Ccl5 Mediates Proper Wiring of Feedforward and Lateral Inhibition Pathways in the Inner Retina.

The ß-chemokine Ccl5 and its receptors are constitutively expressed in neurons of the murine inner retina. Here, we examined the functional and structural significance of this constitutive Ccl5 signaling on retinal development. We compared outcomes of electrophysiology, ocular imaging and retinal morphology in wild-type mice (WT) and mice with Ccl5 deficiency (Ccl5-/- ). Assessment of retinal structure by ocular coherence tomography and histology revealed slight thinning of the inner plexiform layer (IPL) and inner nuclear layer (INL) in Ccl5-/- mice, compared to WT (p < 0.01). Assessment of postnatal timepoints important for development of the INL (P7 and P10) revealed Ccl5-dependent alterations in the pattern and timing of apoptotic pruning. Morphological analyses of major inner retinal cell types in WT, Ccl5-/- , gustducingfp and gustducingfp/Ccl5-/- mice revealed Ccl5-dependent reduction in GNAT3 expression in rod bipolar cells as well as a displacement of their terminals from the IPL into the GCL. RGC dendritic organization and amacrine cell morphology in the IPL was similarly disorganized in Ccl5-/- mice. Examination of the intrinsic electrophysiological properties of RGCs revealed higher spontaneous activity in Ccl5-/- mice that was characterized by higher spiking frequency and a more depolarized resting potential. This hyperactive phenotype could be negated by current clamp and correlated with both membrane resistance and soma area. Overall, our findings identify Ccl5 signaling as a mediator of inner retinal circuitry during development of the murine retina. The apparent role of Ccl5 in retinal development further supports chemokines as trophic modulators of CNS development and function that extends far beyond the inflammatory contexts in which they were first characterized.

Peripherally derived T regulatory and γδ T cells have opposing roles in the pathogenesis of intractable pediatric epilepsy.

The pathophysiology of drug-resistant pediatric epilepsy is unknown. Flow cytometric analysis of inflammatory leukocytes in resected brain tissues from 29 pediatric patients with genetic (focal cortical dysplasia) or acquired (encephalomalacia) epilepsy demonstrated significant brain infiltration of blood-borne inflammatory myeloid cells and memory CD4+ and CD8+ T cells. Significantly, proinflammatory (IL-17- and GM-CSF-producing) γδ T cells were concentrated in epileptogenic lesions, and their numbers positively correlated with disease severity. Conversely, numbers of regulatory T (T reg) cells inversely correlated with disease severity. Correspondingly, using the kainic acid model of status epilepticus, we show ameliorated seizure activity in both γδ T cell- and IL-17RA-deficient mice and in recipients of T reg cells, whereas T reg cell depletion heightened seizure severity. Moreover, both IL-17 and GM-CSF induced neuronal hyperexcitability in brain slice cultures. These studies support a major pathological role for peripherally derived innate and adaptive proinflammatory immune responses in the pathogenesis of intractable epilepsy and suggest testing of immunomodulatory therapies.

Constitutive and Stress-induced Expression of CCL5 Machinery in Rodent Retina.

Signaling by inflammatory cytokines and chemokines is associated with neurodegeneration in disease and injury. Here we examined expression of the ß-chemokine CCL5 and its receptors in the mouse retina and evaluated its relevance in glaucoma, a common optic neuropathy associated with sensitivity to intraocular pressure (IOP). Using quantitative PCR, fluorescent in situ hybridization, immunohistochemistry and quantitative image analysis, we found CCL5 mRNA and protein was constitutively expressed in the inner retina and synaptic layers. CCL5 appeared to associate with Müller cells and RGCs as well as synaptic connections between horizontal cells and bipolar cells in the OPL and amacrine cells, bipolar cells and RGCs in the IPL. Although all three high-affinity receptors (CCR5, CCR3, CCR1) for CCL5 were expressed constitutively, CCR5 expression was significantly higher than CCR3, which was also markedly greater than CCR1. Localization patterns for constitutive CCR5, CCR3 and CCR1 expression differed, particularly with respect to expression in inner retinal neurons. Stress-related expression of CCL5 was primarily altered in aged DBA/2 mice with elevated IOP. In contrast, changes in expression and localization of both CCR3 and CCR5 were evident not only in aged DBA/2 mice, but also in age-matched control mice and young DBA/2 mice. These groups do not exhibit elevated IOP, but possess either the aging stress (control mice) or the genetic predisposition to glaucoma (DBA/2 mice). Together, these data indicate that CCL5 and its high-affinity receptors are constitutively expressed in murine retina and differentially induced by stressors associated with glaucomatous optic neuropathy. Localization patterns further indicate that CCL5 signaling may be relevant for modulation of synapses in both health and disease, particularly in the inner plexiform layer.

The origin and implementation of the Broadening Experiences in Scientific Training programs: an NIH common fund initiative.

Recent national reports and commentaries on the current status and needs of the U.S. biomedical research workforce have highlighted the limited career development opportunities for predoctoral and postdoctoral trainees in academia, yet little attention is paid to preparation for career pathways outside of the traditional faculty path. Recognizing this issue, in 2013, the U.S. National Institutes of Health (NIH) Common Fund issued a request for application titled "NIH Director's Biomedical Research Workforce Innovation Award: Broadening Experiences in Scientific Training (BEST)." These 5-yr 1-time grants, awarded to 17 single or partnering institutions, were designed to develop sustainable approaches to broaden graduate and postgraduate training, aimed at creating training programs that reflect the range of career options that trainees may ultimately pursue. These institutions have formed a consortium in order to work together to develop, evaluate, share, and disseminate best practices and challenges. This is a first report on the early experiences of the consortium and the scope of participating BEST programs. In this report, we describe the state of the U.S. biomedical workforce and development of the BEST award, variations of programmatic approaches to assist with program design without BEST funding, and novel approaches to engage faculty in career development programs. To test the effectiveness of these BEST programs, external evaluators will assess their outcomes not only over the 5 yr grant period but also for an additional 10 yr beyond award completion.

Pressure-Induced Alterations in PEDF and PEDF-R Expression: Implications for Neuroprotective Signaling in Glaucoma.

INTRODUCTION: Alterations in neuron-glia signaling are implicated in glaucoma, a neurodegenerative disease characterized by retinal ganglion cell (RGC) death. Pigment epithelium derived factor (PEDF) is a secreted protein with potential neuroprotective qualities in retinal disease, including chronic ocular hypertension. Here we sought to determine whether moderate, short-term elevations in IOP alter PEDF signaling and whether pressure-induced PEDF signaling directly impacts RGC apoptosis. METHODS: In retina from naïve mice and mice with unilateral, microbead-induced glaucoma, we examined expression and cell type-specific localization of PEDF and its receptor (PEDF-R), using quantitative PCR and immunohistochemistry. Using primary cultures of purified RGCs and Müller cells, we examined cell type-specific expression of PEDF in response to 48 hours of elevated hydrostatic pressure, using multiplex ELISA and immunocytochemistry. We also measured pressure-induced apoptosis of RGCs in the presence or absence of atglistatin, a potent and selective inhibitor of PEDF-R, and recombinant PEDF, using TUNEL assays. RESULTS: PEDF and PEDF-R are constitutively expressed in naïve retina, primarily in the ganglion cell and nerve fiber layers. Elevated IOP increases PEDF and PEDF-R expression, particularly associated with RGCs and Müller cells. Elevated pressure in vitro increased PEDF secretion by 6-fold in RGCs and trended towards an increase in expression by Müller cells, as compared to ambient pressure. This was accompanied by changes in the subcellular localization of PEDF-R in both cell types. Inhibition of PEDF signaling with atglistatin increased pressure-induced apoptosis in RGCs and treatment with recombinant PEDF inhibited pressure-induced apoptosis, both in a dose-dependent manner. CONCLUSION: Our findings suggest that moderate, short-term elevations in IOP promote PEDF signaling via up-regulation of both PEDF and PEDF-R. Based on in vivo and in vitro studies, this PEDF signaling likely arises from both Müller cells and RGCs, and has the potential to directly inhibit RGC apoptosis.

CNS expression of B7-H1 regulates pro-inflammatory cytokine production and alters severity of Theiler's virus-induced demyelinating disease.

The CNS is a unique organ due to its limited capacity for immune surveillance. As macrophages of the CNS, microglia represent a population originally known for the ability to assist neuronal stability, are now appreciated for their role in initiating and regulating immune responses in the brain. Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease is a mouse model of multiple sclerosis (MS). In response to TMEV infection in vitro, microglia produce high levels of inflammatory cytokines and chemokines, and are efficient antigen-presenting cells (APCs) for activating CD4(+) T cells. However, the regulatory function of microglia and other CNS-infiltrating APCs in response to TMEV in vivo remains unclear. Here we demonstrate that microglia increase expression of proliferating cell nuclear antigen (PCNA), and phenotypically express high levels of major histocompatibility complex (MHC)-Class I and II in response to acute infection with TMEV in SJL/J mice. Microglia increase expression of the inhibitory co-stimulatory molecule, B7-H1 as early as day 5 post-infection, while CNS-infiltrating CD11b(+)CD11c(-)CD45(HIGH) monocytes/macrophages and CD11b(+)CD11c(+)CD45(HIGH) dendritic cells upregulate expression of B7-H1 by day 3 post-infection. Utilizing a neutralizing antibody, we demonstrate that B7-H1 negatively regulates TMEV-specific ex vivo production of interferon (IFN)-γ, interleukin (IL)-17, IL-10, and IL-2 from CD4(+) and CD8(+) T cells. In vivo blockade of B7-H1 in SJL/J mice significantly exacerbates clinical disease symptoms during the chronic autoimmune stage of TMEV-IDD, but only has minimal effects on viral clearance. Collectively, these results suggest that CNS expression of B7-H1 regulates activation of TMEV-specific T cells, which affects protection against TMEV-IDD.

The role of antigen presenting cells in multiple sclerosis.

Multiple sclerosis (MS) is a debilitating T cell mediated autoimmune disease of the central nervous system (CNS). Animal models of MS, such as experimental autoimmune encephalomyelitis (EAE) and Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) have given light to cellular mechanisms involved in the initiation and progression of this organ-specific autoimmune disease. Within the CNS, antigen presenting cells (APC) such as microglia and astrocytes participate as first line defenders against infections or inflammation. However, during chronic inflammation they can participate in perpetuating the self-destructive environment by secretion of inflammatory factors and/or presentation of myelin epitopes to autoreactive T cells. Dendritic cells (DC) are also participants in the presentation of antigen to T cells, even within the CNS. While the APCs alone are not solely responsible for mediating the destruction to the myelin sheath, they are critical players in perpetuating the inflammatory milieu. This review will highlight relevant studies which have provided insight to the roles played by microglia, DCs and astrocytes in the context of CNS autoimmunity.

Glial toll-like receptor signaling in central nervous system infection and autoimmunity.

Innate immunity in the CNS depends primarily on the functions of glial cells, astrocytes and microglia, which are important for the early control of pathogen replication and direct the recruitment and activation of cells of the adaptive immune system required for pathogen clearance. Efficient immune responses are required for clearance of an invading pathogen, but dysregulation of a pro-inflammatory response in the CNS could lead to the development of autoimmunity. This review summarizes the activation of toll-like receptors (TLRs) expressed on glial cells and the functional outcome of these interactions for CNS health and disease which depends on a delicate balance of the protective and toxic effects of molecules induced in the CNS following TLR ligation.