Defining the mitochondrial DAMP repertoire: Formyl peptides are not all created equal!

Elzbieta Kaczmarek, Department of Surgery and Center for Vascular Biology Research, Beth Israel Deaconess Medical Center; Harvard Medical School, Boston MA; Michael Marusich, mAbDx, Inc, Eugene, OR; Nicola Sandler , Department of Surgery, Beth Israel Deaconess Medical Center; Harvard Medical School, Boston MA; Yan Campbell , mAbDx, Inc, Eugene, OR; Christopher Barrett, Department of Surgery, Beth Israel Deaconess Medical Center; Harvard Medical School, Boston MA ; Michael Yaffe, Massachusetts Institute of Technology and Department of Surgery, Beth Israel Deaconess Medical Center; Harvard Medical School, Boston MA ; Leo Otterbein, Department of Surgery, Beth Israel Deaconess Medical Center; Harvard Medical School, Boston MA ; Kiyoshi Itagaki, Department of Surgery, Beth Israel Deaconess Medical Center; Harvard Medical School, Boston MA ; Carl Hauser, Beth Israel Deaconess Medical Center

Mitochondria-derived damage-associated molecular patterns (mtDAMPs) are released from injured tissues by trauma and activate innate immunity. Mitochondrial DNA codes for 13 n-formyl peptides (mtFP) that are presumed to be neutrophil (PMN) chemoattractants and activators like canonical bacterial FPs (eg fMLP). If, like fMLP, wound mtFPs create chemoattractantlocal gradients and suppress PMN expression of other chemotactic receptors, they may be critical modulators of systemic bacterial clearance after trauma.

Individual mtFPs may have important functional differences that that will inform their value as biomarkers or therapeutic targets.

The n-terminal peptides of all 13 mtFP were synthesized as were homologous non-formylated mt-peptides. The activity of each peptide was assayed as intracellular calcium flux ([Ca²⁺]_i) in FURA-2AM labeled human PMN and as PMN chemotaxis (CTX) in transwells. Crosstalk with other chemoattractant receptors (BLT1, CXCR2) was assayed as heterologous suppression of responses to LTB₄ and GROα.

Five mtFP induced concentration-dependent [Ca²⁺]_i flux and CTX in native PMN; eight did not. Only mtFPs that induce [Ca²⁺]_i flux induce CTX. Non-formylated mtFP neither induce [Ca²⁺]_i flux/CTX, nor block PMN response to active mtFPs. Cyclosporine H (an inhibitor of FP receptor-1) blocks responses to all mtFP. Exposure to any active mtFP desensitizes PMN [Ca²⁺]_i flux and CTX response to LTB₄, GROα and fMLP. Mice were inoculated with active mtFPs and monoclonal antibodies were generated: mAbs thus created recognize the mtFP sequence alone, in Western blots using the parent protein, and blocked [Ca²⁺]_i responses only to that specific mtFP.

Only five of the 13 mtFP are involved in activation of PMN by tissue injury. These DAMP motifs can act locally as PMN chemoattractants toward dead and dying tissues but they may simultaneously desensitize PMN chemotactic receptors that are needed for systemic innate immune functions. To the extent that this renders the host susceptible to infection, active mtFPs may therefore prove good biomarkers for the severity of trauma and infection risk. The active mtFPs may be key targets for diagnostic and therapeutic agents.