Repetitive Antigen Responses of LDL-Reactive CD4+ T Cells Induce Tr1 Cell-Mediated Immune Tolerance.

BACKGROUND: Inflammation triggered by the deposition of LDL (low-density lipoprotein) in the arterial wall leads to the development of atherosclerosis. Regulatory T (Treg) cells inhibit vascular inflammation through the induction of immune tolerance toward LDL-related antigens. However, tolerogenic mechanisms that promote the generation of LDL-specific Treg cells in vivo remain unclear. METHODS: We identified LDL-specific T cells by activation-induced marker expression and analyzed expression profiles and suppressive functions of TCR (T-cell antigen receptor)-transgenic T cells upon repetitive transfer into antigen-transgenic mice via flow cytometry. RESULTS: We investigated the naturally occurring Treg-cell response against human LDL in standard chow diet-fed mice that are transgenic for human ApoB100 (apolipoprotein B100). We found that IL (interleukin)-10 expression in LDL-specific T cells from spleen increases with age, albeit LDL-specific populations do not enlarge in older mice. To investigate the generation of IL-10-producing LDL-specific T cells, we transferred naive CD4+ T cells recognizing human ApoB100 from TCR-transgenic mice into human ApoB100-transgenic mice. Adoptive transfer of human ApoB100-specific T cells induced immune tolerance in recipient mice and effectively inhibited activation of subsequently transferred naive T cells of the same specificity in vivo. Moreover, repetitive transfers increased the population of Treg type 1 cells that suppress ApoB100-specific responses via IL-10. In a translational approach, LDL-specific Treg type 1 cells from blood of healthy donors suppressed the activation of monocytic THP-1 cells in an IL-10-dependent manner. CONCLUSIONS: We show that repetitive transfer of naive ApoB100-specific T cells and recurrent LDL-specific T-cell stimulation induces Treg type 1 cell-mediated immune tolerance against LDL in vivo. Our results provide insight into the generation of autoantigen-specific anti-inflammatory T cells under tolerogenic conditions.

Liver damage promotes pro-inflammatory T-cell responses against apolipoprotein B-100.

OBJECTIVES: Liver-derived apolipoprotein B-100 (ApoB100) is an autoantigen that is recognized by atherogenic CD4+ T cells in cardiovascular disease (CVD). CVD is a major mortality risk for patients with chronic inflammatory liver diseases. However, the impact of liver damage for ApoB100-specific T-cell responses is unknown. METHODS: We identified ApoB100-specific T cells in blood from healthy controls, nonalcoholic fatty liver disease (NAFLD) patients, and CVD patients by activation-induced marker expression and analyzed their differentiation pattern in correlation to the lipid profile and liver damage parameters in a cross-sectional study. To assess the induction of extrahepatic ApoB100-specific T cells upon transient liver damage in vivo, we performed hydrodynamic tail vein injections with diphtheria toxin A (DTA)-encoding plasmid in human ApoB100-transgenic mice. RESULTS: Utilizing immunodominant ApoB100-derived peptides, we found increased ApoB100-specific T-cell populations in NAFLD and CVD patients compared to healthy controls. In a peptide-specific manner, ApoB100 reactivity in healthy controls was accompanied by expression of the regulatory T (Treg)-cell transcription factor FOXP3. In contrast, FOXP3 expression decreased, whereas expression of pro-inflammatory cytokine interleukin (IL)-17A increased in ApoB100-specific T cells from NAFLD and CVD patients. Dyslipidemia and liver damage parameters in blood correlated with reduced FOXP3 expression and elevated IL-17A production in ApoB100-specific T-cell populations, respectively. Moreover, DTA-mediated transient liver damage in human ApoB100-transgenic mice accumulated IL-17a-expressing ApoB100-specific T cells in the periphery. CONCLUSION: Our results show that liver damage promotes pro-inflammatory ApoB100-specific T-cell populations, thereby providing a cellular mechanism for the increased CVD risk in liver disease patients.

NADPH Oxidases Are Required for Full Platelet Activation In Vitro and Thrombosis In Vivo but Dispensable for Plasma Coagulation and Hemostasis.

OBJECTIVE: Using 3KO (triple NOX [NADPH oxidase] knockout) mice (ie, NOX1-/-/NOX2-/-/NOX4-/-), we aimed to clarify the role of this family of enzymes in the regulation of platelets in vitro and hemostasis in vivo. Approach and Results: 3KO mice displayed significantly reduced platelet superoxide radical generation, which was associated with impaired platelet aggregation, adhesion, and thrombus formation in response to the key agonists collagen and thrombin. A comparison with single-gene knockouts suggested that the phenotype of 3KO platelets is the combination of the effects of the genetic deletion of NOX1 and NOX2, while NOX4 does not show any significant function in platelet regulation. 3KO platelets displayed significantly higher levels of cGMP-a negative platelet regulator that activates PKG (protein kinase G). The inhibition of PKG substantially but only partially rescued the defective phenotype of 3KO platelets, which are responsive to both collagen and thrombin in the presence of the PKG inhibitors KT5823 or Rp-8-pCPT-cGMPs, but not in the presence of the NOS (NO synthase) inhibitor L-NG-monomethyl arginine. In vivo, triple NOX deficiency protected against ferric chloride-driven carotid artery thrombosis and experimental pulmonary embolism, while hemostasis tested in a tail-tip transection assay was not affected. Procoagulatory activity of platelets (ie, phosphatidylserine surface exposure) and the coagulation cascade in platelet-free plasma were normal. CONCLUSIONS: This study indicates that inhibiting NOXs has strong antithrombotic effects partially caused by increased intracellular cGMP but spares hemostasis. NOXs are, therefore, pharmacotherapeutic targets to develop new antithrombotic drugs without bleeding side effects.

Targeting NETs using dual-active DNase1 variants.

Background: Neutrophil Extracellular Traps (NETs) are key mediators of immunothrombotic mechanisms and defective clearance of NETs from the circulation underlies an array of thrombotic, inflammatory, infectious, and autoimmune diseases. Efficient NET degradation depends on the combined activity of two distinct DNases, DNase1 and DNase1-like 3 (DNase1L3) that preferentially digest double-stranded DNA (dsDNA) and chromatin, respectively. Methods: Here, we engineered a dual-active DNase with combined DNase1 and DNase1L3 activities and characterized the enzyme for its NET degrading potential in vitro. Furthermore, we produced a mouse model with transgenic expression of the dual-active DNase and analyzed body fluids of these animals for DNase1 and DNase 1L3 activities. We systematically substituted 20 amino acid stretches in DNase1 that were not conserved among DNase1 and DNase1L3 with homologous DNase1L3 sequences. Results: We found that the ability of DNase1L3 to degrade chromatin is embedded into three discrete areas of the enzyme's core body, not the C-terminal domain as suggested by the state-of-the-art. Further, combined transfer of the aforementioned areas of DNase1L3 to DNase1 generated a dual-active DNase1 enzyme with additional chromatin degrading activity. The dual-active DNase1 mutant was superior to native DNase1 and DNase1L3 in degrading dsDNA and chromatin, respectively. Transgenic expression of the dual-active DNase1 mutant in hepatocytes of mice lacking endogenous DNases revealed that the engineered enzyme was stable in the circulation, released into serum and filtered to the bile but not into the urine. Conclusion: Therefore, the dual-active DNase1 mutant is a promising tool for neutralization of DNA and NETs with potential therapeutic applications for interference with thromboinflammatory disease states.

Absence of Malaria-Associated Coagulopathy in Asymptomatic Plasmodium falciparum Infection: Results From a Cross-sectional Study in the Ashanti Region, Ghana.

Background: Coagulopathy is common in acute symptomatic Plasmodium falciparum malaria, and the degree of coagulation abnormality correlates with parasitemia and disease severity. Chronic asymptomatic malaria has been associated with increased morbidity. However, the role of coagulation activation in asymptomatic, semi-immune individuals remains unclear. This study investigates the potential effect of asymptomatic P falciparum infection on coagulation activation in semi-immune Ghanaian adults. Methods: Blood from asymptomatic Ghanaian adults with P falciparum blood stage infection detectable by polymerase chain reaction (PCR) or by both PCR and rapid diagnostic test and from noninfected individuals, was investigated. Markers of coagulation activation including global coagulation tests, D-dimer, antithrombin III, fibrinogen, and von Willebrand factor antigen were tested. Furthermore, blood count, inflammation markers, and liver and kidney function tests were assessed. Results: Acquired coagulopathy was not found in asymptomatic P falciparum infection. Asymptomatic malaria was associated with significantly lower platelet counts. Systemic inflammation markers and liver and kidney function tests were not altered compared to noninfected controls. Conclusions: There is no laboratory evidence for acquired coagulopathy in adults with asymptomatic P falciparum malaria in highly endemic regions. Lack of laboratory evidence for systemic inflammation and liver and kidney dysfunction indicates that asymptomatic malaria may not be associated with significant morbidity.

An update on factor XII-driven vascular inflammation.

The plasma protein factor XII (FXII) is the liver-derived zymogen of the serine protease FXIIa that initiates an array of proteolytic cascades. Zymogen activation, enzymatic FXIIa activity and functions are regulated by interactions with cell receptors, negatively charged surfaces, other serine proteases, and serpin inhibitors, which bind to distinct protein domains and regions in FXII(a). FXII exerts mitogenic activity, while FXIIa initiates the pro-inflammatory kallikrein-kinin pathway and the pro-thrombotic intrinsic coagulation pathway, respectively. Growing evidence indicates that FXIIa-mediated thrombo-inflammation plays a crucial role in various pathological states besides classical thrombosis, such as endothelial dysfunction. Consistently, increased FXIIa levels are associated with hypercholesterolemia and hypertriglyceridemia. In contrast, FXII deficiency protects from thrombosis but is otherwise not associated with prolonged bleeding or other adverse clinical manifestations. Here, we review current concepts for FXII(a)-driven vascular inflammation focusing on endothelial hyperpermeability, receptor signaling, atherosclerosis and immune cell activation.

Intrinsic coagulation pathway-mediated thrombin generation in mouse whole blood.

Calibrated Automated Thrombography (CAT) is a versatile and sensitive method for analyzing coagulation reactions culminating in thrombin generation (TG). Here, we present a CAT method for analyzing TG in murine whole blood by adapting the CAT assay used for measuring TG in human plasma. The diagnostically used artificial and physiologic factor XII (FXII) contact activators kaolin, ellagic acid and polyphosphate (polyP) stimulated TG in murine blood in a dose-dependent manner resulting in a gradual increase in endogenous thrombin potential and peak thrombin, with shortened lag times and times to peak. The activated FXII inhibitor rHA-Infestin-4 and direct oral anticoagulants (DOACs) interfered with TG triggered by kaolin, ellagic acid and polyP and TG was completely attenuated in blood of FXII- (F12 -/-) and FXI-deficient (F11 -/-) mice. Moreover, reconstitution of blood from F12 -/- mice with human FXII restored impaired contact-stimulated TG. HEK293 cell-purified polyP also initiated FXII-driven TG in mouse whole blood and addition of the selective inhibitor PPX_Δ12 ablated natural polyP-stimulated TG. In conclusion, the data provide a method for analysis of contact activation-mediated TG in murine whole blood. As the FXII-driven intrinsic pathway of coagulation has emerged as novel target for antithrombotic agents that are validated in mouse thrombosis and bleeding models, our novel assay could expedite therapeutic drug development.

Mechanism, Functions, and Diagnostic Relevance of FXII Activation by Foreign Surfaces.

Factor XII (FXII) is a serine protease zymogen produced by hepatocytes and secreted into plasma. The highly glycosylated coagulation protein consists of six domains and a proline-rich region that regulate activation and function. Activation of FXII results from a conformational change induced by binding ("contact") with negatively charged surfaces. The activated serine protease FXIIa drives both the proinflammatory kallikrein-kinin pathway and the procoagulant intrinsic coagulation cascade, respectively. Deficiency in FXII is associated with a prolonged activated partial thromboplastin time (aPTT) but not with an increased bleeding tendency. However, genetic or pharmacological deficiency impairs both arterial and venous thrombosis in experimental models. This review summarizes current knowledge of FXII structure, mechanisms of FXII contact activation, and the importance of FXII for diagnostic coagulation testing and thrombosis.

The contact system in liver injury.

Coagulation is controlled by a delicate balance of prothrombotic and antithrombotic mechanisms, to prevent both excessive blood loss from injured vessels and pathologic thrombosis. The liver plays a pivotal role in hemostasis through the synthesis of plasma coagulation factors and their inhibitors that, in addition to thrombosis and hemostasis, orchestrates an array of inflammatory responses. As a result, impaired liver function has been linked with both hypercoagulability and bleeding disorders due to a pathologic balance of pro- and anticoagulant plasma factors. At sites of vascular injury, thrombus propagation that finally may occlude the blood vessel depends on negatively charged biopolymers, such as polyphosphates and extracellular DNA, that provide a physiological surface for contact activation of coagulation factor XII (FXII). FXII initiates the contact system that drives both the intrinsic pathway of coagulation, and formation of the inflammatory mediator bradykinin by the kallikrein-kinin system. Moreover, FXII facilitates receptor-mediated signalling, thereby promoting mitogenic activities, angiogenesis, and neutrophil stimulation with implications for liver diseases. Here, we summarize current knowledge on the FXII-driven contact system in liver diseases and review therapeutic approaches to target its activities during impaired liver function.

Defective NET clearance contributes to sustained FXII activation in COVID-19-associated pulmonary thrombo-inflammation.

BACKGROUND: Coagulopathy and inflammation are hallmarks of Coronavirus disease 2019 (COVID-19) and are associated with increased mortality. Clinical and experimental data have revealed a role for neutrophil extracellular traps (NETs) in COVID-19 disease. The mechanisms that drive thrombo-inflammation in COVID-19 are poorly understood. METHODS: We performed proteomic analysis and immunostaining of postmortem lung tissues from COVID-19 patients and patients with other lung pathologies. We further compared coagulation factor XII (FXII) and DNase activities in plasma samples from COVID-19 patients and healthy control donors and determined NET-induced FXII activation using a chromogenic substrate assay. FINDINGS: FXII expression and activity were increased in the lung parenchyma, within the pulmonary vasculature and in fibrin-rich alveolar spaces of postmortem lung tissues from COVID-19 patients. In agreement with this, plasmaaac acafajföeFXII activation (FXIIa) was increased in samples from COVID-19 patients. Furthermore, FXIIa colocalized with NETs in COVID-19 lung tissue indicating that NETs accumulation leads to FXII contact activation in COVID-19. We further showed that an accumulation of NETs is partially due to impaired NET clearance by extracellular DNases as DNase substitution improved NET dissolution and reduced FXII activation in vitro. INTERPRETATION: Collectively, our study supports that the NET/FXII axis contributes to the pathogenic chain of procoagulant and proinflammatory responses in COVID-19. Targeting both NETs and FXIIa may offer a potential novel therapeutic strategy. FUNDING: This study was supported by the European Union (840189), the Werner Otto Medical Foundation Hamburg (8/95) and the German Research Foundation (FR4239/1-1, A11/SFB877, B08/SFB841 and P06/KFO306).

Identification of the factor XII contact activation site enables sensitive coagulation diagnostics.

Contact activation refers to the process of surface-induced activation of factor XII (FXII), which initiates blood coagulation and is captured by the activated partial thromboplastin time (aPTT) assay. Here, we show the mechanism and diagnostic implications of FXII contact activation. Screening of recombinant FXII mutants identified a continuous stretch of residues Gln317-Ser339 that was essential for FXII surface binding and activation, thrombin generation and coagulation. Peptides spanning these 23 residues competed with surface-induced FXII activation. Although FXII mutants lacking residues Gln317-Ser339 were susceptible to activation by plasmin and plasma kallikrein, they were ineffective in supporting arterial and venous thrombus formation in mice. Antibodies raised against the Gln317-Ser339 region induced FXII activation and triggered controllable contact activation in solution leading to thrombin generation by the intrinsic pathway of coagulation. The antibody-activated aPTT allows for standardization of particulate aPTT reagents and for sensitive monitoring of coagulation factors VIII, IX, XI.

Targeting isoaspartate-modified Aß rescues behavioral deficits in transgenic mice with Alzheimer's disease-like pathology.

BACKGROUND: Amyloid ß (Aß)-directed immunotherapy has shown promising results in preclinical and early clinical Alzheimer's disease (AD) trials, but successful translation to late clinics has failed so far. Compelling evidence suggests that post-translationally modified Aß peptides might play a decisive role in onset and progression of AD and first clinical trials targeting such Aß variants have been initiated. Modified Aß represents a small fraction of deposited material in plaques compared to pan-Aß epitopes, opening up pathways for tailored approaches of immunotherapy. Here, we generated the first monoclonal antibodies that recognize L-isoaspartate-modified Aß (isoD7-Aß) and tested a lead antibody molecule in 5xFAD mice. METHODS: This work comprises a combination of chemical and biochemical techniques as well as behavioral analyses. Aß peptides, containing L-isoaspartate at position 7, were chemically synthesized and used for immunization of mice and antibody screening methods. Biochemical methods included anti-isoD7-Aß monoclonal antibody characterization by surface plasmon resonance, immunohistochemical staining of human and transgenic mouse brain, and the development and application of isoD7-Aß ELISA as well as different non-modified Aß ELISA. For antibody treatment studies, 12 mg/kg anti-isoD7-Aß antibody K11_IgG2a was applied intraperitoneally to 5xFAD mice for 38 weeks. Treatment controls implemented were IgG2a isotype as negative and 3D6_IgG2a, the parent molecule of bapineuzumab, as positive control antibodies. Behavioral studies included elevated plus maze, pole test, and Morris water maze. RESULTS: Our advanced antibody K11 showed a KD in the low nM range and > 400fold selectivity for isoD7-Aß compared to other Aß variants. By using this antibody, we demonstrated that formation of isoD7-Aß may occur after formation of aggregates; hence, the presence of the isoD7-modification differentiates aged Aß from newly formed peptides. Importantly, we also show that the Tottori mutation responsible for early-onset AD in a Japanese pedigree is characterized by massively accelerated formation of isoD7-Aß in cell culture. The presence of isoD7-Aß was verified by K11 in post mortem human cortex and 5xFAD mouse brain tissue. Passive immunization of 5xFAD mice resulted in a significant reduction of isoD7-Aß and total Aß in brain. Amelioration of cognitive impairment was demonstrated by Morris water maze, elevated plus maze, pole, and contextual fear conditioning tests. Interestingly, despite the lower abundance of the isoD7-Aß epitope, the application of anti-isoD7-Aß antibodies showed comparable treatment efficacy in terms of reduction of brain amyloid and spatial learning but did not result in an increase of plasma Aß concentration as observed with 3D6 treatment. CONCLUSIONS: The present study demonstrates, for the first time, that the antibody-mediated targeting of isoD7-modified Aß peptides leads to attenuation of AD-like amyloid pathology. In conjunction with previously published data on antibodies directed against pGlu-modified Aß, the results highlight the crucial role of modified Aß peptides in AD pathophysiology. Hence, the results also underscore the therapeutic potential of targeting modified amyloid species for defining tailored approaches in AD therapy.