Figure 1.
Improved survival and increased neutrophils in mice treated with CpG-MSCs in P aeruginosa pulmonary infection in the setting of radiation-associated bone marrow hypoplasia. (A) Schematic of experimental model. Mice were irradiated with 5 Gy on day 0. Three days later, mice received an injection of 1) PBS, 2) 5 × 105 MSCs, or 3) 5 × 105 CpG-MSCs. On day 7, the mice were infected intranasally with either 6 to 7 × 106 or 1 to 2 × 106 colony forming units of P aeruginosa and either monitored for 7 additional days for survival or euthanized 24 to 48 hours after infection for functional analyses, respectively. (B) Survival of male CD-1 mice (n = 30 per group) after irradiation with 5 Gy, injection with PBS (green filled circle), 5 × 105 MSC (blue filled square), or 5 × 105 MSC conditioned with CpG-MSC (red filled triangle) and infected with pulmonary P aeruginosa. Data are presented as Kaplan-Meier survival curves and analyzed by log-rank test. *Significant comparisons (CpG-MSC vs PBS, P = .048). Representative hematoxylin and eosin staining of femurs 9 days after irradiation with 5 Gy, (C) without infection vs (D-F) with P aeruginosa pulmonary infection and administration of (D) PBS, (E) 5 × 105 MSCs, or (F) 5 × 105 CpG-MSCs. Images were taken on a Nikon Eclipse 80i microscope using an AmScope 18MP USB 3.0 digital camera at 10× original magnification acquired on NIS-Elements BR 3.2 software and white-balanced in Fiji ImageJ. (G) Single cell suspensions were prepared from the bone marrow of irradiated mice administered PBS (n = 9, green filled circle), MSC (n = 8, blue filled square), or CpG-MSC (n = 10, red filled triangle) 48 hours after infection with P aeruginosa and cell populations were analyzed by cytometry by time of flight. Neutrophil numbers were assessed by analysis of variance (ANOVA), P = .007. *Significant comparisons by Bonferroni’s multiple comparisons test, PBS vs CpG-MSC, P = .029; †PBS vs MSC, P = .012. (H) Whole peripheral blood was collected 48 hours after infection in mice administered PBS (n = 6), MSC (n = 8), and CpG-MSC (n = 8), and total neutrophil and large unstained cell (immature granulocyte) counts were analyzed by Kruskal-Wallis, P = .025. *Significant comparisons by uncorrected Dunn’s post hoc test, PBS vs CpG-MSC, P = .016; §CpG-MSC vs MSC, P = .038. (I) Bronchoalveolar lavage (BAL) was obtained from mice administered PBS (n = 6), MSC (n = 6), or CpG-MSC (n = 5) 24 hours after infection with P aeruginosa and the number of neutrophils (GR-1 cells) analyzed by Kruskal-Wallis, P = .018. *Significant comparisons by Dunn’s post hoc test, PBS vs CpG-MSC, P = .044; §CpG-MSC vs MSC, P = .009.

Improved survival and increased neutrophils in mice treated with CpG-MSCs in P aeruginosa pulmonary infection in the setting of radiation-associated bone marrow hypoplasia. (A) Schematic of experimental model. Mice were irradiated with 5 Gy on day 0. Three days later, mice received an injection of 1) PBS, 2) 5 × 105 MSCs, or 3) 5 × 105 CpG-MSCs. On day 7, the mice were infected intranasally with either 6 to 7 × 106 or 1 to 2 × 106 colony forming units of P aeruginosa and either monitored for 7 additional days for survival or euthanized 24 to 48 hours after infection for functional analyses, respectively. (B) Survival of male CD-1 mice (n = 30 per group) after irradiation with 5 Gy, injection with PBS (green filled circle), 5 × 105 MSC (blue filled square), or 5 × 105 MSC conditioned with CpG-MSC (red filled triangle) and infected with pulmonary P aeruginosa. Data are presented as Kaplan-Meier survival curves and analyzed by log-rank test. *Significant comparisons (CpG-MSC vs PBS, P = .048). Representative hematoxylin and eosin staining of femurs 9 days after irradiation with 5 Gy, (C) without infection vs (D-F) with P aeruginosa pulmonary infection and administration of (D) PBS, (E) 5 × 105 MSCs, or (F) 5 × 105 CpG-MSCs. Images were taken on a Nikon Eclipse 80i microscope using an AmScope 18MP USB 3.0 digital camera at 10× original magnification acquired on NIS-Elements BR 3.2 software and white-balanced in Fiji ImageJ. (G) Single cell suspensions were prepared from the bone marrow of irradiated mice administered PBS (n = 9, green filled circle), MSC (n = 8, blue filled square), or CpG-MSC (n = 10, red filled triangle) 48 hours after infection with P aeruginosa and cell populations were analyzed by cytometry by time of flight. Neutrophil numbers were assessed by analysis of variance (ANOVA), P = .007. *Significant comparisons by Bonferroni’s multiple comparisons test, PBS vs CpG-MSC, P = .029; †PBS vs MSC, P = .012. (H) Whole peripheral blood was collected 48 hours after infection in mice administered PBS (n = 6), MSC (n = 8), and CpG-MSC (n = 8), and total neutrophil and large unstained cell (immature granulocyte) counts were analyzed by Kruskal-Wallis, P = .025. *Significant comparisons by uncorrected Dunn’s post hoc test, PBS vs CpG-MSC, P = .016; §CpG-MSC vs MSC, P = .038. (I) Bronchoalveolar lavage (BAL) was obtained from mice administered PBS (n = 6), MSC (n = 6), or CpG-MSC (n = 5) 24 hours after infection with P aeruginosa and the number of neutrophils (GR-1 cells) analyzed by Kruskal-Wallis, P = .018. *Significant comparisons by Dunn’s post hoc test, PBS vs CpG-MSC, P = .044; §CpG-MSC vs MSC, P = .009.

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