Carmelo Carmona-Rivera

NETs Are a Source of Citrullinated Autoantigens and Stimulate Inflammatory Responses in Rheumatoid Arthritis

Neutrophil NETs may contribute to pathogenesis of rheumatoid arthritis. Autoantigens Slip Through the NET Autoimmune diseases are caused when the body’s immune system attacks the very tissues it’s supposed to protect. Yet, what exactly induces this loss of tolerance to self remains murky. For some autoimmune diseases, autoantigens—cellular targets of the immune response—have been identified, although it remains unclear how these normally intracellular proteins are exposed to the immune response. One hypothesis as to how these proteins may be externalized is through the excretion of neutrophil extracellular traps (NETosis). NETosis is thought to be involved in neutrophil response to bacteria, but the secretion of self-antigens in the context of inflammatory stimuli may boost autoimmune response. Now, Khandpur et al. look at the role of NETosis in rheumatoid arthritis. Autoantibodies to citrullinated antigens (ACPAs) are thought to be pathogenic in rheumatoid arthritis. The authors observed increased NETosis in patients with rheumatoid arthritis compared with both healthy controls and patients with non-autoimmune osteoarthritis. Indeed, NETosis correlated with levels of ACPA, and ACPA actually altered the makeup of the proteins secreted by neutrophils. NETs from rheumatoid arthritis patients contained citrullinated proteins, and these NETs enhanced the inflammatory response in fibroblasts from inflamed joints. Thus, altered NETosis in rheumatoid arthritis patients may contribute to the pathogenesis of disease. The early events leading to the development of rheumatoid arthritis (RA) remain unclear, but formation of autoantibodies to citrullinated protein antigens (ACPAs) is considered a key pathogenic event. Neutrophils isolated from patients with various autoimmune diseases display enhanced neutrophil extracellular trap (NET) formation, a phenomenon that exposes autoantigens in the context of immunostimulatory molecules. We investigated whether aberrant NETosis occurs in RA, determined its triggers, and examined its deleterious inflammatory consequences. Enhanced NETosis was observed in circulating and RA synovial fluid neutrophils compared to neutrophils from healthy controls and from patients with osteoarthritis (OA). Further, netting neutrophils infiltrated RA synovial tissue, rheumatoid nodules, and skin. NETosis correlated with ACPA presence and levels and with systemic inflammatory markers. RA sera and immunoglobulin fractions from RA patients with high levels of ACPA and/or rheumatoid factor significantly enhanced NETosis, and the NETs induced by these autoantibodies displayed distinct protein content. Indeed, during NETosis, neutrophils externalized the citrullinated autoantigens implicated in RA pathogenesis, and anti–citrullinated vimentin antibodies potently induced NET formation. Moreover, the inflammatory cytokines interleukin-17A (IL-17A) and tumor necrosis factor–α (TNF-α) induced NETosis in RA neutrophils. In turn, NETs significantly augmented inflammatory responses in RA and OA synovial fibroblasts, including induction of IL-6, IL-8, chemokines, and adhesion molecules. These observations implicate accelerated NETosis in RA pathogenesis, through externalization of citrullinated autoantigens and immunostimulatory molecules that may promote aberrant adaptive and innate immune responses in the joint and in the periphery, and perpetuate pathogenic mechanisms in this disease.

Neutrophil extracellular traps enriched in oxidized mitochondrial DNA are interferogenic and contribute to lupus-like disease

Neutrophil extracellular traps (NETs) are implicated in autoimmunity, but how they are generated and their roles in sterile inflammation remain unclear. Ribonucleoprotein immune complexes (RNP ICs), inducers of NETosis, require mitochondrial reactive oxygen species (ROS) for maximal NET stimulation. After RNP IC stimulation of neutrophils, mitochondria become hypopolarized and translocate to the cell surface. Extracellular release of oxidized mitochondrial DNA is proinflammatory in vitro, and when this DNA is injected into mice, it stimulates type I interferon (IFN) signaling through a pathway dependent on the DNA sensor STING. Mitochondrial ROS are also necessary for spontaneous NETosis of low-density granulocytes from individuals with systemic lupus erythematosus. This was also observed in individuals with chronic granulomatous disease, who lack NADPH oxidase activity but still develop autoimmunity and type I IFN signatures. Mitochondrial ROS inhibition in vivo reduces disease severity and type I IFN responses in a mouse model of lupus. Together, these findings highlight a role for mitochondria in the generation not only of NETs but also of pro-inflammatory oxidized mitochondrial DNA in autoimmune diseases.

Neutrophil Extracellular Trap–Associated Protein Activation of the NLRP3 Inflammasome Is Enhanced in Lupus Macrophages

Neutrophil extracellular traps (NETs) represent an important defense mechanism against microorganisms. Clearance of NETs is impaired in a subset of patients with systemic lupus erythematosus, and NETosis is increased in neutrophils and, particularly, in low-density granulocytes derived from lupus patients. NETs are toxic to the endothelium, expose immunostimulatory molecules, activate plasmacytoid dendritic cells, and may participate in organ damage through incompletely characterized pathways. To better understand the role of NETs in fostering dysregulated inflammation, we examined inflammasome activation in response to NETs or to LL-37, an antibacterial protein externalized on NETs. Both NETs and LL-37 activate caspase-1, the central enzyme of the inflammasome, in both human and murine macrophages, resulting in release of active IL-1β and IL-18. LL-37 activation of the NLRP3 inflammasome utilizes P2X7 receptor–mediated potassium efflux. NET and LL-37-mediated activation of the inflammasome is enhanced in macrophages derived from lupus patients. In turn, IL-18 is able to stimulate NETosis in human neutrophils. These results suggest that enhanced formation of NETs in lupus patients can lead to increased inflammasome activation in adjacent macrophages. This leads to release of inflammatory cytokines that further stimulate NETosis, resulting in a feed-forward inflammatory loop that could potentially lead to disease flares and/or organ damage.

Neutrophil extracellular traps induce endothelial dysfunction in systemic lupus erythematosus through the activation of matrix metalloproteinase-2

Rationale The structural and functional integrity of the endothelium is crucial in maintaining vascular homeostasis and preventing atherosclerosis. Patients with systemic lupus erythematosus (SLE) have an increased risk of developing endothelial dysfunction and premature cardiovascular disease. Neutrophil extracellular trap (NET) formation is increased in SLE and has been proposed to contribute to endothelial damage, but the mechanism remains unclear. Objective To determine the mechanism by which enhanced NET formation by low-density granulocytes (LDGs) in SLE contributes to endothelial damage and disrupts the endothelium. Results The putative role of NET-externalised matrix metalloproteinases (MMPs) in altering the functional integrity of the endothelium was examined. MMP-9 externalised by lupus LDGs during NET formation specifically impaired murine aortic endothelium-dependent vasorelaxation and induced endothelial cell apoptosis. Endothelial dysfunction correlated with the activation of endothelial MMP-2 by MMP-9 present in NETs, while inhibition of MMP-2 activation restored endothelium-dependent function and decreased NET-induced vascular cytotoxicity. Moreover, immunogenic complexes composed of MMP-9 and anti-MMP-9 were identified in SLE sera. These complexes, as well as anti-MMP-9 autoantibodies, induced NETosis and enhanced MMP-9 activity. Conclusions These observations implicate activation of endothelial MMP-2 by MMP-9 contained in NETs as an important player in endothelial dysfunction, and MMP-9 as a novel self-antigen in SLE. These results further support that aberrant NET formation plays pathogenic roles in SLE.

A BLOC-1 Mutation Screen Reveals that PLDN Is Mutated in Hermansky-Pudlak Syndrome Type 9

Hermansky-Pudlak Syndrome (HPS) is an autosomal-recessive condition characterized by oculocutaneous albinism and a bleeding diathesis due to absent platelet delta granules. HPS is a genetically heterogeneous disorder of intracellular vesicle biogenesis. We first screened all our patients with HPS-like symptoms for mutations in the genes responsible for HPS-1 through HPS-6 and found no functional mutations in 38 individuals. We then examined all eight genes encoding the biogenesis of lysosome-related organelles complex-1, or BLOC-1, proteins in these individuals. This identified a homozygous nonsense mutation in PLDN in a boy with characteristic features of HPS. PLDN is mutated in the HPS mouse model pallid and encodes the protein pallidin, which interacts with the early endosomal t-SNARE syntaxin-13. We could not detect any full-length pallidin in our patients cells despite normal mRNA expression of the mutant transcript. We could detect an alternative transcript that would skip the exon that harbored the mutation, but we demonstrate that if this transcript is translated into protein, although it correctly localizes to early endosomes, it does not interact with syntaxin-13. In our patients melanocytes, the melanogenic protein TYRP1 showed aberrant localization, an increase in plasma-membrane trafficking, and a failure to reach melanosomes, explaining the boys severe albinism and establishing his diagnosis as HPS-9.

Low density granulocytes: a distinct class of neutrophils in systemic autoimmunity

Recent studies have renewed the interest on the potential role that neutrophils play in the development of systemic lupus erythematosus (SLE) and other autoimmune conditions. A distinct subset of proinflammatory, low-density granulocytes (LDGs) isolated from the peripheral blood mononuclear cell fractions of patients with SLE has been described. While the origin and role of LDGs needs to be fully characterized, there is evidence that these cells may contribute to lupus pathogenesis and to the development of end-organ damage through heightened proinflammatory responses, altered phagocytic capacity, enhanced ability to synthesize type I interferons, and to kill endothelial cells. Furthermore, these cells readily form neutrophil extracellular traps, a phenomenon that may promote autoantigen externalization and organ damage. This review examines the biology and potential origin of LDGs, describes the ultrastructural characteristics of these cells, and discusses their putative pathogenic role in systemic autoimmune diseases.

Proteins derived from neutrophil extracellular traps may serve as self-antigens and mediate organ damage in autoimmune diseases

Neutrophils are the most abundant leukocytes in circulation and represent one of the first lines of defense against invading pathogens. Neutrophils possess a vast arsenal of antimicrobial proteins, which can be released from the cell by a death program termed NETosis. Neutrophil extracellular traps (NETs) are web-like structures consisting of decondensed chromatin decorated with granular and cytosolic proteins. Both exuberant NETosis and impaired clearance of NETs have been implicated in the organ damage of autoimmune diseases, such as systemic lupus erythematosus (SLE), small vessel vasculitis (SVV), and psoriasis. NETs may also represent an important source of modified autoantigens in SLE and SVV. Here, we review the autoimmune diseases linked to NETosis, with a focus on how modified proteins externalized on NETs may trigger loss of immune tolerance and promote organ damage.

Neutrophil extracellular trap-derived enzymes oxidize high-density lipoprotein: an additional proatherogenic mechanism in systemic lupus erythematosus.

Oxidative stress and oxidized high‐density lipoprotein (HDL) are implicated as risk factors for cardiovascular disease (CVD) in systemic lupus erythematosus (SLE). Yet, how HDL is oxidized and rendered dysfunctional in SLE remains unclear. Neutrophil extracellular traps (NETs), the levels of which are elevated in lupus, possess oxidant‐generating enzymes, including myeloperoxidase (MPO), NADPH oxidase (NOX), and nitric oxide synthase (NOS). We hypothesized that NETs mediate HDL oxidation, impairing cholesterol efflux capacity (CEC).

Neutrophil-Related Gene Expression and Low-Density Granulocytes Associated With Disease Activity and Response to Treatment in Antineutrophil Cytoplasmic Antibody-Associated Vasculitis

To discover biomarkers involved in the pathophysiology of antineutrophil cytoplasmic antibody–associated vasculitis (AAV) and to determine whether low‐density granulocytes (LDGs) contribute to gene expression signatures in AAV.

Clinical, molecular and cellular features of non-Puerto Rican Hermansky-Pudlak syndrome patients of Hispanic descent

Hermansky-Pudlak syndrome is an autosomal recessive condition characterized by a bleeding diathesis and hypopigmentation of the skin, hair and eyes. Some HPS patients develop other complications such as granulomatous colitis and/or a fatal pulmonary fibrosis. Eight genes have been associated with the condition, resulting in subtypes HPS-1 through HPS-8. The HPS gene products are involved in the biogenesis of specialized lysosome-related organelles such as melanosomes, platelet delta granules and others. HPS1 and HPS4 form a stable complex named BLOC-3, and patients with BLOC-3 or AP-3 deficiency develop pulmonary fibrosis. Therefore, it is important to subtype each HPS patient. HPS type 1 (HPS-1) occurs frequently on the island Puerto Rico due to a founder mutation. Here, we describe seven mutations, six of which are previously unreported, in the HPS1, HPS4 and HPS5 genes among patients of Mexican, Uruguayan, Honduran, Cuban, Venezuelan and Salvadoran ancestries. Our findings demonstrate that the diagnosis of HPS should be considered in Hispanic patients with oculocutaneous albinism and bleeding symptoms. Moreover, such patients should not be assumed to have the HPS-1 subtype typical of northwest Puerto Rican patients. We recommend molecular HPS subtyping in such cases, since it may have significant implications for prognosis and intervention.