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1.
Mol Metab ; 53: 101327, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34428557

RESUMO

OBJECTIVE: The hormone liver-expressed antimicrobial peptide-2 (LEAP2) is a recently identified antagonist and an inverse agonist of the growth hormone secretagogue receptor (GHSR). GHSR's other well-known endogenous ligand, acyl-ghrelin, increases food intake, body weight, and GH secretion and is lowered in obesity but elevated upon fasting. In contrast, LEAP2 reduces acyl-ghrelin-induced food intake and GH secretion and is found elevated in obesity but lowered upon fasting. Thus, the plasma LEAP2/acyl-ghrelin molar ratio could be a key determinant modulating GHSR signaling in response to changes in body mass and feeding status. In particular, LEAP2 may serve to dampen acyl-ghrelin action in the setting of obesity, which is associated with ghrelin resistance. Here, we sought to determine the metabolic effects of genetic LEAP2 deletion. METHODS: We generated the first known LEAP2-KO mouse line. Food intake, GH secretion, and cellular activation (c-fos induction) in different brain regions following s.c. acyl-ghrelin administration in LEAP2-KO mice and wild-type littermates were determined. LEAP2-KO mice and wild-type littermates were submitted to a battery of tests (such as measurements of body weight, food intake, and body composition; indirect calorimetry, determination of locomotor activity, and meal patterning while housed in metabolic cages) over the course of 16 weeks of high-fat diet and/or standard chow feeding. Fat accumulation was assessed in hematoxylin & eosin-stained and oil red O-stained liver sections from these mice. RESULTS: LEAP2-KO mice were more sensitive to s.c. ghrelin. In particular, acyl-ghrelin acutely stimulated food intake at a dose of 0.5 mg/kg BW in standard chow-fed LEAP2-KO mice while a 2× higher dose was required by wild-type littermates. Also, acyl-ghrelin stimulated food intake at a dose of 1 mg/kg BW in high-fat diet-fed LEAP2-KO mice while not even a 10× higher dose was effective in wild-type littermates. Acyl-ghrelin induced a 90.9% higher plasma GH level and 77.2-119.7% higher numbers of c-fos-immunoreactive cells in the arcuate nucleus and olfactory bulb, respectively, in LEAP2-KO mice than in wild-type littermates. LEAP2 deletion raised body weight (by 15.0%), food intake (by 18.4%), lean mass (by 6.1%), hepatic fat (by 42.1%), and body length (by 1.7%) in females on long-term high-fat diet as compared to wild-type littermates. After only 4 weeks on the high-fat diet, female LEAP2-KO mice exhibited lower O2 consumption (by 13%), heat production (by 9.5%), and locomotor activity (by 49%) than by wild-type littermates during the first part of the dark period. These genotype-dependent differences were not observed in high-fat diet-exposed males or female and male mice exposed for long term to standard chow diet. CONCLUSIONS: LEAP2 deletion sensitizes lean and obese mice to the acute effects of administered acyl-ghrelin on food intake and GH secretion. LEAP2 deletion increases body weight in females chronically fed a high-fat diet as a result of lowered energy expenditure, reduced locomotor activity, and increased food intake. Furthermore, in female mice, LEAP2 deletion increases body length and exaggerates the hepatic fat accumulation normally associated with chronic high-fat diet feeding.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Grelina/análogos & derivados , Secretagogos/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/deficiência , Peptídeos Catiônicos Antimicrobianos/genética , Dieta Hiperlipídica/efeitos adversos , Feminino , Grelina/administração & dosagem , Grelina/metabolismo , Hormônio do Crescimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
2.
Infect Immun ; 88(8)2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32513857

RESUMO

Infections caused by multidrug-resistant (MDR) Klebsiella pneumoniae are difficult to treat with conventional antibiotics. Thus, alternative strategies to control the growth of MDR Klebsiella are warranted. We hypothesized that activation of innate effector systems could sensitize MDR K. pneumoniae to conventional antibiotics. Thus, human primary macrophages were stimulated with compounds known to activate innate immunity (vitamin D3, phenylbutyrate [PBA], and the aroylated phenylenediamine HO53) and then infected with MDR Klebsiella in the presence or absence of antibiotics. Antibiotics alone were ineffective against MDR Klebsiella in the cellular model, whereas vitamin D3, PBA, and HO53 reduced intracellular growth by up to 70%. The effect was further improved when the innate activators were combined with antibiotics. Vitamin D3- and PBA-induced bacterial killing was dependent on CAMP gene expression, whereas HO53 needed the production of reactive oxygen species (ROS), as shown in cells where the CYBB gene was silenced and in cells from a patient with reduced ROS production due to a deletion in the CYBB gene and skewed lyonization. The combination of innate effector activation by vitamin D3, PBA, and HO53 was effective in sensitizing MDR Klebsiella to conventional antibiotics in a primary human macrophage model. This study provides new evidence for future treatment options for K. pneumoniae.


Assuntos
Antibacterianos/farmacologia , Colecalciferol/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Fenilbutiratos/farmacologia , Fenilenodiaminas/farmacologia , Peptídeos Catiônicos Antimicrobianos/deficiência , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/imunologia , Sinergismo Farmacológico , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Klebsiella pneumoniae/crescimento & desenvolvimento , Klebsiella pneumoniae/imunologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/microbiologia , Testes de Sensibilidade Microbiana , NADPH Oxidase 2/deficiência , NADPH Oxidase 2/genética , NADPH Oxidase 2/imunologia , Fagocitose/efeitos dos fármacos , Cultura Primária de Células , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/metabolismo , Catelicidinas
3.
Peptides ; 128: 170311, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32278809

RESUMO

The human antimicrobial peptide LL-37 is produced by neutrophils and epithelial cells, and the peptide can be detected in plasma as well as saliva. LL-37 is active against both gram-positive and gram-negative bacteria including oral pathogens such as Porphyromonas gingivalis and Streptococcus mutans. Besides its antimicrobial properties, LL-37 modulates the innate immune system, and furthermore, it also affects host cell viability. Although, both structural and functional properties of LL-37 have been extensively investigated, its physiological/pathophysiological importance in-vivo is not completely understood. In this review, Kostmann disease (morbus Kostmann) is highlighted since it may represent a LL-37 knockdown model which can provide new important information and insights about the functional role of LL-37 in the human in-vivo setting. Patients with Kostmann disease suffer from neutropenia, and although they are treated with recombinant granulocyte colony-stimulating factor (G-CSF) to normalize their levels of neutrophils, they lack or have very low levels of LL-37 in plasma, saliva and neutrophils. Interestingly, these patients suffer from severe periodontal disease, linking LL-37-deficiency to oral infections. Thus, LL-37 seems to play an important pathophysiological role in the oral environment antagonizing oral pathogens and thereby prevents oral infections.


Assuntos
Peptídeos Catiônicos Antimicrobianos/deficiência , Síndrome Congênita de Insuficiência da Medula Óssea/metabolismo , Doenças da Boca/metabolismo , Neutropenia/congênito , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Síndrome Congênita de Insuficiência da Medula Óssea/microbiologia , Síndrome Congênita de Insuficiência da Medula Óssea/patologia , Humanos , Doenças da Boca/sangue , Doenças da Boca/microbiologia , Doenças da Boca/patologia , Neutropenia/metabolismo , Neutropenia/microbiologia , Neutropenia/patologia , Saliva/metabolismo , Saliva/microbiologia , Catelicidinas
4.
Clin Exp Pharmacol Physiol ; 47(4): 677-686, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31868940

RESUMO

The therapeutic potential of the antimicrobial peptide cathelicidin (Camp) administration in sepsis has been widely investigated. However, little is known about the pathophysiological roles of cathelicidin in septic cardiomyopathy. In a lipopolysaccharide (LPS)-induced endotoxaemic model, we found that the mRNA and protein expression of cardiac cathelicidin were induced in C57BL/6J wild-type (WT) mice upon LPS challenge, accompanied by increased circulating cathelicidin levels. We showed that this peptide was mainly derived from neutrophils and monocytes/macrophages. Camp deficiency exacerbated LPS-induced myocardial depression, while the administration of CRAMP (the mature form of mouse cathelicidin) decreased the LPS-induced mortality in a D-galactosamine hydrochloride (D-GalN)-sensitized endotoxin shock model. In vivo, LPS-treated Camp knockout mice had a significant higher protein level of myocardial and circulating tumour necrosis factor-alpha (TNF-α), a major contributing factor to septic cardiomyopathy, compared to LPS-treated WT mice, while CRAMP administration inhibited LPS-induced TNF-α production in the heart and plasma in D-GalN-sensitized endotoxaemic mice. In vitro, CRAMP treatment suppressed LPS-induced Tnf-α mRNA expression in cultured neonatal mouse cardiomyocytes and reduced TNF-α secretion in the culture supernatant. The inhibitory effects of CRAMP on TNF-α production may be related to its neutralizing ability of LPS, since CRAMP application had no effects on another toll-like receptor 4 ligand paclitaxel-induced Tnf-α mRNA expression in cardiomyocytes. These findings suggest that LPS-induced cathelicidin protects the heart against myocardial depression partly through the inhibition of TNF-α production via neutralizing LPS.


Assuntos
Peptídeos Catiônicos Antimicrobianos/deficiência , Endotoxemia/induzido quimicamente , Endotoxemia/fisiopatologia , Coração/efeitos dos fármacos , Coração/fisiopatologia , Lipopolissacarídeos/farmacologia , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Endotoxemia/genética , Endotoxemia/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , RNA Mensageiro/genética , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Catelicidinas
5.
J Proteome Res ; 18(7): 2875-2884, 2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31188604

RESUMO

Alterations in gut bacterial homeostasis result in changes in intestinal metabolites. To investigate the effects of alcohol on fecal metabolites and the role of cathelicidin-related antimicrobial peptide (CRAMP) in alcoholic liver disease (ALD), CRAMP knockout (KO) and their control wild type (WT) mice were fed a Lieber-DeCarli liquid diet with or without alcohol. Polar metabolites in mouse feces were analyzed by GC × GC-MS and 2DLC-MS, and the concentrations of short chain fatty acids (SCFAs) were measured by GC-MS. A total of 95 and 190 metabolites were detected by GC × GC-MS and 2DLC-MS, respectively. Among the significantly changed metabolites, taurine and nicotinic acid were decreased in WT mice fed alcohol, which were also down-regulated in KO mice fed without alcohol. Interestingly, these two metabolites were increased in KO mice fed alcohol compared to them in WT controls. Additionally, SCFAs were significantly decreased in WT mice fed alcohol and in KO mice fed without alcohol, whereas two branched-chain SCFAs were increased by alcohol treatment in KO mice. In summary, the analytical platforms employed in this study successfully dissected the alterations of polar metabolites and SCFAs in fecal samples, which helped understand the effects of alcohol consumption and CRAMP in intestinal metabolism and alcohol-induced liver injury.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Etanol/farmacologia , Fezes/química , Hepatopatias Alcoólicas/etiologia , Animais , Peptídeos Catiônicos Antimicrobianos/deficiência , Peptídeos Catiônicos Antimicrobianos/genética , Etanol/administração & dosagem , Ácidos Graxos Voláteis/análise , Cromatografia Gasosa-Espectrometria de Massas , Camundongos , Camundongos Knockout , Catelicidinas
6.
mBio ; 9(4)2018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-30018109

RESUMO

Macrophages are critical to innate immunity due to their ability to phagocytose bacteria. The macrophage phagolysosome is a highly acidic organelle with potent antimicrobial properties, yet remarkably, ingested Staphylococcus aureus replicates within this niche. Herein we demonstrate that S. aureus requires the GraXRS regulatory system for growth within this niche, while the SaeRS and AgrAC two-component regulatory systems and the α-phenol soluble modulins are dispensable. Importantly, we find that it is exposure to acidic pH that is required for optimal growth of S. aureus inside fully acidified macrophage phagolysosomes. Exposure of S. aureus to acidic pH evokes GraS signaling, which in turn elicits an adaptive response that endows the bacteria with increased resistance to antimicrobial effectors, such as antimicrobial peptides, encountered inside macrophage phagolysosomes. Notably, pH-dependent induction of antimicrobial peptide resistance in S. aureus requires the GraS sensor kinase. GraS and MprF, a member of the GraS regulon, play an important role for bacterial survival in the acute stages of systemic infection, where in murine models of infection, S. aureus resides within liver-resident Kupffer cells. We conclude that GraXRS represents a vital regulatory system that functions to allow S. aureus to evade killing, prior to commencement of replication, within host antibacterial immune cells.IMPORTANCES. aureus can infect any site of the body, including the microbicidal phagolysosome of the macrophage. The ability of S. aureus to infect diverse niches necessitates that the bacteria be highly adaptable. Here we show that S. aureus responds to phagolysosome acidification to evoke changes in gene expression that enable the bacteria to resist phagolysosomal killing and to promote replication. Toxin production is dispensable for this response; however, the bacteria require the sensor kinase GraS, which transduces signals in response to acidic pH. GraS is necessary for phagolysosomal replication and survival of S. aureus in the acute stage of systemic infection. Disruption of this S. aureus adaptation would render S. aureus susceptible to phagocyte restriction.


Assuntos
Interações Hospedeiro-Patógeno , Macrófagos/microbiologia , Fagossomos/química , Fagossomos/microbiologia , Proteínas Quinases/genética , Staphylococcus aureus/crescimento & desenvolvimento , Aminoaciltransferases/genética , Animais , Peptídeos Catiônicos Antimicrobianos/deficiência , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Carga Bacteriana , Proteínas de Bactérias/genética , Células Cultivadas , Farmacorresistência Bacteriana , Concentração de Íons de Hidrogênio , Fígado/microbiologia , Macrófagos/ultraestrutura , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Proteínas Quinases/deficiência , Espécies Reativas de Oxigênio/metabolismo , Regulon , Staphylococcus aureus/efeitos dos fármacos
7.
J Immunol ; 197(5): 1989-99, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27481851

RESUMO

Emergency granulopoiesis refers to the increased production of neutrophils in bone marrow and their release into circulation induced by severe infection. Several studies point to a critical role for G-CSF as the main mediator of emergency granulopoiesis. However, the consequences of G-CSF stimulation on the transcriptome of neutrophils and their precursors have not yet been investigated in humans. In this work, we examine the changes in mRNA expression induced by administration of G-CSF in vivo, as a model of emergency granulopoiesis in humans. Blood samples were collected from healthy individuals after 5 d of G-CSF administration. Neutrophil precursors were sorted into discrete stages of maturation by flow cytometry, and RNA was subjected to microarray analysis. mRNA levels were compared with previously published expression levels in corresponding populations of neutrophil precursors isolated from bone marrow of untreated, healthy individuals. One thousand one hundred and ten mRNAs were differentially expressed >2-fold throughout terminal granulopoiesis. Major changes were seen in pathways involved in apoptosis, cytokine signaling, and TLR pathways. In addition, G-CSF treatment reduced the levels of four of five measured granule proteins in mature neutrophils, including the proantibacterial protein hCAP-18, which was completely deficient in neutrophils from G-CSF-treated donors. These results indicate that multiple biological processes are altered to satisfy the increased demand for neutrophils during G-CSF-induced emergency granulopoiesis in humans.


Assuntos
Expressão Gênica , Fator Estimulador de Colônias de Granulócitos/farmacologia , Leucopoese/genética , Neutrófilos/fisiologia , Peptídeos Catiônicos Antimicrobianos/deficiência , Peptídeos Catiônicos Antimicrobianos/genética , Apoptose/imunologia , Movimento Celular , Citocinas/imunologia , Citocinas/metabolismo , Voluntários Saudáveis , Humanos , Análise em Microsséries , Neutrófilos/efeitos dos fármacos , Proteínas Recombinantes/imunologia , Catelicidinas
8.
FASEB J ; 28(8): 3456-67, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24736410

RESUMO

In humans, LL-37 and eicosanoids are important mediators of inflammation and immune responses. Here we report that LL-37 promotes leukotriene B4 (LTB4) and thromboxane A2 (TXA2) generation by human monocyte-derived macrophages (HMDMs). LL-37 evokes calcium mobilization apparently via the P2X7 receptor (P2X7R), activation of ERK1/2 and p38 MAPKs, as well as cytosolic phospholipase A2 (cPLA2) and 5-lipoxygenase in HMDMs, leading to an early (1 h) release of LTB4. Similarly, TXA2 production at an early time involved the same signaling sequence along an LL-37-P2X7R-cPLA2-cyclooxygenase-1 (COX-1) axis. However, at later (6-8 h) time points, internalized LL-37 up-regulates COX-2 expression, promoting TXA2 production. Furthermore, intraperitoneal injection of mice with murine cathelicidin-related antimicrobial peptide (mCRAMP) induces significantly higher levels of LTB4 and TXA2 in mouse ascites rich in macrophages. Conversely, cathelicidin-deficient (Cnlp(-/-)) mice produce much less LTB4 and TXB2 in vivo in response to TNF-α compared with control mice. We conclude that LL-37 elicits a biphasic release of eicosanoids in macrophages with early, Ca(2+)-dependent formation of LTB4 and TXA2 followed by a late peak of TXA2, generated via induction of COX-2 by internalized LL-37, thus allowing eicosanoid production in a temporally controlled manner. Moreover, our findings provide evidence that LL-37 is an endogenous regulator of eicosanoid-dependent inflammatory responses in vivo.


Assuntos
Peptídeos Catiônicos Antimicrobianos/fisiologia , Eicosanoides/biossíntese , Leucotrieno B4/metabolismo , Macrófagos/efeitos dos fármacos , Peritonite/metabolismo , Tromboxano A2/metabolismo , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/deficiência , Araquidonato 5-Lipoxigenase/metabolismo , Sinalização do Cálcio , Catelicidinas/deficiência , Catelicidinas/fisiologia , Catelicidinas/toxicidade , Células Cultivadas , Humanos , Inflamação/fisiopatologia , Sistema de Sinalização das MAP Quinases , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Peritonite/induzido quimicamente , Peritonite/patologia , Fosfolipases A2 Citosólicas/metabolismo , Fosforilação , Prostaglandina-Endoperóxido Sintases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional , Receptores Purinérgicos P2X7/fisiologia , Proteínas Recombinantes/toxicidade , Fator de Necrose Tumoral alfa/toxicidade
9.
PLoS One ; 8(2): e55331, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23390527

RESUMO

Salmonella, a ubiquitous Gram-negative intracellular bacterium, is a food borne pathogen that infects a broad range of hosts. Infection with Salmonella Typhimurium in mice is a broadly recognized experimental model resembling typhoid fever in humans. Using a N-ethyl-N-nitrosurea (ENU) mutagenesis recessive screen, we report the identification of Ity16 (Immunity to Typhimurium locus 16), a locus responsible for increased susceptibility to infection. The position of Ity16 was refined on chromosome 8 and a nonsense mutation was identified in the ankyrin 1 (Ank1) gene. ANK1 plays an important role in the formation and stabilization of the red cell cytoskeleton. The Ank1(Ity16/Ity16) mutation causes severe hemolytic anemia in uninfected mice resulting in splenomegaly, hyperbilirubinemia, jaundice, extramedullary erythropoiesis and iron overload in liver and kidneys. Ank1(Ity16/Ity16) mutant mice demonstrated low levels of hepcidin (Hamp) expression and significant increases in the expression of the growth differentiation factor 15 (Gdf15), erythropoietin (Epo) and heme oxygenase 1 (Hmox1) exacerbating extramedullary erythropoiesis, tissue iron deposition and splenomegaly. As the infection progresses in Ank1(Ity16/Ity16), the anemia worsens and bacterial load were high in liver and kidneys compared to wild type mice. Heterozygous Ank1(+/Ity16) mice were also more susceptible to Salmonella infection although to a lesser extent than Ank1(Ity16/Ity16) and they did not inherently present anemia and splenomegaly. During infection, iron accumulated in the kidneys of Ank1(+/Ity16) mice where bacterial loads were high compared to littermate controls. The critical role of HAMP in the host response to Salmonella infection was validated by showing increased susceptibility to infection in Hamp-deficient mice and significant survival benefits in Ank1(+/Ity16) heterozygous mice treated with HAMP peptide. This study illustrates that the regulation of Hamp and iron balance are crucial in the host response to Salmonella infection in Ank1 mutants.


Assuntos
Anemia Hemolítica/genética , Anquirinas/genética , Peptídeos Catiônicos Antimicrobianos/genética , Códon sem Sentido/efeitos dos fármacos , Etilnitrosoureia/toxicidade , Sobrecarga de Ferro/genética , Infecções por Salmonella/genética , Anemia Hemolítica/metabolismo , Anemia Hemolítica/microbiologia , Anemia Hemolítica/mortalidade , Animais , Anquirinas/metabolismo , Peptídeos Catiônicos Antimicrobianos/deficiência , Eritrócitos/metabolismo , Eritrócitos/patologia , Eritropoetina/genética , Eritropoetina/metabolismo , Expressão Gênica , Predisposição Genética para Doença , Fator 15 de Diferenciação de Crescimento/genética , Fator 15 de Diferenciação de Crescimento/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Hepcidinas , Heterozigoto , Homozigoto , Ferro/metabolismo , Sobrecarga de Ferro/metabolismo , Sobrecarga de Ferro/microbiologia , Sobrecarga de Ferro/mortalidade , Fígado/metabolismo , Fígado/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Infecções por Salmonella/metabolismo , Infecções por Salmonella/microbiologia , Infecções por Salmonella/mortalidade , Salmonella typhimurium/fisiologia , Análise de Sobrevida
10.
PLoS One ; 7(11): e49588, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23166721

RESUMO

Salmonella enterica serovar Typhimurium (S. Typhimurium) uses two-component regulatory systems (TCRS) to respond to stimuli in the local microenvironment. Upon infection, the Salmonella TCRSs PhoP-PhoQ (PhoPQ) and PmrA-PmrB (PmrAB) are activated by environmental signals in the intestinal lumen and within host cells. TCRS-mediated gene expression results in lipopolysaccharide (LPS) modification and cationic antimicrobial peptide resistance. The PmrA-regulated pmrHFIJKLM operon mediates 4-amino-4-deoxy-L-arabinose (Ara4N) production and attachment to the lipid A of LPS. A ΔpmrF S. Typhimurium strain cannot produce Ara4N, exhibits increased sensitivity to cationic antimicrobial peptide (CAMP)-mediated killing, and attenuated virulence in mice upon oral infection. CAMPs are predicted to play a role in elimination of Salmonella, and may activate PhoPQ and PmrAB in vivo, which could increase bacterial resistance to host defenses. Competition experiments between wild type (WT) and ΔpmrF mutant strains of S. Typhimurium indicated that selection against this mutant first occurs within the intestinal lumen early during infection. However, CRAMP and active cryptdins alone are not responsible for elimination of Ara4N-deficient bacteria in vivo. Investigation into the early immune response to ΔpmrF showed that it differed slightly from the early immune response to WT S. Typhimurium. Further investigation into the early immune response to infection of Peyer's patches suggests a role for IL-13 in the attenution of the ΔpmrF mutant strain. Thus, prominent CAMPs present in the mouse intestine are not responsible for the selection against the ΔpmrF strain in this location, but limited alterations in innate immune induction were observed that affect bacterial survival and virulence.


Assuntos
Amino Açúcares/fisiologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Salmonelose Animal/imunologia , Salmonella typhimurium/imunologia , Salmonella typhimurium/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/deficiência , Feminino , Mucosa Intestinal/metabolismo , Intestinos/microbiologia , Lipopolissacarídeos/química , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Knockout , Salmonelose Animal/genética , Salmonelose Animal/microbiologia , Salmonella typhimurium/patogenicidade , Virulência , alfa-Defensinas/metabolismo
11.
Blood ; 120(18): 3829-36, 2012 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-22990014

RESUMO

The deficiency of hepcidin, the hormone that controls iron absorption and its tissue distribution, is the cause of iron overload in nearly all forms of hereditary hemochromatosis and in untransfused iron-loading anemias. In a recent study, we reported the development of minihepcidins, small drug-like hepcidin agonists. Here we explore the feasibility of using minihepcidins for the prevention and treatment of iron overload in hepcidin-deficient mice. An optimized minihepcidin (PR65) was developed that had superior potency and duration of action compared with natural hepcidin or other minihepcidins, and favorable cost of synthesis. PR65 was administered by subcutaneous injection daily for 2 weeks to iron-depleted or iron-loaded hepcidin knockout mice. PR65 administration to iron-depleted mice prevented liver iron loading, decreased heart iron levels, and caused the expected iron retention in the spleen and duodenum. At high doses, PR65 treatment also caused anemia because of profound iron restriction. PR65 administration to hepcidin knockout mice with pre-existing iron overload had a more moderate effect and caused partial redistribution of iron from the liver to the spleen. Our study demonstrates that minihepcidins could be beneficial in iron overload disorders either used alone for prevention or possibly as adjunctive therapy with phlebotomy or chelation.


Assuntos
Peptídeos Catiônicos Antimicrobianos/agonistas , Peptídeos Catiônicos Antimicrobianos/deficiência , Hemocromatose/prevenção & controle , Anemia Ferropriva/induzido quimicamente , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Hepcidinas , Sobrecarga de Ferro/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
12.
J Innate Immun ; 4(5-6): 569-78, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22759465

RESUMO

BACKGROUND: Dysfunctional immune responses characterize sarcoidosis, but the status of cathelicidin, a potent immunoregulatory and antimicrobial molecule, has not been established in clinical disease activity. METHODS: Alveolar macrophage cathelicidin expression was determined in biopsy-proven sarcoidosis patients classified clinically as 'severe' (requiring systemic treatment) or 'non-severe' (never requiring treatment). Bronchoalveolar lavage (BAL) cells from sarcoidosis patients and healthy controls were analyzed for mRNA expression of cathelicidin, vitamin D receptor (VDR) and the VDR coactivator steroid receptor coactivator-3 (SRC3) by quantitative PCR. Cathelicidin-derived peptide LL-37 was determined by immunocytochemistry. Serum calcidiol (25-hydroxyvitamin D2; vitD2) and calcitriol (1,25-dihydroxyvitamin D3; vitD3) were quantified. RESULTS: The results indicated reduced BAL cell expression of cathelicidin and SRC3 in severe but not non-severe sarcoidosis compared to controls. Serum levels of biologically active vitD3 in both severe and non-severe patients were within the control range even though vitD2 levels in both groups were below the recommended level (30 ng/ml). Sarcoidosis and control alveolar macrophages were studied in vitro to determine cathelicidin responses to vitD3 and tumor necrosis factor-α (TNFα), a vitD3 antagonist elevated in active sarcoidosis. Alveolar macrophage cathelicidin was stimulated by vitD3 but repressed by TNFα, which also repressed SRC3. CONCLUSIONS: These findings suggest that TNFα-mediated repression of SRC3 contributes to alveolar macrophage cathelicidin deficiency in severe sarcoidosis despite healthy vitD3 levels. Deficiency of cathelicidin, a multifunctional regulator of immune cells and proinflammatory cytokines, may impede resolution of inflammation in the lungs of patients with severe sarcoidosis.


Assuntos
Peptídeos Catiônicos Antimicrobianos/deficiência , Macrófagos Alveolares/metabolismo , Coativador 3 de Receptor Nuclear/metabolismo , Sarcoidose Pulmonar/fisiopatologia , Fator de Necrose Tumoral alfa/metabolismo , Vitamina D/análogos & derivados , Adulto , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Líquido da Lavagem Broncoalveolar/citologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Coativador 3 de Receptor Nuclear/genética , Sarcoidose Pulmonar/metabolismo , Índice de Gravidade de Doença , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/farmacologia , Vitamina D/metabolismo , Adulto Jovem , Catelicidinas
13.
Vet Clin North Am Small Anim Pract ; 42(1): 85-96, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22285159

RESUMO

The relatively recent discovery of hepcidin has stimulated renewed research interest in iron metabolism and iron-related disorders, emphasizing the importance of this hormone in many normal and pathologic processes. Important questions still remain to be answered; however, research to date offers promising diagnostic and therapeutic implications for both humans and veterinary species.


Assuntos
Peptídeos Catiônicos Antimicrobianos/fisiologia , Homeostase/fisiologia , Distúrbios do Metabolismo do Ferro/veterinária , Ferro/metabolismo , Animais , Animais Domésticos , Peptídeos Catiônicos Antimicrobianos/deficiência , Hepcidinas , Distúrbios do Metabolismo do Ferro/diagnóstico , Distúrbios do Metabolismo do Ferro/metabolismo
14.
J Clin Invest ; 121(12): 4880-8, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22045566

RESUMO

Iron overload is the hallmark of hereditary hemochromatosis and a complication of iron-loading anemias such as ß-thalassemia. Treatment can be burdensome and have significant side effects, and new therapeutic options are needed. Iron overload in hereditary hemochromatosis and ß-thalassemia intermedia is caused by hepcidin deficiency. Although transgenic hepcidin replacement in mouse models of these diseases prevents iron overload or decreases its potential toxicity, natural hepcidin is prohibitively expensive for human application and has unfavorable pharmacologic properties. Here, we report the rational design of hepcidin agonists based on the mutagenesis of hepcidin and the hepcidin-binding region of ferroportin and computer modeling of their docking. We identified specific hydrophobic/aromatic residues required for hepcidin-ferroportin binding and obtained evidence in vitro that a thiol-disulfide interaction between ferroportin C326 and the hepcidin disulfide cage may stabilize binding. Guided by this model, we showed that 7­9 N-terminal amino acids of hepcidin, including a single thiol cysteine, comprised the minimal structure that retained hepcidin activity, as shown by the induction of ferroportin degradation in reporter cells. Further modifications to increase resistance to proteolysis and oral bioavailability yielded minihepcidins that, after parenteral or oral administration to mice, lowered serum iron levels comparably to those after parenteral native hepcidin. Moreover, liver iron concentrations were lower in mice chronically treated with minihepcidins than those in mice treated with solvent alone. Minihepcidins may be useful for the treatment of iron overload disorders.


Assuntos
Peptídeos Catiônicos Antimicrobianos/agonistas , Sobrecarga de Ferro/tratamento farmacológico , Fragmentos de Peptídeos/farmacologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/deficiência , Peptídeos Catiônicos Antimicrobianos/genética , Sítios de Ligação , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Simulação por Computador , Cisteína/química , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Hepcidinas , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ferro/sangue , Fígado/química , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/uso terapêutico , Conformação Proteica , Mapeamento de Interação de Proteínas , Relação Estrutura-Atividade
15.
Clin Chem ; 57(12): 1650-69, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21989113

RESUMO

BACKGROUND: The peptide hormone hepcidin plays a central role in regulating dietary iron absorption and body iron distribution. Many human diseases are associated with alterations in hepcidin concentrations. The measurement of hepcidin in biological fluids is therefore a promising tool in the diagnosis and management of medical conditions in which iron metabolism is affected. CONTENT: We describe hepcidin structure, kinetics, function, and regulation. We moreover explore the therapeutic potential for modulating hepcidin expression and the diagnostic potential for hepcidin measurements in clinical practice. SUMMARY: Cell-culture, animal, and human studies have shown that hepcidin is predominantly synthesized by hepatocytes, where its expression is regulated by body iron status, erythropoietic activity, oxygen tension, and inflammatory cytokines. Hepcidin lowers serum iron concentrations by counteracting the function of ferroportin, a major cellular iron exporter present in the membrane of macrophages, hepatocytes, and the basolateral site of enterocytes. Hepcidin is detected in biologic fluids as a 25 amino acid isoform, hepcidin-25, and 2 smaller forms, i.e., hepcidin-22 and -20; however, only hepcidin-25 has been shown to participate in the regulation of iron metabolism. Reliable assays to measure hepcidin in blood and urine by use of immunochemical and mass spectrometry methods have been developed. Results of proof-of-principle studies have highlighted hepcidin as a promising diagnostic tool and therapeutic target for iron disorders. However, before hepcidin measurements can be used in routine clinical practice, efforts will be required to assess the relevance of hepcidin isoform measurements, to harmonize the different assays, to define clinical decision limits, and to increase assay availability for clinical laboratories.


Assuntos
Peptídeos Catiônicos Antimicrobianos/fisiologia , Distúrbios do Metabolismo do Ferro/diagnóstico , Ferro/sangue , Animais , Peptídeos Catiônicos Antimicrobianos/análise , Peptídeos Catiônicos Antimicrobianos/deficiência , Biomarcadores/análise , Eritropoese , Hepcidinas , Humanos , Hipóxia/metabolismo , Inflamação/metabolismo , Distúrbios do Metabolismo do Ferro/tratamento farmacológico , Distúrbios do Metabolismo do Ferro/metabolismo , Terapia de Alvo Molecular , Conformação Proteica , Valores de Referência
16.
Cell Metab ; 14(5): 635-46, 2011 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-22019085

RESUMO

The iron exporter ferroportin (Fpn) is essential to transfer iron from cells to plasma. Systemic iron homeostasis in vertebrates is regulated by the hepcidin-mediated internalization of Fpn. Here, we demonstrate a second route for Fpn internalization; when cytosolic iron levels are low, Fpn is internalized in a hepcidin-independent manner dependent upon the E3 ubiquitin ligase Nedd4-2 and the Nedd4-2 binding protein Nfdip-1. Retention of cell-surface Fpn through reductions in Nedd4-2 results in cell death through depletion of cytosolic iron. Nedd4-2 is also required for internalization of Fpn in the absence of ferroxidase activity as well as for the entry of hepcidin-induced Fpn into the multivesicular body. C. elegans lacks hepcidin genes, and C. elegans Fpn expressed in mammalian cells is not internalized by hepcidin but is internalized in response to iron deprivation in a Nedd4-2-dependent manner, supporting the hypothesis that Nedd4-2-induced internalization of Fpn is evolutionarily conserved.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Ferro/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/deficiência , Peptídeos Catiônicos Antimicrobianos/genética , Evolução Biológica , Caenorhabditis elegans , Proteínas de Transporte/genética , Proteínas de Transporte de Cátions/genética , Células Cultivadas , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Células HEK293 , Hepcidinas , Homeostase/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Macrófagos/citologia , Macrófagos/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Ubiquitina-Proteína Ligases Nedd4 , Plasmídeos , RNA Interferente Pequeno , Proteínas Recombinantes de Fusão/genética , Transfecção , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
17.
PLoS One ; 6(9): e25324, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21957487

RESUMO

Oxidative stress plays an important role in neuronal injuries caused by cerebral ischemia. It is well established that free iron increases significantly during ischemia and is responsible for oxidative damage in the brain. However, the mechanism of this ischemia-induced increase in iron is not completely understood. In this report, the middle cerebral artery occlusion (MCAO) rat model was performed and the mechanism of iron accumulation in cerebral ischemia-reperfusion was studied. The expression of L-ferritin was significantly increased in the cerebral cortex, hippocampus, and striatum on the ischemic side, whereas H-ferritin was reduced in the striatum and increased in the cerebral cortex and hippocampus. The expression level of the iron-export protein ferroportin1 (FPN1) significantly decreased, while the expression of transferrin receptor 1 (TfR1) was increased. In order to elucidate the mechanisms of FPN1 regulation, we studied the expression of the key regulator of FPN1, hepcidin. We observed that the hepcidin level was significantly elevated in the ischemic side of the brain. Knockdown hepcidin repressed the increasing of L-ferritin and decreasing of FPN1 invoked by ischemia-reperfusion. The results indicate that hepcidin is an important contributor to iron overload in cerebral ischemia. Furthermore, our results demonstrated that the levels of hypoxia-inducible factor-1α (HIF-1α) were significantly higher in the cerebral cortex, hippocampus and striatum on the ischemic side; therefore, the HIF-1α-mediated TfR1 expression may be another contributor to the iron overload in the ischemia-reperfusion brain.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Ferro/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/deficiência , Peptídeos Catiônicos Antimicrobianos/genética , Encéfalo/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Ferritinas/metabolismo , Técnicas de Silenciamento de Genes , Hepcidinas , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/genética , Interleucina-6/genética , Sobrecarga de Ferro/complicações , Sobrecarga de Ferro/genética , Sobrecarga de Ferro/metabolismo , Camundongos , Ratos , Receptores da Transferrina/metabolismo , Traumatismo por Reperfusão/complicações , Regulação para Cima
18.
Biochem Soc Trans ; 39(4): 994-9, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21787336

RESUMO

BPI (bactericidal/permeability-increasing protein) is a 55 kDa anti-infective molecule expressed in neutrophil and eosinophil granules and on some epithelial cells. BPI's high affinity for the lipid A region of endotoxin targets its opsonizing, microbicidal and endotoxin-neutralizing activities towards Gram-negative bacteria. Several immunocompromised patient populations demonstrate BPI deficiency, including newborns, those with anti-neutrophil cytoplasmic antibodies (as in cystic fibrosis and HIV infection) and those exposed to radiochemotherapy. BPI may be replenished by administering agents that induce its expression or by administration of recombinant BPI congeners, potentially shielding BPI-deficient individuals against Gram-negative bacterial infection, endotoxemia and its toxic sequelae.


Assuntos
Peptídeos Catiônicos Antimicrobianos/deficiência , Proteínas Sanguíneas/deficiência , Infecções por Bactérias Gram-Negativas/imunologia , Hospedeiro Imunocomprometido , Animais , Antibacterianos/uso terapêutico , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas Sanguíneas/metabolismo , Endotoxemia/prevenção & controle , Células Epiteliais/metabolismo , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Humanos , Imunidade Inata , Leucócitos/metabolismo , Lipoxinas/uso terapêutico , Terapia de Alvo Molecular
19.
J Immunol ; 186(2): 1228-39, 2011 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-21148041

RESUMO

Overexpression of the T cell cytokine IL-22 is linked to the development of some chronic diseases, but little is known about IL-22 deficiency in humans. As demonstrated in this study, acne inversa (AI; also designated as Hidradenitis suppurativa) lesions show a relative deficiency of IL-22 and IL-20, but not of IL-17A, IL-26, IFN-γ, IL-24, or IL-1ß. Moreover, AI lesions had reduced expression of membranous IL-22 and IL-20 receptors and increased expression of the natural IL-22 inhibitor, IL-22 binding protein. AI is a chronic inflammatory skin disease with prevalence up to 4% of the population and in which cutaneous bacterial persistence represents an important pathogenetic factor. Accordingly, we also found a relative deficiency of antimicrobial proteins (AMPs) in AI lesions and a positive correlation between lesional IL-22 and IL-20 versus AMP levels. IL-22, like its tissue cell downstream mediator IL-20, upregulated AMPs in reconstituted human epidermis and was critical for increased AMP levels under inflammatory conditions. The relative IL-22 deficiency in AI was not linked to lesional T cell numbers or Th22/Th1/Th17 subset markers and -inducing cytokines. However, IL-10 was highly expressed in AI lesions and correlated negatively with IL-22 expression. Moreover, IL-10 inhibited IL-22 but not IL-17 production in vitro. The IL-10 overexpression, in turn, was not associated with an elevated presence of regulatory T cells but with the enhanced presence of an IL-10-inducing cytokine. We conclude that IL-22 deficiency may contribute to the pathogenesis of certain chronic disorders as postulated in this paper for AI.


Assuntos
Hidradenite Supurativa/imunologia , Hidradenite Supurativa/patologia , Mediadores da Inflamação/fisiologia , Interleucinas/deficiência , Adolescente , Adulto , Idoso , Animais , Peptídeos Catiônicos Antimicrobianos/deficiência , Peptídeos Catiônicos Antimicrobianos/fisiologia , Células Cultivadas , Doença Crônica , Citocinas/biossíntese , Citocinas/deficiência , Feminino , Hidradenite Supurativa/metabolismo , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Interleucinas/genética , Interleucinas/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Regulação para Cima/imunologia , Adulto Jovem , Interleucina 22
20.
Curr Opin Allergy Clin Immunol ; 10(5): 463-8, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20720487

RESUMO

PURPOSE OF REVIEW: We review here the recent discoveries in innate immunity that shed light on the pathophysiology of atopic dermatitis. RECENT FINDINGS: The mechanisms that promote the enhanced susceptibility to cutaneous infections in atopic dermatitis are complex interactions among several factors. They include skin barrier dysfunction, reduced skin lipid content, and abnormalities of the innate immune response. Some of the innate immune defects observed in atopic dermatitis are primary defects, such as epithelial barrier defects and defects in signaling or expression of innate receptors. Others may be secondary to the effects of the adaptive immune response. For example, deficiencies in antimicrobial peptides may be due to the overexpression of T helper 2 cytokines such as interleukin-4 and interleukin-13. However, how all components interact with each other remains to be fully investigated. SUMMARY: To break this vicious circle, a multiprolonged approach directed at healing or protecting the skin barrier and addressing the immune dysregulation is necessary to improve and control the disease.


Assuntos
Dermatite Atópica/imunologia , Infecções/imunologia , Pele/imunologia , Peptídeos Catiônicos Antimicrobianos/deficiência , Peptídeos Catiônicos Antimicrobianos/imunologia , Dermatite Atópica/complicações , Dermatite Atópica/genética , Humanos , Imunidade Inata/genética , Imunomodulação , Infecções/etiologia , Infecções/genética , Metabolismo dos Lipídeos/genética , Metabolismo dos Lipídeos/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Pele/metabolismo , Equilíbrio Th1-Th2
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