Functional analysis of microglial signal regulatory protein β 1 (SIRPβ1)

Microglial cells are the resident macrophages of the central nervous system (CNS) and thus form the interface between the neural parenchyma and the immune system. Although little is known about microglia in the CNS, it is obvious that they are quickly activated in all acute pathological events that might affect the CNS. In this study, the role of signal regulatory protein-β1 (SIRPβ1) was investigated by analyzing the effect of SIRPβ1 engagement on phagocytosis, inflammatory responses as well as on intracellular signalling in microglia. The signal regulatory proteins (SIRPs) are a family of transmembrane glycoproteins that are mainly involved in signal transduction cascade and belong to the immunoglobulin (Ig) superfamily. These proteins are expressed in the hematopoietic cells including granulocytes, monocytes, dendritic cells, lymphocytes and in the cells of the CNS. Although the extracellular domains of SIRPs are highly similar, classical motifs in the cytoplasmic or transmembrane domains distinguish them as either activating (β) or inhibitory (α) isoforms. The activating isoform SIRPβ1 have a short cytoplasmic tail which was reported to physically associate with the immunoreceptor tyrosine-based activation motif (ITAM) containing adaptor protein DAP12. We demonstrated that SIRPβ1 associates with DAP12 activating molecule and is expressed in microglia. Expression of SIRPβ1 and DAP12 were found to be in macrophage, myilod cells and splenocytes but do not found in neurons. Protein expression of SIRPβ1 and DAP12 in microglial cells was analyzed in brain, spinal cord and spleen from 7-8 week old EAE induced mice (mouse model of multiple sclerosis) and from 12 months old APP transgenic mice (mouse model of Alzheimer’s disease) by immunohistochemistry with purified antibodies directed against SIRPβ1. Gene transcript levels of SIRPβ1 and DAP12 significantly increased in the spinal cord of mice upon EAE induction as well as in the brain of APP transgenic mice. Double labelling with antibodies directed against SIRPβ1 and microglia marker Iba1 confirmed the microglial morphology of the cells, which were positively stained for SIRPβ1. We showed that dying neurons, splenocytes as well as β-amyloid and myelin are phagocytosed by microglia and stimulation of SIRPβ1 promotes phagocytosis in microglia by inducing the tyrosine phosphorylation of DAP12. To study the pathophysiological function of SIRPβ1, we used a lentiviral strategy to either knock down SIRPβ1 by RNA interference or to overexpress SIRPβ1. Our results demonstrate that the deficiency of SIRPβ1 results in diminished microglial uptake of apoptotic neurons, splenocytes, Aβ-peptide and myelin which gives additional significant functional input of microglial SIRPβ1 and its associated signalling molecule DAP12 in phagocytosis. This made us to conclude that microglial SIRPβ1 plays an important role in removal of apoptotic cells, suggesting that SIRPβ1 mediated phagocytosis could be a mechanism involved in ongoing response to neurodegeneration. Thus, understanding the role of SIRPβ1 mediated phagocytosis in the neurodegenerative process may help to elucidate the difference between normal microglial homeostasis and the disease state, offering hope for the generation of novel therapeutic compounds

Autoantigen-specific immunosuppression with tolerogenic peripheral blood cells prevents relapses in a mouse model of relapsing-remitting multiple sclerosis

Background: Dendritic cells (DCs) rendered suppressive by treatment with mitomycin C and loaded with the autoantigen myelin basic protein demonstrated earlier their ability to prevent experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis (MS). This provides an approach for prophylactic vaccination against autoimmune diseases. For clinical application such DCs are difficult to generate and autoantigens hold the risk of exacerbating the disease. Methods: We replaced DCs by peripheral mononuclear cells and myelin autoantigens by glatiramer acetate (Copaxone®), a drug approved for the treatment of MS. Spleen cells were loaded with Copaxone®, incubated with mitomycin C (MICCop) and injected into mice after the first bout of relapsing-remitting EAE. Immunosuppression mediated by MICCop was investigated in vivo by daily assessment of clinical signs of paralysis and in in vitro restimulation assays of peripheral immune cells. Cytokine profiling was performed by enzyme-linked immunosorbent assay (ELISA). Migration of MICCop cells after injection was examined by biodistribution analysis of 111Indium-labelled MICCop. The number and inhibitory activity of CD4+CD25+FoxP3+ regulatory T cells were analysed by histology, flow cytometry and in vitro mixed lymphocyte cultures. In order to assess the specificity of MICCop-induced suppression, treated EAE mice were challenged with the control protein ovalbumin. Humoral and cellular immune responses were then determined by ELISA and in vitro antigen restimulation assay. Results: MICCop cells were able to inhibit the harmful autoreactive T-cell response and prevented mice from further relapses without affecting general immune responses. Administered MICCop migrated to various organs leading to an increased infiltration of the spleen and the central nervous system with CD4+CD25+FoxP3+ cells displaying a suppressive cytokine profile and inhibiting T-cell responses. Conclusion: We describe a clinically applicable cell therapeutic approach for controlling relapses in autoimmune encephalomyelitis by specifically silencing the deleterious autoimmune response

Studying M1 and M2 states in adult microglia.

Microglial cell function receives increasing interest. To date, the majority of experiments are performed by using immortalized microglia-like cells or primary microglia prepared from pre- or postnatal rodent brain. As those may not adequately reflect the microglial biology in the adult brain, this protocol advocates a procedure which allows for the isolation, purification, and subsequent analysis of microglial cells. Once isolated, the principal state of activation, M1 or M2, can be determined in adult microglia using fluorescence-activated cell sorting, quantitative PCR, and/or Western blotting. Likewise, adult microglia generated by this protocol can be used for functional analysis through cell cultivation for a limited time

Signal Regulatory Protein-β1 : A Microglial Modulator of Phagocytosis in Alzheimer’s Disease

The signal regulatory protein-β1 (SIRPβ1) is a DAP12-associated transmembrane receptor expressed in a subset of hematopoietic cells. Recently, it was shown that peritoneal macrophages express SIRPβ1, which positively regulated phagocytosis. Here, we found that SIRPβ1 was up-regulated and acted as a phagocytic receptor on microglia in amyloid precursor protein J20 (APP/J20) transgenic mice and in Alzheimer’s disease (AD) patients. Interferon (IFN)-γ and IFN-β stimulated gene transcription of SIRPβ1 in cultured microglia. Activation of SIRPβ1 on cultured microglia by cross-linking antibodies induced reorganization of the cytoskeleton protein β-actin and suppressed lipopolysaccharide-induced gene transcription of tumor necrosis factor-α and nitric oxide synthase-2. Furthermore, activation of SIRPβ1 increased phagocytosis of microsphere beads, neural debris, and fibrillary amyloid-β (Aβ). Phagocytosis of neural cell debris and Aβ was impaired after lentiviral knockdown of SIRPβ1 in primary microglial cells. Thus, SIRPβ1 is a novel IFN-induced microglial receptor that supports clearance of neural debris and Aβ aggregates by stimulating phagocytosis