Macrophages Educated with Exosomes from Primed Mesenchymal Stem Cells Treat Acute Radiation Syndrome by Promoting Hematopoietic Recovery

Abstract

In the setting of radiation-induced trauma, exposure to high levels of radiation can cause an acute radiation syndrome (ARS) causing bone marrow (BM) failure, leading to life-threatening infections, anemia, and thrombocytopenia. We have previously shown that human macrophages educated with human mesenchymal stem cells (MSCs) by coculture can significantly enhance survival of mice exposed to lethal irradiation. In this study, we investigated whether exosomes isolated from MSCs could replace direct coculture with MSCs to generate exosome educated macrophages (EEMs). Functionally unique phenotypes were observed by educating macrophages with exosomes from MSCs (EEMs) primed with bacterial lipopolysaccharide (LPS) at different concentrations (LPS-low EEMs or LPS-high EEMs). LPS-high EEMs were significantly more effective than uneducated macrophages, MSCs, EEMs, or LPS-low EEMs in extending survival after lethal ARS in vivo. Moreover, LPS-high EEMs significantly reduced clinical signs of radiation injury and restored hematopoietic tissue in the BM and spleen as determined by complete blood counts and histology. LPS-high EEMs showed significant increases in gene expression of STAT3, secretion of cytokines like IL-10 and IL-15, and production of growth factors like FLT-3L. LPS-EEMs also showed increased phagocytic activity, which may aid with tissue remodeling. LPS-high EEMs have the potential to be an effective cellular therapy for the management of ARS.

Keywords: Acute radiation syndrome; Exosomes; Hematopoiesis; Macrophages; Mesenchymal stem cells; Radiation injury.

Figure 1:. EVs isolated from human MSCs consist primarily of exosomes sized vesicles.

EVs were isolated from BM-MSC’s, and EVs were analyzed by TEM and two different instruments to quantify the mean particle diameter. (A) Representative TEM of the EV preparations indicated that the particles had the typical cup-shaped vesicular appearance of EVs and the size was generally less than 200 nm. (B) The preparations characterized by resistive pulse sensing using the qNano Nanoparticle instrument indicated the majority of the particles were within the 60–130 nm range. (C) The preparations characterized by dynamic light scattering using the Nanosight NS300 indicated that the size of the majority of particles were 95 nm with a range of 50 to 129 nm and generally matched the same profile as using the qNano Nanoparticle instrument. Based on these characterizations the majority of the particles consisted primarily of exosomes-sized EVs.

Figure 2:. Human LPS-high EEMs express low levels of M1 markers CD16, CD86 and HLA-DR.

Day 7 macrophages were either untreated (Control) or treated with exosomes from MSCs to produce EEMs, LPS-low or high EEMs. The Median Fluorescence Intensity (MFI) of CD14+ positive cells for each marker (+/− SEM) is shown. Results pooled from 2 separate experiments, with 4–13 samples/group. Groups compared by Kruskal-Wallis with a Dunn’s post test. *p

Figure 3:. Treatment with human LPS-high EEMs significantly improves survival, weight loss and clinical scores in mice after lethal radiation injury.

(A-C) On day 0, NSG mice were received 4 Gy of lethal radiation followed by an i.v. treatment 4 hours later with either PBS (vehicle control), 1 × 10⁶ MSCs, 1 × 10⁶ macrophages, 1 × 10⁶ EEMs, 1 × 10⁶ LPS-low EEMs or 1 × 10⁶ LPS-high EEMs. (A) Survival curve of treated mice after radiation. (B) Mean % weight change compared to PBS controls. (C) Mean clinical scores (% weight loss, posture, activity and fur texture) compared to MSC and/or PBS controls. The final mean percent weight change and clinical score were carried over after death to allow for comparison by Kruskal-Wallis with a Dunn’s post test between groups at a given timepoint. Results pooled from 2 separate experiments, with 7–21 mice/group. * p< 0.05, *** p</=0.005, ****p</= 0.0001.

Figure 4:. Human LPS-high EEM treatment protects against tissue damage in the BM and spleen of mice after lethal radiation injury.

On day 0, NSG mice were received either no radiation (normal healthy) or 4 Gy of lethal radiation followed by an i.v. treatment 4 hours later with PBS or with 10⁶ cells of LPS-high EEMs. Histology on tissue preparations of BM from femurs and spleens of healthy mice were compared to day 9 post radiation PBS controls, and LPS-high EEMs, day 31 LPS-high EEM treated mice or day 53 LPS-high EEMs. (A) Representative 20x images of H&E stained femoral BM sections from each group. (B) Representative 20× images of H&E stained spleen sections.

Figure 5:. Human LPS-high EEMs secrete high levels of anti-inflammatory cytokines and growth factors by multiplex ELISA.

Day 7 macrophages were either untreated (control) or treated with exosomes from MSCs for 3 days to produce EEMs and LPS-low or high EEMs. Cells were then washed and supernatants collected after 24 hours and assayed. Samples were run in triplicate and compared by ANOVA with a Dunn’s post test. *p

Figure 6:. LPS-high EEMs are strongly phagocytic using pHrodoGreen E.coli bio particles.

Day 7 macrophages were either untreated (control) or treated for 3 days using exosomes from MSCs to produce EEMs and LPS-low or high EEMs or stimulated with M1 factors; (PMA/IFN-gamma/LPS) to produce M1 stimulated macrophages. Day 10 macrophages were treated with pHrodoGreen E.coli bioparticles and the ratio of CD14+ cells positive for phrodoGreen E.coli bioparticles (designated as percent (%) cells) was determined by flow cytometry. Samples were pooled from 2 separate experiments and compared by ANOVA with a Dunn’s post test, 3–5 samples/group. *p