TLR4-pathway impairs synaptic number and cerebrovascular functions through astrocyte activation following traumatic brain injury

Background and purpose: Activation of astrocytes contributes to synaptic remodelling, tissue repair and neuronal survival following traumatic brain injury (TBI). The mechanisms by which these cells interact to resident/infiltrated inflammatory cells to rewire neuronal networks and repair brain functions remain poorly understood. Here, we explored how TLR4-induced astrocyte activation modified synapses and cerebrovascular integrity following TBI. Experimental approach: To determine how functional astrocyte alterations induced by activation of TLR4 pathway in inflammatory cells regulate synapses and neurovascular integrity after TBI, we used pharmacology, genetic approaches, live calcium imaging, immunofluorescence, flow cytometry, blood-brain barrier (BBB) integrity assessment and molecular and behavioural methods. Key results: Shortly after a TBI, there is a recruitment of excitable and reactive astrocytes mediated by TLR4 pathway activation with detrimental effects on post-synaptic density-95 (PSD-95)/vesicular glutamate transporter 1 (VGLUT1) synaptic puncta, BBB integrity and neurological outcome. Pharmacological blockage of the TLR4 pathway with resatorvid (TAK-242) partially reversed many of the observed effects. Synapses and BBB recovery after resatorvid administration were not observed in IP3 R2-/- mice, indicating that effects of TLR4 inhibition depend on the subsequent astrocyte activation. In addition, TBI increased the astrocytic-protein thrombospondin-1 necessary to induce a synaptic recovery in a sub-acute phase. Conclusions and implications: Our data demonstrate that TLR4-mediated signalling, most probably through microglia and/or infiltrated monocyte-astrocyte communication, plays a crucial role in the TBI pathophysiology and that its inhibition prevents synaptic loss and BBB damage accelerating tissue recovery/repair, which might represent a therapeutic potential in CNS injuries and disorders.This work was supported by grants from the Instituto de Salud Carlos III (ISCIII) (Programa Miguel Servet II Grants CPII19/00005;PI16/00735; PI19/00082 to JE; and PI18/00357 to DC, partiallyfunded by FEDER - European Union ‘Una manera de hacer Europa’) and Fundación Mutua Madrileña to JE; European Union's Horizon2020 research and innovation programme under the H2020 MarieSkłodowska-Curie Actions grant agreement no. 794926 and StopFuga de Cerebros Roche Pharma to JMR; and Ministerio de Ciencia e Innovación RTI2018-094887-B-I00 and RYC-2016-20414 to MN andRYC2019-026870-I to JMR. DC, MCO, VVS and EFL are hired bySESCAM

TLR4-pathway impairs synaptic number and cerebrovascular functions through astrocyte activation following traumatic brain injury.

Background and purpose: Activation of astrocytes contributes to synaptic remodelling, tissue repair and neuronal survival following traumatic brain injury (TBI). The mechanisms by which these cells interact to resident/infiltrated inflammatory cells to rewire neuronal networks and repair brain functions remain poorly understood. Here, we explored how TLR4-induced astrocyte activation modified synapses and cerebrovascular integrity following TBI. Experimental approach: To determine how functional astrocyte alterations induced by activation of TLR4 pathway in inflammatory cells regulate synapses and neurovascular integrity after TBI, we used pharmacology, genetic approaches, live calcium imaging, immunofluorescence, flow cytometry, blood-brain barrier (BBB) integrity assessment and molecular and behavioural methods. Key results: Shortly after a TBI, there is a recruitment of excitable and reactive astrocytes mediated by TLR4 pathway activation with detrimental effects on post-synaptic density-95 (PSD-95)/vesicular glutamate transporter 1 (VGLUT1) synaptic puncta, BBB integrity and neurological outcome. Pharmacological blockage of the TLR4 pathway with resatorvid (TAK-242) partially reversed many of the observed effects. Synapses and BBB recovery after resatorvid administration were not observed in IP3 R2-/- mice, indicating that effects of TLR4 inhibition depend on the subsequent astrocyte activation. In addition, TBI increased the astrocytic-protein thrombospondin-1 necessary to induce a synaptic recovery in a sub-acute phase. Conclusions and implications: Our data demonstrate that TLR4-mediated signalling, most probably through microglia and/or infiltrated monocyte-astrocyte communication, plays a crucial role in the TBI pathophysiology and that its inhibition prevents synaptic loss and BBB damage accelerating tissue recovery/repair, which might represent a therapeutic potential in CNS injuries and disorders.This work was supported by grants from the Instituto de Salud Carlos III (ISCIII) (Programa Miguel Servet II Grants CPII19/00005;PI16/00735; PI19/00082 to JE; and PI18/00357 to DC, partiallyfunded by FEDER - European Union ‘Una manera de hacer Europa’) and Fundación Mutua Madrileña to JE; European Union's Horizon2020 research and innovation programme under the H2020 MarieSkłodowska-Curie Actions grant agreement no. 794926 and StopFuga de Cerebros Roche Pharma to JMR; and Ministerio de Ciencia e Innovación RTI2018-094887-B-I00 and RYC-2016-20414 to MN andRYC2019-026870-I to JMR. DC, MCO, VVS and EFL are hired bySESCAM

Docosahexaenoic Acid Inhibits UVB-Induced Activation of NF-κB and Expression of COX-2 and NOX-4 in HR-1 Hairless Mouse Skin by Blocking MSK1 Signaling

Exposure to ultraviolet-B (UVB) radiation induces inflammation and photocarcinogenesis in mammalian skin. Docosahexaenoic acid (DHA), a representative ω-3 polyunsaturated fatty acid, has been reported to possess anti-inflammatory and chemopreventive properties. In the present study, we investigated the molecular mechanisms underlying the inhibitory effects of DHA on UVB-induced inflammation in mouse skin. Our study revealed that topical application of DHA prior to UVB irradiation attenuated the expression of cyclooxygenase-2 (COX-2) and NAD(P)H:oxidase-4 (NOX-4) in hairless mouse skin. DHA pretreatment also attenuated UVB-induced DNA binding of nuclear factor-kappaB (NF-κB) through the inhibition of phosphorylation of IκB kinase-α/β, phosphorylation and degradation of IκBα and nuclear translocation of p50 and p65. In addition, UVB-induced phosphorylation of p65 at the serine 276 residue was significantly inhibited by topical application of DHA. Irradiation with UVB induced phosphorylation of mitogen and stress-activated kinase-1 (MSK1), extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase, and all these events were attenuated by pretreatment with DHA. Blocking ERK and p38 MAP kinase signaling by U0126 and SB203580, respectively, diminished MSK1 phosphorylation in UVB-irradiated mouse skin. Pretreatment with H-89, a pharmacological inhibitor of MSK1, abrogated UVB-induced activation of NF-κB and the expression of COX-2 and NOX-4 in mouse skin. In conclusion, topically applied DHA inhibits the UVB-induced activation of NF-κB and the expression of COX-2 and NOX-4 by blocking the phosphorylation of MSK1, a kinase downstream of ERK and p38 MAP kinase, in hairless mouse skin

Interferon Regulatory Factor-1 (IRF-1) Shapes Both Innate and CD8+ T Cell Immune Responses against West Nile Virus Infection

Interferon regulatory factor (IRF)-1 is an immunomodulatory transcription factor that functions downstream of pathogen recognition receptor signaling and has been implicated as a regulator of type I interferon (IFN)-αβ expression and the immune response to virus infections. However, this role for IRF-1 remains controversial because altered type I IFN responses have not been systemically observed in IRF-1-/- mice. To evaluate the relationship of IRF-1 and immune regulation, we assessed West Nile virus (WNV) infectivity and the host response in IRF-1-/- cells and mice. IRF-1-/- mice were highly vulnerable to WNV infection with enhanced viral replication in peripheral tissues and rapid dissemination into the central nervous system. Ex vivo analysis revealed a cell-type specific antiviral role as IRF-1-/- macrophages supported enhanced WNV replication but infection was unaltered in IRF-1-/- fibroblasts. IRF-1 also had an independent and paradoxical effect on CD8+ T cell expansion. Although markedly fewer CD8+ T cells were observed in naïve animals as described previously, remarkably, IRF-1-/- mice rapidly expanded their pool of WNV-specific cytolytic CD8+ T cells. Adoptive transfer and in vitro proliferation experiments established both cell-intrinsic and cell-extrinsic effects of IRF-1 on the expansion of CD8+ T cells. Thus, IRF-1 restricts WNV infection by modulating the expression of innate antiviral effector molecules while shaping the antigen-specific CD8+ T cell response

Differential impact of LPG-and PG-deficient Leishmania major mutants on the immune response of human dendritic cells

<div><p>Background</p><p><i>Leishmania major</i> infection induces robust interleukin-12 (IL12) production in human dendritic cells (hDC), ultimately resulting in Th1-mediated immunity and clinical resolution. The surface of <i>Leishmania</i> parasites is covered in a dense glycocalyx consisting of primarily lipophosphoglycan (LPG) and other phosphoglycan-containing molecules (PGs), making these glycoconjugates the likely pathogen-associated molecular patterns (PAMPS) responsible for IL12 induction.</p><p>Methodology/Principal Findings</p><p>Here we explored the role of parasite glycoconjugates on the hDC IL12 response by generating <i>L</i>. <i>major</i> Friedlin V1 mutants defective in LPG alone, (FV1 <i>lpg1-</i>), or generally deficient for all PGs, (FV1 <i>lpg2-</i>). Infection with metacyclic, infective stage, <i>L</i>. <i>major</i> or purified LPG induced high levels of <i>IL12B</i> subunit gene transcripts in hDCs, which was abrogated with FV1 <i>lpg1-</i> infections. In contrast, hDC infections with FV1 <i>lpg2-</i> displayed increased <i>IL12B</i> expression, suggesting other PG-related/<i>LPG2</i> dependent molecules may act to dampen the immune response. Global transcriptional profiling comparing WT, FV1 <i>lpg1-</i>, FV1 <i>lpg2-</i> infections revealed that FV1 <i>lpg1-</i> mutants entered hDCs in a silent fashion as indicated by repression of gene expression. Transcription factor binding site analysis suggests that LPG recognition by hDCs induces IL-12 in a signaling cascade resulting in Nuclear Factor κ B (NFκB) and Interferon Regulatory Factor (IRF) mediated transcription.</p><p>Conclusions/Significance</p><p>These data suggest that <i>L</i>. <i>major</i> LPG is a major PAMP recognized by hDC to induce IL12-mediated protective immunity and that there is a complex interplay between PG-baring <i>Leishmania</i> surface glycoconjugates that result in modulation of host cellular IL12.</p></div

Transcriptional profile of breast muscle in heat stressed layers is similar to that of broiler chickens at control temperature

Abstract Background In recent years, the commercial importance of changes in muscle function of broiler chickens and of the corresponding effects on meat quality has increased. Furthermore, broilers are more sensitive to heat stress during transport and at high ambient temperatures than smaller egg-laying chickens. We hypothesised that heat stress would amplify muscle damage and expression of genes that are involved in such changes and, thus, lead to the identification of pathways and networks associated with broiler muscle and meat quality traits. Broiler and layer chickens were exposed to control or high ambient temperatures to characterise differences in gene expression between the two genotypes and the two environments. Results Whole-genome expression studies in breast muscles of broiler and layer chickens were conducted before and after heat stress; 2213 differentially-expressed genes were detected based on a significant (P < 0.05) genotype × treatment interaction. This gene set was analysed with the BioLayout Express3D and Ingenuity Pathway Analysis software and relevant biological pathways and networks were identified. Genes involved in functions related to inflammatory reactions, cell death, oxidative stress and tissue damage were upregulated in control broilers compared with control and heat-stressed layers. Expression of these genes was further increased in heat-stressed broilers. Conclusions Differences in gene expression between broiler and layer chickens under control and heat stress conditions suggest that damage of breast muscles in broilers at normal ambient temperatures is similar to that in heat-stressed layers and is amplified when broilers are exposed to heat stress. The patterns of gene expression of the two genotypes under heat stress were almost the polar opposite of each other, which is consistent with the conclusion that broiler chickens were not able to cope with heat stress by dissipating their body heat. The differentially expressed gene networks and pathways were consistent with the pathological changes that are observed in the breast muscle of heat-stressed broilers

Randomized trial comparing albumin, dextran 70, and polygeline in cirrhotic patients with ascites treated by paracentesis.

Gastroenterology. 1996 Oct;111(4):1002-10. Randomized trial comparing albumin, dextran 70, and polygeline in cirrhotic patients with ascites treated by paracentesis. Gin\ue8s A, Fern\ue1ndez-Esparrach G, Monescillo A, Vila C, Dom\ue8nech E, Abecasis R, Angeli P, Ruiz-Del-Arbol L, Planas R, Sol\ue0 R, Gin\ue8s P, Terg R, Inglada L, Vaqu\ue9 P, Salerno F, Vargas V, Clemente G, Quer JC, Jim\ue9nez W, Arroyo V, Rod\ue9s J. SourceLiver Unit, Hospital Clinic i Provincial, Barcelona, Spain. Abstract BACKGROUND &#38; AIMS: Paracentesis associated with plasma expanders is widely used for the treatment of ascites in cirrhosis. This study investigated the clinical importance of paracentesis-induced-circulatory dysfunction and compared the efficacy of albumin, dextran 70, and polygeline in preventing this complication. METHODS: A total of 289 cirrhotic patients with ascites were randomized to treatment by total paracentesis plus intravenous albumin (97 patients), dextran 70 (93 patients), or polygeline (99 patients). Postparacentesis circulatory dysfunction was defined as an increase in plasma renin activity on the sixth day after paracentesis of more than 50% of the pretreatment value to a level > 4 ng.mL-1.h-1. RESULTS: Postparacentesis circulatory dysfunction occurred more frequently in patients treated with dextran 70 (34.4%; P = 0.018) or polygeline (37.8%; P = 0.004) than in those receiving albumin (18.5%). The plasma expander used and the volume of ascites removed were independent predictors of this complication. Postparacentesis circulatory dysfunction persisted during follow-up and was associated with a shorter time to first readmission (1.3 +/- 0.5 vs. 3.5 +/- 0.8 months, median +/- SEM; P = 0.03) and shorter survival (9.3 +/- 4.2 vs. 16.9 +/- 4.3 months; P = 0.01). Creatinine and sodium levels in serum, and Child-Pugh score at inclusion, and postparacentesis circulatory dysfunction were independent predictors of survival. CONCLUSIONS: Postparacentesis circulatory dysfunction is not spontaneously reversible and is associated with a shorter time to first readmission and shorter survival. Albumin is the best plasma expander to prevent this complication

Cyclooxygenase-2 expression on ifosfamide-induced hemorrhagic cystitis in rats

Purpose Hemorrhagic cystitis (HC) is a limiting side effect of chemotherapy with ifosfamide (IFS). In this study, we investigated the participation of cyclooxygenase-2 (COX-2) upon ifosfamide-induced HC. Methods Male Wistar rats (150-200 g; six rats per group) were treated with saline, IFS (400 mg/kg, i.p.) and analyzed by changes in bladder wet weight, macroscopic and microscopic parameters, and COX-2 expression. In other groups etoricoxib (selective COX-2 inhibitor), indomethacin (non-selective COX inhibitor), thalidomide (selective TNF-alpha inhibitor), pentoxifyllin (non-selective TNF-alpha inhibitor) were added 1 h before IFS administration. The classical protocol using three doses of Mesna was also evaluated and compared with two extra doses of etoricoxib or indomethacin. Results COX-2 was expressed significantly 24 h after IFS administration mainly in myofibroblasts and mast cells evaluated by immunohistochemistry. Treatment 1 h before IFS injection with etoricoxib, indomethacin, thalidomide, and pentoxifylline reduced COX-2 expression and some macroscopic and microscopic parameters in IFS-induced HC. Moreover, addition of etoricoxib or indomethacin with the last two doses of Mesna was more efficient than three doses of Mesna alone when evaluated microscopically. Conclusions COX-2 participates in the pathogenesis of IFS-induced HC and the treatment with COX and TNF-alpha inhibitors reduced COX-2 expression. The addition of COX-inhibitors to the last two doses of Mesna represents a new therapeutic strategy of preventing HC.National Research Council of Brazil (CNPq), CAPES and Fundação Cearense de Amparo a Pesquisa (FUNCAP), Ceará-Brazil, for financial support