646 - Autophagy Protein Beclin Impacts Mesenchymal Stromal Cell (MSC) function in a Murine Model of Sepsis by Altering Resolution of Inflammation

Sepsis remains a significant cause of morbidity and mortality in neonates. The infecting organisms result in pro-inflammatory responses that can lead to organ dysfunction and death. There is a need for therapeutics that focus on resolution of inflammation to improve survival and neurodevelopmental outcomes in neonates. The immunomodulatory properties of mesenchymal stromal cells (MSCs) make them an ideal candidate for a therapeutic in sepsis. MSC immunomodulation in oxidative environments can be mediated by autophagy, a process that regulates immune responses by facilitating turnover of damaged proteins. We have shown that autophagy deficient MSCs are more susceptible to injury and mitochondrial dysfunction in vitro. However, gaps exist in our understanding of the role of autophagy in MSC immunomodulation in sepsis.

Objective:

We hypothesize that autophagy deficient MSCs will be less effective in murine sepsis.

Design/Methods: Mice underwent cecal ligation and puncture (CLP) and received 2 doses of 500,000 cells (WT MSCs, autophagy deficient Beclin^+/- MSCs, or control fibroblasts). Plasma for Luminex, organs for histology and peritoneal fluid for in vivo phagocytosis and efferocytosis assays was collected at 24 hours. In vitro efferocytosis assays used flow cytometry to study uptake of apoptotic neutrophils by macrophages co-cultured with MSCs. Mass spectroscopy was used to analyze specialized pro-resolving lipid mediator (SPM) production.

Results:

WT, but not Beclin^+/-, MSCs increased survival in mice after CLP as compared with control cells (76 ± 10.3% and 33.3 ± 6.5% respectively) (P< 0.05). Only WT MSCs attenuated liver and kidney injury as well as lung, spleen, and peritoneal fluid inflammation and systemic IL-6 after CLP-induced sepsis. WT MSCs enhanced neutrophil phagocytosis and macrophage efferocytosis in an in vitro environment of oxidative stress and this enhancement was not seen with Beclin^+/- MSCs. Beclin^+/- MSCs produced less SPMs (Resolvin D1 and D4) than WT MSCs and when Beclin^+/- MSCs were conditioned with Resolvin D1 and D4, survival was increased in CLP to the level of WT MSCs (Figure 1).

Conclusion(s):

WT, and not Beclin^+/- MSCs, improved survival, bacterial clearance, organ injury and inflammation in murine sepsis as well as promoted resolution of inflammation in environments of oxidative stress. Autophagy deficient MSCs produced less SPMs. Understanding the role of autophagy in MSC resolution of inflammation will provide valuable knowledge to shape the clinical use of MSCs as enhancing autophagy or SPM production may optimize MSC therapy for sepsis.