In this study, we identified the effects of ER agonists on the transcription of neuroinflammatory genes in the frontal cortex of middle-aged female rats. From the major findings we conclude that 1) ERα agonist 16α-LE2 modulates the expression of a large number of genes related to immunity/inflammation, 2) E2, 16α-LE2 and DPN are potent regulators of neuroinflammatory gene expression, 3) estrogens' effects are mediated by both ERα and ERβ, 4) estrogens target glial cells including microglia, 5) estrogens suppress genes encoding key elements of C-mediated phagocytosis, 6) E2 may alter the lymphokine profile, 7) E2 can reverse age-related repression of ERα.
Expression profiling revealed potent immunomodulatory effects of ERα agonist in the middle-aged female neocortex
In the frontal cortex, ERα agonist 16α-LE2 regulated the expression of numerous genes related to immunity/inflammation. There were similarities between the effects of 16α-LE2 and E2  on immunity/inflammation gene expression. Overlapping effects include down-regulation of C3, Cd74, Fcgr2b and RT1-Aw2. On the other hand, a characteristic feature of the ERα agonist-evoked changes was the up-regulation of genes encoding antimicrobial peptides and S100 proteins. Antimicrobial peptides represent evolutionary ancient weapons of the immune system . Antimicrobial activity of these peptides contributes to the defense mechanism against pathogens. Some of these peptides can chemoattract monocytes and macrophages through CCR2 [37, 38]. S100A8 and S100A9 calcium-binding proteins can form a non-covalent heterocomplex which is involved in diverse functions. In macrophages, the complex regulates microtubule reorganization during phagocyte migration , NADPH oxidase complex assembly and calcium-dependent signaling during phagocyte activation .
Our data indicate that 16α-LE2-induced up-regulation of antimicrobial peptide and S100 protein genes may support defense mechanisms associated with microglia, astrocytes and blood-derived monocytes in the frontal cortex of middle-aged females.
Estrogens are potent modulators of neuroinflammatory gene expression
Profound regulation of immunity/inflammation genes by 16α-LE2 led us to further investigate the effects of estrogens on additional neuroinflammatory genes of primarily glial origin. We identified sixteen E2-regulated changes including up-regulation of defensin Np4 and RatNP-3b, IgG-2a, Il6 and Esr1, and down-regulation of C3 and C4, lymphokine genes Ccl2 and Tgfb1, MHC gene RT1-Aw2, Mpeg1, Cx3cr1, phagocytic and recognition receptor genes Fcgr2b, Itgam, Tlr4 and Tlr9. These data indicate that decreasing levels of E2 result in a significant change in the expression of neuroinflammatory genes which alters the innate immune response in the frontal cortex of aging females.
The effects of 16α-LE2 and DPN showed similarities to the effects of E2. All ER agonists evoked up-regulation of defensin genes, Il6, and down-regulation of complement C3 and some phagocytic receptors. Up-regulation of defensins and down-regulation of C3 and its receptor Cd11b can modulate various glial cell functions. Up-regulation of Il6 can affect a broad range of processes through the widely expressed IL6R in the cerebral cortex .
Both ERα and ERβ are involved in the immunomodulatory effects of E2
The large number of overlapping genes indicated that both ERα and ERβ were involved in the remarkable immunomodulatory effects of E2. These findings are in accord with published results obtained in in vitro  and in vivo [13, 43] LPS and EAE  models.
The significant effect of DPN on neuroinflammatory gene expression we found is in agreement with previous results implicating ERβ in the estrogenic regulation of microglia-mediated inflammation . Very recently, an ERβ-specific transrepression pathway has been identified which is controlled endogenously with 5-androsten-3β,17β-diol . This mechanism inhibits inflammatory responses of microglia and astrocytes . These results highlight the potential of selective ERβ agonists to suppress microglia and astrocytes in various neuroinflammatory diseases .
E2 targets glial cells including microglia in the aging frontal cortex
It is known that estrogens influence the regulatory functions of microglia via ERs [[13, 44, 47]]. We found several genes, such as Mpeg1 , Cx3cr1 , Cd11b , Tlr4  and Tlr9  which are expressed predominantly in microglia, and were suppressed by E2. Down-regulation of Cd11b is in accord with previous observations showing suppression of microglia reactivity by estrogens [53, 54]. It is known that Cd11b expression correlates with microglia reactivity, and accumulating evidence indicates that the microglia phenotype changes during aging [4, 55]. In the aged CNS, microglial cells possess elevated reactivity as characterized by up-regulation of cell surface activation markers . Our findings indicate that estrogens suppress microglia reactivity in the aging female cortex. This is consistent with earlier observations that E2 attenuates LPS-induced microglia reactivity in the rat brain . Transcriptional regulation of the fractalkine and toll-like receptors by E2 is novel finding and may have functional consequences. As fractalkine receptor signaling is involved in the regulation of microglia neurotoxicity , E2 may alter this microglia function via down-regulation of Cx3cr1.
E2 may suppress complement-mediated phagocytosis involved in synapse elimination
In the aging female cortex, we demonstrated down-regulation of C3 in the presence of estrogens. This finding is in line with the presence of 3 ERE sequences in the C3 promoter [57, 58] and estrogenic regulation of C3 in other tissues . Up-regulation of early C components has been reported recently in the aging mouse forebrain . Following activation, C promotes local inflammation and facilitates destruction through opsonization and lysis . Host tissue is protected from C lysis by soluble and membrane-bound regulators, but cortical neurons express low level of C inhibitors which makes them susceptible to C-mediated damage . As a result of the activation of the classical C pathway, C3b fragment is released from C3, which in turn binds to the surfaces of microbes, apoptotic or injured cells to label them for elimination by professional phagocytes . In the CNS, microglial cells recognize C3b or its proteolytic fragments via multiple receptors including Cd11b/Cd18, which leads to phagocytosis of the labeled substance. This C-mediated mechanism is responsible for the elimination of weak or unwanted synapses in the developing and the aging CNS [63, 64]. It is likely that both astrocytes and microglia are involved in this synapse elimination mechanism  which is highly relevant to the layer-specific loss of synapses in the estrogen-deprived, aging female neocortex .
Our results indicate that the expression of C3 and its receptor Cd11b, and the reactivity of microglial cells are suppressed by estrogens which may contribute to their neuroprotective effects in the cerebral cortex [[12, 67–69]].
Estrogens may alter the lymphokine profile
We also followed the effects of estrogens on the expression of Ccl2 and Il6. We found down-regulation of Ccl2 by ER agonists which was in agreement with recent data observed in EAE model . On the other hand, we demonstrated up-regulation of Il6 in the frontal cortex of middle-aged rats by E2 and isotype selective ER agonists. This is in line with publications reporting estrogen-responsiveness of Il6 . However, Il6 shows down-regulation by E2 in osteoblastoma Saos-2 cells , in contrast to the up-regulation we report in the frontal cortex. Different regulation of Il6 in human osteoblastoma and rodent glial or neuronal cells can be a result of tissue- and species-specificity of estrogen effects . A recent publication reports antimicrobial peptide-induced IL6 expression in glial cells via P2Y receptor signaling . This finding suggests that secondary effects may be involved in the transcriptional regulation of Il6 in a chronic treatment paradigm.
In the CNS, the actions of IL6 are complex and diverse that are mediated by the widely expressed IL6R . IL6 regulates neuroimmune and inflammatory responses , neurogenesis , neuronal differentiation, growth and survival . As astrocytes are one of the major sources of chemokines and cytokines in the CNS, astrocytes are likely to contribute to the anti-inflammatory effects of estrogens [69, 70].
E2 can reverse age-related repression of ERα transcription
E2 replacement evoked up-regulation of ERα. This finding suggested that chronic treatment with E2 supported estrogen responsiveness of the cortex. It warrants comprehensive examination of the effects of estrogen replacement in various tissues to correctly estimate the benefits and risks of replacement therapies. Up-regulation of ERα has particular importance as ERα expression decreases during aging , and it might support the hypothesis of 'critical period' to start an effective hormone replacement in postmenopausal women .