Mediation of protection and recovery from experimental autoimmune encephalomyelitis by macrophages expressing the human voltage-gated sodium channel NaV1.5.

Multiple sclerosis (MS) is the most common nontraumatic cause of neurologic disability in young adults. Despite treatment, progressive tissue injury leads to accumulation of disability in many patients. Here, our goal was to develop an immune-mediated strategy to promote tissue repair and clinical recovery in an MS animal model. We previously demonstrated that a variant of the voltage-gated sodium channel NaV1.5 is expressed intracellularly in human macrophages, and that it regulates cellular signaling. This channel is not expressed in mouse macrophages, which has limited the study of its functions. To overcome this obstacle, we developed a novel transgenic mouse model (C57BL6), in which the human macrophage NaV1.5 splice variant is expressed in vivo in mouse macrophages. These mice were protected from experimental autoimmune encephalomyelitis, the mouse model of MS. During active inflammatory disease, NaV1.5-positive macrophages were found in spinal cord lesions where they formed phagocytic cell clusters; they expressed markers of alternative activation during recovery. NaV1.5-positive macrophages that were adoptively transferred into wild-type recipients with established experimental autoimmune encephalomyelitis homed to lesions and promoted recovery. These results suggest that NaV1.5-positive macrophages enhance recovery from CNS inflammatory disease and could potentially be developed as a cell-based therapy for the treatment of MS.

The human macrophage sodium channel NaV1.5 regulates mycobacteria processing through organelle polarization and localized calcium oscillations.

Phagocytosis and intracellular processing of mycobacteria by macrophages are complex cellular processes that require spatial and temporal coordination of particle uptake, organelle movement, activation of signaling pathways, and channel-mediated ionic flux. Recent work demonstrated that human macrophage NaV1.5, an intracellular voltage-gated sodium channel expressed on late endosomes, enhances endosomal acidification and phagocytosis. Here, using bacillus Camille-Guerin (BCG) as a model of mycobacterial infection, we examined how this channel regulates phagocytosis and phagosome maturation in human macrophages. Knockdown of NaV1.5 reduced high capacity uptake of labeled BCG. BCG-containing, NaV1.5-expressing cells demonstrated localization of NaV1.5 and Rab-7 positive endosomes and mitochondria to periphagosome regions that was not observed in NaV1.5-deficient cells. Knockdown of the channel reduced the initial calcium response following bacterial challenge and prevented the generation of prolonged and localized calcium oscillations during phagosome maturation. Inhibition of the mitochondrial Na(+) /Ca(2+) exchanger also prevented prolonged calcium oscillations during phagosome maturation. These results suggest that NaV1.5 and mitochondrial-dependent calcium signaling regulate mycobacteria phagocytosis and phagosome maturation in human macrophages through spatial-temporal coordination of calcium signaling within a unique subcellular region.

Characterization of epithelial V-like antigen in human choroid plexus epithelial cells: potential role in CNS immune surveillance.

Prior work demonstrated that immune surveillance of the brain occurs primarily through the blood-cerebrospinal (CSF) fluid barrier rather than the blood-brain barrier endothelium. Recently, we identified epithelial V-like antigen (EVA), an immunoglobulin-like adhesion molecule, as a regulator of blood-CSF barrier integrity in a mouse model. Here we characterized EVA expression and function in human choroid plexus epithelial cells and analyzed its role in CD4 T lymphocyte adhesion. In human choroid plexus epithelial cells and a subset of CD4 T lymphocytes, EVA is expressed at high levels. Epithelial adhesion of T lymphocytes is inhibited by a blocking monoclonal antibody that recognizes EVA. T cell adhesion elicits calcium flux in choroid plexus epithelial cells that also can be blocked by an EVA-specific antibody. EVA-positive cell-cell contacts between epithelial and T cells are associated with increased complexity of cytoskeletal epithelial morphology. These results demonstrate that EVA is expressed in human choroid plexus epithelial cells and CD4 T lymphocytes and regulates CD4+ T lymphocyte adhesion to human choroid plexus epithelial cells in vitro. These data suggest a novel mechanism to regulate CNS immune surveillance.

Regulation of podosome formation in macrophages by a splice variant of the sodium channel SCN8A.

Voltage-gated sodium channels initiate electrical signaling in excitable cells such as muscle and neurons. They also are expressed in non-excitable cells such as macrophages and neoplastic cells. Previously, in macrophages, we demonstrated expression of SCN8A, the gene that encodes the channel NaV1.6, and intracellular localization of NaV1.6 to regions near F-actin bundles, particularly at areas of cell attachment. Here we show that a splice variant of NaV1.6 regulates cellular invasion through its effects on podosome and invadopodia formation in macrophages and melanoma cells. cDNA sequence analysis of SCN8A from THP-1 cells, a human monocyte-macrophage cell line, confirmed the expression of a full-length splice variant that lacks exon 18. Immunoelectron microscopy demonstrated NaV1.6-positive staining within the electron dense podosome rosette structure. Pharmacologic antagonism with tetrodotoxin (TTX) in differentiated THP-1 cells or absence of functional NaV1.6 through a naturally occurring mutation (med) in mouse peritoneal macrophages inhibited podosome formation. Agonist-mediated activation of the channel with veratridine caused release of sodium from cationic vesicular compartments, uptake by mitochondria, and mitochondrial calcium release through the Na/Ca exchanger. Invasion by differentiated THP-1 and HTB-66 cells, an invasive melanoma cell line, through extracellular matrix was inhibited by TTX. THP-1 invasion also was inhibited by small hairpin RNA knockdown of SCN8A. These results demonstrate that a variant of NaV1.6 participates in the control of podosome and invadopodia formation and suggest that intracellular sodium release mediated by NaV1.6 may regulate cellular invasion of macrophages and melanoma cells.

Expression of the voltage-gated sodium channel NaV1.5 in the macrophage late endosome regulates endosomal acidification.

Voltage-gated sodium channels expressed on the plasma membrane activate rapidly in response to changes in membrane potential in cells with excitable membranes such as muscle and neurons. Macrophages also require rapid signaling mechanisms as the first line of defense against invasion by microorganisms. In this study, our goal was to examine the role of intracellular voltage-gated sodium channels in macrophage function. We demonstrate that the cardiac voltage-gated sodium channel, NaV1.5, is expressed on the late endosome, but not the plasma membrane, in a human monocytic cell line, THP-1, and primary human monocyte-derived macrophages. Although the neuronal channel, NaV1.6, is also expressed intracellularly, it has a distinct subcellular localization. In primed cells, NaV1.5 regulates phagocytosis and endosomal pH during LPS-mediated endosomal acidification. Activation of the endosomal channel causes sodium efflux and decreased intraendosomal pH. These results demonstrate a functionally relevant intracellular voltage-gated sodium channel and reveal a novel mechanism to regulate macrophage endosomal acidification.