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1.
Eur J Clin Invest ; 53(3): e13907, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36377348

RESUMO

AIMS: Pulmonary hypertension (PH) is accompanied by pulmonary vascular remodelling. By targeted delivery of Interleukin-9 (IL9) via the immunocytokine F8IL9, beneficial effects could be demonstrated in a mouse model of PH. This study aimed to compare two immunocytokine formats (single-chain Fv and full IgG) and to identify potential target cells of IL9. METHODS: The Monocrotaline mouse model of PH (PH, n = 12) was chosen to evaluate the treatment effects of F8IL9F8 (n = 12) and F8IgGIL9 (n = 6) compared with sham-induced animals (control, n = 10), the dual endothelin receptor antagonist Macitentan (MAC, n = 12) or IL9-based immunocytokines with irrelevant antigen specificity (KSFIL9KSF, n = 12; KSFIgGIL9 n = 6). Besides comparative validation of treatment effects, the study was focused on the detection and quantification of mast cells (MCs) and regulatory T cells (Tregs). RESULTS: There was a significantly elevated systolic right ventricular pressure (104 ± 36 vs. 45 ± 17 mmHg) and an impairment of right ventricular echocardiographic parameters (RVbasal: 2.52 ± 0.25 vs. 1.94 ± 0.13 mm) in untreated PH compared with controls (p < 0.05). Only the groups treated with F8IL9, irrespective of the format, showed consistent beneficial effects (p < 0.05). Moreover, F8IL9F8 but not F8IgGIL9 treatment significantly reduced lung tissue damage compared with untreated PH mice (p < 0.05). There was a significant increase in Tregs in F8IL9-treated compared with control animals, the untreated PH and the MAC group (p < 0.05). CONCLUSIONS: Beneficial treatment effects of targeted IL9 delivery in a preclinical model of PH could be convincingly validated. IL9-mediated recruitment of Tregs into lung tissue might play a crucial role in the induction of anti-inflammatory and anti-proliferative mechanisms potentially contributing to a novel disease-modifying concept.


Assuntos
Hipertensão Pulmonar , Camundongos , Animais , Hipertensão Pulmonar/tratamento farmacológico , Interleucina-9/efeitos adversos , Pulmão , Modelos Animais de Doenças
2.
Glia ; 70(12): 2309-2329, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-35929192

RESUMO

Astrocytes are increasingly being recognized as contributors to physiological brain function and behavior. Astrocytes engage in glia-synaptic interactions through peripheral astrocyte processes, thus modulating synaptic signaling, for example, by handling glutamate removal from the synaptic cleft and (re)provision to axonal terminals. Peripheral astrocyte processes are ultrafine membrane protrusions rich in the membrane-to-actin cytoskeleton linker Ezrin, an essential component of in vitro filopodia formation and in vivo peripheral astrocyte process motility. Consequently, it has been postulated that Ezrin significantly contributes to neurodevelopment as well as astrocyte functions within the adult brain. However, while Ezrin has been studied in vitro within cultured primary astrocytes, in vivo studies on the role of Ezrin in astrocytes remain to be conducted and consequences of its depletion to be studied. Here, we investigated consequences of Ezrin deletion in the mouse brain starting from early neuronal specification. While Ezrin knockout did not impact prenatal cerebral cortex development, behavioral phenotyping depicted reduced exploratory behavior. Starting with postnatal appearance of glia cells, Ezrin was verified to remain predominantly expressed in astrocytes. Proteome analysis of Ezrin deficient astrocytes revealed alterations in glutamate and ion homeostasis, metabolism and cell morphology - important processes for synaptic signal transmission. Notably, Ezrin deletion in astrocytes provoked (GFAP) glial fibrillary acidic protein upregulation - a marker of astrocyte activation and reactive astrogliosis. However, this spontaneous, reactive astrogliosis exhibited proteome changes distinct from ischemic-induced reactive astrogliosis. Moreover, in experimental ischemic stroke, Ezrin knockout mice displayed reduced infarct volume, indicating a protective effect of the Ezrin deletion-induced changes and astrogliosis.


Assuntos
Astrócitos , Gliose , Animais , Astrócitos/metabolismo , Proteínas do Citoesqueleto , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/metabolismo , Ácido Glutâmico/metabolismo , Camundongos , Camundongos Knockout , Fenótipo , Gravidez , Proteoma/metabolismo , Regulação para Cima
3.
FASEB J ; 35(10): e21939, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34549824

RESUMO

The unfolded protein response (UPR) is associated with hepatic metabolic function, yet it is not well understood how endoplasmic reticulum (ER) disturbance might influence metabolic homeostasis. Here, we describe the physiological function of Cysteine-rich with EGF-like domains 2 (Creld2), previously characterized as a downstream target of the ER-stress signal transducer Atf6. To this end, we generated Creld2-deficient mice and induced UPR by injection of tunicamycin. Creld2 augments protein folding and creates an interlink between the UPR axes through its interaction with proteins involved in the cellular stress response. Thereby, Creld2 promotes tolerance to ER stress and recovery from acute stress. Creld2-deficiency leads to a dysregulated UPR and causes the development of hepatic steatosis during ER stress conditions. Moreover, Creld2-dependent enhancement of the UPR assists in the regulation of energy expenditure. Furthermore, we observed a sex dimorphism in human and mouse livers with only male patients showing an accumulation of CRELD2 protein during the progression from non-alcoholic fatty liver disease to non-alcoholic steatohepatitis and only male Creld2-deficient mice developing hepatic steatosis upon aging. These results reveal a Creld2 function at the intersection between UPR and metabolic homeostasis and suggest a mechanism in which chronic ER stress underlies fatty liver disease in males.


Assuntos
Moléculas de Adesão Celular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Homeostase , Fígado/metabolismo , Resposta a Proteínas não Dobradas , Envelhecimento , Animais , Progressão da Doença , Estresse do Retículo Endoplasmático , Fígado Gorduroso , Humanos , Masculino , Camundongos , Hepatopatia Gordurosa não Alcoólica
4.
Int J Mol Sci ; 22(5)2021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-33806610

RESUMO

Microglia, the innate immune cells of the CNS, exhibit long-term response changes indicative of innate immune memory (IIM). Our previous studies revealed IIM patterns of microglia with opposing immune phenotypes: trained immunity after a low dose and immune tolerance after a high dose challenge with pathogen-associated molecular patterns (PAMP). Compelling evidence shows that innate immune cells adopt features of IIM via immunometabolic control. However, immunometabolic reprogramming involved in the regulation of IIM in microglia has not been fully addressed. Here, we evaluated the impact of dose-dependent microglial priming with ultra-low (ULP, 1 fg/mL) and high (HP, 100 ng/mL) lipopolysaccharide (LPS) doses on immunometabolic rewiring. Furthermore, we addressed the role of PI3Kγ on immunometabolic control using naïve primary microglia derived from newborn wild-type mice, PI3Kγ-deficient mice and mice carrying a targeted mutation causing loss of lipid kinase activity. We found that ULP-induced IIM triggered an enhancement of oxygen consumption and ATP production. In contrast, HP was followed by suppressed oxygen consumption and glycolytic activity indicative of immune tolerance. PI3Kγ inhibited glycolysis due to modulation of cAMP-dependent pathways. However, no impact of specific PI3Kγ signaling on immunometabolic rewiring due to dose-dependent LPS priming was detected. In conclusion, immunometabolic reprogramming of microglia is involved in IIM in a dose-dependent manner via the glycolytic pathway, oxygen consumption and ATP production: ULP (ultra-low-dose priming) increases it, while HP reduces it.


Assuntos
Classe Ib de Fosfatidilinositol 3-Quinase/imunologia , Imunidade Inata/imunologia , Memória Imunológica/imunologia , Trifosfato de Adenosina/imunologia , Animais , Glicólise/imunologia , Tolerância Imunológica/imunologia , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/imunologia , Consumo de Oxigênio/imunologia , Moléculas com Motivos Associados a Patógenos/imunologia , Transdução de Sinais/imunologia
5.
J Neuroinflammation ; 17(1): 292, 2020 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-33028343

RESUMO

BACKGROUND: Sepsis-associated encephalopathy (SAE) is an early and frequent event of infection-induced systemic inflammatory response syndrome. Phosphoinositide 3-kinase γ (PI3Kγ) is linked to neuroinflammation and inflammation-related microglial activity. In homeotherms, variations in ambient temperature (Ta) outside the thermoneutral zone lead to thermoregulatory responses, mainly driven by a gradually increasing sympathetic activity, and may affect disease severity. We hypothesized that thermoregulatory response to hypothermia (reduced Ta) aggravates SAE in PI3Kγ-dependent manner. METHODS: Experiments were performed in wild-type, PI3Kγ knockout, and PI3Kγ kinase-dead mice, which were kept at neutral (30 ± 0.5 °C) or moderately lowered (26 ± 0.5 °C) Ta. Mice were exposed to lipopolysaccharide (LPS, 10 µg/g, from Escherichia coli serotype 055:B5, single intraperitoneal injection)-evoked systemic inflammatory response (SIR) and monitored 24 h for thermoregulatory response and blood-brain barrier integrity. Primary microglial cells and brain tissue derived from treated mice were analyzed for inflammatory responses and related cell functions. Comparisons between groups were made with one-way or two-way analysis of variance, as appropriate. Post hoc comparisons were made with the Holm-Sidak test or t tests with Bonferroni's correction for adjustments of multiple comparisons. Data not following normal distribution was tested with Kruskal-Wallis test followed by Dunn's multiple comparisons test. RESULTS: We show that a moderate reduction of ambient temperature triggers enhanced hypothermia of mice undergoing LPS-induced systemic inflammation by aggravated SAE. PI3Kγ deficiency enhances blood-brain barrier injury and upregulation of matrix metalloproteinases (MMPs) as well as an impaired microglial phagocytic activity. CONCLUSIONS: Thermoregulatory adaptation in response to ambient temperatures below the thermoneutral range exacerbates LPS-induced blood-brain barrier injury and neuroinflammation. PI3Kγ serves a protective role in suppressing release of MMPs, maintaining microglial motility and reinforcing phagocytosis leading to improved brain tissue integrity. Thus, preclinical research targeting severe brain inflammation responses is seriously biased when basic physiological prerequisites of mammal species such as preferred ambient temperature are ignored.


Assuntos
Regulação da Temperatura Corporal/fisiologia , Classe Ib de Fosfatidilinositol 3-Quinase/deficiência , Lipopolissacarídeos/toxicidade , Encefalopatia Associada a Sepse/enzimologia , Encefalopatia Associada a Sepse/fisiopatologia , Animais , Animais Recém-Nascidos , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/enzimologia , Barreira Hematoencefálica/fisiopatologia , Temperatura Corporal/efeitos dos fármacos , Temperatura Corporal/fisiologia , Regulação da Temperatura Corporal/efeitos dos fármacos , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Encefalopatia Associada a Sepse/induzido quimicamente
6.
Basic Res Cardiol ; 114(3): 26, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31016449

RESUMO

Sepsis-induced myocardial depression (SIMD) is an early and frequent consequence of the infection-induced systemic inflammatory response syndrome. In homiotherms, variations in ambient temperature (Ta) outside the thermoneutral zone induce thermoregulatory responses mainly driven by a gradually increased sympathetic activity, which may affect disease severity. We hypothesized that thermoregulatory responses upon reduced Ta exposition aggravate SIMD in mice. Mice were kept at neutral Ta (30 ± 0.5 °C), moderately lowered Ta (26 ± 0.5 °C) or markedly lowered Ta (22 ± 0.5 °C), exposed to lipopolysaccharide- (LPS, 10 µg/g, from Escherichia coli serotype 055:B5, single intraperitoneal injection) evoked shock and monitored for survival, cardiac autonomic nervous system function and left ventricular performance. Primary adult cardiomyocytes and heart tissue derived from treated mice were analyzed for inflammatory responses and signaling pathways of myocardial contractility. We show that a moderate reduction of Ta to 26 °C led to a 40% increased mortality of LPS-treated mice when compared to control mice and that a marked reduction of Ta to 22 °C resulted in an early mortality of all mice. Mice kept at 26 °C exhibited increased heart rate and altered indices of heart rate variability (HRV), indicating sympathovagal imbalance along with aggravated LPS-induced SIMD. This SIMD was associated with reduced myocardial ß-adrenergic receptor expression and suppressed adrenergic signaling, as well as with increased myocardial iNOS expression, nitrotyrosine formation and leukocyte invasion as well as enhanced apoptosis and appearance of contraction band necrosis in heart tissue. While ineffective separately, combined treatment with the ß2-adrenergic receptor (AR) antagonist ICI 118551 (10 ng/gbw) and the inducible nitric oxide synthase (iNOS) inhibitor 1400 W (5 µg/gbw) reversed the increase in LPS-induced mortality and aggravation of SIMD at reduced Ta. Thus, consequences of thermoregulatory adaptation in response to ambient temperatures below the thermoneutral range increase the mortality from LPS-evoked shock and markedly prolong impaired myocardial function. These changes are mitigated by combined ß2-AR and iNOS inhibition.


Assuntos
Sistema Nervoso Autônomo/fisiopatologia , Regulação da Temperatura Corporal , Cardiopatias/induzido quimicamente , Coração/inervação , Abrigo para Animais , Contração Miocárdica , Síndrome de Resposta Inflamatória Sistêmica/induzido quimicamente , Temperatura , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Cardiopatias/metabolismo , Cardiopatias/fisiopatologia , Hemodinâmica , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos , Masculino , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais , Síndrome de Resposta Inflamatória Sistêmica/metabolismo , Síndrome de Resposta Inflamatória Sistêmica/fisiopatologia
7.
Dev Biol ; 425(2): 191-207, 2017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28390801

RESUMO

The Drosophila compound eye develops from a bilayered epithelial sac composed of an upper peripodial epithelium layer and a lower disc proper, the latter giving rise to the eye itself. During larval stages, complex signalling events between the layers contribute to the control of cell proliferation and differentiation in the disc. Previous work in our lab established the gap junction protein Innexin2 (Inx2) as crucial for early larval eye disc growth. By analysing the contribution of other Innexins to eye size control, we have identified Innexin3 (Inx3) as an important growth regulator. Depleting inx3 during larval eye development reduces eye size, while elevating inx3 levels increases eye size, thus phenocopying the inx2 loss- and gain-of-function situation. As demonstrated previously for inx2, inx3 regulates disc cell proliferation and interacts genetically with the Dpp pathway, being required for the proper activation of the Dpp pathway transducer Mad at the furrow and the expression of Dpp receptor Punt in the eye disc. At the developmental timepoint corresponding to eye disc growth, Inx3 colocalises with Inx2 in disc proper and peripodial epithelium cell membranes. In addition, we show that Inx3 protein levels critically depend on inx2 throughout eye development and that inx3 modulates Inx2 protein levels in the larval eye disc. Rescue experiments demonstrate that Inx3 and Inx2 cooperate functionally to enable eye disc growth in Drosophila. Finally, we demonstrate that expression of Inx3 and Inx2 is not only needed in the disc proper but also in the peripodial epithelium to regulate growth of the eye disc. Our data provide a functional demonstration that putative Inx2/Inx3 heteromeric channels regulate organ size.


Assuntos
Conexinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Disco Óptico/crescimento & desenvolvimento , Disco Óptico/metabolismo , Animais , Proliferação de Células , Epitélio/metabolismo , Larva/metabolismo , Disco Óptico/anatomia & histologia , Tamanho do Órgão , Fenótipo , Transdução de Sinais
8.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1863(7): 734-749, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29653252

RESUMO

The replacement of two consecutive histidine residues by alanine residues in the catalytic center of ceramide synthase 2 in a new transgenic mouse mutant (CerS2 H/A) leads to inactivation of catalytic activity and reduces protein level to 60% of the WT level. We show here by qRT-PCR and transcriptome analyses that several transcripts of genes involved in lipid metabolism and cell division are differentially regulated in livers of CerS2 H/A mice. Thus, very long chain ceramides produced by CerS2 are required for transcriptional regulation of target genes. The hepatocellular carcinomata previously described in old CerS2 KO mice were already present in 8-week-old CerS2 H/A animals and thus are caused by the loss of CerS2 catalytic activity already during early life.


Assuntos
Carcinoma Hepatocelular/genética , Divisão Celular/genética , Metabolismo dos Lipídeos/genética , Neoplasias Hepáticas/genética , Esfingosina N-Aciltransferase/genética , Fatores Etários , Animais , Carcinoma Hepatocelular/patologia , Ceramidas/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Fígado/patologia , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Esfingosina N-Aciltransferase/metabolismo
9.
Mol Cell Neurosci ; 78: 1-8, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27825984

RESUMO

Microglial motility is tightly controlled by multitude of agonistic and antagonistic factors. Chemoattractants, released after infection or damage of the brain, provoke directed migration of microglia to the pathogenic incident. In contrast, noradrenaline and other stress hormones have been shown to suppress microglial movement. Here we asked for the signaling reactions involved in the positive and negative control of microglial motility. Using pharmacological and genetic approaches we identified the lipid kinase activity of phosphoinositide 3-kinase species γ (PI3Kγ) as an essential mediator of microglial migration provoked by the complement component C5a and other chemoattractants. Inhibition of PI3Kγ lipid kinase activity by protein kinase A was disclosed as mechanism causing suppression of microglial migration by noradrenaline. Together these data characterize PI3Kγ as a nodal point in the control of microglial motility.


Assuntos
Agonistas alfa-Adrenérgicos/farmacologia , Fatores Quimiotáticos/farmacologia , Quimiotaxia , Microglia/metabolismo , Norepinefrina/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Complemento C5a/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/fisiologia , Fosfatidilinositol 3-Quinases/genética
10.
Glia ; 65(2): 416-428, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27859601

RESUMO

Microglia cells are brain macrophages whose proper functioning is essential for maintenance and repair processes of the central nervous system (CNS). Migration and phagocytosis are critical aspects of microglial activity. By using genetically modified cell lines and knockout mice we demonstrate here that the receptor protein-tyrosine phosphatase (PTP) DEP-1 (also known as PTPRJ or CD148) acts as a positive regulator of both processes in vitro and in vivo. Notably, reduced microglial migration was detectable in brains of Ptprj-/- mice using a wounding assay. Mechanistically, density-enhanced phosphatase-1 (DEP-1) may in part function by inhibiting the activity of the Src family kinase Fyn. In the microglial cell line BV2 DEP-1 depletion by shRNA-mediated knockdown resulted in enhanced phosphorylation of the Fyn activating tyrosine (Tyr420 ) and elevated specific Fyn-kinase activity in immunoprecipitates. Moreover, Fyn mRNA and protein levels were reduced in DEP-1 deficient microglia cells. Consistent with a negative regulatory role of Fyn for microglial functions, which is inhibited by DEP-1, microglial cells from Fyn-/- mice exhibited elevated migration and phagocytosis. Enhanced microglia migration to a site of injury was also observed in Fyn-/- mice in vivo. Taken together our data revealed a previously unrecognized role of DEP-1 and suggest the existence of a potential DEP-1-Fyn axis in the regulation of microglial functions. GLIA 2017;65:416-428.


Assuntos
Movimento Celular/fisiologia , Regulação da Expressão Gênica/genética , Microglia/fisiologia , Fagocitose/genética , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Animais , Animais Recém-Nascidos , Linhagem Celular Transformada , Movimento Celular/genética , Células Cultivadas , Córtex Cerebral/citologia , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose/fisiologia , Proteínas Proto-Oncogênicas c-fyn/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismo
11.
Int J Mol Sci ; 18(8)2017 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-28757567

RESUMO

Pulmonary hypertension (PH) is a heterogeneous disorder associated with a poor prognosis. Thus, the development of novel treatment strategies is of great interest. The enzyme arginase (Arg) is emerging as important player in PH development. The aim of the current study was to determine the expression of ArgI and ArgII as well as the effects of Arg inhibition in a rat model of PH. PH was induced in 35 Sprague-Dawley rats by monocrotaline (MCT, 60 mg/kg as single-dose). There were three experimental groups: sham-treated controls (control group, n = 11), MCT-induced PH (MCT group, n = 11) and MCT-induced PH treated with the Arg inhibitor Nω-hydroxy-nor-l-arginine (nor-NOHA; MCT/NorNoha group, n = 13). ArgI and ArgII expression was determined by immunohistochemistry and Western blot. Right ventricular systolic pressure (RVPsys) was measured and lung tissue remodeling was determined. Induction of PH resulted in an increase in RVPsys (81 ± 16 mmHg) compared to the control group (41 ± 15 mmHg, p = 0.002) accompanied by a significant elevation of histological sum-score (8.2 ± 2.4 in the MCT compared to 1.6 ± 1.6 in the control group, p < 0.001). Both, ArgI and ArgII were relevantly expressed in lung tissue and there was a significant increase in the MCT compared to the control group (p < 0.01). Arg inhibition resulted in a significant reduction of RVPsys to 52 ± 19 mmHg (p = 0.006) and histological sum-score to 5.8 ± 1.4 compared to the MCT group (p = 0.022). PH leads to increased expression of Arg. Arg inhibition leads to reduction of RVPsys and diminished lung tissue remodeling and therefore represents a potential treatment strategy in PH.


Assuntos
Arginase/metabolismo , Arginina/análogos & derivados , Hipertensão Pulmonar/tratamento farmacológico , Monocrotalina/efeitos adversos , Animais , Arginina/administração & dosagem , Arginina/farmacologia , Modelos Animais de Doenças , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/fisiopatologia , Humanos , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/enzimologia , Hipertensão Pulmonar/fisiopatologia , Pulmão/efeitos dos fármacos , Pulmão/enzimologia , Ratos Sprague-Dawley , Resultado do Tratamento
12.
J Dtsch Dermatol Ges ; 13(11): 1165-74, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26513078

RESUMO

BACKGROUND AND OBJECTIVES: Matriptase-1 participates in terminal keratinocyte (KC) differentiation. Knockdown of matriptase-1 in skin equivalent cultures leads to impaired KC differentiation and retention of nuclei in the stratum corneum. Here, we investigated the expression and regulation of matriptase-1 in psoriatic skin and in KC in vitro. PATIENTS AND METHODS: Matriptase-1 expression in healthy and psoriatic skin and its regulation in skin equivalents were analyzed by Western blotting, immunofluorescence staining, qRT-PCR, and activity assays. Involvement of the nuclear factor kappa B (NFκB) signaling pathway was investigated by adenoviral overexpression of a dominant-negative form of IKK2. RESULTS: Matriptase-1 expression was detected in the stratum granulosum of healthy human skin and in skin equivalent cultures. Its expression and activity was strongly reduced in lesional skin of patients with psoriasis. Addition of TNFα to skin equivalent cultures resulted in complete loss of matriptase-1 expression accompanied by disturbed KC differentiation. Mechanistically, we were able to show that TNFα-induced downregulation of matriptase-1 was inhibited by blocking the IKK2/NFκB signaling pathway. CONCLUSIONS: Given that matriptase-1 participates in terminal KC differentiation, its absence in psoriatic skin lesions indicates that this contributes to the barrier disturbances in this disease. Our data suggests that blocking the IKK2/NFκB-pathway represents a potential target for the treatment of psoriasis.


Assuntos
Regulação Enzimológica da Expressão Gênica , Psoríase/metabolismo , Serina Endopeptidases/metabolismo , Pele/metabolismo , Pele/patologia , Fator de Necrose Tumoral alfa/metabolismo , Células Cultivadas , Regulação para Baixo , Humanos , Psoríase/patologia
13.
Blood ; 119(19): 4499-511, 2012 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-22438257

RESUMO

Signal transduction of FMS-like tyrosine kinase 3 (FLT3) is regulated by protein-tyrosine phosphatases (PTPs). We recently identified the PTP DEP-1/CD148/PTPRJ as a novel negative regulator of FLT3. This study addressed the role of DEP-1 for regulation of the acute myeloid leukemia (AML)-related mutant FLT3 internal tandem duplication (ITD) protein. Our experiments revealed that DEP-1 was expressed but dysfunctional in cells transformed by FLT3 ITD. This was caused by enzymatic inactivation of DEP-1 through oxidation of the DEP-1 catalytic cysteine. In intact cells, including primary AML cells, FLT3 ITD kinase inhibition reactivated DEP-1. DEP-1 reactivation was also achieved by counteracting the high levels of reactive oxygen species (ROS) production detected in FLT3 ITD-expressing cell lines by inhibition of reduced NAD phosphate (NADPH)-oxidases, or by overexpression of catalase or peroxiredoxin-1 (Prx-1). Interference with ROS production in 32D cells inhibited cell transformation by FLT3 ITD in a DEP-1-dependent manner, because RNAi-mediated depletion of DEP-1 partially abrogated the inhibitory effect of ROS quenching. Reactivation of DEP-1 by stable overexpression of Prx-1 extended survival of mice in the 32D cell/C3H/HeJ mouse model of FLT3 ITD-driven myeloproliferative disease. The study thus uncovered DEP-1 oxidation as a novel event contributing to cell transformation by FLT3 ITD.


Assuntos
Transformação Celular Neoplásica/genética , Leucemia Mieloide Aguda/genética , Tirosina Quinase 3 Semelhante a fms/genética , Animais , Linhagem Celular Tumoral , Genes Supressores de Tumor/efeitos dos fármacos , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C3H , Oxidantes/farmacologia , Oxirredução/efeitos dos fármacos , Espécies Reativas de Oxigênio/farmacologia , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/antagonistas & inibidores , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismo , Sequências de Repetição em Tandem/genética , Transfecção
14.
Sci Rep ; 14(1): 7224, 2024 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-38538761

RESUMO

Although Alzheimer's disease (AD) is characterized by distinct pathological changes, their precise impact on cortical functions are not well understood. Here we used TASTPM mice as an AD model and asked whether the development of neurodegenerative changes has an impact on the extracellular space (ECS) and neuronal excitability, in particular cortical spreading depolarization (CSD) which requires intact neuron and glial functions. We studied wildtype (WT) and TASTPM mice (3, 6, and 12 months old). TASTPM mice showed progressive proliferation of neocortical Amyloid-beta (Aß) plaques between 3 and 12 months (more deposits in females than in males) and Aß accumulation in cortical vessels. As plaques proliferated, neuroinflammatory microglial reaction (CD68, CD39 and Galectin-3) and astrogliosis (GFAP) developed progressively. The cortical ECS volume shrank significantly to about half the size of the WT. CSD in both WT and TASTPM mice showed considerable heterogeneity but did not correlate with the histological changes. However, CSDs were easier to elicit in TASTPM than in WT mice at 3 months, and also compared to older TASTPM mice. Moreover, TASTPM mice showed more hyperexcitability manifested as clonic-tonic behavior after sodium thiopental anesthesia. Thus, AD pathology was associated with abnormal hyperexcitability but did not homogenously alter CSD susceptibility.


Assuntos
Doença de Alzheimer , Masculino , Feminino , Camundongos , Animais , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide , Camundongos Transgênicos , Camundongos Endogâmicos C57BL , Peptídeos beta-Amiloides , Modelos Animais de Doenças
15.
Front Immunol ; 15: 1432307, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39281680

RESUMO

Background: Limited availability and side effects of opioids have led to an increased use of non-opioid analgesia in animal disease models. However, by affecting the immune-inflammatory reactions, analgesia may disrupt the resolution of the host inflammation and modulate the survival in septic animals. This study used a clinically relevant sepsis mouse model of peritoneal contamination and infection (PCI) to investigate the antinociceptive and anti-inflammatory properties of two non-opioid analgesics. Methods: Adult C57BL/6J mice were intraperitoneally injected with a human feces suspension and received either no analgesics (Non-A), Meloxicam, or Metamizole orally. The mice were monitored for pain and illness. Mortality was assessed at 7 days post-PCI. A separate group of mice was sacrificed 24 hours after infection. Blood, peritoneal lavage fluid (PLF), liver, and spleen were harvested for pathogen load quantification via qPCR, macrophage phenotyping, neutrophil infiltration/activation, and systemic/tissue cytokine release by flow cytometry. Results: Meloxicam but not Metamizole reduced the mortality of septic mice by 31% on day 7 compared to the Non-A group. Both analgesics effectively alleviated pain but did not affect illness severity, body weight, and temperature. Meloxicam quadrupled the bacterial burden in the blood and PLF. In high IL-6 responders, Meloxicam treatment was associated with reduced circulating IL-10 and IL-1ß compared to the Non-A septic group. In low IL-6 responders, Meloxicam increased circulating MCP-1 levels and decreased PGE2 levels compared to Non-A septic mice. Notably, Meloxicam reduced spleen neutrophil infiltration by 20% compared to two other sepsis groups. Conclusion: Metamizole and Meloxicam effectively relieved pain and increased the animals' basal activity in the PCI sepsis model. Meloxicam prolonged survival yet triggered maladaptive responses due to its immunosuppressive features that decreased tissue bacterial clearance during sepsis. In contrast, Metamizole constitutes a safe and effective non-opioid alternative for analgesic control in the non-surgical PCI sepsis model.


Assuntos
Dipirona , Modelos Animais de Doenças , Meloxicam , Camundongos Endogâmicos C57BL , Sepse , Animais , Meloxicam/uso terapêutico , Sepse/tratamento farmacológico , Sepse/imunologia , Sepse/mortalidade , Dipirona/uso terapêutico , Dipirona/farmacologia , Camundongos , Analgésicos/uso terapêutico , Analgésicos/farmacologia , Imunomodulação/efeitos dos fármacos , Anti-Inflamatórios não Esteroides/uso terapêutico , Anti-Inflamatórios não Esteroides/farmacologia , Masculino , Citocinas/metabolismo , Citocinas/sangue , Peritonite/tratamento farmacológico , Peritonite/imunologia , Peritonite/microbiologia , Peritonite/mortalidade , Humanos
16.
Acta Physiol (Oxf) ; 240(5): e14140, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38546351

RESUMO

AIM: Inflammation and calcification are hallmarks in the development of aortic valve stenosis (AVS). Ceramides mediate inflammation and calcification in the vascular tissue. The highly abundant d18:1,16:0 ceramide (C16) has been linked to increased cardiovascular mortality and obesity. In this study, we investigate the role of ceramide synthase 5 (CerS5), a critical enzyme for C16 ceramide synthesis, in the development of AVS, particularly in conjunction with a high-fat/high-cholesterol diet (Western diet, WD). METHODS: We used wild-type (WT) and CerS5-/- mice on WD or normal chow in a wire injury model. We measured the peak velocity to determine AVS development and performed histological analysis of the aortic valve area, immune cell infiltration (CD68 staining), and calcification (von Kossa). In vitro experiments involved measuring the calcification of human aortic valvular interstitial cells (VICs) and evaluating cytokine release from THP-1 cells, a human leukemia monocytic-like cell line, following CerS5 knockdown. RESULTS: CerS5-/- mice showed a reduced peak velocity compared to WT only in the experiment with WD. Likewise, we observed reduced immune cell infiltration and calcification in the aortic valve of CerS5-/- mice, but only on WD. In vitro, calcification was reduced after knockdown of CerS5 in VICs, while THP-1 cells exhibited a decreased inflammatory response following CerS5 knockdown. CONCLUSION: We conclude that CerS5 is an important mediator for the development of AVS in mice on WD and regulates critical pathophysiological hallmarks of AVS formation. CerS5 is therefore an interesting target for pharmacological therapy and merits further investigation.

17.
Cardiovasc Res ; 120(12): 1485-1497, 2024 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-39023231

RESUMO

AIMS: Pulmonary vascular and right ventricular (RV) remodelling processes are important for development and progression of pulmonary hypertension (PH). The current study analysed the functional role of the extra domain A-containing fibronectin (ED-A+ Fn) for the development of PH by comparing ED-A+ Fn knockout (KO) and wild-type (WT) mice as well as the effects of an antibody-based therapeutic approach in a model of monocrotaline (MCT)-induced PH, which will be validated in a model of Sugen 5416/hypoxia-induced PH. METHODS AND RESULTS: PH was induced using MCT (PH mice). Sixty-nine mice were divided into the following groups: sham-treated controls (WT: n = 7; KO: n = 7), PH mice without specific treatment (WT: n = 12; KO: n = 10), PH mice treated with a dual endothelin receptor antagonist (macitentan; WT: n = 6; KO: n = 11), WT PH mice treated with the F8 antibody, specifically recognizing ED-A+ Fn, (n = 8), and WT PH mice treated with an antibody of irrelevant antigen specificity (KSF, n = 8). Compared to controls, WT_PH mice showed a significant elevation of the RV systolic pressure (P = 0.04) and RV functional impairment including increased basal RV (P = 0.016) diameter or tricuspid annular plane systolic excursion (P = 0.008). In contrast, KO PH did not show such effects compared to controls (P = n.s.). In WT_PH mice treated with F8, haemodynamic and echocardiographic parameters were significantly improved compared to untreated WT_PH mice or those treated with the KSF antibody (P < 0.05). On the microscopic level, KO_PH mice showed significantly less tissue damage compared to the WT_PH mice (P = 0.008). Furthermore, lung tissue damage could significantly be reduced after F8 treatment (P = 0.04). Additionally, these findings could be verified in the Sugen 5416/hypoxia mouse model, in which F8 significantly improved echocardiographic, haemodynamic, and histologic parameters. CONCLUSION: ED-A+ Fn is of crucial importance for PH pathogenesis representing a promising therapeutic target in PH. We here show a novel therapeutic approach using antibody-mediated functional blockade of ED-A+ Fn capable of attenuating and partially reversing PH-associated tissue remodelling.


Assuntos
Modelos Animais de Doenças , Fibronectinas , Hipertensão Pulmonar , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monocrotalina , Função Ventricular Direita , Remodelação Ventricular , Animais , Hipertensão Pulmonar/fisiopatologia , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/patologia , Hipertensão Pulmonar/imunologia , Fibronectinas/metabolismo , Fibronectinas/genética , Função Ventricular Direita/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos , Pirimidinas/farmacologia , Artéria Pulmonar/fisiopatologia , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Masculino , Antagonistas dos Receptores de Endotelina/farmacologia , Remodelação Vascular/efeitos dos fármacos , Anticorpos Bloqueadores/farmacologia , Anticorpos Monoclonais/farmacologia , Hipertrofia Ventricular Direita/metabolismo , Hipertrofia Ventricular Direita/fisiopatologia , Hipertrofia Ventricular Direita/patologia , Sulfonamidas/farmacologia
18.
Front Immunol ; 15: 1227355, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38655254

RESUMO

Preconditioning with lipopolysaccharide (LPS) induces neuroprotection against subsequent cerebral ischemic injury, mainly involving innate immune pathways. Microglia are resident immune cells of the central nervous system (CNS) that respond early to danger signals through memory-like differential reprogramming. However, the cell-specific molecular mechanisms underlying preconditioning are not fully understood. To elucidate the distinct molecular mechanisms of preconditioning on microglia, we compared these cell-specific proteomic profiles in response to LPS preconditioning and without preconditioning and subsequent transient focal brain ischemia and reperfusion, - using an established mouse model of transient focal brain ischemia and reperfusion. A proteomic workflow, based on isolated microglia obtained from mouse brains by cell sorting and coupled to mass spectrometry for identification and quantification, was applied. Our data confirm that LPS preconditioning induces marked neuroprotection, as indicated by a significant reduction in brain infarct volume. The established brain cell separation method was suitable for obtaining an enriched microglial cell fraction for valid proteomic analysis. The results show a significant impact of LPS preconditioning on microglial proteome patterns by type I interferons, presumably driven by the interferon cluster regulator proteins signal transducer and activator of transcription1/2 (STAT1/2).


Assuntos
Lipopolissacarídeos , Microglia , Proteoma , Proteômica , Animais , Microglia/metabolismo , Microglia/imunologia , Camundongos , Proteômica/métodos , Masculino , Isquemia Encefálica/metabolismo , Isquemia Encefálica/imunologia , Precondicionamento Isquêmico/métodos , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças
19.
EMBO J ; 28(23): 3706-16, 2009 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-19834458

RESUMO

Ceramide synthases are highly conserved transmembrane proteins involved in the biosynthesis of sphingolipids, which are essential structural components of eukaryotic membranes and can act as second messengers regulating tissue homeostasis. However, the role of these enzymes in development is poorly understood due to the lack of animal models. We identified schlank as a new Drosophila member of the ceramide synthase family. We demonstrate that schlank is involved in the de novo synthesis of a broad range of ceramides, the key metabolites of sphingolipid biosynthesis. Unexpectedly, schlank mutants also show reduction of storage fat, which is deposited as triacylglyerols in the fat body. We found that schlank can positively regulate fatty acid synthesis by promoting the expression of sterol-responsive element-binding protein (SREBP) and SREBP-target genes. It further prevents lipolysis by downregulating the expression of triacylglycerol lipase. Our results identify schlank as a new regulator of the balance between lipogenesis and lipolysis in Drosophila. Furthermore, our studies of schlank and the mammalian Lass2 family member suggest a novel role for ceramide synthases in regulating body fat metabolism.


Assuntos
Proteínas de Drosophila/fisiologia , Drosophila melanogaster/enzimologia , Drosophila melanogaster/crescimento & desenvolvimento , Família Multigênica/fisiologia , Oxirredutases/fisiologia , Tecido Adiposo/enzimologia , Tecido Adiposo/crescimento & desenvolvimento , Tecido Adiposo/metabolismo , Animais , Animais Geneticamente Modificados , Sequência Conservada , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Feminino , Larva/enzimologia , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Lipogênese/fisiologia , Lipólise/fisiologia , Masculino , Oxirredutases/genética
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