Cell competition drives bronchiolization and pulmonary fibrosis

Club cells compete with BLCs to regenerate vs bronchiolize the lung parenchyma upon catastrophic injury to the lung parenchyma

After catastrophic injury to the lung parenchyma by influenza infection, bronchial epithelial stem cells (BESCs) in the airway have been proposed to undergo a binary response to reconstitute epithelial barriers giving rise to either alveolar epithelium or generate more airway epithelium and “bronchiolize” the lung parenchyma. However, it has been unclear whether one particular BESC subpopulation undergoes this binary response or whether there is competition between different BESC populations capable of either promoting alveolar epithelial regeneration or bronchiolization. This is largely because most lineage tracing experiments to target BESCs rely on Sox2^CreERT2;mTmG mice which lineage labels all bronchial epithelial cells.

It is well known that subsets of Club cells (e.g., BASCs) can give rise to both airway and alveolar epithelium^38,39,40 but do not contribute in a significant way to bronchiolization of the lung parenchyma after catastrophic injury mediated by H1N1 influenza²¹. However, because Club cells and especially BASCs are also destroyed by H1N1 influenza it has been difficult to assert whether they can contribute to alveolar epithelial regeneration if they survive the initial injury. To investigate this, we used Scgb1a1^CreERT;mTmG mice to lineage label Club cells, including BASCs and performed H1N1-mediated injury. Our experiments confirm previous reports that Club cells do not participate in the bronchiolization of the lung parenchyma after H1N1-mediated injury (Fig. 1A–D) which is known to be mediated by BLCs under this condition²¹. However, we find that if Club cells survive the initial assault they can contribute to alveolar epithelial regeneration after injury (Fig. 1B–D). Remarkably, Club cells regenerating alveolar epithelium or BLCs bronchiolizing the lung parenchyma are mutually exclusive events i.e. a binary response, suggesting that when Club cells survive the initial assault, they compete with BLCs, preventing them from invading and bronchiolizing the lung parenchyma.

Scgb1a1^CreERT;mTmG and Scgb1a1^CreERT;Myc^f/f;mTmG were placed on tamoxifen containing chow at 8 weeks of age for 3 weeks to inactivate Myc and permanently label all Club cells/BASCs and their offspring with GFP. After a 3 week wash-out period, mice were infected with H1N1 influenza virus, and lungs were harvested at 6 weeks post injury. Coimmunostaining for GFP (lineage label), Keratin 5 (Krt5; basal and BLCs), and Keratin 8 (Krt8; BLCs and transitional cells) on Scgb1a1^CreERT;mTmG (A) and Scgb1a1^CreERT;Myc^f/f;mTmG (E) lung sections. Coimmunostaining for GFP (lineage label), surfactant protein C (Sftpc; AT2 cells), Rage (AT1 cells) on Scgb1a1^CreERT;mTmG (B, C) and Scgb1a1^CreERT;Myc^f/f;mTmG (F, G) lung sections. White boxes in B and F are enlarged in (C, G), respectively. D Diagram demonstrating that normal Club cells inhibit BLCs and give rise to alveolar epithelial cells. H Diagram demonstrating that Myc deficient Club cells are outcompeted by Myc sufficient BLCs. Created in BioRender. Warren (2024) https://BioRender.com/ k46i357. I Nanostring nCounter analysis on RNA from Scgb1a1^CreERT;mTmG (n = 4) and Scgb1a1^CreERT;Myc^f/f;mTmG (n = 7) lungs for BLC genes Keratin 17 (p = 0.03, Log2 fold change = −1.87), Sox9 (p = 0.03, Log2 fold change = −1.04), and Krt5 (p = 0.047, Log2 fold change = −1.86). Data are Log2 normalized. J Lineage tracing analysis on immunostaining in B (n = 7), E (n = 2) using Aivia machine learning software (Sftpc/gfp p = 0.003, RAGE/gfp p = 0.6, Sum/gfp p = 0.03). K Hydroxyproline analysis on Cre- controls (n = 17) and Scgb1a1^CreERT;Myc^f/f;mTmG (n = 20) lungs normalized to control (p = 0.04). L Image analysis of total area of basal cells in control (n = 11) and Scgb1a1^CreERT;Myc^f/f;mTmG (n = 9) lung sections (p = 0.03). Data are presented as mean values +/− SEM. Scale bar: 250 μm. Two two-tailed unpaired T-test was used to determine significance. F test was used to determine equal variances and unreported F values indicate equal variance. *p < 0.05, **p < 0.01.

Full size image

In tissues harboring a mosaic imbalance in Myc protein levels, cells with higher Myc levels expand at the expense of cells with lower levels by eliminating them through apoptosis, inducing senescence, promoting autophagy or directing them to terminal differentiation and sloughing²⁸. To investigate whether Club cells compete with BLCs using the classic cell competition model we inactivated Myc in Club cells specifically using Scgb1a1^CreERT;Myc^f/f;mTmG mice while simultaneously lineage tracing them. We find that upon H1N1 injury alveolar epithelial regeneration by Club cells in Scgb1a1^CreERT;Myc^f/f;mTmG mice is impaired with the majority being outcompeted by BLCs (Fig. 1E–L) and the remainder giving rise preferentially to AT1 rather than AT2 cells compared to Scgb1a1^CreERT;mTmG control mice, that feature normal Myc levels. Scgb1a1^CreERT;Myc^f/f;mTmG lungs featured increased bronchiolization mediated by BLCs (Fig. 1E, I, L) and increased pulmonary fibrosis as measured by hydroxyproline content (Fig. 1K). Together these findings indicate that stem cell competition in the lung is governed by Myc levels.

Subsets of bronchial epithelial stem cells acquire myoepithelial cell characteristics after injury to the lung parenchyma

We next wanted to investigate how Myc levels in BLCs affect bronchiolization. To do this we performed immunostaining for Myc on lungs after H1N1 injury or severe bleomycin injury. We show that after catastrophic H1N1 or severe bleomycin injury some BESC offspring at the periphery or leading edge of the BC-pods feature high Myc levels (Fig. 2A–C, Supplementary Fig. 1A–H). Interestingly, these leading edge BLCs unlike trailing cells in the BC pods also express high levels of Sox9 and Acta2 (Fig. 2D, E), reminiscent of myoepithelial cells (MECs) in the submucosal gland^41,42 (SMG). Immunostaining and scRNAseq analysis of human IPF tissue demonstrate that subsets of BCs in honeycomb cysts of IPF lungs also feature high levels of Myc, Sox9 and/or Acta2 expression reminiscent of myoepithelial cells present in microdissected proximal airways of human donors confirming the proximalization of human IPF containing myoepithelial-like cells in the distal epithelium (Fig. 2F, G, Supplementary Fig. 1I–M).

A–E Coimmunostaining for myoepithelial cell markers Myc (A–C) or Sox9 (D, E), Krt5, and Acta2 (smooth muscle actin) on honeycomb regions in mouse lungs 17 days after bleomycin (A–D) and 14 or 21 days after H1N1 injury (B, C, E). F Coimmunostaining for Myc, Krt17, and Krt5 on honeycomb regions in human IPF tissue. (G) scRNAseq analysis of myoepithelial cell genes Krt5, Krt17, Myc, and Acta2 expression in human IPF vs donor lungs. Uniform Manifold Approximation and Projection (UMAP) of 10x scRNAseq data on human control (Donor Distal) and control including microdissected proximal airways (donor proximal) and IPF distal lungs demonstrating high Myc, Krt5, Krt17, Krt15, Acta2 expression in bronchiolized epithelium in IPF and proximal airway basal cells. Scale bar: 50 μm (A–C), 100 μm (D–F).

Full size image

To investigate if the MEC-like cells at the leading edge of the BC pods are derived from MECs in the SMG, we lineage labeled the latter prior to injury using a Nkx2.1^Flpo;Acta2-Frt-STOP-Frt-CreERT2;mTmG⁴³ intersectional mouse model in which we can specifically lineage label lung epithelial cells that co-express the lung epithelial cell marker Nkx2.1 and the mesenchymal Acta2 (α-SMA) marker. The Acta2-Frt-STOP-Frt-CreERT2 knock-in mouse line, possesses a CreERT2 cassette, inserted in the Acta2 locus, which is preceded by a STOP codon, flanked by Frt sites. As such, when crossed with Nkx2.1^Flpo expressing mice, Acta2-Frt-STOP-Frt-CreERT2 mice permanently express CreERT2 in Acta2 and Nkx2.1^Flpo co-expressing cells as well as their offspring, due to removal of the STOP codon. This then allows for the lineage labeling of MECs in Nkx2.1^Flpo;Acta2-Frt-STOP-Frt-CreERT2;mTmG after tamoxifen treatment.

Using this mouse model we find that SMG MECs do not migrate and give rise to BC pods after H1N1 injury (Fig. 3A, B). However, we can label de novo myoepithelial like cells in BC-pods by treating this same intersectional mouse model with tamoxifen after H1N1 injury (Fig. 3C, D), suggesting that BLCs, other than the MECs in the SMG, can acquire MEC-like characteristics upon catastrophic H1N1 or bleomycin injury. We were able to confirm these findings using two additional intersectional mouse models Trp63^DreERT2;Acta2^CreERT2;RLTG and Nkx2.1^Flpo; Acta2^CreERT2;FLTG (Fig. 3E, G–L).

A, B Nkx2.1^Flpo;Acta2-Frt-STOP-Frt^CreERT2;mTmG mice were place on tamoxifen containing chow for 3 weeks. Following a 3 week washout period, mice were infected with H1N1. At 6 weeks after injury, left lung lobes and trachea were inflation fixed, embedded in paraffin, and sectioned. Coimmunostaining for myoepithelial cell markers Acta2, Krt5, and lineage label GFP on Nkx2.1^Flpo;Acta2-Frt-STOP-Frt^CreERT2;mTmG trachea (E) and lung (F). C, D Nkx2.1^Flpo;Acta2-Frt-STOP-Frt^CreERT2;mTmG mice were intranasally administered H1N1. At 2 weeks after injury mice were placed on tamoxifen containing chow. At 6 weeks after injury, left lung lobes and trachea were inflation fixed, embedded in paraffin, and sectioned. Coimmunostaining for Krt5, Krt8, and GFP on Nkx2.1^Flpo;Acta2-Frt-STOP-Frt^CreERT2;mTmG lungs. E Trp63^DreERT2;Acta2^CreERT2;RLTG mice were intranasally administered H1N1 and placed on tamoxifen containing chow at 2 weeks after injury. tdTomato is induced in only Trp63^DreERT2 expressing cells and GFP is induced only when both Trp63^DreERT2 and Acta2^CreERT2 are expressed (myoepithelial cells). Coimmunostaining for RFP (tdtomato) and GFP on lungs at 6 weeks after injury. F Sox9^CreERT2;tdTomato mice were intranasally administered H1N1 and placed on tamoxifen containing chow at the injury. Coimmunostaining for RFP (tdTomato) and Krt5 on lungs at 6 weeks after injury. G–L Nkx2.1^Flpo; Acta2^CreERT2;FLTG mice were intranasally administered H1N1 and placed on tamoxifen containing chow at 2 weeks after injury with additional tamoxifen shots at 14 and 16 days after injury. tdTomato is induced in only Nkx2.1^Flpo expressing cells and GFP is induced only when both Nkx2.1^Flpo and Acta2^CreERT2 are expressed (myoepithelial cells). G Coimmunostaining for GFP and Krt5 on lungs at 6 weeks after injury and (H) image analysis using Aivia machine learning software (n = 4 animals). Coimmunostaining for GFP and (I) Dclk1 (tuft cells), (J) Foxj1 (ciliated cells), and (K, L) Scgb1a1 (secretory cells) and Krt5 (BLCs). Data are presented as mean values +/− SEM. Scale bar: 200 μm (A) 100 μm (D, F), 50 μm (B, G), 25 μm (C, E, I–L).

Full size image

Using Trp63^DreERT2;Acta2^CreERT2;RLTG we can specifically target MEC-like cells that co-express the basal cell transcription factor Trp63 and the mesenchymal Acta2 (α-SMA) marker. Tamoxifen exposure results in Dre-mediated excision of a polyA signal (STOP) from the RLTG dual recombinase reporter allele (Dre/Cre recombinase reporter) within Trp63 expressing intralobular basal cells, with subsequent Cre-mediated excision of tdTomato-STOP within Acta2-expressing MECs. Outcomes of these recombination events include tracing of Trp63⁺-intralobular basal cells by expression of tdTomato, and Trp63⁺/Acta2⁺ MECs by expression of eGFP (Fig. 3E).

Using Nkx2.1^Flpo; Acta2^CreERT2;FLTG we can specifically target MEC-like cells that co-express the lung epithelial-specific transcription factor Nkx2.1 and the mesenchymal Acta2 (α-SMA) marker. Flpo-mediated excision of a polyA signal (STOP) from the FLTG dual recombinase reporter allele (Flpo/Cre recombinase reporter) occurs within Nkx2.1 expressing lung epithelial cells, with subsequent tamoxifen-induced Cre-mediated excision of tdT-STOP within Acta2-expressing MEC like cells. Outcomes of these recombination events include tracing of Nkx2.1⁺-lung epithelial cells by expression of tdTomato, and Nkx2.1⁺/Acta2⁺ myoepithelial-like cells by expression of eGFP (Fig. 3G–L).

Finally, to investigate whether all cells in BC pods may be derived from these MEC like cells that lead the invasion we gave tamoxifen chow to Sox9^CreERT2;mTmG mice after H1N1 injury and found that all cells in the BC pods were lineage labeled (Fig. 3F), indicating that all cells in basal cell pods either induced Sox9 expression at some point during the invasion of BC pods or are all derived from the MEC-like stem cells at the leading edge of the invasion. Indeed, treating Nkx2.1^Flpo; Acta2^CreERT2;FLTG mice with tamoxifen 2 weeks after H1N1 injury, lineage labels MEC-like cells and their offspring and demonstrates that myoepithelial-like cells do differentiate into conducting airway epithelial cells such as Dclk1⁺ tuft cells (Fig. 3I), Foxj1⁺ ciliated cells (Fig. 3J) and Scgb1a1⁺ club cells (Fig. 3K, L). This is interesting as it indicates that super-competitor myoepithelial like cells may give rise to all trailing cells in the basal cell pods. Our findings further suggest that the process of bronchiolization is reminiscent of the process that drives submucosal gland development, suggesting that BC pods may be considered as de novo submucosal glands^44,45.

Myc drives bronchiolization and fibrosis through the generation of myoepithelial-like cells

To investigate if Myc levels in BESCs affect stem cell competition after severe bleomycin or H1N1 injury we generated Sox2^CreERT2;Myc^f/f;mTmG mice in which we can inactivate Myc in all BESCs, in order to level fitness levels, while simultaneously lineage labeling them. When we perform bleomycin (Fig. 4B–K) or H1N1 (Fig. 4L–U) injury on Sox2^CreERT2;Myc^f/f;mTmG mice, in which we inactivated Myc in BESCs prior to injury (Fig. 4A), BESCs fail to acquire MEC-status and fail to bronchiolize the lung parenchyma, as demonstrated by impaired basal cell pod generation (Fig. 4B, E, K, L, O, U) and reduced expression of bronchial epithelial markers Muc5b, Muc5ac and Krt5 by Nanostring nCounter RNA analysis (Fig. 4I), qPCR analysis (Fig. 4S) and 10x Visium spatial transcriptomics (Supplementary Fig. 2). Interestingly, BESCs in Sox2^CreERT2;Myc^f/f;mTmG lungs, presumably Club cells/BASCs, give rise to more alveolar epithelium after severe bleomycin injury (Fig. 4C, D, F, G, J) but less alveolar epithelium after H1N1 injury (Fig. 4M, N, P, Q, T) compared to control mice. This is likely because inactivation of Myc in Club cells prevents them from giving rise to basal cells and allowing them only to give rise to alveolar epithelium after bleomycin injury resulting in a net increase in regeneration, whereas decreasing Club cell fitness through Myc inactivation likely also makes them more vulnerable to H1N1 infection. As a corollary, Sox2^CreERT2;Myc^f/f;mTmG lungs feature reduced pulmonary fibrosis based on hydroxyproline content (Fig. 4H) after bleomycin injury, but increased pulmonary fibrosis after H1N1 injury (Fig. 4R). However, in the H1N1 injury model in which most BASCs and AT2 stem cells are destroyed we find that that the inability of BLCs to robustly participate in the immediate regenerative response, even though maladaptive, is detrimental to survival (Supplementary Fig. 6I) and likely contributes to the increased pulmonary fibrosis (Fig. 4R) compared to littermate controls. Our theory is that de novo submucosal gland development (basal cell pod development) near terminal bronchioles and lung parenchyma that have been catastrophically damaged, is a way to seal off or plug those damaged conducting airways preventing more air from entering the lung via that route, which would cause further mechanical damage to the lung parenchyma.

A Mice were placed on tamoxifen chow for 3 weeks to inactivate Myc and permanently lineage label bronchial epithelial cells and their offspring. Following a 3-week washout period, mice were injured with intratracheal administration of bleomycin (B–K) or intranasal administration of H1N1 (L–U) and lungs were harvested at 6 weeks post injury. (B, E, L, O) Immunostaining for Krt5 (basal and BLCs) and Acta2 (smooth muscle actin; myofibroblasts) on bleomycin (B, E) and influenza (L, O) injured Sox2^CreERT2;mTmG and Sox2^CreERT2;Myc^f/f;mTmG. (C, F, M, P) Immunostaining for Rage (AT1 cells), GFP (lineage label), and Sftpc (AT2 cells) on Sox2^CreERT2;mTmG and Sox2^CreERT2;Myc^f/f;mTmG. (D, G, N, Q) Models demonstrating that Myc sufficient Club cells inhibit BLCs and give rise to alveolar epithelial cells after bleomycin (D) and influenza (N) injury but Myc insufficient BLCs fail to give rise to basal cell pods (G, Q). Created in BioRender. Warren (2024) https://BioRender.com/j04r129. H Hydroxyproline analysis on bleomycin injured Cre- controls (n = 26) and Sox2^CreERT2;Myc^f/f (n = 19) (p = 0.04). I Nanostring nCounter analysis on RNA from bleomycin injured Cre- controls (n = 17) and Sox2^CreERT2;Myc^f/f;mTmG (n = 6) lungs BLC genes (Muc5ac p = 0.01, Muc5b p = 0.003, Krt5 p = 0.046). Data are Log2 normalized. J Lineage tracing analysis on bleomycin injured lungs from immunostaining in (C) (n = 2), (F) (n = 5) using Aivia machine learning software (p = 0.008). K Image analysis of the total area of basal cells in bleomycin injured control (n = 3) and Sox2^CreERT2;Myc^f/f;mTmG (n = 3) lung sections (p = 0.04). R Hydroxyproline analysis on influenza injured Cre- controls (n = 8) and Sox2^CreERT2;Myc^f/f (n = 5) (p = 0.004). S qPCR analysis on RNA from influenza injured Cre- controls (n = 12) and Sox2^CreERT2;Myc^f/f;mTmG (n = 7) lungs for BLC genes (Krt5 (p = 0.04, F = 3.32×10⁻⁹) and Tp63 (p = 0.01, F = 6.29×10⁻⁸)). T Lineage tracing analysis on influenza-injured lungs from immunostaining in M (n = 4), (P) (n = 5) using Aivia machine learning software (p = 0.04). U Image analysis of total area of basal cells in influenza injured control (n = 5) and Sox2^CreERT2;Myc^f/f;mTmG (n = 13) lung sections (p = 0.007). Data are presented as mean values +/− SEM. Scale bars: 100 µm. Magnification insets are 400x larger. Two tailed unpaired T-test was used to determine significance. F test was used to determine equal variances and unreported F values indicate equal variance. *p < 0.05, **p < 0.01.

Full size image

Inactivation of Myc in basal cell pods post H1N1 injury promotes their differentiation into AT1 cells

So far, our findings suggest that Myc levels in lung stem cells determine their fitness levels and that cells with the lowest Myc levels differentiate into AT1 cells. Since BC-pods are known to persist in the lung long after H1N1 infection, we wondered whether Myc is required for their maintenance and/or expansion post H1N1 injury. To investigate this we infected Krt5^CreERT2;mTmG and Krt5^CreERT2;Myc^f/f;mTmG mice with H1N1 influenza, and lineage labeled their BLCs with or without simultaneous inactivation of Myc, starting at 2 weeks after injury (Fig. 5C). Interestingly, we find that upon inactivation of Myc in BC pods in Krt5^CreERT2;Myc^f/f;mTmG mice, BC-pods are reduced in size, as indicated by less GFP RNA per Krt5 transcript, and fewer basal cells in Krt5^CreERT2;Myc^f/f;mTmG lung sections (Fig. 5A, B, F) compared to H1N1 injured Krt5^CreERT2;mTmG control mice. In addition, we find that compared to H1N1 injured Krt5^CreERT2;mTmG mice, fibrosis is reduced in Krt5^CreERT2;Myc^f/f;mTmG mice, in which we inactivated Myc in BLCs and myoepithelial-like cells after injury (Fig. 5G). More strikingly we find that inactivation of Myc in BC pods post H1N1 injury affects BC stem cell maintenance over time and allows for their differentiation towards the AT1 lineage by 12 weeks after H1N1 injury (Fig. 5I–Q).

C, K Krt5^CreERT2;mTmG and Krt5^CreERT2;Myc^f/f;mTmG were infected with H1N1 at 8 weeks of age. At 2 weeks after injury, mice were placed on tamoxifen chow to inactivate Myc and permanently label all BLCs and their offspring with GFP and lungs were harvested at 6 (A–H) or 12 (I–Q) weeks post injury. A, B Coimmunostaining for Keratin 8 (Krt8; BLCs and transitional cells), GFP (lineage label), and Keratin5 (Krt5; basal and BLCs), (D, E) and coimmunostaining for Muc5b (mucus-producing secretory cells) and GFP (lineage label) on Krt5^CreERT2;mTmG (A, D) and Krt5^CreERT2;Myc^f/f;mTmG (B, E). F qPCR analysis for Gfp, Krt5, and Muc5b Cre- control (n = 5) and Krt5^CreERT2;Myc^f/f (n = 5). Values are graphed as ratios (GFP/Krt5 p = 0.046, GFP/Muc5b p = 0.79, Krt5/Muc5b p = 0.006). G Hydroxyproline analysis on Cre- control (n = 10) and Krt5^CreERT2;Myc^f/f (n = 16) (p = 0.01). H Image analysis of total area of basal cells in influenza injured control (n = 6) and Krt5^CreERT2;Myc^f/f;mTmG (n = 8) lung sections (p = 0.02). I, J Coimmunostaining for Rage (AT1 cells) and GFP (lineage label) on Krt5^CreERT2;mTmG and Krt5^CreERT2;Myc^f/f;mTmG lungs at 12 weeks post injury with magnification in O–Q. L Lineage tracing analysis on influenza injured lungs from immunostaining in I and (J) (n = 3). Area of GFP (p = 0.02), Sftpc (p = 0.03), and Rage (p = 0.02) were determined using Aivia machine learning software. M, N Diagram depicting that Myc deficient basal cells pods are smaller than Myc sufficient basal cell pods and can differentiating into Rage+ AT1 cells at 12 weeks after influenza injury. Created in BioRender. Warren. (2024) https://BioRender.com/ w28w946. Data are presented as mean values +/− SEM. Scale bars: 250 µm (A, B, D, E), 125 µm (I, J), 25 µm (O–Q). The two-tailed unpaired T-test was used to determine significance. F test was used to determine equal variances and unreported F values indicate equal variance. *p < 0.05, **p < 0.01.

Full size image

Overexpression of Myc endows subsets of BESCs with a super competitor myoepithelial like status which can outcompete AT2 stem cells

We next wondered what would happen if we boosted the fitness of Club cells by overexpressing Myc in Club cells after a less severe bleomycin injury. Overexpression of a dominant active version of the Hippo transcriptional effector Yap1^S112A in Club cells has been shown to direct their differentiation along the BLC lineages⁴⁶, and Myc and Yap have both been shown to be important for cell competition^{29,31,36,47,48}.

Interestingly, when we overexpress Myc in Club cells/BASCs after bleomycin injury (Fig. 6A), Club cells/BASCs massively acquire a super-competitor myoepithelial cell (SCMC) like status, coexpressing Krt5, Acta2, Sox9 and Myc (Fig. 6B–M), resulting in the hyper-invasion and apparent destruction of the lung parenchyma including AT2 cells and its replacement with bronchial epithelial cells demonstrated by increased expression of bronchial epithelial markers Krt5, Krt17, and Muc5b, increased pulmonary fibrosis based on hydroxyproline content and reduced expression of alveolar epithelial marker Sftpc by Nanostring nCounter RNA analysis (Fig. 6B–P) and 10x Visium spatial transcriptomics (Supplementary Fig. 3). This suggests that the cell competition program may converge onto a SCMC plastic like state that can be acquired by different BESC populations.

A Mice were placed on tamoxifen chow for 3 weeks and following a 3-week washout period, mice were injured with intratracheal administration of bleomycin and placed on doxycycline containing chow to induce Myc overexpression and harvested at 6 weeks post injury. B–D, H–J Coimmunostaining for markers associated with fibrosis and bronchiolization (Col1a1, Krt5, and Acta2) on control (B–D) and Scgb1a1^CreERT;LSL-rtTA;Tet-Myc (H–J). Coimmunostaining for myoepithelial cell-like markers Sox9, Krt5, and Myc on control (E, F) and Scgb1a1^CreERT;LSL-rtTA;Tet-Myc (K, L). G, M Diagram demonstrating that Myc sufficient Club cells inhibit basal cells and give rise to alveolar epithelial cells after bleomycin injury while Myc overexpressing Club cells dedifferentiate into basal cells and promote their invasion into the alveolus. Created in BioRender. Warren, R. (2024) https://BioRender.com/ l07p577. N Immunostaining for Muc5b (mucus producing secretory cells), Krt5 (BLCs), and Scgb1a1 (Club cells/BASCs) on Scgb1a1^CreERT;LSL-rtTA;Tet-Myc. O Hydroxyproline analysis on Cre- control (n = 31) and Scgb1a1^CreERT;LSL-rtTA;Tet-Myc (n = 30) normalized to control (p = 0.003). P Log2 normalized values for RNA expression for BLC (Krt5, Krt17, Scgb3a2, Muc5b) and AT2 cell (Sftpc) genes from NanoString analysis on control (n = 9 Krt5 p = 0.0000008, Krt17 p = 0.000001, Scgb3a2 p = 0.02, Sftpc p = 0.02 F = 0.02, Muc5b p = 0.002), Scgb1a1^CreERT;LSL-rtTA;Tet-Myc (n = 5), and Scgb1a1^CreERT;Yap^f/f;LSL-rtTA;Tet-Myc (n = 8 Krt5 p = 0.0001, Krt17 p = 0.00006, Scgb3a2 p = 0.0007, Sftpc p = 0.04,Muc5b p = 0.02). Values are normalized to control and p values are compared to Scgb1a1^CreERT;LSL-rtTA;Tet-Myc. Data are presented as mean values +/− SEM. Scale bars: 500 µm (B, G, L) and 100 µm (C–F, H–K). Two tailed unpaired T-test was used to determine significance. F test was used to determine equal variances and unreported F values indicate equal variance. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Full size image

Lung epithelial stem cell fitness levels are determined by their Myc levels, which are tuned by the Hippo pathway

It is well known that Hippo pathway plays an important role in cell competition, and Yap and Myc are thought to work together in this process^{29,31,36,47,48}. It is also well known that Myc is a quintessential target gene of the canonical Wnt signaling pathway⁴⁹ and that the Hippo pathway controls β-catenin stabilization and nuclear localization^50,51. However, how the Hippo pathway affects Myc levels seems to be context dependent.

Interestingly, the Hippo pathway is active in AT2⁵² and Club cells, demonstrated by Merlin expression, phospho-Yap and phospo-Mst1/2 staining (Supplementary Fig. 5A–E), resulting in the degradation and cytoplasmic retention of Yap and Taz the nuclear effectors of the pathway. To investigate how increased Yap and/or Taz levels may affect Myc expression in BESCs cells we inactivated the Hippo kinases Mst1/2 (encoded by Stk4/3) in BESCs, and found that this resulted in decreased Myc expression, decreased bronchiolization, increased AT1 cell regeneration and reduced pulmonary fibrosis based on hydroxyproline content upon severe bleomycin injury (Fig. 7A–E, L, N, O, Supplementary Fig. 4). Interestingly, inactivation of the Hippo pathway via deletion of Nf2 in Club cells or AT2 stem cells in the absence of injury is sufficient to drive their spontaneous differentiation into AT1 cells, consistent with previous reports^53,54 (Supplementary Fig. 5F, G & 8A–H, Q).

A Mice were placed on tamoxifen chow for 3 weeks to inactivate Yap1 and/or Wwtr1 or Stk3/4 and permanently label all Sox2+ cells and their offspring with GFP. Following a 3 week washout period, mice were injured with intratracheal administration of bleomycin and harvested at 6 weeks post injury. Coimmunostaining for Rage (AT1 cells), GFP (lineage label), and Sftpc (AT2 cells) on Sox2^CreERT2;mTmG (B), Sox2^CreERT2;Stk3^f/f;Stk4^f/f;mTmG (D), Sox2^CreERT2;Yap1^f/f;mTmG (F), Sox2^CreERT2;Wwtr1^f/f;mTmG (H), and Sox2^CreERT2;Yap1^f/f;Wwtr1^f/f;mTmG (J) lungs. C, E, G, I, K diagrams illustrating the direction of airway epithelial cell differentiation in figures (B, D, F, H, J). Created in BioRender. Warren (2024) https://BioRender.com/ a02e401. L Lineage tracing analysis on bleomycin injured lungs from immunostaining in B (n = 3) and D (n = 4) using Zeiss Zen Intellesis machine learning software to trace (p = 0.57). M Lineage tracing analysis on bleomycin-injured lungs from immunostaining in B (n = 3), F (n = 5, p = 0.92), H (n = 4, p = 0.002), and J (n = 4, p = 0.004) using Aivia machine learning software. N Hydroxyproline analysis on Cre- control, Sox2^CreERT2;Yap1^f/f;Wwtr1^f/f (n = 19, p = 0.04), Sox2^CreERT2;Yap1^f/f (n = 25, p = 0.01), Sox2^CreERT2;Wwtr1^f/f (n = 9, p = 0.04), and Sox2^CreERT2;Stk3^f/f;Stk4^f/f (n = 18, p = 0.03). O qPCR analysis for bronchiolization and fibrosis genes (Krt5, p63, Col1a1, Col3a1 and Muc5b) on Cre- control (n = 22), Sox2^CreERT2;Yap1^f/f;Wwtr1^f/f (n = 8, Krt5 p = 0.03, F = 0.00001, p63 p = 0.03, F = 0.01, Col1a1 p = 0.2, Col3a1 p = 0.008, and Muc5b p = 0.05), Sox2^CreERT2;Yap1^f/f (n = 5, Krt5 p = 0.01, F = 0.00007, p63 p = 0.002, F = 0.005, Col1a1 p = 0.002, Col3a1 p = 0.0005 and Muc5b p = 0.8), Sox2^CreERT2;Wwtr1^f/f (n = 10, Krt5 p = 0.27, p63 p = 0.05, Col1a1 p = 0.03 F = 0.02, Col3a1 p = 0.004 and Muc5b p = 0.05), and Sox2^CreERT2;Stk3^f/f;Stk4^f/f (n = 10, Krt5 p = 0.009, F = 5.7 × 10^-8, p63 p = 0.01 F = 0.05, Col1a1 p = 0.95, Col3a1 p = 0.16 and Muc5b p = 0.00009 F = 0.003). qPCR for Myc on control (n = 23), Sox2^CreERT2;Yap1^f/f;Wwtr1^f/f (n = 14, p = 0.05) and Sox2^CreERT2;Stk3^f/f;Stk4^f/f (n = 10, p = 0.04). Values are represented as 2^-ΔΔCt normalized to Control. Values are normalized to control. Data are presented as mean values +/− SEM. Scale bar: 200 µm. Two tailed unpaired T-test was used to determine significance. F test was used to determine equal variances and unreported F values indicate equal variance. *p < 0.05, **p < 0.01, ***p < 0.001.

Full size image

To investigate how decreased Yap/Taz levels may affect Myc expression in BESCs, we inactivated Yap1 and Wwtr1 in BESCs, using Sox2^CreERT2;Yap1^f/f;Wwtr1^f/f;mTmG mice, and found that this resulted in increased Myc expression, decreased AT1 cell regeneration and increased pulmonary fibrosis based on hydroxyproline content upon severe bleomycin injury (Fig. 7J, K, M–O). Even though Sox2^CreERT2;Yap1^f/f;Wwtr1^f/f;mTmG mice feature increased Myc levels, their lack of Yap prevents them from BLC-mediated bronchiolization (Fig. 7F, J, K, O). Instead, Sox2^CreERT2;Yap1^f/f;Wwtr1^f/f;mTmG mice featured increased goblet cell differentiation based on Muc5b expression (Fig. 7O), something we also observed in the absence of injury (Supplementary Fig. 5H, I, L), and which is consistent with previous reports⁵⁵ demonstrating the spontaneous differentiation of BESCs into goblet cells upon simultaneous inactivation of Yap1 and Wwtr1.

Together these findings suggest that the Hippo pathway controls Myc levels and therefore stem cell competitiveness by controlling Yap and/or Taz levels. This is consistent with a role for cytoplasmic Taz inhibiting Wnt/β-catenin signaling^50,51. Interestingly, overexpression of a dominant active β-catenin in the airway epithelium has been shown to result in excessive goblet cell differentiation⁵⁶.

Yap-Myc-p63 promote bronchiolization whereas Taz promotes AT1 differentiation

The fact that inactivation of upstream Hippo kinases (Nf2, Stk3/4, Lats1/2) in Club⁵⁷ (Supplementary Fig. 6F, G) or AT2 (Fig. 8A–H)⁵² cells results in their spontaneous differentiation into AT1 cells is intriguing since we and others have previously demonstrated that Yap is required for tracheal BC maintenance^46,58 and overexpression of a dominant active version of the Hippo transcriptional effector Yap1^S112A in Club cells is able to drive their differentiation towards a BLC lineage in cooperation with p63⁴⁶. Together all these findings suggest a role for Yap-Myc-Trp63 in the acquisition of SCMC state whereas an increase in Taz or a lack of Yap-Myc-Trp63 promotes AT1 cell differentiation.

A–P Sftpc^CreERT2;mTmG, Sftpc^CreERT2;Nf2^f/f;mTmG, Sftpc^CreERT2;Nf2^f/f;Yap1^f/f;mTmG, and Sftpc^CreERT2;Nf2^f/f;Wwtr1^f/f;mTmG were placed on tamoxifen containing chow at 8 weeks of age for 3 weeks to inactivate Nf2 and/or Yap1 and/or Wwtr1 and permanently lineage label AT2 stem cells and their offspring. At 9 weeks after being placed on normal chow left lung lobes were inflation fixed, embedded in paraffin, and sectioned. Coimmunostaining for GFP (lineage label), Rage (AT1 cells), and Sftpc (AT2 cells). Q Image analysis for lineage labeled AT1 and AT2 cells in A-P using Aivia machine learning software (Sftpc^CreERT2;mTmG (n = 6), Sftpc^CreERT2;Nf2^f/f;mTmG (n = 8, p = 0.00003), Sftpc^CreERT2;Nf2^f/f;Yap1^f/f;mTmG (n = 5, p = 0.0009), and Sftpc^CreERT2;Nf2^f/f;Wwtr1^f/f;mTmG (n = 6, p = 0.13)). Data are presented as mean values +/− SEM. Two tailed unpaired T-test was used to determine significance. F test was used to determine equal variances. Scale bar: 200μm. *p < 0.05.

Full size image

Interestingly, though we have long favored a role for Taz and not Yap in AT1 cell differentiation and maintenance, some reports seem to suggest a role for Yap in AT1 cell differentiation^59,60. To definitively answer this question we generated Sftpc^CreERT2;Nf2^f/f;Wwtr1^f/f;mTmG and Sftpc^CreERT2;Nf2^f/f;Yap1^f/f;mTmG mice to investigate which nuclear effector of the Hippo pathway is required for the spontaneous differentiation of AT2 cells into AT1 cells upon Nf2 inactivation. We now demonstrate that the simultaneous inactivation of Nf2 and Yap1 in AT2 cells does not inhibit their spontaneous differentiation into AT1 cells (Fig. 8A–L, Q), whereas the simultaneous inactivation of Nf2 and Wwtr1 in AT2 cells completely blocks this process (Fig. 8A–Q).

If Taz promotes AT1 cell differentiation and Yap promotes BLC differentiation, inactivation of Wwtr1 in bronchial epithelium using Sox2^CreERT2;Wwtr1;mTmG mice should mainly impair AT1 cell differentiation but not bronchiolization, whereas inactivation of Yap1 in airways using Sox2^CreERT2;Yap1^f/f;mTmG mice should mainly impair bronchiolization. Interestingly, Sox2^CreERT2;Wwtr1;mTmG mice not only feature impaired AT1 cell differentiation but also increased bronchiolization (Fig. 7B, C, H, I, M, O). The latter is likely because Yap and Taz compete to bind to Tead transcription factors, with a loss of Taz resulting increased Yap/Tead binding. Vice versa, Sox2^CreERT2;Yap1^f/f;mTmG mice fail to generate BLCs and to bronchiolize the lung parenchyma upon severe bleomycin injury or H1N1 injury, with increased AT1 cell differentiation likely due to increased Taz/Tead, and increased pulmonary fibrosis as measured by hydroxyproline content as well as increased mortality (Fig. 7B, C, F, G, M, O & Supplementary Fig. 6A–I).

Since overexpression of dominant active Yap1^S112A in BESCs is sufficient to drive Club cell to BLC differentiation⁴⁶, we wondered if overexpression of dominant active Yap1^S112A alone in BESCs and their offspring after bleomycin injury, using Sox2^CreERT2;LSL-rtTA;Tet-Yap1^S112A mice, is sufficient to promote bronchiolization. Interestingly, while overexpression of a dominant active Yap1^S112A in BESCs is sufficient to drive BLC differentiation and prevent alveolar epithelial differentiation after bleomycin injury (Supplementary Fig. 6H–N), overexpression of dominant active Yap1^S112A did not induce Myc expression, and BESCs failed to acquire SCMC status and as such did not amplify nor invade the lung parenchyma nor destroy the remaining alveolar epithelium (Supplementary Fig. 6H–N).

This is interesting as we have just demonstrated that overexpression of Myc in Club cells after bleomycin injury causes them to acquire a super-competitor myoepithelial cell (SCMC) like status, coexpressing Krt5, Acta2, Sox9 and Myc (Fig. 6). Therefore, to specifically investigate the requirement for Yap and Myc in the acquisition of MEC-status we generated Scgb1a1^CreER;Yap1^f/f;LSL-rtTA;Tet-Myc mice in which we could simultaneously inactivate Yap1 and overexpress Myc in Club cells after bleomycin injury and found that BCs and MEC-like cells development was impaired, compared to Scgb1a1^CreER;LSL-rtTA;Tet-Myc mice, indicating that both Myc and Yap are required for obtaining SCMC status (Fig. 6P).

Finally, to investigate if AT2 stem cells have the capacity to acquire a SCMC state and to bronchiolize the lung parenchyma we generated Sftpc^CreERT2;LSL-rtTA;Tet-Myc, Sftpc^CreERT2;LSL-rtTA;Tet-Yap1^S112A and Sftpc^CreERT2;LSL-rtTA;Tet-Myc;Tet-Yap1^S112A mice in which we could respectively overexpress Myc, a dominant active Yap1^S112A or both in combination in AT2 stem cells and their progeny after bleomycin injury. We found that simultaneous overexpression of both Myc and Yap1^S112A in AT2 cells allowed them to adopt SCMC state and to bronchiolize the lung parenchyma (Supplementary Fig. 7A–D, M–O). However, overexpression of Myc or Yap1^S112A alone was not sufficient (Supplementary Fig. 7E-L).

• SEO Powered Content & PR Distribution. Get Amplified Today.
• PlatoData.Network Vertical Generative Ai. Empower Yourself. Access Here.
• PlatoAiStream. Web3 Intelligence. Knowledge Amplified. Access Here.
• PlatoESG. Carbon, CleanTech, Energy, Environment, Solar, Waste Management. Access Here.
• PlatoHealth. Biotech and Clinical Trials Intelligence. Access Here.
• Source: https://www.nature.com/articles/s41467-024-54997-2