ABSTRACT
BACKGROUND: Convolutional neural network (CNN) can capture the structural features changes of brain aging based on MRI, thus predict brain age in healthy individuals accurately. However, most studies use single feature to predict brain age in healthy individuals, ignoring adding information from multiple sources and the changes in brain aging patterns after mild traumatic brain injury (mTBI) were still unclear. METHODS: Here, we leveraged the structural data from a large, heterogeneous dataset (N = 1464) to implement an interpretable 3D combined CNN model for brain-age prediction. In addition, we also built an atlas-based occlusion analysis scheme with a fine-grained human Brainnetome Atlas to reveal the age-sstratified contributed brain regions for brain-age prediction in healthy controls (HCs) and mTBI patients. The correlations between brain predicted age gaps (brain-PAG) following mTBI and individual's cognitive impairment, as well as the level of plasma neurofilament light were also examined. RESULTS: Our model utilized multiple 3D features derived from T1w data as inputs, and reduced the mean absolute error (MAE) of age prediction to 3.08 years and improved Pearson's r to 0.97 on 154 HCs. The strong generalizability of our model was also validated across different centers. Regions contributing the most significantly to brain age prediction were the caudate and thalamus for HCs and patients with mTBI, and the contributive regions were mostly located in the subcortical areas throughout the adult lifespan. The left hemisphere was confirmed to contribute more in brain age prediction throughout the adult lifespan. Our research showed that brain-PAG in mTBI patients was significantly higher than that in HCs in both acute and chronic phases. The increased brain-PAG in mTBI patients was also highly correlated with cognitive impairment and a higher level of plasma neurofilament light, a marker of neurodegeneration. The higher brain-PAG and its correlation with severe cognitive impairment showed a longitudinal and persistent nature in patients with follow-up examinations. CONCLUSION: We proposed an interpretable deep learning framework on a relatively large dataset to accurately predict brain age in both healthy individuals and mTBI patients. The interpretable analysis revealed that the caudate and thalamus became the most contributive role across the adult lifespan in both HCs and patients with mTBI. The left hemisphere contributed significantly to brain age prediction may enlighten us to be concerned about the lateralization of brain abnormality in neurological diseases in the future. The proposed interpretable deep learning framework might also provide hope for testing the performance of related drugs and treatments in the future.
Subject(s)
Aging , Brain Concussion , Brain , Magnetic Resonance Imaging , Neural Networks, Computer , Humans , Adult , Male , Female , Middle Aged , Magnetic Resonance Imaging/methods , Brain Concussion/diagnostic imaging , Brain/diagnostic imaging , Young Adult , Aged , Cognitive Dysfunction/diagnostic imaging , Deep LearningABSTRACT
Blood proteins are emerging as potential biomarkers for mild traumatic brain injury (mTBI). Molecular pathology of mTBI underscores the critical roles of neuronal injury, neuroinflammation, and vascular health in disease progression. However, the temporal profile of blood biomarkers associated with the aforementioned molecular pathology after CT-negative mTBI, their diagnostic and prognostic potential, and their utility in monitoring white matter integrity and progressive brain atrophy remain unclear. Thus, we investigated serum biomarkers and neuroimaging in a longitudinal cohort, including 103 CT-negative mTBI patients and 66 matched healthy controls (HCs). Angiogenic biomarker vascular endothelial growth factor (VEGF) exhibited the highest area under the curve of 0.88 in identifying patients from HCs. Inflammatory biomarker interleukin-1ß and neuronal cell body injury biomarker ubiquitin carboxyl-terminal hydrolase L1 were elevated in acute-stage patients and associated with deterioration of cognitive function from acute-stage to 6-12 mo post-injury period. Notably, axonal injury biomarker neurofilament light (NfL) was elevated in acute-stage patients, with higher levels associated with impaired white matter integrity in acute-stage and progressive gray and white matter atrophy from 3- to 6-12 mo post-injury period. Collectively, our findings emphasized the potential clinical value of serum biomarkers, particularly NfL and VEGF, in diagnosing mTBI and monitoring disease progression.
Subject(s)
Brain Concussion , Humans , Brain Concussion/diagnostic imaging , Vascular Endothelial Growth Factor A , Neurofilament Proteins , Disease Progression , Biomarkers , Atrophy/pathology , Tomography, X-Ray Computed , Brain/diagnostic imaging , Brain/pathologyABSTRACT
Mild traumatic brain injury (mTBI) disrupts the integrity of white matter microstructure, which affects brain functional connectivity supporting cognitive function. Although the relationship between structural and functional connectivity (SC and FC), here called SC-FC coupling, has been studied on global level in brain disorders, the long-term disruption of SC-FC coupling in mTBI at regional scale was still unclear. The current study investigated the alteration pattern of regional SC-FC coupling in 104 acute mTBI patients (41 with 6-12 months of follow-up) and 56 healthy controls (HCs). SC and FC networks were constructed to measure regional, intra-network, and inter-network SC-FC coupling. Compared with HCs, acute mTBI exhibited altered SC-FC coupling of the sensorimotor network (SMN). The coupling laterality indicators of the SMN can identify mTBI from controls. The persistent SC-FC decoupling of the SMN and the additional decoupling of the default mode network (DMN) were observed in chronic mTBI. Crucially, decoupling of the SMN and DMN predicted better cognitive outcomes. The findings revealed the SC-FC coupling alternations exhibited hierarchical trend originating from the sensorimotor cortex to high-order cognitive regions with the progression of mTBI. The regional and hierarchical SC-FC coupling may be a prognostic biomarker to provide insights into the pathophysiology mechanism of mTBI.
Subject(s)
Brain Concussion , Cognitive Dysfunction , Humans , Brain Concussion/complications , Brain Concussion/diagnostic imaging , Magnetic Resonance Imaging , Nerve Net/diagnostic imaging , Brain/diagnostic imaging , Brain MappingABSTRACT
Traumatic brain injury (TBI) disrupt the coordinated activity of triple-network and produce impairments across several cognitive domains. The triple-network model posits a key role of the salience network (SN) that regulates interactions with the central executive network (CEN) and default mode network (DMN). However, the aberrant dynamic interactions among triple-network and associations with neurobehavioral symptoms in mild TBI was still unclear. In present study, we used brain network interaction index (NII) and dynamic functional connectivity to examine the time-varying cross-network interactions among the triple-network in 109 acute patients, 41 chronic patients, and 65 healthy controls. Dynamic cross-network interactions were significantly increased and more variable in mild TBI compared to controls. Crucially, mild TBI exhibited an increased NII as enhanced integrations between the SN and CEN while reduced coupling of the SN with DMN. The increased NII also implied much severer and multiple domains of cognitive impairments at both acute and chronic mild TBI. Abnormities in time-varying engagement of triple-network is a clinically relevant neurobiological signature of psychopathology in mild TBI. The findings provided align with and advance an emerging perspective on the importance of aberrant brain dynamics associated with highly disparate cognitive and behavioral outcomes in trauma.
Subject(s)
Brain Concussion , Cognitive Dysfunction , Humans , Brain Concussion/complications , Brain Concussion/diagnostic imaging , Magnetic Resonance Imaging , Brain/diagnostic imaging , Brain Mapping , Nerve Net , Cognitive Dysfunction/diagnostic imaging , Cognitive Dysfunction/etiology , Cognitive Dysfunction/pathologyABSTRACT
Integration of signaling and metabolic pathways enables and sustains lymphocyte function. Whereas metabolic changes occurring during T cell activation are well characterized, the metabolic demands of differentiated T lymphocytes are largely unexplored. In this study, we defined the bioenergetics of Th17 effector cells generated in vivo. These cells depend on oxidative phosphorylation (OXPHOS) for energy and cytokine production. Mechanistically, the essential role of OXPHOS in Th17 cells results from their limited capacity to increase glycolysis in response to metabolic stresses. This metabolic program is observed in mouse and human Th17 cells, including those isolated from Crohn disease patients, and it is linked to disease, as inhibiting OXPHOS reduces the severity of murine colitis and psoriasis. These studies highlight the importance of analyzing metabolism in effector lymphocytes within in vivo inflammatory contexts and suggest a therapeutic role for manipulating OXPHOS in Th17-driven diseases.
Subject(s)
Cell Differentiation/immunology , Colitis/immunology , Lymphocyte Activation/immunology , Oxidative Phosphorylation , Th17 Cells/immunology , Animals , Cell Separation , Disease Models, Animal , Gene Expression Profiling , Humans , Mice , Mice, Inbred C57BL , Oligonucleotide Array Sequence Analysis , Real-Time Polymerase Chain Reaction , TranscriptomeABSTRACT
Genetic variations in the ITGAM gene (encoding CD11b) strongly associate with risk for systemic lupus erythematosus (SLE). Here we have shown that 3 nonsynonymous ITGAM variants that produce defective CD11b associate with elevated levels of type I interferon (IFN-I) in lupus, suggesting a direct link between reduced CD11b activity and the chronically increased inflammatory status in patients. Treatment with the small-molecule CD11b agonist LA1 led to partial integrin activation, reduced IFN-I responses in WT but not CD11b-deficient mice, and protected lupus-prone MRL/Lpr mice from end-organ injury. CD11b activation reduced TLR-dependent proinflammatory signaling in leukocytes and suppressed IFN-I signaling via an AKT/FOXO3/IFN regulatory factor 3/7 pathway. TLR-stimulated macrophages from CD11B SNP carriers showed increased basal expression of IFN regulatory factor 7 (IRF7) and IFN-ß, as well as increased nuclear exclusion of FOXO3, which was suppressed by LA1-dependent activation of CD11b. This suggests that pharmacologic activation of CD11b could be a potential mechanism for developing SLE therapeutics.
Subject(s)
CD11b Antigen/immunology , Lupus Erythematosus, Systemic/immunology , Macrophages/immunology , Toll-Like Receptors/immunology , Animals , CD11b Antigen/genetics , Female , Forkhead Box Protein O3/genetics , Forkhead Box Protein O3/immunology , Humans , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/immunology , Interferon Regulatory Factor-7/genetics , Interferon Regulatory Factor-7/immunology , Interferon Type I/genetics , Interferon Type I/immunology , Lupus Erythematosus, Systemic/genetics , Lupus Erythematosus, Systemic/pathology , Macrophages/pathology , Male , Mice , Mice, Inbred MRL lpr , Polymorphism, Single Nucleotide , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/immunology , Toll-Like Receptors/geneticsABSTRACT
OBJECTIVE: Dysregulation of innate and adaptive immune responses contributes to the pathogenesis of systemic lupus erythematosus (SLE) and its associated premature vascular damage. No drug to date targets both systemic inflammatory disease and the cardiovascular complications of SLE. Tofacitinib is a JAK inhibitor that blocks signaling downstream of multiple cytokines implicated in lupus pathogenesis. While clinical trials have shown that tofacitinib exhibits significant clinical efficacy in various autoimmune diseases, its role in SLE and the associated vascular pathology remains to be characterized. METHODS: MRL/lpr lupus-prone mice were administered tofacitinib or vehicle by gavage for 6 weeks (therapeutic arm) or 8 weeks (preventive arm). Nephritis, skin inflammation, serum levels of autoantibodies and cytokines, mononuclear cell phenotype and gene expression, neutrophil extracellular traps (NETs) release, endothelium-dependent vasorelaxation, and endothelial differentiation were compared in treated and untreated mice. RESULTS: Treatment with tofacitinib led to significant improvement in measures of disease activity, including nephritis, skin inflammation, and autoantibody production. In addition, tofacitinib treatment reduced serum levels of proinflammatory cytokines and interferon responses in splenocytes and kidney tissue. Tofacitinib also modulated the formation of NETs and significantly increased endothelium-dependent vasorelaxation and endothelial differentiation. The drug was effective in both preventive and therapeutic strategies. CONCLUSION: Tofacitinib modulates the innate and adaptive immune responses, ameliorates murine lupus, and improves vascular function. These results indicate that JAK inhibitors have the potential to be beneficial in SLE and its associated vascular damage.
Subject(s)
Lupus Erythematosus, Systemic/complications , Lupus Erythematosus, Systemic/drug therapy , Piperidines/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Pyrimidines/therapeutic use , Pyrroles/therapeutic use , Vascular Diseases/drug therapy , Vascular Diseases/etiology , Animals , Female , Lupus Erythematosus, Systemic/immunology , Mice , Mice, Inbred MRL lpr , Vascular Diseases/immunologyABSTRACT
OBJECTIVE: Alterations in phenotype and function of endothelial progenitor cells (EPCs) have been associated with poor vascular outcomes and impaired vascular repair in various conditions. Our hypothesis was that patients with PM and DM have dysregulation of EPCs driven by type I IFN and IL-18 similar to other autoimmune diseases. METHODS: Quantification of circulating EPCs was performed by flow cytometry in patients with PM/DM and matched healthy controls. The ability of EPCs to differentiate into mature endothelial cells was investigated by light and fluorescence microscopy quantification in the presence or absence of PM/DM or control serum, neutralizing antibodies to type I IFN receptor or IL-18. Serum type I IFN activity was quantified by induction of type I IFN-inducible genes in HeLa cells. Circulating IL-18 concentrations were assessed by ELISA. RESULTS: Circulating EPCs were significantly lower in PM/DM patients compared with controls. PM/DM EPCs displayed a decreased capacity to differentiate into mature endothelial cells and PM/DM serum significantly inhibited differentiation of control EPCs. This effect was reversed in the majority of samples with neutralizing antibodies to IL-18 or to type I IFN receptor or by a combination of these antibodies. Patients with associated impairments in EPC function had higher type I IFN serum activity. CONCLUSION: PM/DM is associated with dysregulation of EPC phenotype and function that may be attributed, at least in part, to aberrant IL-18 and type I IFN pathways. The implication of these vasculopathic findings for disease prognosis and complications remains to be determined.
Subject(s)
Dermatomyositis/pathology , Endothelial Progenitor Cells/physiology , Interferon Type I/metabolism , Polymyositis/pathology , Adult , Aged , Cell Differentiation/physiology , Dermatomyositis/metabolism , Enzyme-Linked Immunosorbent Assay , Female , Humans , Interleukin-18/metabolism , Male , Middle Aged , Polymyositis/metabolism , Prospective Studies , Receptor, Interferon alpha-beta/metabolismABSTRACT
NETosis is a newly recognized mechanism of programmed neutrophil death. It is characterized by a stepwise progression of chromatin decondensation, membrane rupture, and release of bactericidal DNA-based structures called neutrophil extracellular traps (NETs). Conventional 'suicidal' NETosis has been described in pathogenic models of systemic autoimmune disorders. Recent in vivo studies suggest that a process of 'vital' NETosis also exists, in which chromatin is condensed and membrane integrity is preserved. Techniques to assess 'suicidal' or 'vital' NET formation in a specific, quantitative, rapid and semiautomated way have been lacking, hindering the characterization of this process. Here we have developed a new method to simultaneously assess both 'suicidal' and 'vital' NETosis, using high-speed multi-spectral imaging coupled to morphometric image analysis, to quantify spontaneous NET formation observed ex-vivo or stimulus-induced NET formation triggered in vitro. The use of imaging flow cytometry allows automated, quantitative and rapid analysis of subcellular morphology and texture, and introduces the potential for further investigation using NETosis as a biomarker in pre-clinical and clinical studies.
Subject(s)
Extracellular Traps/physiology , Neutrophils/physiology , Flow Cytometry/methods , Humans , Image Cytometry/methods , Light , Optical Imaging/methodsABSTRACT
Recent evidence suggests that neutrophils play an important role in the pathogenesis of lupus. The goal of this study was to characterize the epigenetic architecture, by studying the DNA methylome, of neutrophils and low density granulocytes (LDGs) in lupus patients. We studied 15 lupus patients and 15 healthy age, sex, and ethnicity matched controls. Genome-wide DNA methylation was assessed using the Illumina HumanMethylation 450 BeadChip array, which includes over 485,000 methylation sites across the entire genome. Bisulfite DNA sequencing was used to validate the array results. Statistical and bioinformatic analysis was performed to identify and characterize differentially methylated loci and genes. We identified 293 differentially methylated CG sites in neutrophils between lupus patients and controls. The majority (68%) of differentially methylated CG sites were hypomethylated in lupus neutrophils compared to controls, suggesting overall hypomethylation. We found a robust and consistent demethylation of interferon signature genes in lupus neutrophils, and similar demethylation in the same genes in autologous LDGs. Indeed, the DNA methylome in lupus neutrophils and LDGs was almost identical, suggesting similar chromatin architecture in the two granulocyte subsets. A notable exception was the hypomethylation of a CG site in the promoter region of the cytoskeleton-regulating gene RAC1 in LDGs. Our findings demonstrate a pattern of robust demethylation of interferon signature genes in lupus patients supporting a pathogenic role for neutrophils in lupus. We suggest a model whereby DNA from lupus neutrophils and LDGs externalized by NETosis enhance type-I IFN production via TLR-9 stimulation by hypomethylated DNA.
Subject(s)
Epigenesis, Genetic , Interferon Type I/genetics , Lupus Erythematosus, Cutaneous/genetics , Neutrophils/immunology , Adult , Chromatin/metabolism , DNA Methylation , Epigenomics , Female , Gene Expression Profiling , Humans , Microarray Analysis , Middle Aged , Up-Regulation , Young AdultABSTRACT
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.
Subject(s)
Amidines/pharmacology , Extracellular Traps/metabolism , Hydrolases/antagonists & inhibitors , Kidney Diseases/prevention & control , Lupus Erythematosus, Systemic/drug therapy , Vascular Diseases/prevention & control , Animals , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Kidney Diseases/etiology , Lupus Erythematosus, Systemic/complications , Lupus Erythematosus, Systemic/immunology , Mice , Mice, Inbred MRL lpr , Protein-Arginine Deiminases , Vascular Diseases/etiologyABSTRACT
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.
Subject(s)
Endothelium, Vascular/metabolism , Endothelium, Vascular/physiopathology , Extracellular Traps/metabolism , Lupus Erythematosus, Systemic/metabolism , Lupus Erythematosus, Systemic/physiopathology , Matrix Metalloproteinase 2/metabolism , Animals , Aorta/pathology , Arthritis, Rheumatoid , Autoantibodies/immunology , Autoantibodies/metabolism , Cells, Cultured , Endothelium, Vascular/pathology , Female , Granulocytes/metabolism , Granulocytes/pathology , Humans , Lupus Erythematosus, Systemic/pathology , Matrix Metalloproteinase 9/immunology , Matrix Metalloproteinase 9/metabolism , Mice , Mice, Inbred C57BL , Models, Animal , Umbilical Veins/pathology , Up-RegulationABSTRACT
RATIONALE: Neutrophil extracellular trap (NET) formation promotes vascular damage, thrombosis, and activation of interferon-α-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-α 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-α 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.
Subject(s)
Atherosclerosis/prevention & control , Enzyme Inhibitors/therapeutic use , Hydrolases/antagonists & inhibitors , Immunity, Innate/drug effects , Ornithine/analogs & derivatives , Animals , Aortic Diseases/drug therapy , Aortic Diseases/etiology , Aortic Diseases/pathology , Aortic Diseases/prevention & control , Apolipoproteins E/deficiency , Atherosclerosis/drug therapy , Atherosclerosis/enzymology , Atherosclerosis/etiology , Atherosclerosis/immunology , Atherosclerosis/pathology , Autoantibodies/biosynthesis , Autoantibodies/immunology , Citrulline/analysis , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Drug Evaluation, Preclinical , Enzyme Inhibitors/pharmacology , Extracellular Space , Histones/metabolism , Hydrolases/physiology , Interferon-alpha/biosynthesis , Interferon-alpha/genetics , L-Selectin/analysis , Lipids/blood , Mice , Mice, Inbred C57BL , Mice, Knockout , Neutropenia/immunology , Neutrophils/drug effects , Neutrophils/immunology , Neutrophils/ultrastructure , Ornithine/pharmacology , Ornithine/therapeutic use , Photochemical Processes , Protein Processing, Post-Translational/drug effects , Protein-Arginine Deiminase Type 4 , Receptor, Interferon alpha-beta/deficiency , Sinus of Valsalva/pathology , Tunica Intima/pathologyABSTRACT
OBJECTIVE: Systemic lupus erythematosus (SLE) is a systemic autoimmune syndrome associated with organ damage and an elevated risk of cardiovascular disease resulting from activation of both innate and adaptive immune pathways. Recently, increased activation of the inflammasome machinery in SLE has been described. Using the mouse model of pristane-induced lupus, we undertook this study to explore whether caspase 1, the central enzyme of the inflammasome, plays a role in the development of SLE and its associated vascular dysfunction. METHODS: Eight-week-old wild-type (WT) or caspase 1(-/-) mice were injected intraperitoneally with phosphate buffered saline or pristane. Six months after injection, mice were euthanized, and the development of a lupus phenotype and vascular dysfunction was assessed. RESULTS: While WT mice exposed to pristane developed autoantibodies and a strong type I interferon response, mice lacking caspase 1 were significantly protected against these features as well as against pristane-induced vascular dysfunction. Further, the development of immune complex glomerulonephritis, which was prominent after pristane exposure in WT mice, was significantly abrogated in caspase 1(-/-) mice. CONCLUSION: These results indicate that caspase 1 is an essential component in the development of lupus and its associated vascular dysfunction and that it may play an important role in the cross-talk between environmental exposures and autoimmunity development, thus identifying a novel pathway for therapeutic targeting.
Subject(s)
Autoantibodies/immunology , Caspase 1/immunology , Endothelium, Vascular/physiology , Interferon Type I/immunology , Lupus Erythematosus, Systemic/immunology , Vascular Diseases/immunology , Vasodilation/physiology , Animals , Cytokines/immunology , Disease Models, Animal , Glomerulonephritis/immunology , Immune Complex Diseases/immunology , Immunosuppressive Agents , Inflammasomes/immunology , Lupus Erythematosus, Systemic/chemically induced , Lupus Erythematosus, Systemic/complications , Mice , Mice, Knockout , Terpenes , Vascular Diseases/etiology , Vascular Diseases/physiopathologyABSTRACT
OBJECTIVES: SSc is associated with an increased prevalence of atherosclerosis (ATS). This study assessed the prevalence of subclinical ATS as measured by carotid US and explored serum proteins to identify potential biomarkers of SSc-ATS. METHODS: Forty-six SSc female patients and 46 age- and ethnicity-matched controls underwent carotid US to assess the presence of plaque and carotid intima media thickness (CIMT). Abstracted data included demographics, ATS risk factors and serum measurements [cholesterol, proinflammatory high-density lipoprotein (piHDL), CRP, lipoproteins]. Serum cytokines/proteins analyses included circulating type I IFN activity by quantifying IFN-inducible genes, soluble junctional adhesion molecule A (sJAM-A) and 100 serum proteins by using a microplate-based multiplex platform. Proteins significant at P < 0.05 on bivariate analyses for the presence of plaque were used to develop a composite measure. RESULTS: Patients with SSc had more plaque (45.6% vs 19.5%, P = 0.01) but similar CIMT compared with controls. Multiplex analysis detected significant associations between serum proteins of inflammation, vasculopathy and fibrosis with ATS in SSc, including IL-2, IL-6, CRP, keratinocyte growth factor, intercellular adhesion molecule 1, endoglin, plasminogen activator inhibitor 1 and insulin-like growth factor binding protein 3 associated with carotid plaque. Myeloid progenitor inhibitory factor 1, serum amyloid A, thrombomodulin, N-terminal pro-brain natriuretic peptide (BNP), and Clara cell secretory protein 16 kD correlated with CIMT. The median composite score for the plaque group was 6 and for the no plaque group it was 2 (P < 0.0001). CONCLUSION: Patients with SSc have a higher prevalence of carotid plaque than matched controls, and patients with SSc-plaque vs patients without plaque have elevated serum proteins implicated in both vasculopathy and fibrosis. Further studies are needed to evaluate the role of these proteins in SSc compared with healthy controls.
Subject(s)
Carotid Artery Diseases/complications , Plaque, Atherosclerotic/complications , Scleroderma, Systemic/complications , Adult , Antigens, CD/blood , Asymptomatic Diseases , Biomarkers , C-Reactive Protein/metabolism , Carotid Arteries/diagnostic imaging , Carotid Artery Diseases/blood , Carotid Artery Diseases/diagnostic imaging , Carotid Intima-Media Thickness , Case-Control Studies , Chemokines, CC/blood , Cross-Sectional Studies , Endoglin , Female , Fibroblast Growth Factor 7/blood , Humans , Insulin-Like Growth Factor Binding Protein 3/blood , Intercellular Adhesion Molecule-1/blood , Interleukin-2/blood , Interleukin-6/blood , Middle Aged , Natriuretic Peptide, Brain/blood , Peptide Fragments/blood , Plaque, Atherosclerotic/blood , Plaque, Atherosclerotic/diagnostic imaging , Plasminogen Activator Inhibitor 1/blood , Receptors, Cell Surface/blood , Risk Factors , Scleroderma, Systemic/blood , Scleroderma, Systemic/diagnostic imaging , Serum Amyloid A Protein , Thrombomodulin/blood , Uteroglobin/bloodABSTRACT
PPAR-γ agonists can suppress autoimmune responses and renal inflammation in murine lupus but the mechanisms implicated in this process remain unclear. We tested the effect of the PPAR-γ agonist pioglitazone in human lupus and control PBMCs with regard to gene regulation and various functional assays. By Affymetrix microarray analysis, several T cell-related pathways were significantly highlighted in pathway analysis in lupus PBMCs. Transcriptional network analysis showed IFN-γ as an important regulatory node, with pioglitazone treatment inducing transcriptional repression of various genes implicated in T cell responses. Confirmation of these suppressive effects was observed specifically in purified CD4+ T cells. Pioglitazone downregulated lupus CD4+ T cell effector proliferation and activation, while it significantly increased proliferation and function of lupus T regulatory cells. We conclude that PPAR-γ agonists selectively modulate CD4+ T cell function in SLE supporting the concept that pioglitazone and related,-agents should be explored as potential therapies in this disease.
Subject(s)
Hypoglycemic Agents/pharmacology , Lupus Erythematosus, Systemic/immunology , PPAR gamma/agonists , T-Lymphocyte Subsets/drug effects , T-Lymphocytes/drug effects , Thiazolidinediones/pharmacology , Cell Proliferation/drug effects , Cells, Cultured , DNA/metabolism , Gene Expression Profiling , Gene Expression Regulation/drug effects , Humans , Immunoglobulin G/immunology , Leukocytes, Mononuclear/cytology , Oligonucleotide Array Sequence Analysis , Pioglitazone , T-Lymphocyte Subsets/immunology , T-Lymphocytes/immunologyABSTRACT
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.
Subject(s)
Cardiovascular Diseases/prevention & control , Hydrolases/antagonists & inhibitors , Ornithine/analogs & derivatives , Animals , Autoantibodies/blood , Bone Marrow Cells/physiology , Cardiovascular Diseases/enzymology , Cardiovascular Diseases/etiology , Cardiovascular Diseases/immunology , Carotid Artery Thrombosis/prevention & control , Cell Differentiation , Cells, Cultured , Disease Models, Animal , Endothelium/pathology , Female , Humans , Injections, Subcutaneous , Lupus Erythematosus, Systemic/complications , Lupus Erythematosus, Systemic/drug therapy , Lupus Erythematosus, Systemic/enzymology , Lupus Erythematosus, Systemic/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Neutrophils/drug effects , Neutrophils/immunology , Ornithine/administration & dosage , Phenotype , Protein-Arginine Deiminases , Stem Cells/physiology , Vasodilation/drug effectsABSTRACT
OBJECTIVE: Patients with systemic lupus erythematosus (SLE) have a notable increase in atherothrombotic cardiovascular disease (CVD) which is not explained by the Framingham risk equation. In vitro studies indicate that type I interferons (IFNs) may play prominent roles in increased CV risk in SLE. However, the in vivo relevance of these findings, with regard to the development of CVD, has not been characterized. This study was undertaken to examine the role of type I IFNs in endothelial dysfunction, aberrant vascular repair, and atherothrombosis in murine models of lupus and atherosclerosis. METHODS: Lupus-prone New Zealand mixed 2328 (NZM) mice and atherosclerosis-prone apolipoprotein E- knockout (apoE(-/-) ) mice were compared to mice lacking type I IFN receptor (INZM and apoE(-/-) IFNAR(-/-) mice, respectively) with regard to endothelial vasodilatory function, endothelial progenitor cell (EPC) function, in vivo neoangiogenesis, plaque development, and occlusive thrombosis. Similar experiments were performed using NZM and apoE(-/-) mice exposed to an IFNα-containing or empty adenovirus. RESULTS: Loss of type I IFN receptor signaling improved endothelium-dependent vasorelaxation, lipoprotein parameters, EPC numbers and function, and neoangiogenesis in lupus-prone mice, independent of disease activity or sex. Further, acute exposure to IFNα impaired endothelial vasorelaxation and EPC function in lupus-prone and non-lupus-prone mice. Decreased atherosclerosis severity and arterial inflammatory infiltrates and increased neoangiogenesis were observed in apoE(-/-) IFNAR(-/-) mice, compared to apoE(-/-) mice, while NZM and apoE(-/-) mice exposed to IFNα developed accelerated thrombosis and platelet activation. CONCLUSION: These results support the hypothesis that type I IFNs play key roles in the development of premature CVD in SLE and, potentially, in the general population, through pleiotropic deleterious effects on the vasculature.
Subject(s)
Atherosclerosis/metabolism , Interferon Type I/metabolism , Lupus Erythematosus, Systemic/metabolism , Plaque, Atherosclerotic/metabolism , Thrombosis/metabolism , Wound Healing/physiology , Animals , Atherosclerosis/physiopathology , Disease Models, Animal , Disease Progression , Endothelial Cells/metabolism , Endothelium, Vascular/metabolism , Female , Lupus Erythematosus, Systemic/physiopathology , Male , Mice , Vasodilation/physiologyABSTRACT
BACKGROUND: Systemic lupus erythematosus (SLE) patients have a striking increase in cardiovascular (CV) comorbidity not fully explained by the Framingham risk score. Recent evidence from in vitro studies suggests that type I interferons (IFN) could promote premature CV disease (CVD) in SLE. We assessed the association of type I IFN signatures with functional and anatomical evidence of vascular damage, and with biomarkers of CV risk in a cohort of lupus patients without overt CVD. METHODOLOGY/PRINCIPAL FINDINGS: Serum type I IFN activity (induction of five IFN-inducible genes; IFIGs) from 95 SLE patient and 38 controls was quantified by real-time PCR. Flow mediated dilatation (FMD) of the brachial artery and carotid intima media thickness (CIMT) were quantified by ultrasound, and coronary calcification by computed tomography. Serum vascular biomarkers were measured by ELISA. We evaluated the effect of type I IFNs on FMD, CIMT and coronary calcification by first applying principal components analysis to combine data from five IFIGs into summary components that could be simultaneously modeled. Three components were derived explaining 97.1% of the total IFIG variation. Multivariable linear regression was utilized to investigate the association between the three components and other covariates, with the outcomes of FMD and CIMT; zero-inflated Poisson regression was used for modeling of coronary calcification. After controlling for traditional CV risk factors, enhanced serum IFN activity was significantly associated with decreased endothelial function in SLE patients and controls (p<0.05 for component 3), increased CIMT among SLE patients (p<0.01 for components 1 and 2), and severity of coronary calcification among SLE patients (p<0.001 for component 3). CONCLUSIONS: Type I IFNs are independently associated with atherosclerosis development in lupus patients without history of overt CVD and after controlling for Framingham risk factors. This study further supports the hypothesis that type I IFNs promote premature vascular damage in SLE.
Subject(s)
Cardiovascular Diseases/blood , Cardiovascular Diseases/etiology , Interferon Type I/blood , Lupus Erythematosus, Systemic/blood , Lupus Erythematosus, Systemic/complications , Adult , Biomarkers/blood , Cardiovascular Diseases/diagnosis , Cardiovascular Diseases/genetics , Carotid Intima-Media Thickness , Case-Control Studies , Cohort Studies , Female , Humans , Lupus Erythematosus, Systemic/genetics , Male , Middle Aged , Regression Analysis , Risk Factors , Vascular Calcification/diagnosis , Vascular Calcification/etiologyABSTRACT
An abnormal neutrophil subset has been identified in the PBMC fractions from lupus patients. We have proposed that these low-density granulocytes (LDGs) play an important role in lupus pathogenesis by damaging endothelial cells and synthesizing increased levels of proinflammatory cytokines and type I IFNs. To directly establish LDGs as a distinct neutrophil subset, their gene array profiles were compared with those of autologous normal-density neutrophils and control neutrophils. LDGs significantly overexpress mRNA of various immunostimulatory bactericidal proteins and alarmins, relative to lupus and control neutrophils. In contrast, gene profiles of lupus normal-density neutrophils do not differ from those of controls. LDGs have heightened capacity to synthesize neutrophils extracellular traps (NETs), which display increased externalization of bactericidal, immunostimulatory proteins, and autoantigens, including LL-37, IL-17, and dsDNA. Through NETosis, LDGs have increased capacity to kill endothelial cells and to stimulate IFN-α synthesis by plasmacytoid dendritic cells. Affected skin and kidneys from lupus patients are infiltrated by netting neutrophils, which expose LL-37 and dsDNA. Tissue NETosis is associated with increased anti-dsDNA in sera. These results expand the potential pathogenic roles of aberrant lupus neutrophils and suggest that dysregulation of NET formation and its subsequent responses may play a prominent deleterious role.