Figure 4.
Irradiated mice that received CpG-MSCs have less organ damage after infection. (A) Irradiated mice given PBS, MSCs, or CpG-MSCs were euthanized 48 hours after P aeruginosa infection and the whole left lung was harvested for homogenization and quantification of organ bacterial CFU. There was no significant difference by Kruskal-Wallis in bacterial clearance between mice that received PBS (green bars), MSC (blue bars), or CpG-MSC (red bars) (n = 10 per group). TUNEL staining of (B) kidney (n = 6 per group) and (C) left lung (n = 6 per group), standardized to total tissue area, relative to TUNEL staining in mice that received PBS (fold change PBS). Organs were harvested 48 hours after P aeruginosa infection in mice that received PBS (green filled circle), MSC (blue filled square), or CpG-MSC (red filled triangle). Data were assessed by ANOVA, P = .0002 (kidneys), P = .009 (lung). *Significant comparisons by Bonferroni’s multiple comparisons test, PBS vs CpG-MSC, P = .0005 (kidneys), P = .008 (lungs); †PBS vs MSC, P = .001 (kidneys). (D) IL-6 Luminex on plasma samples (n = 7 PBS, n = 8 MSC, n = 9 CpG-MSC) obtained 48 hours after infection analyzed by Kruskal-Wallis, P = .0012. *Significant comparisons by Dunn’s multiple comparisons test, PBS vs CpG, P = .001. (E) Experimental design for the in vitro analysis of NET formation. Mice are irradiated with 5 Gy radiation on day 0. Two days before the assay, MSCs are plated. The following day, CpG-MSCs are stimulated with CpG (3 µg/mL) for 30 minutes. On the day of the assay, bone marrow neutrophils are harvested from irradiated mice by density centrifugation and added to MSCs, CpG-MSCs or media control with opsonized P aeruginosa. (F) Bone marrow-derived neutrophils from irradiated mice were cocultured with HBSS, MSC, or CpG-MSCs and P aeruginosa for 3 hours before assay by Sytox Green. Data were analyzed by pooled 1-way ANOVA (n = 6 per group), P = .0001. *Significant comparisons by Bonferroni’s multiple comparisons test, CpG-MSC vs HBSS, P = .0001; §CpG-MSC vs MSC, P = .009. Immunofluorescence for citrullinated H3 (yellow) was performed on lung tissue sections from irradiated and infected mice given (H) PBS, (I) MSCs, or (J) CpG-MSCs demonstrating (G) decreased citrullinated H3 staining in the lungs of mice given CpG-MSCs. Images were taken on a Nikon Eclipse 80i using an AmScope 18MP USB 3.0 digital camera at 20× and 40× original magnification acquired on NIS-Elements BR 3.2 software with DAPI and AlexaFluor 488 fluorochromes in Vectashield antifade mounting media (Vector); background was subtracted, and brightness/contrast were adjusted to whole channel images before merging the channels in Fiji ImageJ. Coculturing bone marrow-derived neutrophils from irradiated mice with MSCs and CpG-MSCs resulted in (K) more neutrophils phagocytosing at least 1 P aeruginosa. Data were analyzed by Brown-Forsythe ANOVA, P = .003. †Significant comparisons by Dunnett’s multiple comparisons test, PBS vs MSC, P = .001; *PBS vs CpG-MSC, P = .015. There was no significant difference in the (L) mean fluorescence intensity of phagocytosed P aeruginosa.

Irradiated mice that received CpG-MSCs have less organ damage after infection. (A) Irradiated mice given PBS, MSCs, or CpG-MSCs were euthanized 48 hours after P aeruginosa infection and the whole left lung was harvested for homogenization and quantification of organ bacterial CFU. There was no significant difference by Kruskal-Wallis in bacterial clearance between mice that received PBS (green bars), MSC (blue bars), or CpG-MSC (red bars) (n = 10 per group). TUNEL staining of (B) kidney (n = 6 per group) and (C) left lung (n = 6 per group), standardized to total tissue area, relative to TUNEL staining in mice that received PBS (fold change PBS). Organs were harvested 48 hours after P aeruginosa infection in mice that received PBS (green filled circle), MSC (blue filled square), or CpG-MSC (red filled triangle). Data were assessed by ANOVA, P = .0002 (kidneys), P = .009 (lung). *Significant comparisons by Bonferroni’s multiple comparisons test, PBS vs CpG-MSC, P = .0005 (kidneys), P = .008 (lungs); †PBS vs MSC, P = .001 (kidneys). (D) IL-6 Luminex on plasma samples (n = 7 PBS, n = 8 MSC, n = 9 CpG-MSC) obtained 48 hours after infection analyzed by Kruskal-Wallis, P = .0012. *Significant comparisons by Dunn’s multiple comparisons test, PBS vs CpG, P = .001. (E) Experimental design for the in vitro analysis of NET formation. Mice are irradiated with 5 Gy radiation on day 0. Two days before the assay, MSCs are plated. The following day, CpG-MSCs are stimulated with CpG (3 µg/mL) for 30 minutes. On the day of the assay, bone marrow neutrophils are harvested from irradiated mice by density centrifugation and added to MSCs, CpG-MSCs or media control with opsonized P aeruginosa. (F) Bone marrow-derived neutrophils from irradiated mice were cocultured with HBSS, MSC, or CpG-MSCs and P aeruginosa for 3 hours before assay by Sytox Green. Data were analyzed by pooled 1-way ANOVA (n = 6 per group), P = .0001. *Significant comparisons by Bonferroni’s multiple comparisons test, CpG-MSC vs HBSS, P = .0001; §CpG-MSC vs MSC, P = .009. Immunofluorescence for citrullinated H3 (yellow) was performed on lung tissue sections from irradiated and infected mice given (H) PBS, (I) MSCs, or (J) CpG-MSCs demonstrating (G) decreased citrullinated H3 staining in the lungs of mice given CpG-MSCs. Images were taken on a Nikon Eclipse 80i using an AmScope 18MP USB 3.0 digital camera at 20× and 40× original magnification acquired on NIS-Elements BR 3.2 software with DAPI and AlexaFluor 488 fluorochromes in Vectashield antifade mounting media (Vector); background was subtracted, and brightness/contrast were adjusted to whole channel images before merging the channels in Fiji ImageJ. Coculturing bone marrow-derived neutrophils from irradiated mice with MSCs and CpG-MSCs resulted in (K) more neutrophils phagocytosing at least 1 P aeruginosa. Data were analyzed by Brown-Forsythe ANOVA, P = .003. †Significant comparisons by Dunnett’s multiple comparisons test, PBS vs MSC, P = .001; *PBS vs CpG-MSC, P = .015. There was no significant difference in the (L) mean fluorescence intensity of phagocytosed P aeruginosa.

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