Sex as a biological variable

For studies using human and animal samples, sex is specified as appropriate to the design of the given experiment. For preclinical studies, age and sex-matched mice were used throughout, and analysis by individual sex did not reveal dimorphism in phenotype. Thus, data for males and females sexes have been combined.

Mice

STING–/– (MPYS–/–) mice were purchased from The Jackson Laboratory (strain 025805), then crossed with in-house C75BL/6J mice for maintenance. Pairs of STINGfl/fl (strain 035692), VeCad-Cre (strain 006137), LysM-Cre (strain 004781), and SMA-Cre (strain 029925) mice were purchased from The Jackson Laboratory and maintained in house with a C57BL/6J background. Thus, all transgenic mice generated in this study were on a C57BL/6J background. Transgenic mice expressing Cre-recombinase under the control of the VE-cadherin promoter (VeCad-Cre) were crossed with STING mice flanked by 2 loxP sites (STINGfl/fl) to generate Cre-mediated specific deletion of the STING gene in endothelial cells. Similarly, transgenic mice expressing Cre combinase under the control of the LysM promoter (LysM-Cre) or SMA promoter (SMA-Cre) were crossed with STINGfl/fl mice for generation of Cre-mediated specific deletion of the STING gene in myeloid cells and smooth muscle cells, respectively. Breeding was set up such that the STING construct was kept in a homozygous state, while VeCad-Cre, LysM-Cre and SMA-Cre were maintained in a heterozygous state, yielding Cre+ mice with respective cell deletion. Cre– mice were used as littermate controls. Mice were bred and housed in specific pathogen–free conditions. Eight- to 10-week-old sex-matched mice were used for each experiment (4–5 mice/sex/group), after confirmation of appropriate genotype, with genotyping performed for all experiments.

Mouse models of PH

PH was induced in mice with either bleomycin injection or chronic hypoxia exposure.

Bleomycin. Mice received i.p. injections of bleomycin (MilliporeSigma 9041934) at 0.018 U/g twice a week for 4 weeks. Weights of animals were monitored throughout the injection period. A 20% loss of body weight resulted in temporary termination of bleomycin treatment. Injection was resumed when the animal regained at least 10% of lost weight. Euthanasia and data collection were performed 5 days after final injection, on day 33 of the bleomycin injection protocol.

Chronic hypoxia. Mice undergoing chronic hypoxia exposure were placed in a normobaric ventilated chamber, in which the level of O2 is controlled through flow of N2 (ProO2 monitor/controller and chamber, Biospherix). O2 and CO2 concentration was monitored continuously, such that their concentrations remained at 10% and 0.1% respectively. Exposure to normal air was limited to water, food, and cage changes. All mice were sacrificed after 28 days (4 weeks) of exposure for analysis.

Generation of bone marrow chimeric mice

Ten-week-old recipient CD45.1+ and STING–/– male mice were irradiated with 2 doses at 5 Gy (11 minutes, 4-hour interval) and received bone marrow cells from 10-week-old male CD45.2+ or STING–/– donor mice the next day through retro-orbital injection. Antibiotic (TMS, 100 mg/kg) was delivered in drinking water together with soft food for 2 weeks after bone marrow transplantation. Six weeks were allowed for bone marrow reconstitution before mice were subjected to 4 weeks of chronic hypoxia exposure. An experimental design scheme is provided in Figure 2A.

Chimerism was confirmed using flow cytometry staining for CD45.1 or CD45.2 in recipient mouse submandibular blood collected at 6 weeks after transplantation. Chimerism was calculated as: % cells expressing CD45.2/% cells expressing CD45.1.

Treatment with anti–PD-L1 antibody

Eight- to 10-week-old LysM-Cre+/–STINGfl/fl and LysM-Cre–/–STINGfl/fl male mice were given i.p. injections of 500 mg anti–PD-L1 antibody (BioXCell BE0101) or isotype control (BioXCell BE0090) once a week for 4 weeks, concurrently with injection of bleomycin (0.018 U/g twice a week) or PBS according to experimental group.

Primer sequences in mice

See Supplemental Table 3 for primer sequences in mice. SAVI mice genotyping was sent to Transnetyx for confirmation of appropriate genotype.

Pulmonary hemodynamic assessment

Mice were put under deep anesthesia with i.p. injection of 25% avertin (2,2,2-tribromoethanol, Thermo Fisher Scientific AC421432500) in PBS at a 16 mg/kg dose. A 1.4-French pressure-volume microtip catheter (Millar Instruments, SPR-839) was inserted through a right internal jugular incision and threaded down into the right ventricle. The catheter was connected to a signal processor (PowerLab and ADInstruments), and RVSP (mmHg) was recorded digitally and displayed with Chart5. After stable measurements of a minimum of 5 minutes, animals were euthanized, with hearts and lungs removed for subsequent analysis. The right ventricle was separated from the heart after removal of the atria, and the weights of both right ventricle (RV) and left ventricle plus septum (LV+S) were obtained. Right ventricular hypertrophy was later calculated using the ratio RV/LV+S (%) (Fulton index).

Flow cytometry and phagocytosis assay

The left lung was cut into pieces and digested for 1 hour with 3 mL RPMI 1640 with 10% FBS (Gibco A4766801), DNAse I (10 mg/mL, Roche 10104159001), and 5% Liberase (MilliporeSigma 05401127001) at approximately 200g and 37°C. Tissues were triturated with 3 mL syringes and 18G needles until complete dissociation was achieved. Cells were filtered through 70 mm cell strainer, washed 3 times with D-PBS–2% FBS and 1 M EDTA to remove debris. Remaining red blood cells were lysed with ammonium chloride lysis buffer (KD Medical 50-1019080). Single-cell suspensions obtained were stained with fluorochrome-conjugated surface antibodies for 30 minutes on ice, Fixable Viability Dye (eBioscience 65-0865-14) for 30 minutes, or overnight on ice; and fixed, permeabilized, and then incubated with intracellular markers for 30 minutes on ice. Data were acquired using a BD FACSymphony A3 Cytometer (BD Biosciences) with 5 lasers and analyzed with FlowJo version 10 software. A list of antibodies is provided in Supplemental Table 4. Phagocytosis was assessed using the Vybrant Phagocytosis Assay Kit (Thermo Fisher Scientific V6694), per the manufacturer’s instructions, with isolation and macrophage differentiation in vitro as previously published by our group (31).

FACS antibodies

A comprehensive list of FACS antibodies used is presented in Supplemental Table 4.

cDNA library construction and scRNA-Seq

Mouse whole lungs were perfused with 10 mL PBS to remove blood cells. Lung tissues then were cut into small pieces and incubated in 3 mL RPMI 1640 with 10% FBS (Gibco A4766801), DNAse I (10 mg/mL, Roche 10104159001) and 5% Liberase (MilliporeSigma 05401127001) for 1 hour at Approximately 200g and 37°C. Tissues were triturated with 3mL syringes and 18G needles until complete dissociation was achieved. Cells were filtered through 70 mm cell strainer, washed 3 times with D-PBS–2% FBS and 1 M EDTA to remove debris. Remaining red blood cells were lysed with ammonium chloride lysis buffer (KD Medical 50-1019080). Single cells were captured in 10X Genomics Chromium Single Cell 3′ Solution, and RNA-Seq libraries were prepared following the manufacturer’s protocol (10X Genomics). The libraries were subjected to high-throughput sequencing on an Illumina NovaSeq 6000 platform, targeting 6,000–8,000 cells per sample with a sequencing depth of at least 20 million reads of 150 bp paired-end reads.

Process and quality control of the scRNA-Seq data

The raw sequencing reads were aligned with mouse genome mm10 provided on the CellRanger website by 10X Genomics. The mapped reads then were used for unique molecular identifier (UMI) counting, following the standard CellRanger pipeline for quality control as recommended by the manufacturer (10X Genomics). In short, cells with UMI counts lower than 500 or a feature count less than 200 were excluded. In addition, cells with greater than 30% of RNA content made up of the most common genes were excluded, as they accounted for empty droplets with free-floating RNA. Cells with greater than 30% of RNA content mapped to the mitochondrial genome were also eliminated, as they indicated poor quality. Last, cells with abnormally high counts were discarded. Subsequently, the filtered single cells were imported into the R package “Seurat” (version 4.0) for clustering of data and calculating differential gene expression, following the standard pipeline per the manufacturer’s instruction (10X Genomics). Markers for different populations of cells (stromal, endothelial, and myeloid) were obtained from previously described work (62–66).

Mouse sample histological staining

The right lower lobe of mouse lungs, upon harvest, was fixed in formalin overnight. Fixed tissues were paraffin embedded, cut with a Leica RM2235 Microtome, and stained for Masson’s trichrome (MTC) and α-SMA to assess inflammation as well as to identify muscularized pulmonary vessels (21).

MTC staining and semiquantitative inflammation scoring. Lung inflammation was evaluated on trichrome-stained lung sections using a 0–4 scale: score of 0, normal lung architecture; 1, increased thickness of some (<50%) of the interalveolar septa; 2, thickening of >50% of the interalveolar septa without formation of fibrotic foci; 3, thickening of the interalveolar septa with formation of isolated fibrotic foci; and 4, formation of multiple fibrotic foci with total or subtotal distortion of parenchymal architecture. Masked evaluation was performed on 10 randomized sequential, nonoverlapping fields (magnification 10×) of lung parenchyma from each specimen. The mean score for the 10 fields represented the score for each individual specimen.

a-SMA staining and muscularized vessel count. Formalin-fixed lung sections were stained for rabbit polyclonal α-SMA (Abcam ab5694; diluted 1:750 in antibody diluent reagent solution [Life Technologies], not reused; blocking reagent, Background Sniper [Biocare]). Stained lung specimens were then randomized, and lung parenchyma from each specimen was assessed in a masked manner for pulmonary vessel counts in 10 sequential, nonoverlapping fields (magnification, 10×). Partially or completely muscularized pulmonary vessels were visualized in brown. Vessels were considered small if length was less than 50 μm; medium if 50–150 μm; and large if greater than 150 μm.

For immunofluorescence staining, sections were incubated with the primary antibodies CD31 (R&D Systems, AF3628, 1:100) or CD11b (Novus, NB600-1327, 1:100) and STING (Novus, NBP2-24683, 1:100) for 1 hour at room temperature. Sections were then incubated with corresponding secondary antibodies, prior to being mounted using Vectashield Vibrance Antifade DAPI (Vector Laboratories), with imaging as described below.

Image processing and acquisition

Images of representative tissues from the histological staining were taken with a Keyence BZ-X microscope at 10×–20× magnification. Image processing was performed using BZ-X-Analyzer software (Keyence).

Western blot and zymography

Right middle lobes were cut into pieces in 1 mL Pierce RIPA Buffer (Thermo Fisher Scientific 89901), 1X Halt Protease & Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific 1861282), then homogenized using QIAGEN TissuelyzerLT. Protein concentration was determined using Pierce BCA Protein Assay Kits (Thermo Fisher Scientific 23225). 10% Criterion TGX Precast Gels (Bio-Rad 5671034) were used for protein separation (20 μg per sample) in Criterion Midi Cell (Bio-Rad 1656001) with 1× Tris/Glycine Buffer (Bio-Rad 1610771). Proteins were transfered to membrane blots using the Trans-Blot Turbo Transfer System (Bio-Rad 1704150) at standard setting. Membranes were blocked with EveryBlot Blocking Buffer (Bio-Rad 12010020) for 10 minutes at room temperature, then incubated with primary antibodies (1:1,000) against MMP8 (ProteinTech 17874-1), MMP9 (Cell Signaling Technology 24317AS), MMP2 (Cell Signaling Technology 87809S), TIMP3 (Cell Signaling Technology 5673S), STING (Cell Signaling Technology 13647), and β-Actin (Cell Signaling Technology 4967L) at 4°C overnight. Membranes were then washed in TBS 0.1% Tween 20 and incubated with secondary antibodies (1:10,000) for 1 hour at room temperature. Protein expression was detected with Clarity ECL Western Blotting Substrates (Bio-Rad 1705060) and visualized with the ChemiDoc Imaging System (Bio-Rad 12003153). Quantification of protein was performed in Image Lab software (Bio-Rad). All protein levels were normalized to the β-Actin protein level.

Primary vascular SMCs were isolated from smSTING and control mice as previously described (67). Cells were then grown to confluence in 6-well plates before being exposed to hypoxic conditions for 24 hours (10% O2 concentration; O2 Control InVitro Cabinet, Coy Laboratory Products). MMP activity in cell lysates was then analyzed using gel zymography. Protein samples (10 μL each; 6 μg per lane) were mixed in Novex Tris-Glycine Sodium Dodecyl Sulfate (SDS) Sample Buffer (Thermo Fisher Scientific) and then loaded onto Novex 10% Zymogram (Gelatin) protein gel for separation. Gel was electrophoresed at 126 V for 90 minutes. After electrophoresis, the gels were renatured in Zymogram renaturing buffer (Thermo Fisher Scientific) at room temperature for 30 minutes and incubated overnight at 37°C in Zymogram developing buffer (Thermo Fisher Scientific). The gels were stained with Coomassie blue staining solution (0.1% Coomassie R250 in 40% ethanol, 10% acetic acid) for 2 hours and then destained twice for 30 minutes in destaining solution (10% ethanol and 7.5% acetic acid). The presence of a clear band on a dark background indicated MMP activity; images were captured as described above.

Human samples and scRNA-Seq analysis

Blood was obtained from healthy donors and patients with PAH and ILD (with and without PH) at the University of Florida Shands Hospital Pulmonary Hypertension and Interstitial Lung Disease Clinics (Gainesville, Florida). Participants were either healthy individuals (donor) or patients diagnosed with Group 1 idiopathic PAH, Group 3 ILD-associated PH, or ILD without associated PH. PH was diagnosed by right heart catheterization. Exclusion criteria included age less than 18 years and concomitant cardiopulmonary phenotype. Samples were then processed by a specialist into PBMCs and serum, then stored at –80°C until analysis.

PBMCs were subjected to flow cytometry for quantification of STING expression in various cell populations. Extracellular staining was performed with fluorochrome-conjugated surface antibodies for 30 minutes on ice, followed by Fixable Viability Dye (eBioscience 65-0865-14) for 30 minutes on ice. The cells were then fixed, permeabilized, and incubated with intracellular markers for 30 minutes on ice. Data were acquired using a BD FACSymphony A3 Cytometer (BD Biosciences) with 5 lasers and analyzed with FlowJo version 10 software. A list of antibodies used is provided in Supplemental Table 4.

Serum samples were subjected to MILLIPLEX Magnetic Bead Panel (EMD MilliporeSigma MCYTOMAG-70K) for quantification of different interleukins, chemokines, and growth factors, including eotaxin, G-CSF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17, IP-10, KC, LIF, LIX, MCP-1, M-CSF, MIG, MIP-1α, MIP-1β, MIP-2, RANTES, TNF-α, and VEGF.

Patient and healthy donor control lung samples for generation of immunofluorescence images were obtained from the Lung Tissue Research Consortium as previously reported (31) (NIH BioLINCC, HLB02342020a). Upon antigen retrieval, sections were incubated with the primary antibodies CD31 (R&D Systems AF3628; 1:300) or α-SMA (MilliporeSigma A2547; 1:300) and STING (Novus NBP2-24683; 1:200) for 1 hour at room temperature. Sections were then incubated with corresponding secondary antibodies, prior to imaging as described above. Of note, sections were mounted using Vectashield Vibrance Antifade DAPI (Vector Laboratories); thus, DAPI was used as a nuclear counterstain. Images were qualitatively interpreted to identify patterns of coexpression between STING and cell-specific antibodies.

scRNA-Seq data were obtained from the NCBI’s Gene Expression Omnibus database (GEO GSE210248). For the analysis, only samples from patients with PAH and healthy donors (Donor) were selected, specifically the following 6 samples: Donor_1, Donor_2, Donor_3, PAH_1, PAH_2, and PAH_3. Each sample was processed individually using the Seurat package (v5) (68). Quality control (QC) steps included removing cells with over 5% mitochondrial gene content, fewer than 200 detected features (genes), or an abnormally high number of features exceeding 3 times the median absolute deviation. After QC, highly variable genes were identified using the FindVariableFeatures() function with default parameters. Gene expression data were normalized, scaled, and centered using the ScaleData() function. Principal component analysis (PCA) was conducted on the variable genes, and the first 20 principal components were used to construct a shared nearest neighbor (SNN) graph. Clustering was performed using FindClusters() with a resolution parameter of 0.2. For visualization, dimensionality reduction was performed using Uniform Manifold Approximation and Projection (UMAP), based on the same 20 PCs. Cell type annotation was conducted using canonical marker genes, including DCN, CD14, CXCR4, TAGLN, CCL5, S100A8, VWF, NKG7, BGN, TSPAN7, IGKC, CD1C, SFTPC, and TPSAB1 (25). Subsequently, genes encoding cGAS/MB21D1, STING1, type I interferon A and B, involved cGAS/STING pathway, and IL6, CCL7 (C6orf150, CCL5, CXCL10, IRF3, TBK1, TMEM173, STAT1, CCL2, CCL20, IFNA, IFNB, STAT6, DDX41, IFNAR1, IFNAR2, IL6, and CCL7) were used for KEGG pathway enrichment analysis for both the PAH and Donor groups by ShinyGO 0.82 (69) (https://bioinformatics.sdstate.edu/go/). Genes for each KEGG pathway were retrieved using BioServices Python library (70). The expression levels of all genes within each pathway were summed to obtain a pathway-level expression level. For each cell type, differential expression analysis for each pathway between PAH and donor groups was then performed using Wilcoxon’s rank-sum test. P values were adjusted for multiple comparisons using the Benjamini-Hochberg (BH) method. Pathways were considered differentially regulated if the adjusted P value was ≤0.05 and the absolute log fold change (|FC|) was ≥1.5.

Statistics

Statistical analysis was performed using GraphPad Prism 9.0 software. Quantitative data are presented as mean SEM. Each data point on bar graphs represents an individual mouse or human. Violin plots show mean, mode, and interquartile range of the data set. Data from each graph were collected across 1–3 individual experiment(s). Power analysis revealed 80% to more than 90% power to detect a change in the mean with n = 4. Therefore, all data sets were performed with n = 4–10. Data were pooled for biological replications performed in individual experiments for statistical analysis. Mean difference across groups was determined using ANOVA followed by unpaired 2-tailed Welch’s t test (unequal variance assumption), with Dunnett’s test used to compare each experimental group with a control group, accounting for multiple comparisons. For human cytokine data, Bonferroni’s method was used to correct for multiple comparisons, after 2-way ANOVA was utilized. A P value less than 0.05 was considered statistically significant.

Study approval

Animal experiments and maintenance were approved by the IACUC of the University of Florida (protocol 08702). Animal studies are reported in compliance with Animal Research: Reporting of In Vivo Experiments (ARRIVE) and the Guide for Care and Use of Laboratory Animals (National Academies Press, 2011). Ethical approval of biobanking (human tissue collection) and data collection was received from the University of Florida Institutional Review Board (IRB201400744). Tissues were collected after written informed consent was obtained from donors.

Data availability

Values for all data points in graphs are reported in the Supporting Data Values file.scRNA-Seq data were deposited in the NCBI’s Gene Expression Omnibus database (GEO 244864). R codes used for scRNA-Seq analysis are available in the GitHub depository (https://github.com/annt289/A-non-inteferon-dependent-role-of-STING-in-pulmonary-hypertension; commit ID 9be1c65). Additional data, analytical methods, and study materials that support the findings of this study are available from the corresponding author upon reasonable request.