Degradomics Unravels Proteolytically Altered Pathways in Controlling Inflammation

Balancing neutrophil and macrophage infiltration with their clearance is key in the pathogenesis of inflammation. We have uncovered critical roles for the control of inflammation exerted by a family of extracellular matrix metalloproteinases (MMPs) that typically have been previously viewed as being detrimental for inflammation by tissue degradation. We found that ablation of the neutrophil specific MMP8 increases inflammation by reducing neutrophil apoptosis, that in a murine model of chronic arthritis led to worsened disease. Now we have examined the role of macrophage-specific matrix metalloproteinase-12 (MMP12) in murine peritonitis, LPS lung inflammation, and arthritis by proteomics. Notably, Mmp12-/- mice display earlier and dramatic severe inflammation vs wild-type mice characterized by massive neutrophil infiltrations. Degradomics using 8-plex iTRAQ-Terminal Amino Isotopic Labelling of Substrates (TAILS¹) identified 611 in vitro substrates, with many cleavage sites being confirmed in vivo by TAILS. Overall, MMP12 dampens inflammation by concerted cleavages in multiple inflammation regulatory pathways. Mechanistically, we find that MMP12 facilitates macrophage invasion, but inactivates all CXCR2 chemokines responsible for neutrophil recruitment. This terminates neutrophil infiltration so accounting for the masses of neutrophils and the joint destruction in Mmp12-/- arthritis. MMP12 also promotes clotting by activation of prothrombin and inactivation of anti-thrombin-III, reducing clotting times by ~20%. Moreover, MMP12 squashes complement C3 activity at multiple levels: C3a cleavage abolishes receptor binding so halting inflammatory cell recruitment; cleavage of full-length C3 and surface-bound C3b reduces target cell lysis; whereas cleavage of C3b and iC3b increases phagocytosis of C3-opsonized particles to increase target cell elimination. This also contributes to enhanced neutrophil accumulation in these tissues. The importance of MMP12 in removing extracellular nets of actin and fibrin, identified by TAILS as new substrates, was strikingly demonstrated in vivo. Mmp12-/- mice were characterized by huge extracellular DNA/actin/fibrin nets that were absent in wild-type mice. These and multiple other inflammatory protein substrates discovered by TAILS highlights the unexpected control exerted by macrophages and MMP12 in resolving inflammation and the power of TAILS to discover new proteolytic pathways in vivo. Thus the inflammatory cell-specific MMPs dampen inflammation.

¹Kleifeld et al 2010 Nature Biotech 28, 281-288; Nature Protocols 6, 1578-1611