Neutrophil Proteases Promote Experimental Abdominal Aortic Aneurysm via Extracellular Trap Release and Plasmacytoid Dendritic Cell Activation.
ABSTRACT: We previously established that neutrophil-derived dipeptidyl peptidase I (DPPI) is essential for experimental abdominal aortic aneurysm (AAA) development. Because DPPI activates several neutrophil serine proteases, it remains to be determined whether the AAA-promoting effect of DPPI is mediated by neutrophil serine proteases.Using an elastase-induced AAA model, we demonstrate that the absence of 2 neutrophil serine proteases, neutrophil elastase and proteinase-3, recapitulates the AAA-resistant phenotype of DPPI-deficient mice. DPPI and neutrophil serine proteases direct the in vitro and in vivo release of extracellular structures termed neutrophil extracellular traps (NETs). Administration of DNase1, which dismantles NETs, suppresses elastase-induced AAA in wild-type animals and in DPPI-deficient mice reconstituted with wild-type neutrophils. NETs also contain the cathelicidin-related antimicrobial peptide that complexes with self-DNA in recruiting plasmacytoid dendritic cells (pDCs), inducing type I interferons (IFNs) and promoting AAA in DPPI-deficient mice. Conversely, depletion of pDCs or blockade of type I IFNs suppresses experimental AAA. Moreover, we find an abundance of human cathelicidin peptide, a 37 amino acid sequence starting with 2 leucines and the human orthologue of cathelicidin-related antimicrobial peptide, in the vicinity of pDCs in human AAA tissues. Increased type I IFN mRNA expression is observed in human AAA tissues and circulating IFN-? is detected in ?50% of the AAA sera examined.These results suggest that neutrophil protease-mediated NET release contributes to elastase-induced AAA through pDC activation and type I IFN production. These findings increase our understanding of the pathways underlying AAA inflammatory responses and suggest that limiting NET, pDC, and type I IFN activities may suppress aneurysm progression.
Project description:Dipeptidyl peptidase I (DPPI) is a lysosomal cysteine protease critical for the activation of granule-associated serine proteases, including neutrophil elastase, cathepsin G, and proteinase 3. DPPI and granule-associated serine proteases have been shown to play a key role in regulating neutrophil recruitment at sites of inflammation. It has recently been suggested that neutrophils and neutrophil-associated proteases may also be important in the development and progression of abdominal aortic aneurysms (AAAs), a common vascular disease associated with chronic inflammation and destructive remodeling of aortic wall connective tissue. Here we show that mice with a loss-of-function mutation in DPPI are resistant to the development of elastase-induced experimental AAAs. This is in part because of diminished recruitment of neutrophils to the elastase-injured aortic wall and impaired local production of CXC-chemokine ligand (CXCL) 2. Furthermore, adoptive transfer of wild-type neutrophils is sufficient to restore susceptibility to AAAs in DPPI-deficient mice, as well as aortic wall expression of CXCL2. In addition, in vivo blockade of CXCL2 by using neutralizing antibodies directed against its cognate receptor leads to a significant reduction in aortic dilatation. These findings suggest that DPPI and/or granule-associated serine proteases are necessary for neutrophil recruitment into the diseased aorta and that these proteases act to amplify vascular wall inflammation that leads to AAAs.
Project description:The pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated necrotizing crescentic GN (NCGN) is incompletely understood. Dipeptidyl peptidase I (DPPI) is a cysteine protease required for the activation of neutrophil serine proteases (NSPs) cathepsin G, neutrophil elastase, and proteinase 3, which are enzymes that modulate inflammation. We used a mouse model of anti-myeloperoxidase (MPO) antibody-induced NCGN to determine whether active NSPs contribute to its pathogenesis. MPO-deficient animals immunized with murine MPO, irradiated, and transplanted with wild-type bone marrow developed NCGN. In contrast, transplantation with bone marrow that lacked DPPI or lacked both neutrophil elastase and proteinase 3 protected mice from NCGN induced by anti-MPO antibody. The kidneys of mice reconstituted with DPPI-deficient bone marrow generated significantly less IL-1? than did those of mice reconstituted with wild-type bone marrow; similarly, in vitro, DPPI-deficient monocytes produced significantly less IL-1? in response to anti-MPO antibody than did wild-type monocytes. This reduction in IL-1? was NSP dependent; exogenous addition of PR3 restored IL-? production in DPPI-deficient monocytes. Last, the IL-1 receptor antagonist anakinra protected animals against anti-MPO antibody-induced NCGN (16.7%±6.0% versus 2.4%±1.7% crescents), suggesting that IL-1? is a critical inflammatory mediator in this model. These data suggest that the development of anti-MPO antibody-induced NCGN requires NSP-dependent IL-1? generation and that these processes may provide therapeutic targets for ANCA-mediated diseases in humans.
Project description:Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by a breakdown of tolerance to nuclear antigens and the development of immune complexes. Genomic approaches have shown that human SLE leukocytes homogeneously express type I interferon (IFN)-induced and neutrophil-related transcripts. Increased production and/or bioavailability of IFN-? and associated alterations in dendritic cell (DC) homeostasis have been linked to lupus pathogenesis. Although neutrophils have long been shown to be associated with lupus, their potential role in disease pathogenesis remains elusive. Here, we show that mature SLE neutrophils are primed in vivo by type I IFN and die upon exposure to SLE-derived anti-ribonucleoprotein antibodies, releasing neutrophil extracellular traps (NETs). SLE NETs contain DNA as well as large amounts of LL37 and HMGB1, neutrophil proteins that facilitate the uptake and recognition of mammalian DNA by plasmacytoid DCs (pDCs). Indeed, SLE NETs activate pDCs to produce high levels of IFN-? in a DNA- and TLR9 (Toll-like receptor 9)-dependent manner. Our results reveal an unsuspected role for neutrophils in SLE pathogenesis and identify a novel link between nucleic acid-recognizing antibodies and type I IFN production in this disease.
Project description:Neutrophil Extracellular Traps (NETs) are structures consisting of chromatin and antimicrobial molecules that are released by neutrophils during a form of regulated cell death called NETosis. NETs trap invading pathogens, promote coagulation and activate myeloid cells to produce Type I interferons (type I IFN), proinflammatory cytokines that regulate the immune system. The mechanism of NET recognition by myeloid cells is not yet clearly identified. Here we show that macrophages and other myeloid cells phagocytose NETs. Once in phagosomes, NETs translocate to the cytosol, where they activate the DNA sensor cyclic GMP-AMP synthase (cGAS) and induce type I IFN expression. cGAS recognizes the DNA backbone of NETs. Interestingly, the NET associated serine protease Neutrophil Elastase (NE) mediates the activation of the pathway. We confirmed that NETs activate cGAS in vivo. Thus, our findings identify cGAS as a major sensor of NETs, mediating the immune activation during infection and in auto-immune diseases.
Project description:Papillon-Lefèvre syndrome (PLS) results from mutations that inactivate cysteine protease cathepsin C (CTSC), which processes a variety of serine proteases considered essential for antimicrobial defense. Despite serine protease-deficient immune cell populations, PLS patients do not exhibit marked immunodeficiency. Here, we characterized a 24-year-old woman who had suffered from severe juvenile periodontal disease, but was otherwise healthy, and identified a homozygous missense mutation in CTSC indicative of PLS. Proteome analysis of patient neutrophil granules revealed that several proteins that normally localize to azurophil granules, including the major serine proteases, elastase, cathepsin G, and proteinase 3, were absent. Accordingly, neutrophils from this patient were incapable of producing neutrophil extracellular traps (NETs) in response to ROS and were unable to process endogenous cathelicidin hCAP-18 into the antibacterial peptide LL-37 in response to ionomycin. In immature myeloid cells from patient bone marrow, biosynthesis of CTSC and neutrophil serine proteases appeared normal along with initial processing and sorting to cellular storage. In contrast, these proteins were completely absent in mature neutrophils, indicating that CTSC mutation promotes protease degradation in more mature hematopoietic subsets, but does not affect protease production in progenitor cells. Together, these data indicate CTSC protects serine proteases from degradation in mature immune cells and suggest that neutrophil serine proteases are dispensable for human immunoprotection.
Project description:Neutrophils can form neutrophil extracellular traps (NETs) to capture microbes and facilitate their clearance. NETs consist of decondensed chromatin decorated with anti-microbial proteins. Here, we describe the effect of neutrophil proteases on the protein content of NETs. We show that the neutrophil serine proteases degrade several neutrophil proteins associated with NETs. Interestingly, the anti-bacterial proteins associated with NETs, such as myeloperoxidase, calgranulin B and neutrophil elastase (NE), seem to be less susceptible to proteolytic degradation than other NET proteins, such as actin and MNDA. NETs have been proposed to play a role in autoimmune reactions. Our data demonstrate that a large number of the autoepitopes of NET proteins that are recognized by autoantibodies produced by systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) patients are also removed by the proteases. In conclusion, neutrophil serine proteases have a major impact on the NET proteome and the proteolytic changes of NET-associated proteins may counteract autoimmune reactions to NET components.
Project description:Neutrophil extracellular traps (NETs) consist of a decondensed DNA scaffold decorated with neutrophil-derived proteins. The proteome of NETs, or "NETome," has been largely elucidated in vitro. However, components such as plasma and extracellular matrix proteins may affect the NETome under physiological conditions. Here, using a reductionistic approach, we explored the effects of two proteases active during injury and wounding, human thrombin and plasmin, on the NETome. Using high-resolution mass spectrometry, we identified a total of 164 proteins, including those previously not described in NETs. The serine proteases, particularly thrombin, were also found to interact with DNA and bound to NETs in vitro. Among the most abundant proteins were those identified previously, including histones, neutrophil elastase, and antimicrobial proteins. We observed reduced histone (H2B, H3, and H4) and neutrophil elastase levels upon the addition of the two proteases. Analyses of NET-derived tryptic peptides identified subtle changes upon protease treatments. Our results provide evidence that exogenous proteases, present during wounding and inflammation, influence the NETome. Taken together, regulation of NETs and their proteins under different physiological conditions may affect their roles in infection, inflammation, and the host response.
Project description:We determined whether nucleases are deficient in the tear fluid of dry eye disease (DED) patients, and whether this causes extracellular DNA (eDNA) and neutrophil extracellular trap (NET) accumulation in the precorneal tear film, thus causing ocular surface inflammation.Exfoliated cells adhered to Schirmer test strips were collected on glass slides, and immunofluorescence confocal microscopy was used to evaluate neutrophils, eDNA, NETs, and their molecular components. Similar experiments were performed with mucoid films collected from the inferior conjunctival fornix or bulbar conjunctiva. We used quantitative PCR to evaluate eDNA signaling pathways and inflammatory cytokine expression. We also determined the amount of ocular surface eDNA and evaluated tear fluid nuclease activity.eDNA, NETs, and neutrophils were present on the ocular surface in DED patients and abundant in mucoid films. NETs consisted of eDNA, histones, cathelicidin, and neutrophil elastase. Tear fluid nuclease activity was decreased significantly in DED patients, whereas the amount of eDNA on the ocular surface was increased significantly. Expression of genes downstream of eDNA signaling, such as TLR9, MyD88, and type I interferon, as well as the inflammatory cytokines interleukin-6 and tumor necrosis factor-α, was significantly increased in DED patients.Extracellular DNA production and clearance mechanisms are dysregulated in DED. Nuclease deficiency in tear fluid allows eDNA and NETs to accumulate in precorneal tear film, and results in ocular surface inflammation. These findings point to novel therapeutic interventions in severe DED based on clearance of eDNA, NETs, and other molecular components from the ocular surface.
Project description:Neutrophil extracellular traps (NETs) consist of antimicrobial molecules embedded in a web of extracellular DNA. Formation of NETs is considered to be a defense mechanism utilized by neutrophils to ensnare and kill invading pathogens, and has been recently termed NETosis. Neutrophils can be stimulated to undergo NETosis ex vivo, and are predicted to contain high levels of serine proteases, such as neutrophil elastase (NE), cathepsin G (CG) and proteinase 3 (PR3). Serine proteases are important effectors of neutrophil-mediated immunity, which function directly by degrading pathogenic virulent factors and indirectly via proteolytic activation or deactivation of cytokines, chemokines and receptors. In this study, we utilized a diverse and unbiased peptide library to detect and profile protease activity associated with NETs induced by phorbol-12-myristate-13-acetate (PMA). We obtained a "proteolytic signature" from NETs derived from healthy donor neutrophils and used proteomics to assist in the identification of the source of this proteolytic activity. In addition, we profiled each neutrophil serine protease and included the newly identified enzyme, neutrophil serine protease 4 (NSP4). Each enzyme had overlapping yet distinct endopeptidase activities and often cleaved at unique sites within the same peptide substrate. The dominant proteolytic activity in NETs was attributed to NE; however, cleavage sites corresponding to CG and PR3 activity were evident. When NE was immunodepleted, the remaining activity was attributed to CG and to a lesser extent PR3 and NSP4. Our results suggest that blocking NE activity would abrogate the major protease activity associated with NETs. In addition, the newly identified substrate specificity signatures will guide the design of more specific probes and inhibitors that target NET-associated proteases.
Project description:BACKGROUND:Wiskott-Aldrich syndrome (WAS) is a rare primary immunodeficiency caused by mutations in Wiskott-Aldrich syndrome protein (WASp), a key regulator of cytoskeletal dynamics in hematopoietic cells. A high proportion of patients experience autoimmunity caused by a breakdown in T- and B-cell tolerance. Moreover, excessive production of type I interferon (IFN-I) by plasmacytoid dendritic cells (pDCs) contributes to autoimmune signs; however, the factors that trigger excessive innate activation have not been defined. OBJECTIVE:Neutrophil extracellular traps (NETs) emerged as major initiating factors in patients with diseases such as systemic lupus erythematosus and rheumatoid arthritis. In this study we explored the possible involvement of aberrant neutrophil functions in patients with WAS. METHODS:We evaluated the expression of a set of granulocyte genes associated with NETs in a cohort of patients with WAS and the presence of NET inducers in sera. Using a mouse model of WAS, we analyzed NET release by WASp-null neutrophils and evaluated the composition and homeostasis of neutrophils in vivo. By using depletion experiments, we assessed the effect of neutrophils in promoting inflammation and reactivity against autoantigens. RESULTS:Transcripts of genes encoding neutrophil enzymes and antimicrobial peptides were increased in granulocytes of patients with WAS, and serum-soluble factors triggered NET release. WASp-null neutrophils showed increased spontaneous NETosis, induced IFN-I production by pDCs, and activated B cells through B-cell activating factor. Consistently, their depletion abolished constitutive pDC activation, normalized circulating IFN-I levels, and, importantly, abolished production of autoantibodies directed against double-stranded DNA, nucleosomes, and myeloperoxidase. CONCLUSIONS:These findings reveal that neutrophils are involved in the pathogenic loop that causes excessive activation of innate cells and autoreactive B cells, thus identifying novel mechanisms that contribute to the autoimmunity of WAS.