control subjects [63]. These results indicated functional upregulation of sensory neurons in the proximal airways of IPF lungs.

#### **6. Genetic Evidence Indicating Involvement of Bronchial Epithelium in IPF**

IPF is a multifactorial disease where the interplay between environmental exposure and genetic susceptibility plays a central role in disease pathogenesis. Genome-wide association studies (GWAS) on large cohorts of various ethnical backgrounds have provided interesting insights into genetic susceptibility for IPF development and have linked specific genetic variants to poorer outcomes in sporadic IPF and familial pulmonary fibrosis [64]. In this context, single nucleotide polymorphisms (SNPs) conferring a higher risk for IPF were discovered in several genes reported to be expressed in airway epithelial cells, strongly suggesting a role for bronchial and bronchiolar epithelial cells in IPF aetiology [65,66]. These include mucin-5B (*MUC5B*), toll interactive protein (*TOLLIP*), desmoplakin (*DSP*), family with sequence similarity 13 member A (*FAM13A*), and A kinase anchor protein 13 (*AKAP13*). For all but *TOLLIP*, which seems comparably little expressed in airway epithelial cells, scRNA-Seq data confirms variable expression of these genes in bronchial, bronchiolar, and aberrant basaloid cells (Figure 6). While *MUC5B* and *FAM13A* are particularly expressed by secretory cells and ciliated cells, respectively, *DSP* is expressed by all bronchial and bronchiolar epithelial cell types including aberrant basaloid cells, where it is one of the top overexpressed genes relative to all other healthy epithelial cell types (Figure 5A). In contrast, except for *AKAP13*, expression of which is enriched in AT2 and aberrant basaloid cells, alveolar epithelial cells show relatively little expression of these genes (Figure 6).

**Figure 6.** Expression of selected risk factor genes in epithelial cell populations. Using the data set described in Figure 3, expression of selected genes harbouring IPF risk-associated SNPs is given. Selection was based on previous reports on their expression in airway epithelium (see text for more details). pct., percentage; avg. expr., average expression; ab., aberrant.

#### *6.1. MUC5B*

A common promoter SNP in the airway gene *MUC5B* on chromosome 11, rs35705950, is the strongest risk factor for IPF, accounting for 30–35% of the overall risk to develop IPF [29,55]. *MUC5B* encodes mucin-5B, a mucin protein predominantly expressed in serous cells of submucosal glands in healthy lungs, and normally little expressed in airway surface epithelium [67]. In contrast, in IPF lungs, *MUC5B* is overexpressed in secretory cells within honeycomb cysts as well as in bronchioalveolar regions [29,30,39]. A series of elegant in vivo work has demonstrated that overexpression of *MUC5B*, both in proximal and distal airways, aggravates bleomycin-induced lung fibrosis in mice, while MUC5B-deficient mice are protected from the development of lung fibrosis. Interestingly, increased mortality was particularly observed when *MUC5B* was overexpressed in the distal murine airways [56].

#### *6.2. TOLLIP*

The gene *TOLLIP* encodes a ubiquitous protein with essential functions in the innate immune response, epithelial survival, defence against pathogens and further biological processes [68,69]. The *TOLLIP* gene is located adjacent to *MUC5B* and evidence regarding linkage disequilibrium between the *MUC5B* SNP rs35705950 and *TOLLIP* SNPs suggests that *TOLLIP* and *MUC5B* SNPs may not be passed on independently [68]. Three common variants within the *TOLLIP* locus (rs111521887, rs5743894, rs574389) have been shown to associate with higher susceptibility for IPF [66]. The minor alleles for all *TOLLIP* SNPs result in reduced expression by 20–50%, with rs111521887 and rs5743894, which are in high linkage disequilibrium, having stronger effects on expression than rs5743890 [66]. Interestingly, even though all result in reduced expression, the clinical effects of the rs111521887 and rs5743894 minor alleles are opposite to the rs5743890 minor allele: Individuals who carry the minor allele for rs111521887 and rs5743894 are more susceptible to developing IPF, while the minor allele rs5743890 is associated with less susceptibility. However, despite this initial protective effect, mortality in IPF patients with this variant is actually increased [66,68]. In the integrative scRNA-Seq data set that we examined, *TOLLIP* overall was comparably little detected (Figure 6). However, a recent study focusing on *TOLLIP* expression in the lung has demonstrated *TOLLIP* expression in AT2 cells, basal cells, and aberrant basaloid cells, but at the same time reported a global downregulation of *TOLLIP* expression in the IPF lung [70].

#### *6.3. DSP*

Linking intermediate filaments to the plasma membrane, desmoplakin, encoded by *DSP*, is a critical intracellular component of desmosomes, cell–cell adhesive junctions, which are critical for tissue integrity [71]. In the lung, *DSP* is primarily expressed in bronchi and bronchioles, with comparably little expression in alveoli [72]. The latter is also reflected by the scRNA-Seq data shown here (Figure 6). GWAS have linked at least two genetic variations in *DSP* with risk for IPF development, namely the minor alleles of rs2076295 and

rs2744371 [65,72]. Among those, the minor allele of the intronic SNP rs2076295 (intron 5) is established as the strongest causal factor and is associated with an increased risk for IPF development, while the minor allele of rs2744371 confers a protective effect against IPF onset. Paradoxically, while *DSP* expression is increased in IPF lungs, the risk allele rs2076295 correlates with lower *DSP* expression. Some well-designed in vitro experiments using CRISPR/Cas9 gene editing in human bronchial epithelial cells have shown that deletion or disruption of the DNA region spanning rs2076295 as well as introduction of the minor allele (G) led to decreased expression of *DSP*, in agreement with an enhancer function of this region in intron 5 [73]. Decreased *DSP* expression in turn resulted in reduced barrier integrity, enhanced cell migration, and increased expression of markers for EMT and of ECM genes [73].

#### *6.4. FAM13A*

*FAM13A* encodes a so far uncharacterized protein with largely unknown function. Amino acid sequence homology suggests that FAM13A contains a Ras homologous (Rho) GTPase-activating protein (GAP) domain and hence a function in Rho GTPase signalling [74]. In the lung, *FAM13A* is primarily expressed in bronchial epithelial cells, but also by AT2 cells, and macrophages [75,76]. GWAS have identified a genetic risk variant within this gene, intronic rs2609255, that increases susceptibility for COPD and IPF with opposite risk alleles [65,77]. For IPF, this risk variant appears not to be associated with expression changes on transcript level [65]. Owing to its association with COPD and IPF disease risk, experimental studies have been performed in both disease contexts. These studies suggest that, on the one hand, FAM13A, protein levels of which are increased in COPD, may protect from cigarette smoke-induced disruption of airway integrity and neutrophilia [75], but at the same time promote β-catenin degradation, thus inhibit β-catenin signalling and associated repair processes, and increase susceptibility to emphysema [76]. On the other hand, FAM13A deficiency has been reported to exacerbate bleomycin-induced lung fibrosis in the mouse, possibly via induction of EMT-related gene expression [78]. Overall, FAM13A, even though its exact function remains unclear, appears to play an important role in airway epithelial barrier integrity and repair.

#### *6.5. AKAP13*

*AKAP13*, encoding A kinase anchor protein 13, is another gene with a genetic variant, rs62025270, conferring increased risk for development of IPF [79], expression of which is largely confined to the airway epithelium [80]. *AKAP13* is overexpressed in IPF where it localizes to aberrant epithelial regions [79] and functions as a Rho guanine nucleotide exchange factor regulating activation of RhoA [81], known for its involvement in profibrotic pathways.

#### **7. Implicated Mechanisms**

The precise pathogenesis of IPF is still not entirely understood, but the current knowledge on environmental and genetic risk factors strongly suggests epithelial injury-triggered reactivation of developmental pathways which, ultimately, leads to aberrant repair and regeneration resulting in drastic changes in lung structure and function. Therefore, in the following we will recapitulate these processes with a focus on what is known for the contributions of the bronchial and bronchiolar epithelium.

#### *7.1. Types of Epithelial Injury*

The airway epithelium represents the first line defence against inhaled particles, pathogens, and toxicants. Environmental and occupational triggers like cigarette smoke, wood dust, metal dust, pesticides, and herpesvirus infection are established risk factors for IPF [82,83]. Additionally, inhalation of traffic-related air pollutants has been linked to increased incidence of IPF [84]. Furthermore, gastroesophageal reflux (GER) is an overrepresented comorbidity of IPF, suggesting that microaspiration of stomach acids increases

risk for IPF. Moreover, treatment of GER in IPF patients decelerates IPF disease progression and improves survival, indicating that GER also influences disease progression [82,83].

#### *7.2. Epithelial Apoptosis*

Apoptosis of alveolar epithelial cells is a well-established phenomenon in IPF and clearly reflected by the above discussed scRNA-Seq data showing a drastic decrease in normal alveolar type I and II cells in IPF relative to control lung tissue (Figure 3D). Immunofluorescent stainings of pro- and anti-apoptotic proteins in combination with terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) stainings for DNA strand breaks have revealed that bronchiolar epithelial cells, hyperplastic epithelial cells and epithelial cells lining honeycomb cysts in the lungs of IPF patients show distinct signs of ongoing apoptosis [85–88]. While such cells in the past have often been referred to as "hyperplastic AT2 cells" [88], our recently gained more detailed understanding of the arising epithelial subpopulations in IPF, thanks to the above-described scRNA-Seq studies, strongly suggests that these cells also include epithelial cells of a bronchiolar origin like activated hyperplastic basal cells. Moreover, strengthening a potential role of apoptotic SCGBB1A1<sup>+</sup> secretory cells in IPF, a recent report has demonstrated that ablation of programmed cell death 5 (*PDCD5*) expression in these secretory cells, but not in AT2 cells protects from experimental lung fibrosis [89].

#### *7.3. Endoplasmic Reticulum (ER) Stress as Trigger for Epithelial Apoptosis*

ER stress is a well-established trigger of alveolar epithelial apoptosis in IPF [85,90], but has received less attention for bronchial or bronchiolar epithelial cells. Many types of epithelial injury linked to an increased IPF risk, as, e.g., herpesvirus infection, cigarette smoke, and particulate matter, have been shown to cause ER stress and induce the unfolded protein response (UPR), also in cultured bronchial epithelial cells [90–92]. An elegant recent study has provided an intriguing link between the *MUC5B* promoter polymorphism (see Section 6) and ER stress in secretory airway epithelial cells. Chen et al. not only demonstrated that central components of the UPR induced *MUC5B* expression in secretory airway epithelial cells in pulmonary fibrosis, but also were able to show that this induction is dependent on sequences within the promoter variant rs35705950 region which harbours the IPF risk variant. Notably, in a luciferase reporter assay, the minor risk allele T alone increased expression of *MUC5B* by almost two-fold. This study provides another piece of evidence that ER stress and induction of the UPR in bronchiolar cells likely also contributes to expression of *MUC5B*, impaired mucociliary clearance, and the development of IPF [93].

#### *7.4. Ageing and Epithelial Senescence*

IPF predominates in the elderly and is characterized by increased senescence in many cell types, presumably because of replicative exhaustion and/or repetitive injuries to the epithelium [94]. It is by now well established that epithelial cells covering fibroblast foci are positive for senescence-associated β-galactosidase activity, nuclear p16 and p21 [95–99]. In agreement, recent scRNA-Seq-based studies have demonstrated that the above-described basaloid cells as well as hyperplastic basal cell population in bronchiolized regions express genes related to growth arrest and senescence [44,45,100]. This has also been observed for the transient population of Krt8<sup>+</sup> ADI cells in mouse models of lung injury [54]. Collectively, these observations put forward an attractive hypothesis where a specific population of epithelial cells, normally committed to repair an injury of the lung mucosa followed by clearance, persists "locked in repair" in IPF [101]. Notably, senescent epithelial cells from fibrotic tissue have been shown to secrete proinflammatory and profibrotic molecules as components of their senescence-associated secretory phenotype (SASP) [97], suggesting that they may be a direct driver of disease pathogenesis.

#### *7.5. Reactivation of Developmental Pathways*

Reactivation of molecular signalling pathways such as the transforming growth factorβ (TGF-β), WNT, sonic hedgehog (SHH), and Notch pathways are critical players during the developmental stages of lung, remain largely inactive in the postnatal lung except for the maintenance of progenitor cell niches, but can become aberrantly reactivated during an injury repair response and then trigger chronic disease [102]. In the following, the induction and regulation of these developmental pathways during IPF pathogenesis is discussed with a focus in bronchial and bronchiolar epithelial cells.

#### 7.5.1. Transforming Growth Factor-β (TGF-β) Signalling

All three TGF-β isoforms (β1, β2, β3), their receptors TGF-β receptors (TGFBR) I, II, and III, and their signalling mediators SMAD-2, -3, -4, -5, -6 and -7 are involved in embryonic lung development where they regulate branching morphogenesis and alveolarization [102]. TGF-β ligands act by binding to their cognate receptors on target cells, where they trigger intracellular signalling pathways including the canonical SMAD-mediated pathway but also non-canonical signalling pathways [103].

TGF-β is synthesized as an inactive precursor homodimer with N-terminal prodomains, which, after cleavage by the intracellular protease furin, remain non-covalently bound to the TGF-β homodimer as latency-associated peptide (LAP), collectively forming the small latent complex (SLC). Only if this complex is bound to the latent TGF-β-binding protein (LTBP), it will be secreted to the extracellular matrix as a complex called large latent complex (LLC) [104]. Hence, TGF-β is always secreted in a latent form and requires activation in situ by additional triggers.

Out of the three isoforms, TGF-β1 plays a well-recognized central role in IPF pathogenesis [105–107]. Activation of latent TGF-β1 implies the release of active TGF-β1 ligands from the ECM by proteolysis or deformation of their LAP portion. Many potential mechanisms have been observed in vitro, but for many the physiological relevance remains unclear. In vivo activation has been clearly shown for several αv integrins in the context of fibrosis, e.g., avβ1, avβ3, avβ5, and avβ6 [108]. Even though the underlying mechanisms are not fully understood, it appears that cells carrying these integrins can exert a pulling force on the LLC which "unwraps" the LAP and releases active TGF-β1 from the ECM. Other reasonably well-established activators are thrombospondin-1 (TSP1), pregnancy specific glycoproteins, and tenascin X. Additionally, activation by unspecific physico- or biochemical factors like low pH and reactive oxygen species has been described, which may also be physiologically relevant. Finally, proteolytic activation has been described for a variety of proteases, including, e.g., several matrix metalloproteinases (MMPs), calpain, plasmin, kallikrein, and cathepsin D. Interestingly, while deficiency of integrin subunits like αv, β6, and β8 in mice phenocopies the TGF-β1 knockout mouse, this has not been observed for any protease-deficient mouse so far, indicating considerable redundancy in proteolytic activation of TGF-β1 in vivo [108–110].

Bronchial epithelial cells potentially may contribute to TGF-β1-mediated mechanisms in IPF by at least three mechanisms. First, bronchial and bronchiolar epithelial cells express TGF-β1 [111,112], implying that the underlying ECM likely harbours latent TGF-β1. Second, bronchial epithelial express many of the suggested activating factors in fibrosis: Airway epithelial cells express both αvβ6 and αvβ8 integrin heterodimers, and expression of αvβ6 is dramatically increased after injury [113]. Notably, the *ITGAV* transcript for the αv integrin monomer is clearly enriched in aberrant basaloid cells relative to all other healthy epithelial cell types (Figure 5A). Second, airway and aberrant basaloid epithelial cells also have been shown to express activators of latent TGF-β1 in IPF, including MMP-8 [114], MMP-3, MMP-13, MMP14, calpain, and cathepsin D in IPF [44,45] (ipfcellatlas.com), all representing proteases previously proposed to activate latent TGF-β1 [108]. ScRNA-Seq data also demonstrates expression of the thrombospondin 1 precursor by bronchial epithelial cells [44,45] (ipfcellatlas.com). Third, bronchial epithelial cells themselves are reactive to TGF-β1 and have been shown to undergo partial epithelial-to-mesenchymal transition

(pEMT) in response to TGF-β1 [115,116]. Whether EMT contributes to the myofibroblast population in IPF is controversially discussed, as conflicting results have been reported in in vivo models of pulmonary fibrosis—so far neither lineage-tracing experiments nor scRNA-Seq data have provided unambiguous evidence for a complete EMT as a source for myofibroblasts in the lung [117,118]. However, the resulting cell phenotype after pEMT is partly reminiscent of the aberrant basal-like cell phenotype observed in IPF—following pEMT, human bronchiolar epithelial cells lose epithelial morphology and polarity and upregulate mesenchymal markers like type I collagen and fibronectin. On the other hand, downregulation of expression of typical epithelial markers such as E-cadherin and upregulation of vimentin is not evident in the scRNA-Seq data sets published so far [44,45] (ipfcellatlas.com). These discrepancies may reflect the crosstalk between variously activated profibrotic pathways and the complex cellular and ECM environment in end-stage IPF, parameters frequently not considered in studies of EMT. Clearly, further work is warranted to elucidate the role of TGF-β1 in the emergence of aberrant basaloid cells, and how this process relates to pEMT.

#### 7.5.2. WNT Signalling Pathway

Wingless/integrase-1 (WNT) signalling pathways are fundamentally important for tissue morphogenesis including all stages of lung development [119]. The WNT ligand family comprises 19 human members which are characterized by strictly controlled spatiotemporal expression in various organs during development and tissue homeostasis and associated with a constantly growing number of human diseases by upregulation, genetic polymorphisms and mutations [120]. It is well-established that the WNT signalling pathway is reactivated in IPF [119,121] and expression of WNT ligands (WNT1, WNT3a), intracellular downstream inducers (β-catenin, GSK-3β), as well as extracellular inhibitors of canonical WNT signalling (Dickkopf proteins DKK1, DKK4 and the interacting transmembrane receptor Kremen 1) has been demonstrated in bronchial and bronchiolar epithelium in IPF [122,123]. Studies in various models of lung injury have put forward WNT signalling as a critical component for stem cell maintenance, lung regeneration, and repair [119]. WNT signalling is activated during repair after proximal lung injury and dynamically regulates submucosal gland progenitor maintenance, proliferation, and differentiation to other airway epithelial cell types [124–128]. Furthermore, in mice, expression of Wnt7b by basal cells in the proximal airways generates their own stem cell niche via induction of fibroblast growth factor 10 (Fgf10) in adjacent smooth muscle cells [129]. Airway injury induces Wnt7b in the more distal airways, generating new Fgf10-expressing mesenchymal cells and allowing for recruitment of basal cells and/or differentiation of lineage-negative progenitors into the basal progenitor cell lineage [129,130]. Collectively, these studies imply an important role of WNT signalling in aberrant bronchial and bronchiolar repair in IPF.

#### 7.5.3. Sonic Hedgehog Signalling (SHH) Pathway

During lung development, sonic hedgehog (SHH) is expressed in the respiratory epithelium in a gradient with higher levels in the branching tips, presumably providing polarization during branching morphogenesis in the embryonic and pseudoglandular stage. Furthermore, SHH is essential for the coordination of epithelial-mesenchymal compartment growth, also during the alveolarization phase [131,132]. Bolaños et al. systematically assessed expression of SHH signalling pathway components in control lung tissue and IPF and found that expression of all SHH signalling components was induced or drastically increased in IPF. They observed expression of the ligand SHH exclusively in bronchial, bronchiolar, and alveolar epithelial cells, but expression of the receptors transmembrane receptor Patched-1 and the G-protein coupled receptor Smoothened mainly in fibroblasts and inflammatory cells. While the SHH signalling transcription factor glioma-associated oncogene homolog (*GLI*) *1* was expressed ubiquitously, including in fibroblasts, nuclear GLI2 was confined to distal epithelial cells [133]. Furthermore, the authors could show that recombinant SHH increased proliferation, expression of ECM components, and migration

of primary human lung fibroblasts and at the same time inhibited fibroblast apoptosis [133]. These results indicate that SHH generated by distal, bronchiolar and alveolar, epithelial cells activates fibroblasts, which indicates an important profibrotic contribution of epithelialderived SHH in IPF pathogenesis. Interestingly, a more recent study provided evidence that a profibrotic feed-forward mechanism may exist in this context: Gli<sup>+</sup> mesenchymal stromal cells promote differentiation of airway progenitors into aberrant metaplastic Krt5<sup>+</sup> basal cells by antagonizing activation of the bone morphogenetic protein (BMP) pathway [134]. Overall, this suggests that upregulation of epithelial SHH may be an early event in IPF pathogenesis and trigger reciprocal epithelial-mesenchymal interactions that propagate lung fibrogenesis.

#### 7.5.4. Notch Signalling Pathway

In lung development, Notch signalling determines ciliated versus secretory cell fate in conducting airways [135,136]. Following bleomycin injury or influenza infection in mice, Notch signalling has been shown to activate proliferation and migration of a KRT5<sup>+</sup> progenitor cell lineage in the context of repair after injury while blockade of Notch signalling induced an alveolar cell type faith. Importantly, active Notch signalling was detected in IPF honeycomb cysts [130], indicating a role for Notch signalling in aberrant epithelial repair and honeycomb cyst formation. Interestingly, overexpression of Notch can also induce EMT [137]; so, Notch signalling may not only promote aberrant cyst formation, but also contribute to the emergence of the above- described aberrant basaloid cells. In mice, Dlk1-mediated temporal regulation of Notch signalling is required for differentiation of AT2 to AT1 cells during repair [138]. Interestingly, deletion of Dlk1 in AT2 cells led to the accumulation of an intermediate cell population. We may speculate that a similar Notch-dependent mechanism might drive the appearance of aberrant basaloid cells in IPF.

In summary, bronchial and bronchiolar epithelial cells including airway-cell derived disease-specific lineages contribute to the reactivation of developmental pathways in IPF, including central pathways like the TGF-β1, WNT, SHH, and Notch signalling pathways. The collective evidence clearly demonstrates that, via autocrine and paracrine mechanisms, conducting airway epithelial-derived factors induce and modulate developmental programmes in IPF and drive major pathological outcomes in this disease like excessive ECM deposition and honeycomb cyst formation.

#### *7.6. Epigenetic Mechanisms*

Epigenetics traditionally comprises DNA methylation and histone modification, molecular alterations in chromatin which serve as marks for transcriptional activation or repression without affecting the DNA sequence per se. Epigenetic regulation mechanisms are typically persistent, can be inherited, and have the potential to translate environmental exposures into regulation of gene transcription at the level of chromatin structure [139,140]. This applies particularly to the airway mucosa, which represents a direct interface between environment and human body [141,142]. As IPF development seems to be orchestrated by genetic predisposition and environmental risk factors, epigenetic mechanisms may provide important mechanistic links and novel targets for therapy. Indeed, a number of studies have established that epigenetic signatures are changed in IPF, including DNA methylation and expression of DNA methyl transferases [143,144] as well as single histone modification marks [140] and expression of histone modifying enzymes [145]. To the best of our knowledge, genome-wide histone modification studies in IPF are lacking to date.

Our knowledge on epigenetic marks in IPF and their cell type-specific contribution to disease pathogenesis and progression is still very limited. However, it is well-known that IPF risk factors like cigarette smoke or particulate matter, for instance, induce epigenetic alterations in bronchial epithelial cells [146–148], indicating that such changes may be frequent in IPF. Furthermore, increased expression and activity of histone deacetylases in IPF has been localized to myofibroblasts, but also to aberrant basal cells in IPF [145]. Clearly, the role of epigenetic changes in airway epithelial cells requires more attention and

detailed mechanistic studies, and such investigations may ultimately provide interesting novel therapeutic intervention opportunities for early therapy.

#### *7.7. Non-Coding RNAs*

Non-coding RNA (ncRNA), i.e., RNA which is not translated to proteins, constitutes approximately 98% of the total transcribed RNA in humans [149]. NcRNAs include housekeeping RNAs, such as ribosomal, spliceosomal, or transfer RNA, expression of which is constitutive, but also regulatory RNAs, such as long noncoding RNAs (lncRNA) or microRNAs (miRNA), which are expressed in a cell type- and tissue-specific manner and often altered in disease. LncRNA molecules are arbitrarily defined as >200 nucleotides in length and can regulate gene expression by transcriptional interference, chromatin remodelling, promoter inactivation, activation and transport of accessory and transcription factors, epigenetic silencing, and as precursors for small interfering RNAs [150,151]. In contrast, miRNAs are short, approximately 22 nucleotides long, RNA molecules which suppress protein translation by non-perfect complementary binding to regions in the 3′UTR of their target mRNAs.

Even though our knowledge on function and regulation of lncRNAs in general is still very limited, several studies support the concept that lncRNAs contribute to profibrotic cellular mechanisms in IPF [152,153]. While some studies in this context focussed on the function of specific lncRNAs in lung fibroblasts [154], other recent reports highlight altered lncRNA expression and function in bronchial epithelial cells. For instance, increased expression of lncRNA *MEG3* was observed in atypical KRT5<sup>+</sup> p63<sup>+</sup> basal cells in IPF relative to normal donor lung tissue. In vitro studies showed that *MEG3* induced basal cell gene transcription (*KRT14, TP63*) in bronchial cell lines, but also fundamental events of EMT, including increased cellular migration and downregulation of *CDH1* (E-cadherin) [155]. *MEG3* may thus cause or at least contribute to the emergence of the aberrant basal-like cell populations in IPF described above (see Section 4). In contrast, loss of the terminal differentiation-induced lncRNA (TINCR), a lncRNA normally expressed in the bronchial epithelium, but decreased in IPF, has been described to, among others, induce basal cell markers and ECM genes [156,157], reminiscent of gene expression signatures of aberrant basal and basaloid cells in IPF [42,44,45]. Studies in mouse models of lung fibrosis and primary human cells have proposed additional lncRNAs as regulators of EMT in bronchial epithelial cells, but localization in the IPF lung has, to the best of our knowledge, not yet been demonstrated. These include lncRNAs uc.77 and 2700086A05Rik [158] and lncRNA H19 [159]. Collectively, these studies support the concept of bronchial epithelial cell-specific lncRNA expression as an emerging driver in IPF pathogenesis.

To date, few studies have addressed the function of airway epithelial miRNAs in IPF pathogenesis. A pioneering study has globally assessed expression of miRNAs in bronchoscopy-assisted bronchial brushes from fibrotic airways of bronchiolitis obliterans syndrome (BOS) and found that miR-323a-3p was drastically downregulated (>18-fold) in airways of BOS patients relative to control lung transplant patients. The authors also examined miR-323a-3p expression in isolated AT2 cells from IPF lung explants and from fibrotic mouse lungs after bleomycin injury and observed significant downregulation, indicating general downregulation in lung epithelium during fibrogenesis [160]. Furthermore, miR-323a-3p mimics and miR-323a-3p antagomirs suppressed and exacerbated lung fibrogenesis, respectively, in the bleomycin mouse model. In vitro studies suggested that miR-323a-3p directly targets central mediators of TGF-α and TGF-β signalling as well as caspase 3, thereby attenuating key profibrotic mechanisms and epithelial cell apoptosis [160]. Given that miRNA therapeutics are coming of age and, in the case of the lung, can be easily delivered to the epithelium by inhalation, more such studies are warranted to identify further epithelial-specific miRNA-based profibrotic mechanisms.

#### **8. Summary, Conclusions, and Emerging Questions**

The last decade has transformed our understanding of IPF pathogenesis and set forth multiple evidence that strongly argues for a critical role of conducting airway epithelial cell populations in IPF aetiology and disease development (summarized in Figure 7). The discovery of the *MUC5B* promoter polymorphism as the strongest causative factor for IPF onset drew attention from the alveolar department to bronchial and bronchiolar cell contributions to lung fibrogenesis. IPF airways are drastically distorted, and alveolar areas are repopulated by airway-like epithelial cells in a process termed bronchiolization. In agreement, several recent scRNA-Seq analyses of IPF lungs have consistently revealed drastic alterations in epithelial subpopulations including the replacement of alveolar epithelial cells by various airway-like cells that are either directly distal airway-derived or the result of alveolar epithelial cell transdifferentiation or a combination of both. Emerging new evidence suggests that specific mesenchymal niche environments in the IPF patient may promote plasticity of the alveolar epithelium that leads to full transdifferentiation towards airway-like states [38]. Another line of evidence shows that persistent alveolar repair generates intermediate cells, which display features of senescence and p53 activation. In mice, inducing senescence in AT2 cells and thereby shifting them to a state that resembles injury-induced alveolar differentiation intermediates [54,161] and the aberrant basaloid cells [42,44,45] leads to progressive pulmonary fibrosis as seen in IPF patients [162]. Future work needs to leverage histopathological disease grade staging to further clarify the cellular origins of these intermediate cell populations and the natural evolution of epithelial metaplasia and bronchiolization in IPF disease progression.

**Figure 7.** Hypothetical contributions of the airway epithelium to IPF pathogenesis. Summarizing scheme linking established environmental and genetic risk factors via the bronchial and bronchiolar epithelium to IPF-specific disease mechanisms and outcomes like bronchiolization and interstitial scarring. Figure was created with biorender.com.

Critical airway functions like mucociliary clearance and epithelial barrier integrity are also affected in IPF. Genetic risk factors beyond the *MUC5B* promoter polymorphism, in particular the *DSP* and *FAM13A* risk SNPs, argue for airway epithelial cells as central culprits in disease onset. Finally, evidence is accumulating that bronchial epithelial cells directly trigger central profibrotic mechanisms like the reactivation of multiple developmental programmes in an aberrant injury response.

The balance between epithelial proliferation, trans-differentiation, apoptosis and cellular senescence is drastically disturbed in IPF airway epithelial cells. Impaired mucociliary clearance may be a key disease-initiating feature in this context. However, we still understand very little about the mechanisms that trigger the balance to tip from normal alveolar repair towards this aberrant, airway epithelial cell-driven repair process leading to the emergence of epithelial metaplasia and aberrant basaloid cells in the lung periphery. Similarly, the sequence of events that ultimately lead to IPF development remains ill-defined. For instance, is bronchiolization an epiphenomenon and characteristic of end-stage disease, or may pEMT of airway epithelial cells actually precede activation of fibroblasts? What are key mechanisms that can be safely and effectively employed to target profibrotic epithelialmesenchymal cross-talk and regenerate normal stem cell niches? In particular epigenetic mechanisms, the role of epithelial non-coding RNAs, how these affect profibrotic and disease-perpetuating mechanisms, and whether they can be targeted for therapy remains a largely unexplored area. Additionally, the contributions of immune cells to the described processes remain little understood. Evidently, more mechanistic studies are needed to decipher these processes in molecular detail. It is becoming increasingly clear that, for this aim, we need to develop novel animal lung fibrosis models, which recapitulate impaired mucociliary function and environmental exposure. The above-described mouse model derived by conditional deletion of NEDD4-2 from airway epithelial cells represents a great opportunity to study in more detail the mechanisms that trigger fibrosis as a result of impaired mucociliary clearance. The good news about airway epithelial cells as emerging central culprits in IPF pathogenesis is that, finally, targeting airway epithelial cells is a more straightforward task than targeting fibroblasts, because, given that fibrotic areas are ventilated, the inhalatory route would deliver the drug directly and specifically onto the aberrant epithelium.

**Author Contributions:** Writing—review and editing, A.C., M.M., M.A., H.B.S., C.A.S.-W.; supervision, C.A.S.-W.; funding acquisition, H.B.S., C.A.S.-W. All authors have read and agreed to the published version of the manuscript.

**Funding:** Work in the authors' laboratories is supported by the Helmholtz Association, the German Center for Lung Research (DZL), the Deutsche Forschungsgemeinschaft (DFG) within the Research Training Group GRK2338 (grant to C.A.S.-W.), the Federal Institute for Risk Assessment (Bundesinstitut für Risikobewertung, BfR) (#1328-570, grant to C.A.S.-W.), the European Union's Horizon 2020 research and innovation program (grant agreement 874656, to H.B.S.) and the Chan Zuckerberg Initiative (CZF2019-002438, to H.B.S.).

**Institutional Review Board Statement:** For previously unpublished stainings given in Figure 2B, the study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of the Ludwig-Maximilians University of Munich, Germany (Ethic vote #333-10, #382-10). For all other data extracted from previous work, please refer to the original publications.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** Count tables of the Munich single-cell cohort as well as custom preprocessing code can be accessed at https://github.com/theislab/2020\_Mayr (last accessed 22 December 2021). Raw count tables for additional cohorts were retrieved from the Gene Expression Omnibus database by the accession numbers as provided in the original publications (Chicago cohort GSE122960; Nashville cohort GSE135893; New Haven cohort GSE136831).

**Acknowledgments:** We gratefully acknowledge the provision of human biomaterial and clinical data from the CPC-M bioArchive and its partners at the Asklepios Biobank Gauting, the Klinikum der Universität München and the Ludwig-Maximilians-Universität München.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


## *Review* **EGFR Signaling in Lung Fibrosis**

**Fabian Schramm 1,† , Liliana Schaefer <sup>2</sup> and Malgorzata Wygrecka 1, \* ,†**


† Member of the German Center for Lung Research.

**Abstract:** In this review article, we will first provide a brief overview of the ErbB receptor–ligand system and its importance in developmental and physiological processes. We will then review the literature regarding the role of ErbB receptors and their ligands in the maladaptive remodeling of lung tissue, with special emphasis on idiopathic pulmonary fibrosis (IPF). Here we will focus on the pathways and cellular processes contributing to epithelial–mesenchymal miscommunication seen in this pathology. We will also provide an overview of the in vivo studies addressing the efficacy of different ErbB signaling inhibitors in experimental models of lung injury and highlight how such studies may contribute to our understanding of ErbB biology in the lung. Finally, we will discuss what we learned from clinical applications of the ErbB1 signaling inhibitors in cancer in order to advance clinical trials in IPF.

**Keywords:** epidermal growth factor; epidermal growth factor receptor; ErbB-signaling; pulmonary fibrosis; idiopathic pulmonary fibrosis; lung fibrosis; tyrosine kinase inhibitor; TGF-α; TGF-β; amphiregulin; neuregulin 1

#### **1. Introduction**

The epidermal growth factor (EGF) receptor (EGFR), belongs to the family of the ErbB tyrosine kinase receptors [1,2]. EGFR is also known as ErbB1 or HER1. Other members of this family are ErbB2 (HER2) [3], ErbB3 (HER3) [4] and ErbB4 (HER4) [5]. Following ligand binding, ErbB receptors form homo- or heterodimers that are autophosphorylated on tyrosine residues by intrinsic tyrosine kinase activity and mediate signal transduction to the nucleus [6]. Several ErbB ligands have been identified thus far, including EGF [7,8], transforming growth factor-α (TGF-α) [9], amphiregulin (AREG) [10], heparin-binding EGF-like growth factor (HB-EGF) [11], betacellulin (BC) [12], epiregulin (EREG) [13], epigen (EPG) [14] and neuregulins (NRGs) [15]. All ErbB ligands exist as membrane-anchored precursors that are released in an active form by enzymatic cleavage to the extracellular milieu. Matrix metalloproteinases (MMPs) and A disintegrin and metalloproteinases (ADAMs) were found to be responsible for the shedding of ErbB ligands [16–18]. The main sheddases of ErbB ligands are ADAM-10 and -17. ADAM-10 was reported to release EGF and BC [18], whereas ADAM-17 to cleave TGF-α, AREG, HB-EGF and EREG. In addition, MMP-3 was described to release HB-EGF from rat ventral prostate epithelial cells [17].

EGF is a prototype member of the ErbB ligand family. This polypeptide was originally identified in the submaxillary glands of mice and the urine of humans. EGF was discovered by Stanley Cohen while working with Rita Levi-Montalcini on nerve growth factors. For these discoveries, Stanley Cohen and Rita Levi-Montalcini were awarded the Nobel Prize in Physiology or Medicine in 1986 [19]. EGF, TGF-α, AREG and EPG only interact with ErbB1, while EREG, BC and HB-EGF bind to ErbB1 and ErbB4. A heterogeneous group of ligands called neuregulins (NRG) interacts with ErbB3 and ErbB4 [15] (Figure 1). The NRG family is

**Citation:** Schramm, F.; Schaefer, L.; Wygrecka, M. EGFR Signaling in Lung Fibrosis. *Cells* **2022**, *11*, 986. https://doi.org/10.3390/ cells11060986

Academic Editor: Steven G. Gray

Received: 14 February 2022 Accepted: 11 March 2022 Published: 14 March 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

composed of NRG1 isoforms, NRG2 (also known as neural- and thymus-derived activator for ErbB kinases (NTAK)) [11,20], NRG3 [21] and NRG4 [22]. The different NRG1 isoforms can be categorized into three smaller groups: type I NRG1, which includes heregulins (HRGs) [23], neu differentiation factor (NDF) [24,25] and acetylcholine receptor-inducing activity (ARIA) [26]; type II NRG1, which contains the glial growth factors (GGFs) [24] and type III NRG1, which comprises the sensory and motor neuron-derived factor (SMDF) [27]. While NRG1 and NRG2 bind to ErbB3 and ErbB4, NRG3 and NRG4 only interact with ErbB4 [15] (see Figure 1).

TGFα α; PLCγ, phospholipase Cγ. **Figure 1. ErbB receptors and their ligands**. ErbB2 has no known ligand, while ErbB3 lacks intrinsic kinase activity (indicated by a cross). Following ligand binding, the receptors activate several downstream signaling pathways thereby regulating cell growth, proliferation, and survival. These processes play an important role in development, wound healing and tissue regeneration. EGF, epidermal growth factor; TGFα, transforming growth factor-α; EPG, epigen; AREG, amphiregulin; EREG, epiregulin; BC, betacellulin; HB-EGF, heparin-binding EGF-like growth factor; NRG1-4, neuregulin 1-4; STAT, signal transducer and activator of transcription; MAPK/ERK, mitogen-activated protein kinase/extracellular signal-regulated kinase; PIK3/Akt, phosphoinositide 3-kinase/protein kinase B; PLCγ, phospholipase Cγ.

> So far, there is no known ligand for the ErbB2 receptor [28], however, ErbB2 is a preferred partner when forming heterodimers with other ErbB receptors [29]. For instance, binding of EGF to ErbB1 may induce phosphorylation of ErbB2 [30] and thus marked amplification of a signal. Furthermore, binding of NRG1 or NRG2 to ErbB3, which lacks intrinsic catalytic activity, can trigger ErbB3 phosphorylation by the kinase-active partner ErbB2 and thus transduction of a potent mitogenic signal [31].

Upon ligand binding, ErbB receptors trigger phosphorylation of diverse effector proteins and activation of multiple downstream signaling pathways. There is a number of excellent reviews describing downstream signaling pathways initiated by ErbB receptors for those interested in this topic [8,15]. Here, only some of the major ErbB downstream signaling pathways are listed. These include: mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway [32], phosphoinositide 3-kinase (PIK3)/protein kinase B (Akt) pathway [33], phospholipase Cγ (PLCγ) pathway [34], or signal transducers and activators of transcription (STAT) pathway [8]. Activation of the ErbB receptors regulates cell growth, proliferation, and survival and it is associated with a number of biological processes such as organ development, tissue regeneration, and wound healing [35]. The importance of the ErbB-mediated signaling for organogenesis is underscored by the studies showing that inhibition of ErbB1 impairs the development of epithelia in almost every organ including the heart [36], skin [37], lung, brain, kidney and liver [38,39], leading to mouse death shortly after birth [37,39]. Deficiency in single ErbB ligands results in similar pathological changes as the lack of the receptors themselves [40,41].

Besides its role in many developmental and physiological processes, overactivation of ErbB signaling has been widely described in many forms of cancer, including glioblastoma [42] and lung [43], breast and ovarian cancer [44–46]. These findings led to the development of ErbB signaling inhibitors for the treatment of the aforementioned pathologies [47,48]. Emerging interest in the ErbB signaling and its function during carcinogenesis brought attention to the role of the ErbB receptors and their ligands in other hyperproliferative diseases including lung fibrosis [49].

In this review, we will focus on the ErbB signaling in lung fibrosis, with special emphasis on idiopathic pulmonary fibrosis (IPF). We will discuss the implications of the ErbB signaling in processes that are hallmarks of the maladaptive remodeling of lung tissue. Lastly, we will critically discuss recent advances and future perspectives in targeting the ErbB signaling for lung fibrosis therapy.

#### **2. Idiopathic Pulmonary Fibrosis**

Idiopathic pulmonary fibrosis (IPF) is one of the most common forms of diffuse parenchymal lung diseases and is characterized by excessive deposition of extracellular matrix proteins in the lung [50]. IPF is an age-related disease, and with the human population aging worldwide, the economic burden of IPF is expected to constantly increase in the future. The pathomechanism of IPF remains elusive, with preferred concepts of disease pathobiology involving recurrent microinjuries to a genetically predisposed alveolar epithelium, followed by an abnormal activation of mesenchymal cells ((myo)fibroblasts), their expansion and massive accumulation of collagens in the lung. Aggregates of active (myo)fibroblasts, so-called fibroblastic foci, are typical histological features of IPF [50]. Fibroblastic foci are often covered by aberrant basaloid cells [51] and MUC5B-producing airway secretory cells [52]. Repopulation of alveoli by abnormal airway epithelial cells is associated with the formation of honeycomb cysts, which are indicators of advanced fibrosis and poor prognosis [50]. Active involvement of airway epithelial cells in the pathogenesis of IPF is reinforced by the fact that the gain-of-function *MUC5B* promoter variant rs35705950 is the dominant risk factor for disease development [53]. Although, it is not entirely clear how increased expression of MUC5B contributes to IPF pathobiology, the study by Hancock et al. [52] linked MUC5B overexpression to impaired mucociliary clearance accompanied by progressive lung tissue scarring.

Increased expression of several cytokine/growth factors have been considered to drive profibrotic processes in the lung, including transforming growth factor-β1 (TGF-β1), platelet-derived growth factor-BB (PDGF-BB), connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF), and tumor necrosis factor-α (TNF-α). These mediators contribute to lung tissue scarring by deregulating activation, survival, proliferation, and differentiation of a variety of cells, including mesenchymal and epithelial cells [54,55]. For instance, TGF-β1, which is stored in the ECM in a latent form and activated by cell contractile forces, drives the conversion of fibroblasts to matrix-producing myofibroblasts. Excessive deposition of ECM and its stiffening lower the threshold for TGF-β1 activation thereby creating a self-amplifying loop that promotes the expansion of myofibroblasts and fibrosis development [56]. These processes may be enhanced by factors such as interleukin (IL)-6, IL-1β, or TNF-α, which potentiate TGF-β1 expression and activation of the TGFβ1 signaling pathway [57,58]. All these changes are aggravated by the resistance of IPF (myo)fibroblasts to apoptosis [59].

Despite the approval of pirfenidone [60] and nintedanib [61], IPF has a very poor prognosis with a life expectancy of 3–5 years once diagnosed [54]. Thus, lung transplantation still remains the only treatment option that markedly improves the quality of life and survival of IPF patients [50]. Both pirfenidone and nintedanib delay disease progression by exerting pleiotropic effects, which range from the inhibition of inflammatory processes to the blockage of fibroblast proliferation and ECM production. Although the pirfenidone mode of action remains elusive, several studies demonstrated the direct impact of this drug on the Hedgehog and TGF-β signaling pathways [60,62]. In contrast to pirfenidone, nintedanib is a tyrosine kinase inhibitor (TKI) of PDGF, VEGF, and fibroblast growth factor (FGF) receptors. In addition, it also inhibits a narrow range of other targets at pharmacologically-relevant doses including the Src family and Flt-3 kinases. Ligands of PDGF, VEGF, and FGF receptors are known to have potent profibrotic effects [61]. While the approval of pirfenidone and nintedanib was a milestone in the care of IPF, there is still a high and unmet clinical need in this patient group. A multi-targeted approach, potentially with combination therapies and the identification of subsets of IPF patients who may respond more favorably to specific agents, are likely to dominate future clinical studies.

Targeting ErbB receptors and their ligands may serve as a potential therapeutic option for IPF, in particular, that different elements of the ErbB signaling can be pharmacologically targeted. However, a complex ligand–receptor network and the involvement of the ErbB signaling in the tissue regenerative process may encounter some unexpected surprises, thus further studies critically evaluating the role of ErbB receptors and their ligands in adaptive versus maladaptive remodeling of lung tissue are urgently needed.

#### **3. ErbB Receptor–Ligand System in Lung Fibrosis**

ErbB receptors and their ligands are expressed in a large variety of human tissues including the epithelial cells of the lung [63]. Under physiological conditions, ErbB1–4 are expressed in bronchial epithelial and alveolar type II (ATII) cells [64,65], whereas ErbB ligands, such as TGF-α, AREG, and HB-EGF, are expressed in bronchial epithelial cells [65]. Furthermore, TGF-α, AREG, HB-EGF, BTC, and EGF are produced in serous acinar cells from submucosal glands beneath the respiratory epithelium [64]. In cell culture, naïve lung fibroblasts were found to express TGF-α, HB-EGF, HRG and AREG but not BTC [66].

Increased expression of various ErbB ligands is associated with fibrosis development in multiple organs, including, lung, liver, or pancreas. For example, overexpression of HB-EGF and AREG causes pancreatic fibrosis [67,68], while high levels of AREG alone are sufficient to trigger liver fibrosis [69]. Furthermore, increased expression of EPG results in the fibrosis of the nerve system, and overexpression of TGF-α is associated with fibrosis of the lung [70]. Interestingly, deficiency of HB-EGF is linked to liver fibrosis [71], thus pointing towards a dual and organ-specific role of the ErbB receptor–ligand system in the tissue scarring processes. Below, we provide evidence for a dual ("good" versus "bad") role of ErbB receptors and their ligands in lung fibrosis and, in particular, in IPF.

#### *3.1. ErbB1/EGFR Receptor*

Besides being overexpressed in many types of cancer, ErbB1 is also upregulated in lung epithelial cells from patients with different forms of pulmonary fibrosis [72]. In IPF, abundant ErbB1 immunostaining was found in the hyperplastic alveolar epithelium surrounding areas of fibrosis and inflammation. In addition, increased ErbB1 protein levels were reported in IPF fibroblastic foci and in fibroblasts isolated from IPF lungs [73]. Furthermore, IPF lung fibroblast (LF)-derived culture supernatants were found to stimulate expression of ErbB1 in donor LF in an FGF-dependent manner [73]. In addition, a negative correlation between ErbB1 mRNA levels and the indicators of IPF progression, such as forced vital capacity (FVC) and diffusion capacity of the lung for carbon monoxide (DLCO) [72], was reported.

After the introduction of ErbB1 TKI to cancer therapy, the discussion on their repurposing and usage in the treatment of other hyperproliferative diseases, including lung fibrosis, began. Quickly, first studies demonstrated that tyrphostin AG1478 reduces proliferation of LF and attenuates pulmonary fibrosis caused by intratracheal instillation of vanadium pentoxide in rats [74]. Another ErbB1 TKI, gefitinib, suppressed proliferation of LF and diminished pulmonary fibrosis in the bleomycin-treated mice [75,76]. In contrast, Suzuki et al. [77] demonstrated that gefitinib aggravates bleomycin-induced lung fibrosis in mice by reducing the regenerative potential of alveolar epithelial cells. Although the reasons for these contradictory findings are unknown, differences in mouse strains, dosages, intervals and mode of drug application could have played a role.

Development of acute lung injury and ILD in non-small cell lung cancer (NSCLC) patients receiving gefitinib demonstrates possible deleterious effects of the ErbB1 signaling inhibition [78,79]. Interestingly, similar harmful effects were also observed in NSCLC patients treated with another ErbB1 TKI, erlotinib [80,81]. The incidence of ILD in TKItreated NSCLC patients is ~1% worldwide [82]. In the Japanese population, it is significantly higher at ~2%. Despite this observation, the *EGFR* polymorphism leading to the genetic susceptibility to the treatment with ErbB1 TKI in the Japanese was not observed [82]. Preexisting lung disorders, such as interstitial pneumonia or pulmonary fibrosis, male sex and history of smoking, were identified as risk factors for the development of gefitinibassociated ILD [83,84]. Considering the chemical and pharmacological similarities between gefitinib and erlotinib, the same risk factors may apply to the erlotinib-trigger ILD. In addition, radio- and chemotherapy, both used to treat cancer, seem to aggravate ErbB1 TKIinduced ILD [85]. Currently, it is not known what mechanisms lead to the development of ILD in NSCLS patients receiving gefitinib or erlotinib. However, it is increasingly recognized that the border between adaptive and maladaptive repair of the lung tissue is thin and the clue to success is maintaining the balance between all the factors involved [86].

#### *3.2. Transforming Growth Factor-α*

Among all ErbB1 ligands, TGF-α is the one with a well-described function in pulmonary fibrosis. TGF-α was found to be overexpressed in ATII cells, endothelial cells and fibroblasts in the lungs of IPF patients [87]. In addition, its levels were reported to be increased in IPF bronchoalveolar lavage fluid (BALF) [88]. The profibrotic potential of TGF-α was demonstrated in several studies, in which lung-specific overexpression of TGF-α in mice was conducted. For example, chronic production of TGF-α in surfactant protein-C (SP-C)-expressing cells disrupted alveolar and vascular development and caused pulmonary fibrosis and pulmonary hypertension in mice [85]. Similarly, chronic conditional expression of TGF-α driven by the doxycycline-regulatable Clara cell secretory protein (dox-CCSP) promoter triggered progressive vascular adventitial, peribronchial, interstitial, and pleural fibrosis, which was independent of inflammation and TGF-β activation [89]. Further studies demonstrated transcriptional similarities between dox-CCSP-TGF-α-induced lung fibrosis and IPF, thus pointing towards an essential role of the ErbB1–TGF-α axis in the development of IPF. In the rat bleomycin model, increased immunoreactivity for TGF-α and ErbB1 was observed in macrophages, alveolar septal cells and respiratory epithelial cells. Both proteins were predominantly detected in foci of cellular proliferation and in areas of intra-alveolar fibrosis [90]. Accordingly, TGF-α-deficient mice showed reduced hydroxyproline levels and partially preserved lung structure following bleomycin application as compared to wild-type littermates [91]. Interestingly, overexpression of TGF-α under the control of SP-C promoter protected mice against acute lung injury was caused

by inhalation of polytetrafluoroethylene (PTFE; teflon) fumes. Histological hallmarks of this model are pulmonary hemorrhage and inflammation. Indeed, SP-C-TGFα-transgenic mice exhibited reduced levels of IL-6 and macrophage inflammatory protein 2 in lung homogenates and decreased total protein levels and neutrophil numbers in BALF as compared to non-transgenic controls. Altogether, these findings demonstrate the etiology-dependent role of TGF-α in lung pathologies.

In line with these observations, ErbB1 TKI, gefitinib, partially reduced collagen levels and improved lung compliance, tissue and airway elastance, and airway resistance in mice overexpressing TGF-α under tetracycline-inducible CCSP (rtTA-CCSP) promoter [89]. These changes were supported by the decreased expression of several genes associated with lung parenchymal and vascular remodeling. It is worth mentioning here that gefitinib neither induced chronic lung injury nor exacerbated pulmonary fibrosis, thus supporting further studies to determine the role of ErbB1 in human lung fibrotic diseases [85,92]. Furthermore, the same group demonstrated that blockage of the ErbB1 downstream signaling mediator, PI3K, by the PX-866 pan-inhibitor reduced total lung collagen content and improved pulmonary mechanics in rtTA-CCSP-TGF-α overexpressing mice [93]. These results were recapitulated when another ErbB1 signaling pathway, MAPK/ERK, was targeted. Administration of an allosteric MEK inhibitor, ARRY-142886, prevented the progression of established lung fibrosis in rtTA-CCSP-TGF-α overexpressing mice [94]. To sum up, a growing body of evidence suggests that TGF-α-driven activation of the ErbB1 signaling pathways may play an important role in the development of lung fibrosis and that TGF-α might be amenable to targeted therapy.

#### *3.3. Amphiregulin*

Amphiregulin, another ErbB1 ligand, was discussed in the context of maladaptive remodeling of the liver and lung. In this respect, AREG-deficient mice were found to be protected against liver fibrosis induced by chronic administration of carbon tetrachloride (CCl4) [69]. To decipher the underlying molecular mechanism, several studies focused on the link between AREG and a master regulator of fibrogenesis, TGF-β1. Zhou et al. [95] reported that stimulation of fibroblasts with TGF-β1 elevates AREG production, which in turn increases cell proliferation and the expression of profibrotic genes, such as α-smooth muscle actin, collagen 1-α1/α2, fibronectin and tenascin. These effects were reversed by the treatment of TGF-β1-stimulated fibroblasts with AREG siRNA or ErbB1 inhibitors, AG1478 or gefitinib [95]. Consistent with these in vitro findings, AREG expression was markedly increased in the lungs of dox-CC10-TGF-β1 overexpressing mice and administration of AREG siRNA or AG1478 reduced collagen content and attenuated lung fibrosis in these animals. Besides AREG, the increased expression of other ErbB1 ligands, such as EREG and HB-EGF following exposure of fibroblasts to TGF-β1, was reported [96,97]. Andrianifahanana et al. [97] documented that TGFβ-induced AREG, EREG, and HB-EGF production requires the integration of an autocrine signal from a PDGF receptor and engages a positive feedback loop through ErbB1. The same authors demonstrated the pathological relevance of PDGFR-ErbB1 cooperation in the bleomycin model of lung fibrosis. Namely, they observed that simultaneous application of imatinib (a PDGF receptor inhibitor) and lapatinib (an ErbB1/2 inhibitor) is more effective than either treatment alone. Although, there is no evidence that pirfenidone and nintedanib directly interfere with the ErbB1, their ability to inhibit TGFβ and VEGF/PDGF/FGF receptors, respectively, might influence the overall ErbB1 activity in lung fibrosis. Accordingly, Shochet et al. reported that ErbB1 expression in donor LF triggered by IPF LF-culture media depends on FGF and can be reversed by nintedanib [73]. Interconnections of ErbB1 with other signaling pathways have to be considered when designing future IPF therapies.

The complexity of AREG cellular effects is underscored by the recent publication by Stancil et al., who showed the role of the AREG–ErbB1 axis in the jamming–unjamming of airway epithelial cells in IPF. Jamming transition describes a process of epithelial cell transformation from migratory (unjammed) to non-migratory (jammed) status in the absence of

wounding or cell-type changes. This transition is believed to play an important role during embryogenesis, in processes such as axis elongation and tissue development [98–100]. It was also associated with the pathogenesis of carcinomas [101] and asthma [102]. Stancil et al. [103] demonstrated in vitro that the unjammed phase is extended in distal airway epithelial cells of IPF patients and is associated with increased activity of the ErbB-YAP (Yes-Associated Protein) signaling pathway. YAP is a transcriptional co-activator, which was found to regulate epithelial progenitor cell proliferation in the lung [104] and epithelial– mesenchymal transition in lung cancer cells following exposure to TGF-β [105]. These findings are supported by the increased levels of AREG in IPF distal airway epithelial cells and its ability to induce jammed to unjammed transition in controlling distal airway epithelial cells in vitro [103]. Interestingly, the AREG-triggered extended unjammed phase of distal airway epithelial cells correlated with activation of the fibroblasts lying underneath [103], thus providing ample evidence for the contribution of airways epithelial cells repopulating distal parts of IPF lungs to the disease progression. The association between the AREG-driven prolonged unjammed status of distal airway epithelial cells and the gain-of-function *MUC5B* promotor variant underscores this assumption.

Besides its "bad" role in lung fibrosis, AREG was also found to contribute to the restoration of tissue homeostasis after acute lung injury driven by infection. Minutti et al. [106] showed that macrophage-derived AREG promotes TGF-β1 activation and subsequent differentiation of pericytes into collagen-producing myofibroblasts leading to restoration of vascular integrity in injured tissue and wound healing. Thus, not only TGF-β1 may regulate AREG expression but vice versa AREG can control the levels of active TGF-β1. It seems that the first scenario operates in fibrosis and the second under inflammatory conditions. Thus, the function of AREG may depend on its source, concentration and cellular and molecular landscape of the surrounding area. Although further research is needed to decipher the function of AREG in acute versus chronic pathological conditions, it becomes clear that identification of dynamics and causal flows in complex AREG signaling networks is crucial for its use as a therapeutic target. This assumption is supported by the study demonstrating attenuation of bleomycin-induced lung fibrosis upon AREG application during the late inflammatory phase [107]. This observation is in sharp contrast to the lung fibrosis reports mentioned above but it may be explained by the findings of Minutti et al. [106], namely, the AREG effects on blood vessel regeneration and thus epithelial cell survival in acute lung injury [95]. Overall, it seems that AREG properties may depend on the genetic background and the immune system condition, thus preselecting the potential responders prior to the treatment may raise the possibility of the success of an anti-AREG therapy in IPF.

#### *3.4. ErbB2 and ErbB3 Receptors and Their Ligands*

Another ErbB receptor that was linked to pulmonary fibrosis is ErbB2. Besides being an important oncogene in breast and ovarian cancer [44], ErbB2 was found to be involved in epithelial cell recovery upon acute lung injury. While ErbB2 was detected on the basolateral side of airway epithelial cells, HRG-α was only found in the apical membrane of these cells and in the overlying mucus film [108]. When epithelial integrity is disrupted, HRG-α translocates to Erb2 and enables a rapid response to injury. Thus, the Erb2-HRG-α systems sense changes in the extracellular environment and ensure restoration of barrier function that may be critical for survival. As there is no known ligand for ErbB2, this receptor is able to transduce intracellular signaling only upon forming a complex with other ErbB receptors. In pulmonary epithelial cells, ErbB2 is the preferred binding partner for ErbB3. Besides being engaged in the HRG-α-triggered intracellular signaling, the ErbB2–ErbB3 complex also responds to NRG1. Using a dominant-negative mutant of ErbB3 expressed under the SP-C promotor, Nethery et al. [109] demonstrated that SP-C-ErbB3 transgenic mice exhibit reduce collagen levels in the lung and better survival following bleomycin administration. The effect was associated with the inability of NRG1 to signal *via* the nonfunctional ErbB2– ErbB3 complex. These findings are corroborated by Faress et al. [110], who reported

preserved lung structure and diminished lung collagen content upon administration of an anti-ErbB2 antibody (2C4) to bleomycin-treated mice.

The important role of ErbB2 in bronchial epithelial cell differentiation and proliferation was shown by Vermeer et al. [66]. These authors demonstrated that treatment of airway epithelial cells with an anti-ErbB2 antibody, trastuzumab, induces their de-differentiation associated with an increase in the numbers of non-ciliated and metaplastic, flat cells. By contrast, the exposure of the cells to HRG-α preserved normal differentiation of airway epithelial cells. Most interestingly, co-culturing of airway epithelial cells with fibroblasts potentiated epithelial cell differentiation comparable to that achieved following treatment with HRG-α, pointing towards the ability of fibroblasts to produce ErbB ligands. Indeed, further studies demonstrated that normal human LF express TGF-α, HB-EGF, EREG, AREG, and HRG-α [66]. These observations were in line with the clinical case report describing reversible changes in airway epithelial cell differentiation of a breast cancer patient that coincided with the initiation and discontinuation of a trastuzumab therapy [66].

The ErbB3–NRG1-α axis was also discussed in the context of alveolar bronchiolization seen in the lungs of IPF patients. In these patients, NRG1-α was detected in epithelial cells lining honeycombing areas, as well as in normal submucosal glands [111]. In addition, elevated levels of this molecule were measured in IPF BALF [112]. Given the ability of NRG1-α to regulate airway mucus cell differentiation and MUC5B expression, it is worth speculating about its pivotal role in airway epithelial cell reprogramming and thus honeycomb cyst formation in IPF [113].

Taken together, it seems that ErbB2–ErbB3 activation is essential for the differentiation of airway epithelial cells and their integrity. Overactivation of this receptor complex system may induce abnormal behavior of airway epithelial cells thereby contributing to the honeycomb cyst formation and fibrosis progression. Because of the risks associated with the ErbB2–ErbB3 complex inhibition, close patient monitoring and patient categorization have to be taken into account when considering an anti-ErbB2–ErbB3 therapy in IPF.

#### **4. Conclusions**

A growing body of evidence suggests the pivotal role of the ErbB-ligand system in irreversible lung tissue scarring (Figure 2). The ErbB receptors and ligands were found to be overexpressed in IPF lungs and a number of preclinical studies demonstrated their pro-fibrotic properties in the loss-of-function and gain-of-function approaches. In addition, the therapeutic application of ErbB receptor/ligand inhibitors was often associated with favorable outcomes in lung fibrosis models (see Table 1). However, in view of the multifunctionality of the ErbB receptor–ligand system and its role in tissue regeneration, concern remains. Identification of dynamics and causal flows in the ligand–ErbB signaling network in acute versus chronic lung injury will be a prerequisite to maximize the chance of success of anti-ErbB/ligand agents in the clinical trials for IPF.

In addition, a lesson has to be also drawn from the remarkable progress in understanding the ErbB biology in cancer. It is now clear that the results of clinical trials can only be improved by taking into account a number of important issues. First, the effects of the targeted therapy may be weakened because of differences in etiology and heterogeneity. Furthermore, stratification of the patients according to a predominant disease mechanism have to be considered. Molecular endotyping should be integrated into the protocols of clinical trials. This strategy may promote the prudent use of novel targeted therapies. Finally, identification of the factors that can predict drug response or resistance will play a fundamental role to tailor individual ErbB-based therapy regimens.

TGFα α; α, heregulins; TGF β, transforming growth factor β; CCSP, NRG1α 1α; **Figure 2. Overview of the in vitro, in vivo and clinical findings for the role of the ErbB/ligand system in lung fibrosis.** LF, lung fibroblasts; TGFα, transforming growth factor-α; HB-EGF, heparinbinding epidermal growth factor-like growth factor; EREG, epiregulin; *AREG*, amphiregulin; HRG-α, heregulins; TGF-β, transforming growth factor-β; CCSP, Clara cell secretory protein; SP-C, surfactant protein-C; PI3K, phosphoinositide 3-kinase; MEK, mitogen-activated protein kinase kinase; IPF, idiopathic pulmonary fibrosis; ATII cells, alveolar type II cells; NRG1α, neuregulin-1α; BALF, bronchoalveolar lavage fluid; FVC, forced vital capacity; DLCO, diffusion capacity of the lung for carbon monoxide; ILD, interstitial lung disease; NSCLC, non-small-cell lung cancer.

α α α β1


**Table 1.** Anti-ErbB/ligand approaches in preclinical models of lung fibrosis.

<sup>1</sup> PI3K, phosphoinositide 3-kinase; <sup>2</sup> MEK, mitogen-activated protein kinase; <sup>3</sup> AREG, amphiregulin; <sup>4</sup> rtTA-CCSP, tetracycline-inducible Clara cell secretory protein; <sup>5</sup> TGF-α, transforming growth factor α; <sup>6</sup> dox-CC10, doxycycline-regulatable Clara cell 10-kDa protein; <sup>7</sup> TGF-β, transforming growth factor β1.

**Author Contributions:** Conceptualization, F.S.; original draft preparation, F.S. and M.W.; review and editing, F.S., L.S. and M.W.; discussions and suggestions, M.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** M.W. acknowledges financial support from the German Centerfor Lung Research (82 DZL 005A1).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

