Sex-oriented perspectives in immunopharmacology.

Several immunopharmacological agents are effective in the treatment of cancer and immune-mediated conditions, with a favorable impact on life expectancy and clinical outcomes for a large number of patients. Nevertheless, response variation and undesirable effects of these drugs represent major issues, and overall efficacy remains unpredictable. Males and females show a distinct difference in immune system responses, with females generally mounting stronger responses to a variety of stimuli. Therefore, exploring sex differences in the efficacy and safety of immunopharmacological agents would strengthen the practice of precision medicine. As a pharmacological target highlight, programmed cell death 1 ligand 1 (PD-L1) is the first functionally characterized ligand of the coinhibitory programmed death receptor 1 (PD-1). The PD-L1/PD-1 crosstalk plays an important role in the immune response and is relevant in cancer, infectious and autoimmune disease. Sex differences in the response to immune checkpoint inhibitors are well documented, with male patients responding better than female patients. Similarly, higher efficacy of and adherence to tumor necrosis factor inhibitors in chronic inflammatory conditions including rheumatoid arthritis and Crohn's disease have been reported in male patients. The pharmacological basis of sex-specific responses to immune system modulating drugs is actively investigated in other settings such as stroke and type 1 diabetes. Advances in therapeutics targeting the endothelium could soon be wielded against autoimmunity and metabolic disorders. Based on the established sexual dimorphism in immune-related pathophysiology and disease presentation, sex-specific immunopharmacological protocols should be integrated into clinical guidelines.

The immune activity of selective estrogen receptor modulators is gene and macrophage subtype-specific yet converges on Il1b downregulation.

Raloxifene belongs to the family of Selective Estrogen Receptor Modulators (SERMs), which are drugs widely prescribed for Estrogen Receptor alpha (ERα)-related pathologies. Recently, SERMs are being tested in repurposing strategies for ERα-independent clinical indications, including a wide range of microbial infections. Macrophages are central in the fight against pathogen invasion. Despite estrogens have been shown to regulate macrophage phenotype, SERMs activity in these cells is still poorly defined. We investigated the activity of Raloxifene in comparison with another widely used SERM, Tamoxifen, on immune gene expression in macrophages obtained from mouse and human tissues, including mouse peritoneal macrophages, bone marrow-derived macrophages, microglia or human blood-derived macrophages, assaying for the involvement of the ERα, PI3K and NRF2 pathways also under inflammatory conditions. Our data demonstrate that Raloxifene acts by a dual mechanism, which entails ERα antagonism and off-target mediators. Moreover, micromolar concentrations of Raloxifene increase the expression of immune metabolic genes, such as Vegfa and Hmox1, through PI3K and NRF2 activation selectively in peritoneal macrophages. Conversely, Il1b mRNA down-regulation by SERMs is consistently observed in all macrophage subtypes and unrelated to the PI3K/NRF2 system. Importantly, the production of the inflammatory cytokine TNFα induced by the bacterial endotoxin, LPS, is potentiated by SERMs and paralleled by the cell subtype-specific increase in IL1ß secretion. This work extends our knowledge on the biological and molecular mechanisms of SERMs immune activity and indicate macrophages as a pharmacological target for the exploitation of the antimicrobial potential of these drugs.

ERα-Dependent Regulation of Adropin Predicts Sex Differences in Liver Homeostasis during High-Fat Diet.

Non-alcoholic fatty liver disease (NAFLD) represents a public health issue, due to its prevalence and association with other cardiometabolic diseases. Growing evidence suggests that NAFLD alters the production of hepatokines, which, in turn, influence several metabolic processes. Despite accumulating evidence on the major role of estrogen signaling in the sexually dimorphic nature of NAFLD, dependency of hepatokine expression on sex and estrogens has been poorly investigated. Through in vitro and in vivo analysis, we determined the extent to which hepatokines, known to be altered in NAFLD, can be regulated, in a sex-specific fashion, under different hormonal and nutritional conditions. Our study identified four hepatokines that better recapitulate sex and estrogen dependency. Among them, adropin resulted as one that displays a sex-specific and estrogen receptor alpha (ERα)-dependent regulation in the liver of mice under an excess of dietary lipids (high-fat diet, HFD). Under HFD conditions, the hepatic induction of adropin negatively correlates with the expression of lipogenic genes and with fatty liver in female mice, an effect that depends upon hepatic ERα. Our findings support the idea that ERα-mediated induction of adropin might represent a potential approach to limit or prevent NAFLD.

Tamoxifen Twists Again: On and Off-Targets in Macrophages and Infections.

Beyond the wide use of tamoxifen in breast cancer chemotherapy due to its estrogen receptor antagonist activity, this drug is being assayed in repurposing strategies against a number of microbial infections. We conducted a literature search on the evidence related with tamoxifen activity in macrophages, since these immune cells participate as a first line-defense against pathogen invasion. Consistent data indicate the existence of estrogen receptor-independent targets of tamoxifen in macrophages that include lipid mediators and signaling pathways, such as NRF2 and caspase-1, which allow these cells to undergo phenotypic adaptation and potentiate the inflammatory response, without the induction of cell death. Thus, these lines of evidence suggest that the widespread antimicrobial activity of this drug can be ascribed, at least in part, to the potentiation of the host innate immunity. This widens our understanding of the pharmacological activity of tamoxifen with relevant therapeutic implications for infections and other clinical indications that may benefit from the immunomodulatory effects of this drug.

The Use of ERE-Luc Reporter Mice to Monitor Estrogen Receptor Transcriptional Activity in a Spatio-Temporal Dimension.

In spite of the fact that women spend 1/3 of their lives in postmenopause, the search for appropriate therapies able to counteract the derangements associated with the menopause still represents a sort of sought after the "Holy Grail."Nowadays, the combination of estrogens and selective estrogen receptor modulators (SERMs), a class of compounds with a mixed agonist/antagonistic activity on the estrogen receptor (ER) in various tissues, represents the most promising approach to improve postmenopausal women's health, by preserving the benefits while avoiding the side effects of estrogen-based therapy.Given their complex mechanisms of action, the evaluation of SERM activity in combination with conjugated estrogens (CE) requires a multifactorial analysis that takes into account the multifaceted and dynamic effects of these compounds in target tissues, even in relation to the physiological/pathological status.To accomplish such a goal, we took advantage of the ERE-Luc model, a reporter mouse that allows the monitoring of ER transcriptional activity in a spatio-temporal dimension. Cluster analyses performed on in vivo/ex vivo bioluminescence (BLI) data and ex vivo luciferase activity enabled to sustain the combination of CE plus bazedoxifene (TSEC, tissue-selective estrogen complex) as a valuable option for the pharmacological treatment of the postmenopause.

ERα-independent NRF2-mediated immunoregulatory activity of tamoxifen.

Sex differences in immune-mediated diseases are linked to the activity of estrogens on innate immunity cells, including macrophages. Tamoxifen (TAM) is a selective estrogen receptor modulator (SERM) used in estrogen receptor-alpha (ERα)-dependent breast cancers and off-target indications such as infections, although the immune activity of TAM and its active metabolite, 4-OH tamoxifen (4HT), is poorly characterized. Here, we aimed at investigating the endocrine and immune activity of these SERMs in macrophages. Using primary cultures of female mouse macrophages, we analyzed the expression of immune mediators and activation of effector functions in competition experiments with SERMs and 17ß-estradiol (E2) or the bacterial endotoxin LPS. We observed that 4HT and TAM induce estrogen antagonist effects when used at nanomolar concentrations, while pharmacological concentrations that are reached by TAM in clinical settings regulate the expression of VEGFα and other immune activation genes by ERα- and G protein-coupled receptor 1 (GPER1)-independent mechanisms that involve NRF2 through PI3K/Akt-dependent mechanisms. Importantly, we observed that SERMs potentiate cell phagocytosis and modify the effects of LPS on the expression of inflammatory cytokines, such as TNFα and IL1ß, with an overall increase in cell inflammatory phenotype, further sustained by potentiation of IL1ß secretion through caspase-1 activation. Altogether, our data unravel a novel molecular mechanism and immune functions for TAM and 4HT, sustaining their repurposing in infective and other estrogen receptors-unrelated pathologies.

Inhibition of microglial ß-glucocerebrosidase hampers the microglia-mediated antioxidant and protective response in neurons.

BACKGROUND: Homozygotic mutations in the GBA gene cause Gaucher's disease; moreover, both patients and heterozygotic carriers have been associated with 20- to 30-fold increased risk of developing Parkinson's disease. In homozygosis, these mutations impair the activity of ß-glucocerebrosidase, the enzyme encoded by GBA, and generate a lysosomal disorder in macrophages, which changes morphology towards an engorged phenotype, considered the hallmark of Gaucher's disease. Notwithstanding the key role of macrophages in this disease, most of the effects in the brain have been attributed to the ß-glucocerebrosidase deficit in neurons, while a microglial phenotype for these mutations has never been reported. METHODS: We applied the bioluminescence imaging technology, immunohistochemistry and gene expression analysis to investigate the consequences of microglial ß-glucocerebrosidase inhibition in the brain of reporter mice, in primary neuron/microglia cocultures and in cell lines. The use of primary cells from reporter mice allowed for the first time, to discriminate in cocultures neuronal from microglial responses consequent to the ß-glucocerebrosidase inhibition; results were finally confirmed by pharmacological depletion of microglia from the brain of mice. RESULTS: Our data demonstrate the existence of a novel neuroprotective mechanism mediated by a direct microglia-to-neuron contact supported by functional actin structures. This cellular contact stimulates the nuclear factor erythroid 2-related factor 2 activity in neurons, a key signal involved in drug detoxification, redox balance, metabolism, autophagy, lysosomal biogenesis, mitochondrial dysfunctions, and neuroinflammation. The central role played by microglia in this neuronal response in vivo was proven by depletion of the lineage in the brain of reporter mice. Pharmacological inhibition of microglial ß-glucocerebrosidase was proven to induce morphological changes, to turn on an anti-inflammatory/repairing pathway, and to hinder the microglia ability to activate the nuclear factor erythroid 2-related factor 2 response, thus increasing the neuronal susceptibility to neurotoxins. CONCLUSION: This mechanism provides a possible explanation for the increased risk of neurodegeneration observed in carriers of GBA mutations and suggest novel therapeutic strategies designed to revert the microglial phenotype associated with ß-glucocerebrosidase inhibition, aimed at resetting the protective microglia-to-neuron communication.

Reciprocal interference between the NRF2 and LPS signaling pathways on the immune-metabolic phenotype of peritoneal macrophages.

The metabolic and immune adaptation to extracellular signals allows macrophages to carry out specialized functions involved in immune protection and tissue homeostasis. Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that coordinates cell redox and metabolic responses to stressors. However, the individual and concomitant activation of NRF2 and inflammatory pathways have been poorly investigated in isolated macrophages. We here took advantage of reporter mice for the transcriptional activities of NRF2 and nuclear factor-kB (NFκB), a key transcription factor in inflammation, and observe a persisting reciprocal interference in the response of peritoneal macrophages to the respective activators, tert-Butylhydroquinone (tBHQ) and lipopolysaccharide (LPS). When analyzed separately by gene expression studies, these pathways trigger macrophage-specific metabolic and proliferative target genes that are associated with tBHQ-induced pentose phosphate pathway (PPP) with no proliferative response, and with opposite effects observed with LPS. Importantly, the simultaneous administration of tBHQ + LPS alters the effects of each individual pathway in a target gene-specific manner. In fact, this co-treatment potentiates the effects of tBHQ on the antioxidant enzyme, HMOX1, and the antibacterial enzyme, IRG1, respectively; moreover, the combined treatment reduces tBHQ activity on the glycolytic enzymes, TALDO1 and TKT, and decreases LPS effects on the metabolic enzyme IDH1, the proliferation-related proteins KI67 and PPAT, and the inflammatory cytokines IL-1ß, IL-6, and TNFα. Altogether, our results show that the activation of NRF2 redirects the metabolic, immune, and proliferative response of peritoneal macrophages to inflammatory signals, with relevant consequences for the pharmacological treatment of diseases that are associated with unopposed inflammatory responses.

The Role of Sex and Sex Hormones in Neurodegenerative Diseases.

Neurodegenerative diseases (NDs) are a wide class of disorders of the central nervous system (CNS) with unknown etiology. Several factors were hypothesized to be involved in the pathogenesis of these diseases, including genetic and environmental factors. Many of these diseases show a sex prevalence and sex steroids were shown to have a role in the progression of specific forms of neurodegeneration. Estrogens were reported to be neuroprotective through their action on cognate nuclear and membrane receptors, while adverse effects of male hormones have been described on neuronal cells, although some data also suggest neuroprotective activities. The response of the CNS to sex steroids is a complex and integrated process that depends on (i) the type and amount of the cognate steroid receptor and (ii) the target cell type-either neurons, glia, or microglia. Moreover, the levels of sex steroids in the CNS fluctuate due to gonadal activities and to local metabolism and synthesis. Importantly, biochemical processes involved in the pathogenesis of NDs are increasingly being recognized as different between the two sexes and as influenced by sex steroids. The aim of this review is to present current state-of-the-art understanding on the potential role of sex steroids and their receptors on the onset and progression of major neurodegenerative disorders, namely, Alzheimer's disease, Parkinson's diseases, amyotrophic lateral sclerosis, and the peculiar motoneuron disease spinal and bulbar muscular atrophy, in which hormonal therapy is potentially useful as disease modifier.

Identification of new molecular targets for PET imaging of the microglial anti-inflammatory activation state.

Microglia are potential targets for therapeutic intervention in neurological and neurodegenerative diseases affecting the central nervous system. In order to assess the efficacy of therapies aimed to reduce the tissue damaging activities of microglia and/or to promote the protective potential of these cells, suitable pre-clinical and clinical tools for the in vivo analysis of microglia activities and dynamics are required. The aim of this work was to identify new translational markers of the anti-inflammatory / protective state of microglia for the development of novel PET tracers. Methods: New translational markers of the anti-inflammatory/protective activation state of microglia were selected by bioinformatic approaches and were in vitro and ex vivo validated by qPCR and immunohistochemistry in rodent and human samples. Once a viable marker was identified, a novel PET tracer was developed. This tracer was subsequently confirmed by autoradiography experiments in murine and human brain tissues. Results: Here we provide evidence that P2RY12 expression increases in murine and human microglia following exposure to anti-inflammatory stimuli, and that its expression is modulated in the reparative phase of experimental and clinical stroke. We then synthesized a novel carbon-11 labeled tracer targeting P2RY12, showing increased binding in brain sections of mice treated with IL4, and low binding to brain sections of a murine stroke model and of a stroke patient. Conclusion: This study provides new translational targets for PET tracers for the anti-inflammatory/protective activation state of microglia and shows the potential of a rationale-based approach. It therefore paves the way for the development of novel non-invasive methodologies aimed to monitor the success of therapeutic approaches in various neurological diseases.

The estrogen-macrophage interplay in the homeostasis of the female reproductive tract.

BACKGROUND: Estrogens are known to orchestrate reproductive events and to regulate the immune system during infections and following tissue damage. Recent findings suggest that, in the absence of any danger signal, estrogens trigger the physiological expansion and functional specialization of macrophages, which are immune cells that populate the female reproductive tract (FRT) and are increasingly being recognized to participate in tissue homeostasis beyond their immune activity against infections. Although estrogens are the only female gonadal hormones that directly target macrophages, a comprehensive view of this endocrine-immune communication and its involvement in the FRT is still missing. OBJECTIVE AND RATIONALE: Recent accomplishments encourage a revision of the literature on the ability of macrophages to respond to estrogens and induce tissue-specific functions required for reproductive events, with the aim to envision macrophages as key players in FRT homeostasis and mediators of the regenerative and trophic actions of estrogens. SEARCH METHODS: We conducted a systematic search using PubMed and Ovid for human, animal (rodents) and cellular studies published until 2018 on estrogen action in macrophages and the activity of these cells in the FRT. OUTCOMES: Our search identified the remarkable ability of macrophages to activate biochemical processes in response to estrogens in cell culture experiments. The distribution at specific locations, interaction with selected cells and acquisition of distinct phenotypes of macrophages in the FRT, as well as the cyclic renewal of these properties at each ovarian cycle, demonstrate the involvement of these cells in the homeostasis of reproductive events. Moreover, current evidence suggests an association between estrogen-macrophage signaling and the generation of a tolerant and regenerative environment in the FRT, although a causative link is still missing. WIDER IMPLICATIONS: Dysregulation of the functions and estrogen responsiveness of FRT macrophages may be involved in infertility and estrogen- and macrophage-dependent gynecological diseases, such as ovarian cancer and endometriosis. Thus, more research is needed on the physiology and pharmacological control of this endocrine-immune interplay.

Sex-Specific Features of Microglia from Adult Mice.

Sex has a role in the incidence and outcome of neurological illnesses, also influencing the response to treatments. Neuroinflammation is involved in the onset and progression of several neurological diseases, and the fact that estrogens have anti-inflammatory activity suggests that these hormones may be a determinant in the sex-dependent manifestation of brain pathologies. We describe significant differences in the transcriptome of adult male and female microglia, possibly originating from perinatal exposure to sex steroids. Microglia isolated from adult brains maintain the sex-specific features when put in culture or transplanted in the brain of the opposite sex. Female microglia are neuroprotective because they restrict the damage caused by acute focal cerebral ischemia. This study therefore provides insight into a distinct perspective on the mechanisms underscoring a sexual bias in the susceptibility to brain diseases.

Selective proliferative response of microglia to alternative polarization signals.

BACKGROUND: Microglia are resident myeloid cells of the central nervous system (CNS) that are maintained by self-renewal and actively participate in tissue homeostasis and immune defense. Under the influence of endogenous or pathological signals, microglia undertake biochemical transformations that are schematically classified as the pro-inflammatory M1 phenotype and the alternatively activated M2 state. Dysregulated proliferation of M1-activated microglia has detrimental effects, while an increased number of microglia with the alternative, pro-resolving phenotype might be beneficial in brain pathologies; however, the proliferative response of microglia to M2 signals is not yet known. We thus evaluated the ability of interleukin-4 (IL-4), a typical M2 and proliferative signal for peripheral macrophages, to induce microglia proliferation and compared it with other proliferative and M2 polarizing stimuli for macrophages, namely colony-stimulating factor-1 (CSF-1) and the estrogen hormone, 17ß-estradiol (E2). METHODS: Recombinant IL-4 was delivered to the brain of adult mice by intracerebroventricular (i.c.v.) injection; whole brain areas or ex vivo-sorted microglia were analyzed by real-time PCR for assessing the mRNA levels of genes related with cell proliferation (Ki67, CDK-1, and CcnB2) and M2 polarization (Arg1, Fizz1, Ym-1) or by FACS analyses of in vivo BrdU incorporation in microglia. Primary cultures of microglia and astrocytes were also tested for proliferative effects. RESULTS: Our results show that IL-4 only slightly modified the expression of cell cycle-related genes in some brain areas but not in microglia, where it strongly enhanced M2 gene expression; on the contrary, brain delivery of CSF-1 triggered proliferation as well as M2 polarization of microglia both in vivo and in vitro. Similar to IL-4, the systemic E2 administration failed to induce microglia proliferation while it increased M2 gene expression. CONCLUSIONS: Our data show that, in contrast to the wider responsiveness of peripheral macrophages, microglia proliferation is stimulated by selected M2 polarizing stimuli suggesting a role for the local microenvironment and developmental origin of tissue macrophages in regulating self-renewal following alternative activating stimuli.

Influence of Estrogen Modulation on Glia Activation in a Murine Model of Parkinson's Disease.

Epidemiological data suggest a sexual dimorphism in Parkinson disease (PD), with women showing lower risk of developing PD. Vulnerability of the nigrostriatal pathway may be influenced by exposure to estrogenic stimulation throughout fertile life. To further address this issue, we analyzed the progression of nigrostriatal damage, microglia and astrocyte activation and microglia polarization triggered by intrastriatal injection of dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) in male, female and ovariectomized (OVX) mice, as well as in OVX mice supplemented with 17ßestradiol (OVX+E). Animals were sacrificed at different time points following 6-OHDA injection and brain sections containing striatum and substantia nigra pars compacta (SNc) underwent immunohistochemistry for tyrosine hydroxylase (TH) (dopaminergic marker), immunofluorescence for IBA1 and GFAP (markers of microglia and astrocyte activation, respectively) and triple immunoflorescent to identify polarization of microglia toward the cytotoxic M1 (DAPI/IBA1/TNFα) or cytoprotective M2 (DAPI/IBA1/CD206) phenotype. SNc damage induced by 6-OHDA was significantly higher in OVX mice, as compared to all other experimental groups, at 7 and 14 days after surgery. Astrocyte activation was higher in OVX mice with respect the other experimental groups, at all time points. Microglial activation in the SNc was detected at earlier time points in male, female and OVX+E, while in OVX mice was detected at all time-points. Microglia polarization toward the M2, but not the M1, phenotype was detected in female and OVX+E mice, while the M1 phenotype was observed only in male and OVX mice. Our results support the protective effects of estrogens against nigrostriatal degeneration, suggesting that such effects may be mediated by an interaction with microglia, which tend to polarize preferentially toward an M2, cytoprotective phenotype in the presence of intense estrogenic stimulation.

Self-renewal and phenotypic conversion are the main physiological responses of macrophages to the endogenous estrogen surge.

Beyond the physiology of reproduction, estrogen controls the homeostasis of several tissues. Although macrophages play a key role in tissue remodeling, the interplay with estrogen is still ill defined. Using a transcriptomic approach we first obtained a comprehensive list of genes that are differentially expressed in peritoneal macrophages in response to physiological levels of 17ß-estradiol (E2) injected in intact female mice. Our data also showed the dynamic nature of the macrophage response to E2 and pointed to specific biological programs induced by the hormone, with cell proliferation, immune response and wound healing being the most prominent functional categories. Indeed, the exogenous administration of E2 and, more importantly, the endogenous hormonal surge proved to support macrophage proliferation in vivo, as shown by cell cycle gene expression, BrdU incorporation and cell number. Furthermore, E2 promoted an anti-inflammatory and pro-resolving macrophage phenotype, which converged on the induction of genes related to macrophage alternative activation and on IL-10 expression in vivo. Hormone action was maintained in an experimental model of peritoneal inflammation based on zymosan injection. These findings highlight a direct effect of estrogen on macrophage expansion and phenotypic adaptation in homeostatic conditions and suggest a role for this interplay in inflammatory pathologies.

Estrogens, Neuroinflammation, and Neurodegeneration.

Inflammatory activation of microglia is a hallmark of several disorders of the central nervous system. In addition to protecting the brain against inflammatory insults, microglia are neuroprotective and play a significant role in maintaining neuronal connectivity, but the prolongation of an inflammatory status may limit the beneficial functions of these immune cells. The finding that estrogen receptors are present in monocyte-derived cells and that estrogens prevent and control the inflammatory response raise the question of the role that this sex steroid plays in the manifestation and progression of pathologies that have a clear sex difference in prevalence, such as multiple sclerosis, Parkinson's disease, and Alzheimer's disease. The present review aims to provide a critical review of the current literature on the actions of estrogen in microglia and on the involvement of estrogen receptors in the manifestation of selected neurological disorders. This current understanding highlights a research area that should be expanded to identify appropriate replacement therapies to slow the progression of such diseases.

Estrogen accelerates the resolution of inflammation in macrophagic cells.

Although 17ß-estradiol (E2) anti-inflammatory activity has been well described, very little is known about the effects of this hormone on the resolution phase of the inflammatory process. Here, we identified a previously unreported ERα-mediated effect of E2 on the inflammatory machinery. The study showed that the activation of the intracellular estrogen receptor shortens the LPS-induced pro-inflammatory phase and, by influencing the intrinsic and extrinsic programs, triggers the resolution of inflammation in RAW 264.7 cells. Through the regulation of the SOCS3 and STAT3 signaling pathways, E2 facilitates the progression of the inflammatory process toward the IL10-dependent "acquired deactivation" phenotype, which is responsible for tissue remodeling and the restoration of homeostatic conditions. The present study may provide an explanation for increased susceptibility to chronic inflammatory diseases in women after menopause, and it suggests novel anti-inflammatory treatments for such disorders.

Alternative activation of human macrophages is rescued by estrogen treatment in vitro and impaired by menopausal status.

CONTEXT AND OBJECTIVE: During their reproductive years, women are generally protected from cardiovascular disease events by their estrogen-replete status. Our starting hypothesis was that lower estrogen levels after menopause are associated with macrophage activation profiles skewed toward proinflammatory phenotypes. Research Design and Setting: This was an in vitro and ex vivo study in human blood-derived macrophages. SUBJECTS: We obtained blood from 12 healthy male donors for the in vitro study and from 5 premenopausal and 8 postmenopausal women for the ex vivo study. OUTCOME: We measured macrophage immunophenotypes in the resting state and after activation with M1-associated (lipopolysaccharide [LPS]/interferon-γ [IFN-γ]) or M2-associated (IL-4/IL-13) stimuli and expression of estrogen receptors (ERs) and other transcription factors. RESULTS: Unpolarized macrophages expressed both ERα and ERß, and ERα but not ERß levels were decreased by M1 stimuli. LPS/IFN-γ also induced down-regulation of CD163 and CD206, markers of alternative activation, and increased cell-bound TNF-α and IL-10. These effects were prevented by 17ß-estradiol treatment through impaired nuclear factor-κB liberation. In agreement with a role for 17ß-estradiol in attenuating the inflammatory response, M1/M2 subpopulations in monocytes and unstimulated macrophages from premenopausal and postmenopausal donors were similar. In contrast, M2 activation appeared to be blunted in macrophages from postmenopausal women, leading to an increased M1/M2 response ratio. CONCLUSIONS: Estrogen treatment prevented LPS/IFN-γ action on human M2 macrophage markers and cytokine production, whereas menopausal estrogen loss was associated with an impaired response to alternative activation, suggesting that these mechanisms affect the cardiovascular risk profile in relation to menopausal status.

Heterogeneous induction of microglia M2a phenotype by central administration of interleukin-4.

BACKGROUND: Acquisition of the M1 or M2 phenotypes by microglia has been shown to occur during the development of pathological conditions, with M1 activation being widely involved in neurotoxicity in relation with the anatomical localization and the reactivity of subtypes of microglia cells. On the contrary, little is known on the ability of microglia to undergo M2 polarization by interleukin-4 (IL4), the typical M2a polarization signal for peripheral macrophages. METHODS: Recombinant mouse IL4 was injected in the third cerebral ventricle of mice to induce brain alternative polarization. The mRNA levels of Fizz1, Arg1, and Ym1 genes, known to be up-regulated by IL4 in peripheral macrophages, together with additional polarization markers, were evaluated in the striatum and frontal cortex at different time intervals after central administration of IL4; in parallel, M2a protein expression was evaluated in tissue extracts and at the cellular level. RESULTS: Our results show that the potency and temporal profile of IL4-mediated M2a gene induction vary depending on the gene analyzed and according to the specific brain area analyzed, with the striatum showing a reduced M2a response compared with the frontal cortex, as further substantiated by assays of polarization protein levels. Of notice, Fizz1 mRNA induction reached 100-fold level, underscoring the potency of this specific IL4 signaling pathway in the brain. In addition, immunochemistry assays demonstrated the localization of the M2 response specifically to microglia cells and, more interestingly, the existence of a subpopulation of microglia cells amenable to undergoing M2a polarization in the healthy mouse brain. CONCLUSIONS: These results show that the responsiveness of brain macrophages to centrally administered IL4 may vary depending on the gene and brain area analyzed, and that M2a polarization can be ascribed to a subpopulation of IL4-responsive microglia cells. The biochemical pathways that enable microglia to undergo M2a activation represent key aspects for understanding the physiopathology of neuroinflammation and for developing novel therapeutic and diagnostic agents.

A lack of ovarian function increases neuroinflammation in aged mice.

Although several lines of evidence have indicated that menopause is associated with increased susceptibility to neurological disorders, the mechanisms involved in this phenomenon remain to be elucidated. Because neuroinflammation is a common feature of a number of brain diseases, we hypothesized that the cessation of ovarian functions and the consequent decrease in estrogen receptor (ER)-mediated antiinflammatory activity may represent a trigger for postmenopausal brain dysfunctions. The aim of the present study was to investigate the effects of aging and surgical menopause on the activity of ER in neuroinflammation. The present study shows that ER genes are expressed in the hippocampus, but ER transcriptional activity decreases significantly beginning at 12 months of age in intact and ovariectomized mice. With ovariectomy, we observe an age-dependent accumulation of mRNA encoding inflammatory mediators (e.g. TNFα, IL1ß, and macrophage inflammatory protein-2) and changes in the morphology of astroglia and microglia. In addition, we show that aging itself is coupled with an exaggerated response to acute inflammatory stimuli with a major accumulation of TNFα, IL1ß, macrophage inflammatory protein-2, and macrophage chemoattractant protein-1 mRNA in response to lipopolysaccharide administration. The response to acute inflammatory stimuli appears to be differentially modulated by the duration of hormone deprivation in 12-month-old mice. Taken together, the present results show that aging is associated with decreased ER activity, despite continuous ER synthesis, and that age-dependent neuroinflammation is strongly influenced by hormone deprivation.