Investigation of the therapeutic potential of mesenchymal stem/stromal cells in preclinical models of systemic sepsis
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Introduction: Sepsis is a syndrome of life-threatening physiologic, pathologic, and biochemical abnormalities resulting from infection 1. The host immune response, specifically the loss of immune homeostasis induced by the pathogen, is of critical importance to the initiation, evolution, and outcome, of sepsis 2. With knowledge of this imbalance in the early phase of sepsis, altering this cascade of events may be both beneficial to the patient, in terms of dampening the deleterious effects of some of the cytokines involved on the body systems, and in aiding elimination of the pathogen. Using in vitro experiments, we elucidated how this immune homeostatic imbalance, specifically the cytokine milieu, could be modified, using Mesenchymal Stem/Stromal Cells (MSCs), to eradicate the offending pathogens more efficiently. Replication of systemic sepsis in animal experimental models can prove challenging. The essential attributes of an appropriate model include a focal infection that is polymicrobial in nature with resultant bacteraemia, and a release of bacterial products into the periphery 3. Caecal ligation and puncture (CLP), which disrupts the continuity of the bowel lining and replicates perforated visci, such as appendicitis or diverticulitis, has been the gold standard model for sepsis research since 1980 4. In vivo, using this model, we determined and compared the efficacy of three sub-populations of MSCs using our established CLP Model of Systemic Sepsis. Objectives: 1. To demonstrate the ability of MSCs, in co-culture with human monocytes, to kill relevant sepsis-inducing pathogens, such as E. coli. The specific mechanism(s) by which this occurs was elucidated. 2. To determine the optimal degree of injury using the CLP model of systemic sepsis. We proposed that a 25% caecal ligation, with a single 19 Gauge through-and-through puncture would create a suitable model, as had been used previously in our laboratory. 3. To determine if either the cryopreserved umbilical cord-derived MSCs are comparable, or more efficacious, than our bone-marrow-derived MSC population using our established CLP Model of Systemic Sepsis. Methods: In vitro, pre-activated MSCs, in co-culture with THP-1 human monocytes, were used to assess phagocytitic potential against E. coli bacteria in vitro. Enzyme-linked immunosorbent assay (ELISA) was used to assess MSC activation (MSC IL-6 and 8 production), and spectrophotometry was used to assess E. coli phagocytosis by the above co-cultures, under a variety of MSC pre-activated conditions (Cytomix-activated or not). Phosphate buffered saline (PBS) and Medical Research Council cell strain 5 (MRC5) -fibroblast controls were used throughout all experiments. Transwell plate technology was used to elucidate the effects of contact and non-contact dependent mechanisms on the experiment. For CLP model re-establishment, sepsis was induced in rodents using varying degrees of caecal ligation and puncture (ligation of 25-60%, puncture of 19G-3mm) until multi-organ injury was observed, in keeping with organ injury from septic shock. Animals were monitored every 4 hours, and assessed for indices of physiologic and metabolic derangement, 48 hours following injury. In vivo, sepsis was induced in rodents using our re-established CLP model of systemic sepsis. Umbilical cord and bone marrow MSCs were administered intravenously immediately following the induction of CLP. Vehicle control (PBS) was used. Animals were monitored every 4 hours, and assessed for indices of physiologic and metabolic derangement, 48 hours following injury. Results: 1. Activation of MSCs resulted in significantly increased Interleukin (IL)-6 and IL-8 levels. In the spectrophotometry experiments, both umbilical cord (UC)- and bone marrow (BM)-MSCs, pre-activated with Cytomix, and co-cultured with differentiated THP-1 monocytes, enhanced macrophage E. coli phagocytosis, whether cell-cell contact was inhibited or not. 2. We concluded that, using our novel puncture method, a caecal ligation of 60% and puncture of 2mm provided significant injury in this pilot series. 3. In vivo, MSC therapy significantly reduced bacterial growth in both liver and spleen homogenate cultures. A significant increase in survival duration, and overall survival, was subsequently seen in animals receiving MSC therapy. Secondary analysis showed comparable efficacy of cryopreserved MSCs versus freshly harvested MSCs. Conclusion: In vitro experimentation provided evidence that our MSCs significantly enhance pathogen killing, both directly (contact-dependent), and indirectly (contact-independent). This allowed us to confidently progress to in vivo experimentation with our therapeutic. In vivo, MSC therapy significantly reduced organ bacterial growth, with evidence of an improved shock state in the injured animals, and increased survival. Cryopreserved MSCs are as efficacious as freshly harvested cells. These results will enable further testing of our cryopreserved therapeutic, prior to ultimate clinical translation. Limitations of this series of experiments included inconsistencies in the re-establishment of the CLP model (potential inter-operator variability, potential animal batch variability, and a mandated change to anaesthesia drug regimens) and the lack of a series assessing administration of MSCs using a therapeutic window.