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This is an Open Access article distributed under the terms of the Creative Commons Attribution License. ; [Background]: Activation of microglia, the primary component of the innate immune response in the brain, is a hallmark of neuroinflammation in neurodegenerative disorders, including Alzheimer's disease (AD) and other pathological conditions such as stroke or CNS infection. In response to a variety of insults, microglial cells produce high levels of inflammatory cytokines that are often involved in neuronal injury, and play an important role in the recognition, engulfment, and clearance of apoptotic cells and/or invading microbes. Secreted phospholipase A2-IIA (sPLA2-IIA), an enzyme that interacts with cells involved in the systemic immune/inflammatory response, has been found up-regulated in the cerebrospinal fluid and brain of AD patients. However, despite several approaches, its functions in mediating CNS inflammation remain unknown. In the present study, the role of sPLA2-IIA was examined by investigating its direct effects on microglial cells. [Methods]: Primary and immortalized microglial cells were stimulated by sPLA2-IIA in order to characterize the cytokine-like actions of the phospholipase. The hallmarks of activated microglia analyzed include: mitogenic response, phagocytic capabilities and induction of inflammatory mediators. In addition, we studied several of the potential molecular mechanisms involved in those events. [Results]: The direct exposure of microglial cells to sPLA2-IIA stimulated, in a time- and dose-dependent manner, their phagocytic and proliferative capabilities. sPLA2-IIA also triggered the synthesis of the inflammatory proteins COX-2 and TNFα. In addition, EGFR phosphorylation and shedding of the membrane-anchored heparin-binding EGF-like growth factor (pro-HB-EGF) ectodomain, as well as a rapid activation/phosphorylation of the classical survival proteins ERK, P70S6K and rS6 were induced upon sPLA2-IIA treatment. We further demonstrated that the presence of an EGFR inhibitor (AG1478), a matrix metalloproteinase inhibitor (GM6001), an ADAM inhibitor (TAPI-1), and a HB-EGF neutralizing antibody abrogated the phenotype of activated microglia induced by the sPLA2-IIA. [Conclusion]: These results support the hypothesis that sPLA2-IIA may act as a potent modulator of microglial functions through its ability to induce EGFR transactivation and HB-EGF release. Accordingly, pharmacological modulation of EGFR might be a useful tool for treating neuroinflammatory diseases characterized by sPLA2-IIA accumulation. ; This work was supported by grants SAF2009-08407 from the Spanish Ministry of Science and Innovation, and CSI11A08 from the Government of Castilla y León. Claudia Cordova was funded by the FPI Program from the Government of Castilla y León (co-funded by FSE). ; Peer Reviewed

Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are inflammatory demyelinating diseases that develop as a result of deregulated immune responses causing glial activation and destruction of CNS tissues. Oleanolic acid and erythrodiol are natural triterpenes that display strong anti-inflammatory and immunomodulatory activities. Oleanolic acid beneficially influences the course of established EAE. We now extend our previous observations to erythrodiol and address the efficacy of both compounds to protect against EAE, given under different regimens. ; The utility of both triterpenes in disease prevention was evaluated at a clinical and molecular level: in vivo through their prophylactic administration to myelin oligodendrocyte protein-immunized C57BL/6 mice, and in vitro through their addition to stimulated-BV2 microglial cells. ; These triterpenes protected against EAE by restricting infiltration of inflammatory cells into the CNS and by preventing blood–brain barrier disruption. Triterpene-pretreated EAE-mice exhibited less leptin secretion, and switched cytokine production towards a Th2/regulatory profile, with lower levels of Th1 and Th17 cytokines and higher expression of Th2 cytokines in both serum and spinal cord. Triterpenes also affected the humoral response causing auto-antibody production inhibition. In vitro, triterpenes inhibited ERK and rS6 phosphorylation and reduced the proliferative response, phagocytic properties and synthesis of proinflammatory mediators induced by the addition of inflammatory stimuli to microglia. ; Both triterpenes restricted the development of the characteristic features of EAE. We envision these natural products as novel helpful tools for intervention in autoimmune and neurodegenerative diseases including MS. ; This work was supported by grants SAF2009-08407 from the Spanish Ministry of Science and Innovation, and CSI11A08 from the Autonomous Government of Castilla y León. ; Peer reviewed

Glioblastoma, the most aggressive type of primary brain tumour, shows worse prognosis linked to diabetes or obesity persistence. These pathologies are chronic inflammatory conditions characterized by altered profiles of inflammatory mediators, including leptin and secreted phospholipase A2-IIA (sPLA2-IIA). Both proteins, in turn, display diverse pro-cancer properties in different cell types, including astrocytes. Herein, to understand the underlying relationship between obesity and brain tumors, we investigated the effect of leptin, alone or in combination with sPLA2-IIA on astrocytoma cell functions. sPLA2-IIA induced up-regulation of leptin receptors in 1321N1 human astrocytoma cells. Leptin, as well as sPLA2-IIA, increased growth and migration in these cells, through activation/phosphorylation of key proteins of survival cascades. Leptin, at concentrations with minimal or no activating effects on astrocytoma cells, enhanced growth and migration promoted by low doses of sPLA2-IIA. sPLA2-IIA alone induced a transient phosphorylation pattern in the Src/ERK/Akt/mTOR/p70S6K/rS6 pathway through EGFR transactivation, and co-addition of leptin resulted in a sustained phosphorylation of these signaling regulators. Mechanistically, EGFR transactivation and tyrosine- and serine/threonine-protein phosphatases revealed a key role in this leptin-sPLA2-IIA cross-talk. This cooperative partnership between both proteins was also found in primary astrocytes. These findings thus indicate that the adipokine leptin, by increasing the susceptibility of cells to inflammatory mediators, could contribute to worsen the prognosis of tumoral and neurodegenerative processes, being a potential mediator of some obesity-related medical complications. ; This work was supported by the FPI Program from the Autonomous Government of Castilla y Leon (to RM and CC) co-funded by FSE, and by the Spanish Ministry of Science and Innovation (grant SAF2009-08407 and SAF2016-81063). ; Peer Reviewed

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al. ; [Background]: The natural triterpenes, erythrodiol and uvaol, exert anti-inflammatory, vasorelaxing and anti-proliferative effects. Angiotensin II is a well-known profibrotic and proliferative agent that participates in the cardiac remodeling associated with different pathological situations through the stimulation and proliferation of cardiac fibroblasts. Therefore, the aim of the study was to investigate the preventive effects of the natural triterpenes erythrodiol and uvaol on the proliferation and collagen production induced by angiotensin II in cardiac myofibroblasts. Their actions on cardiac hypertrophy triggered by angiotensin II were also studied. [Methodology/Principal Findings]: The effect of erythrodiol and uvaol on angiotensin II-induced proliferation was evaluated in cardiac myofibroblasts from adult rats in the presence or the absence of the inhibitors of PPAR-γ, GW9662 or JNK, SP600125. The effect on collagen levels induced by angiotensin II was evaluated in cardiac myofibroblasts and mouse heart. The presence of low doses of both triterpenes reduced the proliferation of cardiac myofibroblasts induced by angiotensin II. Pretreatment with GW9662 reversed the effect elicited by both triterpenes while SP600125 did not modify it. Both triterpenes at high doses produced an increase in annexing-V binding in the presence or absence of angiotensin II, which was reduced by either SP600125 or GW9662. Erythrodiol and uvaol decreased collagen I and galectin 3 levels induced by angiotensin II in cardiac myofribroblasts. Finally, cardiac hypertrophy, ventricular remodeling, fibrosis, and increases in myocyte area and brain natriuretic peptide levels observed in angiotensin II-infused mice were reduced in triterpene-treated animals. [Conclusions/Significance]: Erythrodiol and uvaol reduce cardiac hypertrophy and left ventricle remodeling induced by angiotensin II in mice by diminishing fibrosis and myocyte area. They also modulate growth and survival of cardiac myofibroblasts. They inhibit the angiotensin II-induced proliferation in a PPAR-γ-dependent manner, while at high doses they activate pathways of programmed cell death that are dependent on JNK and PPAR-γ. © 2012 Martín et al. ; This work was supported by grants from Fondo de Investigaciones Sanitarias (PI09/0871), and Junta de Castilla y León (CSI11A08). Maria Miana and Raquel Jurado-López were paid with a Grant from Red Cardiovascular del FIS (RD06/0014/0007). Rubén Martin was paid with a Grant from La Fundación General de la Universidad de Valladolid (FGUVa) (060/053186). Claudia Cordova was funded by the FPI Program from the Government of Castilla y León (co-funded by FSE). All the authors are members of the Red Cardiovascular del FIS (RECAVA, RD06/0014/0007 and RD06/0014/0000). ; Peer Reviewed

10 páginas, 5 figuras. ; We have investigated mechanisms that contribute to reinforce the relationship between inflammation and cancer. Secreted phospholipase A2 group IIA (sPLA2-IIA) is a molecule relevant in inflammatory events and has been proposed as a marker for some of these. Previously, we reported the mitogenic properties of this sPLA2 in the human astrocytoma cell line 1321N1. Here, we go deeper into the mechanisms that link this inflammatory protein with proliferation in one of the most aggressive types of tumors. We found that phosphorylation of the extracellular regulated kinase (ERK) was preceded by the activation of the small GTPase Ras, and both failed to be activated by inhibiting protein kinase C (PKC). Fractionation and immunofluorescence studies revealed translocation of PKC alpha, delta, and epsilon to the membrane fraction upon stimulation with sPLA2-IIA. Immunoprecipitation analysis showed that sPLA2-IIA induces phosphorylation of the epidermal growth factor receptor (EGFR) through a PKC-dependent pathway. We found that phosphorylation of this receptor contributed to Ras and ERK activation and that inhibition of ERK, PKC, and EGFR blocked the mitogenic response induced by sPLA2-IIA. This study showed that sPLA2-IIA is able to bring into play EGFR to trigger its signaling and that PKC leads the distribution of resources. Interestingly, we found that this is not a cell-specific response, because sPLA2-IIA was also able to transactivate EGFR in MCF7 human breast cancer cells. Therefore, this mechanism could contribute to worsen the prognosis of a tumor in an inflammatory microenvironment. We also present more links of the tumor chain possibly susceptible to targeting. ; This work was supported by the Ramón y Cajal Program (to M.H.), F.P.I. Program from the Autonomous Government of Castilla y León. (to R.M.), both cofunded by F.S.E and by the ISCIII, Red de Centros RECAVA, C03/01 (to P.M.H. and M.D.G.C.), and by grants SAF2005-01242 and SAF2009-08407 from the Spanish Ministry of Science and Innovation, and CSI11A08 from the Autonomous Government of Castilla y León. ; Peer reviewed

[Background]: Triterpene alcohols and acids are multifunctional compounds widely distributed throughout the plant kingdom that exhibit a variety of beneficial health properties, being synthetic analogs of oleanolic acid under clinical evaluation as anti-tumoral therapeutic agents. However, the antineoplastic activity of two natural occuring triterpenoid alcohols extracted from olive oil, erythrodiol (an intermediate from oleanolic acid), and its isomer, uvaol, has barely been reported, particularly on brain cancer cells. Astrocytomas are among the most common and aggressive type of primary malignant tumors in the neurological system lacking effective treatments, and in this study, we addressed the effect of these two triterpenic diols on the human 1321N1 astrocytoma cell line. ; [Principal Findings]: Erythrodiol and uvaol effectively affected cell proliferation, as well as cell cycle phases and induced 1321N1 cell death. Both triterpenes successfully modulated the apoptotic response, promoting nuclear condensation and fragmentation. They caused retraction and rounding of cultured cells, which lost adherence from their supports, while F-actin and vimentin filaments disappeared as an organized cytoplasmic network. At molecular level, changes in the expression of surface proteins associated with adhesion or death processes were also observed. Moreover, triterpene exposure resulted in the production of reactive oxygen species (ROS) with loss of mitochondrial transmembrane potential, and correlated with the activation of c-Jun N-terminal kinases (JNK). The presence of catalase reversed the triterpenic diols-induced mitochondrial depolarization, JNK activation, and apoptotic death, indicating the critical role of ROS in the action of these compounds. ; [Conclusions]: Overall, we provide a significant insight into the anticarcinogenic action of erythrodiol and uvaol that may have a potential in prevention and treatment of brain tumors and other cancers. ; This study has been funded by a grants SAF2005-01242, SAF2008-00245 and AGL2008-022845 from MICINN, and CSI11A08 from the Government of Castilla y Leon. MH is under the Ramon y Cajal Program (Co-funded by F.S.E.). E.I., R.M. and J.C-T. are supported by a fellowship from the Spanish Ministerio de Educación y Ciencia. ; Peer reviewed

Human group IIA secreted phospholipase A2 (sPLA2-IIA) has been characterized in numerous inflammatory and neoplastic conditions. sPLA2-IIA can either promote or inhibit cell growth depending on the cellular type and the specific injury. We have previously demonstrated that exogenous sPLA2-IIA, by engagement to a membrane structure, induces proliferation and activation of mitogen-activated protein kinases cascade in human astrocytoma cells. In this study, we used human astrocytoma 1321N1 cells to investigate the key molecules mediating sPLA2-IIA-induced cell proliferation. We found that sPLA2-IIA promoted reactive oxygen species (ROS) accumulation, which was abrogated in the presence of allopurinol and DPI, but not by rotenone, discarding mitochondria as a ROS source. In addition, sPLA2-IIA triggered Ras and Raf-1 activation, with kinetics that paralleled ERK phosphorylation, and co-immunoprecipitation assays indicated an association between Ras, Raf-1 and ERK. Additionally, Akt, p70 ribosomal protein S6 kinase, and S6 ribosomal protein were also phosphorylated upon sPLA2-IIA treatment, effect that was abrogated by N-acetylcysteine or LY294002 treatment indicating that ROS and phosphatidylinositol 3 kinase are upstream signaling regulators. As the inhibitors N-acetylcysteine, PD98059, LY294002 or rapamycin blocked sPLA 2-IIA-induced proliferation without activation of the apoptotic program, we suggest that inhibition of these intracellular signal transduction elements may represent a mechanism of growth arrest. Our results reveal new potential targets for therapeutic intervention in neuroinflammatory disorders and brain cancer in particular. © 2009 International Society for Neurochemistry. ; This work was supported by the Ramón y Cajal Program (to M. H.), F. P. I. Program from the Government of Castilla y Leon. (to R. M.) both co-funded by F. S. E, Grants SAF2005-01242 and SAF2008-00245 from the Ministry of Science and Technology, and Grant CSI11A08 from the Government of Castilla y León. ; Peer Reviewed

This is an open-access article distributed under the terms of the Creative Commons Attribution License. ; Pollen is the most common aeroallergen to cause seasonal conjunctivitis. The result of allergen exposure is a strong Th2-mediated response along with conjunctival mast cell degranulation and eosinophilic infiltration. Oleanolic acid (OA) is natural a triterpene that displays strong anti-inflammatory and immunomodulatory properties being an active anti-allergic molecule on hypersensitivity reaction models. However, its effect on inflammatory ocular disorders including conjunctivits, has not yet been addressed. Hence, using a Ragweed pollen (RWP)-specific allergic conjunctivitis (EAC) mouse model we study here whether OA could modify responses associated to allergic processes. We found that OA treatment restricted mast cell degranulation and infiltration of eosinophils in conjunctival tissue and decreased allergen-specific Igs levels in EAC mice. Th2-type cytokines, secreted phospholipase A2 type-IIA (sPLA2-IIA), and chemokines levels were also significantly diminished in the conjunctiva and serum of OA-treated EAC mice. Moreover, OA treatment also suppressed RWP-specific T-cell proliferation. In vitro studies, on relevant cells of the allergic process, revealed that OA reduced the proliferative and migratory response, as well as the synthesis of proinflammatory mediators on EoL-1 eosinophils and RBL-2H3 mast cells exposed to allergic and/or crucial inflammatory stimuli such as RWP, sPLA2-IIA or eotaxin. Taken together, these findings demonstrate the beneficial activity of OA in ocular allergic processes and may provide a new intervention strategy and potential therapy for allergic diseases. © 2014 Córdova et al. ; The study was supported by grants from the Spanish MINECO (reference SAF2009-08407). Patricia Gallego-Muñoz and Claudia Cordova were funded by the FPI Program from the Government of Castilla y León (co-funded by FSE). Rubén Martín was supported by the Sara Borrell Program from the Spanish MINECO. ; Peer Reviewed

Background: Multiple sclerosis (MS) is a chronic demyelinating autoimmune disease affecting the CNS. Recent studies have indicated that intestinal alterations play key pathogenic roles in the development of autoimmune diseases, including MS. The triterpene oleanolic acid (OA), due to its anti-inflammatory properties, has shown to beneficially influence the severity of the experimental autoimmune encephalomyelitis (EAE), a preclinical model of MS. We herein investigate EAE-associated gut intestinal dysfunction and the effect of OA treatment. Methods: Mice with MOG-induced EAE were treated with OA or vehicle from immunization day and were daily analyzed for clinical deficit. We performed molecular and histological analysis in serum and intestinal tissues to measure oxidative and inflammatory responses. We used Caco-2 and HT29-MTX-E12 cells to elucidate OA in vitro effects. Results: We found that OA protected from EAE-induced changes in intestinal permeability and preserved the mucin-containing goblet cells along the intestinal tract. Serum levels of the markers for intestinal barrier damage iFABP and monocyte activation sCD14 were consistently and significantly reduced in OA-treated EAE mice. Beneficial OA effects also included a decrease of pro-inflammatory mediators both in serum and colonic tissue of treated-EAE mice. Moreover, the levels of some immunoregulatory cytokines, the neurotrophic factor GDNF, and the gastrointestinal hormone motilin were preserved in OA-treated EAE mice. Regarding oxidative stress, OA treatment prevented lipid peroxidation and superoxide anion accumulation in intestinal tissue, while inducing the expression of the ROS scavenger Sestrin-3. Furthermore, short-chain fatty acids (SCFA) quantification in the cecal content showed that OA reduced the high iso-valeric acid concentrations detected in EAE-mice. Lastly, using in vitro cell models which mimic the intestinal epithelium, we verified that OA protected against intestinal barrier dysfunction induced by injurious agents produced in both EAE and MS. Conclusion: These findings reveal that OA ameliorates the gut dysfunction found in EAE mice. OA normalizes the levels of gut mucosal dysfunction markers, as well as the pro- and anti-inflammatory immune bias during EAE, thus reinforcing the idea that OA is a beneficial compound for treating EAE and suggesting that OA may be an interesting candidate to be explored for the treatment of human MS. ; This work was supported by Ministerio de Economía y Competitividad (SAF2016-81063 and PID2019-111788RB-I00) and Instituto de Salud Carlos III-Fondo Europeo de Desarrollo Regional (FEDER) (PI18/00257), CIBERCV a way to build Europe, and BG and IG were funded by the FPI Program from the Government of Castilla y León (co-funded by FSE).

Background: The objective of this study is to determine the role of mitochondrial oxidative stress in the dysbiosis associated with a high fat diet in rats. In addition, the impact of gut microbiota (GM) in the cardiometabolic consequences of diet-induced obesity in rats has been evaluated. Methods: Male Wistar rats were fed either a high fat diet (HFD) or a control (CT) one for 6 weeks. At the third week, one-half of the animals of each group were treated with the mitochondrial antioxidant MitoTempo (MT; 0.7 mgKg−1 day−1 i.p). Results: Animals fed an HFD showed a lower microbiota evenness and diversity in comparison to CT rats. This dysbiosis is characterized by a decrease in Firmicutes/Bacteroidetes ratio and relevant changes at family and genera compared with the CT group. This was accompanied by a reduction in colonic mucin-secreting goblet cells. These changes were reversed by MT treatment. The abundance of certain genera could also be relevant in the metabolic consequences of obesity, as well as in the occurrence of cardiac fibrosis associated with obesity. Conclusions: These results support an interaction between GM and mitochondrial oxidative stress and its relation with development of cardiac fibrosis, suggesting new approaches in the management of obesity-related cardiometabolic consequences. ; This work was supported by Instituto de Salud Carlos III-Fondo Europeo de Desarrollo Regional (FEDER) (PI18/00257; CIBERCV) a way to build Europe, Ministerio de Economía y Competitividad (SAF2016-81063). EMM was supported by a contract from CAM (Atracción de talento). BG was funded by the FPI Program from the Government of Castilla y León (co-funded by FSE).