Johnson, Renee K.

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 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 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.