O03 – Expression of MiR-21 is advantageous in the Immune Response to Peritonitis

Author(s):

Rebecca Barnett, Ernesto Sepulveda, William Cheadle, University of Louisville

Background: Peritonitis may trigger sepsis and organ failure, with most mortality due to the latter. Innate immune modulation allows a directed response to pathogens and minimal collateral organ injury. MicroRNA-21 (MiR-21) is an short non-coding RNA with targets in the toll-like receptor (TLR) signaling pathway, and may facilitate the switch from pro- to anti-inflammatory phases of the innate response to peritonitis.

Hypothesis: Macrophage MiR-21 modulates the innate immune response, resulting in a reduction of pro-inflammatory cytokines.

Methods: 8-12week WT and miR-21-/- C57BL/6 mice were given peritonitis by intra-peritoneal injection of lipopolysaccharide (LPS). Temperature, daily weight and survival were recorded. Bone Marrow Derived Macrophages from WT and miR-21-/- mice were plated and stimulated with LPS. MiR-21 and cytokine levels were measured at 0,3,6,12,24 and 48hours. Peritoneal macrophages were transfected with miR-21 mimics and antagomirs for 24hours, and stimulated with LPS. Cytokines were measured in the supernatant after 6hours.

Results: Knockouts did not express miR-21. Although WT mice began to die earlier, more miR-21-/- mice died overall (p=0.002). MiR-21-/- macrophages produced significantly less IL-10 at 3, 6 and 12 hours after stimulation (p<0.05). IL-6/TNF-α was significantly higher at 12 and 48hours (p<0.05) in the miR-21-/- cultures. The increase in TNF-α/IL-6 were also seen after transfection with miR-21 antagomir and subsequent LPS stimulation (p=0.02 and 0.1 respectively). An increase in IL-10 was seen after transfection of miR-21 mimic and LPS stimulation (p=0.02).

Conclusions: Expression of miR-21 conferred a survival benefit from LPS peritonitis, related to targets in the TLR pathway and ultimately cytokine production of the innate response. Identification of patients with overwhelming pro-inflammatory response could allow treatment to reduce the increased inflammatory load and improve organ damage and outcomes.