Fetal microchimeric cells influence maternal lung health following term and preterm births

Animal care
All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Texas Medical Branch at Galveston. For ascending infection and immune cell-trafficking studies, a cyclic recombinase (Cre)-reporter mouse model previously described by Sheller-Miller et al.25 was used. Wild-type C57BL/6J (Strain #000664) and transgenic B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J (Strain #007676) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). This transgenic mouse, referred to as mT/mG, has a two-color fluorescent Cre-reporter allele targeting the plasma membrane of all cells and tissues26. In the absence of cyclic recombinase, tandem dimer Tomato (tdTomato) red fluorescent protein (RFP) is expressed in the plasma membrane of all cells and tissues. As these mice are fertile, all cells and tissues of the progeny express tdTomato when homozygous males are mated with wild-type C57BL/6 females, while the cells and tissues of maternal origin do not express this RFP. This study was conducted and reported in accordance with ARRIVE guidelines (https://arriveguidelines.org)27. The mice were housed in a temperature- and humidity-controlled facility with 12:12-h light and dark cycles. Regular chow and drinking solutions were provided ad libitum. Eight- to twelve-week-old wild-type females were mated with mT/mG homozygous males. Females observed with vaginal plugs in the morning, indicating gestational day 0.5 (E0.5), were housed separately from the males. A gain of at least 1.75 g by E10.5 confirmed pregnancy. A cohort of female mice not mated with males was maintained as the non-pregnant control (n = 5).
Polymerase chain reaction analysis
To determine the origin of fetal-specific cells in maternal tissues, polymerase chain reaction (PCR) was performed for the SRY gene on the Y chromosome. RNA was extracted from isolated maternal tissues (lungs, placenta, uterus, and kidney) using TRIzol® reagent (Invitrogen, Life Technologies), followed by chloroform (Fisher Scientific) and isopropanol (Acros Organics) treatments to purify total RNA. Total RNA was reverse-transcribed into complementary DNA (cDNA) with a high-capacity RNA-to-cDNA kit (Applied bio-systems). cDNA was then amplified by PCR using the RED Taq®Ready MixTM PCR reaction mix (Sigma) with the specific primers (for SRY gene, forward: 5′-TGGCGATTAAGTCAAATTCGC-3′ and reverse: 5′-CCCTAGTACCCTGACAATGTATT-3′; for GAPDH gene, forward: 5′-ACCACAGTCCATGCCATCAC-3′ and reverse:5′-TCCACCACCCTGTTGCTGTA-3′), according to the manufacturer’s instructions. The PCR amplification was conducted using an iCycler thermal cycler (Bio-Rad) under the following conditions: 5 min at 94 °C denaturation, followed by 40 cycles at 94 °C for 30 s, 58 °C for 45 s, and 72 °C for 45 s. The PCR products (137 bp for SRY, 452 bp for GAPDH) were then analyzed with 1.5% agarose gel electrophoresis. The lung tissues collected from a male mT+ served as a positive control.
Model of ascending infection-induced preterm birth (PTB)
The detailed protocol for inducing ascending infection by E. coli has been previously described28,29. In brief, varying doses of E. coli (104 CFU) in 40-µL nutrient broth (Difco, Cat. # 234000, BD Biosciences, Franklin Lakes, NJ, USA) were vaginally administered to pregnant C57BL/6J mice using a blunt 200-µL pipette tip on gestational day E15. The timing of delivery, defined as the delivery of the first pup, was monitored using live cameras (Shenzen Wansview Technology, Shenzen, China). Delivery on or before E18.5 was considered preterm. A subset of pregnant C57BL/6 mice (n = 6) was euthanized at E16 via carbon dioxide inhalation, following IACUC and the American Veterinary Medical Association guidelines, to collect the maternal lung tissues.
Immunofluorescent imaging for mT signal in the maternal lung tissues
Tissue samples were collected from pregnant mice on E16 and were fixed in 4% paraformaldehyde and stored overnight at 4 °C before being washed twice with 1× PBS and transferred to a 15% sucrose solution overnight at 4 °C. The samples were then transferred to 30% sucrose and were stored at 4 °C until they were embedded in the optimal cutting temperature compound and cut into 5-mm sections. The sections were incubated at room temperature for 30 min and were then washed twice in water to remove the optimal cutting temperature compound. The sections were incubated with DAPI for nuclear staining for 2 min at room temperature and were then washed twice in water. To reduce the autofluorescence, tissues were incubated for 10 s with TrueVIEW Autofluorescence Quenching Kit (Vector Laboratories, Burlingame, CA), then washed twice with 1× Tris-buffered saline + Tween 20 (TBS-T). The slides were air-dried at room temperature for 10 min and then mounted using a mounting medium.
Immune cell isolation from the maternal lung tissue
The collected postpartum-term and preterm maternal lung tissues were cleaned using fine forceps to remove excess fat and were washed in cold 1× phosphate-buffered saline (PBS) (pH 7.4). Using fine scissors, the lung tissues were cut into small pieces and were digested with (30,31,32,33) Accutase cell-detachment solution (Corning, Corning, NY, USA) for 35 min at 37 °C with gentle rocking. While on ice, the tissue samples were strained through a 70-µm cell strainer and were washed twice with 10.0 mL of 1× PBS. After centrifugation at 1250 g for 10 min at 4 °C, the resulting cell pellets were resuspended in 2.0 mL of 1× red blood cell lysis buffer (BioLegend, San Diego, CA, USA) and incubated for 10 min at room temperature. Following lysis, the cell suspension was centrifuged at 1250 g for 10 min at room temperature. The supernatant was removed, and the cell pellet was resuspended in 1.0 mL of serum-free DMEM/Nutrient Mixture F-12 medium (DMEM/F12; Mediatech, Manassas, VA, USA). The cell suspension was overlaid gently on 500 µL of neat fetal bovine serum (FBS) (Sigma-Aldrich., Burlington, MA, USA) and the resulting mixture was centrifuged at 1100 g without brake for 10 min at room temperature. After carefully discarding the supernatant, the cell pellet was resuspended in 1.0 mL of DMEM/F-12 supplemented with 10% FBS.
High-dimensional single-cell profiling of fetal membrane tissues by mass cytometry
The CyTOF panel was designed based on the high-throughput screening results and also included proteins known to regulate myeloid cell functions (such as MerTK and Axl), transcription factors, and signaling molecules known to be relevant to inflammation during pregnancy (NF-κB and MAPK pathway). A summary of antibodies used for each panel is shown in Table 1. The antibodies were sourced from the MD Anderson Cancer Research Center Flow core facilities (MDACC, Texas, Houston), or were custom conjugated using the Maxpar antibody conjugation kit (Fluidigm, Markham, ON, Canada), following the manufacturer’s protocol. After being labeled with their corresponding metal conjugate, the percentage yield was determined by measuring their absorbance at 280 nm using a Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE). The antibodies were diluted to 0.3 mg/mL using the Candor PBS antibody stabilization solution (Candor Bioscience GmbH, Wangen, Germany) and then stored at 4 °C.
Antibody staining
Single-cell suspension samples were resuspended in Maxpar staining buffer for 10 min at room temperature on a shaker to block Fc receptors. The cells were mixed with a cocktail of metal-conjugated surface marker antibodies (Table 1), yielding 500-μL of final reaction volumes, and then were stained at room temperature for 30 min on a shaker. Following staining, the cells were washed twice with Maxpar staining buffer. Next, the cells were permeabilized with Max Perm Buffer for 10 min at 4 °C. The cells were then washed twice in Maxpar staining buffer to remove the remaining Max Perm, and then stained with intracellular antibodies in 500 μL for 30 min at room temperature on a shaker. The samples were then washed twice in Maxpar staining buffer. The cells were incubated overnight at 4 °C with 1 mL of 1:4000 191/193Ir DNA intercalator (Standard BioTools, Inc., Markham, ON) diluted in Maxpar fix/perm. The following day, the cells were washed once with Maxpar staining buffer and then two times with double-deionized water (ddH20).
Mass cytometry
Prior to analysis, the stained and intercalated cell pellet was resuspended in ddH2O containing polystyrene normalization beads containing lanthanum-139, praseodymium-141, terbium-159, thulium-169, and lutetium-175, as described previously 59. Stained cells were analyzed on a CyTOF 2 (Standard BioTools Inc, Markham, ON) outfitted with a Super Sampler sample introduction system (Victorian Airship & Scientific Apparatus, Alamo, CA) at an event rate of 200–300 cells per second. All mass cytometry files were normalized using the mass cytometry data normalization algorithm freely available for download from https://github.com/nolanlab/bead-normalization.
Data analysis
CyTOF data sets were first manually gated using the Standard BioTools/Fluidigm clean-up procedure with Gaussian discrimination (Markham, ON) in FlowJo V10 (FlowJo LLC). Each sample was given a unique Sample ID and then all samples were concatenated into a single .fcs file. This concatenated file was further analyzed by T-Distributed Stochastic Neighbor Embedding (t-SNE) in FlowJo V10, using equal numbers of cells from preterm delivered, term delivered mouse lungs tissue, all surface markers, and the following settings: iterations, 3000; perplexity, 50; and eta (learning rate), 4105. Heat maps of marker expression were generated using the Color Map Axis function. To explore the phenotypic diversity of immune cell populations in the different groups of mouse FM tissues, we applied a K-nearest-neighbor density-based clustering algorithm called Phenograph. This algorithm allows for the unsupervised clustering analysis of data from single cells. The output is organized using a cluster explorer tool to visualize the phenotypic continuum of cell populations. This tool creates an interactive cluster profile graph and a heat map and displays the cluster populations on a tSNE plot.
HDME-induced allergic lung inflammation models
For this study, House dust mice extract (HDME) induced murine models of allergic lung inflammation were employed (Fig. 3A) following established protocols31,32. Briefly, mice were challenged with seven doses of HDME (50 ug) on every alternate day. To investigate potential pregnancy-associated effects, mice of the same age group, including both term delivery and preterm delivery mice, were categorized into four distinct groups and were subjected to specific treatments. Group 1 (n = 5) consisted of healthy, pregnant mice treated with PBS, Group 2 (n = 5) involved exposing healthy, pregnant mice to an HDME challenge, Group 3 (n = 5) comprised preterm, pregnant mice treated with PBS, and Group 4 (n = 5) entailed preterm mice challenged with HDME.
Following the final HDME challenge, mice were anesthetized using a mixture of 100 mg/kg of ketamine, 10 mg/kg of xylazine, and 3 mg/kg of acepromazine. A tracheostomy was performed, and the forced oscillation technique was employed to measure airway mechanics, utilizing a small animal ventilator (flexiVent, SCIREQ Scientific Respiratory Equipment, Montreal, QC, Canada). Central airway resistance (Rn) was assessed upon stimulation with methacholine (Mch). Afterward, blood, bronchoalveolar lavage fluid (BALF), and lung tissues were collected for subsequent endpoint analyses.
Quantitative real-time PCR
RNA from mouse lungs was reverse transcribed into cDNA using the high-capacity cDNA Reverse Transcription Kit or the TaqMan Advanced miRNA cDNA Synthesis Kit (Applied Biosystems, Foster City, CA). Real-time quantitative PCR was performed using Quant Studio 3 (Applied Biosystems) with validated TaqMan primers and the Fast Advanced Master Mix. Relative gene expression data (fold change) between samples were accomplished using the 2-ΔΔCt method. GAPDH (for gene expression) was used as the internal control.
Evaluation of lung inflammation and goblet cell hyperplasia
Lung sections were prepared and were subjected to staining with H&E to examine lung inflammation and periodic acid-Schiff (PAS) to assess goblet cell hyperplasia31,32. The methodology involved the infusion of the lungs with 10% buffered formalin through the trachea. After excision, the lungs were immersed in fresh 10% formalin and left overnight. Subsequently, the samples were embedded in paraffin, sliced into sections with a thickness of 5 mm, and stained with either H&E or PAS. To capture digital images of the sections, a Nikon Eclipse 50i microscope equipped with an Infinity-3 Digital Color Camera from Teledyne Lumenera and Infinity Analyze 6.5.4 software were utilized. The sections were anonymized and assessed in a blinded manner for scoring.
Inflammation scoring
For inflammation scoring, multiple images encompassing the entire lung section were acquired at a magnification of 4× to determine the total count of bronchioles demonstrating the presence of infiltrated inflammatory leukocytes. Each bronchiole was assigned a score ranging from 0 to 4. Scores of 0–1 denoted no inflammation or the sporadic occurrence of inflammatory cells, while scores of 2–4 indicated bronchioles surrounded by a thin layer (two to four cells) of inflammatory cells. Scores exceeding 4 indicated bronchioles enclosed by a thick layer (more than five cells) of inflammatory cells. The number of bronchioles falling into each category (0–1, 2–4, or > 4) was then divided by the total number of inflamed bronchioles, resulting in the percentage of bronchioles within each inflammation category. To calculate the percentage severity of inflammation, the number of inflamed bronchioles scoring 4 or higher was divided by the total number of inflamed bronchioles. Goblet cell hyperplasia was evaluated on lung sections stained with PAS32. Each lung sample was divided into nine imaginary sections, and digital images were captured at a magnification of 10× to ensure consistent observation of identical regions across all samples and experiments. The intensity of PAS staining was analyzed using ImageJ software to determine the count and percentage area of PAS-positive cells in each section.
In vitro functional assay of fetal microchimeric cells
Cell isolation and culture
FMCs were sorted from mice of both term and preterm deliveries by FACS, utilizing a transgenic model in which fetal cells expressed the mT+ fluorescent marker. This enabled the efficient identification and isolation of fetal-specific cells (Fig. 4A). The isolated FMCs were cultured and maintained under appropriate conditions.
Co-culture experiment
Human small airway epithelial cells (HSAECs) were cultured according to the supplier’s instructions (Lonza, Basel, Switzerland). FMCs (10,000 cells) obtained from the term and preterm deliveries from the previously isolated cell population were co-cultured with HSAECs in 24-well plates, as shown in Fig. 4A. This co-culture system aimed to mimic the interaction between FMCs and lung epithelial cells in a controlled environment.
Challenge with HDME
To assess the response of the co-cultured cells, HSAECs were challenged with HDME at a concentration of 5 μg/mL. HDME was chosen to induce a relevant inflammatory challenge in the HSAECs. This challenge was performed both with and without the presence of FMCs to investigate the potential modulatory effects of FMCs on the cellular response to HDME (Fig. 4B).
Supernatant collection and cytokine analysis
Following the incubation period of 24 h, the supernatant from the co-culture experiments was collected. The supernatant contained secreted factors that could reflect cellular responses. Cytokine analysis was performed on the collected supernatant samples to evaluate the inflammatory status of the lung epithelial cells. This analysis aimed to uncover any variations in cytokine profiles that might result from the interaction between FMCs and HSAECs (Fig. 4B).
The entire experimental procedure aimed to simulate the interaction between FMCs and HSAECs under different conditions to assess the potential impact of FMCs on HSAEC inflammation. The co-culture approach provided insights into the intricate interplay between FMCs and lung health, while the cytokine analysis allowed for the quantification of inflammatory responses, contributing to a comprehensive understanding of this cellular interaction.
Statistical analysis
Statistical analysis was performed using the GraphPad Prism 10.0 software (GraphPad, San Diego, CA). Statistical parameters associated with the figures are reported in the figure legends. All data are reported as the mean ± SEM. The statistical significance in differences between experimental groups and controls was assessed as follows: non-parametric and unpaired t-test for E. coli dose-dependent PTB study and Fisher’s exact test for the rates of PTB. Significance was considered at p < 0.05.
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