RESUMO
BACKGROUND AND AIMS: Intestinal fibrotic stenosis is a major reason for surgery in Crohn's disease [CD], but the mechanism is unknown. Thus, we asked whether intestinal adipocytes contribute to intestinal fibrosis. Adipocytes were found to transdifferentiate into myofibroblasts and confirmed to be involved in mesenteric fibrosis in our recent study. Here, we investigated the role and possible mechanisms of intestinal adipocytes in intestinal fibrosis in CD. METHODS: The intestinal tissue of patients with CD with or without fibrotic stenosis [CDS or CDN] and normal intestinal tissue from individuals without CD were obtained to assess alterations in submucosal adipocytes in CDS and whether these cells transdifferentiated into myofibroblasts and participated in the fibrotic process. Human primary adipocytes and adipose organoids were used to evaluate whether adipocytes could be induced to transdifferentiate into myofibroblasts and to investigate the fibrotic behaviour of adipocytes. LPS/TLR4/TGF-ß signalling was also studied to explore the underlying mechanism. RESULTS: Submucosal adipocytes were reduced in number or even absent in CDS tissue, and the extent of the reduction correlated negatively with the degree of submucosal fibrosis. Interestingly, submucosal adipocytes in CDS tissue transdifferentiated into myofibroblast-like cells and expressed collagenous components, possibly due to stimulation by submucosally translocated bacteria. Lipopolysaccharide [LPS]-stimulated human primary adipocytes and adipose organoids also exhibited transdifferentiation and profibrotic behaviour. Mechanistically, TLR4-mediated TGF-ß signalling was associated with the transdifferentiation and profibrotic behaviour of intestinal adipocytes in CDS tissue. CONCLUSIONS: Intestinal adipocytes transdifferentiate into myofibroblasts and participate in the intestinal fibrosis process in CD, possibly through LPS/TLR4/TGF-ß signalling.
Assuntos
Adipócitos , Transdiferenciação Celular , Doença de Crohn , Fibrose , Miofibroblastos , Doença de Crohn/patologia , Doença de Crohn/metabolismo , Humanos , Miofibroblastos/patologia , Adipócitos/patologia , Fator de Crescimento Transformador beta/metabolismo , Masculino , Adulto , Receptor 4 Toll-Like/metabolismo , Transdução de Sinais , Feminino , Constrição Patológica/patologia , Lipopolissacarídeos/farmacologia , Mucosa Intestinal/patologiaRESUMO
Until recently, the development of new human adjuvants was held back by a poor understanding of their mechanisms of action. The field was revolutionized by the discovery of the toll-like receptors (TLRs), innate immune receptors that directly or indirectly are responsible for detecting pathogen-associated molecular patterns (PAMPs) and respond to them by activating innate and adaptive immune pathways. Hundreds of ligands targeting various TLRs have since been identified and characterized as vaccine adjuvants. This work has important implications not only for the development of vaccines against infectious diseases but also for immuno-therapies against cancer, allergy, Alzheimer's disease, drug addiction and other diseases. Each TLR has its own specific tissue localization and downstream gene signalling pathways, providing researchers the opportunity to precisely tailor adjuvants with specific immune effects. TLR agonists can be combined with other TLR or alternative adjuvants to create combination adjuvants with synergistic or modulatory effects. This review provides an introduction to the various classes of TLR adjuvants and their respective signalling pathways. It provides an overview of recent advancements in the TLR field in the past 2-3 years and discusses criteria for selecting specific TLR adjuvants based on considerations, such as disease mechanisms and correlates of protection, TLR immune biasing capabilities, route of administration, antigen compatibility, new vaccine technology platforms, and age- and species-specific effects.
Assuntos
Neoplasias , Vacinas , Adjuvantes Imunológicos/farmacologia , Adjuvantes de Vacinas , Humanos , Neoplasias/tratamento farmacológico , Moléculas com Motivos Associados a Patógenos , Receptores Toll-Like , Vacinas/uso terapêuticoRESUMO
The interleukin-1 receptor-related kinase 4 (IRAK4), downstream of myd88, plays an essential role in hyperactive TLR signalling seen in some B-cell lymphomas. In particular, efficient IRAK4 inhibitors of activated B-cell subtype of human diffuse large B-Cell lymphoma (DLBCL) are being developed. However, the anticancer effect of IRAK-4 inhibitors in veterinary medicine has not been elucidated. It is therefore explored in this study involving the GL-1 and CL-1 canine lymphoma cell lines in vitro. MyD88 expression was analysed using polymerase chain reaction. GL-1 and CL-1 cells were subjected to concentration- and time-dependent treatment with an IRAK-4 inhibitor and assessed for viability, TLR signalling association and apoptosis using a cell counting Kit-8 assay, Western blotting and flow cytometry. The GL-1 and CL-1 cells exhibited enhanced MyD88 expression, however, canine peripheral blood mononuclear cells (cPBMCs) did not. The IRAK-4 inhibitor reduced cell viability in a dose- and time-dependent manner, significantly reduced the phosphorylation of molecules associated with TLR signalling at IC50 such as IRAK1, IRAK4, NF-κB and STAT3, and induced apoptosis in GL-1 and CL-1 cells. The anticancer effect of the IRAK-4 inhibitor on canine lymphoma cells is mediated by apoptosis via downregulation of TLR signalling.
Assuntos
Doenças do Cão , Linfoma Difuso de Grandes Células B , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Doenças do Cão/tratamento farmacológico , Cães , Humanos , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Leucócitos Mononucleares , Linfoma Difuso de Grandes Células B/veterinária , Fator 88 de Diferenciação Mieloide/metabolismoRESUMO
We aimed to identify key genes and pathways associated with different immune statuses of hepatitis B virus (HBV) infection. The gene expression and DNA methylation profiles were analysed in different immune statuses of HBV infection. Differentially expressed genes (DEGs) and differentially methylated genes (DMGs) were identified, followed by their functional and integrative analyses. The differential expression of IgG Fc receptors (FcγRs) in chronic HBV-infected patients and immune cells during different stages of HBV infection was investigated. Toll-like receptor (TLR) signalling pathway (including TLR6) and leucocyte transendothelial migration pathway (including integrin subunit beta 1) were enriched during acute infection. Key DEGs, such as FcγR Ib and FcγR Ia, and interferon-alpha inducible protein 27 showed correlation with alanine aminotransferase levels, and they were differentially expressed between acute and immune-tolerant phases and between immune-tolerant and immune-clearance phases. The integrative analysis of DNA methylation profile showed that lowly methylated and highly expressed genes, including cytotoxic T lymphocyte-associated protein 4 and mitogen-activated protein kinase 3 were enriched in T cell receptor signalling pathway during acute infection. Highly methylated and lowly expressed genes, such as Ras association domain family member 1 and cyclin-dependent kinase inhibitor 2A were identified in chronic infection. Furthermore, differentially expressed FcγR Ia, FcγR IIa and FcγR IIb, CD3- CD56+ CD16+ natural killer cells and CD14high CD16+ monocytes were identified between immune-tolerant and immune-clearance phases by experimental validation. The above genes and pathways may be used to distinguish different immune statuses of HBV infection.
Assuntos
Vírus da Hepatite B/imunologia , Hepatite B Crônica/genética , Hepatite B Crônica/imunologia , Hepatite B/genética , Hepatite B/imunologia , Transdução de Sinais/genética , Metilação de DNA/genética , Metilação de DNA/imunologia , Feminino , Expressão Gênica/genética , Hepatite B Crônica/virologia , Humanos , Células Matadoras Naturais/imunologia , Masculino , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de IgG/genética , Receptores de IgG/imunologia , Linfócitos T Citotóxicos/imunologiaRESUMO
Worldwide approximately 68 million people are infected with lymphatic filariasis (Lf), provoked by Wuchereria bancrofti, Brugia malayi and Brugia timori. This disease can lead to massive swelling of the limbs (elephantiasis) and disfigurement of the male genitalia (hydrocele). Filarial induced immune regulation is characterised by dominant type 2 helper T cell and regulatory immune responses. In vitro studies have provided evidence that signalling via Toll-like receptor-mediated pathways is triggered by filarial associated factors. Nevertheless, until now, less is known about the role of the adapter molecule TRIF during in vivo infections. Here, we used the rodent-specific nematode Litomosoides sigmodontis to investigate the role of TLR signalling and the corresponding downstream adapter and regulatory molecules TRIF, MyD88, IRF1 and IRF3 during an ongoing infection in semi-susceptible C57BL/6 mice. Interestingly, lack of the central adapter molecule TRIF led to higher worm burden and reduced overall absolute cell numbers in the thoracic cavity (the site of infection) 30 days post-infection. In addition, frequencies of macrophages and lymphocytes in the TC were increased in infected TRIF-/- C57BL/6 mice, whereas frequencies of eosinophils, CD4+ and CD8+ T cells were reduced. Nevertheless, cytokine levels and regulatory T cell populations remained comparable between TRIF-deficient and wildtype C57BL/6 mice upon 30 days of L. sigmodontis infection. In summary, this study revealed a crucial role of the adapter molecule TRIF on worm recovery and immune cell recruitment into the site of infection 30 days upon L. sigmodontis infection in C57BL/6 mice.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Filariose/imunologia , Filariose/parasitologia , Filarioidea/crescimento & desenvolvimento , Filarioidea/imunologia , Transdução de Sinais , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Citocinas/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T Reguladores/imunologia , Células Th2/imunologiaRESUMO
OBJECTIVES: To investigate the effect of dimethyl fumarate (DMF) on Toll-like receptor (TLR) signalling pathway in isoproterenol (ISO)-induced cardiac hypertrophy in rats. METHODS: Sixty adult male Sprague-Dawley rats were randomly allocated into three groups. group I: rats received the vehicles only; group II: rats were treated with ISO (5 mg/kg per day S.C.) to induce cardiac hypertrophy for 7 days; and group III: rats were given DMF (25 mg/kg per 12 h P.O.) for 28 days, and at the last 7 days, they were treated with ISO (5 mg/kg per day S.C.). KEY FINDINGS: Pretreatment with DMF decreased heart-to-body weight ratio, heart rate and blood pressure and improved the electrocardiographic patterns when compared with ISO group. DMF exhibited cardioprotective effect as evidenced by the reduction in cardiac troponin I, creatine kinase-MB and atrial natriuretic peptide levels. Moreover, DMF alleviated the changed oxidative stress and inflammatory biochemical markers through its anti-inflammatory and antioxidant effects. DMF interfered with TLR signalling pathway, evidenced by decreased levels of the TLR adaptor protein MyD88 and p-ERK1/2 and increased p-Akt level. CONCLUSIONS: Dimethyl fumarate exerted cardioprotective effect against ISO-induced cardiac hypertrophy. This effect is suggested to be through interfering with TLR signalling pathway.
Assuntos
Cardiomegalia/prevenção & controle , Fumarato de Dimetilo/farmacologia , Isoproterenol , Fator 88 de Diferenciação Mieloide/metabolismo , Miocárdio/metabolismo , Receptores Toll-Like/metabolismo , Remodelação Ventricular/efeitos dos fármacos , Animais , Cardiomegalia/induzido quimicamente , Cardiomegalia/metabolismo , Cardiomegalia/fisiopatologia , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Masculino , Miocárdio/patologia , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacosRESUMO
Infection and tissue damage induces assembly of supramolecular organizing centres (SMOCs)), such as the Toll-like receptor (TLR) MyDDosome, to co-ordinate inflammatory signaling. SMOC assembly is thought to drive digital all-or-none responses, yet TLR activation by diverse microbes induces anything from mild to severe inflammation. Using single-molecule imaging of TLR4-MyDDosome signaling in living macrophages, we find that MyDDosomes assemble within minutes of TLR4 stimulation. TLR4/MD2 activation leads only to formation of TLR4/MD2 heterotetramers, but not oligomers, suggesting a stoichiometric mismatch between activated receptors and MyDDosomes. The strength of TLR4 signalling depends not only on the number and size of MyDDosomes formed but also how quickly these structures assemble. Activated TLR4, therefore, acts transiently nucleating assembly of MyDDosomes, a process that is uncoupled from receptor activation. These data explain how the oncogenic mutation of MyD88 (L265P) assembles MyDDosomes in the absence of receptor activation to cause constitutive activation of pro-survival NF-κB signalling.
Assuntos
Antígeno 96 de Linfócito/metabolismo , Multimerização Proteica , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Animais , Camundongos , Células RAW 264.7 , Imagem Individual de MoléculaRESUMO
Preterm birth continues to be a significant global health care issue, due to our lack of understanding of the mechanisms that drive human labour and delivery. Toll-like receptors (TLRs) are essential in triggering an inflammatory response in human gestational tissues, leading to the production of pro-inflammatory and pro-labour mediators, and thus preterm birth. The aims of this study were to determine whether the adaptor molecules associated with TLR2, TLR3 and TLR5 signalling are involved in human myometrium. Primary human myometrial cells were transfected with siRNA against TIRAP, IRAK1, IRAK4, TAK1and stimulated with bacterial product fsl-1 (TLR2); TRIF, TRADD, TRAF6, RIP1, TAK1 and stimulated with dsRNA viral analogue poly(I:C) (TLR3); IRAK1, IRAK4, TAK1 and stimulated with bacterial product flagellin (TLR5), and assayed for production of pro-inflammatory and pro-labour mediators. Cells transfected with TIRAP, IRAK1, IRAK4 or TAK1 all showed a decrease in fsl-1-induced expression of cytokines (IL-1α, IL-1ß, IL-6), chemokines (GRO-α, IL-8, MCP-1), adhesion molecule ICAM-1, cyclooxygenase (COX)-2 mRNA and release of PGF2α and MMP-9 expression. Cells transfected with TRIF, TRAF6, RIP1 or TAK1 all decreased production of poly(I:C)-induced IL-1α, IL-1ß, IL-6, GRO-α, IL-8, MCP-1, ICAM-1 and MMP-9 expression. Cells transfected with IRAK1, IRAK4 or TAK1 all showed decreased expression of flagellin-induced cytokine and chemokine expression, ICAM-1 and MMP-9 expression. Lastly, transfection with these siRNAs decreased fsl-1, poly(I:C) and flagellin-induced NF-κB transcriptional activity. Our study signifies that these adaptor molecules are necessary for the proper production of cytokines, chemokines and pro-labour mediators after TLR ligation.
Assuntos
Trabalho de Parto/imunologia , Miométrio/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor 3 Toll-Like/metabolismo , Receptor 5 Toll-Like/metabolismo , Idoso , Células Cultivadas , Diglicerídeos/imunologia , Feminino , Flagelina/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Quinases Associadas a Receptores de Interleucina-1/genética , Metaloproteinase 9 da Matriz/metabolismo , Glicoproteínas de Membrana/genética , Miométrio/citologia , Miométrio/imunologia , NF-kappa B/metabolismo , Oligopeptídeos/imunologia , Poli I-C/imunologia , Gravidez , Cultura Primária de Células , RNA Interferente Pequeno/genética , Receptores de Interleucina-1/genética , Receptores de Esteroides/genética , Receptores dos Hormônios Tireóideos/genética , Transdução de SinaisRESUMO
Toll-like receptors (TLRs) are fundamental components of innate immunity that play significant roles in the defence against pathogen invasion. In this study, we present the molecular characterization of the full-length coding sequence of tlr1, tlr2a and tlr2b from common carp (Cyprinus carpio). Each is encoded within a single exon and contains a conserved number of leucine-rich repeats, a transmembrane region and an intracellular TIR domain for signalling. Indeed, sequence, phylogenetic and synteny analysis of carp tlr1, tlr2a and tlr2b support that these genes are orthologues of mammalian TLR1 and TLR2. The tlr genes are expressed in various immune organs and cell types. Furthermore, the carp sequences exhibited a good three-dimensional fit with the heterodimer structure of human TLR1-TLR2, including the potential to bind to the ligand Pam3CSK4. This supports the possible formation of carp Tlr1-Tlr2 heterodimers. However, we were unable to demonstrate Tlr1/Tlr2-mediated ligand binding in transfected cell lines through NF-κB activation, despite showing the expression and co-localization of Tlr1 and Tlr2. We discuss possible limitations when studying ligand-specific activation of NF-κB after expression of Tlr1 and/or Tlr2 in human but also fish cell lines and we propose alternative future strategies for studying ligand-binding properties of fish Tlrs.
Assuntos
Carpas/genética , Carpas/imunologia , Proteínas de Peixes/genética , Imunidade Inata , Receptor 1 Toll-Like/genética , Receptor 2 Toll-Like/genética , Sequência de Aminoácidos , Animais , Carpas/classificação , Carpas/metabolismo , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Filogenia , Alinhamento de Sequência , Sintenia , Receptor 1 Toll-Like/química , Receptor 1 Toll-Like/imunologia , Receptor 2 Toll-Like/química , Receptor 2 Toll-Like/imunologiaRESUMO
The heterodimeric mycobacterial receptors, macrophage C-type lectin (MCL) and macrophage inducible C-type lectin (Mincle), are upregulated at the cell surface following microbial challenge, but the mechanisms underlying this response are unclear. Here we report that microbial stimulation triggers Mincle expression through the myeloid differentiation primary response gene 88 (MyD88) pathway; a process that does not require MCL. Conversely, we show that MCL is constitutively expressed but retained intracellularly until Mincle is induced, whereupon the receptors form heterodimers which are translocated to the cell surface. Thus this "two-step" model for induction of these key receptors provides new insights into the underlying mechanisms of anti-mycobacterial immunity.
Assuntos
Interações Hospedeiro-Patógeno , Lectinas Tipo C/metabolismo , Proteínas de Membrana/metabolismo , Mycobacterium/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Imunológicos/metabolismo , Animais , Células Cultivadas , Expressão Gênica , Lectinas Tipo C/genética , Macrófagos/imunologia , Macrófagos/microbiologia , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Receptores de Superfície Celular/genética , Receptores Imunológicos/genéticaRESUMO
Extracellular vesicles (EVs), including exosomes, microvesicles and apoptotic bodies, participate in intercellular communication, and particularly, in paracrine and endocrine signalling. The EVs and their specific contents have been considered hallmarks of different diseases. It has been recently discovered that EVs can co-transport nucleic acids such as DNAs, ribosomal RNAs, circular RNAs (circRNAs), long noncoding RNAs (lnRNAs) and microRNAs (miRNAs). miRNAs are important regulators of gene expression at the post-transcriptional level, although they may also play other roles. Recent evidence supports the hypothesis that miRNAs can activate Toll-like receptors (TLRs) under certain circumstances. TLRs belong to a multigene family of immune system receptors and have been recently described in the nervous system. In the immune system, TLRs are important for the recognition of the invading microorganisms, whereas in the nervous system, they recognise endogenous ligands released by undifferentiated or necrotic/injured cells. In the neuronal disease field, TLRs activity has been associated with amyotrophic lateral sclerosis (ALS), stroke, Alzheimer's and Parkinson's disease. Herein, we reviewed the current knowledge of the relationship between miRNA release by EVs and the inflammation signalling triggered by TLRs in neighbouring cells or during long-distance cell-to-cell communication. We highlight novel aspects of this communication mechanism, offering a valuable insight into such pathways in health and disease.
Assuntos
Encefalopatias/metabolismo , Exossomos/metabolismo , MicroRNAs/metabolismo , Receptores Toll-Like/metabolismo , Animais , Vesículas Extracelulares/metabolismo , Humanos , Transporte de RNARESUMO
NOD-like receptors (NLR) are a family of cytosolic pattern recognition receptors that include many key drivers of innate immune responses. NLRP12 is an emerging member of the NLR family that is closely related to the well-known inflammasome scaffold, NLRP3. Since its discovery, various functions have been proposed for NLRP12, including the positive regulation of dendritic cell (DC) and neutrophil migration and the inhibition of NF-κB and ERK signalling in DC and macrophages. We show here that NLRP12 is poorly expressed in murine macrophages and DC, but is strongly expressed in neutrophils. Using myeloid cells from WT and Nlrp12(-/)(-) mice, we show that, contrary to previous reports, NLRP12 does not suppress LPS- or infection-induced NF-κB or ERK activation in myeloid cells, and is not required for DC migration in vitro. Surprisingly, we found that Nlrp12 deficiency caused increased rather than decreased neutrophil migration towards the chemokine CXCL1 and the neutrophil parasite Leishmania major, revealing NLRP12 as a negative regulator of directed neutrophil migration under these conditions.
Assuntos
Movimento Celular/imunologia , Quimiocina CXCL1/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Neutrófilos/imunologia , Animais , Movimento Celular/efeitos dos fármacos , Quimiocina CXCL1/genética , Células Dendríticas/imunologia , Células Dendríticas/parasitologia , Feminino , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leishmania major/imunologia , Lipopolissacarídeos/farmacologia , Macrófagos/imunologia , Macrófagos/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/imunologia , NF-kappa B/genética , NF-kappa B/imunologia , Neutrófilos/parasitologia , Especificidade de Órgãos , Transdução de SinaisRESUMO
Toll-like receptors (TLRs) recognise invading pathogens and initiate an innate immune response by recruiting intracellular adaptor proteins via heterotypic Toll/interleukin-1 receptor (TIR) domain interactions. Of the five TIR domain-containing adaptor proteins identified, Sterile α- and armadillo-motif-containing protein (SARM) is functionally unique; suppressing immune signalling instead of promoting it. Here we demonstrate that the recombinantly expressed and purified SARM TIR domain interacts with both the major human TLR adaptors, MyD88 and TRIF. A single glycine residue located in the BB-loop of the SARM TIR domain, G601, was identified as essential for interaction. A short peptide derived from this motif was also found to interact with MyD88 in vitro. SARM expression in HEK293 cells was found to significantly suppress lipopolysaccharide (LPS)-mediated upregulation of inflammatory cytokines, IL-8 and TNF-α, an effect lost in the G601A mutant. The same result was observed with cytokine activation initiated by MyD88 expression and stimulation of TLR2 with lipoteichoic acid (LTA), suggesting that SARM is capable of suppressing both TRIF- and MyD88- dependent TLR signalling. Our findings indicate that SARM acts on a broader set of target proteins than previously thought, and that the BB-loop motif is functionally important, giving further insight into the endogenous mechanisms used to suppress inflammation in immune cells.
Assuntos
Proteínas do Domínio Armadillo/metabolismo , Proteínas do Citoesqueleto/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Receptores de Interleucina-1/metabolismo , Receptores Toll-Like/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Proteínas do Domínio Armadillo/genética , Sítios de Ligação/genética , Western Blotting , Proteínas do Citoesqueleto/genética , Células HEK293 , Humanos , Interleucina-8/metabolismo , Lipopolissacarídeos/farmacologia , Microscopia Confocal , Mutação , Fator 88 de Diferenciação Mieloide/genética , Ligação Proteica , Receptores de Interleucina-1/genética , Transdução de Sinais/efeitos dos fármacos , Ácidos Teicoicos/farmacologia , Receptores Toll-Like/genética , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Infection in male and female genital tracts can lead to infertility. The underlying mechanisms of this process remain unclear. Toll-like receptors (TLRs) recognize conserved structures and respond to pathogens by initiating signals that activate inflammatory gene transcription. Here, we demonstrate that TLR activation in sperm reduces sperm motility via signalling through myeloid differentiation factor 88 (MyD88), phosphatidylinositol 3-kinase (PI3K), and glycogen synthase kinase (GSK)-3α. Upon TLR activation, phosphorylated forms of PI3K and GSK3α were detected in the mitochondria, and the mitochondrial membrane potential was impaired in sperm. In addition, mitochondrial ATP levels were decreased after TLR agonist stimulation. Furthermore, blocking PI3K or GSK3α activation abrogated these effects and reversed the TLR-induced reduction in sperm motility. These results identify a previously unrecognized TLR signalling pathway that leads to dysfunctional sperm mitochondria, which reduce sperm motility. Our study reveals a novel mechanism by which pathogenic infection affects sperm motility and possibly leads to infertility.
Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Mitocôndrias/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Espermatozoides/metabolismo , Receptores Toll-Like/metabolismo , Trifosfato de Adenosina/metabolismo , Androstadienos/farmacologia , Animais , Feminino , Humanos , Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/deficiência , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Fosfatidilinositol 3-Quinase/genética , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Motilidade dos Espermatozoides/efeitos dos fármacos , Receptores Toll-Like/agonistas , Wortmanina , Zimosan/farmacologiaRESUMO
In this study, the full-length complementary (c)DNA of interleukin-1 receptor-associated kinase 1 gene (irak1) was cloned from common carp Cyprinus carpio. The complete open reading frame of irak1 contained 2109 bp encoding a protein of 702 amino acid residues that comprised a death domain, a ProST region, a serine-threonine-specific protein kinase catalytic domain and a C-terminal domain. The amino-acid sequence of C. carpio Irak1 protein shared sequence homology with grass carp Ctenopharyngodon idellus (84.5%). The phylogenetic tree of IRAKs separated the polypeptides into four clades, comprising IRAK1s, IRAK2s, IRAK3s and IRAK4s. Cyprinus carpio Irak1 fell into the cluster with previously reported IRAK1s including teleost Irak1s. The irak1 gene was highly expressed in gills, followed by brain, skin, hindgut, buccal epithelium, spleen, foregut, head kidney and liver, and was expressed at lowest levels in gonad and muscle. The irak1 messenger (m)RNA expression was up-regulated in liver, spleen, head kidney, foregut, hindgut, gills and skin after stimulation with Vibrio anguillarum and poly(I:C), and significantly high up-regulated expression was observed in liver and spleen. These results implied that irak1 might participate in antibacterial and antiviral innate immunity. These findings gave the indications that irak1 may participate in antibacterial and antiviral immunity.
Assuntos
Carpas/genética , Proteínas de Peixes/metabolismo , Imunidade Inata , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Sequência de Aminoácidos , Animais , Infecções Bacterianas/imunologia , Carpas/imunologia , Clonagem Molecular , DNA Complementar/genética , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/imunologia , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , RNA Mensageiro/metabolismoRESUMO
Dendritic cells (DCs) are key regulators of both innate and adaptive immunity. During infection, DCs recognise pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs) including the Toll-like receptor (TLR) family. TLRs mainly signal via the adaptor protein MyD88. This signalling pathway is required for immune protection during many infections, which are lethal in the absence of MyD88. However, the cell type specific importance of this pathway during both innate and adaptive immune responses against pathogens in vivo remains ill-defined. We discuss recent findings from conditional KO or gain-of-function mouse models targeting TLR/MyD88 signalling pathways in DCs and other myeloid cells during infection. While the general assumption that MyD88-dependent recognition by DCs is essential for inducing protective immunity holds true in some instances, the results surprisingly indicate a much more complex context-dependent requirement for this pathway in DCs and other myeloid or lymphoid cell-types in vivo. Furthermore, we highlight the advantages of Cre-mediated DC targeting approaches and their possible limitations. We also present future perspectives on the development of new genetic mouse models to target distinct DC subsets in vivo. Such models will serve to understand the functional heterogeneity of DCs in vivo.
Assuntos
Candidíase/imunologia , Células Dendríticas/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Transdução de Sinais/imunologia , Receptores Toll-Like/imunologia , Toxoplasmose/imunologia , Imunidade Adaptativa , Animais , Candida albicans/imunologia , Candidíase/genética , Candidíase/microbiologia , Células Dendríticas/microbiologia , Células Dendríticas/parasitologia , Regulação da Expressão Gênica , Imunidade Inata , Integrases/genética , Integrases/metabolismo , Camundongos , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide/genética , Regiões Promotoras Genéticas , Transdução de Sinais/genética , Receptores Toll-Like/genética , Toxoplasma/imunologia , Toxoplasmose/genética , Toxoplasmose/parasitologiaRESUMO
The innate immune system provides the host's first line of defence against invading pathogens. Key to the stimulation of the innate immune response is pattern-recognition receptors (PRRs), such as Toll-like receptors (TLRs), which recognize microbial-associated molecular patterns (MAMPs). Binding of MAMPs to TLRs triggers a signalling cascade resulting in the production of pro-inflammatory mediators. Central to this TLR signalling pathway are heterotypic protein-protein interactions mediated through Toll/interleukin-1 receptor (TIR) domains found in both the cytoplasmic regions of TLRs and several key adaptor proteins. Interestingly, TIR-domain containing proteins (Tcps) do not seem to be unique to the mammalian TLR system, but occurs in abundance in many biological forms. Recent evidence suggests that pathogenic bacteria have developed a range of ingenuous strategies to evade the host immune mechanisms involving Tcps. There is increasing evidence to suggest that these pathogen-encoded Tcps interfere directly with the TLR signalling pathway and thus inhibit the activation of NF-κB, with different modes of action and roles in virulence. Here, we review the current state of knowledge on the possible roles and mechanisms of action of bacterial encoded Tcp.