Proteolysis of Annexin A2 Leads to Inflammasome-Mediated Proinflammatory Response in Human Sepsis

Sepsis is a systemic inflammatory response that arises in the setting of severe infection and results in tissue injury beyond the site of the original insult. Sepsis carries high risk of morbidity and mortality with hospital mortality ranging from 30-50% due to organ failure from immune dysregulation. The basic pathophysiology of the exaggerated inflammatory response is poorly understood, and treatment remains non-specific and limited to supportive care. Recent scientific advances have shown that there is a significant crosstalk between coagulation proteases and inflammatory mediators in sepsis, but the role of fibrinolytic proteins has not been extensively studied.

Annexin A2 (A2) is a profibrinolytic, calcium-dependent, phospholipid-binding protein that, with protein S100A10, binds plasminogen and tissue plasminogen activator and increases the catalytic efficiency of plasminogen activation by 60-fold. Plasmin generation by the cell-surface A2 complex leads to fibrin clearance and facilitates monocyte migration through extracellular matrices. A2 also serves an important role in inflammation, orchestrating endosomal membrane repair as a membrane anchoring protein to prevent leakage of endosomal cathepsin and subsequent inflammasome activation. It is reported that Anxa2^-/- mice displayed more severe disease in the cecal ligation and puncture (CLP) model of sepsis. Therefore, we hypothesized that A2 may be dysregulated in human sepsis and this leads to overactivation of immune response.

We have found that A2 undergoes an orderly, reproducible pattern of proteolysis in peripheral blood mononuclear cells (PBMCs) from human subjects with documented sepsis. Semiquantitative immunoblot analysis of PBMC lysates revealed 79% decreased A2 expression in septic subjects compared to healthy controls (p=0.0001). Degradation of A2 was observed in all 25 sepsis samples, and complete depletion of the intact protein was evident in 10 out of 12 septic shock subjects. A2-associated plasmin generation was also strikingly impaired with 64% decrease at the cell surface of PBMCs from subjects with sepsis (p=0.007). We found that A2, but not other annexin family proteins, was specifically degraded by a serine-protease activated in human sepsis, but not in other critical care conditions associated with severe hypotension such as hemorrhagic shock and pancreatitis. Further investigation into the mechanism of A2 degradation revealed proteolysis of membrane-bound A2 by a serine protease activated in septic environment.

Interleukin(IL)-18, a key proinflammatory cytokine in sepsis pathophysiology that also correlates with sepsis severity, was significantly elevated in sepsis and septic shock patients compared to healthy controls in our study (p=0.05 and 0.01, respectively). Levels of less stable acute phase cytokines such as IL-1β and interferon(IFN)-γ did not show significant difference between the cohorts. IL-18 levels correlated inversely with A2 levels, suggesting an increased inflammatory response when A2 decreases. Immunofluorescence analysis showed 50% reduction in anti-A2 immunoreactive area in sepsis PBMCs (vs healthy control, p=0.003) with relative localization to membrane-bound structures, and redistribution of cathepsin to the cytosol suggesting lysosomal leak.

We identified a sepsis-specific pattern of A2 degradation mediated via a membrane-bound serine protease and observed significant rise in IL-18 level and cathepsin leak in sepsis PBMC cytosol. Taken together, the proinflammatory consequence of A2 degradation in sepsis may be driven by impairment of A2-dependent lysosomal membrane repair functions, which leads to spillage of cathepsins that activate the NLRP3 inflammasome and release of proinflammatory IL-18. To our knowledge, this is the first reported evidence of specific, inflammation-activated modulation of A2 with consequential immune dysregulation. Elucidating a better understanding of the A2-dependent pathway of immune regulation may engender novel therapeutic and biomarker approaches in sepsis.