Mice
Six- to eight-week-old C57BL/6J female mice (stock no: 000664) were purchased from Jackson Laboratories (Bar Harbor, ME). All animal studies have been approved by the Northwestern University IACUC.
Wound induction
Mice in the control and experimental treatment groups were acclimated for 2 days before their dorsal area was shaved and chemically depilated using Nair hair removal gel while they were under inhalational anesthesia (VetEquip V1 small animal anesthesia system with isoflurane). Two days after depilation, mice were anesthetized via intraperitoneal injection (125–250 mg/kg) of freshly prepared and filter sterilized 1.25% solution of Avertin (Sigma T48402, pH 7). A 12 mm circular template was drawn on the depilated area and baseline skin thickness measurements were taken. A 0.5% solution of Mechlorethamine hydrochloride (nitrogen mustard, NM) (Sigma 122564) in 1.5% DMSO-PBS was applied into the template area in a chemical hood. Mice were kept under observation on a warming pad until the NM dried and were then returned to their cages. The cages were kept in a chemical fume hood for 2 h before they were returned to the mouse facility.
PLGA particle preparation
Poly(lactic-co-glycolic acid) (PLGA) nanoparticles were obtained from COUR Pharmaceuticals (Skokie, IL) and prepared with proprietary formulations. Briefly, polymer was dissolved in a proprietary organic solvent, subsequently mixed with an aqueous surfactant before being sonicated to make a single oil-in-water emulsion. After removing the solvent by evaporation, PLGA nanoparticles of an average 500 nm in size and a zeta-potential of −75 mV were obtained. The resulting nanoparticles were washed and freeze-dried for storage as a lyophilized powder. Before use, PLGA nanoparticles were washed and dissolved at a final concentration of 5 mg/mL in PBS for injection.
Interventions
PLGA-IMPs were diluted to 5 mg/ml concentration in 1X PBS and 200 µl per mouse (1 mg) per day was delivered intravenously via tail vein injection. The particles were administered daily for up to 4 days, with the first dose given 2–3 h following skin application of NM. For Treg inactivation, 500 µg of PC-61.5.3 anti-CD25 (BioXCell, Lebanon, NH) or control IgG isotype-matched antibody HRPN (BioXCell) were given via intraperitoneal injection 4 days and 2 days before NM application.
Skin monitoring
Mice were observed daily after induction of NM skin injury while they were under inhalational anesthesia using a VetEquip V1 small animal anesthesia system with isoflurane. The following measurements were taken for up to 5 days: The bifold skin thickness within the 12 mm template area was measured using a digital caliper (Mitutoyo, PK0505CPX). Photographs of the injured area were taken using an iPhone camera mounted on a box. Mice were weighed using a mini scale. Wound areas were traced, analyzed, and quantified using ImageJ software (National Institutes of Health, Bethesda, MD).
RNA expression and cell type enrichment
Skin injury and wound healing–related gene expression profiling was measured with real-time quantitative PCR. Skin was collected from euthanized mice under Avertin anesthesia. Total RNA was extracted from mouse skin tissue using TRIzol reagent (Invitrogen,15596026), per manufacturer’s instructions. TaqMan gene expression assays were used for measuring the relative expression levels of interleukin 1β (IL-1β; Mm00434228_m1), tumor necrosis factor-alpha (TNF-α; Mm99999068_m1), matrix metallopeptidase9 (MMP9; Mm00442991_m1), and nitric oxide2 (iNOS; Mm01309902_m1). 18 s (Hs99999901_s1) was used as the control housekeeping gene. 100 ng of each RNA was amplified using TaqMan Fast Virus 1-Step Master Mix (4444434). The relative expression was calculated using the ddCt method as fold change compared to controls.
Bulk RNA-seq counts (GEO accession number GSE218810) were created from skin biopsies taken as a part of our clinical trial NCT0296844620. Biopsies were collected before NM exposure and 72 h post-exposure were normalized and subjected to the “raw Enrichment Analysis” from the “xCell”34 package in R for cell type enrichment analysis of curated cell type gene sets. Enrichment scores were then submitted to the interactive xCell heatmap viewer https://comphealth.ucsf.edu/app/xcellview/. Weak signatures were filtered out and enrichment scores were percentiled. The column dendrogram was generated using Euclidean distance and Ward.D linkage.
Flow cytometry
Mouse skin samples were mechanically disrupted followed by digestion using Liberase TL (Sigma, 1 h incubation at 37 °C) at concentration 0.25 mg/mL dissolved in RPMI-1640 with l-glutamine (Corning), supplemented with 1 mM sodium pyruvate, MEM nonessential amino acids, and 20 mM HEPES (Gibco). The resulting cellular suspension was filtered through 70 then 40 μM cell strainers and centrifuged at 500 × g for 10 min. at room temperature. The cell pellet was collected, washed with a PBS solution containing 5% FCS serum (PBS/FCS), and resuspended in fluorescence‐activated cell sorting (FACS) buffer (PBS with 2% fetal calf serum), and cells counted. Spleens were triturated, passed through 100 μm cell strainers, spun at 500 × g for 10 min, and washed with PBS/FCS. Red blood cells were lysed with Tris-ammonium chloride (0.16 M) for 5 min at room temperate, washed with PBS/FCS, and spun at 500 × g for 10 min. The cell pellet was resuspended in FACS buffer. The numbers of each subpopulation in the skin and spleen were determined by multiplying the percentage of lineage marker–positive cells by the total number of mononuclear cells isolated from the corresponding tissue.
Single cells were incubated with Fc block (anti-mouse CD16/32, 0.25 μg; eBioscience, San Diego, CA) for 30 min. at 4 °C. Cells were then washed with FACS buffer containing PBS with 2.5% (volume/volume) fetal bovine serum and 0.1% (weight/volume) NaN3 (Sigma‐Aldrich). Cells were then stained for surface markers (at a dilution range of 1:100 to 1:250) for 30 min at 4 °C using the specified antibodies. These antibodies included CD45, CD11b, CD11c, Ly6C, Ly6G, MHC class II, CD80, CD86, B220, CD3, CD4, CD8, CD49b, PD-1, CD122, IL-10, ST2, TGF-β, IL-6 and MARCO. Cells were then washed with PBS, and viability staining was performed using the LIVE/DEAD fixable dead cell stain kit (Invitrogen, Carlsbad, CA). Following viability staining, cells were washed with PBS and were either resuspended in FACS buffer for flow cytometric analysis or were subjected to intracellular staining to detect inducible nitric oxide synthase (iNOS), CD206, or FoxP3. For intracellular staining, cells were fixed and permeabilized using the FoxP3 staining buffer kit (eBioscience) and then intracellularly stained. Briefly, cells were incubated in fixation buffer from eBioscience for 45 min at 4 °C, washed with Permeabilization buffer, stained with a cocktail of antibodies specific for intracellular cytokines for 45 min at 4 °C, washed again and analyzed on a cytometer. As controls, fluorescence minus one was used to place the gates for analysis.
For flow cytometric analysis, cells were first gated according to forward and side scatter and then restricted to single cells and live cells. Tissue infiltrating myeloid cells were identified as CD45+CD11b+CD3− and infiltrating lymphoid cells as CD45+CD11b−CD3+ for T cells and CD45+CD3−CD11b−B220+CD11c− for B cells. On the infiltrating lymphoid population, cells were gated on CD3+CD4+CD8− or CD3+CD8+CD4− to evaluate the different T lymphocyte subpopulations. For infiltrating myeloid cells, Ly6G+ neutrophils were first gated and excluded from the infiltrating myeloid subpopulations. The Ly6G− myeloid cells were divided into CD11c+ myeloid dendritic cells (mDCs) and CD11c− monocytes/macrophages. Finally, the monocytes/macrophages were further divided into Ly6Chi inflammatory monocytes and Ly6clo noninflammatory monocytes. From these subpopulations, expression of CD86 and MHC II were evaluated. Expression of iNOS, eGR2, and CD206 were evaluated on the monocyte/macrophage subpopulation.
Mouse-specific antibodies applied to the mixture of cell types segregated as described above are listed in the key resources Table 1 (Anti-mouse antibodies used for flow cytometry). A 6-laser Fortessa flow cytometer (BD Biosciences) was used to enumerate cell populations, and the data was analyzed using FlowJo software (TreeStar, Ashland, OR).
Histology and microscopy
Half of the 12 mm wound area from euthanized mice were fixed in 10% formalin solution for 24 h. Samples were then embedded in paraffin, sectioned at 4-μm thickness, and stained with hematoxylin and eosin Y. Slides were scanned in a brightfield microscope and images were captured using a mounted camera. For immunohistochemistry, slides were incubated with anti-human MARCO (Abcam, ab231046), anti-mouse MARCO (Abcam, ab256822) antibodies, or isotype-matched control IgG (eBioscience, San Diego, CA) followed by HRP-conjugated secondary antibody, which was visualized with diaminobenzidine substrate, and counterstained with hematoxylin.
Statistics
Data are presented as means ± SEM. Two-tailed Student’s t-test or Mann Whitney U test was used for comparisons between 2 groups. If experiment involved more than three groups, 1-way ANOVA followed by Tukey or Sidak’s analysis was used to examine for statistical significance. The Chi Square analysis was used for categorical variables. A p-value less than 0.05 denotes a statistically significant difference. The Pearson correlation for continuous variables and the Spearman correlation for ordinal variables were used to evaluate relationships between two variables. Data were analyzed and graphs were created using GraphPad prism (GraphPad Prism Software version 7.03, GraphPad Software Inc.). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. The percent variation of skin thickness, weight, and wound area was calculated and graphed using GraphPad Prism software. The built-in statistics in Prism software was used to calculate the significance of the results. Fold change values of RNA expression were plotted using GraphPad Prism software, and statistical significance was calculated with built-in One-way ANOVA method.
Study approval
All animal studies were conducted in accordance with NIH guidelines for the care and use of laboratory animals and protocols were approved by the Institutional Animal Care and Use Committee of Northwestern University. Studies involving human subjects were approved by the IRB of Northwestern University, all participants provided written informed consent, and study complied with all relevant ethical regulations including the Declaration of Helsinki.
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- Source: https://www.nature.com/articles/s41536-024-00381-z