B Cell Tolerance to Deiminated Histones in BALB/c, C57BL/6, and Autoimmune-Prone Mouse Strains

https://doi.org/10.3389/fimmu.2017.00362 Deimination, a posttranslational modification of arginine to citrulline carried out by peptidylarginine deiminases, may compromise tolerance of self-antigens. Patients with connective tissue autoimmunity, particularly rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), or Felty’s syndrome, present with autoantibodies to deiminated histones (dH), which thus form a category of antibodies to citrullinated protein antigens (ACPA). In general, ACPA are a sensitive diagnostic for RA and may form in response to the release of nuclear chromatin (DNA plus dH) from granulocytes, usually referred to as neutrophil extracellular traps. The aim of this study was to examine spontaneously autoimmune mice for autoantibodies and T cell responses to dH. We compared IgG binding to deiminated and non-deiminated histones (nH) by ELISA and Western blotting in spontaneously autoimmune strains of (NZB × NZW) F1 and NZM2410 together with their derivative congenic strains, C57BL/6.Sle1 and C57BL/6.Sle1.Sle3, which display profound autoreactivity against nuclear self-antigens. The splenocyte proliferation against the two antigens was determined in the spontaneously autoimmune (NZB × NZW) F1 strain from which other autoimmune strains used in the study were derived. Immunizations with dH and nH were attempted in BALB/c mice to assess their splenocyte response. Splenocytes from BALB/c mice and from autoimmune mice at the time of conversion to autoimmunity proliferated strongly in response to dH, yet serum IgG from autoimmune (NZB × NZW) F1, NZM2410, and C57BL/6.Sle1.Sle3 mice displayed a remarkable bias against binding to dH. At the time of seroconversion, the antibodies already exhibited preference for nH, and only nH were recovered from circulating immune complexes. Analysis of histone deimination showed constitutive deimination in thymic extracts from C57BL/6 and C57BL/6.Sle1.Sle2.Sle3 triply congenic mice and in spleens of autoimmune triply congenic mice. Our study demonstrates that tolerance mechanisms against dH are intact in BALB/c and C57BL/6 mice and continue to be effective in mice with overt autoimmunity to nH. We conclude that, in contrast to human RA and SLE patients, where we frequently observe autoantibodies against dH, autoimmune mice maintain strong tolerance mechanisms to prevent the development of autoantibodies to dH

The Mitochondrion-lysosome Axis in Adaptive and Innate Immunity: Effect of Lupus Regulator Peptide P140 on Mitochondria Autophagy and NETosis

Mitochondria deserve special attention as sensors of cellular energy homeostasis and metabolic state. Moreover, mitochondria integrate intra- and extra-cellular signals to determine appropriate cellular responses that range from proliferation to cell death. In autoimmunity, as in other inflammatory chronic disorders, the metabolism of immune cells may be extensively remodeled, perturbing sensitive tolerogenic mechanisms. Here, we examine the distribution and effects of the therapeutic 21-mer peptide called P140, which shows remarkable efficacy in modulating immune responses in inflammatory settings. We measured P140 and control peptide effects on isolated mitochondria, the distribution of peptides in live cells, and their influence on the levels of key autophagy regulators. Our data indicate that while P140 targets macro- and chaperone-mediated autophagy processes, it has little effect, if any, on mitochondrial autophagy. Remarkably, however, it suppresses NET release from neutrophils exposed to immobilized NET-anti-DNA IgG complexes. Together, our results suggest that in the mitochondrion-lysosome axis, a likely driver of NETosis and inflammation, the P140 peptide does not operate by affecting mitochondria directly

FCRL1 immunoregulation in B cell development and malignancy

Immunotherapeutic targeting of surface regulatory proteins and pharmacologic inhibition of critical signaling pathways has dramatically shifted our approach to the care of individuals with B cell malignancies. This evolution in therapy reflects the central role of the B cell receptor (BCR) signaling complex and its co-receptors in the pathogenesis of B lineage leukemias and lymphomas. Members of the Fc receptor-like gene family (FCRL1-6) encode cell surface receptors with complex tyrosine-based regulation that are preferentially expressed by B cells. Among them, FCRL1 expression peaks on naïve and memory B cells and is unique in terms of its intracellular co-activation potential. Recent studies in human and mouse models indicate that FCRL1 contributes to the formation of the BCR signalosome, modulates B cell signaling, and promotes humoral responses. Progress in understanding its regulatory properties, along with evidence for its over-expression by mature B cell leukemias and lymphomas, collectively imply important yet unmet opportunities for FCRL1 in B cell development and transformation. Here we review recent advances in FCRL1 biology and highlight its emerging significance as a promising biomarker and therapeutic target in B cell lymphoproliferative disorders

Patients with COVID-19: in the dark-NETs of neutrophils.

SARS-CoV-2 infection poses a major threat to the lungs and multiple other organs, occasionally causing death. Until effective vaccines are developed to curb the pandemic, it is paramount to define the mechanisms and develop protective therapies to prevent organ dysfunction in patients with COVID-19. Individuals that develop severe manifestations have signs of dysregulated innate and adaptive immune responses. Emerging evidence implicates neutrophils and the disbalance between neutrophil extracellular trap (NET) formation and degradation plays a central role in the pathophysiology of inflammation, coagulopathy, organ damage, and immunothrombosis that characterize severe cases of COVID-19. Here, we discuss the evidence supporting a role for NETs in COVID-19 manifestations and present putative mechanisms, by which NETs promote tissue injury and immunothrombosis. We present therapeutic strategies, which have been successful in the treatment of immunο-inflammatory disorders and which target dysregulated NET formation or degradation, as potential approaches that may benefit patients with severe COVID-19

Current Challenges and Limitations in Antibody-Based Detection of Citrullinated Histones

Studies on NETosis demand reliable and convenient markers to monitor the progress of this form of cell death. Because a determining step in the release of nuclear chromatin NETs requires the conversion of arginine residues to citrulline residues in histones by peptidylarginine deiminase, citrullinated histones can provide such a marker. Here, we evaluate antibody reagents for the detection of citrulline residues in histones and observe alarming differences between commercial antisera and mouse and rabbit monoclonal antibodies in their ability to detect their nominal target residues. Differences between antibodies that are currently used to detect citrulline residues in histones could jeopardize efforts to reach a scientific consensus and instead lead to inconsistent and even conflicting conclusions regarding the regulation of histone deimination. Our results will assist others in planning their initial or ongoing studies on peptidylarginine deiminase activity with the use of currently available antibodies. Further, we argue that, along with the careful attention to experimental conditions and calcium concentrations, validated antibody reagents are urgently needed to avoid possible setbacks in the research on NETosis

Electrospun Template Architecture and Composition Regulate Neutrophil NETosis in Vitro and in Vivo

Mounting evidence indicates that neutrophils, first responders to an implanted biomaterial, prime the microenvironment for recruited immune cells by secreting factors and releasing neutrophil extracellular traps (NETs) through NETosis. In this study, we investigated the role of electrospun template architecture and composition in regulating NETosis. Electrospun polydioxanone (PDO), collagen type I (COL), and blended PDO-COL templates (PC) were fabricated with small-diameter (0.25-0.35 μm) and large-diameter (1.0-2.00 μm) fibers. Neutrophil-template interactions were evaluated in vitro for 3 and 24 h with human neutrophils, and the PDO templates were studied in vivo (rat subcutaneous model) for 1 and 7 days. Template-bound NETs were quantified by fluorescent microscopy and an On-cell Western assay. The in vitro results indicate that larger fiber diameters reduced NETosis on PDO templates, whereas the incorporation of COL attenuated NETosis independent of fiber diameter. The in vivo results similarly revealed a lower degree of NETs on large-diameter PDO templates at 1 day, resulting in marginal tissue integration of the templates at 7 days. In contrast, the small-diameter PDO templates, which were coated in a large amount of NETs at 24 h in vivo, were surrounded by capsule-like tissue at 7 days. These preliminary in vivo results validate the in vitro model and signify NETosis as a potentially significant physiological response and a critical preconditioning event for the innate immune response to templates. In conclusion, these results demonstrate the importance of characterizing the neutrophil\u27s acute confrontation with biomaterials to engineer templates capable of promoting in situ regeneration

Localized Delivery of Cl-Amidine From Electrospun Polydioxanone Templates to Regulate Acute Neutrophil NETosis: A Preliminary Evaluation of the PAD4 Inhibitor for Tissue Engineering

Upon interaction, neutrophils can potentially release neutrophil extracellular traps (NETs) on the surface of an implanted electrospun template, which may be a significant preconditioning event for implantable biomaterials of yet unknown consequences. In this study, we investigated the potential of polydioxanone templates as a delivery vehicle for Cl-amidine, an inhibitor of peptidyl arginase deiminase 4 (PAD4), and if drug elution could attenuate PAD4-mediated NETosis in the vicinity of implanted templates. Electrospun polydioxanone templates were fabricated with distinct architectures, small diameter (0.4 μm) or large diameter (1.8 μm) fibers, and incorporated with 0–5 mg/mL Cl-amidine to examine dose-dependent effects. Acute neutrophil-template interactions were evaluated in vitro with freshly isolated human neutrophils and in vivo with a rat subcutaneous implant model. The in vitro results suggest large diameter templates with 0 mg/mL Cl-amidine significantly attenuate NETosis compared to small diameter templates. As the drug concentration increased, NETosis was significantly decreased on small diameter templates in a dose-dependent manner. The opposite was observed for large diameter templates, indicating multiple mechanisms of NETosis may be regulating neutrophil template preconditioning. Similar results were observed in vivo, verifying local NETosis inhibition by Cl-amidine eluting templates in a physiological environment. Importantly, large diameter templates with Cl-amidine enhanced neutrophil invasion and survival, supporting the potential for long-term modulation of tissue integration and regeneration. This preliminary study demonstrates a novel delivery vehicle for Cl-amidine that can be used to regulate acute NETosis as the potential critical link between the innate immune response, inflammation, and template-guided tissue regeneration

Deimination of linker histones links neutrophil extracellular trap release with autoantibodies in systemic autoimmunity

Autoantibodies to nuclear antigens arise in human autoimmune diseases, but a unifying pathogenetic mechanism remains elusive. Recently we reported that exposure of neutrophils to inflammatory conditions induces the citrullination of core histones by peptidylarginine deiminase 4 (PAD4) and that patients with autoimmune disorders produce autoantibodies that recognize such citrullinated histones. Here we identify histone H1 as an additional substrate of PAD4, localize H1 within neutrophil extracellular traps, and detect autoantibodies to citrullinated H1 in 6% of sera from patients with systemic lupus erythematosus and Sjogren\u27s syndrome. No preference for deiminated H1 was observed in healthy control sera and sera from patients with scleroderma or rheumatoid arthritis. We map binding to the winged helix of H1 and determine that citrulline 53 represents a key determinant of the autoantibody epitope. In addition, we quantitate RNA for H1 histone subtypes in mature human neutrophils and identify citrulline residues by liquid chromatography and tandem mass spectrometry. Our results indicate that deimination of linker histones generates new autoantibody epitopes with enhanced potential for stimulating autoreactive human B cells.-Dwivedi, N., Neeli, I., Schall, N., Wan, H., Desiderio, D. M., Csernok, E., Thompson, P. R., Dali, H., Briand, J.-P., Muller, S., Radic, M. Deimination of linker histones links neutrophil extracellular trap release with autoantibodies in systemic autoimmunity