Embrionic–Derived MYB¯ Macrophages Improve Recovery and Reduce Bacterial Burden in Rodent Polymicrobial Abdominal Sepsis
CCCF ePoster library. Jerkic M. Nov 9, 2018; 233360
Dr. Mirjana Jerkic
Dr. Mirjana Jerkic
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Abstract
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Introduction & Objective: Sepsis and septic shock are characterized by a dysregulated immune response to microbial invasion (1). Bacterial infection of the lung and abdominal cavity constitute the commonest causes of sepsis (2). In the peritoneal cavity two types of macrophages (Mɸ) are recognized: large Embryonic derived (Ed) Myb independent Mɸ or LPMs, crucial for tissue homeostasis, mainly replaced by pro-inflammatory small Mɸ or SPMs originated from circulating monocytes under inflammatory conditions. Myb¯ Mɸ derived from pluripotent stem cells (PSCs) have demonstrated therapeutic benefit in experimental pulmonary transplantation and animal models of acute and chronic airway diseases (3). We wished to determine the potential of intra-peritoneal Ed-Mɸ delivery in a rodent polymicrobial abdominal sepsis.



Methods: Directed differentiation of murine PSCs was used to produce expandable Ed-Mɸ conditioned to a ‘LPM-peritoneal-like’ phenotype with granulocyte–macrophage colony–stimulating factor. Polymicrobial sepsis was developed in rats by instilling cecal slurry intraperitoneally (i.p., 0.5g/kg). Ed-Mɸ (10x10^6 cells/kg) or vehicle (PBS) were given i.p. to the rats 4h later and the animals were monitored for 48 or 72 hours. Lung functional parameters, broncho-alveolar lavage (BAL), peritoneal lavage fluid (PLF) and major organs were collected for infiltration and bacterial count assessment. Macrophages (Mf) and neutrophils (Nf) were isolated from the peritoneum of septic animals using Ficoll gradient and their phagocytic function (using serum opsonized zymosan), activation state, reactive oxygen species (ROS) production, as well as Mf efferocytosis determined by confocal microscopy. Nf apoptotic markers were assessed by Western blot analysis. 

 

Results: Ed-Mɸ treatment significantly attenuated the increase in alveolar and peritoneal white blood cell and neutrophil infiltration induced following abdominal fecal sepsis. Ed-Mɸ-treated rats cleared bacteria more efficiently, with lower bacterial counts (CFU/mL in the lung, liver & spleen compared to vehicle-treated animals). Peritoneal Mɸ isolated from septic rats treated with Ed-Mɸ showed better attachment, more effective phagocytosis and higher ROS production, compared to Mɸ’s from vehicle treated animals. In contrast, peritoneal  Nf isolated from septic rats treated with vehicle were more active and produced more ROS than Nf isolated from septic rats treated with Ed-Mɸ. Apoptotic markers (Caspase-3 and Bax) were significantly more expressed in PLF Nf from Ed-Mɸ treated than vehicle treated septic rats 72h after sepsis induction. That indicates prolonged life span of PLF Nf from PBS-treated rats, as another sign of more severe sepsis (4) in this animal group.



Conclusions: Intra-peritoneal Ed-Mf therapy attenuated the severity of fecal sepsis, and reduced bacterial load, and may have therapeutic potential for systemic sepsis.

 


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