Cytokine polarized, alternatively activated bone marrow neutrophils drive axon regeneration.

The adult central nervous system (CNS) possesses a limited capacity for self-repair. Severed CNS axons typically fail to regrow. There is an unmet need for treatments designed to enhance neuronal viability, facilitate axon regeneration and ultimately restore lost neurological functions to individuals affected by traumatic CNS injury, multiple sclerosis, stroke and other neurological disorders. Here we demonstrate that both mouse and human bone marrow neutrophils, when polarized with a combination of recombinant interleukin-4 (IL-4) and granulocyte colony-stimulating factor (G-CSF), upregulate alternative activation markers and produce an array of growth factors, thereby gaining the capacity to promote neurite outgrowth. Moreover, adoptive transfer of IL-4/G-CSF-polarized bone marrow neutrophils into experimental models of CNS injury triggered substantial axon regeneration within the optic nerve and spinal cord. These findings have far-reaching implications for the future development of autologous myeloid cell-based therapies that may bring us closer to effective solutions for reversing CNS damage.

Respiratory tract Moraxella catarrhalis and Klebsiella pneumoniae can promote pathogenicity of myelin-reactive Th17 cells.

The respiratory tract is home to a diverse microbial community whose influence on local and systemic immune responses is only beginning to be appreciated. The airways have been linked with the trafficking of myelin-specific T-cells in the preclinical stages of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Th17 cells are important pathogenic effectors in MS and EAE but are innocuous immediately following differentiation. Upregulation of the cytokine GM-CSF appears to be a critical step in their acquisition of pathogenic potential, but little is known about the mechanisms that mediate this process. Here, primed myelin-specific Th17 cells were transferred to congenic recipient mice prior to exposure to various human respiratory tract-associated bacteria and T-cell trafficking, phenotype and the severity of resulting EAE were monitored. Disease was exacerbated in mice exposed to the Proteobacteria Moraxella catarrhalis and Klebsiella pneumoniae, but not the Firmicute Veillonella parvula, and this was associated with significantly increased GM-CSF+ and GM-CSF+IFNγ+ ex-Th17-like donor CD4 T cells in the lungs and central nervous system (CNS) of these mice. These findings support the concept that respiratory bacteria may contribute to the pathophysiology of CNS autoimmunity by modulating pathogenicity in crucial T-cell subsets that orchestrate neuroinflammation.

Characterization of Zymosan-Modulated Neutrophils With Neuroregenerative Properties.

Recent studies using advanced techniques such as single cell RNA sequencing (scRNAseq), high parameter flow cytometry, and proteomics reveal that neutrophils are more heterogeneous than previously appreciated. Unique subsets have been identified in the context of bacterial and parasitic infections, cancer, and tissue injury and repair. The characteristics of infiltrating neutrophils differ depending on the nature of the inflammation-inciting stimulus, the stage of the inflammatory response, as well as the tissue microenvironment in which they accumulate. We previously described a new subpopulation of immature Ly6Glow neutrophils that accumulate in the peritoneal cavity 3 days following intraperitoneal (i.p.) administration of the fungal cell wall extract, zymosan. These neutrophils express markers of alternative activation and possess neuroprotective/regenerative properties. In addition to inducing neurite outgrowth of explanted neurons, they enhance neuronal survival and axon regeneration in vivo following traumatic injury to the optic nerve or spinal cord. In contrast, the majority of neutrophils that accumulate in the peritoneal fluid 4 hours following i.p. zymosan injection (4h NΦ) have features of conventional, mature Ly6Ghi neutrophils and lack neuroprotective or neuroregenerative properties. In the current study, we expand upon on our previously published observations by performing a granular, in-depth analysis of these i.p. zymosan-modulated neutrophil populations using scRNAseq and high parameter flow cytometry. We also analyze cell lysates of each neutrophil population by liquid chromatography/mass spectrometry. Circulating blood neutrophils, harvested from naive mice, are analyzed in parallel as a control. When samples were pooled from all three groups, scRNAseq revealed 11 distinct neutrophil clusters. Pathway analyses demonstrated that 3d NΦ upregulate genes involved in tissue development and wound healing, while 4h NΦ upregulate genes involved in cytokine production and perpetuation of the immune response. Proteomics analysis revealed that 3d NΦ and 4h NΦ also express distinct protein signatures. Adding to our earlier findings, 3d NΦ expressed a number of neuroprotective/neuroregenerative candidate proteins that may contribute to their biological functions. Collectively, the data generated by the current study add to the growing literature on neutrophil heterogeneity and functional sub-specialization and might provide new insights in elucidating the mechanisms of action of pro-regenerative, neuroprotective neutrophil subsets.

Encephalitogenic and Regulatory CD8 T Cells in Multiple Sclerosis and Its Animal Models.

Multiple sclerosis (MS), a neuroinflammatory disease that affects millions worldwide, is widely thought to be autoimmune in etiology. Historically, research into MS pathogenesis has focused on autoreactive CD4 T cells because of their critical role in the animal model, experimental autoimmune encephalomyelitis, and the association between MS susceptibility and single-nucleotide polymorphisms in the MHC class II region. However, recent studies have revealed prominent clonal expansions of CD8 T cells within the CNS during MS. In this paper, we review the literature on CD8 T cells in MS, with an emphasis on their potential effector and regulatory properties. We discuss the impact of disease modifying therapies, currently prescribed to reduce MS relapse rates, on CD8 T cell frequency and function. A deeper understanding of the role of CD8 T cells in MS may lead to the development of more effective and selective immunomodulatory drugs for particular subsets of patients.

Analysis of the immune response to sciatic nerve injury identifies efferocytosis as a key mechanism of nerve debridement.

Sciatic nerve crush injury triggers sterile inflammation within the distal nerve and axotomized dorsal root ganglia (DRGs). Granulocytes and pro-inflammatory Ly6Chigh monocytes infiltrate the nerve first and rapidly give way to Ly6Cnegative inflammation-resolving macrophages. In axotomized DRGs, few hematogenous leukocytes are detected and resident macrophages acquire a ramified morphology. Single-cell RNA-sequencing of injured sciatic nerve identifies five macrophage subpopulations, repair Schwann cells, and mesenchymal precursor cells. Macrophages at the nerve crush site are molecularly distinct from macrophages associated with Wallerian degeneration. In the injured nerve, macrophages 'eat' apoptotic leukocytes, a process called efferocytosis, and thereby promote an anti-inflammatory milieu. Myeloid cells in the injured nerve, but not axotomized DRGs, strongly express receptors for the cytokine GM-CSF. In GM-CSF-deficient (Csf2-/-) mice, inflammation resolution is delayed and conditioning-lesion-induced regeneration of DRG neuron central axons is abolished. Thus, carefully orchestrated inflammation resolution in the nerve is required for conditioning-lesion-induced neurorepair.

Multiple sclerosis relapse risk in the postoperative period: Effects of invasive surgery and anesthesia.

BACKGROUND: Postoperative multiple sclerosis (MS) relapses are a concern among patients and providers. OBJECTIVE: To determine whether MS relapse risk is higher postoperatively. METHODS: Data were extracted from medical records of MS patients undergoing surgery at a tertiary center (2000-2016). Conditional logistic regression estimated within-patient unadjusted and age-adjusted odds of postoperative versus preoperative relapse. RESULTS: Among 281 patients and 609 surgeries, 12 postoperative relapses were identified. The odds of postoperative versus preoperative relapse in unadjusted (odds ratio (OR) = 0.56, 95% confidence interval (CI) = 0.18-1.79; p = 0.33) or age-adjusted models (OR = 0.66, 95% CI = 0.20-2.16; p = 0.49) were not increased. CONCLUSIONS: Surgery/anesthesia exposure did not increase postoperative relapse risk. These findings require confirmation in larger studies.

GM-CSF Promotes Chronic Disability in Experimental Autoimmune Encephalomyelitis by Altering the Composition of Central Nervous System-Infiltrating Cells, but Is Dispensable for Disease Induction.

GM-CSF has been portrayed as a critical cytokine in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) and, ostensibly, in multiple sclerosis. C57BL/6 mice deficient in GM-CSF are resistant to EAE induced by immunization with myelin oligodendrocyte glycoprotein (MOG)35-55 The mechanism of action of GM-CSF in EAE is poorly understood. In this study, we show that GM-CSF augments the accumulation of MOG35-55-specific T cells in the skin draining lymph nodes of primed mice, but it is not required for the development of encephalitogenic T cells. Abrogation of GM-CSF receptor signaling in adoptive transfer recipients of MOG35-55-specific T cells did not alter the incidence of EAE or the trajectory of its initial clinical course, but it limited the extent of chronic CNS tissue damage and neurologic disability. The attenuated clinical course was associated with a relative dearth of MOG35-55-specific T cells, myeloid dendritic cells, and neutrophils, as well as an abundance of B cells, within CNS infiltrates. Our data indicate that GM-CSF drives chronic tissue damage and disability in EAE via pleiotropic pathways, but it is dispensable during early lesion formation and the onset of neurologic deficits.

Myeloid cell plasticity in the evolution of central nervous system autoimmunity.

OBJECTIVE: Myeloid cells, including macrophages and dendritic cells, are a prominent component of central nervous system (CNS) infiltrates during multiple sclerosis (MS) and the animal model experimental autoimmune encephalomyelitis (EAE). Although myeloid cells are generally thought to be proinflammatory, alternatively polarized subsets can serve noninflammatory and/or reparative functions. Here we investigate the heterogeneity and biological properties of myeloid cells during central nervous system autoimmunity. METHODS: Myeloid cell phenotypes in chronic active MS lesions were analyzed by immunohistochemistry. In addition, immune cells were isolated from the CNS during exacerbations and remissions of EAE and characterized by flow cytometric, genetic, and functional assays. RESULTS: Myeloid cells expressing inducible nitric oxide synthase (iNOS), indicative of a proinflammatory phenotype, were detected in the actively demyelinating rim of chronic active MS lesions, whereas macrophages expressing mannose receptor (CD206), a marker of alternatively polarized human myeloid cells, were enriched in the quiescent lesion core. During EAE, CNS-infiltrating myeloid cells, as well as microglia, shifted from expression of proinflammatory markers to expression of noninflammatory markers immediately prior to clinical remissions. Murine CNS myeloid cells expressing the alternative lineage marker arginase-1 (Arg1) were partially derived from iNOS+ precursors and were deficient in activating encephalitogenic T cells compared with their Arg1- counterparts. INTERPRETATION: These observations demonstrate the heterogeneity of CNS myeloid cells, their evolution during the course of autoimmune demyelinating disease, and their plasticity on the single cell level. Future therapeutic strategies for disease modification in individuals with MS may be focused on accelerating the transition of CNS myeloid cells from a proinflammatory to a noninflammatory phenotype. Ann Neurol 2018;83:131-141.

CD4+ T Cells Orchestrate Lethal Immune Pathology despite Fungal Clearance during Cryptococcus neoformans Meningoencephalitis.

Cryptococcus neoformans is a major fungal pathogen that disseminates to the central nervous system (CNS) to cause fatal meningoencephalitis, but little is known about immune responses within this immune-privileged site. CD4+ T cells have demonstrated roles in anticryptococcal defenses, but increasing evidence suggests that they may contribute to clinical deterioration and pathology in both HIV-positive (HIV+) and non-HIV patients who develop immune reconstitution inflammatory syndrome (IRIS) and post-infectious inflammatory response syndrome (PIIRS), respectively. Here we report a novel murine model of cryptococcal meningoencephalitis and a potential damaging role of T cells in disseminated cryptococcal CNS infection. In this model, fungal burdens plateaued in the infected brain by day 7 postinfection, but activation of microglia and accumulation of CD45hi leukocytes was significantly delayed relative to fungal growth and did not peak until day 21. The inflammatory leukocyte infiltrate consisted predominantly of gamma interferon (IFN-γ)-producing CD4+ T cells, conventionally believed to promote fungal clearance and recovery. However, more than 50% of mice succumbed to infection and neurological dysfunction between days 21 and 35 despite a 100-fold reduction in fungal burdens. Depletion of CD4+ cells significantly impaired IFN-γ production, CD8+ T cell and myeloid cell accumulation, and fungal clearance from the CNS but prevented the development of clinical symptoms and mortality. These findings conclusively demonstrate that although CD4+ T cells are necessary to control fungal growth, they can also promote significant immunopathology and mortality during CNS infection. The results from this model may provide important guidance for development and use of anti-inflammatory therapies to minimize CNS injury in patients with severe cryptococcal infections.IMPORTANCE CNS infection with the fungal pathogen Cryptococcus neoformans often results in debilitating brain injury and has a high mortality rate despite antifungal treatment. Treatment is complicated by the fact that immune responses needed to eliminate infection are also thought to drive CNS damage in a subset of both HIV+ and non-HIV patients. Thus, physicians need to balance efforts to enhance patients' immune responses and promote microbiological control with anti-inflammatory therapy to protect the CNS. Here we report a novel model of cryptococcal meningoencephalitis demonstrating that fungal growth within the CNS does not immediately cause symptomatic disease. Rather, accumulation of antifungal immune cells critically mediates CNS injury and mortality. This model demonstrates that antifungal immune responses in the CNS can cause detrimental pathology and addresses the urgent need for animal models to investigate the specific cellular and molecular mechanisms underlying cryptococcal disease in order to better treat treat patients with CNS infections.

IL-12-polarized Th1 cells produce GM-CSF and induce EAE independent of IL-23.

CD4(+) T-helper (Th) cells reactive against myelin antigens mediate the mouse model experimental autoimmune encephalomyelitis (EAE) and have been implicated in the pathogenesis of multiple sclerosis (MS). It is currently debated whether encephalitogenic Th cells are heterogeneous or arise from a single lineage. In the current study, we challenge the dogma that stimulation with the monokine IL-23 is universally required for the acquisition of pathogenic properties by myelin-reactive T cells. We show that IL-12-modulated Th1 cells readily produce IFN-γ and GM-CSF in the CNS of mice and induce a severe form of EAE via an IL-23-independent pathway. Th1-mediated EAE is characterized by monocyte-rich CNS infiltrates, elicits a strong proinflammatory cytokine response in the CNS, and is partially CCR2 dependent. Conversely, IL-23-modulated, stable Th17 cells induce EAE with a relatively mild course via an IL-12-independent pathway. These data provide definitive evidence that autoimmune disease can be driven by distinct CD4(+) T-helper-cell subsets and polarizing factors.

Neutrophil-related factors as biomarkers in EAE and MS.

A major function of T helper (Th) 17 cells is to induce the production of factors that activate and mobilize neutrophils. Although Th17 cells have been implicated in the pathogenesis of multiple sclerosis (MS) and the animal model experimental autoimmune encephalomyelitis (EAE), little attention has been focused on the role of granulocytes in those disorders. We show that neutrophils, as well as monocytes, expand in the bone marrow and accumulate in the circulation before the clinical onset of EAE, in response to systemic up-regulation of granulocyte colony-stimulating factor (G-CSF) and the ELR(+) CXC chemokine CXCL1. Neutrophils comprised a relatively high percentage of leukocytes infiltrating the central nervous system (CNS) early in disease development. G-CSF receptor deficiency and CXCL1 blockade suppressed myeloid cell accumulation in the blood and ameliorated the clinical course of mice that were injected with myelin-reactive Th17 cells. In relapsing MS patients, plasma levels of CXCL5, another ELR(+) CXC chemokine, were elevated during acute lesion formation. Systemic expression of CXCL1, CXCL5, and neutrophil elastase correlated with measures of MS lesion burden and clinical disability. Based on these results, we advocate that neutrophil-related molecules be further investigated as novel biomarkers and therapeutic targets in MS.

In vitro polarization of T-helper cells.

In vitro polarization of CD4(+) T cells along distinct T-helper (Th) lineages is critical for defining the factors and properties that determine the differentiation, stability, and effector functions of each Th subset. Furthermore, polarized cells can be transferred into naïve syngeneic mice to investigate their trafficking patterns and pathological or therapeutic roles in the setting of infection, autoimmunity, and neoplasia. In this chapter, we describe methods for generating and characterizing a spectrum of CD4(+) Th cell lines in vitro. Protocols are provided that use naïve wild-type or T cell receptor (TCR) transgenic CD4+ T cells, or a polyclonal population of primed CD4+ T cells from immunized mice.

Neurosarcoidosis: diagnostic approaches and therapeutic strategies.

PURPOSE OF REVIEW: The aims of this article are to discuss the epidemiology, pathophysiology and clinical phenomenology of neurosarcoidosis, as well as current approaches to diagnosis and treatment. This review focuses on central nervous system (CNS) complications of sarcoidosis. RECENT FINDINGS: Neurosarcoidosis is a rare disorder with diverse clinical manifestations and outcomes. It is often difficult to diagnose and even more difficult to treat. New diagnostic approaches include the use of [¹8F]-fluorodeoxyglucose PET to identify potential biopsy sites. Success has been reported in the treatment of steroid refractory cases with disease-modifying therapies that were originally designed to manage other chronic inflammatory conditions by neutralizing key cytokines or depleting leukocyte subsets. SUMMARY: The diagnosis and management of neurosarcoidosis can be challenging. Currently, the disorder is treated with corticosteroids in combination with global immunosuppressant agents and/or immunomodulatory monoclonal antibodies, such as infliximab. The development of novel CNS penetrant drugs that are particularly effective at inhibiting granuloma formation would represent a significant therapeutic advance. Future progress will be informed by a deeper understanding of the pathways underlying the granulomatous inflammation characteristic of sarcoidosis and by an increased appreciation of how sarcoid lesions evolve in the CNS microenvironment.

Experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease of the central nervous system that is induced in laboratory animals by the generation of an immune response against myelin epitopes. It has been used as a prototype of Th1- and/or Th17-driven, organ-specific autoimmunity and as a model for the human disease, multiple sclerosis. In this chapter we describe two classic protocols for EAE induction (active immunization and adoptive transfer of Th1- or Th17-polarized cells) in Subheadings 3.1 and 3.2, respectively. Subheading 3.3 describes methods for rating clinical disease in symptomatic animals. Subheading 3.4 includes instructions for the isolation of mononuclear cells from the inflamed spinal cords of mice with EAE. Subheading 3.5 describes a method for performing the enzyme-linked immunospot assay.

The unwavering commitment of regulatory T cells in the suppression of autoimmune encephalomyelitis: another aspect of immune privilege in the CNS.

FoxP3(+) regulatory T (Treg) cells accumulate in the central nervous system (CNS) during experimental autoimmune encephalomyelitis and have been shown to limit the extent of neuroinflammation and to facilitate clinical recovery. The recent demonstration that Treg cells lose FoxP3 expression and assume effector cell characteristics upon stimulation with proinflammatory cytokines has raised questions about their stability in the inflamed CNS. In this issue of the European Journal of Immunology, O'Connor et al. [Eur. J. Immunol. 2012. 42: 1164-1173] show that CNS-infiltrating Treg cells maintain their suppressor phenotype by downregulating the IL-6 receptor. This commentary discusses the finding particularly with relevance to therapy of multiple sclerosis.

Lymphoid chemokines in the CNS.

Lymphoid chemokines, including CCL19, CCL21 and CXCL13, are critical in the development and organization of secondary lymphoid tissues and in the generation of adaptive immune responses. These molecules have also been implicated in the development of ectopic lymphoid structures in the setting of chronic inflammation. Here we review current knowledge on the production of lymphoid chemokines in the central nervous system during both homeostatic conditions and in disease states. Accumulating evidence suggests that constitutive expression of CCL19 plays a critical immunosurveillance role in healthy individuals. In contrast, aberrant induction of CCL19, CCL21 and CXCL13 may support the establishment of chronic autoimmunity and hematopoietic tumors within the CNS.

EAE mediated by a non-IFN-γ/non-IL-17 pathway.

Previous studies have shown that EAE can be elicited by the adoptive transfer of either IFN-γ-producing (Th1) or IL-17-producing (Th17) myelin-specific CD4(+) T-cell lines. Paradoxically, mice deficient in either IFN-γ or IL-17 remain susceptible to EAE following immunization with myelin antigens in CFA. These observations raise questions about the redundancy of IFN-γ and IL-17 in autoimmune demyelinating disease mediated by a diverse, polyclonal population of autoreactive T cells. In this study, we show that an atypical form of EAE, induced in C57BL/6 mice by the adoptive transfer of IFN-γ-deficient effector T cells, required IL-17 signaling for the development of brainstem infiltrates. In contrast, classical EAE, characterized by predominant spinal cord inflammation, occurred in the combined absence of IFN-γ and IL-17 signaling, but was dependent on GM-CSF and CXCR2. Our findings contribute to a growing body of data, indicating that individual cytokines vary in their importance across different models of CNS autoimmunity.

GM-CSF-dependent, CD103+ dermal dendritic cells play a critical role in Th effector cell differentiation after subcutaneous immunization.

Dendritic cells (DCs) play an important role in CD4(+) T helper (Th) cell differentiation and in the initiation of both protective and pathogenic immunity. Granulocyte/macrophage colony-stimulating factor (GM-CSF) is a DC growth factor critical for the induction of experimental autoimmune encephalomyelitis (EAE) and other autoimmune diseases, yet its mechanism of action in vivo is not fully defined. We show that GM-CSF is directly required for the accumulation of radiosensitive dermal-derived langerin(+)CD103(+) DCs in the skin and peripheral lymph nodes under steady-state and inflammatory conditions. Langerin(+)CD103(+) DCs stimulated naive myelin-reactive T cells to proliferate and produce IFN-gamma and IL-17. They were superior to other DC subsets in inducing expression of T-bet and promoting Th1 cell differentiation. Ablation of this subset in vivo conferred resistance to EAE. The current report reveals a previously unidentified role for GM-CSF in DC ontogeny and identifies langerin(+)CD103(+) DCs as an important subset in CD4(+) T cell-mediated autoimmune disease.

Circulating Ly-6C+ myeloid precursors migrate to the CNS and play a pathogenic role during autoimmune demyelinating disease.

Mature myeloid cells (macrophages and CD11b(+) dendritic cells) form a prominent component of neuroinflammatory infiltrates in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). The mechanism by which these cells are replenished during relapsing and chronic neuroinflammation is poorly understood. Here we demonstrate that CD11b(+)CD62L(+)Ly6C(hi) monocytes with colony-forming potential are mobilized into the bloodstream by a granulocyte-macrophage colony-stimulating factor-dependent pathway immediately before EAE relapses. Circulating Ly6C(hi) monocytes traffic across the blood-brain barrier, up-regulate proinflammatory molecules, and differentiate into central nervous system dendritic cells and macrophages. Enrichment of Ly6C(hi) monocytes in the circulating pool is associated with an earlier onset and increased severity of clinical EAE. Our studies indicate that granulocyte-macrophage colony-stimulating factor-driven release of Ly6C(hi) precursors from the bone marrow prevents exhaustion of central nervous system myeloid populations during relapsing or chronic autoimmune demyelination, suggesting a novel pathway for therapeutic targeting.

Treatment of CNS sarcoidosis with infliximab and mycophenolate mofetil.

OBJECTIVE: To describe the effects of the anti-tumor necrosis factor neutralizing antibody, infliximab, and the antiproliferative immunosuppressant, mycophenolate mofetil, in refractory neurosarcoidosis. METHODS: We treated patients with biopsy-proven sarcoidosis and CNS involvement, who had failed treatment with steroids, with infliximab (5 mg/kg on weeks 0, 2, and 6, and then every 6-8 weeks thereafter). Six out of seven patients were co-treated with mycophenolate mofetil (1,000 mg PO BID). Patients underwent a review of symptoms and complete neurologic examination every 3 months and MRI scanning before and after 3-4 infusions of infliximab. RESULTS: All patients reported significant symptomatic improvement by the fourth infusion of infliximab, including relief of headache and neuropathic pain, reversal of motor, sensory, or coordination deficits, and control of seizure activity. Furthermore, infliximab therapy was universally associated with a decrease in lesion size or suppression of gadolinium enhancement as documented by MRI. A positive treatment response was attained irrespective of location or distribution of CNS involvement by sarcoidosis (dural/leptomeningeal based vs intraparenchymal; cord vs brain; single lesion vs multifocal). There were no serious adverse effects in a follow-up period spanning 6-18 months. CONCLUSIONS: Combination treatment with mycophenolate mofetil and infliximab is a promising therapeutic approach for neurosarcoidosis.