Human bone marrow-derived MSC
Human bone marrow-derived MSC were obtained from a National Institutes of Health repository at Texas A&M Health Science Center, which fulfill the criteria for MSC as defined by the International Society of Cellular Therapy. MSC were cultured in Minimal Essential Medium alpha without nucleosides containing 16.5% fetal bovine serum, 0.2 M L-glutamine, and 1% penicillin-streptomycin. To obtain MSC EV, confluent MSC were cultured in condition medium (deprived of fetal bovine serum and supplemented with 0.5% bovine serum albumin) for 48 h as previously described . The viability and quantity of serum-starved MSC were evaluated by trypan blue exclusion using hemacytometer. The supernatants from MSC were centrifuged at 3000 rpm for 20 mins to remove cellular debris and then underwent ultracentrifugation at 100,000g at 4 °C for 1 h twice. The pellets were re-suspended and washed with phosphate-buffered saline in between ultracentrifugation. MSC EV were re-suspended according to the final cell counts after 48 h serum starvation (10 μl of EV were equivalent to the vesicles released by 1 × 106 cells over 48 h) and stored at – 80 °C. The size and number of MSC EV were analyzed using NanoSight NS300 (Malvern, Inc.). We previously characterized MSC EV using the current isolation technique [8, 11] as recommended by the International Society of Extracellular Vesicles .
Binding ability of different molecular weights of HA to MSC EV
Glass slides were coated with PBS or LMW (40 KDa), HMW (1 MDa), or HMW (1.5 MDa) HA at a concentration of 10 mg/ml and dried in room temperature overnight [20, 21]. PKH26-labeled MSC EV (50 μl) were then added onto the chamber slides. Two hours later, chamber slides were washed extensively with PBS to remove unbound MSC EV. The slides were examined under fluorescence microscopy, and the intensity of fluorescent areas was analyzed using Image J software. MSC EV were labeled with PKH26 according to the manufacturer’s protocol, washed with PBS, and subjected to the same ultracentrifugation steps to isolate the EV. For the PBS control, the same volume of PBS as MSC EV was incubated with PKH26 and subjected to the same washing and ultracentrifugation steps to demonstrate that the ultracentrifugation step removed free PKH26.
Uptake of MSC EV preincubated with different doses or molecular weights of HA by LPS stimulated human blood monocytes
Human blood monocytes were collected from fresh whole blood from healthy donors as previously described . Monocytes were re-suspended in RPMI 1640 medium with 10% FBS and plated in 4-well chamber slides (5 × 105 cells/well) at 37 °C in 5% CO2 incubator overnight. In order to compare the differences in uptake of MSC EV preincubated with different doses of HMW HA (1 MDa) by LPS stimulated monocytes, the cells were exposed to LPS (1 μg/ml) and (a) MSC EV (50 μl), (b) MSC EV + 0.2 μg/ml HMW HA (1.0 MDa), (c) MSC EV + 1 μg/ml HMW HA (1.0 MDa), or (d) MSC EV + 5 μg/ml HMW HA (1.0 MDa). MSC EV were labeled with PKH67 before incubation with HMW HA. MSC EV were then incubated with HA for 30 min prior to administration. After 24 h, the monocytes were stained with mounting media and DAPI (Vectashield, Vector Laboratories, CA). The uptake of MSC EV by monocytes was measured using fluorescence microscopy. In separate experiments, to determine the effect of different molecular weights of HA on the uptake of MSC EV by LPS stimulated monocytes, the monocytes were exposed to LPS and (a) MSC EV (50 μl), (b) LMW HA primed MSC EV [MSC EV preincubated with LMWHA (40 KDa)], (c) HMW HA(1.0 MDa) primed MSC EV [MSC EV preincubated with HMW HA (1.0 MDa)], or (d) HMW HA (1.5 MDa) primed MSC EV [MSC EV preincubated with HMW HA (1.5 MDa)]. The slides were fixed in 4% paraformaldehyde, and the uptake of MSC EV by human monocytes was measured using fluorescence microscopy. In all experiments described, MSC EV were incubated with HA for 30 min at 37 °C before administration onto cells or into mice.
Phagocytosis of PA103 bacteria by LPS stimulated monocytes treated with HA primed MSC EV
Human blood monocytes were exposed to LPS (1 μg/ml) and (a) PBS, (b) MSC EV (90 μl), (c) LMW HA (40 KDa) primed MSC EV at a dose of 5 μg/ml or 0.5 μg total, (d) HMW HA (1.0 MDa) primed MSC EV, or (e) HMW HA (1.5 MDa) primed MSC EV for 24 h. Opsonized PA103 bacteria (107 colony forming units (CFU)) were then added into each well and incubated at 37 °C for 90 min. Aliquots of the culture medium, serially diluted, were removed and plated on 5% sheep blood agar plates to count PA103 CFU levels in the supernatant. In separate experiments, the monocytes were injured with opsonized PA103 bacteria and incubated at 37 °C for 60 min. Gentamycin was then added to the culture medium. After 30 min, the monocytes were washed with D-PBS twice, and 1% Triton X-100 was added into each well. The cell lysates were collected and plated on 5% sheep blood agar plates with serial dilution to count the intracellular PA103 CFU load.
In separate experiments, the impact of CD44 siRNA pretreatment of MSC EV prior to incubation with HMW HA on bacterial phagocytosis by monocytes was investigated. MSC EV (90 μl) were transfected with CD44 siRNA or scrabbled siRNA (Negative Control No.1 siRNA, Ambion) for 24 h. MSC EV were washed extensively with PBS and isolated by ultracentrifugation. Then, human monocytes were exposed to LPS (1 μg/ml) and (a) PBS, (b) CD44 siRNA transfected MSC EV + HMW HA (1.0 MDa), or (c) scrabbled siRNA transfected MSC EV + HMW HA (1.0 MDa) for 24 h. Intracellular and extracellular PA 103 CFU levels were then measured.
Severe PA103 pneumonia in mice
The Institutional Animal Care and Use Committee at the University of California San Francisco approved all experimental protocols. Male C57BL/6 mice (8–12 weeks of age, ~ 25 g; Jackson Laboratory, Bar Harbor, ME) were used in all the experiments. Mice were anesthetized with isoflurane, and then PA103 bacteria was instilled intratracheally (5 × 104 CFU per mice). Four hours later, various treatments were instilled through the retro-orbital vein: (1) PBS as a carrier control, (2) MSC EV (90 μl), (3) HMW HA (1.0 MDa, dose 5 μg/ml or 0.5 μg total) primed MSC EV, (4) HMW HA alone (1.0 MDa, dose 5 μg/ml), (5) MSC (5 × 105 cells/mouse), or (6) MSC preincubated with HMW HA (1.0 MDa, dose, 5 μg/ml). Mice were euthanized after 24 h, and the blood and bronchoalveolar lavage fluid (BALF) were collected for assessment of cell counts, bacterial CFU levels, inflammatory cytokines, and histology. In separate experiments, additional groups were studied: (1) CD44 siRNA transfected MSC EV + HMW HA (1.0 MDa, dose 5 μg/ml or 0.5 μg total), (2) scrabbled siRNA transfected MSC EV + HMW HA (1.0 MDa, dose 5 μg/ml or 0.5 μg total). The total cell counts and differential were measured by using the Hemavet HV950FS (Drew Scientific, Miami Lakes, FL). Mouse TNFα and IL-6 levels in the BALF and plasma were measured using ELISA kits (R&D Systems, Minneapolis, MN). For histology, mice lungs were fixed in 10% formalin and embedded in paraffin, cut into 4 μm sections, and stained with H&E. Levels of lung injury were determined by using semi-quantitative lung injury score as previously described . For each mouse, 20 fields of the left lung at × 20 magnification were examined. Scoring was performed by grading as follows: infiltration or aggregation of inflammatory cells in air space or vessel wall: 1 = only wall, 2 = few cells (1–5 cells) in air space, 3 = intermediate, 4 = severe (air space congested); interstitial congestion and hyaline membrane formation: 1 = normal lung, 2 = moderate (50% of lung section); hemorrhage: 0 = absent, 1 = present.
Trafficking of MSC EV in mice with PA103 pneumonia
Male C57BL/6 mice were intratracheally injured with PA103 bacteria (5 × 104 CFU per mice). Four hours later, PKH26-labeled MSC EV (90 μl) or HMW HA (1.0 MDa) primed MSC EV was intravenously instilled into the retro-orbital vein. Six hours later, mice were euthanized, and the lungs, liver, and spleen were harvested. The organs were washed twice with cold PBS and fixed in 4% paraformaldehyde for 24 h at 4 °C. The organs were embedded in O.C.T (Tissue-Teck O.C.T. Compound, Sakura Finetek) and immediately frozen at − 80 °C. Frozen 10-μm slices were stained with mounting media with DAPI (Vectashield). The intensity of PKH26-labeled MSC EV in the organs was measured using Cytation 5 cell imaging multi-mode reader (Bio Tek Instruments).
In separate experiments, the role of MSC EV CD44 expression in trafficking of HMW HA (1.0 MDa) primed MSC EV was studied. MSC EV were transfected with a CD44 siRNA or scrabbled control siRNA. PKH26-labeled CD44 or scrabbled siRNA-treated MSC EV (90 μl) incubated with HMW HA (1.0 MDa) were intravenously instilled 4 h following pneumonia. Six hours later, mice lungs, liver, and spleen were harvested, processed, and analyzed as before.
Data were presented as the mean ± SD or median with interquartile range (IQR). Shapiro-Wilk normality test was used to determine if the values were from a Gaussian distribution. Comparisons between the two groups were made using Student’s t test or the Mann-Whitney U test. Comparisons between more than two groups were made using ANOVA with the Bonferroni’s correction or Kruskal-Wallis test with Dunn’s correction. P < 0.05 was considered statistically significant. Graphpad Prism 8 software was used for statistical analysis.