K. P. Samy1, L. Lin1, F. Feng1, B. Parker1, Y. Yang2, A. D. Kirk1, T. V. Brennan1 1Duke University Medical Center,Department Of Surgery,Durham, NC, USA 2Duke University Medical Center,Departments Of Medicine And Immunology,Durham, NC, USA
Introduction: Tissue injury during surgery or trauma can expose free mitochondria to surrounding tissues. Mitochondria are unique organelles that evolved from a bacterial origin and have retained immunologic epitopes that activate the innate immune system similar to bacterial pathogens. We examined the effect of mitochondria as endogenous innate immune activators ex vivo and in vivo.
Methods: Mitochondria isolated from mouse fibroblast (LMTK) lysate was performed by differential centrifugation methods followed by affinity purification using TOM22 specific antibody coated magnetic beads. Purity of mitochondrial fractions was confirmed by western blot and qPCR for mitochondrial specific proteins and DNA, respectively. Cultured dendritic cell (DC2.4) and macrophage (RAW 264.7) cell lines were incubated with increasing concentrations of mitochondria and assessed for upregulation of costimulatory molecules (CD40, CD80, and CD86) by flow cytometry. Cell culture supernatant was assessed for inflammatory cytokines (TNF-alpha, IFN-gamma, and MCP-1) by cytometric bead assay. In vivo assay of the inflammatory response to mitochondria was performed by injecting mitochondria derived from LMTK cells or rho zero (mitochondrial DNA-deficient) LMTK cells into WT or MyD88-ko C57BL/6 mice and enumerating neutrophil and monocytes amongst peritoneal exudate cells (PECs).
Results: Exposure of macrophage and dendritic cells to highly purified mitochondria resulted in increased expression of costimulatory molecules and increased inflammatory cytokine production in both cell lines. Intraperitoneal injection of mitochondria led to robust PEC accumulation consisting of activated neutrophils and inflammatory monocytes. The PEC inflammatory response was greatly reduced in MyD88-ko mice compared to WT. PECs were also diminished when mitochondria lacking mitochondrial-DNA (mtDNA) were injected.
Conclusion: These data support a role for mitochondria in the inflammatory response to tissue injury. By serving as endogenous innate immune activators, mitochondria can activate antigen-presenting cells and provoke the infiltration of neutrophils and monocytes. The components of mitochondria that are key to these processes remains to be determined, but our data suggests they are dependent on mitochondrial DNA, and/or the products of mitochondrial gene expression.