Paul R. Thompson

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.

Regulation of the p300 HAT domain via a novel activation loop

The transcriptional coactivator p300 is a histone acetyltransferase (HAT) whose function is critical for regulating gene expression in mammalian cells. However, the molecular events that regulate p300 HAT activity are poorly understood. We evaluated autoacetylation of the p300 HAT protein domain to determine its function. Using expressed protein ligation, the p300 HAT protein domain was generated in hypoacetylated form and found to have reduced catalytic activity. This basal catalytic rate was stimulated by autoacetylation of several key lysine sites within an apparent activation loop motif. This post-translational modification and catalytic regulation of p300 HAT activity is conceptually analogous to the activation of most protein kinases by autophosphorylation. We therefore propose that this autoregulatory loop could influence the impact of p300 on a wide variety of signaling and transcriptional events.

The structural basis of protein acetylation by the p300/CBP transcriptional coactivator

The transcriptional coactivator p300/CBP (CREBBP) is a histone acetyltransferase (HAT) that regulates gene expression by acetylating histones and other transcription factors. Dysregulation of p300/CBP HAT activity contributes to various diseases including cancer. Sequence alignments, enzymology experiments and inhibitor studies on p300/CBP have led to contradictory results about its catalytic mechanism and its structural relation to the Gcn5/PCAF and MYST HATs. Here we describe a high-resolution X-ray crystal structure of a semi-synthetic heterodimeric p300 HAT domain in complex with a bi-substrate inhibitor, Lys-CoA. This structure shows that p300/CBP is a distant cousin of other structurally characterized HATs, but reveals several novel features that explain the broad substrate specificity and preference for nearby basic residues. Based on this structure and accompanying biochemical data, we propose that p300/CBP uses an unusual ‘hit-and-run’ (Theorell–Chance) catalytic mechanism that is distinct from other characterized HATs. Several disease-associated mutations can also be readily accounted for by the p300 HAT structure. These studies pave the way for new epigenetic therapies involving modulation of p300/CBP HAT activity.

N-α-Benzoyl-N5-(2-Chloro-1-Iminoethyl)-l-Ornithine Amide, a Protein Arginine Deiminase Inhibitor, Reduces the Severity of Murine Collagen-Induced Arthritis

Rheumatoid arthritis is associated with the development of autoantibodies to citrullinated self-proteins. Citrullinated synovial proteins, which are generated via the actions of the protein arginine deiminases (PADs), are known to develop in the murine collagen-induced arthritis (CIA) model of inflammatory arthritis. Given these findings, we evaluated whether N-α-benzoyl-N5-(2-chloro-1-iminoethyl)-l-ornithine amide (Cl-amidine), a recently described pan-PAD inhibitor, could affect the development of arthritis and autoimmunity by treating mice in the CIA model with Cl-amidine on days 0–35. Cl-amidine treatment reduced total synovial and serum citrullination, decreased clinical disease activity by ∼50%, and significantly decreased IgG2a anti-mouse type II collagen Abs. Additionally, histopathology scores and total complement C3 deposition were significantly lower in Cl-amidine–treated mice compared with vehicle controls. Synovial microarray analyses demonstrated decreased IgG reactivity to several native and citrullinated epitopes compared with vehicle controls. Cl-amidine treatment had no ameliorative effect on collagen Ab-induced arthritis, suggesting its primary protective mechanism was not mediated through effector pathways. Reduced levels of citrullinated synovial proteins observed in mice treated with Cl-amidine are consistent with the notion that Cl-amidine derives its efficacy from its ability to inhibit the deiminating activity of PADs. In total, these results suggested that PADs are necessary participants in the autoimmune and subsequent inflammatory processes in CIA. Cl-amidine may represent a novel class of disease-modifying agents that modulate aberrant citrullination, and perhaps other immune processes, necessary for the development of inflammatory arthritis.

Peptidylarginine deiminase inhibition is immunomodulatory and vasculoprotective in murine lupus

Recent evidence suggests that enhanced neutrophil extracellular trap (NET) formation activates plasmacytoid dendritic cells and serves as a source of autoantigens in SLE. We propose that aberrant NET formation is also linked to organ damage and to the premature vascular disease characteristic of human SLE. Here, we demonstrate enhanced NET formation in the New Zealand mixed 2328 (NZM) model of murine lupus. NZM mice also developed autoantibodies to NETs as well as the ortholog of human cathelicidin/LL37 (CRAMP), a molecule externalized in the NETs. NZM mice were treated with Cl-amidine, an inhibitor of peptidylarginine deiminases (PAD), to block NET formation and were evaluated for lupus-like disease activity, endothelial function, and prothrombotic phenotype. Cl-amidine treatment inhibited NZM NET formation in vivo and significantly altered circulating autoantibody profiles and complement levels while reducing glomerular IgG deposition. Further, Cl-amidine increased the differentiation capacity of bone marrow endothelial progenitor cells, improved endothelium-dependent vasorelaxation, and markedly delayed time to arterial thrombosis induced by photochemical injury. Overall, these findings suggest that PAD inhibition can modulate phenotypes crucial for lupus pathogenesis and disease activity and may represent an important strategy for mitigating cardiovascular risk in lupus patients.

Regulation of p53 target gene expression by peptidylarginine deiminase 4

ABSTRACT Histone Arg methylation has been correlated with transcriptional activation of p53 target genes. However, whether this modification is reversed to repress the expression of p53 target genes is unclear. Here, we report that peptidylarginine deiminase 4, a histone citrullination enzyme, is involved in the repression of p53 target genes. Inhibition or depletion of PAD4 elevated the expression of a subset of p53 target genes, including p21/CIP1/WAF1, leading to cell cycle arrest and apoptosis. Moreover, the induction of p21, cell cycle arrest, and apoptosis by PAD4 depletion is p53 dependent. Protein-protein interaction studies showed an interaction between p53 and PAD4. Chromatin immunoprecipitation assays showed that PAD4 is recruited to the p21 promoter in a p53-dependent manner. RNA polymerase II (Pol II) activities and the association of PAD4 are dynamically regulated at the p21 promoter during UV irradiation. Paused RNA Pol II and high levels of PAD4 were detected before UV treatment. At early time points after UV treatment, an increase of histone Arg methylation and a decrease of citrullination were correlated with a transient activation of p21. At later times after UV irradiation, a loss of RNA Pol II and an increase of PAD4 were detected at the p21 promoter. The dynamics of RNA Pol II activities after UV treatment were further corroborated by permanganate footprinting. Together, these results suggest a role of PAD4 in the regulation of p53 target gene expression.

Inhibition of PAD4 activity is sufficient to disrupt mouse and human NET formation

PAD4 has been strongly implicated in the pathogenesis of autoimmune, cardiovascular and oncological diseases, through clinical genetics and gene disruption in mice. Novel, selective PAD4 inhibitors binding to a calcium-deficient form of the PAD4 enzyme have, for the first time, validated the critical enzymatic role of human and mouse PAD4 in both histone citrullination and neutrophil extracellular trap formation. The therapeutic potential of PAD4 inhibitors can now be explored.

Identification of farnesoid X receptor beta as a novel mammalian nuclear receptor sensing lanosterol

ABSTRACT Nuclear receptors are ligand-modulated transcription factors. On the basis of the completed human genome sequence, this family was thought to contain 48 functional members. However, by mining human and mouse genomic sequences, we identified FXRβ as a novel family member. It is a functional receptor in mice, rats, rabbits, and dogs but constitutes a pseudogene in humans and primates. Murine FXRβ is widely coexpressed with FXR in embryonic and adult tissues. It heterodimerizes with RXRα and stimulates transcription through specific DNA response elements upon addition of 9-cis-retinoic acid. Finally, we identified lanosterol as a candidate endogenous ligand that induces coactivator recruitment and transcriptional activation by mFXRβ. Lanosterol is an intermediate of cholesterol biosynthesis, which suggests a direct role in the control of cholesterol biosynthesis in nonprimates. The identification of FXRβ as a novel functional receptor in nonprimate animals sheds new light on the species differences in cholesterol metabolism and has strong implications for the interpretation of genetic and pharmacological studies of FXR-directed physiologies and drug discovery programs.

Peptidylarginine Deiminase Inhibition Reduces Vascular Damage and Modulates Innate Immune Responses in Murine Models of Atherosclerosis

Rationale: Neutrophil extracellular trap (NET) formation promotes vascular damage, thrombosis, and activation of interferon-&agr;–producing plasmacytoid dendritic cells in diseased arteries. Peptidylarginine deiminase inhibition is a strategy that can decrease in vivo NET formation. Objective: To test whether peptidylarginine deiminase inhibition, a novel approach to targeting arterial disease, can reduce vascular damage and inhibit innate immune responses in murine models of atherosclerosis. Methods and Results: Apolipoprotein-E (Apoe)−/− mice demonstrated enhanced NET formation, developed autoantibodies to NETs, and expressed high levels of interferon-&agr; in diseased arteries. Apoe−/− mice were treated for 11 weeks with daily injections of Cl-amidine, a peptidylarginine deiminase inhibitor. Peptidylarginine deiminase inhibition blocked NET formation, reduced atherosclerotic lesion area, and delayed time to carotid artery thrombosis in a photochemical injury model. Decreases in atherosclerosis burden were accompanied by reduced recruitment of netting neutrophils and macrophages to arteries, as well as by reduced arterial interferon-&agr; expression. Conclusions: Pharmacological interventions that block NET formation can reduce atherosclerosis burden and arterial thrombosis in murine systems. These results support a role for aberrant NET formation in the pathogenesis of atherosclerosis through modulation of innate immune responses.

Peptidylarginine deiminase inhibition disrupts NET formation and protects against kidney, skin and vascular disease in lupus-prone MRL/lpr mice

Objectives An imbalance between neutrophil extracellular trap (NET) formation and degradation has been described in systemic lupus erythematosus (SLE), potentially contributing to autoantigen externalisation, type I interferon synthesis and endothelial damage. We have demonstrated that peptidylarginine deiminase (PAD) inhibition reduces NET formation and protects against lupus-related vascular damage in the New Zealand Mixed model of lupus. However, another strategy for inhibiting NETs—knockout of NOX2—accelerates lupus in a different murine model, MRL/lpr. Here, we test the effects of PAD inhibition on MRL/lpr mice in order to clarify whether some NET inhibitory pathways may be consistently therapeutic across models of SLE. Methods NET formation and autoantibodies to NETs were characterised in lupus-prone MRL/lpr mice. MRL/lpr mice were also treated with two different PAD inhibitors, Cl-amidine and the newly described BB-Cl-amidine. NET formation, endothelial function, interferon signature, nephritis and skin disease were examined in treated mice. Results Neutrophils from MRL/lpr mice demonstrate accelerated NET formation compared with controls. MRL/lpr mice also form autoantibodies to NETs and have evidence of endothelial dysfunction. PAD inhibition markedly improves endothelial function, while downregulating the expression of type I interferon-regulated genes. PAD inhibition also reduces proteinuria and immune complex deposition in the kidneys, while protecting against skin disease. Conclusions PAD inhibition reduces NET formation, while protecting against lupus-related damage to the vasculature, kidneys and skin in various lupus models. The strategy by which NETs are inhibited will have to be carefully considered if human studies are to be undertaken.