Benjamin Segal, M.D. - US grants

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune demyelinating disease of the central nervous system (CNS) induced by CD4+ T cells reactive with myelin antigens, including myelin basic protein (MBP). It is widely used as an animal model of multiple sclerosis (MS). There is growing evidence for a role of the Th1 polarizing monokine, IL-12, in the pathogenesis of EAE as well as MS. We have recently found that primed MBP-reactive CD4+ Th1 cells are prevented from inducing disease in naive syngeneic recipients when coinjected with a neutralizing antibody against IL-12. Spinal cords from the protected mice are free of infiltrates. In this proposal we plan to expand upon these findings to elucidate the mechanism of action of anti-IL-12 in suppressing CNS inflammation. To do so, we will use an adoptive transfer protocol in which donor T cells can be identified since they express a congenic Thy marker. The goal of Aim 1 is to distinguish between two hypotheses: (i) anti- IL-12 blocks the passage of effector T cells across the blood- brain-barrier; or (ii) anti-IL-12 triggers the premature death of effector T cells. Based on the results of preliminary experiments, we will measure the effects of IL-12 on the expression of candidate adhesion and chemotactic molecules or pro- and anti-apoptotic mediators. In Aim2 we will assess the efficacy of anti-IL-12 therapy when initiated following the onset of clinical signs. These experiments will test the hypothesis that autoimmune effector cells depend on IL-12 to maintain their biological activities relatively late in the pathogenic process. We will assess whether anti-IL-12 suppresses the production of myelinotoxic molecules, such as TNFalpha or Lymphotoxinalpha, or induces the production of immunosuppressive factors, such as IL- 10 or TGFbeta, in the CNS. Furthermore, we will determine whether IL-12 neutralization blocks determinant spreading in the adoptive transfer recipients, thereby suppressing future relapses. These studies may have therapeutic relevance to MS as well as provide insights into the pathways by which IL-12 regulates differentiated effector T cells.

[unreadable] DESCRIPTION (provided by applicant): Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease of the central nervous system (CNS) that is widely used as an animal model of multiple sclerosis (MS). In this proposal, we will test a novel model of the immunopathogenesis of EAE focusing on factors that drive chronic inflammation in the CNS. In preliminary studies we demonstrated that CD11c ( dendritic-like cells accumulate in white matter infiltrates during EAE. Furthermore, the lymphoid chemokines, CCL19, CCL21 and CXCL13, are upregulated in spinal cords of mice at EAE onset and steadily rise during disease progression. Based on these findings, we propose the following hypotheses: (i) CNS CD11c ( cells differentiate from resident microglia under the influence of GM-CSF that, in turn, is secreted by infiltrating T cells, (ii) CD11c ( cells then secrete proinflammatory cytokines (such as IL-12) and chemokines including CXCL13, CCL19 and CCL21; (iii) CXCL13, CCL19 and CCL21 act to recruit leukocytes to the CNS and help shape the cellular composition of perivascular infiltrates; (iv) local production of lymphoid chemokines also triggers lymphoid neogenesis (the development of lymph node-like structures) and B cell activation within the CNS; (v) ultimately, CNS lymphoid chemokines contribute to the severity and chronicity of clinical EAE. We will test each step of the above hypothetical scheme using novel experimental approaches. In Aim 1 we will construct bone marrow chimeras with GFP-transgenic or GM-CSF deficient mice to determine the lineage of CNS CD11c ( cells. In Aims 2 and 3, immunocompetent and lymphoid chemokine deficient mice will be compared with regard to susceptibility to adoptively transferred EAE, recruitment of leukocytes to white matter lesions, and lymphoid neogenesis and B cell activation within the CNS. Our studies might provide new insights into the pathogenesis of autoimmune demyelination and lead to innovative therapies such as reagents that antagonize lymphoid chemokines and/or block CNS dendritic cell accumulation. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Blood borne myeloid cells (macrophages and dendritic cells) comprise a major component of the inflammatory infiltrates in patients with multiple sclerosis (MS) and animals with experimental autoimmune encephalomyelitis (EAE). They have been implicated in the demyelination and axonal loss that cause disability in these disorders. However, relatively little is known about how myeloid cells are regulated in the periphery and CNS during autoimmune demyelinating disease. We have found that myeloid progenitor cells are mobilized from the bone marrow at an accelerated rate immediately prior to EAE exacerbations. Newly exported Ly6ChiCD11b+ monocytes infiltrate the CNS during the preclinical stage and give rise to the CD11c+MHC ClassIIhi DC that constitute a significant percent of neuroinflammatory cells during the symptomatic stage. In Aim 1 we will investigate the cytokine pathways and molecular mechanisms underlying the expansion and mobilization of bone marrow myeloid cells during EAE. Our working hypothesis is that GM-CSF and MIP-1a, induced by myelin-specific T cells, stimulate bone marrow stromal cells to produce G-CSF and CXCL1/2. These factors, in turn, activate resident neutrophils to secrete proteases that degrade chemokines and adhesion molecules critical for the sequestration of hematopoietic precursor cells in intramedullary niches. Interruption of any step in this pathway (ex, by neutralizing G-CSF or by inactivating proteases) will prevent myeloid cell release and ultimately exhaust the peripheral monocyte pools that provide a source of CNS infiltrating cells during relapses. In Aim 2 we will investigate the factors that stimulate Ly6ChiCD11b+ monocytes to differentiate into CD11c+MHC Class IIhi DC within the CNS. We propose that CNS infiltrating monocytes acquire characteristics of myeloid dendritic cells consequent to direct interactions with myelin- specific T cells. The roles of candidate soluble factors (such as GM-CSF and TNF) and cell surface molecules (such as RANKL, CD40L and Lymphotoxin-[unreadable]) will be assessed. Our studies are likely to provide insights into the pathways driving the mobilization and development of pathogenic myeloid cells during autoimmunity. The results are expected to suggest novel therapeutic targets and biomarkers in MS related to myeloid cell dysregulation. PUBLIC HEALTH RELEVANCE: Although recent advancements in the immunotherapy of multiple sclerosis (MS) have focused on the T cell component of the autoimmune response, myeloid cells (including macrophages and dendritic cells) compose the majority of CNS-infiltrating cells in MS and its model, experimental autoimmune encephalomyelitis (EAE), and mediate direct damage to neurons and glia. The purpose of this proposal is to elucidate the cytokine pathways and molecular mechanisms that underlie the mobilization of myeloid cells from the bone marrow and that drive their differentiation into pathogenic effector cells during autoimmune demyelinating disease. It is anticipated that our results will lead to the discovery of novel biomarkers and therapeutic targets related to myeloid cell dysregulation in MS.

Multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS), is the most common cause of non-traumatic neurological disability in young adults in the Western Hemisphere. Significant progress has been made in the development of disease modifying therapies (DMT) that decrease the frequency of clinical MS relapses by blocking or depleting pathogenic lymphocytes. However, none of the approved DMT are curative, and none are effective in all patients. There are no treatments that slow, or reverse, progressive forms of MS. The current proposal is based on the contention that myeloid cells, and the factors that modulate them, should be considered as candidate therapeutic targets for the treatment of relapsing or progressive forms of MS that are unresponsive to currently used DMT. Myeloid cells (including macrophages, dendritic cells and microglia) comprise a major component of the neuroinflammatory infiltrates in patients with MS and in mice with experimental autoimmune encephalomyelitis (EAE, widely used as an animal model of MS). Abnormalities of myeloid cells have been documented in relapsing-remitting as well as progressive forms of MS. The overall goal of this proposal is to investigate the characteristics of the myeloid cells that accumulate in the central nervous system (CNS) during different stages of autoimmune demyelinating disease. Bone marrow derived macrophages have been broadly classified as pro-inflammatory M1 cells that express inducible nitric synthase (iNOS), and reparative M2 cells that express arginase-1 (Arg1). Our preliminary studies show that the phenotypes of CNS-infiltrating myeloid cells evolve with the progression and remission of EAE. During the preclinical stage, a significant percentage of CNS myeloid cells express iNOS, but none express Arg1. At clinical onset, iNOS+Arg1+ double positive and Arg1+ single positive myeloid cells appear in CNS infiltrates. As mice enter the remission phase only Arg1+ single positive myeloid cells remain. In Aim 1 we will use a panel of genetically engineered mice to elucidate the pathways that drive iNOS+ versus Arg1+ myeloid cell polarization in the inflamed CNS during EAE. In Aims 2 and 3 we will characterize the transcriptomes and biological properties of the CNS-infiltrating myeloid subsets, and determine the clinical and pathological consequences of depleting, skewing or blocking the functions of Arg1+ or iNOS+ CNS myeloid cells, respectively. In Aim 4 we will investigate the density and distribution of myeloid cell subsets in a panel of active, chronic active and relapsing MS lesions in autopsied human brain sections. We are hopeful that this research will ultimately lead to the development of novel MS therapies that suppress pathogenic myeloid subsets, while enhancing reparative subsets, with the goal of mitigating, and even reversing, disability in individuals with relapsing-remitting and progressive forms of MS.

Axonopathy is an early and prominent pathological feature of glaucoma, optic neuritis and traumatic optic nerve injury. Permanent loss of vision in all of these conditions is secondary, in large part, to a failure of retinal ganglion cells (RGC), the output neurons of the optic nerve, to survive and regenerate their axons. There is a dire need to develop novel therapeutic interventions that overcome barriers to repair in the eye, promote RGC survival and RGC axonal regrowth, thereby mitigating, or even reversing, visual loss. In this proposal we investigate a novel subset of neutrophils that accumulate in the vitreous body following intraocular (i.o.) injection of mice with the fungal cell wall extract, zymosan, and are associated with the regrowth of severed RGC axons. In preliminary studies we have demonstrated that adoptive transfer of zymosan-elicited neutrophils directly into the vitreous of mice with optic nerve crush (ONC) injury is sufficient to rescue RGC and stimulate RGC axon regrowth. These neutrophils are characterized by ring-form nuclei and the cell surface phenotype CD14+Ly6Glow. They express high levels of transcripts for arginase-1 and CD206. Our major goals are to elucidate the factors that drive the differentiation of reparative neutrophils and develop protocols to generate them in vitro for therapeutic application in individuals with optic neuropathy. In Aim 1 we will test our hypothesis that IL-4 and granulocyte-colony stimulating factor (G-CSF) act synergistically to drive the differentiation of pro-regenerative neutrophils in vivo in mice subjected to i.o. zymosan injection and ONC injury. A role of IL-4 was suggested by our finding that CD14+Ly6Glow neutrophils express high levels of IL-4 signaling molecules, and IL-4 protein is produced in the vitreous following i.o. administration of zymosan. G- CSF is also upregulated in the vitreous and it was recently shown to induce IL-4 receptor expression on neutrophils. We will determine the kinetics and cellular source of IL-4, IL-4 receptor chains, G-CSF and G-CSF receptor in zymosan injected eyes. Loss and gain of function experiments, using a panel of conditional knock- out mice and bone marrow chimeras, will be performed to assess the roles of IL-4 and G-CSF signaling in the development of CD14+Ly6Glow neutrophils, RGC survival and axonal regeneration following i.o. zymosan injection and ONC. This research could ultimately lead to the development of novel, or the repurposing of established, immunomodulators to promote the differentiation and expansion of neuroregenerative neutrophils in patients with optic neuropathy secondary to glaucoma, optic neuritis or trauma. Our exploratory experiments have shown that murine bone marrow neutrophils acquire characteristics of zymosan-elicited CD14+Ly6Glow neutrophils, including the ability to drive axonal regeneration, following in vitro polarization with IL-4 and G- CSF. The overall goal of Aim 2 is to optimize protocols for the generation of pro-regenerative neutrophils from murine bone marrow precursors ex vivo. The goal of Aim 3 is to assess the neuroregenerative potential of IL-4 modulated human neutrophils that could, potentially, be re-infused into patients with optic neuropathy, as an autologous cell therapy.

Abstract Multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS), is the most common cause of non-traumatic neurological disability in young adults in the Western Hemisphere. Disease modifying agents (DMA) that deplete lymphocytes, or block their migration to the CNS, have been shown to reduce the frequency of MS relapses in Phase 3 clinical trials. However, none of those drugs are curative and none are effective in all patients. Granulocyte-macrophage colony-stimulating factor (GM-CSF), a myeloid cell growth factor, has emerged as a promising therapeutic target in MS. GM-CSF producing T cells occur at a relatively high frequency in untreated relapsing remitting MS patients, and have been identified in MS brain tissue. Deficiency or neutralization of GM-CSF confers resistance against experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The mechanism of action of GM-CSF in autoimmune demyelination has yet to be definitively demonstrated. In preliminary studies we found that GM-CSF signaling is necessary for the development of chronic neurological deficits in mice with EAE induced by the adoptive transfer of myelin-specific Th17 cells. GM-CSF blockade or deficiency resulted in a decrease in the numbers and percentages of CNS-infiltrating donor T cells and granulocytes, while CD88 (C5a receptor) expressing myeloid cells were enriched. We found that CNS CD88+ monocytes and myeloid dendritic cells (mDC) are poor antigen presenting cells for myelin-reactive T cells. In animal models of cancer and asthma, CD88+ myeloid cells have immunoregulatory properties. Based on these observations, we propose to investigate two potential mechanisms of action of GM-CSF during the effector phase of EAE. In Aim 1, we will test our hypothesis that GM-CSF deficiency accelerates the conversion of pro-inflammatory iNOS+ myeloid cells in EAE infiltrates to an immunosuppressive CD88+ arginase-1+ phenotype. We will compare cell surface marker expression, antigen presenting capacity, cytokine profiles and immunoregulatory properties of GM-CSF receptor deficient (GM-CSFR-/-) versus WT myeloid cells isolated from the CNS of GM-CSFR-/-/ WT ? WT mixed bone marrow chimeric mice at peak EAE. We will also determine whether treatment of GM-CSFR-/- adoptive transfer recipients with CD88 antagonists exacerbates EAE and promotes chronic disability. In Aim 2 we will investigate the role of GM-CSF in the recruitment of neutrophils, that mediate blood-brain-barrier breakdown, to the CNS. We will determine if forced CNS expression of CXCL1 (a neutrophil attracting chemokine), or treatment with recombinant granulocyte-colony stimulating factor (G-CSF), prevents EAE remission in GM- CSFR-/- hosts. The proposed research could increase our understanding of the immunopathogenesis of EAE and MS, and eventually lead to the development of novel myeloid cell modulating drugs for the treatment of MS patients who do not respond to lymphocyte-targeting DMA.

Axonopathy is an early and prominent pathological feature of many central nervous system (CNS) disorders, including brain and spinal cord trauma, optic neuropathy, Multiple Sclerosis, early stage Alzheimer?s disease, and subcortical ischemia. Poor clinical outcomes in all of these neurological conditions are due, in large part, to the limited regenerative capacity of adult CNS neurons, including retinal ganglion cells (RGC, the neurons that give rise to the optic nerve). There is a dire need to develop novel therapeutic interventions that overcome barriers to repair in the adult CNS and promote axonal regrowth. The studies proposed here are based on our discovery of a novel subset of pro-regenerative neutrophils, characterized by the cell surface phenotype Ly6GlowCD14+, that accumulate in the posterior chamber of the eye or the peritoneal cavity following local administration of the yeast cell wall extract, zymosan. These neutrophils bear a ring-form nucleus and express high levels of pattern recognition receptor, dectin-1, as well as transcripts for arginase-1 and CD206. In preliminary studies we demonstrated that adoptive transfer of zymosan-elicited Ly6GlowCD14+ neutrophils directly into the vitreous of mice with optic nerve crush (ONC) injury is sufficient to rescue RGC from cell death and to stimulate the regrowth of severed RGC axons. Furthermore, conditioned media harvested from cultures of Ly6GlowCD14+neutrophils induce neurite outgrowth of dissociated RGC and dorsal root ganglion neurons in vitro. The overall goal of the current proposal is to elucidate the pathways that underlie the differentiation, survival and mechanism of action of these unconventional reparative neutrophils, and to leverage the knowledge gained for the development of immunomodulatory therapies that mitigate, or even reverse, damage to CNS neurons and axons. In Aim 1 we will test our hypothesis that transforming growth factor (TGF)-? drives the differentiation of Ly6GlowCD14+ neutrophils in vivo following the administration of zymosan. In Aim 2 we will determine the role of hypoxia induced factor (HIF)-1? in the stabilization, survival and biological functions of Ly6GlowCD14+ neutrophils. In Aim 3 we will optimize protocols for the generation of pro-regenerative neutrophils from bone marrow precursors ex vivo. Selected neutrophil lines will be infused into mice with ONC injury to assess their efficacy as an autologous cellular therapy. In addition, we will use proteomic and genetic approaches to characterize the soluble factors present in Ly6Glow neutrophil-conditioned media that are responsible for enhanced neurite outgrowth. A future direction will be to administer candidate neuroregenerative factors to mice with axonopathy as disease modifying agents. We are hopeful that the data generated by our study will ultimately lead to the development of innovative cell based therapies and/ or immunomodulatory drugs with neuroprotective/ regenerative properties that restore lost neurological functions in patients with CNS trauma or other conditions characterized by axonopathy.