Differential pulmonary toxicity and autoantibody formation in genetically distinct mouse strains following combined exposure to silica and diesel exhaust particles.

BACKGROUND: Inhalation of airborne particulate matter, such as silica and diesel exhaust particles, poses serious long-term respiratory and systemic health risks. Silica exposure can lead to silicosis and systemic autoimmune diseases, while DEP exposure is linked to asthma and cancer. Combined exposure to silica and DEP, common in mining, may have more severe effects. This study investigates the separate and combined effects of occupational-level silica and ambient-level DEP on lung injury, inflammation, and autoantibody formation in two genetically distinct mouse strains, thereby aiming at understanding the interplay between genetic susceptibility, particulate exposure, and disease outcomes. Silica and diesel exhaust particles were administered to mice via oropharyngeal aspiration. Assessments of lung injury and host response included in vivo lung micro-computed tomography, lung function tests, bronchoalveolar lavage fluid analysis including inflammatory cytokines and antinuclear antibodies, and histopathology with particle colocalization. RESULTS: The findings highlight the distinct effects of silica and diesel exhaust particles (DEP) on lung injury, inflammation, and autoantibody formation in C57BL/6J and NOD/ShiLtJ mice. Silica exposure elicited a well-established inflammatory response marked by inflammatory infiltrates, release of cytokines, and chemokines, alongside mild fibrosis, indicated by collagen deposition in the lungs of both C57BL/6J and NOD/ShilLtJ mice. Notably, these strains exhibited divergent responses in terms of respiratory function and lung volumes, as assessed through micro-computed tomography. Additionally, silica exposure induced airway hyperreactivity and elevated antinuclear antibody levels in bronchoalveolar lavage fluid, particularly prominent in NOD/ShiLtJ mice. Moreover, antinuclear antibodies correlated with extent of lung inflammation in NOD/ShiLTJ mice. Lung tissue analysis revealed DEP loaded macrophages and co-localization of silica and DEP particles. However, aside from contributing to airway hyperreactivity specifically in NOD/ShiLtJ mice, the ambient-level DEP did not significantly amplify the effects induced by silica. There was no evidence of synergistic or additive interaction between these specific doses of silica and DEP in inducing lung damage or inflammation in either of the mouse strains. CONCLUSION: Mouse strain variations exerted a substantial influence on the development of silica induced lung alterations. Furthermore, the additional impact of ambient-level DEP on these silica-induced effects was minimal.

Reduced miR-99a-3p levels in systemic lupus erythematosus may promote B cell proliferation via NCAPG and the PI3K/AKT signaling pathway.

BACKGROUND: Systemic lupus erythematosus is an immunologically dysregulated disease characterized by the presence of multiple autoantibodies. In SLE, B lymphocytes contribute to the dysregulated production of autoantibodies and cytokines. Recently, we discovered that miR-99a-3p binds to both EIF4EBP1 and NCAPG mRNA and that lowering miR-99a-3p can promote B cell autophagy in SLE by increasing EIF4EBP1 expression. However, the functions of miR-99a-3p and NCAPG in SLE have not been extensively investigated. OBJECTIVE: This work aims to evaluate the levels of miR-99a-3p and NCAPG expression in SLE B cells and to determine whether the aberrant expression of miR-99a-3p and NCAPG contributes to the pathological mechanisms in SLE. METHODS: B lymphocytes were obtained through immunomagnetic negative selection. Using RT-qPCR, miR-99a-3p and NCAPG mRNA expressions in B lymphocytes and in the BALL-1 cell line were measured. To determine the relative abundance of NCAPG, PI3K, p-PI3K, AKT, and p-AKT, we normalize them to the level of ß-actin using Western blotting. Evaluation of miR-99a-3p and NCAPG's impact on cell proliferation was done utilizing CCK-8 assay. Using flow cytometry, the cell cycle and apoptosis were both measured. RESULTS: Comparing SLE B cells to healthy controls, miR-99a-3p expression was significantly downregulated. Additionally, it was observed that SLE B cells had significantly higher NCAPG mRNA expression. Blocking miR-99a-3p expression in BALL-1 cells with an antagomir elevated NCAPG expression, facilitated PI3K/AKT pathway activation, improved cell proliferation, raised the fraction of S-phase cells, and prevented cell apoptosis. The opposite effects of upregulated miR-99a-3p levels on BALL-1 cells were observed by using an agomir. Furthermore, the effect of decreased miR-99a-3p expression on cell proliferation was partially mediated by elevating NCAPG levels and activating the PI3K/AKT pathway. CONCLUSION: Our research indicates that lower miR-99a-3p expression in SLE B cells appears to boost B cell number via the NCAPG and PI3K/AKT pathways.

Comparative Effects of Domain-Specific Human Monoclonal Antibodies Against LGI1 on Neuronal Excitability.

BACKGROUND AND OBJECTIVES: Autoantibodies to leucine-rich glioma inactivated protein 1 (LGI1) cause an autoimmune limbic encephalitis with frequent focal seizures and anterograde memory dysfunction. LGI1 is a neuronal secreted linker protein with 2 functional domains: the leucine-rich repeat (LRR) and epitempin (EPTP) regions. LGI1 autoantibodies are known to interfere with presynaptic function and neuronal excitability; however, their epitope-specific mechanisms are incompletely understood. METHODS: We used patient-derived monoclonal autoantibodies (mAbs), which target either LRR or EPTP domains of LGI1 to investigate long-term antibody-induced alteration of neuronal function. LRR- and EPTP-specific effects were evaluated by patch-clamp recordings in cultured hippocampal neurons and compared with biophysical neuron modeling. Kv1.1 channel clustering at the axon initial segment (AIS) was quantified by immunocytochemistry and structured illumination microscopy techniques. RESULTS: Both EPTP and LRR domain-specific mAbs decreased the latency of first somatic action potential firing. However, only the LRR-specific mAbs increased the number of action potential firing together with enhanced initial instantaneous frequency and promoted spike-frequency adaptation, which were less pronounced after the EPTP mAb. This also led to an effective reduction in the slope of ramp-like depolarization in the subthreshold response, suggesting Kv1 channel dysfunction. A biophysical model of a hippocampal neuron corroborated experimental results and suggests that an isolated reduction of the conductance of Kv1-mediated K+ currents largely accounts for the antibody-induced alterations in the initial firing phase and spike-frequency adaptation. Furthermore, Kv1.1 channel density was spatially redistributed from the distal toward the proximal site of AIS under LRR mAb treatment and, to a lesser extant, under EPTP mAb. DISCUSSION: These findings indicate an epitope-specific pathophysiology of LGI1 autoantibodies. The pronounced neuronal hyperexcitability and SFA together with dropped slope of ramp-like depolarization after LRR-targeted interference suggest disruption of LGI1-dependent clustering of K+ channel complexes. Moreover, considering the effective triggering of action potentials at the distal AIS, the altered spatial distribution of Kv1.1 channel density may contribute to these effects through impairing neuronal control of action potential initiation and synaptic integration.

Astrocyte-Derived Exosomes Contribute to Pathologies of Neuromyelitis Optica Spectrum Disorder in Rodent Model.

OBJECTIVE: Neuromyelitis optica spectrum disorder (NMOSD) is an inflammatory demyelinating disease that leads to severe disability. A large proportion of NMOSD patients are seropositive for aquaporin-4 autoantibodies (AQP4-IgG, named as NMO-IgG) targeting AQP4, which is selectively expressed on astrocytes in the central nervous system. This study tests the hypothesis that in response to NMO-IgG, the pathogenic astrocyte-derived exosomes are released and injure the neighboring cells. METHODS: IgG purified from serum of either NMOSD patients or healthy controls was used to generate astrocyte-derived exosomes (AST-ExosNMO vs AST-ExosCON ) in cultured rat astrocytes. The exosomes were respectively delivered to cultured rat oligodendrocytes in vitro, tissue culture of rat optic nerve ex vivo, and rat optic nerve in vivo to evaluate the pathogenic roles of AST-ExosNMO . The microRNA (miRNA) sequencing of AST-Exos and verification were performed to identify the key pathogenic miRNA. The custom-designed adeno-associated virus (AAV) antagonizing the key miRNA was evaluated for its therapeutic effects in vivo. Moreover, the serum levels of the key exosomal miRNA were measured between NMOSD patients and healthy controls. RESULTS: AST-ExosNMO led to notable demyelination in both cultured oligodendrocytes and optic nerve tissue. Exosomal miR-129-2-3p was identified as the key miRNA mediating the demyelinating pathogenesis via downstream target gene SMAD3. AAV antagonizing miR-129-2-3p protected against demyelination in an NMOSD rodent model. The serum exosomal miR-129-2-3p level was significantly elevated in NMOSD patients and correlated with disease severity. INTERPRETATION: Astrocytes targeted by NMO-IgG release pathogenic exosomes that could potentially be used as therapeutic targets or disease monitoring biomarkers in NMOSD. ANN NEUROL 2023;94:163-181.

IgG Autoantibodies Against IgE from Atopic Dermatitis Can Induce the Release of Cytokines and Proinflammatory Mediators from Basophils and Mast Cells.

IgE-mediated release of proinflammatory mediators and cytokines from basophils and mast cells is a central event in allergic disorders. Several groups of investigators have demonstrated the presence of autoantibodies against IgE and/or FcεRI in patients with chronic spontaneous urticaria. By contrast, the prevalence and functional activity of anti-IgE autoantibodies in atopic dermatitis (AD) are largely unknown. We evaluated the ability of IgG anti-IgE from patients with AD to induce the in vitro IgE-dependent activation of human basophils and skin and lung mast cells. Different preparations of IgG anti-IgE purified from patients with AD and rabbit IgG anti-IgE were compared for their triggering effects on the in vitro release of histamine and type 2 cytokines (IL-4, IL-13) from basophils and of histamine and lipid mediators (prostaglandin D2 and cysteinyl leukotriene C4) from human skin and lung mast cells. One preparation of human IgG anti-IgE out of six patients with AD induced histamine release from basophils, skin and lung mast cells. This preparation of human IgG anti-IgE induced the secretion of cytokines and eicosanoids from basophils and mast cells, respectively. Human monoclonal IgE was a competitive antagonist of both human and rabbit IgG anti-IgE. Human anti-IgE was more potent than rabbit anti-IgE for IL-4 and IL-13 production by basophils and histamine, prostaglandin D2 and leukotriene C4 release from mast cells. Functional anti-IgE autoantibodies rarely occur in patients with AD. When present, they induce the release of proinflammatory mediators and cytokines from basophils and mast cells, thereby possibly contributing to sustained IgE-dependent inflammation in at least a subset of patients with this disorder.

Selective Capture of Anti-N-glucosylated NTHi Adhesin Peptide Antibodies by a Multivalent Dextran Conjugate.

Tentacle-like polymers decorated with several copies of peptide antigens can be interesting tools for increasing the ability to capture circulating antibodies in patient sera, using cooperative effects for stronger avidity. We previously showed that antibodies from multiple sclerosis (MS) patient sera preferentially recognize hyperglucosylated adhesin protein HMW1ct of non-typeable Haemophilus influenzae (NTHi). We selected the C-terminal HMW1ct(1347-1354) minimal epitope and prepared the diglucosylated analogue Ac-KAN(Glc)VTLN(Glc)TTG-K(N3 )-NH2 to graft a 40 kDa dextran scaffold modified with glycidyl-propargyl moieties to perform a copper catalyzed alkyne-azide coupling reaction (CuAAC). Quantitative NMR measurements allowed the characterization of the peptide loading (19.5 %) on the multivalent dextran conjugate. This novel polymeric structure displayed optimal capturing properties of both IgG and, more interestingly, IgM antibodies in MS sera. Specific antibodies from a representative MS serum, were successfully depleted using a Sepharose resin bearing the new glucosylated multivalent conjugate, as confirmed by ELISA. These results may offer a promising proof-of-concept for the selective purification of high affinity autoantibodies from sera of autoimmune patients, in general, and of specific high affinity antibodies against a minimally glcosylated epitope Asn(Glc) from sera of multiple sclerosis (MS) patients, in particular.

Angiotensin II type 1 receptor agonistic autoantibody blockade improves postpartum hypertension and cardiac mitochondrial function in rat model of preeclampsia.

Women with preeclampsia (PE) have a greater risk of developing hypertension, cardiovascular disease (CVD), and renal disease later in life. Angiotensin II type I receptor agonistic autoantibodies (AT1-AAs) are elevated in women with PE during pregnancy and up to 2-year postpartum (PP), and in the reduced uterine perfusion pressure (RUPP) rat model of PE. Blockade of AT1-AA with a specific 7 amino acid peptide binding sequence ('n7AAc') improves pathophysiology observed in RUPP rats; however, the long-term effects of AT1-AA inhibition in PP is unknown. Pregnant Sprague Dawley rats were divided into three groups: normal pregnant (NP) (n = 16), RUPP (n = 15), and RUPP + 'n7AAc' (n = 16). Gestational day 14, RUPP surgery was performed and 'n7AAc' (144 µg/day) administered via osmotic minipump. At 10-week PP, mean arterial pressure (MAP), renal glomerular filtration rate (GFR) and cardiac functions, and cardiac mitochondria function were assessed. MAP was elevated PP in RUPP vs. NP (126 ± 4 vs. 116 ± 3 mmHg, p < 0.05), but was normalized in in RUPP + 'n7AAc' (109 ± 3 mmHg) vs. RUPP (p < 0.05). PP heart size was reduced by RUPP + 'n7AAc' vs. RUPP rats (p < 0.05). Complex IV protein abundance and enzymatic activity, along with glutamate/malate-driven respiration (complexes I, III, and IV), were reduced in the heart of RUPP vs. NP rats which was prevented with 'n7AAc'. AT1-AA inhibition during pregnancy not only improves blood pressure and pathophysiology of PE in rats during pregnancy, but also long-term changes in blood pressure, cardiac hypertrophy, and cardiac mitochondrial function PP.

New BBB Model Reveals That IL-6 Blockade Suppressed the BBB Disorder, Preventing Onset of NMOSD.

BACKGROUND AND OBJECTIVES: To evaluate the pathophysiology of neuromyelitis optica spectrum disorder (NMOSD) and the therapeutic mechanism and levels of interleukin-6 (IL-6) blockade (satralizumab), especially with respect to blood-brain barrier (BBB) disruption with the new in vitro and ex vivo human BBB models and in vivo model. METHODS: We constructed new static in vitro and flow-based ex vivo models for evaluating continued barrier function, leukocyte transmigration, and intracerebral transferability of neuromyelitis optica-immunoglobulin G (NMO-IgG) and satralizumab across the BBB using the newly established triple coculture system that are specialized to closely mimic endothelial cell contact of pericytes and endfeet of astrocytes. In the in vivo study, we assessed the effects of an anti-IL-6 receptor antibody for mice (MR16-1) on in vivo BBB disruption in mice with experimental autoimmune encephalomyelitis in which IL-6 concentration in the spinal cord dramatically increases. RESULTS: In vitro and ex vivo experiments demonstrated that NMO-IgG increased intracerebral transferability of satralizumab and NMO-IgG and that satralizumab suppressed the NMO-IgG-induced transmigration of T cells and barrier dysfunction. In the in vivo study, the blockade of IL-6 signaling suppressed the migration of T cells into the spinal cord and prevented the increased BBB permeability. DISCUSSION: These results suggest that (1) our triple-cultured in vitro and in ex vivo BBB models are ideal for evaluating barrier function, leukocyte transmigration, and intracerebral transferability; (2) NMO-IgG increased the intracerebral transferability of NMO-IgG via decreasing barrier function and induced secretion of IL-6 from astrocytes causing more dysfunction of the barrier and disrupting controlled cellular infiltration; and (3) satralizumab, which can pass through the BBB in the presence of NMO-IgG, suppresses the BBB dysfunction and the infiltration of inflammatory cells, leading to prevention of onset of NMOSD.

Anti-AQP4 autoantibodies promote ATP release from astrocytes and induce mechanical pain in rats.

BACKGROUND: Intractable neuropathic pain is a common symptom of neuromyelitis optica spectrum disorder (NMOSD). However, the underlying mechanism of NMOSD pain remains to be elucidated. In this study, we focused on ATP, which is one of the damage-associated molecular patterns, and also a well-recognized molecule involved in peripheral neuropathic pain. METHODS: We assessed the development of pain symptoms by injecting anti-AQP4 recombinant autoantibodies (rAQP4 IgG) into rat spinal cords. We incubated HEK293 cells expressing AQP4 (HEK-AQP4) and rat astrocytes with rAQP4 IgG and assessed the level of ATP in the supernatant. We performed transcriptome analysis of the spinal cords injected with rAQP4 IgG. Pharmacological inhibition was also applied to investigate the involvement of ATP in the development of neuropathic pain in our rat model. The ATP concentration within the cerebrospinal fluid was examined in patients with NMOSD and other neurological diseases. RESULTS: Development of mechanical allodynia was confirmed in rAQP4 IgG-treated rats. AQP4-Ab-mediated extracellular ATP release from astrocytes was observed in vitro, and pharmacological inhibition of ATP receptor reversed mechanical allodynia in the rAQP4 IgG-treated rats. Furthermore, transcriptome analysis revealed elevation of gene expressions related to several ATP receptors including P2rx4 and IL1B in the spinal cord of rAQP4 IgG-treated rats. In patients, CSF ATP concentration was significantly higher in the acute and remission phase of NMOSD than in multiple sclerosis or other neurological disorders. CONCLUSION: Anti-AQP4 antibody was shown to induce the release of extracellular ATP from astrocytes. The ATP-mediated development of mechanical allodynia was also suggested in rats treated with anti-AQP4 antibody. Our study indicates the pivotal role of ATP in the pain mechanism of NMOSD.

ENT2 facilitates brain endothelial cell penetration and blood-brain barrier transport by a tumor-targeting anti-DNA autoantibody.

The blood-brain barrier (BBB) prevents antibodies from penetrating the CNS and limits conventional antibody-based approaches to brain tumors. We now show that ENT2, a transporter that regulates nucleoside flux at the BBB, may offer an unexpected path to circumventing this barrier to allow targeting of brain tumors with an anti-DNA autoantibody. Deoxymab-1 (DX1) is a DNA-damaging autoantibody that localizes to tumors and is synthetically lethal to cancer cells with defects in the DNA damage response. We found that DX1 penetrated brain endothelial cells and crossed the BBB, and mechanistic studies identify ENT2 as the key transporter. In efficacy studies, DX1 crosses the BBB to suppress orthotopic glioblastoma and breast cancer brain metastases. ENT2-linked transport of autoantibodies across the BBB has potential to be exploited in brain tumor immunotherapy, and its discovery raises hypotheses on actionable mechanisms of CNS penetration by neurotoxic autoantibodies in CNS lupus.

Agonistic ß2-Adrenergic Receptor Autoantibodies Characterize the Aqueous Humor of Patients With Primary and Secondary Open-Angle Glaucoma.

Purpose: Agonistic ß2-adrenergic receptor autoantibodies (ß2-agAAbs) were recently observed in sera of patients with ocular hypertension (OHT), primary (POAG), and secondary open-angle glaucoma (SOAG), yet not in healthy controls (HCs). It was the aim of the present study to investigate the presence of ß2-agAAb in aqueous humor (AH) samples of OAG patients and to correlate these with the corresponding ß2-agAAb serum data. Material and Methods: Thirty-nine patients (21 male, 18 female) were recruited from the Department of Ophthalmology, University of Erlangen-Nürnberg: twenty-one POAG, 18 SOAG. Aqueous humor samples were collected during minimal invasive glaucoma surgery. Serum and AH samples were analyzed for ß2-agAAb by a bioassay quantifying the beating rate of cultured cardiomyocyte (cut-off: 2 U). Results: Thirty-six of 39 (92.3%) and 34 of 39 (87.2%) of OAG patients showed a ß2-agAAb in their sera and AH samples, respectively. All ß2-agAAb AH-positive OAG patients were also seropositive. We also observed a ß2-agAAb seropositivity in 95 and 89% of patients with POAG and SOAG, respectively. Beta2-agAAbs were seen in 86% (POAG) and 78% (SOAG) of AH samples. The ß2-agAAb adrenergic activity was increased in the AH of patients with POAG (6.5 ± 1.5 U) when compared with those with SOAG (4.1 ± 1.1 U; p = 0.004). Serum ß2-agAAb adrenergic activity did not differ between the cohorts [POAG (4.5 ± 1.5 U); SOAG (4.6 ± 2.1 U; p=0.458)]. No correlation of the beating rates were observed between serum and AH samples for group and subgroup analyses. Conclusion: The detection of ß2-agAAb in systemic and local circulations supports the hypothesis of a direct functional impact of these agAAbs on ocular G-protein coupled receptors. The high prevalence of ß2-agAAb in serum and AH samples of patients with POAG or SOAG suggests a common role of these AAbs in the etiopathogenesis of glaucoma, independent of open-angle glaucoma subtype.

Autoantibody against angiotensin II type I receptor induces pancreatic ß-cell apoptosis via enhancing autophagy.

Autoantibody against the angiotensin II type I receptor (AT1-AA) has been found in the serum of patients with diabetes mellitus (DM). However, it remains unclear whether AT1-AA induces ß-cell apoptosis and participates in the development of DM. In this study, an AT1-AA-positive rat model was set up by active immunization, and AT1-AA IgG was purified. INS-1 cells were treated with AT1-AA, and cell viability, apoptosis, and autophagy-related proteins were detected by Cell Counting Kit-8 assay, flow cytometry, and western blot analysis, respectively. Results showed that existence of AT1-AA impaired the islet function and increased the apoptosis of pancreatic islet cells in rats, and the autophagy level in rat pancreatic islet tissues tended to increase gradually with the prolongation of immunization time. AT1-AA markedly reduced INS-1 cell viability, promoted cell apoptosis, and decreased insulin secretion in vitro. In addition, the autophagy level was gradually increased along with the prolongation of AT1-AA treatment time. Meanwhile, it was determined that treatment with autophagy inhibitor 3-methyladenine and angiotensin II type 1 receptor (AT1R) blocker telmisartan could improve insulin secretion and apoptosis in vitro and in vivo. In conclusion, it is deduced that upregulation of autophagy contributed to the AT1-AA-induced ß-cell apoptosis and islet dysfunction, and AT1R mediated the signal transduction.

Autoantibodies Targeting AT1- and ETA-Receptors Link Endothelial Proliferation and Coagulation via Ets-1 Transcription Factor.

Scleroderma renal crisis (SRC) is an acute life-threatening manifestation of systemic sclerosis (SSc) caused by obliterative vasculopathy and thrombotic microangiopathy. Evidence suggests a pathogenic role of immunoglobulin G (IgG) targeting G-protein coupled receptors (GPCR). We therefore dissected SRC-associated vascular obliteration and investigated the specific effects of patient-derived IgG directed against angiotensin II type 1 (AT1R) and endothelin-1 type A receptors (ETAR) on downstream signaling events and endothelial cell proliferation. SRC-IgG triggered endothelial cell proliferation via activation of the mitogen-activated protein kinase (MAPK) pathway and subsequent activation of the E26 transformation-specific-1 transcription factor (Ets-1). Either AT1R or ETAR receptor inhibitors/shRNA abrogated endothelial proliferation, confirming receptor activation and Ets-1 signaling involvement. Binding of Ets-1 to the tissue factor (TF) promoter exclusively induced TF. In addition, TF inhibition prevented endothelial cell proliferation. Thus, our data revealed a thus far unknown link between SRC-IgG-induced intracellular signaling, endothelial cell proliferation and active coagulation in the context of obliterative vasculopathy and SRC. Patients' autoantibodies and their molecular effectors represent new therapeutic targets to address severe vascular complications in SSc.

Semaphorin 4A antibody alleviates arsenic-induced hepatotoxicity in mice via inhibition of AKT2/NF-κB inflammatory signaling.

Semaphorin (Sema) 3A and Sema 4A are immunomodulatory molecules with a common receptor, neuropilin-1 (NRP-1), on the immune cells. Sema 3A binds to NRP-1 and inhibits T cell activation and inflammation, while Sema 4A binds to NRP-1 and promotes T cell activation and inflammation. These molecules are associated closely with the regulation of protein kinase B (AKT)/nuclear factor-kappaB (NF-κB) signaling, which are poorly understood in arsenic toxicity. The present study explored the role of Sema 3A or Sema 4A in arsenic-induced hepatotoxicity in mice. Arsenic exposure induced hepatic injury and resulted in the activations of p-AKT2, NF-κB p65, and NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, downregulation of Sema 3A, and upregulation of Sema 4A or NRP-1. Interestingly, intervention with anti-Sema 4A antibody showed the mitigation of arsenic-induced hepatotoxicity, accompanied by the downregulation of Sema 4A, rebound of Sema 3A, and upregulation of NRP-1. And, the inflammatory signaling p-AKT2 or NF-κB p65, and NLRP3 inflammasome showed a downregulation compared with arsenic treatment group. In contrast, anti-Sema 3A antibody intervention did not show the significant effect in the histopathological features compared with arsenic treatment group. In conclusion, the anti-Sema 4A antibody antagonizes arsenic-induced hepatotoxicity in mice and may be involved in the inhibitions of AKT2/NF-κB and NLRP3 inflammatory signaling mediated synergistically by Sema 4A or Sema 3A and their receptor NRP-1.

Effect of NMO-IgG on the interleukin-6 cascade in astrocytes via activation of the JAK/STAT3 signaling pathway.

AIMS: Astrocytes expressing the aquaporin-4 (AQP4) water channel are pathogenic, disease specific immunoglobulins (IgG) found in neuromyelitis optica spectrum disorder (NMOSD), referred to as NMO-IgG, which targets astrocytic AQP4. The interleukin-6 (IL-6) signaling when astrocytes were exposed to NMO-IgG present in the serum of NMOSD patients was evaluated. MAIN METHODS: Serum or human-IgG from NMOSD or healthy controls were exposed to astrocytes. The selectivity and immuno-pathological consequences of Ig binding to surface epitopes were measured by confocal microscopy. Astrocytes were exposed to medium, IL-6, soluble IL-6 receptor (sIL-6R), IL-6 + sIL-6R (IL-6/R), NMO-IgG or control-IgG, NMO-IgG + IL-6/R. The expression of key proteins in IL-6 signaling pathway, IL-6 cytokine and mRNA levels were evaluated by western blotting, enzyme-linked immunosorbent assay and quantitative polymerase chain reaction, respectively. KEY FINDINGS: Serum or NMO-IgG from NMOSD patients both induced the rapid downregulation of AQP4 expression on the surface of astrocytes. Stimulation of astrocytes with NMO-IgG, IL-6/R, and NMO-IgG + IL-6/R resulted in the enhancement of IL-6 mRNA expression. Meanwhile, the exogenous addition of NMO-IgG elicited an inflammatory transcriptional response that involved signaling through the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway. Inhibition of the IL-6/JAK/STAT3 pathway with the JAK1/2 specific inhibitor, AZD1480, reversed the associated increase of IL-6. SIGNIFICANCE: Our findings suggest that NMO-IgG can stimulate the astrocytic JAK1/2/STAT3-dependent inflammatory response, which represents one of the important events in NMO pathogenesis. Inhibition of the JAK1/2 signaling pathway may be a novel promising therapy for NMOSD.

Dynamics of Cardiac Neutrophil Diversity in Murine Myocardial Infarction.

RATIONALE: After myocardial infarction, neutrophils rapidly and massively infiltrate the heart, where they promote both tissue healing and damage. OBJECTIVE: To characterize the dynamics of circulating and cardiac neutrophil diversity after infarction. METHODS AND RESULTS: We employed single-cell transcriptomics combined with cell surface epitope detection by sequencing to investigate temporal neutrophil diversity in the blood and heart after murine myocardial infarction. At day 1, 3, and 5 after infarction, cardiac Ly6G+ (lymphocyte antigen 6G) neutrophils could be delineated into 6 distinct clusters with specific time-dependent patterning and proportions. At day 1, neutrophils were characterized by a gene expression profile proximal to bone marrow neutrophils (Cd177, Lcn2, Fpr1), and putative activity of transcriptional regulators involved in hypoxic response (Hif1a) and emergency granulopoiesis (Cebpb). At 3 and 5 days, 2 major subsets of Siglecfhi (enriched for eg, Icam1 and Tnf) and Siglecflow (Slpi, Ifitm1) neutrophils were found. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) analysis in blood and heart revealed that while circulating neutrophils undergo a process of aging characterized by loss of surface CD62L and upregulation of Cxcr4, heart infiltrating neutrophils acquired a unique SiglecFhi signature. SiglecFhi neutrophils were absent from the bone marrow and spleen, indicating local acquisition of the SiglecFhi signature. Reducing the influx of blood neutrophils by anti-Ly6G treatment increased proportions of cardiac SiglecFhi neutrophils, suggesting accumulation of locally aged neutrophils. Computational analysis of ligand/receptor interactions revealed putative pathways mediating neutrophil to macrophage communication in the myocardium. Finally, SiglecFhi neutrophils were also found in atherosclerotic vessels, revealing that they arise across distinct contexts of cardiovascular inflammation. CONCLUSIONS: Altogether, our data provide a time-resolved census of neutrophil diversity and gene expression dynamics in the mouse blood and ischemic heart at the single-cell level, and reveal a process of local tissue specification of neutrophils in the ischemic heart characterized by the acquisition of a SiglecFhi signature.

NMDAR Antibodies Alter Dopamine Receptors and Cause Psychotic Behavior in Mice.

OBJECTIVE: The aim was to demonstrate that antibodies from patients with anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis alter the levels of dopamine 1 receptor (D1R) and dopamine 2 receptor (D2R) and cause psychotic-like features in mice. METHODS: Cultured rat hippocampal neurons were treated with cerebrospinal fluid (CSF) from patients with anti-NMDAR encephalitis or controls, and the effects on clusters of D1R and D2R were quantified. In vivo studies included 71 C57BL/6J mice that were chronically infused with CSF from patients or controls through ventricular catheters connected to subcutaneous osmotic pumps. Prepulse inhibition of the acoustic startling reflex (PPI; a marker of psychotic-like behavior), memory, locomotor activity, and the density of cell-surface and synaptic D1R, D2R, and NMDAR clusters were examined at different time points using reported techniques. RESULTS: In cultured neurons, CSF from patients, but not from controls, caused a significant decrease of cell-surface D1R and an increase of D2R clusters. In mice, CSF from patients caused a significant decrease of synaptic and total cell-surface D1R clusters and an increase of D2R clusters associated with a decrease of PPI. These effects were accompanied by memory impairment and a reduction of surface NMDARs, as reported in this model. The psychotic-like features, memory impairment, and changes in levels of D1R, D2R, and NMDAR progressively improved several days after the infusion of CSF from patients stopped. INTERPRETATION: In addition to memory deficits and reduction of NMDARs, CSF antibodies from patients with anti-NMDAR encephalitis cause reversible psychotic-like features accompanied by changes (D1R decrease, D2R increase) in cell-surface dopamine receptor clusters. ANN NEUROL 2020 ANN NEUROL 2020;88:603-613.

Increased AT2R expression is induced by AT1R autoantibody via two axes, Klf-5/IRF-1 and circErbB4/miR-29a-5p, to promote VSMC migration.

Vascular remodeling can be caused by angiotensin II type 1 receptor (AT1R) autoantibody (AT1-AA), although the related mechanism remains unknown. Angiotensin II type 2 receptor (AT2R) plays multiple roles in vascular remodeling through cross-talk with AT1R in the cytoplasm. Here, we aimed to explore the role and mechanism of AT2R in AT1-AA-induced vascular smooth muscle cell (VSMC) migration, which is a key event in vascular remodeling. In vitro and in vivo, we found that AT2R can promote VSMC migration in AT1-AA-induced vascular remodeling. Moreover, AT2R expression was upregulated via Klf-5/IRF-1-mediated transcriptional and circErbB4/miR-29a-5p-mediated posttranscriptional mechanisms in response to AT1-AA. Our data provide a molecular basis for AT1-AA-induced AT2R expression by transcription factors, namely, a circular RNA and a microRNA, and showed that AT2R participated in AT1-AA-induced VSMC migration during the development of vascular remodeling. AT2R may be a potential target for the treatment of AT1-AA-induced vascular diseases.

N-Methyl-D-Aspartate Receptor Antibodies in Autoimmune Encephalopathy Alter Oligodendrocyte Function.

OBJECTIVE: Antibodies against neuronal N-methyl-D-aspartate receptors (NMDARs) in patients with anti-NMDAR encephalitis alter neuronal synaptic function and plasticity, but the effects on other cells of the nervous system are unknown. METHODS: Cerebrospinal fluid (CSF) of patients with anti-NMDAR encephalitis (preabsorbed or not with GluN1) and a human NMDAR-specific monoclonal antibody (SSM5) derived from plasma cells of a patient, along the corresponding controls, were used in the studies. To evaluate the activity of oligodendrocyte NMDARs and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in vitro after exposure to patients' CSF antibodies or SSM5, we used a functional assay based on cytosolic Ca2+ imaging. Expression of the glucose transporter (GLUT1) in oligodendrocytes was assessed by immunocytochemistry. RESULTS: NMDAR agonist responses were robustly reduced after preincubation of oligodendrocytes with patients' CSF or SSM5 but remained largely unaltered with the corresponding controls. These effects were NMDAR specific, as patients' CSF did not alter responses to AMPA receptor agonists and was abrogated by preabsorption of CSF with HEK cells expressing GluN1 subunit. Patients' CSF also reduced oligodendrocyte expression of glucose transporter GLUT1 induced by NMDAR activity. INTERPRETATION: Antibodies from patients with anti-NMDAR encephalitis specifically alter the function of NMDARs in oligodendrocytes, causing a decrease of expression of GLUT1. Considering that normal GLUT1 expression in oligodendrocytes and myelin is needed to metabolically support axonal function, the findings suggest a link between antibody-mediated dysfunction of NMDARs in oligodendrocytes and the white matter alterations reported in patients with this disorder. ANN NEUROL 2020;87:670-676.

Human Cerebrospinal Fluid Monoclonal LGI1 Autoantibodies Increase Neuronal Excitability.

OBJECTIVE: Leucine-rich glioma-inactivated 1 (LGI1) encephalitis is the second most common antibody-mediated encephalopathy, but insight into the intrathecal B-cell autoimmune response, including clonal relationships, isotype distribution, frequency, and pathogenic effects of single LGI1 antibodies, has remained limited. METHODS: We cloned, expressed, and tested antibodies from 90 antibody-secreting cells (ASCs) and B cells from the cerebrospinal fluid (CSF) of several patients with LGI1 encephalitis. RESULTS: Eighty-four percent of the ASCs and 21% of the memory B cells encoded LGI1-reactive antibodies, whereas reactivities to other brain epitopes were rare. All LGI1 antibodies were of IgG1, IgG2, or IgG4 isotype and had undergone affinity maturation. Seven of the overall 26 LGI1 antibodies efficiently blocked the interaction of LGI1 with its receptor ADAM22 in vitro, and their mean LGI1 signal on mouse brain sections was weak compared to the remaining, non-ADAM22-competing antibodies. Nevertheless, both types of LGI1 antibodies increased the intrinsic cellular excitability and glutamatergic synaptic transmission of hippocampal CA3 neurons in slice cultures. INTERPRETATION: Our data show that the patients' intrathecal B-cell autoimmune response is dominated by LGI1 antibodies and that LGI1 antibodies alone are sufficient to promote neuronal excitability, a basis of seizure generation. Fundamental differences in target specificity and antibody hypermutations compared to the CSF autoantibody repertoire in N-methyl-D-aspartate receptor encephalitis underline the clinical concept that autoimmune encephalitides are very distinct entities. Ann Neurol 2020;87:405-418.