Studies were conducted in four (two adult and two aged) baboons from the conventional colony at The University of Oklahoma Health Sciences Center (OUHSC) [Figure 1]. All animals were housed in Animal Biosafety Level 1 (ABSL1) facilities at OUHSC for the duration of the study. All procedures were approved by the University Institutional Animal Care and Use Committee at OUHSC and the OUHSC Biosafety Committee. Animals were vaccinated with Mycobacterium bovis BCG (Pasteur strain, ATCC, Manassas, VA) via the intradermal route in the upper arm at a dose of 5x105 CFUs. Skin tests were performed on animals at the times indicated in Fig. 1 by injecting 100µL of tuberculin (Colorado Serum Company, Denver, CO) containing 5 tuberculin units of purified protein derivative (PPD). Saline injections (100µL of 0.9% NaCl, Baxter International Inc., Deerfield, IL) were performed in animals to serve as negative controls. All skin tests were performed in the chest skin of animals according to the diagram in Fig. 1. All animals had positive TST responses in the study as determined by visual observation (Fig. 1C). At 72 hours and 7-days post-skin test challenge, 8mm skin punch biopsies (ThermoFisher Scientific, Waltham, MA) were obtained from the sites of tuberculin and saline injection (schematic in Fig. 1). All biopsies were shipped overnight to Texas Biomedical Research Institute (Texas Biomed) for downstream processing. Blood was collected in sodium heparin-vacutainers according to the timeline in Fig. 1 and shipped overnight to Texas Biomed for processing. All procedures were performed under anesthesia (10mg/kg Ketamine and 0.05-0.5mg/kg Acepromazine, Covetrus, Portland, ME), and included monitoring of weight, body temperature, heart rate, respiration rate and capillary refill time.
Preparation and storage of skin punch biopsies
Immediately following collection at OUHSC, each 8mm skin biopsy was cut in half using a pathology blade and biopsy halves were stored in 10% neutral buffered formalin (ThermoFisher Scientific), snap-frozen in liquid nitrogen, or prepared for electron paramagnetic resonance (EPR) analysis. Remaining skin tissue was banked for future analysis. For EPR analysis, skin biopsies were further divided into two pieces, weighed, and incubated with the following: 1) 400µM CMH-hydrochloride (Enzo Life Sciences, Farmingdale, NY) or 2) 400µM CMH + 500nM rotenone (MP Biomedicals, Santa Ana, CA) + 100µM antimycin A from Streptomyces sp. (MilliporeSigma, Burlington, MA) (CMH + RA) for 30 minutes at 37°C. After 30-minute incubation, CMH or CMH + RA solution was removed from the tissue and stored in a cryovial. Both tissue and CMH solutions were snap-frozen in liquid nitrogen. Biopsies were shipped on dry ice (snap-frozen tissues) or at room temperature (tissues in formalin) to Texas Biomed for downstream processing.
Preparation of Tissue Homogenates from Skin Biopsies
Skin punch biopsies (snap-frozen in liquid nitrogen) were homogenized in Lysing Matrix D tubes (MP Biomedicals) in Tissue Extraction Reagent I (ThermoFisher Scientific) with cOmplete Mini Protease Inhibitor Cocktail (MilliporeSigma). Protein content was determined by Pierce BCA protein assay kit (ThermoFisher Scientific), according to kit instructions.
Luminex Assay Measurement of Immune Mediators
Immune mediators were measured in cell supernatants, plasma, and tissue homogenates using the following Luminex assays cross-reactive with baboons: NHP XL Cytokine for detection of CCL5, CCL20, CXCL10, IL1β, IL2, IL6, TNFα, and IL8, and Human XL Cytokine for detection of CCL2, CXCL10, IL8, IL1β, and IL6 (R&D Systems, Minneapolis, MN), according to kit instructions. For detection of IFN-γ and IL2, monkey interferon-gamma (IFN-γ) ELISA kit and monkey IL-2 ELISA kit (Mabtech, Inc., Cincinnati, OH) were performed, according to kit instructions. For tissue homogenates, analyte levels were normalized to protein content in homogenates (pg analyte per µg protein), as determined by BCA assay.
Measurement of Tissue Oxidation Levels
For superoxide determination by EPR, CMH and CMH + RA tissue supernatants were thawed and loaded into a quartz cell. Superoxide levels in the samples were determined by EPR. EPR spectra were obtained on the Bruker EMXnano ESR system (Bruker Corporation, MA, USA) using the following parameters: Frequency, 9.636541 GHz; Center Field, 3435.30 G; Modulation Amplitude, 2.000 G; Power 0.3162 mW; Conversion Time of 40.00 ms; Time Constant of 1.28 ms; Sweep Width of 100.0 G; Receiver Gain at 40 dB; and 1 total number of scans. Samples were baseline corrected relative to CMH only (control). Area under the curve for each sample was then determined.
Protein carbonyls in skin tissue homogenates were determined using a Oxiselect Protein Carbonyl ELISA kit (Cell Biolabs, Inc., San Diego, CA), according to kit instructions. Carbonyl levels were normalized to protein content in homogenates, as determined by BCA assay.
Reduced glutathione (GSH) was determined in skin tissue homogenates using a GSH/GSSG Ratio Detection Assay kit (Abcam, Cambridge, MA), per kit instructions. Glutathione levels were normalized to protein content in homogenates, as determined by BCA assay.
Isolation of PBMCs from whole blood
Blood was diluted with 1X PBS and Lymphocyte Separation Media (Corning Life Sciences, Tewksbury, MA) was slowly dispensed underneath the blood, followed by centrifugation at 950 x g for 20 minutes at room temperature with disconnected brake. After centrifugation, the interface containing PBMCs was transferred to a new tube, washed once with 1X PBS, and red blood cells were lysed (freshly prepared lysis solution containing 0.15M NH4Cl, 10mM KHCO3, 0.1mM Na2-EDTA). Cells were washed twice to remove lysis solution and re-suspended in complete medium: 1X RPMI 1640 supplemented with 25mM HEPES (MilliporeSigma), 10% heat-inactivated fetal bovine serum (Atlas Biologicals, Fort Collins, CO), 1% HyClone, 1% L-glutamine, and 1% MEM Non-Essential Amino Acids (all from ThermoFisher Scientific).
For cell stimulations, PBMCs were plated at a final concentration of 250,000 c/well. Cells were stimulated for up to 5 days in the presence of media or Mycobacterium tuberculosis culture filtrate protein (CFP), which contains BCG cross-reactive antigens (BEI Resources, Manassas, VA). For 24–28 hour incubations, CFP was used at a concentration of 20µg/mL. For 5 day incubations, CFP was used at a concentration of 10µg/mL. Supernatants were collected at the end of the incubation and stored at -80°C.
Freezing and thawing of PBMCs
To prepare PBMCs for freezing, cells were re-suspended at a concentration of 10x106 c/mL in freezing medium: 85% heat-inactivated FBS + 10% DMSO + 5% of glucose 45% solution (MilliporeSigma). Cryovials containing 1mL cell suspension were transferred to a pre-chilled Mr. Frosty and stored at -80°C for no longer than 48 hours. Vials were then transferred to liquid nitrogen for long term storage. To thaw PBMCs, pre-warmed complete medium was supplemented with 0.2 µl/ml of Benzonase HC (MilliporeSigma). Cryovials containing frozen PBMCs were quickly thawed in a 37°C water bath. After thaw, 1mL cell medium + 0.2 µl/ml Benzonase was added to each cryovial and the volume was transferred to a 15mL conical tube. Cells were centrifuged at 250 x g for 7 minutes at room temperature. Cells were then re-suspended in complete cell medium and counted for downstream analyses.
PBMC Migration to Skin Tissue Homogenates
PBMC migration was determined using the CytoSelect 96-Well Cell Migration Assay (5µM, Fluorometric Format [Cell Biolabs, Inc]), according to manufacturer’s instructions. Briefly, skin tissue homogenates with known protein content according to BCA assay were prepared at 50µg protein in a final volume of 150µL serum-free medium (1X RPMI 1640 supplemented with 25mM HEPES, 1% HyClone, 1% L-glutamine, and 1% MEM Non-Essential Amino Acids). Homogenates were added to the bottom chamber of the cell migration plate according to homogenous (Adult to Adult; Aged to Aged) or heterogeneous (Adult to Aged; Aged to Adult) experimental design. Then 500,000 PBMCs in serum-free media were added to the top chamber of the cell migration plate and incubated for 6 hours at 37°C. At the end of the incubation period, cell detachment solution was added to the cell harvesting plate. Next, media in the top chamber wells from cell migration plate containing non-migrating cells was discarded and the cell migration top chamber plate was inserted into the cell harvesting tray for 30 minutes at 37°C to detach cells. The bottom chambers of the cell migration plate were set aside. After the cell detachment incubation, 75µL of detachment media (from harvesting plate) and 75µL of media (from bottom chambers of the cell migration plate) was added to a clear 96-well plate. Lysis buffer and dye solution (from kit) was added to each well of the clear 96-well plate and incubated for 20 minutes at room temperature. After the incubation, 150µL from the clear 96-well plate was moved to a clear bottom, black plate and fluorescent was read in a plate reader with a 485 nm/ 538 nm filter and 530 nm cutoff.
Histological Analysis of Skin Tissue
Skin biopsies fixed in 10% neutral buffered formalin were paraffin-embedded, sectioned at 4µm thickness, stained with hematoxylin and eosin using standard methods, and evaluated by a board-certified veterinary pathologist at Texas Biomed. For percent affected inflammation, the following scores were used for grading: 1 = < 10%, 2 = < 25%, 3 = < 50%, 4 = < 75%, 5 = > 75%. Paraffin-embedded skin biopsies were also processed for trichrome elastin staining per standard methods, and similarly evaluated. Tissue images were viewed using Image Scope x64 software.
Data analysis, graphing, and statistical analysis was performed using GraphPad Prism version 7–9 (La Jolla, CA). For statistical analysis the following were used as described in the figure legends: One-way ANOVA and Tukey’s post hoc correction for multiple-testing and Student’s t test for testing of means between two groups. Statistical differences between groups were reported significant when the p-value is less than or equal to 0.05. The data are presented in mean ± SEM for n = 2 animals per age group.