GM-CSF Promotes the Survival of Peripheral-Derived Myeloid Cells in the Central Nervous System for Pain-Induced Relapse of Neuroinflammation.

We recently discovered a (to our knowledge) new neuroimmune interaction named the gateway reflex, in which the activation of specific neural circuits establishes immune cell gateways at specific vessel sites in organs, leading to the development of tissue-specific autoimmune diseases, including a multiple sclerosis (MS) mouse model, experimental autoimmune encephalomyelitis (EAE). We have reported that peripheral-derived myeloid cells, which are CD11b+MHC class II+ and accumulate in the fifth lumbar (L5) cord during the onset of a transfer model of EAE (tEAE), play a role in the pain-mediated relapse via the pain-gateway reflex. In this study, we investigated how these cells survive during the remission phase to cause the relapse. We show that peripheral-derived myeloid cells accumulated in the L5 cord after tEAE induction and survive more than other immune cells. These myeloid cells, which highly expressed GM-CSFRα with common ß chain molecules, grew in number and expressed more Bcl-xL after GM-CSF treatment but decreased in number by blockade of the GM-CSF pathway, which suppressed pain-mediated relapse of neuroinflammation. Therefore, GM-CSF is a survival factor for these cells. Moreover, these cells were colocalized with blood endothelial cells (BECs) around the L5 cord, and BECs expressed a high level of GM-CSF. Thus, GM-CSF from BECs may have an important role in the pain-mediated tEAE relapse caused by peripheral-derived myeloid cells in the CNS. Finally, we found that blockade of the GM-CSF pathway after pain induction suppressed EAE development. Therefore, GM-CSF suppression is a possible therapeutic approach in inflammatory CNS diseases with relapse, such as MS.

Regulation of activated microglia and macrophages by systemically administered DNA/RNA heteroduplex oligonucleotides.

Microglial activation followed by recruitment of blood-borne macrophages into the central nervous system (CNS) aggravates neuroinflammation. Specifically, in multiple sclerosis (MS) as well as in experimental autoimmune encephalomyelitis (EAE), a rodent model of MS, activated microglia and macrophages (Mg/Mφ) promote proinflammatory responses and expand demyelination in the CNS. However, a potent therapeutic approach through the systemic route for regulating their functions has not yet been developed. Here, we demonstrate that a systemically injected DNA/RNA heteroduplex oligonucleotide (HDO), composed of an antisense oligonucleotide (ASO) and its complementary RNA, conjugated to cholesterol (Chol-HDO) distributed more efficiently to demyelinating lesions of the spinal cord in EAE mice with significant gene silencing than the parent ASO. Importantly, systemic administration of Cd40-targeting Chol-HDO improved clinical signs of EAE with significant downregulation of Cd40 in Mg/Mφ. Furthermore, we successfully identify that macrophage scavenger receptor 1 (MSR1) is responsible for the uptake of Chol-HDO by Mg/Mφ of EAE mice. Overall, our findings demonstrate the therapeutic potency of systemically administered Chol-HDO to regulate activated Mg/Mφ in neuroinflammation.

DNA/RNA heteroduplex oligonucleotide technology for regulating lymphocytes in vivo.

Manipulating lymphocyte functions with gene silencing approaches is promising for treating autoimmunity, inflammation, and cancer. Although oligonucleotide therapy has been proven to be successful in treating several conditions, efficient in vivo delivery of oligonucleotide to lymphocyte populations remains a challenge. Here, we demonstrate that intravenous injection of a heteroduplex oligonucleotide (HDO), comprised of an antisense oligonucleotide (ASO) and its complementary RNA conjugated to α-tocopherol, silences lymphocyte endogenous gene expression with higher potency, efficacy, and longer retention time than ASOs. Importantly, reduction of Itga4 by HDO ameliorates symptoms in both adoptive transfer and active experimental autoimmune encephalomyelitis models. Our findings reveal the advantages of HDO with enhanced gene knockdown effect and different delivery mechanisms compared with ASO. Thus, regulation of lymphocyte functions by HDO is a potential therapeutic option for immune-mediated diseases.