Biomedical research institute

    Research project 2



    Pathophysiopathology of ANCA-associated vasculitis: role of proteinase 3

    Our aim is to study the mechanisms of neutrophil activation and the role of proteinase 3 in autoimmune necrotizing vasculitis and especially in granulomatosis with polyangitis (GPA, formerly called Wegener’s granulomatosis) (Figure 7)

    Figure 7 : Role of neutrophils in the pathophysiology of ANCA-associated vasculitis

    The local neutrophil accumulation in ANCA-associated vasculitis is initiated by the priming of circulating neutrophils by low concentrations of cytokines resulting from local infection. Priming causes PR3 and MPO to be expressed on neutrophil membrane, where binding with antibodies is facilitated, thus resulting in the production of reactive oxygen species and release of proteolytic enzymes. The activated neutrophils will also activate and ultimately damage the endothelium. In addition, failure in the phagocytosis by macrophages of apoptotic neutrophils results in a defect in the resolution of inflammation which is a key feature in autoimmune vasculitis. Apoptotic neutrophils expressed membrane associated PR3 which disturbed the mechanisms of the resolution of inflammation (Mouthon et al 2012 Presse Médicale ; Millet et al . Ann Rheum Dis. 2013)

    Our research program comprises :

    1) The functional study of neutrophils from patients with ANCA-associated vasculitis within the NEUTRO-VASC (National PH-RC directed by Luc Mouthon) to identify new biomarkers for the disease. Study of interaction between neutrophils and other innate immune cells and B cells will alos be studied.

    2) the analysis of structure/activity to target specific domain of PR3 in order to modulate its function (Figure 8)
    (WEB NEWS : Institut Cochin novembre 2015)

    3) the molecular analysis of the activity of PR3 expressed during apoptosis as a danger signal in the modulation of the innate immunity (Figure 8).
    (WEB NEWS : Institut Cochin avril 2016)

    Figure 8 : Molecular structure of PR3 showing its membrane insertion site and its potential pathophysiological consequences.

    Proteinase 3 has the typical fold of chymotrypsin-like serine proteases (Hajjar et al. FEBS J. 2010): two beta-barrels made each of six anti-parallel beta-sheets (purple) and a C-terminal alpha helix (yellow). The active catalytic triad site is located between the two barrels (his71, asp118 and ser203 in green). The substrate (red) is positioned optimally in the active site. PR3 has an hydrophobic patch to insert into the membrane (Hajjar et al Proteins 2008) (shown in yellow) and bind to phosphatidylserine (whose atoms are shown in balls). PR3 could bind to phosphatidylserine expressed on apoptotic cells or on microvesicles to further modulate and disseminate inflammation (Martin et al J Biol Chem, 2016) (WEB NEWS : Institut Cochin 2016).


    Figure 9 : The autoantigen PR3 is a danger signal to activate the immune system

    We have recently discovered a novel pathway by which PR3 expressed on apoptotic neutrophils could initiate a cascade of proinflammatory reaction. Our study show for the first time that autoimmune vasculitis could have an autoinflammatory component involving interleukin-1 beta.

    (Video: Journal of Clinical Investigation)

    Nature Review Rheumatology 2015

    Proteinase 3 (PR3) is stored into the azurophil granules of circulating neutrophils.  During vascular inflammation, neutrophils are activated by ANCA and undergo apoptosis. During this process, they can express PR3 at the plasma membrane, which can interfere with phagocytosis (Kantari et al Blood 2007) and activate macrophages through the MYD88/interleukin 1 pathway inducing the production of inflammatory cytokines and chemokines (Gabillet et al J Immunol 2012). PR3 mimics a danger signal for macrophages resulting in a microenvironment favouring activation of pDCs, which are key cells in the immune silencing associated with the phagocytosis of apoptotic cells. Phagocytosis of apoptotic cells expressing PR3 results in an inhibition of the generation of regulatory T cells and a polarization of CD4 positive T helper cells into a Th9 profile. In addition, anti-PR3 ANCA further enhances the generation of Th17 cells thus potentiating inflammation. Generation of G-CSF potentiates PR3 synthesis in myeloid precursors leading to increased PR3 expression in mature neutrophils, and thus in turn potentiating inflammation (Millet et al J Clin Invest 2015) 

    (Video: Journal of Clinical Investigation)