Prophylactic therapy with human amniotic fluid stem cells improved survival in a rat model of lipopolysaccharide-induced neonatal sepsis through immunomodulation via aggregates with peritoneal macrophages

Abstract

Background Despite recent advances in neonatal care, sepsis remains a leading cause of mortality in neonates. Mesenchymal stem cells derived from various tissues, such as bone marrow, umbilical cord, and adipose tissue, have beneficial effects on adult sepsis. Although human amniotic fluid stem cells (hAFSCs) have mesenchymal stem cell properties, the efficacy of hAFSCs on neonatal sepsis is yet to be elucidated. This study aimed to investigate the therapeutic potential of hAFSCs on neonatal sepsis using a rat model of lipopolysaccharide (LPS)-induced sepsis. Methods hAFSCs were isolated as CD117-positive cells from human amniotic fluid. Three-day-old rat pups were intraperitoneally treated with LPS to mimic neonatal sepsis. hAFSCs were administered either 3 h before or at 0, 3, or 24 h after LPS exposure. Serum inflammatory cytokine levels, gene expression profiles from spleens, and multiple organ damage were analyzed. hAFSC localization was determined in vivo. In vitro LPS stimulation tests were performed using neonatal rat peritoneal macrophages co-cultured with hAFSCs in a cell-cell contact-dependent/independent manner. Immunoregulation in the spleen was determined using a DNA microarray analysis. Results Prophylactic therapy with hAFSCs improved survival in the LPS-treated rats while the hAFSCs transplantation after LPS exposure did not elicit a therapeutic response. Therefore, hAFSC pretreatment was used for all subsequent studies. Inflammatory cytokine levels were elevated after LPS injection, which was attenuated by hAFSC pretreatment. Subsequently, inflammation-induced damages in the brain, lungs, and liver were ameliorated. hAFSCs aggregated with peritoneal macrophages and/or transiently accumulated in the liver, mesentery, and peritoneum. Paracrine factors released by hAFSCs induced M1-M2 macrophage polarization in a cell-cell contact-independent manner. Direct contact between hAFSCs and peritoneal macrophages further enhanced the polarization. Microarray analysis of the spleen showed that hAFSC pretreatment reduced the expression of genes involved in apoptosis and inflammation and subsequently suppressed toll-like receptor 4 signaling pathways. Conclusions Prophylactic therapy with hAFSCs improved survival in a rat model of LPS-induced neonatal sepsis. These effects might be mediated by a phenotypic switch from M1 to M2 in peritoneal macrophages, triggered by hAFSCs in a cell-cell contact-dependent/independent manner and the subsequent immunomodulation of the spleen.

Prophylactic Therapy with Human Amniotic Fluid Stem Cells Improves Long-Term Cognitive Impairment in Rat Neonatal Sepsis Survivors

“…Although concerns have been raised that improved survival of infants treated with hAFSCs might be accompanied by an increase in disabling morbidity in neonatal sepsis survivors, in this study, we found that prophylactic therapy with hAFSCs improved cognitive impairment in neonatal sepsis survivors at adolescence in rats. Together with our findings in a previous study [ 14 ], herein, we prove that hAFSC treatment improves mortality and morbidity in neonatal sepsis. The treatment suppresses acute reactive gliosis, such as astrocyte and microglial activation, and subsequently reduces astrogliosis in the hippocampal region for a long time, and could result in a favorable long-term neurological outcome.…”

“…LPS administration induced neuroinflammation in the rat brain, as indicated by GFAP-and ionized calcium-binding adapter molecule 1 (Iba-1)-positive cells. As reported previously [14], LPS injection elicited neuroinflammation 48 h after LPS exposure, whereas the inflammatory changes were significantly attenuated by hAFSC treatment. Although we previously reported a reduction in neuroinflammation in the entire hippocampus after hAFSC treatment, in this study, we analyzed neuroinflammation in three hippocampal regions, viz., CA1, CA3, and dentate gyrus (DG).…”

“…The hAFSCs used in this study were evaluated for differentiation potential and surface markers to ensure that they met the definition of MSCs. As described in our previous report [14], hAFSCs did not express hematopoietic surface markers (CD34, CD14, and CD45) but expressed mesenchymal markers (CD90, CD73, and CD105). They also exhibited the ability to differentiate into osteocytes, chondrocytes, and adipocytes.…”

“…We previously reported that LPS treatment increased the number of Iba-1-positive cells in the rat brain, 48 h after LPS treatment [14]. In the present study, as described above, we also determined the Iba-1-positive area in the hippocampal CA1, CA3, and DG regions.…”

“…Recently, we reported that prophylactic therapy with hAFSCs improved survival in a rat model of lipopolysaccharide (LPS)-induced neonatal sepsis through immunomodulation. The treatment was effective in reducing systemic inflammation and in improving mortality in neonatal rats 48 h after LPS exposure [ 14 ]. Besides improving mortality, increasing survival without major morbidities in neonatal sepsis is another important goal of neonatal intensive care.…”