Koji Shinoda (1, 2) – Ayman Rezk (1, 2) – Rui Li (1, 2) – Amit Bar-Or (1, 2 3)
Department of Neurology, Perelman School of Medicine, University of Pennsylvania, PA, USA (1) – Center for Neuroinflammation and Experimental Therapeutics, Perelman School of Medicine, University of Pennsylvania, PA, USA (2) – Children’s Hospital of Philadelphia, University of Pennsylvania, PA, USA (3)
Anti-CD20 therapy targets a broad range of B cells and a small subset of T cells, and has emerged as a high efficacy treatment for patients with multiple sclerosis (MS). Circulating B cells in untreated patients with MS exhibit an abnormal balance of pro-inflammatory and regulatory cytokine-producing B cell subpopulations. While anti-CD20 therapy has been thought to deplete both these subpopulations from the circulation relatively indiscriminately, differential impact of the depletion phase on smaller yet potentially relevant subsets has not been examined. In addition, relatively little is known about the profiles and kinetics of B-cell subset reconstitution, even after short anti-CD20 exposure. Here, we aimed to clarify the phenotypic and functional changes in circulating B cells following treatment initiation with the humanized anti-CD20 ocrelizumab, by comparing serial (baseline, 3-month and 6-month) profiles using deep immunophenotyping and flow cytometry of peripheral blood mononuclear cells in treatment-naive MS patients. B cells were largely but not completely depleted, 3 months after treatment initiation. Specifically, we found that CD11c+ B cells (implicated as pro-inflammatory in other systemic autoimmune diseases) in addition to plasmablasts, were less efficiently depleted. By the time of the 6-month infusion, B cells were partially repopulated, though to differing degrees across individuals. In general, CD10+ transitional B cells (implicated as anti-inflammatory), as well as a subset of memory B cells, preferentially repopulated by 6 months. The repopulating B cells exhibited decreased levels of surface activation markers, and increased levels of surface regulatory markers. The ratios of IL-6/IL-10-producing B cells (previously implicated in driving pro-inflammatory Th17 T cell responses) were significantly diminished at month 6 compared to the treatment-naïve baseline. Our data unexpectedly demonstrates diminished susceptibility of the CD11c+ B cell subset to anti-CD20 therapy. The kinetics of B cell reconstitution exhibit a degree of heterogeneity across MS patients, with an overall improved inflammatory/regulatory balance, even after an initial cycle of anti-CD20 therapy.
Wolfgang Trillo Alvarez (1) – Joshua Andree Medina Suarez (1) – Maria de Los Angeles Ibañez Mogrovejo (1) – Marineives Lizeth Mejia Vega (1) – Renato Alonso Caceres Amado (1) – Joaquin Jesus Molina Acosta (1) – Claudio Roberto Ibañez Escalante (1) – Jonatan David Escalante Ortiz (1) – Adriana Zaida Escalante Mercado (1) – Miguel Angel Manchego Bautista (1) – Juan Ignacio Santiago Valdivia Pino (1)
SINAPSIS, UCSM School of Medicine, Arequipa, Peru (1)
Objective: Report extended honeymoon period in a patient with Polyneuropathy – type 1A diabetes mellitus. Background: Type1 diabetes mellitus causes the destruction of pancreatic beta cells. Follicular helper T cells play an important role in the maturation and antibody production of beta cells. T follicular helper cells number is increased in type 1 diabetes mellitus, and their count correlates with fasting C peptide. Rituximab, a monoclonal antibodies against CD20, reduces Tfh cells number, IL6 and IL21 levels in peripheral blood. Rituximab has also proven to reduce the autoimmune destruction of beta cells by coupling to B cells and induce immune tolerance thus avoiding the progress of the immune reaction. Methods: a 15 years old latin male with past medical history of urine ketones at age 7, hyperglycemia up to 580 mg/dl was brought to the clinic with polyuria, polydipsia and headache. At physical was positive for signs of polyneuropathy with distal paresthesia, burning pain in palms and soles, Raynaud phenomenon, diminished tendon reflexes, and diminished sensation in four extremities. Laboratory results were relevant for basal glucose: 285.3 mg/dl, HbA1c: 9, anti GAD65: positive, EMG: positive for sensory motor axonal neuropathy. Polyneuropathy – Type 1 diabetes Mellitus diagnosis was stablished. Results: Treatment with an intensive low carbohydrate diet, with Rituximab 1 grm Q/15 days and 1 grm Q/6 months, IV pulse methylprednisolone, hydroxychloroquine 200mg Q/day and Azathioprine 50mgQ/day was started. After treatment anti GAD antibodies became negative and have stayed negative for 65 weeks since, HbA1c is 5.9 and symptoms of polyneuropathy resolved including normal tendon reflexes. Conclusion: Transitory depletion of B-lymphocytes with Rituximab lowers B cell mediated beta cell lysis in patients with type 1 diabetes mellitus. In human essays one can predict that monoclonal antibodies against CD20, anti CD22, and anti T cell antibodies would be most efficacious in newly diagnosed type 1 diabetes or during the Honeymoon period. We report a case of a 15 years old latin male with stablished type 1A diabetes and polyneuropathy that reached 65 weeks extended Honeymoon Period with a combined diet and anti CD20 antibody treatment.
Ayman Rezk (1, 2, 4) – Koji Shinoda (1, 2) – Rui Li (1, 2) – Amit Bar-Or (1, 2, 3, 4)
Department of Neurology, Perelman School of Medicine, University of Pennsylvania, PA, USA (1) – Center for Neuroinflammation and Experimental Therapeutics, University of Pennsylvania, PA, USA (2) – Division of Neurology, Children’s Hospital of Philadelphia, Perelman School of Medicine, University of Pennsylvania, PA, USA (3) – Neuroimmunology Unit, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada (4)
Anti-CD20 antibodies target and deplete a broad range of B cells (and a small subset of CD20-expressing T cells), substantially reducing new disease activity in multiple sclerosis (MS). Recent work has demonstrated important bi-directional interactions between B cells and CD4+ T cells that contribute to their abnormal immune responses in MS. This is supported by the observation that B-cell depletion attenuates CD4+ T cell responses in patients with MS. Curiously, B-cell depletion was noted to also attenuate CD8+ T cell responses in the same patients, suggesting a possible role for B cell:CD8+ T cell interactions in vivo. Though relatively less studied than CD4+ T cells, CD8+ T cells are strongly implicated in the inflamed MS central nervous system (CNS), with presence at sites of inflammation and injury of both CD8+ mucosal associated invariant T (MAIT) cells, and non-MAIT (conventional) CD8+ T cells. Here we set out to study potential interactions between B cells and both MAIT and conventional CD8+ T cells. We found that, in vitro, human B cells can suppress the proliferation of conventional CD8+ T cells yet significantly promote MAIT cell expansion. This differential B cell effect on the CD8+ T cell subsets was rather dependent on the differentiation stage of the B cells. B cells also differentially enhanced CD8+ T-cell subset cytotoxicity, an effect that did not require cell: cell contact, as it could be mediated through B-cell secreted products. B cells also modulated cytokine production in CD8+ T cells, by selectively enhancing IFN-γ production in MAIT cells, and suppressing GM-CSF and TNF production in non-MAIT cells. Further, anti-CD20 treatment resulted in an in vivo shift in the profile of circulating CD8+ T cells, including a reduction in MAIT cells, and diminished CD8+ T cell pro-inflammatory cytokine production, that in part mirrored our in vitro findings. Together this data indicates that disease-relevant cross-talk may exist between B cells and CD8+ T cells, including the capacity of B cells to reciprocally impact distinct CD8+ T cell subsets. The particular capacity of B cells to induce activation and effector responses of MAIT cells may be particularly relevant to MS pathophysiology and possibly to the therapeutic mode of action of anti-CD20 therapy.
Olivia Smith (1) – Courtney Bannerman (1) – Jihoon Choi (1) – Margot Gunning (1) – Scott Duggan (2) – Qingling Duan (1) – Nader Ghasemlou (1)
Queen’s University, Department of Biomedical and Molecular Sciences, Kingston, Canada (1) – Anesthesiology Department, Kingston General Hospital, Kingston, Canada (2)
Chronic pain affects 60-80% of people living with spinal cord injury (SCI), making day-to-day life exceedingly more difficult. Current treatment options, with opioids as the gold-standard, are ineffective at best and can carry risks including addiction, tolerance, and dependence. There is therefore a need for more specific and safer treatment strategies. We have now identified specific genes in circulating immune cells of SCI patients who develop chronic pain using Weighted Gene Co-expression Network Analysis (WGCNA) and KEGG pathway analysis. This human dataset is used to identify those genes/pathways expressed in the mouse. To this end, we developed a modified SCI contusion injury model using the Infinite Horizons spinal cord impactor that results in increased mechanical and thermal hypersensitivity after compression, in comparison to contusion injury alone. Expression patterns of specific genes and pathways of interest from the human dataset are assessed using quantitative PCR in our pain/no pain models of SCI. Our work seeks to better understand the mechanisms underlying the development and maintenance of chronic SCI pain, with a focus on the contribution of the immune response to functional outcomes after injury.
Ben Korin (1, 2) – Shimrit Avraham (3) – Hilla Azulay-Debby (1, 2) – Dorit Farfara (1, 2) – Fahed Hakim (5, 6) – Asya Rolls (1, 2, 4)
Department of Neuroscience (1) – Department of Immunology (2) – Department of Cell Biology and Cancer Science, Rappaport Faculty of Medicine, Technion – Israel Institute of Technology, Haifa, Israel (3) – Technion Integrated Cancer Center (TICC), Technion – Israel Institute of Technology, Haifa, Israel (4) – Pediatric Pulmonary Unit, Rambam Health Care Campus, Haifa, Israel (5) – Cancer Research Center, EMMS Hospital, Nazareth, Israel (6)
Increasing evidence highlight the involvement of immune cells in brain activity and its dysfunction. The brain’s immune compartment is a dynamic ensemble of cells that can fluctuate even in naïve animals. However, the dynamics and factors that can affect the composition of immune cells in the naïve brain are largely unknown. Here we examine whether acute sleep deprivation can affect the brain’s immune compartment (parenchyma, meninges and choroid plexus). Using high-dimensional mass cytometry analysis we broadly characterize the effects of short-term sleep deprivation on the immune composition in the mouse brain. We find that after 6 hours of sleep deprivation there is a significant increase in the abundance of B cells in the brain compartment. This effect can be accounted for, at least in part, by the elevated expression of the migration-related receptor, CXCR5, on B cells and its ligand, cxcl13, in the meninges following sleep deprivation. Thus, our study reveals that short-term sleep deprivation affects the brain’s immune compartment, offering a new insight into how sleep disorders can affect brain function and potentially contribute to neurodegeneration and neuroinflammation.
Maryam Nakhaei-Nejad (1) – Dianhui Zhang (1) – David Barilla (1) – Fabrizio Giuliani (1)
University of Alberta, Department of Medicine, Edmonton, Canada (1)
Multiple Sclerosis (MS) is an autoimmune disease of the Central Nervous System characterized by immune-mediated demyelination and axonal injury. In MS patients, in addition to the loss of neurites, synaptic loss has been reported in both lesions and normal-appearing tissue. Synaptic changes have been associated with C3 complement-dependent phagocytosis in the hippocampus of MS patients. The process of phagocytosis is tightly regulated via a number of positive (“eat me”) and negative (“don’t eat me”) signals that lead to proper uptake of damaged cells or foreign molecules while preserving healthy cells. One of these “don’t eat me” signals is mediated by the interaction between CD47 on target cells and its receptor SIRPalpha on phagocytes, which has been shown to be involved in preventing excess synaptic pruning by microglia in developmental stages. Although a decrease in CD47 has been observed in MS lesions, it is not clear how CD47 is downregulated and if this decreased expression contributes to phagocyte-mediated synaptic loss and myelin stripping in MS. Thus, in our study we aimed to identify conditions that could mediate CD47 downregulation on neurons. To determine the effect of pro-inflammatory conditions on neuronal CD47 expression, primary human neurons (HN), and neurons differentiated from human induced pluripotent stem cells (iPS-Nn) were incubated with activated human lymphocyte supernatant (via CD3 and CD28 costimulation) or specific inflammatory cytokines, IFN-g and TNF-a. To our surprise, we detected an increase in CD47 mRNA levels (2.66-fold and 1.95-fold increase) in activated PBMC supernatant treated iPS-Nns and HNs, respectively, and a 4.36-fold increase in IFN-g treated iPS-Nns. TNF-alpha did not affect CD47 expression at mRNA levels. We are currently investigating the importance of CD47 upregulation in neurons using CD47 RNAi mediated knock down. Next, we tested whether CD47-SIRPalpha interaction is important in myelin uptake. Human myelin fractions were labelled with a pH sensitive dye, pHrodo, and myelin uptake and its localization to lysosomes was measured by flow cytometry. CD47 blockade significantly increased myelin uptake by phagocytes. Overall, unexpectedly, we have observed that under inflammatory conditions, neurons upregulate CD47 expression, which might have protective functions in situations of cellular stress. Also, CD47 interaction with SIRPalpha is involved in myelin debris uptake as its inhibition increases myelin uptake. The role of CD47 expression in myelin stripping needs further investigation.
Solène Pradeloux (1, 2) – Mélissa Côté (1) – Catherine Fontaine-Lavallée (1) – Katherine Coulombe (2) – Frédéric Calon (1) – Denis Soulet (2)
Faculty of Pharmacy, Laval University (1) – CHU de Québec – Laval University research centre (2)
Patients with Parkinson’s disease have motor symptoms, which are often preceded by gastrointestinal disorders associated with the alteration of dopaminergic neurons located in the myenteric plexus. Our laboratory has previously demonstrated the importance of the inflammatory response in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced toxicity on dopaminergic neurons of the enteric nervous system. MPTP is a neurotoxin used to mimic the loss of dopaminergic neurons observed in Parkinson’s disease. In this study, we aim to evaluate the impact of aging on enteric and central nervous systems neuroprotection and immunomodulation in a murine model of Parkinson’s disease, in order to determine whether aging affects the myenteric neurons sensitivity to MPTP. NF-kappa B-EGFP mice ranging from two months to one year old received four intraperitoneal injections of saline or MPTP (at 8mg/kg) at two-hour intervals. Mice were euthanised 5 or 10 days later, and the brain and gut were removed. Immunofluorescence and immunohistochemistry markings with anti-tyrosine hydroxylase (TH) and anti-ionised calcium binding adapter molecule 1 antibodies (Iba1) were performed on the substantia nigra, the striatum and the myenteric plexus for a stereological count of dopaminergic neurons (TH+) and macrophages/microglia (Iba+). Our preliminary results reveal that density in dopaminergic neurons and macrophages are respectively decreased and increased in young mice (about 100 days old) treated with MPTP. In contrast, MPTP injection in aged mice (approximately 300 days old) did not alter the number of dopaminergic neurons and macrophages compared to saline mice. The older the saline mice are, the lower the density of TH+ neurons and the higher the inflammation are. These results suggest that only young dopaminergic neurons are sensitive to MPTP.
Moein Yaqubi (1) – Kelly Perlman (1) – Qiao-Ling Cui (1) – Florian Pernin (1) – Ioaniss Ragousssis (2) – Carlo Santaguida (3) – Jack Antel (1) – Luke Healy (1)
Neuroimmunology Unit, Montreal Neurological Institute, McGill University (1) – Department of Human Genetics, McGill University, Montréal, Canada (2) – McGill University health center, Montreal, Canada (3)
Microglia as the main resident immune cells of the central nervous system (CNS) have critical, underappreciated roles in development and in the maintenance of brain homeostasis. Microglia also become highly reactive throughout the course of all neurological disease including multiple sclerosis. To gain a clear insight regarding the role of microglia in CNS pathology we first need to understand the molecular mechanisms underlying development of these cells. Furthermore, it has been shown that animal model organisms consistently fail to mimic human physiological conditions. In the present study we isolate microglia from human surgical brain tissues from three different developmental timepoints including fetal, pediatric and adult. We investigate temporal changes in gene expression profiles and study heterogeneity within the microglia populations across the developmental trajectory using a single cell RNA-sequencing approach. Preliminary analysis shows that each time point has its own specific expression profile, with fetal and pediatric samples being more similar as compared to the adult population. HSPA1A, APOC1 and HLA-DPA1 are the most up regulated genes in fetal, pediatric and adult samples. In addition, the list of transcription factors (TFs) which control expression of genes have been identified in each time point. We also analyze the expression profiles of microglia isolated from human spinal cord autopsy samples which reveals unique transcriptional signatures of brain and spinal cord microglia. Microglia are implicated in the pathogenesis of most neurodegenerative and neuroimmunological diseases. Understanding the exact molecular mechanisms which underlie their development will likely provide insights into the pivotal roles these cells in normal brain development and disease.
Gonzalo Piñero (1) – Marianela Vence (2) – Paula Soto (1) – Vanina Usach (1) – Patricia Setton-Avruj (1)
CONICET, Universidad de Buenos Aires, CABA, Argentina (1) – CONICET, IQUIFIB, CABA, Argentina (2)
Bone marrow cells include different cell types containing a minority multipotent fraction. For this reason, they have recently become a therapeutic alternative to mesenchymal stem cells, as culture is not required and phenotypic transformations can be hence avoided.
Wallerian degeneration induced by nerve sectioning or compression is a simple and extremely useful experimental approach to study the pathophysiology of peripheral nervous system degenerative disorders. In this model, we have shown systemically transplanted bone marrow cells to spontaneously migrate to and remain in the injured nerve for as long as 60 days. A small number of these cells upregulated markers unexpressed before transplant, leading to cell phenotypic changes and transdifferentiation to Schwann cells, while a significantly larger proportion left the tissue once the inflammatory phase had finished. They also enhanced axonal regeneration and remyelination, promoted functional recovery and prevented lesion-induced hyperalgesia.
The aim of the present work is to evaluate whether transplanted bone marrow cells exert their well-established beneficial effect on sciatic nerve regeneration through immunomodulation. Adult C57BL/6 mice received intravenous transplantation of either bone marrow cells or vehicle after 8-second sciatic nerve crush. Along recovery, functional aspects were evaluated through hot plate and walking track tests. Animals were then sacrificed for immunohistochemistry, ELISA, qPCR and flow cytometry studies. The mouse model resembles results obtained in rats in terms of cell migration, histological and functional recovery. In order to study the immunomodulatory effects of cell therapy, we assessed the profile of the main cytokines involved and the phenotype of macrophages in the lesion area. So far, qPCR and flow cytometry assays have shown that cell transplant reduces the expression of pro-inflammatory markers and anticipates the expression of anti-inflammatory ones. Nevertheless, further studies are required to fully corroborate immunomodulation effects.
Beatriz Marton Freire (1) – Juliana Terzi Maricato (1) – Alexandre Salgado Basso (1)
Federal University of São Paulo, Immunology Division, EPM, São Paulo, Brazil (1)
Introduction: The ability of the nervous system to modulate immune responses is well known. It happens, for example, through activation of adrenergic receptors (AR) on immune cells such as macrophages (MO). MO is an evolutionarily conserved cell type; its effector functions are diverse and includes orchestration of inflammation and promotion of tissue repair. Methods: Macrophages were differentiated in vitro from bone marrow progenitor cells. For activation of AR in macrophages, cells were treated with 1 micromolar of a Beta2 AR (B2AR) specific agonist 15 minutes prior to stimulation with 500 microgram of LPS plus 1 nanogram of IFN-gamma. Transcripts were measured by RT-qPCR assay, the production of cytokines was measured using ELISA and the expression of cell surface molecules was analyzed by flow cytometry. Results: Analysis of relative gene expression showed that treatment with the B2AR specific agonist, prior to stimulation with LPS+IFN-gamma, decreased the expression of pro-inflammatory genes, such as Inos, Il12p35, Il12p40, Tnfa and Il6. Additionally, activation of B2AR promotes increase in the gene expression of Arg1. Accordingly, the amount of IL6, TNF-alfa and nitric oxide (NO) produced by MO treated with B2AR agonist was also decreased. Macrophages were treated with noradrenaline, an endogenous ligand of B2AR and the decrease in IL6, TNF-alfa and NO was also observed. Blockage of PKA and EPAC, which are proteins known to participate in the signaling pathway of B2AR, did not revert the downregulation of IL6 and TNF-alfa production and secretion. However, it was not observed difference in the expression of activation markers (CD80/CD86, MHCII and CCR2) on macrophages after B2AR activation. Moreover, activation of B2AR did not affect phagocytosis activity in macrophages. Conclusions: We found that activation of B2AR could modulate macrophage activity, promoting downregulation of genes and proteins linked to a pro inflammatory response. This modulation is independent of EPAC and PKA. Financial Support: FAPESP; CAPES