Howe, Charles L.

AbstractNew onset refractory status epilepticus (NORSE), including its subtype with a preceding febrile illness known as febrile infection‐related epilepsy syndrome (FIRES), is one of the most severe forms of status epilepticus. The exact causes of NORSE are currently unknown, and there is so far no disease‐specific therapy. Identifying the underlying pathophysiology and discovering specific biomarkers, whether immunologic, infectious, genetic, or other, may help physicians in the management of patients with NORSE. A broad spectrum of biomarkers has been proposed for status epilepticus patients, some of which were evaluated for patients with NORSE. Nonetheless, none has been validated, due to significant variabilities in study cohorts, collected biospecimens, applied analytical methods, and defined outcome endpoints, and to small sample sizes. The NORSE Institute established an open NORSE/FIRES biorepository for health‐related data and biological samples allowing the collection of biospecimens worldwide, promoting multicenter research and sharing of data and specimens. Here, we suggest standard operating procedures for biospecimen collection and biobanking in this rare condition. We also propose criteria for the appropriate use of previously collected biospecimens. We predict that the widespread use of standardized procedures will reduce heterogeneity, facilitate the future identification of validated biomarkers for NORSE, and provide a better understanding of the pathophysiology and best clinical management for these patients.

AbstractObjectiveTherapeutic strategies for patients with febrile infection‐related epilepsy syndrome (FIRES) are limited, ad hoc, and frequently ineffective. Based on evidence that inflammation drives pathogenesis in FIRES, we used ex vivo stimulation of peripheral blood mononuclear cells (PBMCs) to characterize the monocytic response profile before and after therapy in a child successfully treated with dexamethasone delivered intrathecally six times between hospital Day 23 and 40 at 0.25 mg/kg/dose.MethodsPBMCs were isolated from serial blood draws acquired during refractory status epilepticus (RSE) and following resolution associated with intrathecal dexamethasone therapy in a previously healthy 9‐year‐old male that presented with seizures following Streptococcal pharyngitis. Cells were stimulated with bacterial or viral ligands and cytokine release was measured and compared to responses in age‐matched healthy control PBMCs. Levels of inflammatory factors in the blood and CSF were also measured and compared to pediatric healthy control ranges.ResultsDuring RSE, serum levels of IL6, CXCL8, HMGB1, S100A8/A9, and CRP were significantly elevated. IL6 was elevated in CSF. Ex vivo stimulation of PBMCs collected during RSE revealed hyperinflammatory release of IL6 and CXCL8 in response to bacterial stimulation. Following intrathecal dexamethasone, RSE resolved, inflammatory levels normalized in serum and CSF, and the PBMC hyperinflammatory response renormalized.SignificanceFIRES may be associated with a hyperinflammatory monocytic response to normally banal bacterial pathogens. This hyperinflammatory response may induce a profound neutrophil burden and the consequent release of factors that further exacerbate inflammation and drive neuroinflammation. Intrathecal dexamethasone may resolve RSE by resetting this inflammatory feedback loop.

Abstract We recently reported normalization of CSF cytokine levels following successful treatment of drug-resistant epilepsy (DRE) with recombinant interleukin-1 receptor antagonist (rIL1RA). To further understand the mechanism of this therapeutic outcome, we interrogated the ability of serum and CSF factors that are elevated in drug resistant-seizure disorders to promote inflammatory responses in astrocytes and human brain microvessel endothelial cells (HBMVECs). We report that CCL2, TNFa, IL6, and CXCL8/1 were strongly induced in 1) astrocytes and HBMVECs stimulated with IL1β (blocked by rIL1RA) and 2) astrocytes but not HBMVECs in response to DRE patient serum but not control serum (partially blocked by rIL1RA). This suggested that other serum factors contribute to astrocyte activation in this DRE. HMGB1 and IL18 were also dysregulated in DRE patient serum; however, IL18 failed to stimulate cytokine secretion from astrocytes. In contrast, HMGB1 induced cytokine secretion from astrocytes, which was blocked by the TLR4 inhibitor TAK242. Remarkably, TAK242 also prevented cytokine production by astrocytes in response to serum from some patients with DRE. Finally, IL1β induced the polarized uptake and secretion of HMGB1 across transwell inserts harboring confluent monolayers of HBMVECs suggesting that IL1b signaling may cause endothelial cells to transport serum HMGB1 into the perivascular space—promoting TLR4-dependent cytokine release from astrocytes. This translation of an “outside” inflammatory signal into an “inside” inflammatory response may underlie seizure disorders that are resistant to anti-seizure drugs.

Abstract In diseases such as multiple sclerosis and autoimmune encephalitides, activated CD8+ cytotoxic T cells infiltrate the brain and are found in clonally expanded clusters that outnumber CD4+ cells in progressive stages of disease. At present, the pathophysiological role of these cells is poorly understood and therapeutic strategies for preventing or ameliorating autoinflammatory disease progression are limited. T cells exhibit profound metabolic reprogramming that facilitates the energetic and proliferative demands of effector T cells. Key metabolic pathways in this reprogramming include upregulation of glycolysis and the pentose phosphate pathway (PPP). Here we show that 1) pharmacologic inhibition of the PPP suppresses effector phenotype acquisition and proinflammatory cytokine secretion by CD8+ T cells; 2) engagement of the PPP is enhanced by pharmacologic Nrf2 activation, implicating reactive oxygen species-induced activation as a transcriptional regulator of PPP-dependent immune activation; 3) Nrf2-driven PPP flux promotes the acquisition of specific CD8+ effector phenotypes; 4) limiting the metabolic flux of glucose-derived metabolites through the PPP prevents CD8+ T cell-mediated injury of neurons expressing cognate antigen and preserves their electrophysiological function; and 5) pharmacologic inhibition of the PPP in B6 mice at symptom onset is sufficient to delay and ameliorate motor disability in the MOG EAE preclinical model of MS. Overall, these results underscore the potential of immunometabolic therapies to limit T cell mediated injury in autoinflammatory and autoimmune diseases across organ systems including the prevention of neurologic deficits in patients with T cell driven neurologic disease. This work was supported by the NIH (R01NS115126 to CLH; T32GM065841 to EMG), the Mayo Clinic Graduate School of Biomedical Sciences, the Mayo Clinic Center for MS and Autoimmune Neurology, and generous donations from Eugene and Marcia Applebaum, Donald J. and Frances I. Herdrich, Bob and Loralee Marvin, and Rob and Debbie Mudge.

Abstract Neurovirulent picornaviral infections are a significant health risk in developing nations. Unfortunately, the mechanisms of picornaviral clearance from the central nervous system (CNS) are less understood and therapeutic approaches are limited. We have established a mouse model of neurovirulent picornavirus infection using the Theiler’s murine encephalomyelitis virus (TMEV) which replicates during the first 12 days postinfection (dpi) primarily in the hippocampus but is cleared from the brain by 21 dpi. Intracerebral infection of TMEV triggers the early infiltration of natural killer cells (NKCs) followed by CD8+ T cells (CTLs) into the brain. We observed that both brain-infiltrating NKCs and CTLs express the NKG2D activating receptor on their surface and 50% of the the CTLs specific to the VP2121–130 viral capsid peptide also expressed NKG2D. Concomitantly, brains of infected mice expressed NKG2D ligands such as RAE-1, H-60 and MULT-1 and microglia were the cellular locus of these ligands. Importantly, systemic treatment with the NKG2D function blocking antibody CX5 resulted in a significant increase in viral load in the brain. These results suggest that activated and infected microglia stimulate NKCs and CTLs via NKG2D in a manner that restricts viral replication. NKG2D may thus serve as a potential therapeutic target to enhance viral clearance responses during CNS infection. This work was supported by a grant from the National MS Society and by Donald and Frances Herdrich.

Abstract Cognitive impairment in MS is associated with diffuse gray matter injury, which remains poorly understood. Given that axons span both white and gray matter regions, it’s possible that signals within these axons contribute to the spread of diffuse injury in the brain. We show that retrograde IFNg signaling in axons (in vitro human and murine) and demyelination (murine in vivo) cause transcriptional / translational changes in neuronal cell bodies. Chief among the candidate signaling pathways identified is ISGylation, a process whereby a multitude of cellular proteins are modified by the attachment of one or more ISG15 molecules. Using custom adeno-associated viral vectors we tested how altering ISGylation in neurons affects neuronal synaptic function and how it affects neuronal responsiveness to specific inflammatory factors in cell cultures. We also measured ISGylation in MS brain tissues to see if neuronal ISGylation correlates with gray matter injury in MS. We report early evidence that increased neuron ISGylation alters the composition of neuron-derived extracellular vesicles. Microglia treated with extracellular vesicles from neurons overexpressing ISGylation pathway genes exhibited morphology consistent with increased phagocytic activity and increased mRNA expression of inflammatory cytokines including: CCL2, IL1b, TNFa, iNOS, CXCL10, CCL5, and IL6. At this point, it is still unclear how these changes are related to gray matter pathology. However, it is possible that microglial activation by these vesicles is involved with driving synapse loss as activated microglia are known to be capable of stripping neuronal synapses.

Abstract Cytotoxic CD8+ T cells outnumber CD4+ T cells within multiple sclerosis (MS) lesions, yet the pathogenic role for these cells and the relevant target antigen(s) involved in neuropathology remain unknown. We predict that a subset of lesion-infiltrating CD8+ T cells are specific for neuronal antigens and contribute to axon or neuron injury, thereby driving disease progression and accumulation of disability. To test this, we are developing protocols to measure MS patient-derived T cell reactivity against neuronal and control antigens. Peripheral blood mononuclear cells (PBMCs) isolated from whole blood were incubated with GM-CSF and IL-4 to selectively expand dendritic cells (DCs) or with IL-7 to promote survival and maintenance of T cells. After resting, patient T cells were combined 1:1 with autologous DCs in the presence of vaccine antigens or peptides derived from common pathogens. We tested responses against a pool of cytomegalovirus, Epstein Barr virus, and influenza peptides (CEF) and against mumps viral protein extract. We also tested responses in T cells derived from patients with autoimmune neurological disorders following incubation with autologous DCs and disease-specific proteins. Activation profiles were assessed using CD69, CD44, and LFA-1 by conventional flow cytometry. Cellular responses were also assessed using mass cytometry (cytometry by time of flight; CyTOF). Our next steps include testing MS patient-derived T cell responses against neuronal and axonal protein libraries, with the goal of identifying potential autoantigens that underlie disease progression.

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.

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.