Citations

Abstract The immunological response to CNS viral infection must balance the need for effective viral clearance with the need to preserve irreplaceable neurons. Failure to achieve this balance may result in death or severe cognitive impairment. We have observed hippocampal injury, compromised cognitive function, and seizures in C57BL/6 mice following acute infection with TMEV. We established that an initial innate immune response controlled by inflammatory monocyte infiltration into the brain is responsible for this neural injury. To better understand the pathogenic mechanisms involved in monocyte recruitment to the CNS we asked whether reducing the initial viral inoculum impacted the evolution of neural injury. We found that mice infected with standard (2×105 PFU) or low inoculum (1.25×104 PFU) TMEV exhibited a graded inflammatory response. Notably, a 16-fold reduction in viral inoculum resulted in 80% attenuation of the inflammatory monocyte response, concomitant with a 90% attenuation in acute production of CCL2 and complete abrogation of acute increases in TNFα and IL-6 in the hippocampus. Cognitive performance was preserved in low inoculum mice and seizure burden was highly attenuated. Despite the introduction of 16-times less virus, viral load in the brain was equivalent at 3 dpi between groups, indicating that the amount of replicating virus was not associated with reduced neuropathology. Our findings support a model in which the earliest host response to pathogen in the CNS shapes the size of the inflammatory response and constrains the downstream pathological sequelae.

Abstract MR imaging suggests an anatomic correspondence between WM and GM lesions in progressing MS patients, suggesting an underlying neuronal-signaling or neurodegenerative element to the progressive disease state. While retrograde signals evolving from stressed axons that alter soma gene expression have been previously described in models of axonal transection or crush injury, those resulting from inflammation and demyelination are unknown. Using microfluidic chambers that segregate neuronal soma and axons, we have previously demonstrated increased axonal MHC I following treatment with IFNg, a cytokine prominent in demyelinating MS lesions. Furthermore, expression of antigen-loaded MHC I predisposed axons to injury by antigen-specific CD8+ T cells in vitro. Here we report that neurons also upregulate expression of MHC class Ib mRNAs in response to axonal stimulation with IFNg. Genes most highly upregulated following cytokine treatment included H2-M3, H2-Q5, H2-Q6, H2-Q7, H2-Q8, H2-T10 , and H2-T23. We further report the development of an in vivo model system for measuring axonal vulnerability to antigen-specific immune attack following demyelination using an AAV vector to induce neuron-specific expression of axonally-targeted neoantigen (ovalbumin) tagged with cleavable eGFP. We also present optimized methods using AAV vectors in mice for infecting retinal, motor cortex, and hippocampal neurons to induce target gene expression in axons projecting to optic nerve and corpus collosum.

Background and ObjectivesWe sought to determine clinical significance of neuronal septin autoimmunity and evaluate for potential IgG effects.MethodsSeptin‐IgGs were detected by indirect immunofluorescence assays (IFAs; mouse tissue and cell based) or Western blot. IgG binding to (and internalization of) extracellular septin epitopes were evaluated for by live rat hippocampal neuron assay. The impact of purified patient IgGs on murine cortical neuron function was determined by recording extracellular field potentials in a multielectrode array platform.ResultsSeptin‐IgGs were identified in 23 patients. All 8 patients with septin‐5‐IgG detected had cerebellar ataxia, and 7 had prominent eye movement disorders. One of 2 patients with co‐existing septin‐7‐IgG had additional psychiatric phenotype (apathy, emotional blunting, and poor insight). Fifteen patients had septin‐7 autoimmunity, without septin‐5‐IgG detected. Disorders included encephalopathy (11; 2 patients with accompanying myelopathy, and 2 were relapsing), myelopathy (3), and episodic ataxia (1). Psychiatric symptoms (≥1 of agitation, apathy, catatonia, disorganized thinking, and paranoia) were prominent in 6 of 11 patients with encephalopathic symptoms. Eight of 10 patients with data available (from 23 total) improved after immunotherapy, and a further 2 patients improved spontaneously. Staining of plasma membranes of live hippocampal neurons produced by patient IgGs (subclasses 1 and 2) colocalized with pre‐ and post‐synaptic markers. Decreased spiking and bursting behavior in mixed cultures of murine glutamatergic and GABAergic cortical neurons produced by patient IgGs were attributable to neither antigenic crosslinking and internalization nor complement activation.InterpretationSeptin‐IgGs are predictive of distinct treatment‐responsive autoimmune central nervous system (CNS) disorders. Live neuron binding and induced electrophysiologic effects by patient IgGs may support septin‐specific pathophysiology. ANN NEUROL 2022;92:1090–1101

Abstract Same day processing of biospecimens such as blood is not always feasible, which presents a challenge for research programs seeking to study a broad population or to characterize patients with rare diseases. Recruiting sites may not be equipped to process blood samples and variability in timing and technique employed to isolate peripheral blood mononuclear cells (PBMCs) at local sites may compromise reproducibility across patients. One solution is to send whole blood collected by routine phlebotomy via overnight courier to the testing site under ambient conditions. Determining the impact of shipping on subsequent leukocyte responses is a necessary prerequisite to any experimental analysis derived from transported samples. To this end, whole blood was collected from healthy control subjects and processed fresh or at 6, 24 and 48 hours after collection and handling under modeled shipping conditions. At endpoint, whole blood was assessed via a complete blood count with differential and immunophenotyped using a standardized panel of antibodies [HLADR, CD66b, CD3, CD14, CD16]. PBMCs and neutrophils were isolated from whole blood and subjected to ex vivo stimulation with lipopolysaccharide and heat-killed Staphylococcus aureus. Stimulated release of cytokines and chemokines was assessed by cytometric bead array. RNA was also isolated from PBMCs to analyze transcriptional changes induced by shipping. The complete blood count with differential revealed that most parameters were maintained in shipped blood held for 24 hours at ambient temperature. Immunophenotyping indicated preservation of cellular profiles at 24 hours, although with broadening of some populations and a decrease in CD16 intensity on classical monocytes. At the transcriptional level, RNAseq analysis identified upregulation of a transcription factor module associated with inflammation in unstimulated PBMCs derived from whole blood shipped overnight. However, these changes were limited in both scale and number of impacted genes. Ex vivo stimulation of PBMCs further revealed preservation of functional responses in cells isolated from shipped blood held for 24 hours at ambient temperature. However, neutrophil responses were largely abrogated by this time. By 48 hours neither cell population responded within normal parameters. These findings indicate that robust immunophenotyping and PBMC stimulated response profiles are maintained in whole blood shipped overnight and processed within 24 hours of collection, yielding results that are representative of those obtained from the sample immediately following venipuncture. This methodology is feasible for many patient recruitment sites to implement and allows for sophisticated immunological analysis of patient populations derived from large geographic areas. With regard to rare disease research, this meets a universal need to enroll patients in sufficient numbers for immunoprofiling and discovery of underlying pathogenic mechanisms.

Abstract Neuromyelitis optica is an autoimmune inflammatory disorder targeting aquaporin-4 water channels in CNS astrocytes. Histopathological descriptions of astrocytic lesions reported in neuromyelitis optica so far have emphasized a characteristic loss of aquaporin-4, with deposition of IgG and complement and lysis of astrocytes, but sublytic reactions have been underappreciated. We performed a multi-modality study of 23 neuromyelitis optica autopsy cases (clinically and/or pathologically confirmed; 337 tissue blocks). By evaluating astrocytic morphology, immunohistochemistry and AQP4 RNA transcripts, and their associations with demyelinating activity, we documented a spectrum of astrocytopathy in addition to complement deposition, microglial reaction, granulocyte infiltration and regenerating activity. Within advanced demyelinating lesions, and in periplaque areas, there was remarkable hypertrophic astrogliosis, more subtle than astrocytic lysis. A degenerative component was suggested by ‘dystrophic’ morphology, cytoplasmic vacuolation, Rosenthal fibres and associated stress protein markers. The abundance of AQP4 mRNA transcripts in sublytic reactive astrocytes devoid of aquaporin-4 protein supported in vivo restoration following IgG-induced aquaporin-4 endocytosis/degradation. Astrocytic alterations extending beyond demyelinating lesions speak to astrocytopathy being an early and primary event in the evolving neuromyelitis optica lesion. Focal astrocytopathy observed without aquaporin-4 loss or lytic complement component deposition verifies that astrocytic reactions in neuromyelitis optica are not solely dependent on IgG-mediated aquaporin-4 loss or lysis by complement or by IgG-dependent leucocyte mediators. We conclude that neuromyelitis optica reflects a global astrocytopathy, initiated by binding of IgG to aquaporin-4 and not simply definable by demyelination and astrocytic lysis. The spectrum of astrocytic morphological changes in neuromyelitis optica attests to the complexity of factors influencing the range of astrocytic physiological responses to a targeted attack by aquaporin-4-specific IgG. Sublytic astrocytic reactions are no doubt an important determinant of the lesion’s evolution and potential for repair. Pharmacological manipulation of the astrocytic stress response may offer new avenues for therapeutic intervention.

Natural killer cells (NKC) and cytotoxic T lymphocytes (CTL) express NKG2D, a lectin‐like molecule that serves as a direct stimulatory receptor in the innate immune system and as a co‐stimulatory receptor in the adaptive system. In mice, the ligands for NKG2D are stress‐inducible molecules such as Rae‐1, H‐60 and MULT‐1. These MHC class I‐like molecules are generally absent in normal tissues but are upregulated by inflammation and virus infection. We have found that acute intracerebral infection with the Theiler's murine encephalomyelitis virus (TMEV) elicits robust infiltration of NKG2D‐positive CTL and NKC into the brain coincident with the zenith and resolution of viral load. Likewise, using quantitative RT‐PCR we found that MULT‐1 was upregulated coincident with the peak of viral load while Rae‐1 (only epsilon and delta tested) and H‐60 remained undetectable. Therefore, we hypothesized that CTL and NKC recognition of MULT‐1 on neurons is required for efficient viral clearance. However, in vivo immunodepletion of NKC and CTL beginning one day prior to infection and continuing throughout the first 3 weeks postinfection did not prevent efficient viral clearance despite greater than 90% reduction in NKG2D‐positive cells in the brain immune infiltrate as assessed by flow cytometry. Thus, the role of MULT‐1 expression following TMEV infection remains unclear and experiments are ongoing to determine the cellular locus of MULT‐1 expression and recognition. This work was supported by grant RG3636 from the National Multiple Sclerosis Society and by Donald and Frances Herdrich.

Stress‐dependent regulation of the immune response is a general repair and maintenance system. Members of the MHC class I superfamily serve as indicators of cellular stress. Nonclassical class I molecules such as H60, MULT‐1 and the RAE‐1 family are generally absent in normal tissues. We found that intracerebral infection with TMEV upregulated expression of these stress‐response molecules in the brain at timepoints that coincided with the control of viral load. Moreover, we found that about 50% of the brain‐ infiltrating cytotoxic T cells (CTLs) specific for the TMEV peptide VP2121–130 were positive for the NKG2D activating receptor at 7 days postinfection. NKG2D binds to RAE‐1, H60, and MULT‐1 and costimulates CTLs. When we blocked NKG2D using the CX5 function blocking antibody we found a significant increase in viral load in the brain. Therefore, we hypothesize that cellular stress associated with viral infection induces NKG2D ligand expression on target cells that in turn costimulates NKG2D‐bearing VP2‐specific CTLs to promote viral clearance. Our study may ultimately impact the health of people at risk from encephalitis and neurologic complications associated with picornaviral infections and may provide new ways to promote viral clearance in individuals suffering from persistent virus infections of the brain.

Objective This study was undertaken to describe a novel biomarker of germ cell tumor and associated paraneoplastic neurological syndrome (PNS). Methods Archival sera from patients with germ cell tumor–associated PNS were evaluated. We identified a common autoantigen in a human testicular cancer cell line (TCam‐2) by Western blot and mass spectrometry. Its identity was confirmed by recombinant‐protein Western blot, enzyme‐linked immunosorbent assay (ELISA), and cell‐based assay. Autoantibody specificity was confirmed by analyzing assorted control sera/cerebrospinal fluid. Results Leucine zipper 4 (LUZP4)–immunoglobulin G (IgG) was detected in 28 patients' sera, 26 of whom (93%) were men. The median age at neurological symptom onset was 45 years (range = 28–84). Median titer (ELISA) was 1:300 (1:50 to >1:6,400, normal value < 1:50). Coexistent kelchlike protein 11–IgG was identified in 18 cases (64%). The most common presenting phenotype was rhombencephalitis (17/28, 61%). Other presentations included limbic encephalitis (n = 5, 18%), seizures and/or encephalitis (n = 2, 7%), and motor neuronopathy/polyradiculopathy (n = 4, 14%). The most common malignancy among cancer‐evaluated PNS patients was seminoma (21/27, 78%). Nine of the 21 seminomas detected by whole‐body fluorodeoxyglucose positron emission tomography scan (43%) were extratesticular. Both female patients had ovarian teratoma. Regressed testicular germ cell tumors were found in 4 patients. Exposure of T‐cell–dendritic‐cell cocultures from chronic immunosuppression‐naïve LUZP4‐IgG–seropositive patients to recombinant LUZP4 protein evoked a marked increase in CD69 expression on both CD4+ and CD8+ T cells when compared to vehicle‐exposed and healthy control cultures. Interpretation LUZP4‐IgG represents a novel serological biomarker of PNS and has high predictive value for germ cell tumors. The demonstrated antigen‐specific T‐cell responses support a CD8+ T‐cell–mediated cytotoxic paraneoplastic and antitumor potential. ANN NEUROL 2021;89:1001–1010

We previously applied Systematic Evolution of Ligands by EXponential enrichment (SELEX) technology to identify myelin-specific DNA aptamers, using crude mouse central nervous system myelin as bait. This selection identified a 40-nucleotide aptamer (LJM-3064). Multiple biotinylated LJM-3064 molecules were conjugated to a streptavidin core to mimic a multimeric immunoglobulin M (IgM) antibody, generating 3064-BS-streptavidin (Myaptavin-3064). We previously showed that Myaptavin-3064 induces remyelination in the Theiler’s murine encephalomyelitis virus (TMEV) model of chronic spinal cord demyelination. While details of target binding and the mechanism of action remain unclear, we hypothesized that Myaptavin-3064 induces remyelination by binding to oligodendrocytes (OLs). We now report the results of binding assays using the human oligodendroglioma (HOG) cell line, applying both flow cytometry and immunocytochemistry (IC) to assay aptamer conjugate binding to cells. IC assays were applied to compare aptamer conjugate binding to primary embryonic mouse mixed cortical cultures and primary adult rat mixed glial cultures. We show that Myaptavin-3064 binds to HOG cells, with increased binding upon differentiation. In contrast, a negative control aptamer conjugate, 3060-BS, which did not promote central nervous system (CNS) remyelination, does not bind to HOG cells. Myaptavin-3064 did not bind to lung (L2) or kidney (BHK) cell lines. Total internal reflection fluorescence (TIRF) imaging indicates that Myaptavin-3064 binds at the cell membrane of live cells. In addition to HOG cells, Myaptavin-3064 binds to adult rat OLs, but not to embryonic mouse mixed cortical cultures. These data support the hypothesis that Myaptavin-3064 binds to a surface molecule on both rodent and human OLs in a manner that triggers a remyelination signal pathway.