Upregulation of IFN-ß induced by Sema4D-dependent partial Erk1/2 inhibition promotes NO production in microglia.

Interactions between Sema4D and its receptors, PlexinB1 and CD72, induce various functions, including axon guidance, angiogenesis, and immune activation. Our previous study revealed that Sema4D is involved in the upregulation of nitric oxide production in microglia after cerebral ischemia. In this study, we investigated the underlying mechanisms of the enhancement of microglial nitric oxide production by Sema4D. Primary microglia expressed PlexinB1 and CD72, and cortical microglia expressed CD72. Sema4D promoted nitric oxide production and slightly inhibited Erk1/2 phosphorylation in microglia. Partial Erk1/2 inhibition enhanced microglial nitric oxide production. Inhibition of Erk1/2 phosphorylation induced the expression of Ifn-ß mRNA, and IFN-ß promoted nitric oxide production in microglia. In the ischemic cortex, the expression of Ifn-ß mRNA was downregulated by Sema4D deficiency. These findings indicated that the enhancement of nitric oxide production by Sema4D is involved in partial Erk1/2 inhibition and upregulation of IFN-ß.

Changes in L-arginine metabolism by Sema4D deficiency induce promotion of microglial proliferation in ischemic cortex.

Cerebral ischemia induces neuroinflammation and microglial activation, in which activated microglia upregulate their proliferative activity and change their metabolic states. In activated microglia, l-arginine is metabolized competitively by inducible nitric oxide synthase (iNOS) and arginase (Arg), which then synthesize NO or polyamines, respectively. Our previous study demonstrated that Sema4D deficiency inhibits iNOS expression and promotes proliferation of ionized calcium-binding adaptor molecule 1 (Iba1)-positive (Iba1+) microglia in the ischemic cortex, although the underlying mechanisms were unclear. Using middle cerebral artery occlusion, we tested the hypothesis that Sema4D deficiency alters the balance of l-arginine metabolism between iNOS and Arg, leading to an increase in the production of polyamines, which are an essential factor for cell proliferation. In the peri-ischemic cortex, almost all iNOS+ and/or Arg1+ cells were Iba1+ microglia. In the peri-ischemic cortex of Sema4D-deficient (Sema4D-/-) mice, the number of iNOS+ Arg1- Iba1+ microglia was smaller and that of iNOS- Arg1+ Iba1+ microglia was greater than those of wild-type (WT) mice. In addition, urea and polyamine levels in the ischemic cortex of Sema4D-/- mice were higher than those of WT mice; furthermore, the presence of Sema4D inhibited polyamine production in primary microglia obtained from Sema4D-/- mice. Finally, microglia cultured under polyamine putrescine-supplemented conditions demonstrated increased proliferation rates over non-supplemented controls. These findings indicate that Sema4D regulates microglial proliferation at least in part by regulating the competitive balance of l-arginine metabolism.

Transcription factor FOXO1 promotes cell migration toward exogenous ATP via controlling P2Y1 receptor expression in lymphatic endothelial cells.

Sprouting migration of lymphatic endothelial cell (LEC) is a pivotal step in lymphangiogenic process. However, its molecular mechanism remains unclear including effective migratory attractants. Meanwhile, forkhead transcription factor FOXO1 highly expresses in LEC nuclei, but its significance in LEC migratory activity has not been researched. In this study, we investigated function of FOXO1 transcription factor associated with LEC migration toward exogenous ATP which has recently gathered attentions as a cell migratory attractant. The transwell membrane assay indicated that LECs migrated toward exogenous ATP, which was impaired by FOXO1 knockdown. RT-PCR analysis showed that P2Y1, a purinergic receptor, expression was markedly reduced by FOXO1 knockdown in LECs. Moreover, P2Y1 blockage impaired LEC migration toward exogenous ATP. Western blot analysis revealed that Akt phosphorylation contributed to FOXO1-dependent LEC migration toward exogenous ATP and its blockage affected LEC migratory activity. Furthermore, luciferase reporter assay and ChIP assay suggested that FOXO1 directly bound to a conserved binding site in P2RY1 promoter and regulated its activity. These results indicated that FOXO1 serves a pivotal role in LEC migration toward exogenous ATP via direct transcriptional regulation of P2Y1 receptor.

Homology analysis detects topological changes of Iba1 localization accompanied by microglial activation.

The state of microglial activation provides important information about the central nervous system. However, a reliable index of microglial activation in histological samples has yet to be established. Here, we show that microglial activation induces topological changes of Iba1 localization that can be detected by analysis based on homology theory. Analysis of homology was applied to images of Iba1-stained tissue sections, and the 0-dimentional Betti number (b0: the number of solid components) and the 1-dimentional Betti number (b1: the number of windows surrounded by solid components) were obtained. We defined b1/b0 as the Homology Value (HV), and investigated its validity as an index of microglial activation using cerebral ischemia model mice. Microglial activation was accompanied by changes to Iba1 localization and morphology of microglial processes. In single microglial cells, the change of Iba1 localization increased b1. Conversely, thickening or retraction of microglial processes decreased b0. Consequently, microglial activation increased the HV. The HV of a tissue area increased with proximity to the ischemic core and showed a high degree of concordance with the number of microglia expressing activation makers. Furthermore, the HV of human metastatic brain tumor tissue also increased with proximity to the tumor. These results suggest that our index, based on homology theory, can be used to correctly evaluate microglial activation in various tissue images.