RESUMO
Growth differentiation factor 15 (GDF15) is believed to be a major causative factor for cancer-induced cachexia. Recent elucidation of the central circuits involved in GDF15 function and its signaling through the glial cell-derived neurotrophic factor family receptor α-like (GFRAL) has prompted the interest of targeting the GDF15-GFRAL signaling for energy homeostasis and body weight regulation. Here, we applied advanced peptide technologies to identify GDF15 peptide fragments inhibiting GFRAL signaling. SPOT peptide arrays revealed binding of GDF15 C-terminal peptide fragments to the extracellular domain of GFRAL. Parallel solid-phase peptide synthesis allowed for generation of complementary GDF15 peptide libraries and their subsequent functional evaluation in cells expressing the GFRAL/RET receptor complex. We identified a series of C-terminal fragments of GDF15 inhibiting GFRAL activity in the micromolar range. These novel GFRAL peptide inhibitors could serve as valuable tools for further development of peptide therapeutics towards the treatment of cachexia and other wasting disorders.
Assuntos
Caquexia , Obesidade , Humanos , Caquexia/metabolismo , Obesidade/metabolismo , Fator 15 de Diferenciação de Crescimento/farmacologia , Fator 15 de Diferenciação de Crescimento/metabolismo , Fragmentos de Peptídeos/farmacologia , Peso Corporal/fisiologiaRESUMO
Adherent-invasive Escherichia coli (AIEC) are reported to inhabit the gut mucosa in Crohn's disease (CD), however, little is known about the importance of host factors for the interplay between AIEC and the human gut. To examine if differences in bacterial adhesion patterns are disease associated, the AIEC-prototype strain LF82 was evaluated for its ability to adhere to ileal and colonic biopsies from CD and healthy controls (HC). Moreover, the efficacy of the non-pathogenic E. coli Nissle 1917 (ECN) in averting LF82 adhesion to ileal mucosa was assessed. Similar numbers of LF82 adhered to biopsies from CD and HC. A significantly greater LF82 attachment to ileal versus colonic mucosa was found in HC (P < 0.01), however, not in CD. ECN did not reduce the adhesion of LF82 to ileal specimens in CD or HC. These results show that enhanced bacterial adhesion ability is unlikely to play any significant role in CD, thus implying that other host protective factors may be impaired in CD. Further, exclusion of LF82 attachment by ECN co-incubation does not appear to represent a relevant treatment regimen.
Assuntos
Aderência Bacteriana , Doença de Crohn/microbiologia , Escherichia coli/patogenicidade , Mucosa Intestinal/microbiologia , Adolescente , Adulto , Carga Bacteriana , Biópsia , Colo/microbiologia , Feminino , Humanos , Íleo/microbiologia , Masculino , Pessoa de Meia-Idade , Técnicas de Cultura de Órgãos , Adulto JovemRESUMO
A cooperative dialogue between natural killer (NK) cells and dendritic cells (DCs) has been elucidated in the last years. They help each other to acquire their complete functions, both in the periphery and in the secondary lymphoid organs. Thus, NK cells' activation by dendritic cells allows the killing of transformed or infected cells in the periphery but may also be important for the generation of adaptive immunity. Indeed, it has been shown that NK cells may play a key role in polarizing a Th1 response upon interaction with DCs exposed to microbial products. This regulatory role of DC/NK cross-talk is of particular importance at mucosal surfaces such as the intestine, where the immune system exists in intimate association with commensal bacteria such as lactic acid bacteria (LAB). We here review NK/DC interactions in the presence of gut-derived commensal bacteria and their role in bacterial strain-dependent immunomodulatory effects. We particularly aim to highlight the ability of distinct species of commensal bacterial probiotics to differently affect the outcome of DC/NK cross-talk and consequently to differently influence the polarization of the adaptive immune response.
Assuntos
Comunicação Celular/imunologia , Células Dendríticas/imunologia , Imunomodulação , Células Matadoras Naturais/imunologia , Lactobacillus/imunologia , Probióticos/metabolismo , Bactérias/imunologia , Bactérias/metabolismo , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Imunidade Celular , Fatores Imunológicos , Células Matadoras Naturais/citologia , Células Matadoras Naturais/metabolismo , Lactobacillus/metabolismoRESUMO
Humans and other mammals coexist with a diverse array of microbes colonizing the intestine, termed the microflora. The relationship is symbiotic, with the microbes benefiting from a stable environment and nutrient supply, and the host gaining competitive exclusion of pathogens and continuously maintenance of the gut immune homeostasis. Here we report novel crosstalk mechanisms between the human enterocyte cell line, Caco2, and underlying human monocyte-derived DC in a transwell model where Gram-positive (G+) commensals prevent Toll-like receptor-4 (TLR4)-dependent Escherichia coli-induced semimaturation in a TLR2-dependent fashion. These findings add to our understanding of the hypo-responsiveness of the gut epithelium towards the microflora. Gut DC posses a more tolerogenic phenotype than conventional DC. Here we show that Caco2 spent medium (SM) induces tolerogenic DC with lower expression of maturation markers, interleukin (IL)-12p70, and tumour necrosis factor-alpha when matured with G+ and Gram-negative (G-) commensals, while IL-10 production is enhanced in DC upon encountering G+ commensals and reduced upon encountering G- bacteria. The Caco2 SM-induced tolerogenic phenotype is also seen in DC priming of naive T cells with elevated levels of transforming growth factor-beta (TGF-beta) and markedly reduced levels of bacteria-induced interferon-gamma production. Caco2 cell production of IL-8, thymic stromal lymphopoietin (TSLP) and TGF-beta increases upon microbial stimulation in a strain dependent manner. TSLP and TGF-beta co-operate in inducing the tolerogenic DC phenotype but other mediators might be involved.
Assuntos
Bactérias/imunologia , Citocinas/imunologia , Mucosa Intestinal/microbiologia , Simbiose/imunologia , Fator de Crescimento Transformador beta1/imunologia , Células CACO-2 , Diferenciação Celular/imunologia , Meios de Cultivo Condicionados , Citocinas/biossíntese , Células Dendríticas/imunologia , Células Epiteliais/imunologia , Escherichia coli/imunologia , Bactérias Gram-Positivas/imunologia , Humanos , Tolerância Imunológica , Imunidade nas Mucosas , Interleucina-8/biossíntese , Mucosa Intestinal/imunologia , Lactobacillus/imunologia , Células Th1/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Fator de Crescimento Transformador beta1/biossíntese , Linfopoietina do Estroma do TimoRESUMO
The gut microbiota is vital in the maintenance of homeostasis in the gut immune system. Its diversity and composition play major roles in relation to allergies and inflammatory bowel diseases, and administration of lactic acid bacteria (LAB), such as lactobacilli and bifidobacteria, has positive effects on these pathologies. However, the mechanisms behind the beneficial effects are largely unknown. Here we reveal divergent roles played by Toll-like receptor-2 (TLR2) and nucleotide-binding oligomerization domain-2 (NOD2) in dendritic cell (DC) recognition of LAB. Murine bone-marrow-derived DC lacking NOD2 produce higher levels of interleukin-10 (IL-10) and reduced levels of IL-12 and tumour necrosis factor-alpha (TNF-alpha) in response to LAB. This indicates that peptidoglycan is partly responsible for the T helper type 1 skewing effect of certain LAB. Dendritic cells that are TLR2-/- produce less IL-12 and TNF-alpha and more IL-10 in response to some strains of lactobacilli, while they produce more IL-12 and less IL-10 in response to bifidobacteria. The same tendency was found in human monocyte-derived DC. We have previously reported that the weak IL-12-inducing and TNF-alpha-inducing bifidobacteria inhibit the T helper type 1 skewing effect induced by strong immunostimulatory lactobacilli. Here we show that this immunoinhibitory effect of bifidobacteria is dependent on TLR2 and independent of NOD2. Moreover, independently of the cytokine pattern induced by intact LAB, cell wall fractions of all LAB, as well as synthetic lipoproteins possess immunoinhibitory capacities in both human and murine DC. These novel findings suggest that LAB act as immunoregulators through interaction of lipoprotein with TLR2 and as immunostimulators through interaction of peptidoglycan with NOD2.
Assuntos
Bifidobacterium/imunologia , Células Dendríticas/imunologia , Lactobacillus/imunologia , Proteína Adaptadora de Sinalização NOD2/imunologia , Receptor 2 Toll-Like/imunologia , Animais , Moléculas de Adesão Celular/imunologia , Diferenciação Celular/imunologia , Parede Celular/imunologia , Células Cultivadas , Meios de Cultivo Condicionados , Humanos , Tolerância Imunológica , Lactobacillus/classificação , Lactobacillus acidophilus/imunologia , Lectinas Tipo C/imunologia , Camundongos , Camundongos Endogâmicos , Receptores de Superfície Celular/imunologia , Especificidade da EspécieRESUMO
Intestinal cytochrome P450 subclass 1A1 (CYP1A1) contributes to a metabolic "shield" protecting the host from ingested carcinogens such as polycyclic aromatic hydrocarbons (PAH). The expression of CYP1 (including CYP1A2 and CYP1B1) is considered to depend solely on a heterodimeric transcription factor consisting of the arylhydrocarbon receptor (AHR) and the AHR nuclear translocator (ARNT). So far, no interference has been noted between the regulation of CYP1 and the activation of Toll-like receptor 2 (TLR2), which modulates the inflammatory response to bacterial cell wall components in immune cells and enterocytes. Here we report that intestinal CYP1A1 is silenced in TLR2-deficient mice, even when under exposure to the carcinogenic PAH benzo[a]pyrene (BaP). In contrast, hepatic CYP1A1 was moderately induced in TLR2-deficient mice without restoring their ability to clear BaP from systemic circulation, as present in wild-type animals. After feeding of BaP for 21 days, only TLR2(-/-) mice, but not their wild type littermates developed polyps in the colon. Gene expressions and protein concentrations of AHR and ARNT in the intestine did not differ between the genotypes. In conclusion, the presence of ligands for TLR2 of bacterial origin seems to be crucial for detoxication of luminal carcinogens by CYP1A1 in the intestine. This unprecedented finding indicates a complex interplay between the immune system of the host and intestinal bacteria with detoxication mechanisms. This highlights the relevance of intestinal microbiota when trying to unravel pathways present in mammals and opens new perspectives for research in human health.
Assuntos
Benzo(a)pireno/farmacocinética , Carcinógenos/farmacocinética , Citocromo P-450 CYP1A1/metabolismo , Intestinos/enzimologia , Receptor 2 Toll-Like/metabolismo , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Benzo(a)pireno/farmacologia , Carcinógenos/farmacologia , Pólipos do Colo/enzimologia , Pólipos do Colo/genética , Pólipos do Colo/patologia , Citocromo P-450 CYP1A1/genética , Humanos , Inativação Metabólica/genética , Intestinos/microbiologia , Fígado/enzimologia , Camundongos , Camundongos Knockout , Especificidade de Órgãos , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Receptor 2 Toll-Like/genéticaRESUMO
BACKGROUND: Macrophage-derived factors contribute to whole-body insulin resistance, partly by impinging on metabolically active tissues. As proof of principle for this interaction, conditioned medium from macrophages treated with palmitate (CM-PA) reduces insulin action and glucose uptake in muscle cells. However, the mechanism whereby CM-PA confers this negative response onto muscle cells remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: L6-GLUT4myc myoblasts were exposed for 24 h to palmitate-free conditioned medium from RAW 264.7 macrophages pre-treated with 0.5 mM palmitate for 6 h. This palmitate-free CM-PA, containing selective cytokines and chemokines, inhibited myoblast insulin-stimulated insulin receptor substrate 1 (IRS1) tyrosine phosphorylation, AS160 phosphorylation, GLUT4 translocation and glucose uptake. These effects were accompanied by a rise in c-Jun N-terminal kinase (JNK) activation, degradation of Inhibitor of κBα (IκBα), and elevated expression of proinflammatory cytokines in myoblasts. Notably, CM-PA caused IRS1 phosphorylation on Ser1101, and phosphorylation of novel PKCθ and ε. Co-incubation of myoblasts with CM-PA and the novel and conventional PKC inhibitor Gö6983 (but not with the conventional PKC inhibitor Gö6976) prevented PKCθ and ε activation, JNK phosphorylation, restored IκBα mass and reduced proinflammatory cytokine production. Gö6983 also restored insulin signalling and glucose uptake in myoblasts. Moreover, co-silencing both novel PKC θ and ε isoforms in myoblasts by RNA interference, but not their individual silencing, prevented the inflammatory response and restored insulin sensitivity to CM-PA-treated myoblasts. CONCLUSIONS/CLINICAL SIGNIFICANCE: The results suggest that the block in muscle insulin action caused by CM-PA is mediated by novel PKCθ and PKCε. This study re-establishes the participation of macrophages as a relay in the action of fatty acids on muscle cells, and further identifies PKCθ and PKCε as key elements in the inflammatory and insulin resistance responses of muscle cells to macrophage products. Furthermore, it portrays these PKC isoforms as potential targets for the treatment of fatty acid-induced, inflammation-linked insulin resistance.
Assuntos
Resistência à Insulina , Isoenzimas/metabolismo , Macrófagos/metabolismo , Células Musculares/metabolismo , Palmitatos/farmacologia , Proteína Quinase C-épsilon/metabolismo , Proteína Quinase C/metabolismo , Animais , Linhagem Celular , Meios de Cultivo Condicionados/farmacologia , Ácidos Graxos , Glucose/metabolismo , Inflamação , Macrófagos/efeitos dos fármacos , Camundongos , Células Musculares/efeitos dos fármacos , Proteína Quinase C-thetaRESUMO
We here present a method based on qRT-PCR to quantify E. coli LF82 in intestinal human samples. Two different primer-probe sets were designed to detect LF82, and a third to target total E. coli. The assay showed high robustness and specificity for detection of LF82 in the presence of intestinal tissue.