Astrocytes provide structural and functional support for neurons, as well as display neurotoxic or neuroprotective phenotypes depending upon the presence of an immune or inflammatory microenvironment. This study was undertaken to characterize multiple phenotypes of activated astrocytes and to investigate the regulatory mechanisms involved. We report that activated astrocytes in culture exhibit two functional phenotypes with respect to pro- or anti-inflammatory gene expression, glial fibrillary acidic protein expression, and neurotoxic or neuroprotective activities. The two distinct functional phenotypes of astrocytes were also demonstrated in a mouse neuroinflammation model, which showed pro- or anti-inflammatory gene expression in astrocytes following challenge with classical or alternative activation stimuli; similar results were obtained in the absence of microglia. Subsequent studies involving recombinant lipocalin-2 (LCN2) protein treatment or Lcn2-deficient mice indicated that the pro- or anti-inflammatory functionally polarized phenotypes of astrocytes and their intracellular signaling pathway were critically regulated by LCN2 under in vitro and in vivo conditions. Astrocyte-derived LCN2 promoted classical proinflammatory activation of astrocytes but inhibited IL-4–STAT6 signaling, a canonical pathway involved in alternative anti-inflammatory activation. Our results suggest that the secreted protein LCN2 is an autocrine modulator of the functional polarization of astrocytes in the presence of immune or inflammatory stimuli and that LCN2 could be targeted therapeutically to dampen proinflammatory astrocytic activation and related pathologies in the CNS.

Lipocalin-2 (LCN2) has diverse functions in multiple pathophysiological conditions; however, its pathogenic role in vascular dementia (VaD) is unknown. Here, we investigated the role of LCN2 in VaD using rodent models of global cerebral ischemia and hypoperfusion with cognitive impairment and neuroinflammation. Mice subjected to transient bilateral common carotid artery occlusion (tBCCAo) for 50 min showed neuronal death and gliosis in the hippocampus at 7 days post-tBCCAo. LCN2 expression was observed predominantly in the hippocampal astrocytes, whereas its receptor was mainly detected in neurons, microglia, and astrocytes. Furthermore, Lcn2-deficient mice, compared with wild-type animals, showed significantly weaker CA1 neuronal loss, cognitive decline, white matter damage, blood-brain barrier permeability, glial activation, and proinflammatory cytokine production in the hippocampus after tBCCAo. Lcn2 deficiency also attenuated hippocampal neuronal death and cognitive decline at 30 days after unilateral common carotid artery occlusion (UCCAo). Furthermore, intracerebroventricular (i.c.v) injection of recombinant LCN2 protein elicited CA1-neuronal death and a cognitive deficit. Our studies using cultured glia and hippocampal neurons supported the decisive role of LCN2 in hippocampal neurotoxicity and microglial activation, and the role of the HIF-1α-LCN2-VEGFA axis of astrocytes in vascular injury. Additionally, plasma levels of LCN2 were significantly higher in patients with VaD than in the healthy control subjects. These results indicate that hippocampal damage and cognitive impairment are mediated by LCN2 secreted from reactive astrocytes in VaD.

Activated microglia are thought to undergo apoptosis as a self-regulatory mechanism. To better understand molecular mechanisms of the microglial apoptosis, apoptosis-resistant variants of microglial cells were selected and characterized. The expression of lipocalin 2 (lcn2) was significantly down-regulated in the microglial cells that were resistant to NO-induced apoptosis. lcn2 expression was increased by inflammatory stimuli in microglia. The stable expression of lcn2 as well as the addition of rLCN2 protein augmented the sensitivity of microglia to the NO-induced apoptosis, while knockdown of lcn2 expression using short hairpin RNA attenuated the cell death. Microglial cells with increased lcn2 expression were more sensitive to other cytotoxic agents as well. Thus, inflammatory activation of microglia may lead to up-regulation of lcn2 expression, which sensitizes microglia to the self-regulatory apoptosis. Additionally, the stable expression of lcn2 in BV-2 microglia cells induced a morphological change of the cells into the round shape with a loss of processes. Treatment of primary microglia cultures with the rLCN2 protein also induced the deramification of microglia. The deramification of microglia was closely related with the apoptosis-prone phenotype, because other deramification-inducing agents such as cAMP-elevating agent forskolin, ATP, and calcium ionophore also rendered microglia more sensitive to cell death. Taken together, our results suggest that activated microglia may secrete LCN2 protein, which act in an autocrine manner to sensitize microglia to the self-regulatory apoptosis and to endow microglia with an amoeboid form, a canonical morphology of activated microglia in vivo.

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Activated macrophages are classified into two different forms: classically activated (M1) or alternatively activated (M2) macrophages. The presence of M1/M2 phenotypic polarization has also been suggested for microglia. Here, we report that the secreted protein lipocalin 2 (LCN2) amplifies M1 polarization of activated microglia. LCN2 protein (EC 1 μg/ml), but not glutathione S-transferase used as a control, increased the M1-related gene expression in cultured mouse microglial cells after 8-24 h. LCN2 was secreted from M1-polarized, but not M2-polarized, microglia. LCN2 inhibited phosphorylation of STAT6 in IL-4-stimulated microglia, suggesting LCN2 suppression of the canonical M2 signaling. In the lipopolysaccharide (LPS)-induced mouse neuroinflammation model, the expression of LCN2 was notably increased in microglia. Primary microglial cultures derived from LCN2-deficient mice showed a suppressed M1 response and enhanced M2 response. Mice lacking LCN2 showed a markedly reduced M1-related gene expression in microglia after LPS injection, which was consistent with the results of histological analysis. Neuroinflammation-associated impairment in motor behavior and cognitive function was also attenuated in the LCN2-deficient mice, as determined by the rotarod performance test, fatigue test, open-field test, and object recognition task. These findings suggest that LCN2 is an M1-amplifier in brain microglial cells.