Circadian regulation of lung repair and regeneration

Optimal lung repair and regeneration are essential for recovery from viral infections, including influenza A virus (IAV). We have previously demonstrated that acute inflammation and mortality induced by IAV is under circadian control. However, it is not known whether the influence of the circadian clock persists beyond the acute outcomes. Here, we utilize the UK Biobank to demonstrate an association between poor circadian rhythms and morbidity from lower respiratory tract infections, including the need for hospitalization and mortality after discharge; this persists even after adjusting for common confounding factors. Furthermore, we use a combination of lung organoid assays, single-cell RNA sequencing, and IAV infection in different models of clock disruption to investigate the role of the circadian clock in lung repair and regeneration. We show that lung organoids have a functional circadian clock and the disruption of this clock impairs regenerative capacity. Finally, we find that the circadian clock acts through distinct pathways in mediating lung regeneration — in tracheal cells via the Wnt/β-catenin pathway and through IL-1β in alveolar epithelial cells. We speculate that adding a circadian dimension to the critical process of lung repair and regeneration will lead to novel therapies and improve outcomes.

gavage, for 5 consecutive days.Tamoxifen was reconstituted to 100mg/ml solution with ethanol and corn oil and thawed at 55 o C prior to administration.Bmal1 creERT2neg , Sftpc CreERT2neg : Bmal1 fl/fl littermates treated with tamoxifen served as controls.A 2-week washout period was used for all experiments.Club cell specific Bmal1 knockout Cite (Scgb1a1 Cre/+ : Bmal1 fl/fl (3) ) was generated by crossing Scgb1a1-cre + , with Bmal1 fl/fl mice, Scgb1a1 Creneg :Bmal1 fl/fl served as controls (4) [S Fig 9D].PER2::LUC mice (B6.129S6-Per2 tm1Jt /J), in which the luciferase gene is fused in-frame to the 3′ end of the endogenous mPer2 gene (one of the core clock genes), were purchased from Jackson Laboratory animal facility (stock: 006852).For luciferase assay, tracheal cells, CD104 + distal lung cells or AT2s were harvested from these mice.Cry1 -/-Cry2 -/-(Cry1,2 DKO) mice were a gift from Dr. Amita Sehgal, HHMI, University of Pennsylvania, Philadelphia (5,6).Male and female mice were used in approximately equal proportion for all experiments.In all experiments with clock mutant models, WT littermates were used as controls.Unless specified otherwise tissues were harvested at ZT1-2.

Lung Injury
For influenza infections, mice were lightly anesthetized with isoflurane and infected intranasally (i.n.) with sub-lethal dose of IAV (H1N1:PR8,sub-lethal dose), in a volume of 40 µl at the times and under light-dark conditions as indicated in the specific experimental strategy.

Organoid assays
General information for organoid assays: Only two animals (KO vs control) were sorted at each time to ensure that the general health of the cells was optimized.All harvests were done between ZT1-3, based on availability of sorter in the Flowcytometry Core.
Bronchial epithelial organoids and AT2 organoids: Lungs were harvested after PBS perfusion followed by digestion with DNAse II and Liberase at 37°C for 20 mins.Dissociated lung tissue was passed through a 70 µm cell strainer (BD Biosciences), followed by centrifugation and RBC lysis.After magnetic bead-based depletion of leukocytes and endothelial cells (CD45 -CD31 -), the single cell suspension was sorted for alveolar epithelial cells (AT2 as DRAQ7 - EPCAM + CD104 -) and CD104 + bronchial epithelial cells (DRAQ7 -EPCAM + CD104 + ) on BD FACSJazz™ (S.Fig 2A).Using this strategy, the alveolar fraction, is likely to contain some AT1 along with AT2 cells and the CD104+ fraction has a mix of bronchial epithelial cells.Epcam+ CD104 -population was used as a surrogate for AT2 organoids.This was confirmed on histology.Similarly, the CD104 + organoids, represented the regenerative capacity of a mix of basal and club cells.
Thereafter they were plated and serially passaged in DMEM/F12 (11320033 Life Technologies) supplemented with 10% FBS and penicillin-streptomycin (P433310,000 U/mL, Sigma) 3 times.15,000-30,000 cells/well of CD104 + basal cells sorted from distal lung, were cultured in C12 media supplanted with growth factors onto growth factor-reduced, phenol-free matrigel depending on the yield of cells per experiment per experimental condition in 96 well plate.
For alveolar organoid assays: For each experiment, 5 × 10 3 Epcam + CD104 -were isolated as described above and mixed with 5 x10 4 lung fibroblasts.Cells were then suspended media similar to tracheal organoids and growth factor-reduced, phenol-free matrigel at 1:1 concertation.90μl of the cell/media/matrigel mixture was then aliquoted into individual 24-well cell culture inserts and allowed to solidify at 37 0 C. Complete SAGM was added to each well.
Rock inhibitor (Y27632 Sigma) was included in the media for the first two days.Cultures were maintained at 37 0 C, 5% CO2.Media was replenished every 48h until day 8 for tracheal and CD104+organoids, and day 21 for AT2 organoids as described previously (7,8).

Real-time bioluminescence recording of PER2::LUC organoids
For luciferase assay, tracheal cells, CD104 + distal lung cells or AT2s were harvested from mPer2 luc mice (Stock No: 006852).Bioluminescence was recorded without any synchronization agents.Bioluminescence outputs were recorded by adding beetle luciferin potassium salt (E1602 Promega) to organoid media at a final concentration of 1 mM.Cultures were monitored for light output using a custom-made bioluminescence recording system (Cairn Research Ltd, UK) composed of charge-coupled device camera (Andor iKon-M 934) mounted on the top of an Eppendorf Galaxy 170R CO2 incubator.Since the tracheal and CD104+ organoids mature by day 7 and AT2 organoids mature by day 21, they were placed in the bioluminescence chamber on days 4 and 18 respectively.Background was subtracted and bioluminescence data traces were analyzed with a modified R script CellulaRhythm (9).

Flowcytometry:
As described previously, lungs were perfused with PBS through the right ventricle and digested using DNAse II (Roche) and Liberase (Roche) at 37 o C for 30 mins.The left lung was harvested and dissociated tissue was passed through a 70 µm cell strainer, followed by centrifugation and RBC lysis.Cells were washed and re-suspended in PBS with 2% FBS.2-3 x 10 6 cells were blocked with anti-CD16/32 antibody and stained with indicated antibodies on ice for 20-30 minutes.No fixatives were used.Flowcytometric data was acquired using FACS Canto flow cytometer and analyzed using FlowJo software (Tree Star, Inc.).All cells were pre-gated on size as singlet, live cells.All subsequent gating was on CD45 + in lung only.Neutrophils were defined as CD45 + Ly6G + , inflammatory monocytes as CD45 + CD11b + Ly6C hi Ly6G -cells and virus specific T cells as PA-224 + CD62L lo CD44 + CD8 + CD45 + cells (kind gift from John Wherry, University of Pennsylvania, Philadelphia originally obtained from NIH tetramer facility).
H & E staining scoring: Lung injury post 30 days IAV infection was assessed by H&E staining.
We used Image J to overlay a grid on the whole lung cross section and allocated each square contained in the grid into one of four zones based on the severity of the injury as defined previously; Zone 1-minimal injury, Zone 2-minor injury with mild interstitial thickening, Zone 3severe alveolar injury or Zone 4-complete alveolar destruction (10).

Single cell preparation, and sorting
Lungs from embryonic Bmal1 -/-, Bmal1 +/+ controls, Bmal1 creERT2/+ , and Bmal1 creERT2neg littermates (n=1 per genotype) were dissected, and single-cell preparation was obtained as described above (under organoid assay).Single cell suspension was sorted as DRAQ7 -CD31 -CD45 -lung cells [Suppl Fig 4A] The sorted cells were loaded onto a Chromium Controller instrument (10× Genomics, Pleasanton, CA, USA) to generate single-cell barcoded droplets (GEMs) according to the manufacture's protocol using the 10× Single Cell 3' v1 chemistry.The resulting libraries were uniquely indexed using the Chromium i7 Sample Index Kit, pooled, and sequenced were sequenced on the Illumina NovaSeq 6000 sequencer using an SP 100 cycles flow cell in a paired-end, single indexing run.Sequencing for each library targeted 25,000 mean reads per cell.Data was then processed using the Cell ranger pipeline (10x genomics, v.3.1.0)for demultiplexing and alignment of sequencing reads to the mouse mm10 transcriptome and creation of feature-barcode matrices.Individual single cell RNAseq libraries were aggregated using the cell ranger aggr pipeline.Libraries were normalized for sequencing depth across all libraries during aggregation.Seurat and Loup browser were used for further processing and downstream analysis (11).Differentially expressed genes from both Bmal1 models were analyzed for enriched ontology clusters on mescape.org.
Bronchoalveolar Lavage: Flu infected and control mice were euthanized by CO2 asphyxiation on day 8 post infection, and their tracheas cannulated with a 20 G flexible catheter (Surflo, Terumo, Philippines).The lungs were gently lavaged with 600 µl of PBS in four passes.The supernatant from the first pass was collected for BAL total protein analysis (BCA Assay; Pierce Biotechnology, Rockford, IL, USA. Quantification of lung collagen content.Total right lung acid soluble collagen content was determined using the Sircol assay (Biocolor Ltd.) according to the manufacturer's instructions.

Histology (lung and organoids)
Lungs harvested at the end of the experiment were fixed by inflation with 10% buffered formalin at 20 mm H2O of pressure, paraffin embedded, and stained with H&E stain.Stained slides were digitally scanned at 40x magnification using an Aperio CS-O slide scanner (Leica Biosystems, Chicago IL).Representative images were taken from scanned slides using Aperio ImageScope v12.2.2.5015 (Leica Biosystems, Chicago, IL).Three-four random fields per lung section were imaged for Ki67 scoring.
Picrosirius red staining.Staining for collagen was performed using the Picrosirius Red Stain Kit (Polysciences Inc.) according to the manufacturer's instructions.Following staining of lung sections, the whole lung was annotated using the color deconvulation macros from Aperio Imagescope for analysis.Quantitation was performed using the Aperio software.Data represent intensity of total section area/ total area in mm 2 .

Quantitative PCR:
RNA was isolated/extracted from the inferior lobe of the mouse lung using TRIzol (Life Technologies).RNA was further purified using the RNeasy Mini Elute Clean Up Kit (Qiagen).
The quantity and quality of RNA was assessed using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc) and cDNA prepared with TaqMan.SYBR Green gene-expression assays were used to measure mRNA levels for genes of interest.Eukaryotic 18S rRNA (Life Technologies) and 28S (Sigma) were used as an internal control for TaqMan and SYBR Green assays, respectively.The samples were run on a Viia7 real-time PCR thermal cycler (Roche), and the relative ratio of the expression of each gene was calculated using the 2 ΔΔCt method.We used a web interface, Nitecap (nitecap.org)(12) to visualize and compare circadian behaviors of Wnt2, Wnt3a, Wnt5a, Wnt5b, Il1b, Fgf7 gene expression from the mouse lung tissues of Bmal1 creERT2/+ , and Bmal1 creERT2neg littermates.
Primers: All primers were procured from Thermofisher life sciences.Catalog numbers provided in source file 3.

Western Blot Analysis
Total protein was extracted from lungs harvested and stored at -80 o C. Protein was determined by the BCA assay (Thermo Scientific) assay and equal amounts of protein was loaded.20-50 µg of protein from each sample was resolved on SDS-PAGE gels followed by immunoblotting after PVDF membrane transfer, probed overnight with β-catenin (1:500, 610154 BD Biosciences), β actin (1:3000, ab8227 abcam), and secondary antibodies anti-rabbit HRP-linked IgG (1:5000 7074P2, Cell signaling) or anti-mouse IgG (1:5000 A2228 Sigma) and developed

Supplemental Figure 2 :Supplemental Figure 3 : 8 :
(A) Demographic characteristics of study cohort.(B) Distribution of risk factors across the quintiles of RA scores.(C) Relative Hazards ratio for the common risk factors including age, gender, smoking status (codified as ever smoker or non-smoker), medication use and prior history of hospitalization for respiratory infection and overall activity or exercise (a-referred to as total acceleration on actigraphy measurements) Histopathological analyses after 30 days post IAV infection Histological sections were prepared from lungs of C57Bl6J mice infected at either ZT23 or at ZT11 30 days post infection.Representative micrographs are shown in the figures for H&Estained lung sections on day 30 post-infection depicting (A) Zone 1-minimal injury, Zone 2-minor injury with mild interstitial thickening, Zone 3-severe alveolar injury or Zone 4-complete alveolar destruction.Scale Bar: 50 µm Graphs depecting gender spicifc differences in histopathology 30 days post IAV infection (B) Males (**p=0.001)(C) Females( **p=0.006).The data are represented as mean ± SEM, Two-way ANOVA with Bonferroni's multiple comparisons test.Collagen deposition assays 30 days IAV infection Histological sections were prepared from lungs of C57Bl6J mice infected at either ZT23 or at ZT11 30 days post infection.Representative micrographs for collagen deposition are shown by (A)Trichome staining (photomicrograph bar = 100µm) and (B) summarized statistical analysis, **p=0.006(C) Sirius red staining showing the deposition of collagen (black arrows).Box is enlarged in the next panel.Scale bar = 3mm and 200 µm (inset) (D) Quantitation performed using the Aperio software.*p=0.04,Soluble collagen in right lung homogenates measured by sircol assay for (E) C57Bl6J mice infected at either ZT23 or at ZT11 (n=4) and (F) Bmal1 creERT2/+ and their Cre neg littermates infected at CT23 (n=4).*p= 0.02, *p=0.01.Data are represented as mean ± SEM, n=4-9 mice per circadian time point, Unpaired t test with Welch's correction.(G) Representative KRT5 staining images 30days post infection, scale bar: 50 µm Supplemental Figure 5: IAV induced lung injury and lung function C57Bl6J mice were infected at as depecited in figure 2A.(A) Vascular permeability was reported as total protein content from bronchioalveolar lavage fluid (n=3-6) (B) Virus specific CD8 + T cells in lung tissues 10 days post infection were analyzed by flowcytometry (n= 5), *p=0.01,Unpaired t test, Welch's correction Supplemental Figure Differentially expressed genes in scRNA data (A)Gating strategy for isolation of lung cells for single cell sequencing from Bmal1 +/+ , Bmal1 -/-, Bmal1 creERT2/+ , and Bmal1 creERT2neg .Single cell suspension was sorted on DARQ7 -CD31 -CD45 - lung cells for sequencing.(B) The dot plot showing the percentage of cells expressing the respective selected marker gene based on dot size (C) Violin plots of differentially expressed genes in the Bmal1 knockout models