Exuberant fibroblast activity compromises lung function via ADAMTS-4 (2024)

The publisher's final edited version of this article is available at Nature

Mice

B6.129P2-Adamts4tm1Dgen/J (ADAMTS-4^−/−) mice were obtained from Jackson Laboratories. These mice were back-crossed with C57BL/6/J mice for at least 10 generations to generate congenic ADAMTS-4^−/− and ADAMTS-4^+/+ mice. Mice were housed at temperature, light/dark cycle, humidity.

Studies involving mice were approved by the St. Jude Children’s Research Hospital Committee on Care and Use of Animals. All of the mice that were tested in this study were bred and maintained at the St. Jude Children’s Research Hospital under specific-pathogen free conditions.

Single-cell RNA sequencing

Whole lungs were collected from murine pathogen-free C57BL/6J mice (Taconic Biosciences) at 0, 1, 3, 6, and 21 days after initial infection. Four or five mice for each time point were infected with 2500 EID50 of mouse -adapted A/Puerto Rico/8/1934 (PR8) intranasally in 30 μL of 1x PBS³¹. Lungs were perfused with 10 mL of cold 1x PBS injecting into the right ventricle of the heart and then minced and digested in lung digestion buffer containing 400 U/mL of Collagenase I (Worthington Biochemical Corporation) and 50 U/ml of DNaseI (ThermoFisher) in Hanks Balanced Salt Solution (HBSS) for 75 minutes at 37 degrees C. Digested lungs were filtered through a 100 μm cell strainer and washed with HBSS. Red blood cell lysis was performed and cells were resuspended at 1×10⁶ cells/mL. Cells were incubated with Ghost Dye Violet 510 (Tonbo) (1:100 dilution) and APC anti-mouse CD45.2 (clone 104, Biolegend) (1:100 dilution) for 30 minutes at room temperature. Live, CD45-cells were sorted into 1.7 mL microfuge tubes and then centrifuged at 400 × g for 5 minutes. Cells were then incubated with PE anti-mouse H-2 antibody (1:100, clone M1/42, Biolegend) for 30 minutes on ice.

Human lung biopsy samples from de-identified, deceased patients were obtained through the National Disease Research Interchange (NDRI). Cause of death for these patients was unrelated to respiratory tract infection or lung damage, and patients did not have a history of tobacco use or lung cancer. Fresh 5 cm × 5 cm × 5cm lung biopsy samples were minced and digested in 400 U/ml of Collagenase I (Worthington Biochemical Corporation) and 50 U/ml of DNaseI (ThermoFisher) in Hanks Balanced Salt Solution (HBSS) for 90 minutes at 37 degrees. Lung samples were first filtered through sterile gauze and then through 100 μm cells strainer and washed with HBSS, and red blood cell lysis was performed. Single cell suspensions of total lung cells were frozen in freeze media (90% FBS/ 10% DMSO) at a concentration of 5×10^6 cells/mL. For scRNAseq using 10x Genomics, vials of cells were thawed and incubated with Ghost Dye Violet 510 (Tonbo) (1:100 dilution) and APC anti-human CD45 (1:200) for 30 minutes at room temperature. Live, CD45-cells and live, CD45+ cells were sorted into 1.7 mL microfuge tubes and then centrifuged at 400 × g for 5 minutes. Following sorting, cells were incubated with PE anti-human β2 microglobulin (1:100) and PE anti-human CD45 (1:100) antibodies for 30 minutes on ice.

For both mouse and human lung samples, cells were washed three times and then incubated with custom-designed hashtag oligos (HTO) conjugated to anti-PE antibodies (Thunder-Link PLUS Oligo Conjugation System, Expedeon) unique for each individual mouse. After incubation, cells were washed three times and then pooled within each time point. ~25,000 cells per sample were loaded onto the Chromium controller (10x Genomics) to partition single cells into gel beads. Single cell transcriptomic libraries were generated using the 5’ Gene Expression Kit (V2, 10x Genomics) according to the manufacturer’s instructions with the addition of primers to amplify HTOs during cDNA amplification. Sequencing was performed on the Illumina NovaSeq to generate approximately 500M reads per sample.

Single-cell gene expression analyses

10x gene expression data were first processed using CellRanger (v3.0.2, 10x Genomics). Human data were processed using version 3.0.0 of the GRCh38 10x reference, whereas mouse data were processed using the mm10 reference altered to include the PR8 influenza genome. CellRanger was then used to aggregate species-specific samples, normalizing by the number of mapped reads per identified cell. Normalized feature-barcode matrices including both gene expression and HTO counts were then imported into Seurat (v3.0.0.900) for downstream analysis³².

Data were first filtered by excluding any gene that was not present in at least 0.1% of total called cells and then by excluding cells that exhibited extremes in the species-specific distributions of: the number of genes expressed (mouse: <100 or >5,550; human: <100 or >4,500), the number of mRNA molecules (mouse: >30,000; human: >20,000), or the percent of expression owed to mitochondrial genes (mouse and human: >7.5%). Gene expression counts were log-normalized using a scaling factor of 1e4, variable features targeted for downstream analysis were identified using the ‘vst’ method with default parameters, and cell cycle scores were generated for each cell using markers identified elsewhere³³. Gene expression was then scaled to regress out the effects of total transcript expression, percent of mitochondrial expression, and cell cycle scores. For first-pass analyses of entire datasets, we selected principal components (PCs) visually using an elbow plot of the PC standard deviations (mouse: 25; human: 19). These PCs were then used for t-SNE dimensionality reduction and cell clustering with Seurat’s shared nearest neighbor modularity algorithm in order to broadly characterize the cell types in the entirety of each sample. Individual cell subsets were annotated using known markers from the literature.

For more detailed analyses of fibroblast differentiation states, we generated a subset of the identified fibroblast clusters, identified the top variable features within this subset, and re-scaled that data as described above. For the mouse fibroblast data, we also regressed out the effects of days post infection (DPI) in order to more precisely characterize the variance among fibroblast clusters and distinguish cellular responses to experimental infection. After re-conducting Principal Component Analysis, PCs were scored for significance (FDR-adjusted p-value <0.05) using random permutations as implemented in Seurat. Differential gene expression was assessed among clusters for all genes expressed in at least one percent of cells within a cluster using a generalized linear hurdle model that incorporates both expression frequency and abundance³⁴. For pairwise cluster comparisons of interest, genes were then ranked as a function of the product of their average log fold change, the absolute value of the difference in percent expression, and the inverse of the scaled, FDR-adjusted p-value. These gene rank lists were then analyzed using preranked Gene Set Enrichment Analysis³⁵.

Meta-analysis of publicly available human single-cell gene expression datasets

Human single-cell gene expression data from published reports (Extended Data 6a) were obtained from online data repositories. From Vieira Braga et al¹⁷ we were only able to obtain data from four healthy controls, as other data, including the data from asthma patients, were not publicly available. Each dataset was filtered to exclude cells with more than 20% mitochondrial content, fewer than 200 genes, or fewer than 200 RNA molecules before being normalized, scaled, and assessed for variable features using the SCTransform wrapper in Seurat³². Focal analyses of specific datasets were conducted on these resulting data objects. We also compiled the datasets for meta-analysis using reference-based integration with 3,000 integration features, with data from Habermann et al¹² as the reference, again using the SCTransform normalization method. Downstream analyses were focused on the subset of cells that contained at least one ADAMTS4 transcript. Dimensionality reduction and clustering were conducted as described previously, but in this case using the integration assay. Differential gene expression analyses were conducted on the RNA assay of the integrated object using default parameters in Seurat.

Flow Cytometry for Lung Stromal Cells and FACS-based Sorting

At the indicated time point after infection, lungs were perfused with 10 mL of cold 1x PBS injecting into the right ventricle of the heart and then minced and digested in lung digestion buffer containing 400 U/mL of Collagenase I (Worthington Biochemical Corporation) and 50 U/ml of DNaseI (ThermoFisher) in HBSS for 30 minutes at 37 degrees C. Digested lungs were filtered through a 100 μm cell strainer and washed with HBSS. Red blood cell lysis was performed, and cells were washed and resuspended in FACS buffer (1% FBS and 2 mM EDTA in 1x PBS). CD45+ cells were depleted using mouse CD45 Microbeads (Miltenyi) according to the manufacturer’s instructions. Briefly, cells were incubated with microbeads for 15 minutes on ice, washed with FACS buffer, and resuspended in 500 μl of buffer. CD45+ cells were then depleted using a Miltenyi LS column, and the flow through (CD45-cells) were collected. For antibody staining, 1×10⁶ cells were plated and then incubated with Fc block for 10 minutes at room temperature. Cells were washed one time with FACS buffer and then resuspended in 100 μl of an antibody co*cktail containing the following: Ghost Dye Violet 510 (1:100), BV605 anti-mouse CD45 (1:200), BV421 anti-mouse EpCAM (1:200), PerCP/Cy5.5 anti-mouse CD31 (1:200), FITC anti-mouse BST2 (1:100), PE anti-mouse CD9 (1:100), APC anti-mouse CD49e (1:100). Cells were incubated with the antibody co*cktail for 30 minutes at room temperature and then washed twice with FAC buffer before resuspending in a final volume of 150 μl. For single cell suspensions from human lung biopsy samples, cells were washed one time with FACS buffer and then resuspended in 100 μl of an antibody co*cktail containing the following: Ghost Dye Violet 510 (1:100), BV605 anti-human CD45 (1:100), BV785 anti-human EpCAM (1:100), APC Fire 750 anti-human CD31 (1:100), PE/Cy7 anti-human CD140a (1:100), APC anti-human CD49e (1:100), and PE anti-human CD9 (1:100). Cells were incubated with the antibody co*cktail for 30 minutes at room temperature and then washed twice with FAC buffer before resuspending in a final volume of 150 μl. Cells were analyzed using a BD LSRII Fortessa and BD FACSDIVA software (Becton Dickinson). Data were analyzed using FlowJo software (FlowJo).

High-throughput qPCR using Fluidigm Biomark

For in vitro stimulation of primary murine lung fibroblasts, fibroblasts were isolated from C57BL6/J. Lungs were collected, minced, and then digested in lung digestion buffer containing 0.1% Collagenase I (Worthington Biochemical Corporation) and 2.4 U/ml dispase (Thermo Fisher) for 90 minutes at 37 degrees C. Digested lungs were filtered through a 100 μm cells strainer and then washed with 15 mL of 0.05 M EDTA in 1x PBS. Cells were centrifuged at 400 × g for 10 minutes and then re-suspended in complete Dulbecco’s Modified Eagle Medium (cDMEM) and plated in a T75 tissue-culture flask (Corning). For cytokine and virus stimulations, 2×10⁵ cells were plated in each well of a 24-well plate 24 hours prior to stimulation. Fibroblasts were stimulated with the following murine cytokines: IL-1B (10 ng/mL), IL-1A (10 ng/mL), IL-33 (10 ng/mL), IL-18 (10 ng/mL), TNFα (200 ng/mL), IFNA (1000 U/mL), IL-6 (40 ng/mL), TGFB (2 ng/mL), IL-17A (50 ng/mL), IL-27 (50 ng/mL), and GM-CSF (40 ng/mL). For the virus stimulations, cells were infected at a multiplicity of infection (MOI) of 2. Virus inoculum was incubated with the cells for 1 hour at 37 degrees C. The following viruses were used for the stimulations: A/Puerto Rico/8/1934 (PR8), A/California/04/2009 (CA09), and A/Perth/16/09 (Perth). The supernatant was removed, and cells were washed three times with cDMEM. The wells were replenished with 1 ml of cDMEM, and the cells were incubated at 37 degrees C for 24 hours. To collect bronchoalveolar lavage (BAL) fluid, a catheter was inserted into the trachea, and 3 ml of 1x PBS was used to wash the lungs. BAL fluid was centrifuged at 400 × g for 5 minutes to pellet cells, and then the cells were resuspended in 350 μl Trizol reagent.

For co-culture experiments, normal human lung fibroblasts (Lonza) were plated at a density of 2,500 cells/cm² in the basal chamber of a 24-well plate and cultured in fibroblast growth medium (Lonza) overnight at 37 degrees C and 5% CO₂. Normal human bronchial epithelial cells (NHBEs) were seeded at a density of 10,000 cells/cm² kn 12 mm trans-well inserts (Corning) coated with human placental collagen and then added to plates containing fibroblasts. Co-cultures were cultured in bronchial epithelial growth medium (BEGM) (Lonza) at 37 degrees C and 5% CO₂ for 7 – 10 days. Co-cultures were then taken to the air-liquid interface and basolateral media was exchanged for media consisting of 50% DMEM and 50% BEGM supplemented with the BEGM BulletKit (Lonza). Media were changed every 2 – 3 days until the cells were fully differentiated (2 – 3 weeks). For infections, NHBEs were inoculated at a multiplicity of infection (MOI) of 2 using the following influenza A virus strains: H3N2 A/Perth/16/09, H5N6 A/Oriental Magpie Robin/Hong Kong/6154/2015, and H7N9 A/Hong Kong/125/2017. Infections with avian strains were performed in an ABSL3+ facility.

For monoculture experiments, NHBEs from a single donor were plated in 24-well transwell plates (Corning) and cultured in Bronchial Epithelial Basal Medium (BEBM) (Lonza) supplemented with bovine pituitary extract, insulin, hydrocortisone, gentamicin/amphotericin, transferrin, triiodothyronine, epinephrine, epidermal growth factor, and retinoic acid until wells were confluent and cells formed tight junctions. Supernatant was then removed from the apical side of the trans-well and cells were taken into the air-liquid interface for 2 – 3 weeks. NHBEs were then stimulated with the following human cytokines adding each factor to media in the basal chamber: IL-1B (10 ng/mL), IL-1A (10 ng/mL), IL-33 (10 ng/mL), IL-18 (10 ng/mL), TNFA (200 ng/mL), IFNA (1000 U/mL), IL-6 (40 ng/mL), TGFB (2 ng/mL), IL-17A (50 ng/mL), IL-27 (50 ng/mL), and GM-CSF (40 ng/mL). For virus stimulations, cells were infected at an MOI of 2. Virus inoculum was incubated with the cells in the apical chamber for 1 hour at 37 degrees C. The supernatant was removed, and cells were washed three times with BEBM, and incubated at 37 degrees C for 24 hours. Normal human bronchial fibroblasts (NHBFs) (Matek) were plated in 24-well plates at a density of 2×10⁵ cells/well and cultured in cDMEM. NHBFs were stimulated with the cytokines and viruses listed above. Following incubation, the supernatant was removed and cells were collected in 350 μl Trizol Reagent (Thermo Fisher). RNeasy spin columns (Qiagen) were then used to isolate RNA from the samples according to the manufacturer’s instructions. Following RNA isolation, the amount of RNA input was normalized (200 ng), and cDNA was generated using the iScript cDNA synthesis kit (Biorad). cDNA was then prepared for quantitative polymerase chain reaction (qPCR) using the Fluidigm Biomark (Fluidigm). Data were collected using the Fluidigm Biomark data collection software (Fluidigm). Exon-spanning qPCR primers were designed to target a panel of 96 ECM-related genes for humans (Supplemental Table 1) and mice (Supplemental Table 2).

For whole lung hom*ogenates from mice, lungs were collected at 0, 1, 3, 6, 9, 12, 15, 30, and 40 days after infection. C57BL6/J mice were infected with 2500 EID50 PR8. Whole lungs were dissected and hom*ogenized in 750 μl Trizol reagent using the TissueLyser II (Qiagen). RNA was isolated according to the manufacturer’s protocol.

Spatial Transcriptomics

C57BL/6J (Jackson Laboratories) were infected intranasally with 2500 EID₅₀ of IAV PR8 in 30 μl of 1x PBS. Lungs were dissected from the mice 10 days after infection. Sections obtained from four distinct mice were imaged and processed for spatially resolved gene expression using the Visium Spatial Transcriptomics kit (10x Genomics). Lungs were inflated with 300ul of 50% OCT and 50% PBS v/v and immediately snap-frozen in OCT using isopentane that was cooled in a liquid nitrogen bath. For each lung, the same left lobe was harvested and stored at −80°C until cryosectioning. For cryosectioning, samples were equilibrated to −22°C. Exploratory sections were stained with a fluorescent lectin and dapi to determine areas of immune infiltration and remodeling, and then blocks were trimmed to 5×5mm to encompass normal and remodeled areas prior to sectioning directly onto the spatial transcriptomics kit slide. Tissue permeabilization was optimized at 12 minutes on independent lung sections. Libraries were sequenced on the Illumina NovaSeq platform at 28×120 basepairs, and resulting data were processed using SpaceRanger (v1.0.0, 10x Genomics) with manual alignment of fiducial markers, manual tissue identification, and r2-length trimmed to 91bp. Downstream analyses were conducted with Seurat (v3.1.4.9901)³².

ADAMTS-4^−/− and ADAMTS-4^+/+ Survival Experiments

Mice were infected intranasally with 6000 EID50 of PR8 in 30 μl 1x PBS after anesthetizing with 2,2,2-tribromoethanol by intraperitoneal (i.p.) injection. Following infection, mice were monitored twice daily and weight was recorded. Mice were euthanized if they become severely moribund based on body index score and substantial weight loss³¹. Righting reflex of severely moribund animals was assessed to determine if immediate euthanasia was necessary. Mice who passed 20% weight loss were placed on their backs, and if unable to right themselves within 10 seconds, were euthanized. Animals were identified by ear tag numbers, and investigators assessing morbidity were blinded to the genotype.

Plaque Titers

BAL fluid was used to obtain viral titers inoculating Madin-Darby Canine Kidney (MDCK) cells. MDCKs were plated in six-well tissue culture plates at a density of 4×10⁵ cells/well. Twenty-four hours after plating, cells were washed three times with 1x PBS and then incubated with 1 mL of BAL fluid, after performing six 10-fold dilutions in serum-free MEM. Cells were incubated with inoculum for 10 minutes at 4 degrees Celsius and then for 50 minutes at 37 degrees Celsius. Inoculum was removed and 3 mL of MEM containing 0.9% agarose and 1 mg/ml of L-1-tosylamido-2phenylethyl chloromethyl ketone-treated (TPCK) trypsin was overlaid onto the cell monolayer. Plates were then incubated at 37 degrees for 72 hours, and then plaques were counted after staining the cells with crystal violet.

Pulse Oximetry

Mice were inoculated intranasally with 6000 EID₅₀ of IAV PR8 and arterial oxygen saturation was measured using the MouseOx system (STARR Life Sciences). Following infection, fur was removed around the neck of the animal, and arterial oxygen saturation (SpO₂) was measured using a throat collar sensor. After acclimatization, oxygen saturation measurements were recorded for three minutes, and measurements were averaged over the measurement period.

Histology and Morphometry Viral Spread Analysis

Lungs were dissected at 3 and 6 days after infection with 2500 EID50 of PR8. Lungs were inflated with 1 mL of 10% neutral-buffered formalin, and then placed in a 15 mL conical tube containing 3 mL more of formalin. Lungs were incubated in formalin at room temperature for at least 72 hours to ensure complete fixation of the tissue. Lungs were embedded in paraffin blocks and then sectioned onto glass slides. Serial tissue sections were stained with hematoxylin and eosin (H&E) for histology or immunohistochemical labeling of viral antigen was completed by using a primary goat polyclonal antibody (US Biological, Swampscott, MA) against influenza A, USSR (H1N1) at 1:1000 and a secondary biotinylated donkey anti-goat antibody (catalog number sc-2042; Santa Cruz Biotechnology, Santa Cruz, CA) at 1:200 on tissue sections subjected to antigen retrieval for 30 minutes at 98°C. The extent of virus spread was quantified by first capturing digital images of whole-lung sections stained for viral antigen using an Aperio ScanScope XT Slide Scanner (Aperio Technologies, Vista, CA) and then manually outlining fields with the alveolar areas containing virus antigen-positive pneumocytes. The percentage of each lung field with infection/lesions was calculated using the Aperio ImageScope software. For histologic grading of lesions, a pathologist blinded to treatment group identity evaluated pulmonary lesions in H&E-stained histologic sections and assigned scores based on their severity and extent as follows: 0 = no lesions; 1 = minimal, focal to multifocal, barely detectable; 15 = mild, multifocal, small but conspicuous; 40 = moderate, multifocal, prominent; 80 = marked, multifocal coalescing, lobar; 100 = severe, diffuse, with extensive disruption of normal architecture and function.

Assessment of IAV-infected cells by flow cytometry

Lungs were perfused with 10 mL of cold 1x PBS injecting into the right ventricle of the heart and then minced and digested in lung digestion buffer containing 400 U/mL of Collagenase I (Worthington Biochemical Corporation) and 50 U/ml of DNaseI (ThermoFisher) in HBSS for 30 minutes at 37 degrees C. Digested lungs were filtered through a 100 μm cell strainer and washed with HBSS. Red blood cell lysis was performed, and cells were washed and resuspended in FACS buffer. Cells were washed and then incubated with Trustain FcX anti-mouse CD16/CD32 (Fc Block) at a dilution of 1:100 for 10 minutes at room temperature. For surface staining, cells were then washed and incubated in 100 μl of FACS buffer (1% FBS/1 mM EDTA in 1x PBS) containing Ghost Dye Violet 510 (1:100), PE/Cy7 anti-mouse CD45.2 (1:200), PacBlue anti-mouse CD11c (1:100), APC/Cy7 anti-mouse CD11b (1:100), BV650 anti-mouse Gr-1 (1:200), APC anti-mouse CD326/EpCAM (1:100), PE anti-mouse CD90.2 (1:100), PerCP/Cy5.5 anti-mouse CD31 (1:100) for 30 minutes at room temperature. For intracellular staining, cells were fixed and membranes permeabilized by incubating in 100 μl fixation/permeabilization solution (BD Biosciences) for 20 minutes on ice. Cells were washed with permeabilization buffer and then incubated with FITC anti-influenza A (1:100).

Assessment of CD8+ T cell responses

In order to assess immune cell populations by flow cytometry, whole lungs were dissected, minced, and incubated with spleen dissociation media (STEMCELL) for 30 minutes at 37 degrees C. Red blood cell lysis was performed, and cells were washed with HBSS and counted using a Vi-CELL XR cell counter (Beckman Coulter). For ex vivo stimulation and intracellular cytokine staining, 1×10⁶ total lung cells were plated in a 96-well plate for each condition. Cells were stimulated with Cell Stimulation co*cktail (Biolegend), containing PMA/ionomycin and Brefeldin A, at a dilution of 1:500 or with influenza peptides at a concentration of 1 μM for 4 hours. Following stimulation, cells were washed and then incubated with Trustain FcX anti-mouse CD16/CD32 (Fc Block) at a dilution of 1:100 for 10 minutes at room temperature. For surface staining, cells were then washed and incubated in 100 μl of FACS buffer (1% FBS/1 mM EDTA in 1x PBS) containing Live/Dead Aqua (1:100), APC anti-mouse CD45.2 (1:100), FITC anti-mouse CD3 (1:100), BV650 anti-mouse CD8 (1:200), and APC/Cy7 anti-mouse CD4 (1:200) for 30 minutes at room temperature. For intracellular staining, cells were fixed and membranes permeabilized by incubating in 100 μl fixation/permeabilization solution (BD Biosciences) for 20 minutes on ice. Cells were washed with permeabilization buffer and then incubated with PE/Cy7 anti-mouse IFNG (1:100) for 30 minutes on ice.

For IAV peptide:MHC tetramer staining, 1×10⁶ total lung cells were plated in a 96-well plate for each sample. Cells were incubated with APC PB1_703–711 tetramer (1:750), PE PA_224–233 tetramer (1:750), and BV785 NP_366–375 tetramer (1:250) for 1 hour on ice. Then, cells were surface stained with FITC anti-mouse CD3 (1:100), BV650 anti-mouse CD8 (1:200), APC/Cy7 anti-mouse CD4 (1:200) for 30 minutes on ice.

Lung Tissue Immunofluorescence

Lungs were dissected 9 days after infection with 2500 EID50 of PR8. Lungs were inflated with 1 mL of fixative containing 2% paraformaldehyde, 0.1% Triton-100 and 1% DMSO and then transferred to a 15 mL conical tube containing 3 mL of fixative for 24h prior to cryoprotection with 30% sucrose in PBS for an additional 24h. Tissues were cryosectioned at 10μm thickness and blocked in buffer comprised of PBS containing 2% bovine serum albumin and 5% donkey serum. Tissues were stained overnight in blocking buffer containing 4μg/mL rat anti-CD3 (Biolegend, clone 17A2), and 4μg/mL rabbit anti-mouse versican GAG beta domain (Millipore Sigma, AB1033). Sections were washed in PBS prior to incubation with CF488-conjugated donkey anti-rat IgG (Biotium, 20027, lot 16C0301, 2μg/mL), CF568-conjugated donkey anti-rabbit IgG (Biotium, 20098, lot 19C0110, 2μg/mL) and DAPI [10uM] for 1 hr at RT. Slides were washed with PBS and mounted with prolong diamond hardset mounting medium (ThermoFisher). High resolution images were acquired using a Marianis spinning disk confocal microscope (Intelligent Imaging Innovations) equipped with a 40X 1.3NA Plan-Neofluar objective, 405nm, 488nm and 561nm laser lines and Prime 95B CMOS camera (Photometrics), and analyzed using Slidebook software (Intelligent Imaging Innovations). For quantification of CD3+ cells, 3 – 4 fields of view in the areas of tissue remodeling were collected for each lung.

Lung Function Test

For lung function studies, mice were infected with 2500 EID50 of PR8. At 10 days post-infection, mice were anesthetized with 2,2,2-tribromoethanol by intraperitoneal (i.p.) injection and tracheal tube was inserted. Airway resistance and dynamic lung compliance was measured using the Buxco Finepointe Resistance and Compliance system (Data Sciences International). Data were collected using Buxco Finepointe software (Data Sciences International). Mice were mechanically ventilated at a rate of 140 breaths/minute. Five methacholine challenges were performed at doses of 1.5625, 3.125, 6.25, 12.5, and 25 mg/ml, administering the methacholine in 0.010 ml of PBS. Following each methacholine challenge, resistance and compliance were measured for 3 minutes allowing the mice to recover for 1 minute after each measurement. Measurements for resistance and compliance were averaged over the 3 minute measurement period for each methacholine challenge and baseline measurements.

T cell Migration Assays

For migration assays, CD8+ T cells were isolated from spleens of C57BL/6J mice. To enrich for CD8+ T cells, samples were magnetically depleted using a co*cktail of the following biotinylated antibodies: anti-mouse B220 (1:50), anti-mouse MHC Class II (1:50), anti-mouse CD11b (1:100), anti-mouse CD49b (1:200), and anti-mouse CD4 (1:100). Up to 10⁹ total spleen cells were incubated with the antibody co*cktail for 15 minutes on ice. Cells were washed with FACS buffer and then incubated with Miltenyi Streptavidin microbeads (1:25) for 15 minutes on ice. The cells were centrifuged at 500 × g for 5 minutes and then resuspended in 1 mL of FACS buffer. The sample was added to a pre-equilibrated Miltenyi LS column and the flow-through enriched by CD8+ T cells was collected. CD8+ T cells were then activated with anti-mouse CD3e (1 μg/mL) and anti-mouse CD28 (3 μg/mL) antibodies in complete RPMI at 37 degrees C for 72 hours. 48 hours prior to the migration assay, primary murine lung fibroblasts, which were plated at a concentration of 10⁵ cells/well in a 24-well plate the previous night, were stimulated with IL-1B (10 ng/mL) and TNFα (200 ng/mL). For activation states (resting, DRFib, IRFib), sorted fibroblasts were plated at a density of 10⁴ cells/well 48 hours prior to the migration assay. 6.5 mm polycarbonate trans-well inserts with 8.0 μm pore size (Corning) were coated with 10 μg/mL of versican purified from bovine aorta²⁹ for 2 hours at 37 degrees C. Versican was aspirated and trans-well inserts were transferred to the 24-well plate with stimulated fibroblasts. To test migration, 10⁵ activated CD8+ T cells were added to the apical chamber of the trans-well in a volume of 100 μl of complete DMEM. Cells were allowed to migrate to the basal chamber for 4 hours at 37 degrees C. After 4 hours, supernatants from the basal chamber were collected and the number of lymphocytes were manually counted.

Quantification of Proteins in Mouse Lung hom*ogenates and Bronchoalveolar Lavage Fluid

Cytokine levels in lung tissue hom*ogenates were measured using LEGENDplex cytokine bead arrays (Biolegend). Lungs were dissected from PR8-infected mice at 6 days post-infection and hom*ogenized in 500 μl of PBS. hom*ogenized lung samples were centrifuged at 4,000 × rpm for 10 minutes, and 25 μl of undiluted supernatant was tested in the assay. The assay was performed according to the manufacturer’s instructions.

BAL washes were performed using 500 μl of PBS. Matrix protease levels in mouse BAL fluid were quantified using ELISA or a multiplexed, Luminex-based fluorescent assay. A custom Luminex kit (R&D Systems) was used to assay the following analytes: MMP-2, MMP-8, MMP-9, MMP-12, TIMP-1, and TIMP-4. Duoset ELISA kits (R&D Systems) were used to assay the following analytes: ADAMTS-4, MMP-3, MMP-13, and TIMP-3.

Human Cohorts

For the PICFLU cohort, the study was approved by the lead site Boston Children’s Hospital (IRB X08-11-0534) and then at each enrolling site’s IRB (enrolling sites are listed in acknowledgments) and written informed consent was obtained from at least one parent or guardian. Patients were excluded before enrollment if they had an underlying condition predisposing them to severe influenza infection as has been described previously³⁶. Additional cohort information is presented in Extended Data Figure 10a. Endotracheal aspirate samples were obtained using standardized methods with 1.5 ml of sterile normal saline down the ETT then 3–4 breaths prior to obtaining the sample with 2 suctioning attempts aiming to collect at least 2 ml of aspirate. The sample was put on ice and transferred immediately to the laboratory for processing. One aliquot was immediately frozen (−80°C) and the rest was centrifuged and the supernatant was aliquoted and frozen at −80°C.

For the Guangzhou cohort, the study was approved by the ethics committee of the First Affiliated Hospital of Guangzhou Medical University (No: 2016–78), and informed consent was obtained from all patients or their guardians. Additional cohort information is presented in Extended Data Figure 10c.

For the Taiwan cohort, the human subjects’ protocol was reviewed and approved by the Johns Hopkins School of Medicine and the Chang Gung Memorial Hospital Institutional Review Boards (IRB00091667). Informed consent was obtained from all patients or their guardians. This protocol allowed for the collection of residual bronchoalveolar lavage (BAL) and sputum samples that were initially collected for clinical purposes from influenza positive patients. Extended Data Figure 10c.

For lung samples from de-identified, deceased donors, the tissues utilized were procured by the National Disease Research Interchange (NDRI) with support from NIH grant U42OD11158. All NDRI consent forms and protocols are reviewed and approved by the Institutional Review Board at the University of Pennsylvania. Research involving these tissues was determined to not involve human subjects by the Institutional Review Board at St. Jude Children’s Research Hospital.

Quantification of matrix protease and cytokine/chemokine protein levels in human samples

Cytokine and chemokine levels were measured using Milliplex Human Cytokine/Chemokine Magentic Bead Panel - Premixed 41-plex. MMP and TIMP protein levels were quantified using the following Milliplex bead-based multiplexed assay, Human MMP Magnetic Bead Panel 1 (MMP-3, MMP-12, MMP-13), Human MMP Magnetic Bead Panel 2 (MMP-1, MMP-2, MMP-7, MMP-9, MMP-10), and Human TIMP Magnetic Bead Panel 2 (TIMP-1, TIMP-2, TIMP-3, TIMP-4). ADAMTS-4 and ADAMTS-5 were measured using Duoset ELISA kits (R&D Systems).

Cytokine correlation matrices

Correlation matrices were generated using samples analyzed in severity analyses, with cytokines log10 transformed. To control for potential effects of age and day of sample collection (days after symptom onset; DAO), we computed partial pairwise spearman correlations and assessed their statistical significance using the R psych package³⁷. Correlations were visualized using the R corrplot package³⁸, and correlations with p-values <0.05 after FDR adjustment were excluded.

Severity analyses

For analysis of association of ADAMTS-4 levels with severity of infection in the PICFlu cohort, we used three distinct indicators of severity: prolonged multiple organ dysfunction syndrome (PrMODS) or death, prolonged acute hypoxic respiratory failure (PrAHRF) or death, and ventilator-free days (VFDS) less than 10. In survivors, “prolonged” was defined as being present on or after day 7 of PICU admission or being on extracorporeal membrane oxygenation (ECMO) on or after day 7. PrAHRF was defined as PaO₂/FiO₂ ratio <200 and mechanical ventilator support, which are criteria for moderate-severe ARDS¹. Ventilator-free days (VFDS) were defined as days alive and free of invasive or non-invasive mechanical ventilation up to 28 days³⁹. Due to the bimodal distribution of VFDS, we used a cutoff of 10 VFDS to classify cases as severe (Extended Data Fig. 10b) ADAMTS-4 was modeled as a predictor of these categorical outcomes using logistic regressions in R that explicitly included gender, age, baseline health (“Previously Healthy”), and steroid administration. FDR adjustments for multiple testing were applied independently to each model. Patients were considered “Previously Healthy” if they were, upon admission, otherwise healthy, on no prescription medications, without underlying medical conditions, and not dependent on any medical devices prior to initial admission to the hospital for flu. No violations of the model assumptions were found in any reported results after specifically checking for: 1) the assumption that the relationship between continuous predictor variables and the logit of the outcome is linear, 2) evidence of extreme values in continuous predictors (as indicated by standard residuals with an absolute value over three), and 2) evidence of multicollinearity among variables (as indicated by VIF). Odds ratios were visualized using the sjPlot package in R⁴⁰.

Statistical Analyses

For experiments involving mice, statistical analyses were performed using Graphpad Prism version 7.0d software. For analysis of human samples, statistical analyses were performed in R statistical software version 3.5.0 and as detailed in the sections above.

Extended Data Figure 1.

Extended Data Figure 2..

Extended Data Figure 3.

Extended Data Figure 4.

Extended Data Figure 5.

Extended Data Figure 6.

Extended Data Figure 7.

Extended Data Figure 8.

Extended Data Figure 9.

Extended Data Figure 10.

(A) Demographic information for the Pediatric Intensive Care Influenza (PICFLU) cohort. (B) Histogram of VFDS for the PICFlu cohort. (C) Odds ratios for PRMODS and PRAHRF, as well as gender, previously healthy, age, and steroids as covariates. (D) Demographic information for influenza infection cohorts in Taiwan and Guangzhou. (E) Compartmentalization of ADAMTS-4 in samples from upper (nasal wash) and lower (sputum, BALF, ETA) respiratory tracts (NW, n = 131; sputum, n = 48; BALF, n = 25; ETA, n =34). Box plots were generated for samples in which ADAMTS-4 protein was detectable. Center line indicates median, bottom and top of boxes indicate first and third quartiles, and whiskers extend to 1.5 times the interquartile range. (F) Odds ratios for VFDS<10, including gender and age as covariates, in combined analysis of three human cohorts. Odds ratios (C,F) determined using logistic regression with FDR adjustments for multiple testing.

Supp Table 1

Supp Table 2

Supp Table 3

Supp Table 4

We thank the participants in each of the cohorts included in this study. We also thank Hongxia Zhou, Lee-Ann Van De Velde, Aisha Souquette, Greig Lennon, and Dahui You for technical assistance in conducting experiments; Tim Flerlage for critical reading of the manuscript; the St. Jude Animal Resource Center for the care of the animals used in this study. We acknowledge the use of tissues procured by the National Disease Research Interchange (NDRI) with support from NIH grant U42OD11158. Schematics in figures were made using an academic license of Biorender software.

The authors gratefully acknowledge the collaboration and support of PALISI PICFLU Study Site Investigators who assisted with study design, enrolled patients and made other major contributions to the PICFLU Study, specified as follows: Michele Kong, MD (Children’s of Alabama, Birmingham, AL); Ronald C. Sanders, Jr. MD, MS, Olivia K. Irby, MD (Arkansas Children’s Hospital, Little Rock, AR); Katri Typpo, MD (Diamond Children’s Medical Center, Tucson, AZ); Barry Markovitz, MD (Children’s Hospital Los Angeles, Los Angeles, CA); Natalie Cvijanovich, MD, Heidi Flori, MD (UCSF Benioff Children’s Hospital Oakland, Oakland, CA); Adam Schwarz, MD, Nick Anas, MD (Children’s Hospital of Orange County, Orange, CA); Peter Mourani, MD, Angela Czaja, MD (Children’s Hospital Colorado, Aurora, CO); Gwenn McLaughlin, MD (Holtz Children’s Hospital, Miami, FL); Matthew Paden, MD, Keiko Tarquinio, MD (Children’s Healthcare of Atlanta at Egleston, Atlanta, GA); Bria M. Coates, MD (Ann & Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL); Neethi Pinto, MD, Juliane Bubeck Wardenburg,MD, PhD, (The University of Chicago Medicine Comer Children’s Hospital, Chicago, IL); Adrienne G. Randolph, MD, MSc, Anna A. Agan, MPH, Tanya Novak, PhD, Margaret M. Newhams, MPH (Boston Children’s Hospital, Boston, MA); Stephen C. Kurachek, MD (Children’s Hospital and Clinics of Minnesota, Minneapolis, MN); Mary E. Hartman, MD, Allan Doctor, MD (St. Louis Children’s Hospital, St. Louis, MO); Edward J. Truemper, MD, Sidharth Mahapatra, MD, PhD (Children’s Hospital of Nebraska, Omaha, NE); Kate G. Ackerman, MD, L. Eugene Daugherty, MD (Golisano Children’s Hospital, Rochester, NY); Mark W. Hall, MD (Nationwide Children’s Hospital, Columbus, OH); Neal Thomas, MD (Penn State Hershey’s Children’s Hospital, Hershey, PA); Scott L. Weiss, MD, Julie Fitzgerald, MD, PhD (The Children’s Hospital of Philadelphia, Philadelphia, PA); Renee Higgerson, MD (Dell Children’s Medical Center of Central Texas, Austin, TX); Laura L. Loftis, MD (Texas Children’s Hospital, Houston, TX); Rainer G. Gedeit, MD (Children’s Hospital of Wisconsin, Milwaukee, WI); Marc-André Dugas, MD (Centre Hospitalier de l’Université Laval, Quebec, Quebec, Canada).

This work was funded by the National Institute of Allergy and Infectious Diseases under HHS contract HHSN27220140006C-OPT18I for the St. Jude Center of Excellence for Influenza Research and Surveillance (P.G.T), JHCEIRS contract HHSN272201400007C, the National Institutes of Health R01AI084011 and R21HD095228 (A.G.R), the National Institutes of Health and the National Natural Science Foundation of China under NIH-NSFC joint project 5R01AI128805-02, the National Natural Science Foundation of China under 81761128014, and ALSAC. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Competing Interests

PT is a consultant for Cytoagents Inc. A patent has been filed on work related to but not directly based on the findings in this manuscript. The authors declare no other competing interests.

Data Availability

Cohort cytokine and ADAMTS4 data can be found in Supplemental Tables 3 and 4. Single-cell gene expression data and spatial gene expression data are available on the NCBI Short Read Archive under BioProjects PRJNA612345 and PRJNA613670. Hallmark gene sets from the Molecular Signatures Database were used for gene set enrichment analysis (https://www.gsea-msigdb.org/gsea/msigdb/index.jsp). The following publicly available datasets were used for meta-analysis: 20180822_PolypAll_cleaned_rawdata.txt.zip (http://shaleklab.com/wp-content/uploads/2018/08/20180822_PolypAll_cleaned_rawdata.txt.zip) and 20180822_PolypAll_cleaned_metadata.txt (http://shaleklab.com/wp-content/uploads/2018/06/20180822_PolypAll_cleaned_metadata.txt); celseq_matrix_ru1_molecules.tsv.725583.gz and celseq_meta_unfiltered.tsv.725587.gz (https://browser.immport.org/browser?path=SDY998%2FResultFiles%2FRNA_sequencing_resu lt); GSE145926_RAW.tar (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE145926), GSE135893_barcodes.tsv.gz, GSE135893_genes.tsv.gz, and GSE135893_IPF_metadata.csv.gz (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE135893); GSE130148_raw_counts.csv.gz and GSE130148_barcodes_cell_types.txt.gz (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE130148); GSE122960_RAW.tar (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE122960); GSE128169_RAW.tar (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE128169); GSE132771_RAW.tar (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE132771).

Code Availability

All analyses were conducted using publicly available software as detailed in the methods. All code is available upon request.

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