Study of M2 polarization in mouse brain

Microglia are the resident immunocompetent cells of the central nervous system (CNS), which immediately react to any neurological insult to remove the damaging stimulus and restore tissue homeostasis. Similarly to peripheral macrophages, infective stimuli induce these cells to undergo a classical proinflammatory response (M1 activation), characterized by the production of inflammatory mediators, while in response to IL-4/IL-13 an \u201calternative\u201d activation state is induced (M2 polarization), which is associated with the expression of anti-inflammatory molecules that lead to tissue repair and reconstruction. Several studies suggest the existence of heterogeneous populations of microglia in different areas of the brain, with differences reported in cell density and morphology, proliferation rate, expression of immunoregolatory proteins and in response to TNF-\u3b1. The region-specific difference in microglial phenotypes has been ascribed to microenvoirmental signals and is suggestive of specific microglial functions. Since several CNS diseases involve specific brain regions, the region-specific reactivity of microglia could play a key role in preventing or potentiating disease progression. The aim of this study was thus to evaluate whether microglia show a region-specific M2 response. We induced M2 polarization in vivo by injecting IL-4 in the cerebroventricular space (icv), using different IL-4 concentrations and length of treatments. Through realtime-PCR, Western blot and immunohistochemistry analyses we evaluated the expression of M2 markers, such as Arg1, Fizz-1 and YM-1 in different brain areas. Our preliminary results show that our icv IL-4 model provides a novel and reliable manner to study M2 activation in brain; more importantly, our data show that the expression profile of M2 markers in glial cells might be region-specific. These results suggest that glial populations residing in different cerebral areas undergo specific M2 responses, with interesting consequences on the role of microglia in neurodegenerative diseases

Selective agonists of estrogen receptor isoforms : new perspectives for cardiovascular disease

The cloning of estrogen receptors (ERs) and generation of ER-deficient mice have increased our understanding of the molecular mechanisms underlying the cardiovascular effects of estrogen. It is conceivable that clinical trials of estrogens so far failed to improve cardiovascular health because of the poor ER isoform selectivity and tissue specificity of endogenous hormones as well as incorrect treatment timing and regimens. Tissue-selective ER modulators (SERMs) may be safer agents than endogenous estrogens for cardiovascular disease. Yet, designing isoform-selective ER ligands (I-SERMs) with agonist or antagonist activity is required to pursue improved pharmacological control of ERs, especially taking into account emerging evidence for the beneficial role of vascular ER alpha activation. Ideally, the quest for unique ER ligands targeted to the vascular wall should lead to compounds that merge the pharmacological profiles of SERM and I-SERM agents. This review highlights the current bases for and approaches to selective ER modulation in the cardiovascular system

Role of innate immunity in the neuroprotective effect of estrogens

Background and Purpose \u2013 Activation of the brain inflammatory response plays a key role in the pathogenesis and progression of neurodegenerative diseases, such as Parkinson\u2019s disease (PD)1, although the specific contribution of pro and anti-inflammatory phenotypes of microglia is still unclear. Several lines of evidence show a different female/male ratio in the incidence of neurodegenerative diseases, including PD, which has been at least partially ascribed to the neuroprotective activity of estrogen hormones2. Although it is known that there is a gender-related dimorphism in innate immunity, which drives the inflammatory response, the interplay between the neuroprotective effects of estrogens and hormone action in inflammatory cells is still poorly understood. Our previous data showed that 17\u3b2-estradiol (E2) is able to reduce the pro-inflammatory response of the brain induced by LPS, a potent inflammatory stimulus, or by amyloid deposition in the APP23 mice3. Thus, the aim of the study is to better characterize the response of macrophage cells to estrogens, by analyzing gene expression and cell polarization through a genome wide approach, and to evaluate the relevance of dampening neuroinflammation for the efficacy of neuroprotective strategies by using an experimental model of PD. Methods and Results \u2013 Female mice were treated subcutaneously with vehicle or 5 \ub5g/kg E2 for 4 hr. Peritoneal macrophages were isolated by magnetic beads preloaded with an antibody against CD11b, RNA extracted and assayed for gene expression by realtime PCR. E2 treatment resulted in an increase in selected mRNAs, such as Tgm-2 and ApoE, known to be under estrogen control in other tissues, thus showing that peripheral macrophages in the intact animal are responsive to this hormone. The hormonal responsiveness of microglia cells is underway as we recently optimized the isolation of microglia cells from the adult mouse brain. Polarization of microglia cells was first analyzed by setting up a protocol of intracerebroventricular (icv) injections of IL-4, a well-known inducer of the M2 phenotype, followed by gene expression and immunological analyses of known M2 markers. Results will be presented. Conclusions \u2013 We observed that estrogens are able to modify the gene expression programme of macrophage cells in vivo, corroborating the hypothesis that these hormones are able to regulate the inflammatory response. These preliminary results sustain further analyses of hormone action in neuroinflammatory cells and in experimental models of neurodegenerative diseases associated with inflammation and macrophage polarization

Estrogen anti-inflammatory activity in brain: a therapeutic opportunity for menopause and neurodegenerative diseases

Recent studies highlight the prominent role played by estrogens in protecting the central nervous system (CNS) against the noxious consequences of a chronic inflammatory reaction. The neurodegenerative process of several CNS diseases, including Multiple Sclerosis, Alzheimer's and Parkinson's Diseases, is associated with the activation of microglia cells, which drive the resident inflammatory response. Chronically stimulated during neurodegeneration, microglia cells are thought to provide detrimental effects on surrounding neurons. The inhibitory activity of estrogens on neuroinflammation and specifically on microglia might thus be considered as a beneficial therapeutic opportunity for delaying the onset or progression of neurodegenerative diseases; in addition, understanding the peculiar activity of this female hormone on inflammatory signalling pathways will possibly lead to the development of selected anti-inflammatory molecules. This review summarises the evidence for the involvement of microglia in neuroinflammation and the anti-inflammatory activity played by estrogens specifically in microglia

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 (NF\u3baB), 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-1\u3b2, IL-6, and TNF\u3b1. 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

Estrogens, Neuroinflammation and Neurodegeneration

Inflammatory activation of microglia is a hallmark of several disorders of the CNS. In addition to protecting the brain against inflammatory insults, microglia are neuroprotective and play a significant role in maintaining neuronal connectivity; therefore, the prolongation and inflammatory status may limit the beneficial functions of these immune cells. The findings 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 hormone 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 17\u3b2-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 ER\u3b1-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

ER alpha-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 17β-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 IL1β, with an overall increase in cell inflammatory phenotype, further sustained by potentiation of IL1β secretion through caspase-1 activation