RESUMO
Antigen processing and antigen-specific CD8 T cell activation form part and parcel of cell-mediated immunity to infections. Yet, several lacunae remain in our understanding of how antigen processing and CD8 T cell response are coordinated. In this study, using mouse bone marrow-derived dendritic cells (BMDC) as antigen-presenting cells and Ovalbumin (OVA)/DQ-Ovalbumin (DQ-OVA) as model antigen we demonstrated that Wnt5A signaling in BMDC supports antigen processing/presentation and concomitant CD8 T cell activation through regulation of actin and proteasome dynamics. Recombinant Wnt5A conditioning of BMDC and associated actin assembly facilitated DQ-OVA processing, which was inhibited by the proteasome inhibitor MG132. Moreover, Wnt5A depletion led to a significant reduction in OVA processing and presentation. Impaired DQ-OVA processing in Wnt5A depleted BMDC correlated with altered dynamics of both actin and the proteasome regulator PA28α-PA28ß, and reduced association of DQ-OVA with actin and proteasome subunits. Inhibited OVA processing/presentation in the Wnt5A depleted BMDC also resulted in subdued activation of OVA-sensitized CD8 T cells in co-culture with the BMDC. In concurrence with these findings, we demonstrated reduced OVA processing and impaired CD8 T cell response to OVA immunization in Wnt5A heterozygous mice lacking a copy of the Wnt5A gene in comparison to the wild-type cohorts. Taken together, our results reveal a crucial requirement of Wnt5A signaling in antigen processing/presentation and CD8 T cell activation, thus unveiling a vital regulatory node of cell-mediated immunity, unidentified thus far.
Assuntos
Apresentação de Antígeno , Linfócitos T CD8-Positivos , Proteína Wnt-5a , Actinas/imunologia , Animais , Apresentação de Antígeno/imunologia , Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina , Complexo de Endopeptidases do Proteassoma , Proteína Wnt-5a/imunologiaRESUMO
Accruing research shows the implications of long non-coding RNAs (lncRNAs) in the progression of various autoimmune diseases including systemic lupus erythematosus (SLE). The present study aimed to identify the expression pattern of LINC00176 in SLE and to explore its effects on CD4+T cell adhesion in this context. The biological functions of LINC00176, WIF1 and WNT5a on CD4+T cells in SLE were evaluated via gain- and loss-of-function experiments, following delivery of pcDNA3-LINC00176, siRNA-LINC00176, pcDNA3-WIF1 and WNT-sFRP5 (an inhibitor for the WNT5a signaling pathway). High LINC00176 expression was evident in the CD4+T cells of SLE patients. Additionally, WIF1 was identified as a potential target gene of LINC00176, and was negatively regulated by LINC00176. The overexpression of LINC00176 could promote proliferation and adhesion of CD4+T cells in SLE. Such alternations were reversed following up-regulation of WIF1 or inhibition of the WNT5a signaling pathway. Taken together, the key findings of our study highlight the ability of LINC00176 to potentially promote the proliferation and adhesion of CD4+T cells in SLE by down-regulating WIF1 and activating the WNT5a signaling pathway, providing new insight and a theoretical basis for translation in SLE therapy.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Linfócitos T CD4-Positivos/imunologia , Regulação da Expressão Gênica/fisiologia , Lúpus Eritematoso Sistêmico/imunologia , RNA Longo não Codificante/imunologia , Proteína Wnt-5a/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adolescente , Adulto , Linfócitos T CD4-Positivos/metabolismo , Adesão Celular/imunologia , Feminino , Humanos , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/metabolismo , Masculino , RNA Longo não Codificante/metabolismo , Transdução de Sinais/fisiologia , Proteína Wnt-5a/metabolismo , Adulto JovemRESUMO
Wnt5a is the prototypical activator of the non-canonical Wnt pathways, and its overexpression has been implicated in the progression of several tumor types by promoting cell motility, invasion, EMT, and metastasis. Recent evidences have revealed a novel role of Wnt5a in the phosphorylation of the NF-κB subunit p65 and the activation of the NF-κB pathway in cancer cells. In this article, we review the molecular mechanisms and mediators defining a Wnt5a/NF-κB signaling pathway and propose that the aberrant expression of Wnt5a in some tumors drives a Wnt5a/NF-κB/IL-6/STAT3 positive feedback loop that amplifies the effects of Wnt5a. The evidences discussed here suggest that Wnt5a has a double effect on the tumor microenvironment. First, it activates an autocrine ROR1/Akt/p65 pathway that promotes inflammation and chemotaxis of immune cells. Then, Wnt5a activates a TLR/MyD88/p50 pathway exclusively in myelomonocytic cells promoting the synthesis of the anti-inflammatory cytokine IL-10 and a tolerogenic phenotype. As a result of these mechanisms, Wnt5a plays a negative role on immune cell function that contributes to an immunosuppressive tumor microenvironment and would contribute to resistance to immunotherapy. Finally, we summarized the development of different strategies targeting either Wnt5a or the Wnt5a receptor ROR1 that can be helpful for cancer therapy by contributing to generate a more immunostimulatory tumor microenvironment.
Assuntos
Neoplasias/imunologia , Via de Sinalização Wnt/imunologia , Proteína Wnt-5a/imunologia , Animais , Humanos , Tolerância Imunológica , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , NF-kappa B/imunologia , NF-kappa B/metabolismo , Metástase Neoplásica , Neoplasias/metabolismo , Neoplasias/patologia , Microambiente Tumoral/imunologia , Proteína Wnt-5a/metabolismoRESUMO
Asthma is a heterogeneous disease characterized by chronic inflammation and structural changes in the airways. The airway smooth muscle (ASM) is responsible for airway narrowing and an important source of inflammatory mediators. We and others have previously shown that WNT5A mRNA and protein expression is higher in the ASM of asthmatics compared to healthy controls. Here, we aimed to characterize the functional role of (smooth muscle-derived) WNT5A in asthma. We generated a tet-ON smooth-muscle-specific WNT5A transgenic mouse model, enabling in vivo characterization of smooth-muscle-derived WNT5A in response to ovalbumin. Smooth muscle specific WNT5A overexpression showed a clear trend towards enhanced actin (α-SMA) expression in the ASM in ovalbumin challenged animals, but had no effect on collagen content. WNT5A overexpression in ASM also significantly enhanced the production of the Th2-cytokines IL4 and IL5 in lung tissue after ovalbumin exposure. In line with this, WNT5A increased mucus production, and enhanced eosinophilic infiltration and serum IgE production in ovalbumin-treated animals. In addition, CD4+ T cells of asthma patients and healthy controls were stimulated with WNT5A and changes in gene transcription assessed by RNA-seq. WNT5A promoted expression of 234 genes in human CD4+ T cells, among which the Th2 cytokine IL31 was among the top 5 upregulated genes. IL31 was also upregulated in response to smooth muscle-specific WNT5A overexpression in the mouse. In conclusion, smooth-muscle derived WNT5A augments Th2 type inflammation and remodelling. Our findings imply a pro-inflammatory role for smooth muscle-derived WNT5A in asthma, resulting in increased airway wall inflammation and remodelling.
Assuntos
Remodelação das Vias Aéreas/imunologia , Asma/imunologia , Linfócitos T CD4-Positivos/imunologia , Pulmão/imunologia , Músculo Liso/imunologia , Proteína Wnt-5a/imunologia , Actinas/genética , Actinas/imunologia , Remodelação das Vias Aéreas/genética , Alérgenos/administração & dosagem , Animais , Asma/induzido quimicamente , Asma/genética , Asma/patologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/patologia , Movimento Celular , Eosinófilos/imunologia , Eosinófilos/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Imunoglobulina E/biossíntese , Interleucina-4/genética , Interleucina-4/imunologia , Interleucina-5/genética , Interleucina-5/imunologia , Interleucinas/genética , Interleucinas/imunologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Músculo Liso/química , Músculo Liso/patologia , Ovalbumina/administração & dosagem , Cultura Primária de Células , Transgenes , Proteína Wnt-5a/genética , Proteína Wnt-5a/farmacologiaRESUMO
Wnt5A signaling facilitates the killing of several bacterial pathogens, but not the non-pathogen E. coli DH5α. The basis of such pathogen vs. non-pathogen distinction is unclear. Accordingly, we analyzed the influence of Wnt5A signaling on pathogenic E. coli K1 in relation to non-pathogenic E. coli K12-MG1655 and E. coli DH5α eliminating interspecies variability from our study. Whereas cell internalized E. coli K1 disrupted cytoskeletal actin organization and multiplied during Wnt5A depletion, rWnt5A mediated activation revived cytoskeletal actin assembly facilitating K1 eradication. Cell internalized E. coli K12-MG1655 and E. coli DH5α, which did not perturb actin assembly appreciably, remained unaffected by rWnt5A treatment. Phagosomes prepared separately from Wnt5A conditioned medium treated K1 and K12-MG1655 infected macrophages revealed differences in the relative levels of actin and actin network promoting proteins, upholding that the Wnt5A-Actin axis operates differently for internalized pathogen and non-pathogen. Interestingly, exposure of rWnt5A treated K1 and K12-MG1655/DH5α infected macrophages to actin assembly inhibitors reversed the scenario, blocking killing of K1, yet promoting killing of both K12-MG1655 and DH5α. Taken together, our study illustrates that the state of activation of the Wnt5A/Actin axis in the context of the incumbent bacteria is crucial for directing host response to infection.
Assuntos
Infecções por Escherichia coli/imunologia , Escherichia coli K12/imunologia , Macrófagos/imunologia , Transdução de Sinais/imunologia , Proteína Wnt-5a/imunologia , Animais , Infecções por Escherichia coli/genética , Macrófagos/microbiologia , Camundongos , Células RAW 264.7 , Transdução de Sinais/genética , Proteína Wnt-5a/genéticaRESUMO
The wingless and integration site growth factor-5a (Wnt5a) is a ligand of the receptor tyrosine kinase-like orphan receptor-1 (ROR1). Because both Wnt5a and ROR1 are expressed in circulating chronic lymphocytic leukemia (CLL) cells, and because in other cell types, STAT3, which is constitutively activated in CLL, induces Wnt5a signaling, we wondered whether STAT3 induces the expression of Wnt5a in CLL cells. Sequence analysis detected four putative STAT3 binding sites in close proximity to the Wnt5a gene promoter's start codon. Chromatin immunoprecipitation and EMSA revealed that STAT3 binds to the Wnt5a gene promoter, and a luciferase assay showed that STAT3 activates the Wnt5a gene. Additionally, transfection of peripheral blood CLL cells with STAT3 short hairpin RNA downregulated Wnt5a mRNA and protein levels, suggesting that STAT3 binds to the Wnt5a gene promoter and induces the expression of Wnt5a in CLL cells. Flow cytometry and confocal microscopy determined that both Wnt5a and its receptor ROR1 are coexpressed on the surface of CLL cells, and Western immunoblotting showed an inverse correlation between Wnt5a and ROR1 protein levels, implying that, regardless of CLL cells' ROR1 levels, blocking the interaction between Wnt5a and ROR1 might be beneficial to patients with CLL. Indeed, transfection of CLL cells with Wnt5a small interfering RNA reduced Wnt5a mRNA and protein levels and significantly increased the spontaneous apoptotic rate of CLL cells. Taken together, our data unravel an autonomous STAT3-driven prosurvival circuit that provides circulating CLL cells with a microenvironment-independent survival advantage.
Assuntos
Leucemia Linfocítica Crônica de Células B/imunologia , Fator de Transcrição STAT3/imunologia , Proteína Wnt-5a/imunologia , Sobrevivência Celular , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Ligação Proteica , Transdução de Sinais/imunologia , Proteína Wnt-5a/genéticaRESUMO
Coculture of nurse-like cells (NLCs) with chronic lymphocytic leukemia (CLL) cells induced leukemia cell phosphorylation of STAT3 (pSTAT3), which could be blocked by anti-Wnt5a antibodies or the anti-ROR1 monoclonal antibody, cirmtuzumab. Time-course studies revealed Wnt5a could induce activation of NF-κB within 30 minutes, but required more than 3 hours to induce pSTAT3. Culture of isolated CLL cells for 24 hours revealed Wnt5a-induced expression of interleukin 6 (IL-6), IL-8, CCL2, CCL3, CCL4, and CXCL1, which in turn could induce pSTAT3 in unstimulated CLL cells within 30 minutes. We found that Wnt5a could induce CLL cell expression of NF-κB target genes, including IL-6, and that this effect could be blocked by cirmtuzumab or drugs that inhibit NF-κB. Examination of CLL cells and plasma collected from patients treated with cirmtuzumab revealed reduced levels of phosphorylated p65 and diminished expression of NF-κB and STAT3 target genes in CLL cells, as well as lower plasma levels of IL-6, in the samples after therapy. Collectively, these studies indicate that Wnt5a/ROR1-dependent signaling contributes to CLL cell activation of NF-κB, which in turn causes autocrine IL-6-induced activation of pSTAT3. As such, this study demonstrates that cirmtuzumab can inhibit leukemia cell activation of both NF-κB and STAT3 in patients with CLL.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos Imunológicos/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , NF-kappa B/imunologia , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/imunologia , Proteína Wnt-5a/imunologia , Humanos , Leucemia Linfocítica Crônica de Células B/imunologia , Fator de Transcrição STAT3/imunologia , Células Tumorais CultivadasRESUMO
INTRODUCTION: WNT5A (Wnt family member 5A) belongs to the WNT family of secreted signaling glycoproteins that play essential role in developmental, physiological and pathological processes. WNT5A was shown to take part in carcinogenesis process playing both oncogenic and suppressor functions in various types of human malignancies. This study aimed to assess the expression of the WNT5A gene at the mRNA and protein levels in the specimens derived from endometrial cancer (EC) or unchanged control endometrium. The associations between the WNT5A expression levels and clinicopathological characteristics and survival of EC patients were evaluated. MATERIALS AND METHODS: Total RNA was isolated in order to assess the relative amounts of WNT5A mRNA by quantitative polymerase chain reaction (QPCR) in samples of unchanged endometrial control (n = 8) and tumor samples of EC patients (n = 28). Immunohistochemistry (IHC) was used to determine the presence of WNT5A protein in the sections of formalin-fixed, paraffin-embedded tissue specimens derived from unchanged endome-trial controls (n = 6) and EC tumors (n = 19). Significance of differences in WNT5A expression levels between the studied groups of EC patients and correlations between the WNT5A and demographic data, pathological features, hematological parameters and overall survival of the patients were evaluated by statistical analysis. RESULTS: The level of WNT5A mRNA was decreased in EC in comparison to unchanged endometrium. WNT5A expression was associated with primary tumor invasion status exhibiting reduced level of transcripts in EC that involved organs beyond the uterus when compared to the uterus-confined cancers. WNT5A immunoreactivity was visualized in the cytoplasm and nuclei of EC cells as well as in the luminal and glandular epithelial cells of unchanged endometrium. WNT5A mRNA expression levels correlated negatively with cytoplasmic, and positively with nuclear immunoreactivity of the WNT5A protein in the EC cells. In addition, the relationships between blood leucocyte count (in particular granulocytes and lymphocytes) of patients with EC and their WNT5A mRNA and protein expression levels were established. A positive correlation between the nuclear immunoexpression of WNT5A protein in the cancer cells in cell nuclei and mean platelet volume in blood was also found. CONCLUSIONS: The results of the first study of WNT5A expression at the transcript and protein levels indicate that it could be considered as a potential marker of molecular changes that take place during EC development.
Assuntos
Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , RNA Mensageiro/metabolismo , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismo , Adulto , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Biomarcadores Tumorais/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Regulação para Baixo , Neoplasias do Endométrio/diagnóstico , Neoplasias do Endométrio/mortalidade , Endométrio/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Prognóstico , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Proteína Wnt-5a/imunologiaRESUMO
Mycobacterium tuberculosis, which primarily infects mononuclear phagocytes, remains the leading bacterial cause of enormous morbidity and mortality because of bacterial infections in humans throughout the world. The IL-1 family of cytokines is critical for host resistance to M. tuberculosis As a newly discovered subgroup of the IL-1 family, although IL-36 cytokines have been proven to play roles in protection against M. tuberculosis infection, the antibacterial mechanisms are poorly understood. In this study, we demonstrated that IL-36γ conferred to human monocyte-derived macrophages bacterial resistance through activation of autophagy as well as induction of WNT5A, a reported downstream effector of IL-1 involved in several inflammatory diseases. Further studies showed that WNT5A could enhance autophagy of monocyte-derived macrophages by inducing cyclooxygenase-2 (COX-2) expression and in turn decrease phosphorylation of AKT/mTOR via noncanonical WNT signaling. Consistently, the underlying molecular mechanisms of IL-36γ function are also mediated by the COX-2/AKT/mTOR signaling axis. Altogether, our findings reveal a novel activity for IL-36γ as an inducer of autophagy, which represents a critical inflammatory cytokine that control the outcome of M. tuberculosis infection in human macrophages.
Assuntos
Interleucina-1/imunologia , Macrófagos/imunologia , Tuberculose Pulmonar/imunologia , Proteína Wnt-5a/imunologia , Autofagia/imunologia , Humanos , Interleucina-1/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Mycobacterium tuberculosis/imunologia , Transdução de Sinais/imunologia , Tuberculose Pulmonar/metabolismo , Proteína Wnt-5a/metabolismoRESUMO
Innate immune responses are rapid, dynamic and highly regulated to avoid overt reactions. This regulation is executed by innate immune tolerance mechanisms that remain obscure. Wnt5a is a signalling protein mainly involved in developmental processes and cancer. The effect of Wnt5a on inflammatory myeloid cells is controversial. Here, we combine primary cell cultures, in vitro binding studies, mass spectrometry and Drosophila protein modelling to show that Wnt5a is a direct ligand of toll-like receptor (TLR) 2 and 4. The binding promotes a MyD88-non-canonical nuclear factor of kappa B (NFκB) and AP-1 signalling cascade, with contradictory profiles in mouse (pro-inflammatory) and human (anti-inflammatory) myeloid immune cells. These data reveal that the true nature of Wnt5a in inflammatory cells, is to regulate TLR signals, and in human myeloid cells it acts as an endogenous, tolerance-associated molecular pattern (TAMP), inducing IL-10 and innate immune tolerance.
Assuntos
Células Mieloides/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Proteína Wnt-5a/imunologia , Animais , Células Cultivadas , Citocinas/biossíntese , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Humanos , Tolerância Imunológica , Imunidade Inata , Interleucina-10/biossíntese , Interleucina-10/genética , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ligantes , Camundongos , Modelos Imunológicos , Modelos Moleculares , Células Mieloides/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptores Toll-Like/química , Receptores Toll-Like/metabolismo , Transcrição Gênica , Proteína Wnt-5a/metabolismoRESUMO
Resveratrol, a phytochemical, acts several cellular signaling pathways and has anti-inflammatory potentials. The purpose of this study is to research the therapeutic effect of resveratrol in collagen-induced arthritis (CIA) model in rats and whether resveratrol affects the activities of signaling pathways those are potent pathogenic actors of rheumatoid arthritis. Arthritis was induced by intradermal injection of chicken type II collagen combined with incomplete Freund's adjuvant in Wistar albino rats. One day after the onset of arthritis (day 14), resveratrol (20 mg/kg/day) was given via oral gavage, until day 29. The paws of the rats were obtained for further analysis. Tissue Wnt5a, mitogen-activated protein kinase (MAPK), Src tyrosine kinase and signal transducer, and activator of transcription-3 (STAT3) mRNA expressions were determined by real-time polymerase chain reaction. Resveratrol ameliorated the clinical and histopathological (perisynovial inflammation and cartilage-bone destruction) findings of inflammatory arthritis. The tissue mRNA expressions of Wnt5a, MAPK3, Src kinase, and STAT3 were increased in the sham group compared to the control group. Resveratrol supplement decreased their expressions. The present study shows that Src kinase, STAT3, and Wnt signaling pathway are active in the CIA model. Resveratrol inhibits these signaling pathways and ameliorates inflammatory arthritis. © 2018 BioFactors, 45(1):69-74, 2019.
Assuntos
Anti-Inflamatórios/farmacologia , Artrite Experimental/tratamento farmacológico , Resveratrol/farmacologia , Fator de Transcrição STAT3/genética , Via de Sinalização Wnt/efeitos dos fármacos , Quinases da Família src/genética , Administração Oral , Animais , Artrite Experimental/genética , Artrite Experimental/imunologia , Artrite Experimental/patologia , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/imunologia , Osso e Ossos/patologia , Cartilagem/efeitos dos fármacos , Cartilagem/imunologia , Cartilagem/patologia , Esquema de Medicação , Feminino , Regulação da Expressão Gênica , Membro Posterior , Inflamação , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/imunologia , Ratos , Ratos Wistar , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/imunologia , Proteína Wnt-5a/genética , Proteína Wnt-5a/imunologia , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/imunologiaRESUMO
Wnt5a signalling plays pathological roles in synovial inflammation and bone destruction. In the present study, we designed four human Wnt5a-based DNA recombinants and detected their effects on immunogenicity and anti-rheumatism in a collagen-induced arthritis (CIA) model. Histomorphometry and micro-CT scanning showed that the phWnt5a-NL was superior to other recombinants because it resulted in decreased severity of arthritis, histopathological scores of synovial inflammation and bone erosion in CIA mice. In addition, ELISA and TRAP staining showed that the phWnt5a-NL-immunized CIA mice had reductions in the serum concentrations of the rheumatoid-associated cytokines IL-1ß and RANKL and in osteoclastogenesis. Furthermore, flow cytometry showed that the phWnt5a-NL treatment increased the percentage of Treg cells. Finally, western blotting analysis showed that the phWnt5a-NL-immunization interrupted ß-catenin and JNK expression in osteoclast precursors derived from the CIA mice. The results suggest that depleting the carboxy-terminus in hWnt5a-based DNA recombinants may be beneficial for the treatment of chronic inflammatory disorders involving bone resorption.
Assuntos
Artrite Experimental/imunologia , Imunização/métodos , Proteínas Recombinantes/imunologia , Proteína Wnt-5a/imunologia , Animais , Artrite Experimental/diagnóstico por imagem , Artrite Experimental/patologia , Citocinas/sangue , Citocinas/imunologia , Humanos , Interleucina-1beta/sangue , Interleucina-1beta/imunologia , Proteínas Quinases JNK Ativadas por Mitógeno/imunologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Osteoclastos/citologia , Osteoclastos/imunologia , Osteoclastos/metabolismo , Osteogênese/imunologia , Proteínas Recombinantes/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismo , Microtomografia por Raio-X/métodosRESUMO
Fungal keratitis causes devastating corneal ulcers which can result in significant visual impairment and even blindness. As a ligand that activates the non-canonical Wnt signaling pathways, Wnt5a triggers the production of important inflammatory chemokines and the chemotactic migration of neutrophils. In this study we aimed to characterize the role of Wnt5a production, in situ, in vivo and in vitro in response to fungal keratitis. Wnt5a expression in corneas of Aspergillus fumigatus (A. fumigatus) keratitis patients was determined by quantitative polymerase chain reaction (qRT-PCR) and immunofluorescence. In vivo and in vitro experiments were then performed in mouse models and THP-1 macrophages cell cultures infected with A. fumigatus, respectively. C57BL/6 mice were pretreated with siRNAs or neutralizing antibodies for dectin-1, LOX-1 and Wnt5a, or inhibitors of erk1/2 and JNK. Changes in Wnt5a expression were assessed by clinical evaluation, qRT-PCR, immunofluorescence, western blot and bioluminescence imaging system image acquisition. We confirmed that corneal Wnt5a expression increased with A. fumigatus keratitis in patients and a murine model. Wnt5a production was dependent on dectin-1 and LOX-1 expression with contributions by Erk1/2 and JNK pathways. Additionally, Wnt5a knockdown revealed decreased levels of MPO, lower neutrophil recruitment, and a higher fungal load in mouse models. Compared with controls, Wnt5a knockdown impaired pro-inflammatory cytokine IL-1ß production in response to A. fumigatus exposure. Wnt5a also produces dectin-1 and LOX-1 induced inflammatory signature via effective neutrophil recruitment and inflammatory cytokine production in response to A. fumigatus keratitis. These findings demonstrate that Wnt5a is a critical component of the antifungal immune response.
Assuntos
Aspergilose/imunologia , Aspergillus fumigatus/imunologia , Resistência à Doença/imunologia , Infecções Oculares Fúngicas/imunologia , Ceratite/imunologia , Proteína Wnt-5a/fisiologia , Animais , Modelos Animais de Doenças , Feminino , Humanos , Interleucina-1beta/imunologia , Lectinas Tipo C/imunologia , Sistema de Sinalização das MAP Quinases , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores Depuradores Classe E/imunologia , Células THP-1 , Via de Sinalização Wnt , Proteína Wnt-5a/genética , Proteína Wnt-5a/imunologiaRESUMO
Bacterial pathogens are associated with severe infections (e.g., sepsis) and exacerbation of debilitating conditions such as chronic obstructive pulmonary disease (COPD). The interactions of bacterial pathogens with macrophages, a key component of innate immunity and host defense, are not clearly understood and continue to be intensively studied. Having previously demonstrated a role of Wnt5A signaling in phagocytosis, we proceeded to decipher the connection of Wnt5A signaling with infection by pathogenic bacteria, namely Pseudomonas aeruginosa (PA) and Streptococcus pneumoniae (SP), which are related with the progression of COPD and sepsis. We found that during the initial hours of infection with PA and SP, there is decrease in the steady state levels of the Wnt5A protein in macrophages. Suppression of Wnt5A signaling, moreover, impairs macrophage clearance of the bacterial infection both in vitro and in vivo. Activation of Wnt5A signaling, on the other hand, enhances clearance of the infection. Macrophage-mediated containment of bacterial infection in our study is dependant on Wnt5A-induced Rac1/Disheveled activation and cytochalasin D inhibitable actin assembly, which is associated with ULK1 kinase activity and LC3BII accumulation. Our experimental findings are consistent with Wnt5A signaling-dependent induction of autophagic killing (xenophagy) of PA and SP, as further substantiated by transmission electron microscopy. Overall, our study unveils the prevalence of a Wnt5A-Rac1-Disheveled-mediated actin-associated autophagy circuit as an important component of innate immunity in host macrophages that may turn out crucial for restricting infection by leading bacterial pathogens.
Assuntos
Proteínas Desgrenhadas/imunologia , Interações Hospedeiro-Patógeno/imunologia , Neuropeptídeos/imunologia , Infecções Pneumocócicas/imunologia , Infecções por Pseudomonas/imunologia , Proteína Wnt-5a/imunologia , Proteínas rac1 de Ligação ao GTP/imunologia , Animais , Linhagem Celular , Macrófagos/imunologia , Camundongos Endogâmicos BALB C , Peritonite/imunologia , Pseudomonas aeruginosa , Infecções Respiratórias/imunologia , Streptococcus pneumoniaeRESUMO
With regard to post-surgical pain, the incidence of chronic post-thoracotomy pain (CPTP) is second only to that caused by amputation and the underlying mechanism remains elusive. The emerging role of Wnts has been confirmed in the pathogenesis of neuropathic and inflammatory pain, both of which are known components of CPTP. We investigated whether Wnt3a and Wnt5a were involved in the development of CPTP, concerning their regulation of inflammatory responses in a previously established rat model. We observed up regulated protein levels of Wnt3a, Wnt5a, ß-catenin, and TLR4, along with activated astrocytes and pro-inflammatory cytokines, in both dorsal root ganglia and the spinal cord dorsal horn. Furthermore, intrathecal inhibition of Wnt5a but not Wnts relieved mechanical hyperalgesia, down regulated expression of TLR4, and inactivated astrocytes and pro-inflammatory cytokines. These results suggest Wnt5a, but not Wnts, contributes to the development of CPTP, possibly by regulating the inflammatory response.
Assuntos
Dor Crônica/imunologia , Dor Crônica/prevenção & controle , Dor Pós-Operatória/imunologia , Dor Pós-Operatória/prevenção & controle , Toracotomia/efeitos adversos , Proteína Wnt-5a/imunologia , Animais , Dor Crônica/etiologia , Fatores Imunológicos/imunologia , Masculino , Ratos , Ratos Sprague-Dawley , Proteínas Wnt/imunologiaRESUMO
WNT5A is a secreted, noncanonical WNT signaling protein that has been reported to promote progression of several types of cancer, including oral squamous cell carcinoma. Many WNT5A antibodies are available commercially for immunohistochemistry (IHC) and western blot analysis. Validation of the primary antibodies, however, is often neglected. We characterized antibodies for detecting WNT5A by IHC and western blot analysis. We evaluated one polyclonal and three monoclonal commercially available WNT5A antibodies. After optimization of the IHC assay, all four antibodies showed cytoplasmic WNT5A expression in tissue samples; in contrast, only one antibody detected WNT5A in western blots. A pre-absorption test with recombinant WNT5A showed that AF645 and 3A4 antibodies specifically detected WNT5A in different assays. We suggest that the monoclonal 3A4 antibody is the most appropriate for use with IHC, while the polyclonal AF645 antibody is the best for western blot analysis.
Assuntos
Anticorpos/imunologia , Proteína Wnt-5a/imunologia , Biomarcadores Tumorais , Western Blotting , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Reprodutibilidade dos Testes , Coloração e Rotulagem , Proteína Wnt-5a/químicaRESUMO
Breast cancer is the most common malignancy in women. Altered expression of MUC18, a cell surface receptor, and its interaction with Wnt-5a as its ligand, affects the motility and invasiveness of breast cancer cells. In this study, we explored the Wnt-5a binding site and designed an antigenic epitope on the MUC18 receptor using in silico methods. A specific single-chain variable fragment (scFv) was isolated against the epitope by several panning processes. The binding ability of the scFv to the related epitope was evaluated in ELISA and flow cytometry. The inhibitory effects of the selected scFv on MUC18 positive cell line, MDA-MB231, was assessed by migration and invasion assays. The results demonstrated isolation of specific scFv with frequency of 40 % which showed significant binding with the epitope in both ELISA and fluorescence-activated cell sorting (FACS) analyses. The antibody inhibited the migration (76 %) and invasion (67 %) of MUC18 positive cell line. The results suggest the specific anti-MUC18 scFv as an effective antibody for breast cancer immunotherapy.
Assuntos
Neoplasias da Mama/imunologia , Anticorpos de Cadeia Única/uso terapêutico , Proteína Wnt-5a/imunologia , Sítios de Ligação , Neoplasias da Mama/genética , Neoplasias da Mama/terapia , Antígeno CD146/genética , Antígeno CD146/imunologia , Linhagem Celular Tumoral , Movimento Celular/imunologia , Simulação por Computador , Epitopos/genética , Epitopos/imunologia , Feminino , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Invasividade Neoplásica/imunologia , Ligação Proteica , Anticorpos de Cadeia Única/imunologia , Proteína Wnt-5a/genéticaRESUMO
Regulatory T cells (Treg) attenuate dendritic cell (DC) maturation and stimulatory function. Current knowledge on the functional impact of semimature DC is limited to CD4+ T cell proliferation and cytokine production. Little is known about the molecular basis underpinning the functional effects of Treg-treated DC (Treg-DC). We present novel evidence that Treg-DC skewed CD4+ naive T cell polarization toward a regulatory phenotype and impaired CD8+ T cell allo-reactive responses, including their ability to induce target tissue damage in a unique in vitro human graft-versus-host disease skin explant model. Microarray analysis clustered Treg-DC as a discrete population from mature-DC and immature-DC, with 51 and 93 genes that were significantly over- or underexpressed, respectively, compared with mature-DC. Quantitative real-time PCR analysis revealed an intermediate expression level of CD38, CD83, CD80 and CD86 mRNA in Treg-DC, lower than mature-DC, higher than immature-DC. We also observed an attenuation of NF-κB pathway, an upstream regulator of the aforementioned genes, concomitant with reduced expression of two NF-κB-signaling related genes RELB and NFκBIZ, in the Treg-DC, together with an increased expression of Wnt5a, a negative regulator of DC differentiation. We further confirmed that the Treg-DC-mediated skewed CD4+ naive T cell polarization resulted from decreased IL-12 secretion by Treg-DC, which may be post-transcriptionally modulated by decreased expression of microRNA-155 in Treg-DC. To our knowledge, this is the first study demonstrating a transcriptional modulation of DC function by human Treg, partially via attenuation of the NF-κB signaling pathway and upregulation of Wnt5a, suggesting Treg may interfere with DC reprogramming during maturation, thereby modulating DC function.
Assuntos
Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Linfócitos T Reguladores/imunologia , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/citologia , Citometria de Fluxo , Regulação da Expressão Gênica/imunologia , Humanos , Ativação Linfocitária/imunologia , NF-kappa B/imunologia , NF-kappa B/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/imunologia , Transcrição Gênica , Proteína Wnt-5a/imunologia , Proteína Wnt-5a/metabolismoRESUMO
BACKGROUND: Airway remodeling is a detrimental and refractory process showing age-dependent clinical manifestations that are mechanistically undefined. The leukotriene (LT) and wingless/integrase (Wnt) pathways have been implicated in remodeling, but age-specific expression profiles and common regulators remained elusive. OBJECTIVE: We sought to study the activation of the LT and Wnt pathways during early- or late-onset allergic airway inflammation and to address regulatory mechanisms and clinical relevance in normal human bronchial epithelial cells (NHBEs) and nasal polyp tissues. METHODS: Mice were sensitized with house dust mite (HDM) allergens from days 3, 15, or 60 after birth. Remodeling factors in murine bronchoalveolar lavage fluid, lung tissue, or human nasal polyp tissue were analyzed by means of Western blotting, immunoassays, or histology. Regulatory mechanisms were studied in cytokine/HDM-stimulated NHBEs and macrophages. RESULTS: Bronchoalveolar lavage fluid LT levels were increased in neonatal and adult but reduced in juvenile HDM-sensitized mice. Lungs of neonatally sensitized mice showed increased 5-lipoxygenase levels, whereas adult mice expressed more group 10 secretory phospholipase A2, Wnt5a, and transglutaminase 2 (Tgm2). Older mice showed colocalization of Wnt5a and LT enzymes in the epithelium, a pattern also observed in human nasal polyps. IL-4 promoted epithelial Wnt5a secretion, which upregulated macrophage Tgm2 expression, and Tgm2 inhibition in turn reduced LT release. Tgm2, group 10 secretory phospholipase A2, and LT enzymes in NHBEs and nasal polyps were refractory to corticosteroids. CONCLUSION: Our findings reveal age differences in LT and Wnt pathways during airway inflammation and identify a steroid-resistant cascade of Wnt5a, Tgm2, and LTs, which might represent a therapeutic target for airway inflammation and remodeling.