LGI1 Autoantibodies Enhance Synaptic Transmission by Presynaptic Kv1 Loss and Increased Action Potential Broadening.

BACKGROUND AND OBJECTIVES: Autoantibodies against the protein leucine-rich glioma inactivated 1 (LGI1) cause the most common subtype of autoimmune encephalitis with predominant involvement of the limbic system, associated with seizures and memory deficits. LGI1 and its receptor ADAM22 are part of a transsynaptic protein complex that includes several proteins involved in presynaptic neurotransmitter release and postsynaptic glutamate sensing. Autoantibodies against LGI1 increase excitatory synaptic strength, but studies that genetically disrupt the LGI1-ADAM22 complex report a reduction in postsynaptic glutamate receptor-mediated responses. Thus, the mechanisms underlying the increased synaptic strength induced by LGI1 autoantibodies remain elusive, and the contributions of presynaptic molecules to the LGI1-transsynaptic complex remain unclear. We therefore investigated the presynaptic mechanisms that mediate autoantibody-induced synaptic strengthening. METHODS: We studied the effects of patient-derived purified polyclonal LGI1 autoantibodies on synaptic structure and function by combining direct patch-clamp recordings from presynaptic boutons and somata of hippocampal neurons with super-resolution light and electron microscopy of hippocampal cultures and brain slices. We also identified the protein domain mediating the presynaptic effect using domain-specific patient-derived monoclonal antibodies. RESULTS: LGI1 autoantibodies dose-dependently increased short-term depression during high-frequency transmission, consistent with increased release probability. The increased neurotransmission was not related to presynaptic calcium channels because presynaptic Cav2.1 channel density, calcium current amplitude, and calcium channel gating were unaffected by LGI1 autoantibodies. By contrast, application of LGI1 autoantibodies homogeneously reduced Kv1.1 and Kv1.2 channel density on the surface of presynaptic boutons. Direct presynaptic patch-clamp recordings revealed that LGI1 autoantibodies cause a pronounced broadening of the presynaptic action potential. Domain-specific effects of LGI1 autoantibodies were analyzed at the neuronal soma. Somatic action potential broadening was induced by polyclonal LGI1 autoantibodies and patient-derived monoclonal autoantibodies targeting the epitempin domain, but not the leucin-rich repeat domain. DISCUSSION: Our results indicate that LGI1 autoantibodies reduce the density of both Kv1.1 and Kv1.2 on presynaptic boutons, without actions on calcium channel density or function, thereby broadening the presynaptic action potential and increasing neurotransmitter release. This study provides a molecular explanation for the neuronal hyperactivity observed in patients with LGI1 autoantibodies.

NMDA receptor autoantibodies primarily impair the extrasynaptic compartment.

Autoantibodies directed against the N-methyl-D-aspartate receptor (NMDAR-Ab) are pathogenic immunoglobulins detected in patients suffering from NMDAR encephalitis. NMDAR-Ab alter the receptor membrane trafficking, synaptic transmission and neuronal network properties, leading to neurological and psychiatric symptoms in patients. Patients often have very little neuronal damage but rapid and massive (treatment-responsive) brain dysfunctions related to an unknown early mechanism of NMDAR-Ab. Our understanding of this early molecular cascade remains surprisingly fragmented. Here, we used a combination of single molecule-based imaging of membrane proteins to unveil the spatiotemporal action of NMDAR-Ab on live hippocampal neurons. We first demonstrate that different clones of NMDAR-Ab primarily affect extrasynaptic (and not synaptic) NMDARs. In the first minutes, NMDAR-Ab increase extrasynaptic NMDAR membrane dynamics, declustering its surface interactome. NMDAR-Ab also rapidly reshuffle all membrane proteins located in the extrasynaptic compartment. Consistent with this alteration of multiple proteins, effects of NMDAR-Ab were not mediated through the sole interaction between the NMDAR and EphB2 receptor. In the long term, NMDAR-Ab reduce the NMDAR synaptic pool by slowing down receptor membrane dynamics in a cross-linking-independent manner. Remarkably, exposing only extrasynaptic NMDARs to NMDAR-Ab was sufficient to produce their full-blown effect on synaptic receptors. Collectively, we demonstrate that NMDAR-Ab initially impair extrasynaptic proteins, then the synaptic ones. These data thus shed new and unsuspected light on the mode of action of NMDAR-Ab and, probably, our understanding of (extra)synaptopathies.

Positive Allosteric Modulation of NMDARs Prevents the Altered Surface Dynamics Caused by Patients' Antibodies.

OBJECTIVES: A positive allosteric modulator of the NMDAR, SGE-301, has been shown to reverse the alterations caused by the antibodies of patients with anti-NMDAR encephalitis (NMDARe). However, the mechanisms involved beyond receptor modulation are unclear. In this study, we aimed to investigate how this modulator affects NMDAR membrane dynamics. METHODS: Cultured hippocampal neurons were treated with SGE-301 or vehicle, alongside with immunoglobulins G (IgG) from patients with NMDARe or healthy controls. NMDAR surface dynamics were assessed with single-molecule imaging by photoactivated localization microscopy. RESULTS: NMDAR trajectories from neurons treated with SGE-301 were less confinement, with increased diffusion coefficients. This effect mainly occurred at synapses because extrasynaptic diffusion and confinement were minimally affected by SGE-301. Treatment with patients' IgG reduced NMDAR surface dynamics and increased their confinement. Remarkably, SGE-301 incubation antagonized patients' IgG effects in both synaptic and extrasynaptic membrane compartments, restoring diffusion and confinement values similar to those from neurons exposed to control IgG. DISCUSSION: We demonstrate that SGE-301 upregulates NMDAR surface diffusion and antagonizes the pathogenic effects of patients' IgG on NMDAR membrane organization. These findings suggest a potential therapeutic strategy for NMDARe.

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.

Inhibition of angiotensin II type 1 receptor agonistic autoantibodies by direct binding does not impact reduced uterine perfusion pressure offspring birthweight and blood pressure at adulthood.

BACKGROUND: Preeclampsia, a new-onset hypertension with end-organ damage in pregnancy, is associated with maternal death and morbidity, low birthweight, and B cells producing agonistic autoantibodies to the angiotensin II type 1 receptor. Angiotensin II type 1 receptor agonistic autoantibodies are produced during pregnancy and after delivery and are in the fetal circulation of women with preeclampsia. Angiotensin II type 1 receptor agonistic autoantibodies are shown to contribute to endothelial dysfunction, renal dysfunction, hypertension, fetal growth restriction, and chronic inflammation in women with preeclampsia. The reduced uterine perfusion pressure rat model of preeclampsia exhibits these features. In addition, we have shown that the administration of a 'n7AAc', which blocks the actions of the angiotensin II type 1 receptor autoantibodies, improves preeclamptic features in the rat with reduced uterine perfusion pressure. However, the effect of a 'n7AAc' on the long-term health of the offspring of rats with reduced uterine perfusion pressure is unknown. OBJECTIVE: This study aimed to test the hypothesis that inhibition of angiotensin II type 1 receptor autoantibodies during pregnancy will improve offspring birthweight and prevent increased cardiovascular risk in offspring in adulthood. STUDY DESIGN: To test our hypothesis, a 'n7AAc' (24 µg/d) or vehicle (saline) was given on gestation day 14 via miniosmotic pumps to sham-operated (sham) and Sprague-Dawley rat dams with reduced uterine perfusion pressure. Dams were allowed to deliver naturally, and pup weights were recorded within 12 hours after birth. Pups were aged to 16 weeks, at which time mean arterial pressure was measured and whole blood was collected to measure immune cells by flow cytometry, cytokines by enzyme-linked immunosorbent assay, and angiotensin II type 1 receptor autoantibodies by bioassay. A 2-way analysis of variance with the Bonferroni multiple comparison posthoc test was used for statistical analysis. RESULTS: There was no significant change in offspring birthweight of 'n7AAc'-treated male (5.63±0.09 g) or female (5.66±0.14 g) offspring from reduced uterine perfusion pressure dams compared with vehicle male (5.51±0.17 g) or female (5.74±0.13 g) offspring from reduced uterine perfusion pressure dams. In addition, 'n7AAc' treatment did not affect the birthweight of sham male (5.83±0.11 g) or female (5.64±0.12) offspring compared with vehicle sham male (5.811±0.15 g) or female (5.40±0.24 g) offspring. At adulthood, mean arterial pressure was unchanged in 'n7AAc' treated-male (133±2 mm Hg) and female (127±3 mm Hg) offspring from reduced uterine perfusion pressure dams compared with vehicle male (142±3 mm Hg) and female (133±5 mm Hg) offspring from reduced uterine perfusion pressure dams, the 'n7AAc'-treated sham male (133±3 mm Hg) and female (135±3 mm Hg) offspring, and vehicle sham male (138±4 mm Hg) and female (130±5 mm Hg) offspring. The circulating angiotensin II type 1 receptor autoantibodies were increased in vehicle male (10±2 ΔBPM) and female (14±2 ΔBPM) offspring from reduced uterine perfusion pressure dams and 'n7AAc'-treated male (11±2 ΔBPM) and female (11±2 ΔBPM) offspring from reduced uterine perfusion pressure dams compared with vehicle sham male (1±1 ΔBPM) and female (-1±1 ΔBPM) offspring and 'n7AAc'-treated sham male (-2±2 ΔBPM) and female (-2±2 ΔBPM) offspring. CONCLUSION: Our findings indicated that perinatal 7-amino acid sequence peptide treatment does not negatively impact offspring survival or weight at birth. Perinatal 'n7AAc' treatment did not prevent increased cardiovascular risk in offspring, but it also did not cause an increased cardiovascular risk in offspring with reduced uterine perfusion pressure compared with controls. Furthermore, perinatal 'n7AAc' treatment did not affect endogenous immunologic programming as observed by no change in circulating angiotensin II type 1 receptor autoantibodies in either sex of adult offspring from reduced uterine perfusion pressure dams.

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.

Unexpected Crosslinking Effects of a Human Thyroid Stimulating Monoclonal Autoantibody, M22, with IGF1 on Adipogenesis in 3T3L-1 Cells.

To study the effects of the crosslinking of IGF1 and/or the human thyroid-stimulating monoclonal autoantibody (TSmAb), M22 on mouse adipocytes, two- and three-dimensional (2D or 3D) cultures of 3T3-L1 cells were prepared. Each sample was then subjected to the following analyses: (1) lipid staining, (2) a real-time cellular metabolic analysis, (3) analysis of the mRNA expression of adipogenesis-related genes and extracellular matrix (ECM) molecules including collagen (Col) 1, 4 and 6, and fibronectin (Fn), and (4) measurement of the size and physical properties of the 3D spheroids with a micro-squeezer. Upon adipogenic differentiation (DIF+), lipid staining and the mRNA expression of adipogenesis-related genes in the 2D- or 3D-cultured 3T3-L1 cells substantially increased. On adding IGF1 but not M22 to DIF+ cells, a significant enhancement in lipid staining and gene expressions of adipogenesis-related genes was detected in the 2D-cultured 3T3-L1 cells, although some simultaneous suppression or enhancement effects by IGF1 and M22 against lipid staining or Fabp4 expression, respectively, were detected in the 3D 3T3-L1 spheroids. Real-time metabolic analyses indicated that monotherapy with IGF1 or M22 shifted cellular metabolism toward energetic states in the 2D 3T3-L1 cells upon DIF+, although no significant metabolic changes were induced by DIF+ alone in 2D cultures. In addition, some synergistical effects on cellular metabolism by IGF1 and M22 were also observed in the 2D 3T3-L1 cells as well as in cultured non-Graves' orbitopathy-related human orbital fibroblasts (n-HOFs), but not in Graves' orbitopathy-related HOFs (GHOFs). In terms of the physical properties of the 3D 3T3-L1 spheroids, (1) their sizes significantly increased upon DIF+, and this increase was significantly enhanced by the presence of both IGF1 and M22 despite downsizing by monotreatment, and (2) their stiffness increased substantially, and no significant effects by IGF-1 and/or M22 were observed. Regarding the expression of ECM molecules, (1) upon DIF+, significant downregulation or upregulation of Col1 and Fn (3D), or Col4 and 6 (2D and 3D) were observed, and (2) in the presence of IGF-1 and/or M22, the mRNA expression of Col4 was significantly downregulated by M22 (2D and 3D), but the expression of Col1 was modulated in different manners by monotreatment (upregulation) or the combined treatment (downregulation) (3D). These collective data suggest that the human-specific TSmAb M22 induced some unexpected simultaneous crosslinking effects with IGF-1 with respect to the adipogenesis of 2D-cultured 3T3-L1 cells and the physical properties of 3D 3T3-L1 spheroids.

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.

Autoantibodies directed against glutamate decarboxylase interfere with glucose-stimulated insulin secretion in dispersed rat islets.

Islet autoantibodies, including autoantibodies directed against the 65kDa isoform of glutamate decarboxylase (GAD65Ab), are present in the majority of patients with newly diagnosed type 1 diabetes (T1D). Whereas these autoantibodies are historically viewed as an epiphenomenon of the autoimmune response with no significant pathogenic function, we consider in this study the possibility that they impact the major islet function, namely glucose-stimulated insulin secretion. Two human monoclonal GAD65Ab (GAD65 mAb) (b78 and b96.11) were investigated for uptake by live rat beta cells, subcellular localization and their effect on glucose-stimulated insulin secretion. The GAD65 mAbs were internalized by live pancreatic beta cells, where they localized to subcellular structures in an epitope-specific manner. Importantly, GAD65 mAb b78 inhibited, while GAD65 mAb b96.11 enhanced, glucose-stimulated insulin secretion (GSIS). These opposite effects on GSIS rule out non-specific effects of the antibodies and suggest that internalization of the antibody leads to epitope-specific interaction with intracellular machinery regulating insulin granule release. The most likely explanation for the alteration of GSIS by GAD65 Abs is via changes in GABA release due to inhibition or change in GAD65 enzyme activity. This is the first report indicating an active role of GAD65Ab in the pathogenesis of T1D.

Chronic exposure to the anti-M3 muscarinic acetylcholine receptor autoantibody in pemphigus vulgaris contributes to disease pathophysiology.

Pemphigus vulgaris (PV) is a potentially lethal autoimmune mucocutaneous blistering disease characterized by binding of IgG autoantibodies (AuAbs) to keratinocytes (KCs). In addition to AuAbs against adhesion molecules desmogleins 1 and 3, PV patients also produce an AuAb against the M3 muscarinic acetylcholine (ACh) receptor (M3AR) that plays an important role in regulation of vital functions of KCs upon binding endogenous ACh. This anti-M3AR AuAb is pathogenic because its adsorption eliminates the acantholytic activity of PV IgG; however, the molecular mechanism of its action is unclear. In the present study, we sought to elucidate the mode of immunopharmacologic action of the anti-M3AR AuAb in PV. Short-term exposures of cultured KCs to PV IgG or the muscarinic agonist muscarine both induced changes in the expression of keratins 5 and 10, consistent with the inhibition of proliferation and upregulated differentiation and in keeping with the biological function of M3AR. In contrast, long-term incubations induced a keratin expression pattern consistent with upregulated proliferation and decreased differentiation, in keeping with the hyperproliferative state of KCs in PV. This change could result from desensitization of the M3AR, representing the net antagonist-like effect of the AuAb. Therefore, chronic exposure of KCs to the anti-M3AR AuAb interrupts the physiological regulation of KCs by endogenous ACh, contributing to the onset of acantholysis. Since cholinergic agents have already demonstrated antiacantholytic activity in a mouse model of PV and in PV patients, our results have translational significance and can guide future development of therapies for PV patients employing cholinergic drugs.

Gypenosides regulate autophagy through Sirt1 pathway and the anti-inflammatory mechanism of mitochondrial autophagy in systemic lupus erythematosus.

To study the mechanism of gynostemma pentaphyllum saponins (GpS) regulating mitochondrial autophagy and anti-inflammatory through Sirtuin 1 (Sirt1) pathway in systemic lupus erythematosus (SLE). JURKAT cells were cultured in vitro, RT-PCR and western blotting (WB) were utilized to identify the expression of related-proteins in Sirt1 pathway and global autophagy and mitochondrial autophagy markers in JURKAT before and after GpS treatment induced by ultraviolet B (UVB), and the related-mechanism of GpS regulation of autophagy was analyzed. The SLE model was established to analyze the alleviating effects of GpS on various symptoms of lupus mice. Sirt1/AMPK/mTOR pathway was activated in UVB induced JURKAT cells. After the addition of GpS, WB revealed that the phosphorylation of AMPK decreased, the phosphorylation of mTOR increased, the expression of Sirt1 protein decreased, and the activation of the pathway was inhibited. Moreover, autophagy of JURKAT cells wasinhibited. In order to further verify the role of Sirt1 pathway, we activated Sirt1 expression in cells by constructing lentiviral vectors, and the therapeutic effect of GpS was significantly reduced. These results indicate GpS can exert autophagy regulation by inhibiting the activity of Sirt1 pathway. To treat SLE. GpS can significantly reduce the level of autoantibodies, kidney inflammation, immune complex deposition and urinary protein excretion, improve kidney function in lupus-prone mice. GpS can regulate autophagy and mitochondrial autophagy through Sirt1 pathway, which may be a potential mechanism for GpS to reduce the level of autoantibodies, kidney inflammation, immune complex deposition and urinary protein excretion, improve kidney function in lupus-prone mice.

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.