RESUMO
Influenza A virus (IAV) infection during pregnancy initiates significant aortic endothelial and vascular smooth muscle dysfunction, with inflammation and T cell activation, but the details of the mechanism are yet to be clearly defined. Here we demonstrate that IAV disseminates preferentially into the perivascular adipose tissue (PVAT) of the aorta in mice. IAV mRNA levels in the PVAT increased at 1-3 days post infection (d.p.i) with the levels being ~4-8 fold higher compared with the vessel wall. IAV infection also increased Ly6Clow patrolling monocytes and Ly6Chigh pro-inflammatory monocytes in the vessel wall at 3 d.p.i., which was then followed by a greater homing of these monocytes into the PVAT at 6 d.p.i. The vascular immune phenotype was characteristic of a "vascular storm"- like response, with increases in neutrophils, pro-inflammatory cytokines and oxidative stress markers in the PVAT and arterial wall, which was associated with an impairment in endothelium-dependent relaxation to acetylcholine. IAV also triggered a PVAT compartmentalised elevation in CD4+ and CD8+ activated T cells. In conclusion, the PVAT of the aorta is a niche that supports IAV dissemination and a site for perpetuating a profound innate inflammatory and adaptive T cell response. The manifestation of this inflammatory response in the PVAT following IAV infection may be central to the genesis of cardiovascular complications arising during pregnancy.
Assuntos
Vírus da Influenza A , Tecido Adiposo , Animais , Aorta , Endotélio Vascular , Feminino , Inflamação/genética , Camundongos , GravidezRESUMO
Influenza A virus (IAV) infection during pregnancy can increase the risk for neurodevelopmental disorders in the offspring, however, the underlying neurobiological mechanisms are largely unknown. To recapitulate viral infection, preclinical studies have traditionally focused on using synthetic viral mimetics, rather than live IAV, to examine consequences of maternal immune activation (MIA)-dependent processes on offspring. In contrast, few studies have used live IAV to assess effects on global gene expression, and none to date have addressed whether moderate IAV, mimicking seasonal influenza disease, alters normal gene expression trajectories in different brain regions across different stages of development. Herein, we show that moderate IAV infection during pregnancy, which causes mild maternal disease and no overt foetal complications in utero, induces lasting effects on the offspring into adulthood. We observed behavioural changes in adult offspring, including disrupted prepulse inhibition, dopaminergic hyper-responsiveness, and spatial recognition memory deficits. Gene profiling in the offspring brain from neonate to adolescence revealed persistent alterations to normal gene expression trajectories in the prefronal cortex, hippocampus, hypothalamus and cerebellum. Alterations were found in genes involved in inflammation and neurogenesis, which were predominately dysregulated in neonatal and early adolescent offspring. Notably, late adolescent offspring born from IAV infected mice displayed altered microglial morphology in the hippocampus. In conclusion, we show that moderate IAV during pregnancy perturbs neurodevelopmental trajectories in the offspring, including alterations in the neuroinflammatory gene expression profile and microglial number and morphology in the hippocampus, resulting in behavioural changes in adult offspring. Such early perturbations may underlie the vulnerability in human offspring for the later development of neurodevelopmental disorders, including schizophrenia. Our work highlights the importance of using live IAV in developing novel preclinical models that better recapitulate the real-world impact of inflammatory insults during pregnancy on offspring neurodevelopmental trajectories and disease susceptibility later in life.
Assuntos
Encéfalo , Vírus da Influenza A , Infecções por Orthomyxoviridae , Efeitos Tardios da Exposição Pré-Natal , Animais , Feminino , Gravidez , Camundongos , Encéfalo/metabolismo , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Infecções por Orthomyxoviridae/metabolismo , Complicações Infecciosas na Gravidez/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Hipocampo/metabolismo , Transtornos do Neurodesenvolvimento/metabolismo , Transtornos do Neurodesenvolvimento/genética , Expressão Gênica , Modelos Animais de DoençasRESUMO
Maternal influenza A virus (IAV) infection during pregnancy can affect offspring immune programming and development. Offspring born from influenza-infected mothers are at increased risk of neurodevelopmental disorders and have impaired respiratory mucosal immunity against pathogens. The gut-associated lymphoid tissue (GALT) represents a large proportion of the immune system in the body and plays an important role in gastrointestinal (GI) homeostasis. This includes immune modulation to antigens derived from food or microbes, gut microbiota composition, and gut-brain axis signaling. Therefore, in this study, we investigated the effect of maternal IAV infection on mucosal immunity of the GI tract in the offspring. There were no major anatomical changes to the gastrointestinal tract of offspring born to influenza-infected dams. In contrast, maternal IAV did affect the mucosal immunity of offspring, showing regional differences in immune cell profiles within distinct GALT. Neutrophils, monocytes/macrophages, CD4+ and CD8+ T cells infiltration was increased in the cecal patch offspring from IAV-infected dams. In the Peyer's patches, only activated CD4+ T cells were increased in IAV offspring. IL-6 gene expression was also elevated in the cecal patch but not in the Peyer's patches of IAV offspring. These findings suggest that maternal IAV infection perturbs homeostatic mucosal immunity in the offspring gastrointestinal tract. This could have profound ramifications on the gut-brain axis and mucosal immunity in the lungs leading to increased susceptibility to respiratory infections and neurological disorders in the offspring later in life.NEW & NOTEWORTHY Influenza A virus (IAV) infection during pregnancy is associated with changes in gut-associated lymphoid tissue (GALT) in the offspring in a region-dependent manner. Neutrophils and monocytes/macrophages were elevated in the cecal patch of offspring from infected dams. This increase in innate immune cell infiltration was not observed in the Peyer's patches. T cells were also elevated in the cecal patch but not in the Peyer's patches.
Assuntos
Vírus da Influenza A , Influenza Humana , Gravidez , Feminino , Camundongos , Animais , Humanos , Nódulos Linfáticos Agregados , Imunidade nas Mucosas , Linfócitos T CD8-PositivosRESUMO
Influenza A virus (IAV) infection during pregnancy causes severe maternal and perinatal complications, despite a lack of vertical transmission of IAV across the placenta. Here, we demonstrate a significant alteration in the maternal vascular landscape that underpins the maternal and downstream fetal pathology to IAV infection in mice. In IAV infection of nonpregnant mice, the local lung inflammatory response was contained to the lungs and was self-resolving, whereas in pregnant mice, virus dissemination to major maternal blood vessels, including the aorta, resulted in a peripheral "vascular storm," with elevated proinflammatory and antiviral mediators and the influx of Ly6Clow and Ly6Chigh monocytes, plus neutrophils and T cells. This vascular storm was associated with elevated levels of the adhesion molecules ICAM and VCAM and the pattern-recognition receptors TLR7 and TLR9 in the vascular wall, resulting in profound vascular dysfunction. The sequalae of this IAV-driven vascular storm included placental growth retardation and intrauterine growth restriction, evidence of placental and fetal brain hypoxia, and increased circulating cell free fetal DNA and soluble Flt1. In contrast, IAV infection in nonpregnant mice caused no obvious alterations in endothelial function or vascular inflammation. Therefore, IAV infection during pregnancy drives a significant systemic vascular alteration in pregnant dams, which likely suppresses critical blood flow to the placenta and fetus. This study in mice provides a fundamental mechanistic insight and a paradigm into how an immune response to a respiratory virus, such as IAV, is likely to specifically drive maternal and fetal pathologies during pregnancy.
Assuntos
Imunidade Adaptativa/genética , Imunidade Inata/genética , Inflamação/genética , Vírus da Influenza A/genética , Animais , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Feminino , Feto/imunologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Inflamação/virologia , Vírus da Influenza A/patogenicidade , Influenza Humana/genética , Influenza Humana/imunologia , Influenza Humana/virologia , Glicoproteínas de Membrana/genética , Camundongos , Monócitos/metabolismo , Monócitos/patologia , Placenta/irrigação sanguínea , Placenta/imunologia , Placenta/virologia , Gravidez , Linfócitos T/imunologia , Linfócitos T/virologia , Receptor 7 Toll-Like/genética , Receptor Toll-Like 9/genéticaRESUMO
There are currently no effective treatments to prevent spontaneous preterm labor. The precise upstream biochemical pathways that regulate the transition between uterine quiescence during pregnancy and contractility during labor remain unclear. It is well known however that intrauterine inflammation, including infection, is commonly associated with preterm labor. In this study, we identified the immunoproteasome subunit low-molecular-mass protein (LMP)7 mRNA expression to be significantly upregulated in laboring human myometrium. Silencing LMP7 using siRNA-targeted knockdown of LMP7 and its inhibitor ONX-0914 in human myometrial cells and tissues decreased proinflammatory cytokines (IL-6), cell chemotaxis (CXCL8, CCL2 expression, and THP-1 migration), cell to cell adhesion (ICAM1 expression and myometrial adhesion), contraction-associated proteins (PTGS2, FP, PGE2, and PGF2α), as well as suppressing contractions in myometrial cells and in myometrial tissues obtained from laboring women. In addition, LMP7 silencing reduced NF-κB RelA activity. ONX-0914 alleviated inflammation (CCL3, CXCL1, PTGS2, and IL-6) in myometrium, placenta, fetal brain, amniotic fluid, and maternal serum induced by LPS in pregnant mice. Collectively, our data suggest a novel role for ONX-014 to suppress uterine activation and contractility associated with preterm labor.
Assuntos
Miométrio/metabolismo , Trabalho de Parto Prematuro/prevenção & controle , Oligopeptídeos/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Contração Uterina/efeitos dos fármacos , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quimiocina CCL2/metabolismo , Feminino , Humanos , Inflamação/tratamento farmacológico , Inflamação/imunologia , Inflamação/patologia , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Gravidez , Complexo de Endopeptidases do Proteassoma/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Células THP-1 , Fator de Transcrição RelA/metabolismoRESUMO
Preeclampsia affects 5% of all pregnancies and is a serious disorder of pregnancy, characterised by high maternal blood pressure, placental hypoxia, fluid retention (oedema) and proteinuria. Women with preeclampsia are associated with exaggerated levels of pro-inflammatory cytokines, chemokines and anti-angiogenic factors such as soluble fms-like tyrosine kinase-1 (sFLT1). Studies in non-gestational tissues have described the bromodomain (BRD) and extraterminal family of proteins, in particular BRD4 to play a critical role in propagating inflammation and is currently a therapeutic target for treating cancer, lung inflammation and asthma. The aims of this study were to: (i) determine the effect of severe early-onset preeclampsia on placental BRD4 expression; (ii) the effect of loss of BRD4 function by siRNA-targeted knockdown or with the BRD inhibitor JQ1 in human primary trophoblast cells and human umbilical vein endothelial cells (HUVECs) on TNF-stimulated production of pro-inflammatory mediators, cell adhesion molecules and anti-angiogenic markers and (iii) the effect of BRD4 suppression on placental sFLT1 secretion under hypoxia conditions and in preeclampic placenta. BRD4 mRNA expression was significantly increased (sevenfold) in severe early-onset preeclampsia placenta. BRD4 silencing resulted in a significant reduction in TNF-induced IL6, CXCL8, CCL2, CXCL1 and sFLT1-e15a mRNA expression and IL6, CXCL8, CCL2, CXCL1 and sFLT1 secretion in primary trophoblast and HUVECs. Additionally, JQ1 treatment significantly reduced placental sFLT1 secretion under hypoxic conditions and in preterm preeclamptic placenta. In conclusion, these findings suggest BRD4 may play a central role in propagating inflammation and endothelial dysfunction associated with the pathophysiology of early-onset preeclampsia.
Assuntos
Biomarcadores/metabolismo , Proteínas Nucleares/metabolismo , Placenta/metabolismo , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/patologia , Fatores de Transcrição/metabolismo , Trofoblastos/metabolismo , Trofoblastos/patologia , Adulto , Idade de Início , Estudos de Casos e Controles , Proteínas de Ciclo Celular , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , GravidezRESUMO
Preterm birth remains one of the leading causes of neonatal death. Inflammation and maternal infection are two of the leading aetiological factors for preterm birth. Labour is associated with increased production of proinflammatory cytokines, chemokines and prolabour mediators in human gestational tissues. In non-gestational tissues, synthesis of proinflammatory and prolabour mediators is regulated by components of the protein synthesis machinery. Therefore, in the present study we investigated the effect of human labour on the expression of three protein synthesis markers, namely eukaryotic elongation factor 2 kinase (EEF2K), mitogen-activated protein kinase interacting protein kinase 1 (MKNK1) and eukaryotic translation initiation factor 4E (EIF4E), and their role in regulating inflammation in human gestational tissues. In fetal membranes and myometrium, EEF2K expression was significantly lower, whereas MKNK1 expression was significantly higher withterm and preterm labourcompared to term nolabour. In contrast, EIF4E expression did not change in fetal membranes or myometrium with labour. In primary myometrial cells, loss-of-function studies using specific chemical inhibitors of EEF2K (A484954) and MKNK1 (CGP57380) demonstrated that MKNK1, but not EEF2K, was required for polyinosinic-polycytidylic acid (poly(I:C); a viral double-stranded RNA mimetic) and interleukin (IL)-1ß-induced production of IL6, C-X-C motif chemokine ligand 8 (CXCL8), prostaglandin-endoperoxide synthase 2 (PTGS2) and prostaglandin F2α. In conclusion, spontaneous term and preterm labour is associated with decreased EEF2K and increased MKNK1 expression in fetal membranes and myometrium. Moreover, MKNK1 is involved in the genesis of proinflammatory and prolabour mediators that is mediated by inflammation or infection. However, further studies are required to elucidate the role of EEF2K in human labour.
Assuntos
Parto Obstétrico , Quinase do Fator 2 de Elongação/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Membranas Extraembrionárias/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Trabalho de Parto , Miométrio/metabolismo , Complicações na Gravidez/metabolismo , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Biomarcadores/metabolismo , Células Cultivadas , Corioamnionite/genética , Corioamnionite/metabolismo , Quinase do Fator 2 de Elongação/genética , Fator de Iniciação 4E em Eucariotos/genética , Membranas Extraembrionárias/fisiopatologia , Feminino , Ruptura Prematura de Membranas Fetais/genética , Ruptura Prematura de Membranas Fetais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Interleucina-1beta/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Miométrio/efeitos dos fármacos , Miométrio/fisiopatologia , Poli I-C/farmacologia , Gravidez , Complicações na Gravidez/genética , Complicações na Gravidez/fisiopatologia , Nascimento Prematuro/genética , Nascimento Prematuro/metabolismo , Cultura Primária de Células , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Heightened placental inflammation and dysfunction are commonly associated in pregnant obese women compared to their pregnant lean counterparts. The small GTPase superfamily members known as the rat sarcoma viral oncogene homolog (Ras) proteins, in particular, the K-Ras and H-Ras isoforms, have been implicated to regulate inflammation. The aims were to determine the placental Ras expression and activity with maternal obesity and its role in regulating placental inflammation. Human placenta was obtained at term Caesarean section from lean and obese pregnant women to determine the effect of maternal obesity on Ras protein expression and activity. To determine the effect of Ras on inflammation induced by bacterial endotoxin LPS and proinflammatory cytokines TNF-α or IL-1ß, the chemical inhibitor lonafarnib (total Ras inhibitor) and siRNA (siKRAS and siHRAS) were used. Total Ras protein expression together with combined K-Ras and H-Ras activity was significantly increased in the placenta of obese pregnant women and when stimulated with LPS, IL-1ß, or TNF-α. Lonafarnib significantly suppressed LPS-, IL-1ß-, or TNF-α-induced IL-6, IL-8, MCP-1, and GRO-α expression and secretion in placental tissue. Primary trophoblast cells transfected with siKRAS or siHRAS demonstrated only K-Ras silencing significantly decreased IL-1ß-, TNF-α-, or LPS-induced IL-6, IL-8, and MCP-1 expression and secretion. Furthermore, siKRAS significantly reduced downstream ERK-1/2 activation induced by LPS. In trophoblast cells, ERK-1/2 signalling is required for IL-6, IL-8, MCP-1, and GRO-α secretion. These studies implicate a role for K-Ras in regulating inflammation in human placenta. Suppressing overactive placental K-Ras function may prevent adverse fetal outcomes complicated by maternal obesity.
Assuntos
Inflamação/imunologia , Inflamação/metabolismo , Obesidade/imunologia , Obesidade/metabolismo , Placenta/imunologia , Placenta/metabolismo , Proteínas ras/metabolismo , Western Blotting , Cesárea/efeitos adversos , Feminino , Humanos , Técnicas Imunoenzimáticas , Interleucina-6/metabolismo , Piperidinas/farmacologia , Placenta/efeitos dos fármacos , Gravidez , Piridinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/metabolismo , Proteínas ras/antagonistas & inibidoresRESUMO
Preterm birth is the largest single cause of neonatal death and morbidity. By activating cytokine- and Toll-like receptor (TLR)-signaling pathways, infection and/or inflammation are strongly associated with preterm delivery. Interferon regulatory factor-1 (IRF1) is an important regulator of the inflammatory response. The aims of this study were to establish the effect of 1) labor on IRF1 expression in human fetal membranes and myometrium, 2) prolabor mediators on IRF1 expression and activity, and 3) IRF1 small interfering RNA on the expression of prolabor mediators. IRF1 expression was higher in fetal membranes and myometrium after spontaneous term labor and in preterm fetal membranes with infection. The proinflammatory cytokine IL1B, the bacterial product fsl-1, and viral analog polyinosinic:polycytidylic acid (poly [I:C]) significantly increased IRF1 mRNA expression and transcriptional activity in human primary myometrial cells. In addition, IL1B increased IRF1 activity in primary amnion cells. IRF1 silencing in myometrial cells decreased IL1B-, fsl-1-, and poly (I:C)-induced cytokine (IL6, TNF, IL1B) and chemokine (CXCL8, CCL2) mRNA expression and IL6, CXCL8, and CCL2 release. IL1B-, fsl-1-, and poly (I:C)-induced PTGS2 mRNA expression and IL1B-induced prostaglandin release was also decreased by IRF1 silencing. In conclusion, IRF1 upregulation in fetal membranes and myometrium after term labor indicates a proinflammatory role for IRF1 in human parturition. IRF1 is involved in TLR- and cytokine-mediated signaling in human myometrium. These data provide new insights into the mechanisms associated with inflammation- and infection-associated preterm birth. IRF1 inhibitors as therapeutics for the management of spontaneous preterm birth warrants further investigation.
Assuntos
Membranas Extraembrionárias/metabolismo , Fator Regulador 1 de Interferon/metabolismo , Miométrio/metabolismo , Trabalho de Parto Prematuro/metabolismo , Quimiocinas/metabolismo , Citocinas/metabolismo , Feminino , Inativação Gênica , Humanos , Fator Regulador 1 de Interferon/genética , Trabalho de Parto/metabolismo , GravidezRESUMO
Specific protein receptors that mediate internalization and entry of influenza A virus (IAV) have not been identified for any cell type. Sialic acid (SIA), the primary attachment factor for IAV hemagglutinin, is expressed by numerous cell surface glycoproteins and glycolipids, confounding efforts to identify specific receptors involved in virus infection. Lec1 Chinese hamster ovary (CHO) epithelial cells express cell surface SIA and bind IAV yet are largely resistant to infection. Here, we demonstrate that expression of the murine macrophage galactose-type lectin 1 (MGL1) by Lec1 cells enhanced Ca(2+)-dependent IAV binding and restored permissivity to infection. Lec1 cells expressing MGL1 were infected in the presence or absence of cell surface SIA, indicating that MGL1 can act as a primary receptor or as a coreceptor with SIA. Lec1 cells expressing endocytosis-deficient MGL1 mediated Ca(2+)-dependent IAV binding but were less sensitive to IAV infection, indicating that direct internalization via MGL1 can result in cellular infection. Together, these studies identify MGL1 as a cell surface glycoprotein that can act as an authentic receptor for both attachment and infectious entry of IAV.
Assuntos
Assialoglicoproteínas/metabolismo , Vírus da Influenza A/fisiologia , Influenza Humana/metabolismo , Lectinas Tipo C/metabolismo , Proteínas de Membrana/metabolismo , Receptores Virais/metabolismo , Ligação Viral , Internalização do Vírus , Animais , Assialoglicoproteínas/genética , Células CHO , Cálcio/metabolismo , Linhagem Celular , Cricetinae , Cricetulus , Humanos , Vírus da Influenza A/genética , Influenza Humana/genética , Influenza Humana/virologia , Lectinas Tipo C/genética , Macrófagos/metabolismo , Macrófagos/virologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Receptores Virais/genéticaRESUMO
Increasing evidence indicates that endoplasmic reticulum (ER) stress is involved in various diseases. In nongestational tissues, several markers of the unfolded protein response (UPR) have been shown to regulate the inflammatory response. Thus, the aim of this study was to determine the effect of human labor on markers of ER stress in fetal membranes and myometrium. In addition, the effect of ER stress inhibition on the expression and secretion of proinflammatory and prolabor mediators was also assessed. The markers of ER stress, GRP78, IRE1, and spliced XBP1 (XBP1s), were significantly increased in fetal membranes and myometrium after term and preterm labor compared to nonlaboring samples. Given that inflammation is considered to be one of the leading causes of spontaneous preterm birth, here we used bacterial endotoxin lipopolysaccharide (LPS) as a model for infection-induced preterm birth. In term nonlabored fetal membranes and myometrium, LPS induced UPR activation as evidenced by a significant increase in the expression of GRP78, IRE1, and XBP1s in fetal membranes and myometrium. The use of the chemical chaperones 4-phenylbutyric acid (4-PBA) and tauroursodeoxycholic acid (TUDCA) alleviated ER stress induced by LPS. 4-PBA and TUDCA also ameliorated the increase in LPS-induced prolabor mediators. Our data suggest that the UPR may regulate the inflammatory responses associated with labor or infection in fetal membranes and myometrium of pregnant term and preterm women. Thus, the use of ER stress inhibitors, in particular 4-PBA or TUDCA, may be a potential therapeutic strategy for the prevention of infection-mediated spontaneous preterm birth.
Assuntos
Estresse do Retículo Endoplasmático , Membranas Extraembrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Trabalho de Parto/metabolismo , Miométrio/metabolismo , Trabalho de Parto Prematuro/metabolismo , Regulação para Cima , Adulto , Processamento Alternativo , Biomarcadores/metabolismo , Cesárea , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endorribonucleases/genética , Endorribonucleases/metabolismo , Membranas Extraembrionárias/efeitos dos fármacos , Membranas Extraembrionárias/imunologia , Membranas Extraembrionárias/patologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Miométrio/efeitos dos fármacos , Miométrio/imunologia , Miométrio/patologia , Trabalho de Parto Prematuro/imunologia , Trabalho de Parto Prematuro/patologia , Fenilbutiratos/farmacologia , Gravidez , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição de Fator Regulador X , Ácido Tauroquenodesoxicólico/farmacologia , Técnicas de Cultura de Tecidos , Tocolíticos/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Proteína 1 de Ligação a X-BoxRESUMO
Influenza A virus (IAV) infection in pregnancy resembles a preeclamptic phenotype characterised by vascular dysfunction and foetal growth retardation. Given that low dose aspirin (ASA) is safe in pregnancy and is used to prevent preeclampsia, we investigated whether ASA or NO-conjugated aspirin, NCX4016, resolve vascular inflammation and function to improve offspring outcomes following IAV infection in pregnant mice. Pregnant mice were intranasally infected with a mouse adapted IAV strain (Hkx31; 104 plaque forming units) and received daily treatments with either 200µg/kg ASA or NCX4016 via oral gavage. Mice were then culled and the maternal lungs and aortas collected for qPCR analysis, and wire myography was performed on aortic rings to assess endothelial and vascular smooth muscle functionality. Pup and placentas were weighed and pup growth rates and survival assessed. IAV infected mice had an impaired endothelial dependent relaxation response to ACh in the aorta, which was prevented by ASA and NCX4016 treatment. ASA and NCX4016 treatment prevented IAV dissemination and inflammation of the aorta as well as improving the pup placental ratios in utero, survival and growth rates at post-natal day 5. Low dose ASA is safe to use during pregnancy for preeclampsia and this study demonstrates that ASA may prove a promising treatment for averting the significant vascular complications associated with influenza infection during pregnancy.
Assuntos
Aspirina/análogos & derivados , Vírus da Influenza A , Influenza Humana , Nitratos , Pré-Eclâmpsia , Doenças Vasculares , Humanos , Camundongos , Feminino , Gravidez , Animais , Placenta , Aspirina/farmacologia , Inflamação , AortaRESUMO
Prostate cancer is ranked second in the world for cancer-related deaths in men, highlighting the lack of effective therapies for advanced-stage disease. Toll-like receptors (TLRs) and immunity have a direct role in prostate cancer pathogenesis, but TLR9 has been reported to contribute to both the progression and inhibition of prostate tumorigenesis. To further understand this apparent disparity, we have investigated the effect of TLR9 stimulation on prostate cancer progression in an immune-competent, syngeneic orthotopic mouse model of prostate cancer. Here, we utilized the class B synthetic agonist CPG-1668 to provoke a TLR9-mediated systemic immune response and demonstrate a significant impairment of prostate tumorigenesis. Untreated tumors contained a high abundance of immune-cell infiltrates. However, pharmacological activation of TLR9 resulted in smaller tumors containing significantly fewer M1 macrophages and T cells. TLR9 stimulation of tumor cells in vitro had no effect on cell viability or its downstream transcriptional targets, whereas stimulation in macrophages suppressed cancer cell growth via type I IFN. This suggests that the antitumorigenic effects of CPG-1668 were predominantly mediated by an antitumor immune response. This study demonstrated that systemic TLR9 stimulation negatively regulates prostate cancer tumorigenesis and highlights TLR9 agonists as a useful therapeutic for the treatment of prostate cancer.
Assuntos
Neoplasias da Próstata , Receptor Toll-Like 9 , Humanos , Masculino , Animais , Camundongos , Neoplasias da Próstata/tratamento farmacológico , Carcinogênese , Próstata , Transformação Celular NeoplásicaRESUMO
The ability to recognise women who are at-risk of preterm labour (PTL) is often difficult. Over 50% of women who are identified with factors associated with an increased risk of preterm birth will ultimately deliver at term. The cervicovaginal fluid (CVF) comprises a range of proteins secreted by gestational tissues, making it an ideal candidate for the screening of differentially expressed proteins associated with PTL. CVF samples were collected from at-risk asymptomatic women. Two-dimensional gel electrophoresis techniques were used to examine the CVF proteome of women who spontaneously delivered preterm 11-22 days later compared with gestation-matched women who delivered at term. Five candidate biomarkers were selected for further validation in a larger independent cohort of asymptomatic women. Thioredoxin (TXN) and interleukin 1 receptor antagonist (IL1RN) concentrations in the CVF were found to be significantly reduced up to 90 days prior to spontaneous PTL compared with women who subsequently delivered at term. TXN was able to predict spontaneous PTL within 28 days after sampling with a high positive predictive value (PPV) and negative predictive value (NPV) of 75.0% and 96.4% respectively. IL1RN also showed comparable PPV and NPV of 72.7% and 95.7% respectively. The discovery of these differentially expressed proteins may assist in the development of a new predictive bedside test in identifying asymptomatic women who have an increased risk of spontaneous PTL.
Assuntos
Biomarcadores/metabolismo , Líquidos Corporais/metabolismo , Trabalho de Parto Prematuro/diagnóstico , Nascimento Prematuro/diagnóstico , Proteoma/análise , Vagina/metabolismo , Adulto , Estudos de Casos e Controles , Estudos de Coortes , Eletroforese em Gel Bidimensional , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Recém-Nascido , Trabalho de Parto Prematuro/metabolismo , Gravidez , Nascimento Prematuro/metabolismo , Proteômica , Fatores de Risco , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Adulto JovemRESUMO
A significant obstetric complication facing contemporary materno-fetal medicine is preterm premature rupture of the fetal membranes (preterm PROM), which occurs in 30% of all preterm births. The objective of this study was to identify differentially expressed proteins in the cervicovaginal fluid of asymptomatic women before the clinical manifestation of preterm PROM. The preterm PROM group comprised of women with samples collected 6-23 days before PROM, who subsequently delivered preterm (n=5). Women who spontaneously delivered at term served as gestation-matched controls (n=10). Two-dimensional difference in-gel electrophoresis was used to distinguish differential expression between the pooled groups and fold changes were subsequently confirmed by two-dimensional PAGE of individual samples. Spots of interest were identified by mass spectrometry. Proteins that were significantly reduced with impending preterm PROM included the following: thioredoxin (2.7-fold), interleukin 1 receptor antagonist (1.7-fold), fatty acid-binding protein 5 (2.1-fold), cystatin A (dimer; 1.9-fold), monocyte/neutrophil elastase inhibitor (1.6-fold), squamous cell carcinoma antigen-1 (2.1-fold) and γ-glutamyl cyclotransferase (3.0-fold). By contrast, annexin A3 (3.7-fold) and vitamin D binding protein (3.9-fold) were significantly increased with impending preterm PROM. Western blot analysis was also performed on an independent cohort of preterm PROM and control samples to validate these candidate biomarkers. These proteins have known biological functions in oxidative balance, anti-inflammatory activity, metabolism or protease inhibition that may facilitate membrane rupture.
Assuntos
Líquidos Corporais/química , Colo do Útero/metabolismo , Ruptura Prematura de Membranas Fetais/diagnóstico , Proteoma/análise , Vagina/metabolismo , Adulto , Líquidos Corporais/metabolismo , Estudos de Casos e Controles , Feminino , Ruptura Prematura de Membranas Fetais/metabolismo , Humanos , Gravidez , Proteoma/metabolismo , Proteômica , Manejo de Espécimes , Fatores de Tempo , Estudos de Validação como Assunto , Adulto JovemRESUMO
Respiratory syncytial virus (RSV) commonly infects the upper respiratory tract (URT) of humans, manifesting with mild cold or flu-like symptoms. However, in infants and the elderly, severe disease of the lower respiratory tract (LRT) often occurs and can develop into chronic airway disease. A better understanding of how an acute RSV infection transitions to a LRT chronic inflammatory disease is critically important to improve patient care and long-term health outcomes. To model acute and chronic phases of the disease, we infected wild-type C57BL/6 and toll-like receptor 7 knockout (TLR7 KO) mice with RSV and temporally assessed nasal, airway and lung inflammation for up to 42 days post-infection. We show that TLR7 reduced viral titers in the URT during acute infection but promoted pronounced pathogenic and chronic airway inflammation and hyperreactivity in the LRT. This study defines a hitherto unappreciated molecular mechanism of lower respiratory pathogenesis to RSV, highlighting the potential of TLR7 modulation to constrain RSV pathology to the URT.
Assuntos
Asma , Infecções por Vírus Respiratório Sincicial , Receptor 7 Toll-Like , Animais , Camundongos , Brônquios/patologia , Inflamação/patologia , Camundongos Endogâmicos C57BL , Receptor 7 Toll-Like/genética , Camundongos KnockoutRESUMO
Influenza A virus (IAV) infection during pregnancy disrupts maternal and fetal health through biological mechanisms, which are to date poorly characterised. During pregnancy, the viral clearance mechanisms from the lung are sub-optimal and involve hyperactive innate and adaptive immune responses that generate wide-spread inflammation. Pregnancy-related adaptations of the immune and the cardiovascular systems appear to result in delayed recovery post-viral infection, which in turn promotes a prolonged inflammatory phenotype, increasing disease severity, and causing maternal and fetal health problems. This has immediate and long-term consequences for the mother and fetus, with complications including acute cardiopulmonary distress syndrome in the mother that lead to perinatal complications such as intrauterine growth restriction (IUGR), and birth defects; cleft lip, cleft palate, neural tube defects and congenital heart defects. In addition, an increased risk of long-term neurological disorders including schizophrenia in the offspring is reported. In this review we discuss the pathophysiology of IAV infection during pregnancy and its striking similarity to other well-established complications of pregnancy such as preeclampsia. We discuss general features of vascular disease with a focus on vascular inflammation and define the "Vascular Storm" that is triggered by influenza infection during pregnancy, as a pivotal disease mechanism for short and long term cardiovascular complications.
Assuntos
Doenças Transmissíveis , Vírus da Influenza A , Influenza Humana , Infecções por Orthomyxoviridae , Gravidez , Humanos , Feminino , InflamaçãoRESUMO
Endosomal NOX2 oxidase-dependent ROS production promotes influenza pathogenicity, but the role of NOX4 oxidase, which is highly expressed in the lung endothelium, is largely unknown. The aim of this study was to determine if endothelial NOX4 expression can influence viral pathology in vivo, using a mouse model of influenza infection. WT and transgenic endothelial NOX4 overexpressing mice (NOX4 TG) were infected intranasally with the Hong Kong H3N2 X-31 influenza A virus (104 PFU; HK x-31) or PBS control. Mice were culled at either 3 or 7 days post-infection to analyse: airway inflammation by bronchoalveolar lavage fluid (BALF) cell counts; NOX4, as well as inflammatory cytokine and chemokine gene expression by QPCR; and ROS production by an L-012-enhanced chemiluminescence assay. Influenza A virus infection of WT mice resulted in a significant reduction in lung NOX4 mRNA at day 3, which persisted until day 7, when compared to uninfected mice. Influenza A virus infection of NOX4 TG mice resulted in significantly less weight loss than that of WT mice at 3-days post infection. Viral titres were decreased in infected NOX4 TG mice compared to the infected WT mice, at both 3- and 7-days post infection and there was significantly less lung alveolitis, peri-bronchial inflammation and neutrophil infiltration. The oxidative burst from BALF inflammatory cells extracted from infected NOX4 TG mice was significantly less than that in the WT mice. Expression of macrophage and neutrophil chemoattractants CXCL10, CCL3, CXCL1 and CXCL2 in the lung tissue were significantly lower in NOX4 TG mice compared to the WT mice at 3-days post infection. We conclude that endothelial NOX4 oxidase is protective against influenza morbidity and is a potential target for limiting influenza A virus-induced lung inflammation.
Assuntos
NADPH Oxidase 4 , Infecções por Orthomyxoviridae , Pneumonia , Animais , Endotélio/metabolismo , Endotélio/patologia , Inflamação/metabolismo , Vírus da Influenza A Subtipo H3N2 , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Morbidade , NADPH Oxidase 4/metabolismo , Infecções por Orthomyxoviridae/patologia , Oxirredutases/metabolismo , Pneumonia/patologia , Pneumonia/virologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Background and Objective: Neurocognitive dysfunction is present in up to â¼61% of people with chronic obstructive pulmonary disease (COPD), with symptoms including learning and memory deficiencies, negatively impacting the quality of life of these individuals. As the mechanisms responsible for neurocognitive deficits in COPD remain unknown, we explored whether chronic cigarette smoke (CS) exposure causes neurocognitive dysfunction in mice and whether this is associated with neuroinflammation and an altered neuropathology. Methods: Male BALB/c mice were exposed to room air (sham) or CS (9 cigarettes/day, 5 days/week) for 24 weeks. After 23 weeks, mice underwent neurocognitive tests to assess working and spatial memory retention. At 24 weeks, mice were culled and lungs were collected and assessed for hallmark features of COPD. Serum was assessed for systemic inflammation and the hippocampus was collected for neuroinflammatory and structural analysis. Results: Chronic CS exposure impaired lung function as well as driving pulmonary inflammation, emphysema, and systemic inflammation. CS exposure impaired working memory retention, which was associated with a suppression in hippocampal microglial number, however, these microglia displayed a more activated morphology. CS-exposed mice showed changes in astrocyte density as well as a reduction in synaptophysin and dendritic spines in the hippocampus. Conclusion: We have developed an experimental model of COPD in mice that recapitulates the hallmark features of the human disease. The altered microglial/astrocytic profiles and alterations in the neuropathology within the hippocampus may explain the neurocognitive dysfunction observed during COPD.
RESUMO
Macrophages undergo a metabolic switch from oxidative phosphorylation to glycolysis when exposed to gram-negative bacterial lipopolysaccharide (LPS), which modulates antibacterial host defence mechanisms. Here, we show that LPS treatment of macrophages increased the classical oxidative burst response via the NADPH oxidase (NOX) 2 enzyme, which was blocked by 2-deoxyglucose (2-DG) inhibition of glycolysis. The inhibition of the pentose phosphate pathway with 6-aminonicotinamide (6-AN) also suppressed the LPS-induced increase in NOX2 activity and was associated with a significant reduction in the mRNA expression of NOX2 and its organizer protein p47phox. Notably, the LPS-dependent enhancement in NOX2 oxidase activity was independent of both succinate and mitochondrial reactive oxygen species (ROS) production. LPS also increased type I IFN-ß expression, which was suppressed by 2-DG and 6-AN and, therefore, is dependent on glycolysis and the pentose phosphate pathway. The type I IFN-ß response to LPS was also inhibited by apocynin pre-treatment, suggesting that NOX2-derived ROS promotes the TLR4-induced response to LPS. Moreover, recombinant IFN-ß increased NOX2 oxidase-dependent ROS production, as well as NOX2 and p47phox expression. Our findings identify a previously undescribed molecular mechanism where both glycolysis and the pentose phosphate pathway are required to promote LPS-induced inflammation in macrophages.