Sentinel p16INK4a+ cells in the basement membrane form a reparative niche in the lung

Abstract

We engineered an ultrasensitive reporter of p16^INK4a, a biomarker of cellular senescence. Our reporter detected p16^INK4a-expressing fibroblasts with certain senescent characteristics that appeared shortly after birth in the basement membrane adjacent to epithelial stem cells in the lung. Furthermore, these p16^INK4a+ fibroblasts had enhanced capacity to sense tissue inflammation and respond through their increased secretory capacity to promote epithelial regeneration. In addition, p16^INK4a expression was required in fibroblasts to enhance epithelial regeneration. This study highlights a role for p16^INK4a+ fibroblasts as tissue-resident sentinels in the stem cell niche that monitor barrier integrity and rapidly respond to inflammation to promote tissue regeneration.

Figure 1.. INKBRITE identifies p16^INK4A+ cells with senescent characteristics in vivo.

(A) Target construct design for INKBRITE. (B) FACS analysis of GFP+ cells from INKBRITE lungs and qPCR of sorted GFP+ and GFP−neg cells (n=2). (C) Wholemount image of the airway from thick-sectioned INKBRITE lung rendered on Imaris. (D to F) Immunohistochemistry (IHC) and quantification of BrdU incorporation into PDGFRα+ and GFP– (white arrows) or PDGFRα+ and GFP+ (green arrows) cells during alveologenesis or homeostasis (n=4 alveologenesis, n=5 homeostasis). (G and H) IHC and quantification of freshly sorted of GFP+ and GFP– fibroblasts for polynucleation (2 experiments, n=29–31 images per cell type). (I) FACS data with quantification of % GFP+ cells and mean GFP+ intensity of PDGFRα+ fibroblasts over the lifespan of INKBRITE animals (n; PND0=7, PND7=7, 2m=5, 22m=5). (J) Histogram display the of GFP intensity in PND0, PND7, 2m, and 22m PDGFRα+ lung fibroblasts (n= 5 per timepoint, 2 experiments). AW=airway, BM=basement membrane, PND=Postnatal day. Scale bars 100υm. Each point in graph represents one animal or one distinct image for in vitro studies with mean ± s.e.m. All p values determined by one-tailed t-test and two-way ANOVA when applicable. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Figure 2.. Range of p16^INK4a expression correlates with proliferative cell cycle arrest.

(A) Sorting strategy for GFP−high, low, and negative (hi/lo/neg) fibroblasts cells from uninjured INKBRITE lungs using GFP fluorescent intensity. (B) qPCR of p16^INK4A transcript in GFPhi/lo/neg populations (n=4, >2 experiments). (C) Histogram of CellTrace Far Red (CTFR) intensity in INKBRITE lung fibroblasts. (D) quantification of % cell cycle arrest based on percentage of cells with CTFR intensity of serum-deprived cells (n=4, >2 experiments). (E to I) Histology, FACS and quantification of CTFR+ transplanted INKBRITE fibroblasts into Bleomycin injured NGS™ mice (2 experiments, n=4). (J to L) IHC and quantification of BrdU incorporation into PDGFRα+ and GFP–(white arrows) or PDGFRα+ and GFP+ (green arrows) cells in vehicle or naphthalene-injured (14 dpi) lungs (n = 3 for vehicle, 6 for naphthalene). (M) GFP intensity distribution of EdU+ and EdU− fibroblast isolated from naphthalene injured lungs (14 dpi). (N) EdU uptake Index (%EdU+/%EdU−) of GFPhi/lo/neg fibroblast populations in vehicle and naphthalene treated (14 dpi) fibroblasts (n = 5 per condition, 2 experiments). (O) CTFR retention in GFP+ fibroblasts of 2m, 12m, and 25m old INKBRITE lungs (n = 3 per timepoint, 2 experiments). AW = airway, dpi = days post injury. Each point in graph represents one animal with mean ± s.e.m. All p values determined by one-tailed t-test or two-way ANOVA when applicable. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Figure 3.. Single cell and bulk RNA sequencing analysis of p16^INK4A+ cells in the lung.

(A) Bulk RNAseq of p16^INK4A+ and – fibroblasts during homeostasis (0 dpi) and injury (7,14,28 dpi) (n=3 animals per timepoint). (B) Gene correlation plot showing Cdkn2a expression. (C) Hypergeometric probability test for enrichment of differentially expressed genes (DEGs) between GFP+ and GFP−neg fibroblasts at each timepoint with SASP genes as quantified by Representation Factor and p-value. (D and E) Single cell analysis of all p16^INK4A+ cells in 0 dpi and 14 dpi INKBRITE lungs. (F) Clustering of p16^INK4A+ fibroblasts into distinct subsets in 0 dpi and 14 dpi INKBRITE lungs. (G) SASP gene expression at the single cell level in the 4 fibroblast subsets. (H to J) Histologic analysis of F4/80+ and GFP+ and F4/80+ macrophage/monocyte population in 0 dpi and 14 dpi INKBRITE lungs (0, 14, and 28 dpi; n=3). AW = airway, dpi = day post injury. Each point in graph represents one animal with mean ± s.e.m. All p values determined by one-tailed t-test. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Figure 4.. Injured p16^INK4A+ fibroblast enhances epithelial progenitor proliferation ex vivo.

(A) 3D organoid assay combining uninjured Scgb1a1+ airway stem cells (tdTomato+) with p16^INK4Ahi/lo/neg fibroblasts isolated from INKBRITE lungs at 0 or 14 dpi. (B) Quantification of Scgb1a1+ organoid numbers and (C) size (n=3 triplicate wells per condition, >2 experiments). (D) qPCR of Ereg on sorted GFP+ or – fibroblasts from INKBRITE lungs at 0 or 14 dpi (n = 9 per timepoint, >2 experiments). (E and F) Histologic quantification of airway stem cell regeneration in Ereg knockout (KO) vs. WT lungs at 14 dpi (n = 4 per genotype, 2 experiments) (G to I) IHC quantification of GFP+ cells, (J to L) SCGB1A1+ cells, and (M to O) pERK+SCGB1A1+ cells in vehicle vs. D&Q treated INKBRITE lungs after naphthalene injury (14 dpi, vehicle n=4, D&Q n=7; 2 experiments). AW = airway, D&Q = dasatnib/quercetin. Each point in graph represents one technical replicate (bronchosphere) or one animal with mean ± s.e.m. All p values determined by one-tailed t-test and two-way ANOVA when applicable. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Figure 5.. p16^INK4A+ fibroblast senses inflammatory stimuli to augment epithelial regeneration.

(A and B) Quantification of nuclear p65/RelA in p16^INK4A+ or – fibroblasts from uninjured INKBRITE lungs (PND60) after 1 hour of vehicle or LPS treatment (n=3 wells, 14–15 images per condition). (C) SASP gene expression in p16^INK4Ahi/lo/neg fibroblasts from uninjured INKBRITE lungs after 6 hours of PBS or LPS incubation (n=3 wells per condition, 2 experiments). (D and E) Scgb1a1+ 3D organoid assay with p16^INK4A+/- fibroblasts isolated from LPS injured INKBRITE lungs and treated with vehicle or BAY11-7082 (n=3 wells per conditions, 2 experiments). (F) NicheNet interactome analysis of p16^INK4A+ fibroblasts and monocyte/interstitial monocytes from scRNAseq of injured INKBRITE lungs (14 dpi). (G) Expression of Il1b and Il1r1 in p16^INK4A+ cells in the lung. (H and I) Quantification of Scgb1a1+ organoids co-cultured with p16^INK4A+ or – fibroblasts from IL-1B-treated INKBRITE lungs (n=4 wells per conditions). Scale bars 100υm. Each point in graph represents one triplicate well or distinct image with mean ± s.e.m. All p values determined by one-tailed t-test or two-way ANOVA when applicable. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Scale bars 100υm.

Figure 6.. Mesenchymal p16^INK4A expression is required for epithelial regeneration in vivo.

(A) qPCR of Ereg and Il6 in control or p16^INK4A shRNA treated GFP+ fibroblasts after stimulation with vehicle or LPS for 6 hours (n=3 wells per conditions, 2 experiments). (B) qPCR of Ereg and Il6 in adeno-control or adeno-Cre treated p16^flox/flox fibroblasts after stimulation with vehicle or LPS (n=3 wells per conditions, >2 experiments). (C to G) Images and quantification of the airways of control and Dermo1^p16CKO lungs with and without naphthalene injury. (H) qPCR of Scgb1a1 of whole lung RNA (uninjured: n=6 Control, n=5 Dermo1^p16CKO; injured: n=6 Control, n=10 Dermo1^p16CKO,2 experiments ). (I to L) Images, histological quantification of SCGB1A1 club cells, and qPCR of Scgb1a1 on control and Gli1^p16CKO lungs after naphthalene injury (n=7 Control, n=6 Gli1^p16CKO). (M) qPCR of Ereg and Il6 of sorted Gli1 Lin+ cells after naphthalene (n=4 Control; n=4 Gli1^p16CKO). (N to P) Images and quantification of pERK+ and SCGB1A1+ cells in control and Gli1^p16CKO lungs after naphthalene injury (n=7 Control, n=6 Gli1^p16CKO). AW=airway. Scale bars 100υm. Each point in graph represents one animal with mean ± s.e.m. All p values determined by one-tailed t-test and two-way ANOVA when applicable. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.