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.

A new neutrophil subset promotes CNS neuron survival and axon regeneration.

Transected axons typically fail to regenerate in the central nervous system (CNS), resulting in chronic neurological disability in individuals with traumatic brain or spinal cord injury, glaucoma and ischemia-reperfusion injury of the eye. Although neuroinflammation is often depicted as detrimental, there is growing evidence that alternatively activated, reparative leukocyte subsets and their products can be deployed to improve neurological outcomes. In the current study, we identify a unique granulocyte subset, with characteristics of an immature neutrophil, that had neuroprotective properties and drove CNS axon regeneration in vivo, in part via secretion of a cocktail of growth factors. This pro-regenerative neutrophil promoted repair in the optic nerve and spinal cord, demonstrating its relevance across CNS compartments and neuronal populations. Our findings could ultimately lead to the development of new immunotherapies that reverse CNS damage and restore lost neurological function across a spectrum of diseases.

A phenotypic screening platform for identifying chemical modulators of astrocyte reactivity.

Disease, injury and aging induce pathological reactive astrocyte states that contribute to neurodegeneration. Modulating reactive astrocytes therefore represent an attractive therapeutic strategy. Here we describe the development of an astrocyte phenotypic screening platform for identifying chemical modulators of astrocyte reactivity. Leveraging this platform for chemical screening, we identify histone deacetylase 3 (HDAC3) inhibitors as effective suppressors of pathological astrocyte reactivity. We demonstrate that HDAC3 inhibition reduces molecular and functional characteristics of reactive astrocytes in vitro. Transcriptional and chromatin mapping studies show that HDAC3 inhibition disarms pathological astrocyte gene expression and function while promoting the expression of genes associated with beneficial astrocytes. Administration of RGFP966, a small molecule HDAC3 inhibitor, blocks reactive astrocyte formation and promotes neuroprotection in vivo in mice. Collectively, these results establish a platform for discovering modulators of reactive astrocyte states, inform the mechanisms that control astrocyte reactivity and demonstrate the therapeutic benefits of modulating astrocyte reactivity for neurodegenerative diseases.

TAM receptor signaling dictates lesion location and clinical phenotype during experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE), induced by the adoptive transfer of Th17 cells, typically presents with ascending paralysis and inflammatory demyelination of the spinal cord. Brain white matter is relatively spared. Here we show that treatment of Th17 transfer recipients with a highly selective inhibitor to the TAM family of tyrosine kinase receptors results in ataxia associated with a shift of the inflammatory infiltrate to the hindbrain parenchyma. During homeostasis and preclinical EAE, hindbrain microglia express high levels of the TAM receptor Mer. Our data suggest that constitutive TAM receptor signaling in hindbrain microglia confers region-specific protection against Th17 mediated EAE.

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 (BM) neutrophils, when polarized with a combination of recombinant interleukin (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 BM 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.

Biological aging of CNS-resident cells alters the clinical course and immunopathology of autoimmune demyelinating disease.

Biological aging is the strongest factor associated with the clinical phenotype of multiple sclerosis (MS). Relapsing-remitting MS typically presents in the third or fourth decade, whereas the mean age of presentation of progressive MS (PMS) is 45 years old. Here, we show that experimental autoimmune encephalomyelitis (EAE), induced by the adoptive transfer of encephalitogenic CD4+ Th17 cells, was more severe, and less likely to remit, in middle-aged compared with young adult mice. Donor T cells and neutrophils were more abundant, while B cells were relatively sparse, in CNS infiltrates of the older mice. Experiments with reciprocal bone marrow chimeras demonstrated that radio-resistant, nonhematopoietic cells played a dominant role in shaping age-dependent features of the neuroinflammatory response, as well as the clinical course, during EAE. Reminiscent of PMS, EAE in middle-aged adoptive transfer recipients was characterized by widespread microglial activation. Microglia from older mice expressed a distinctive transcriptomic profile suggestive of enhanced chemokine synthesis and antigen presentation. Collectively, our findings suggest that drugs that suppress microglial activation, and acquisition or expression of aging-associated properties, may be beneficial in the treatment of progressive forms of inflammatory demyelinating disease.

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.

Role of Insulin-Like Growth Factor Binding Protein-3 in the Pathogenesis of Herpes Stromal Keratitis.

Purpose: The goal of this study was to determine the role of insulin-like growth factor-binding protein-3 (IGFBP-3) in the pathogenesis of herpes stromal keratitis (HSK). Methods: In an unbiased approach, a membrane-based protein array was carried out to determine the level of expression of pro- and anti-angiogenic molecules in uninfected and HSV-1 infected corneas. Quantitative RT-PCR and ELISA assays were performed to measure the amounts of IGFBP-3 at mRNA and protein levels. Confocal microscopy documented the localization of IGFBP-3 in uninfected and infected corneal tissue. Flow cytometry assay showed the frequency of immune cell types in infected corneas from C57BL/6J (B6) and IGFBP-3 knockout (IGFBP-3-/-) mice. Slit-lamp microscopy was used to quantitate the development of opacity and neovascularization in infected corneas from both groups of mice. Results: Quantitation of protein array dot blot showed an increased level of IGFBP-3 protein in HSV-1 infected than uninfected corneas and was confirmed with ELISA and quantitative RT-PCR assays. Cytosolic and nuclear localization of IGFBP-3 were detected in the cells of corneal epithelium, whereas scattered IGFBP-3 staining was evident in the stroma of HSK developing corneas. Increased opacity and hemangiogenesis were noted in the corneas of IGFBP-3-/- than B6 mice during the clinical period of HSK. Furthermore, an increased number of leukocytes comprising of neutrophils and CD4 T cells were found in HSK developing corneas of IGFBP-3-/- than B6 mice. Conclusions: Our data showed that lack of IGFBP-3 exacerbates HSK, suggesting the protective effect of IGFBP-3 protein in regulating the severity of HSK.