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Preterm birth is often associated with chorioamnionitis and increased risk of neurodevelopmental disorders. Preterm birth also leads to cerebellar underdevelopment but the mechanisms of disrupted cerebellar development in preterm infants are little understood. Here, we leveraged single-nuclei RNA-sequencing of the cerebellum in a rhesus macaque model of chorioamnionitis and preterm birth, to show that chorioamnionitis leads to Purkinje cell loss and disrupted maturation of granule cells and oligodendrocytes in the fetal cerebellum at late gestation. Purkinje cell loss is accompanied by decreased sonic hedgehog signaling from Purkinje cells to granule cells, which show an accelerated maturation. Chorioamnionitis also accelerated pre-oligodendrocyte maturation into myelinating oligodendrocytes, confirmed by increased expression of myelin basic protein in the cerebellum of chorioamnionitis-exposed fetuses. These findings are consistent with reported histopathological findings in individuals with autism and suggest a novel mechanism through which perinatal inflammation contributes to neurodevelopmental disorders.

Many inflammation-associated diseases, including cancers, increase in women after menopause and with obesity. In contrast to anti-inflammatory actions of 17β-estradiol, we find estrone, which dominates after menopause, is pro-inflammatory. In human mammary adipocytes, cytokine expression increases with obesity, menopause, and cancer. Adipocyte:cancer cell interaction stimulates estrone- and NFκB-dependent pro-inflammatory cytokine upregulation. Estrone- and 17β-estradiol-driven transcriptomes differ. Estrone:ERα stimulates NFκB-mediated cytokine gene induction; 17β-estradiol opposes this. In obese mice, estrone increases and 17β-estradiol relieves inflammation. Estrone drives more rapid ER+ breast cancer growth in vivo. HSD17B14, which converts 17β-estradiol to estrone, associates with poor ER+ breast cancer outcome. Estrone and HSD17B14 upregulate inflammation, ALDH1 activity, and tumorspheres, while 17β-estradiol and HSD17B14 knockdown oppose these. Finally, a high intratumor estrone:17β-estradiol ratio increases tumor-initiating stem cells and ER+ cancer growth in vivo. These findings help explain why postmenopausal ER+ breast cancer increases with obesity, and offer new strategies for prevention and therapy., This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 845104

In the brain neuropil, translocator protein 18 kDa (TSPO) is a stress response protein that is upregulated in microglia and astrocytes in diverse central nervous system pathologies. TSPO is widely used as a biomarker of neuroinflammation in preclinical and clinical neuroimaging studies. However, there is a paucity of knowledge on the function(s) of TSPO in glial cells. In this study, we explored a putative interaction between TSPO and NADPH oxidase 2 (NOX2) in microglia. We found that TSPO associates with gp91phox and p22phox, the principal subunits of NOX2 in primary murine microglia. The association of TSPO with gp91phox and p22phox was observed using co-immunoprecipitation, confocal immunofluorescence imaging, and proximity ligation assay. We found that besides gp91phox and p22phox, voltage-dependent anion channel (VDAC) also co-immunoprecipitated with TSPO consistent with previous reports. When we compared lipopolysaccharide (LPS) stimulated microglia to vehicle control, we found that a lower amount of gp91phox and p22phox protein co-immunoprecipitated with TSPO suggesting a disruption of the TSPO-NOX2 subunits association. TSPO immuno-gold electron microscopy confirmed that TSPO is present in the outer mitochondrial membrane but it is also found in the endoplasmic reticulum (ER), mitochondria-associated ER membrane (MAM), and in the plasma membrane. TSPO localization at the MAM may represent a subcellular site where TSPO interacts with gp91phox and p22phox since the MAM is a point of communication between outer mitochondria membrane proteins (TSPO) and ER proteins (gp91phox and p22phox) where they mature and form the cytochrome b558 (Cytb558) heterodimer. We also found that an acute burst of reactive oxygen species (ROS) increased TSPO levels on the surface of microglia and this effect was abrogated by a ROS scavenger. These results suggest that ROS production may alter the subcellular distribution of TSPO. Collectively, our findings suggest that in microglia, TSPO is associated with the major NOX2 subunits gp91phox and p22phox. We hypothesize that this interaction may regulate Cytb558 formation and modulate NOX2 levels, ROS production, and redox homeostasis in microglia.

Renal ischemia and reperfusion injury (IRI) is considered an inflammatory syndrome. To move forward in its pathogenesis, we exploited the role of several cytokines on renal damages triggered by IRI. Specifically to evaluate the role of Th1 immune profile in this system, IL-12, IFN-gamma, and IFN-gamma/IL-12 deficient (KO) mice on C57BL/6 background and their controls were subjected to IRI. In each group, blood and kidney samples were harvested. Renal function was evaluated by serum creatinine and renal morphometric analyses. Gene expression of IL-6 and HO-1 were also investigated by Q-PCR. IFN-gamma KO animals presented the highest impairment in renal function compared to controls. Conversely, IL-12 KO animals were absolutely protected and, in a lesser extent, IFN-gamma/IL-12 KO double knockout was also protected from IRI. Gene expression analyses showed higher expression of HO-1, a cytoprotective gene, and IL-6, a pro-inflammatory cytokine, in IFN-gamma deficient animals subjected to IRI. Our results confirm that Th1 related cytokines such as IL-12 and IFN-gamma are critically involved in renal ischemia and reperfusion injury.

A lesão renal induzida pela I/R é a principal causa de IRA nos rins nativos e nos rins transplantados e estudos enfatizam a participação de células inflamatórias na sua patogênese, através da caracterização de lesão endotelial, infiltração leucocitária e a geração de mediadores inflamatórios pelas células epiteliais tubulares. Evidências recentes mostram que as células T CD4+ exercem um papel fundamental como mediadoras da agressão renal na I/R, ressaltando-se o envolvimento do paradigma Th1/Th2 como um possível mecanismo efetor. O presente estudo foi realizado com o objetivo de estudar a participação de algumas citocinas Th1 e Th2 no desenvolvimento da lesão de I/R renal. Para tanto, nós nos propusemos a desenvolver um modelo experimental de I/R renal em camundongos deficientes em IL-12, IFN-, e duplo deficientes em IFN- e IL-12 (representando defeito da via de ativação Th1), camundongos deficientes em IL-4 e IL-10 (representando defeito da via de ativação Th2) e duplo deficientes em IL-10 e IL-12, tendo como controles camundongos normais (selvagens). Todos os animais foram submetidos a uma lesão de I/R por ligadura reversível do pedículo renal por 45 minutos seguidos de 24 horas de reperfusão. Após a indução da isquemia, nós analisamos as alterações funcionais (creatinina e uréia por método bioquímico colorimétrico) e morfológicas renais (morfometria renal), além de investigar a expressão molecular de HO-1 (um gene de proteção tecidual), de t-bet (um transcrito envolvido na diferenciação Th1), de GATA-3 (um transcrito envolvido na diferenciação Th2), citocina pró-inflamatória IL-6 e de uma quimiocina pró-inflamatória MCP-1, visando caracterizar a influência da polarização Th1/Th2 da resposta imune na lesão renal induzida pela I/R. Nós mostramos que os camundongos deficientes em IL-4, IFN-, IL-10/IL-12 e IL-10 apresentaram uma disfunção renal importante, caracterizada por altos níveis séricos de creatinina e uréia e por um alto grau de agressão morfológica renal, avaliada pela percentagem de área de necrose tubular. Todos estes resultados foram significativamente mais acentuados que obtidos nos camundongos deficientes em IL-12 e IFN-/IL-12, sendo comparáveis aos animais selvagens. Por outro lado, a capacidade de reparação renal, medida pela percentagem de área de regeneração tubular, foi mais precoce nos camundongos deficientes em IFN-, quando comparada com os camundongos deficientes em IL-12 e IFN-/IL-12. A análise molecular quantitativa, utilizando-se o PCR em tempo real mostrou uma expressão significantimente maior de RNAm de HO-1, IL-6 MCP-1 e do fator transcricional pata Th2 (GATA-3) nos camundongos deficientes em IL-4, IFN-, IL-10/IL-12 e IL-10, em comparação com os camundongos deficientes em IL-12, IFN-/IL-12 que apresentaram baixa expressão do fator transcricional para Th1 (T-bet), em 24 horas após a I/R renal. Quando fomos fazer a transferência adotiva de médula óssea de animais deficientes em IL-12 e IL-4 para animais selvagens previamente com depleção subletal celular, esses animais vieram apresentar resultados semelhantes aos animais deficientes em IL-12 e IL-4, onde os animais selvagens reconstituídos com medula óssea dos animais IL-12 apresentaram menor níveis de uréia sérica e menor expressão de RNAm IL-6, enquanto que os animais selvagens recontituídos com medula óssea dos animais IL-4 apresentaram maior níveis de uréia sérica e maior expressão de RNAm IL-6, esses dados comprovam que realmente a citocina IL-12 parece ser a principal citocina envolvida no processo inflamatório desencadeado pela lesão de isquemia e reperfusão renal. Tais achados favorecem a hipótese dos efeitos deletérios das células com per_l Th1 e o papel protetor das células Th2 na lesão isquêmica renal. Estes novos episódios podem fornecer a base para uma melhor compreensão fisiopatológicos, bem como para o desenvolvimento de métodos preventivos e terapêuticos para a insuficiência renal aguda isquêmica. Renal ischemia/reperfusion injury (I/R) is the major cause of acute renal failure (ARF) in the native as well as in the transplanted kidneys, with a complex pathogenesis that involves many components of inflammatory response such as leukocyte infiltration and generation of inflammatory mediators by tubular cells. Recent evidences show a critical role of the CD4+ T cell, with the Th1/Th2 paradigm as a possible ejectors mechanism. The present study has the objective to investigate the participation of some main Th1 and Th2 cytokines in the development of the renal of I/R. For that, we developed an experimental model of renal I/R in deficient in IL-12, IFN-, double deficient IFN- and IL-12 (representing defect of the pathway Th1), IL-4 and IL-10 (representing defect of the pathway Th2) and in double deficient IL-10 and IL-12 knockout mice, having wild-type mice as controls. All animals were submitted to renal I/R by reversible ligation of the renal pedicles for 45 minutes followed by 24 hours of reperfusion. After induction of the ischemia, we analyzed renal function and morphometric histological analyzes. Furthermore, we quantified the expression of HO-1 (cytoprotection gene), t-bet (transcription factor involved in the differentiation Th1), GATA-3 (transcription factor involved in the differentiation Th2), and cytokine pro-inflammatory IL-6 and chemokine pro-inflammatory MCP-1, aiming to characterize the influence of Th1/Th2 polarization in the immune response driven by the renal I/R. We showed that the IL-4, IFN-, IL-10 and IL-10/IL-12-de_cient mice presented an important kidney dysfunction, characterized by higher levels serum creatinine and tubular necrosis, being comparable to the wild-type animals. The molecular analyses, utilizing the real-time PCR, showed a significantly higher expression of HO-1, IL-6, MCP-1 and transcription factor involved in the differentiation Th2 (GATA-3) in the in IL-4, IFN-, IL-10 and IL-10/IL-12-defficient mice, in comparison with the in IL-12, IFN-/IL-12-de_cient mice showed lower expression transcription factor involved in the differentiation Th1 (T-bet) . Moreover, the renal dysfunction seen in Th2-de_cient mice was followed by a higher expression of IL-6. When we make the transfer adoptive of bone marrow of animals deficient in IL-12 and IL-4 to wild animals previously with sublethal cell depletion, these animals produce similar results to animals deficient in IL-12 and IL-4, where the animals reconstituted wild animals with bone marrow IL-12 showed lower levels of serum urea and lower expression mRNA of IL-6, while the wild animals reconstituted with bone marrow of animals IL-4 showed higher levels of serum urea and greater expression of mRNA IL-6. These data show that really the cytokine IL-12 appears to be the main cytokine involved in the inflammatory process triggered by the injury of ischemia and reperfusion kidney. Such _ndings favor the hypothesis of the deleterious effects of Th1 cells with prole and the protective role of Th2 cells in ischemic renal injury. These novel insights may provide basis for the better pathophysiological understanding, as well as for development of preventive and therapeutic methods for the ischemic acute renal failure.

In response to different cellular stresses, a family of protein kinases phosphorylates eIF2alpha (alpha subunit of eukaryotic initiation factor-2), contributing to regulation of both general and genespecific translation proposed to alleviate cellular injury or alternatively induce apoptosis. Recently, we reported eIF2alpha(P) (phosphorylated eIF2alpha) in the brain during SE (status epilepticus) induced by pilocarpine in mice, an animal model of TLE (temporal lobe epilepsy) [Carnevalli, Pereira, Longo, Jaqueta, Avedissian, Mello and Castilho (2004) Neurosci. Lett. 357, 191-194]. We show in the present study that one eIF2alpha kinase family member, PKR (double-stranded-RNA-dependent protein kinase), is activated in the cortex and hippocampus at 30 min of SE, reflecting the levels of eIF2alpha(P) in these areas. In PKR-deficient animals subjected to SE, eIF2alpha phosphorylation was clearly evident coincident with activation of a secondary eIF2alpha kinase, PEK/PERK (pancreatic eIF2alpha kinase/RNA-dependent-protein-kinase-like endoplasmic reticulum kinase), denoting a compensatory mechanism between the two kinases. The extent of eIF2alpha phosphorylation correlated with the inhibition of protein synthesis in the brain, as determined from polysome profiles. We also found that C57BL/6 mice, which enter SE upon pilocarpine administration but are more resistant to seizure-induced neuronal degeneration, showed very low levels of eIF2alpha(P) and no inhibition of protein synthesis during SE. These results taken together suggest that PKR-mediated phosphorylation of eIF2alpha contributes to inhibition of protein synthesis in the brain during SE and that sustained high levels of eIF2alpha phosphorylation may facilitate ensuing cell death in the most affected areas of the brain in TLE.

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundacao de Auxilio aos Docentes e Alunos-UNIFESP (FADA) Complex Fluids INCT Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The Th1/Th2 balance represents an important factor in the pathogenesis of renal ischemia-reperfusion injury (IRI). in addition, IRI causes a systemic inflammation that can affect other tissues, such as the lungs. To investigate the ability of renal IRI to modulate pulmonary function in a specific model of allergic inflammation, C57Bl/6 mice were immunized with ovalbumin/albumen on days 0 and 7 and challenged with an ovalbumin (OA) aerosol on days 14 and 21. After 24 h of the second antigen challenge, the animals were subjected to 45 minutes of ischemia. After 24 h of reperfusion, the bronchoalveolar lavage (BAL) fluid, blood and lung tissue were collected for analysis. Serum creatinine levels increased in both allergic and non-immunized animals subjected to IRI. However, BAL analysis showed a reduction in the total cells (46%) and neutrophils (58%) compared with control allergic animals not submitted to IRI. in addition, OA challenge induced the phosphorylation of ERK and Akt and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lung homogenates. After renal IRI, the phosphorylation of ERK and expression of COX-2 and iNOS were markedly reduced; however, there was no difference in the phosphorylation of Akt between sham and ischemic OA-challenged animals. Mucus production was also reduced in allergic mice after renal IRI. IL-4, IL-5 and IL-13 were markedly down-regulated in immunized/challenged mice subjected to IRI. These results suggest that renal IRI can modulate lung allergic inflammation, probably by altering the Th1/Th2 balance and, at least in part, by changing cellular signal transduction factors. Copyright (C) 2012 S. Karger AG, Basel Universidade Federal de São Paulo, Dept Ciencias Biol, Diadema, SP, Brazil Univ São Paulo, Fac Ciencias Farmaceut, Dept Anal Clin & Toxicol, São Paulo, Brazil Universidade Federal de São Paulo, Disciplina Nefrol, Lab Imunol Clin & Expt, São Paulo, Brazil Univ São Paulo, Inst Ciencias Biomed, Dept Imunol, BR-05508 São Paulo, Brazil Universidade Federal de São Paulo, Dept Ciencias Biol, Diadema, SP, Brazil Universidade Federal de São Paulo, Disciplina Nefrol, Lab Imunol Clin & Expt, São Paulo, Brazil FAPESP: 07/07139-3 FAPESP: 10/01404-0 Web of Science

Ischemia and reperfusion injury (IRI) are mainly caused by leukocyte activation, endothelial dysfunction and production of reactive oxygen species. Moreover, IRI can lead to a systemic response affecting distant organs, such as the lungs.The objective was to study the pulmonary inflammatory systemic response after renal IRI.Male C57Bl/6 mice were subjected to 45 min of bilateral renal ischemia, followed by 4, 6, 12, 24 and 48 h of reperfusion. Blood was collected to measure serum creatinine and cytokine concentrations. Bronchoalveolar lavage fluid (BALF) was collected to determine the number of cells and PGE(2) concentration. Expressions of iNOS and COX-2 in lung were determined by Western blot. Gene analyses were quantified by real time PCR.Serum creatinine increased in the IRI group compared to sham mainly at 24 h after IRI (2.57 +/- 0.16 vs. 0.43 +/- 0.07, p0.01). The total number of cells in BAL fluid was higher in the IRI group in comparison with sham, 12 h (100 x 10(4) +/- 15.63 vs. 18.1 x 10(4) +/- 10.5, p0.05) 24 h (124 x 10(4) +/- 8.94 vs. 23.2 x 10(4) +/- 3.5, p0.05) and 48 h (79 x 10(4) +/- 15.72 vs. 22.2 x 10(4) +/- 4.2, p0.05), mainly by mononuclear cells and neutrophils. Pulmonary COX-2 and iNOS were up-regulated in the IRI group. TNF-alpha, IL-1beta, MCP-1, KC and IL-6 mRNA expression were up-regulated in kidney and lungs 24 h after renal IRI. ICAM-1 mRNA was up-regulated in lungs 24 h after renal IRI. Serum TNF-alpha, IL-1beta and MCP-1 and BALF PGE(2) concentrations were increased 24 h after renal IRI.Renal IRI induces an increase of cellular infiltration, up-regulation of COX-2, iNOS and ICAM-1, enhanced chemokine expression and a Th1 cytokine profile in lung demonstrating that the inflammatory response is indeed systemic, possibly leading to an amplification of renal injury.