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1.
Nature ; 578(7796): 605-609, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32051584

RESUMO

The activation of adenosine monophosphate-activated protein kinase (AMPK) in skeletal muscle coordinates systemic metabolic responses to exercise1. Autophagy-a lysosomal degradation pathway that maintains cellular homeostasis2-is upregulated during exercise, and a core autophagy protein, beclin 1, is required for AMPK activation in skeletal muscle3. Here we describe a role for the innate immune-sensing molecule Toll-like receptor 9 (TLR9)4, and its interaction with beclin 1, in exercise-induced activation of AMPK in skeletal muscle. Mice that lack TLR9 are deficient in both exercise-induced activation of AMPK and plasma membrane localization of the GLUT4 glucose transporter in skeletal muscle, but are not deficient in autophagy. TLR9 binds beclin 1, and this interaction is increased by energy stress (glucose starvation and endurance exercise) and decreased by a BCL2 mutation3,5 that blocks the disruption of BCL2-beclin 1 binding. TLR9 regulates the assembly of the endolysosomal phosphatidylinositol 3-kinase complex (PI3KC3-C2)-which contains beclin 1 and UVRAG-in skeletal muscle during exercise, and knockout of beclin 1 or UVRAG inhibits the cellular AMPK activation induced by glucose starvation. Moreover, TLR9 functions in a muscle-autonomous fashion in ex vivo contraction-induced AMPK activation, glucose uptake and beclin 1-UVRAG complex assembly. These findings reveal a heretofore undescribed role for a Toll-like receptor in skeletal-muscle AMPK activation and glucose metabolism during exercise, as well as unexpected crosstalk between this innate immune sensor and autophagy proteins.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteína Beclina-1/metabolismo , Músculo Esquelético/metabolismo , Condicionamento Físico Animal/fisiologia , Receptor Toll-Like 9/metabolismo , Animais , Autofagia , Ativação Enzimática , Exercício , Glucose/metabolismo , Humanos , Masculino , Camundongos , Modelos Animais , Músculo Esquelético/enzimologia , Fosfatidilinositol 3-Quinase/metabolismo , Receptor Toll-Like 9/deficiência , Receptor Toll-Like 9/genética , Proteínas Supressoras de Tumor/metabolismo
2.
Cell Prolif ; 53(1): e12722, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31737959

RESUMO

OBJECTIVES: The mechanisms underlying the effects of Toll-like receptor 9 (TLR9) and autophagy on rheumatoid arthritis (RA)-aggravated periodontitis are unclear. We aimed to explore a novel target, cathepsin K (Ctsk)-mediated TLR9-related autophagy, during the progress of periodontitis with RA. MATERIALS AND METHODS: DBA/J1 mouse model of periodontitis with RA was created by local colonization of Porphyromonas gingivalis (Pg) and injection of collagen. The expression of Ctsk was inhibited by adeno-associated virus (AAV). Micro-CT, immunohistochemistry (IHC), Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expression of TLR9-related autophagy in periodontitis with RA. Small interfering RNA (siRNA) and CpG oligodeoxynucleotides (CpG ODN) were applied in macrophages. Western blot, immunofluorescence (IF) and qRT-PCR were used to verify the in vivo results. RESULTS: RA can promote periodontitis bone destruction in the lesion area, while inhibiting Ctsk could effectively alleviate this effect. The infiltration of macrophages, TLR9, autophagy proteins (TFEB and LC3) and inflammatory cytokines increased in the periodontitis-with-RA group and was reduced by the inhibition of Ctsk in the periodontal region. Macrophage stimulation confirmed the in vivo results. With the activation of TLR9 by CpG ODN, inhibition of Ctsk could suppress both TLR9 downstream signalling proteins and autophagy-related proteins. CONCLUSIONS: This study advanced a novel role for Ctsk in TLR9 and autophagy to explain the interaction between periodontitis and RA.


Assuntos
Artrite Reumatoide/tratamento farmacológico , Catepsina K/antagonistas & inibidores , Regulação para Baixo/efeitos dos fármacos , Oligodesoxirribonucleotídeos/farmacologia , Periodontite/tratamento farmacológico , Receptor Toll-Like 9/imunologia , Animais , Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/imunologia , Catepsina K/genética , Catepsina K/imunologia , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos DBA , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/imunologia , Oligodesoxirribonucleotídeos/genética , Periodontite/genética , Periodontite/imunologia , Periodontite/patologia , Receptor Toll-Like 9/genética
3.
Immunology ; 158(2): 61-62, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31515802

RESUMO

Immunologists are sometimes guilty of describing the innate immune response as 'non-specific'. What we really mean is that the pattern recognition receptors of innate immune cells are not able to recombine and mutate to bind the spectacular range of molecular patterns that can be recognised by B and T cells. So, while it may be accurate to describe the innate immune response as less specific than adaptive immunity, even this belies the emerging complexity of the receptors and receptor complexes that control inflammatory responses. This complexity is necessary to recognise danger, and therefore successfully initiate proportionate inflammatory responses to cellular damage or against potential pathogens.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Imunidade Inata , Glicoproteínas de Membrana/imunologia , Receptores de Complemento/imunologia , Linfócitos T/imunologia , Receptor Toll-Like 9/imunologia , Animais , Células Apresentadoras de Antígenos/microbiologia , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/microbiologia , Ilhas de CpG , Regulação da Expressão Gênica , Humanos , Glicoproteínas de Membrana/genética , Receptores de Complemento/genética , Linfócitos T/microbiologia , Receptor Toll-Like 9/genética
4.
Int J Mol Sci ; 20(18)2019 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-31546763

RESUMO

Systemic lupus erythematosus (SLE) is a chronic, multifactorial autoimmune disease that predominantly affects young females. Dysregulation of different immune cell populations leads to self-tolerance breakdown and subsequent multiple organ damage as the disease develops. Plasmacytoid dendritic cells (pDCs) are potent producers of type I interferon (IFN), while myeloid dendritic cells (mDCs) are more specialized in antigen presentations. We have previously reported that bone-marrow (BM)-derived pDCs from the murine lupus model New Zealand black/white F1 (BWF1) possess abnormalities. Therefore, this study continues to investigate what aberrant properties peripheral pDCs and mDCs possess in BWF1 and how they mediate SLE progression, by comparing their properties in pre-symptomatic and symptomatic mice. Results showed that CD11chiCD11b+ myeloid DCs expanded during the disease state with down-regulation of co-stimulatory molecules and major histocompatibility complex class II molecules (MHC II), but their capacity to stimulate T cells was not hampered. During the disease state, this subset of mDCs displayed heightened toll-like receptors 7 and 9 (TLR 7/9) responses with increased interleukin 10 (IL-10) and C-X-C motif chemokine ligand 13 (CXCL13) expressions. Moreover, the expressions of myeloid differentiation primary response 88 (Myd88) and nuclear factor kappa B subunit 1 (Nfkb1) were higher in CD11chiCD11b+ DCs at the disease stage, leading to higher nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 phosphorylation activity. In summary, we reported aberrant phenotypic properties with enhanced TLR7/9 responses of CD11chiCD11b+ DCs in SLE mediated by aberrant NF-κB signaling pathway. Our findings add additional and novel information to our current understanding of the role of DCs in lupus immunopathogenesis. Lastly, molecular candidates in the NF-κB pathway should be exploited for developing therapeutic targets for SLE.


Assuntos
Antígenos CD11/imunologia , Antígeno CD11b/imunologia , Quimiocina CXCL13/imunologia , Células Dendríticas/imunologia , Interleucina-10/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Glicoproteínas de Membrana/imunologia , Subunidade p50 de NF-kappa B/imunologia , Receptor 7 Toll-Like/imunologia , Receptor Toll-Like 9/imunologia , Animais , Antígenos CD11/genética , Antígeno CD11b/genética , Quimiocina CXCL13/genética , Células Dendríticas/patologia , Modelos Animais de Doenças , Feminino , Interleucina-10/genética , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/patologia , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Células Mieloides/imunologia , Células Mieloides/patologia , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Subunidade p50 de NF-kappa B/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptor 7 Toll-Like/genética , Receptor Toll-Like 9/genética
5.
Asian Pac J Cancer Prev ; 20(8): 2299-2302, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31450898

RESUMO

Toll-like receptor 9 (TLR9) is a cellular DNA receptor of the innate immune system which plays a pivotal role in inflammatory response. Recently, changing expression levels of TLR9 has been observed in a wide range of cancer cells; however, there is little information about colorectal polyps. Herein, we assessed the mRNA expression of TLR9 in different colorectal polyp types compared to normal group in order to investigate its expression level during CRC initiation. Fifty-four biopsy samples from colorectal polyp patients and from 20 healthy subjects were collected. The mucosal mRNA expression level of TLR9 gene was identified by real time PCR. Fold change of gene expression was evaluated by 2-ΔΔct method. There was a significant relationship between the lower expression of TLR9 gene in the polyp cases compared to normal individuals (P value = 0.0005), Also, decreased TLR9 mRNA expression was obtained in adenomas in contrast to hyperplastic and normal groups (P value = 0.0008). Based on the current results, we hypothesized that aberrant surface expression of TLR9 on tumor cells may promote the growth and invasion of colorectal polyps. Further, TLR9 modulation may have an important impact on the development of novel therapeutic strategies.


Assuntos
Adenocarcinoma/patologia , Biomarcadores Tumorais/genética , Pólipos do Colo/patologia , Neoplasias Colorretais/patologia , Hiperplasia/patologia , RNA Mensageiro/genética , Receptor Toll-Like 9/genética , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Biomarcadores Tumorais/metabolismo , Estudos de Casos e Controles , Pólipos do Colo/genética , Pólipos do Colo/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Feminino , Seguimentos , Humanos , Hiperplasia/genética , Hiperplasia/metabolismo , Masculino , Estadiamento de Neoplasias , RNA Mensageiro/metabolismo , Receptor Toll-Like 9/metabolismo
6.
EBioMedicine ; 45: 328-340, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31300344

RESUMO

BACKGROUND: TLR9 agonists are being developed as immunotherapy against malignancies and infections. TLR9 is primarily expressed in B cells and plasmacytoid dendritic cells (pDCs). TLR9 signalling may be critically important for B cell activity in lymph nodes but little is known about the in vivo impact of TLR9 agonism on human lymph node B cells. As a pre-defined sub-study within our clinical trial investigating TLR9 agonist MGN1703 (lefitolimod) treatment in the context of developing HIV cure strategies (NCT02443935), we assessed TLR9 agonist-mediated effects in lymph nodes. METHODS: Participants received MGN1703 for 24 weeks concurrent with antiretroviral therapy. Seven participants completed the sub-study including lymph node resection at baseline and after 24 weeks of treatment. A variety of tissue-based immunologic and virologic parameters were assessed. FINDINGS: MGN1703 dosing increased B cell differentiation; activated pDCs, NK cells, and T cells; and induced a robust interferon response in lymph nodes. Expression of Activation-Induced cytidine Deaminase, an essential regulator of B cell diversification and somatic hypermutation, was highly elevated. During MGN1703 treatment IgG production increased and antibody glycosylation patterns were changed. INTERPRETATION: Our data present novel evidence that the TLR9 agonist MGN1703 modulates human lymph node B cells in vivo. These findings warrant further considerations in the development of TLR9 agonists as immunotherapy against cancers and infectious diseases. FUND: This work was supported by Aarhus University Research Foundation, the Danish Council for Independent Research and the NovoNordisk Foundation. Mologen AG provided study drug free of charge.


Assuntos
Diferenciação Celular/efeitos dos fármacos , DNA/administração & dosagem , Infecções por HIV/tratamento farmacológico , Receptor Toll-Like 9/genética , Adulto , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Células Dendríticas/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Glicosilação/efeitos dos fármacos , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Humanos , Interferon-alfa/genética , Linfonodos , Ativação Linfocitária/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Receptor Toll-Like 9/agonistas
7.
Immunology ; 158(2): 85-93, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31335975

RESUMO

Bacterial DNA contains CpG oligonucleotide (ODN) motifs to trigger innate immune responses through the endosomal receptor Toll-like receptor 9 (TLR9). One of the cell surface receptors to capture and deliver microbial DNA to intracellular TLR9 is the C-type lectin molecule DEC-205 through its N-terminal C-type lectin-like domain (CTLD). CD93 is a cell surface protein and member of the lectin group XIV with a CTLD. We hypothesized that CD93 could interact with CpG motifs, and possibly serve as a novel receptor to deliver bacterial DNA to endosomal TLR9. Using ELISA and tryptophan fluorescence binding studies we observed that the soluble histidine-tagged CD93-CTLD was specifically binding to CpG ODN and bacterial DNA. Moreover, we found that CpG ODN could bind to CD93-expressing IMR32 neuroblastoma cells and induced more robust interleukin-6 secretion when compared with mock-transfected IMR32 control cells. Our data argue for a possible contribution of CD93 to control cell responsiveness to bacterial DNA in a manner reminiscent of DEC-205. We postulate that CD93 may act as a receptor at plasma membrane for DNA or CpG ODN and to grant delivery to endosomal TLR9.


Assuntos
DNA Bacteriano/imunologia , Regulação da Expressão Gênica/imunologia , Glicoproteínas de Membrana/imunologia , Oligodesoxirribonucleotídeos/imunologia , Receptores de Complemento/imunologia , Receptor Toll-Like 9/imunologia , Antígenos CD/genética , Antígenos CD/imunologia , Transporte Biológico/genética , Transporte Biológico/imunologia , Linhagem Celular Tumoral , Clonagem Molecular , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Endossomos/imunologia , Endossomos/metabolismo , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Inflamação , Interleucina-6/genética , Interleucina-6/imunologia , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/imunologia , Modelos Biológicos , Neurônios/imunologia , Neurônios/metabolismo , Neurônios/patologia , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Ligação Proteica , Domínios Proteicos , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Receptores de Complemento/genética , Receptores de Complemento/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Receptor Toll-Like 9/genética
8.
Immunol Invest ; 48(8): 860-874, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31185757

RESUMO

Toll-like receptors (TLRs) are inevitable elements for immunity development and antibody production. TLRs are in close interaction with Bruton's tyrosine kinase which has been found mutated and malfunctioned in the prototype antibody deficiency disease named X-linked agammaglobulinemia (XLA). TLRs' ability was evaluated to induce transcription of TLR-negative regulators, including suppressor of cytokine signaling 1 (SOCS1), interleukin-1 receptor-associated kinase 3 (IRAK-M), tumor necrosis factor alpha-induced protein 3 (TNFAIP3, A20), and Ring finger protein 216 (RNF216), and Tumor necrosis factor-α (TNF-α) and Interferon-α (IFN-α) production via Lipopolysaccharides (LPS) and CpG-A oligodeoxynucleotides (CpG-A ODN). Measured by TaqMan real-time polymerase chain reaction (PCR), meaningfully increased transcripts of SOCS1 and RNF216 were found in XLA peripheral blood mononuclear cells (PBMCs). Also, TLR inductions of XLA have led to similar downregulations in the regulator's transcription which was different from that in healthy donors. Cytokine measurement by enzyme-linked immunosorbent assay (ELISA) revealed a significant lower TNF-α production both before and after LPS. By selected molecules in this study, TLRs' potential defectiveness range expands TLRs expression, downstream signaling, and cytokine production. The results show new potential elements that could play a part in TLRs defect and pathogenesis of agammaglobulinemia as well.


Assuntos
Agamaglobulinemia/metabolismo , Citocinas/metabolismo , Leucócitos Mononucleares/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Receptores Toll-Like/metabolismo , Adolescente , Agamaglobulinemia/sangue , Agamaglobulinemia/genética , Células Cultivadas , Criança , Humanos , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Masculino , Oligodesoxirribonucleotídeos/farmacologia , Proteína 1 Supressora da Sinalização de Citocina/genética , Proteína 1 Supressora da Sinalização de Citocina/metabolismo , Receptor 4 Toll-Like/genética , Receptor Toll-Like 9/genética , Receptores Toll-Like/genética , Transcrição Genética/efeitos dos fármacos , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Adulto Jovem
9.
Genet Test Mol Biomarkers ; 23(6): 373-379, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31066581

RESUMO

Objective: Hip osteoarthritis (HOA) is one of the most common types of osteoarthritis and affects nearly 10% of men and 18% of women who are >60 years of age worldwide. It has been demonstrated to be a genetic disease with a 50% heritability risk. Recently, the TLR-9 gene has been associated with knee OA in both Turkish and Chinese populations, but the relationship between the TLR-9 gene and HOA has not been evaluated. In this study, we aimed to evaluate the relationship between the common genetic variants in the TLR-9 gene and the predisposition of Han Chinese individuals to HOA. Methods: A total of 730 HOA patients and 1220 healthy controls were recruited in a hospital-based case-control study. Six common single nucleotide polymorphisms (SNPs) of the TLR-9 gene were selected for genotyping, and genetic association analyses were performed using both single-marker and haplotype-based methods. Results: The SNP rs187084 was found to be significantly associated with the risk of HOA after a Bonferroni correction (adjusted allelic p-values with age, gender, and body mass index [BMI] = 0.0008). The results indicated that the A allele of rs187084 is a risk allele for HOA and is likely to be a predisposing factor leading to an increased risk of HOA (adjusted odds ratio with age, gender, and BMI = 1.26, 95% confidence interval = 1.10-1.43). The results of the haplotype analyses confirmed a similar pattern to the SNP analyses. Conclusions: Our study provides strong evidence that variations in the TLR-9 gene are closely linked with genetic susceptibility to HOA in the Han Chinese population. This finding furthers the role of TLR-9 in the development and occurrence of OA in general.


Assuntos
Osteoartrite do Quadril/genética , Receptor Toll-Like 9/genética , Idoso , Idoso de 80 Anos ou mais , Alelos , Grupo com Ancestrais do Continente Asiático/genética , Estudos de Casos e Controles , China , Grupos Étnicos/genética , Feminino , Frequência do Gene/genética , Predisposição Genética para Doença/genética , Variação Genética/genética , Genótipo , Haplótipos/genética , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , Fatores de Risco
10.
Tuberculosis (Edinb) ; 116S: S131-S137, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31085128

RESUMO

Mycobacterium tuberculosis (Mtb) is a facultative intracellular pathogen that infects macrophages where it avoids elimination by interfering with host defense mechanisms, including phago-lysosome fusion. Endosomal Toll-like receptors (TLRs) generate Type I Interferons (IFNs), which are associated with active tuberculosis (TB). We aimed to explore if DNA from different Mtb lineages lead to differences in the inflammatory response of human monocytic/macrophage cells. THP-1 cells which express two inducible reporter constructs for interferons (IFNs) as well as for NF-κB, were stimulated via endosomal delivery of Mtb DNA as a nanocomplex with PEI. DNA from different Mtb phylogenetic lineages elicited differential inflammatory responses in human macrophages. An initial relatively weak IRF-mediated response to DNA from HN878 and H37Rv increased if the cells were pre-treated with Vitamin D (Vit D) for 72 h. RNAseq of THP-1 under different transformation conditions showed that pre-treatment with Vit D upregulated several TLR9 variants, as well as genes involved in inflammatory immune response to infection, immune cell activation, Type I IFN regulation, and regulation of inflammation. Vit D appears to be important in increasing low IRF responses to DNA from certain lineages of Mtb. Variations in the IRF-mediated response to DNA derived from different Mtb genotypes are potentially important in the pathogenesis of tuberculosis since Type I IFN responses are associated with active disease. The role of Vit D in these responses could also translate into future therapeutic approaches.


Assuntos
Calcitriol/farmacologia , DNA Bacteriano/metabolismo , Macrófagos/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/metabolismo , DNA Bacteriano/genética , Interações Hospedeiro-Patógeno , Humanos , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Interferon gama/farmacologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Mycobacterium tuberculosis/genética , NF-kappa B/genética , NF-kappa B/metabolismo , Células THP-1 , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismo
11.
Microb Pathog ; 132: 166-177, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31054870

RESUMO

The macrophage innate immune response is outlined through recognition of the components of Mycobacterium tuberculosis. DNA of M. tuberculosis (MtbDNA) is recognized by macrophages, but the implications of this recognition are poorly characterized. Stimulation of murine macrophages with MtbDNA induces autophagy, a process that promotes elimination of intracellular pathogens. However, it remains unknown whether this or other phenomena also occur in human cells. In this work, we studied the innate response profiles of human macrophages after stimulation with DNA from virulent M. tuberculosis H37Rv. Human monocyte-derived macrophages were polarized into M1 and M2 phenotypes and stimulated with MtbDNA. The plasma membrane markers of the phenotype, production of TNF-α, and induction of autophagy were evaluated. Our results indicate that MtbDNA induced phenotypical changes, the significant production of TNF-α, and autophagy confirmed by the augmented expression of immunity related GTPase M (IRGM) and autophagy related ATG16L1 genes in M1 macrophages, whereas M2 macrophages exhibited limited responses. In addition, MtbDNA activation was TLR-9-dependent. Although TLR-9 expression was similar between M1 and M2 macrophages, only M1 macrophages were fully responsive to MtbDNA. In conclusion, MtbDNA recognition enhanced the antimicrobial mechanisms of M1 macrophages.


Assuntos
Autofagia , DNA Bacteriano/isolamento & purificação , Macrófagos/metabolismo , Mycobacterium tuberculosis/genética , Fator de Necrose Tumoral alfa/metabolismo , DNA Bacteriano/genética , Humanos , Imunidade Inata , Monócitos , Mycobacterium tuberculosis/metabolismo , Fenótipo , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismo
12.
Mediators Inflamm ; 2019: 6217548, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30944547

RESUMO

Liver X receptors (LXRs) have emerged as important regulators of inflammatory gene expression. Previously, we had reported that an LXRα gene promoter polymorphism (-1830 T > C) is associated with systemic lupus erythematosus (SLE). Therefore, we assessed cytokine expression in relation to LXRα polymorphism in monocyte-derived macrophages from patients with SLE. Macrophages were obtained after 72 hours of culture of human monocytes supplemented with phorbol 12-myristate 13-acetate. Cells were transfected with LXRα promoter constructs. Additionally, peripheral blood mononuclear cell- (PBMC-) derived macrophages from the patients were evaluated for proinflammatory cytokines in relation to the genotypes of LXRα -1830 T > C. The expression of LXRα was increased in macrophages; levels of proinflammatory cytokines were decreased with LXRα expression. Production of proinflammatory cytokines varied depending on LXRα -1830 T > C genotype. In particular, expression of LXRα was decreased and that of proinflammatory cytokines was increased for LXRα -1830 TC genotype compared to that for TT genotype. The data were consistent in PBMC-derived macrophages from patients with SLE. Increased proinflammatory cytokines is related to TLR7 and TLR9 expression. These data suggest that the expression levels of LXRα, according to LXRα -1830 T > C genotype, may contribute to the inflammatory response by induction of inflammatory cytokines in SLE.


Assuntos
Leucócitos Mononucleares/metabolismo , Receptores X do Fígado/genética , Receptores X do Fígado/metabolismo , Macrófagos/metabolismo , Benzoatos/farmacologia , Benzilaminas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular Tumoral , Citocinas/metabolismo , Genótipo , Humanos , Hidrocarbonetos Fluorados/farmacologia , Immunoblotting , Leucócitos Mononucleares/efeitos dos fármacos , Receptores X do Fígado/agonistas , Macrófagos/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Sulfonamidas/farmacologia , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/metabolismo , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismo
13.
J Cutan Med Surg ; 23(4): 370-379, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31010295

RESUMO

BACKGROUND: 5-aminolevulinic acid photodynamic therapy (PDT) for genital warts is effective, safe, and can prevent recurrence. It is believed that PDT can induce immune responses, but the mechanism is not completely understood. OBJECTIVES: The objectives of this article are to confirm the effect of PDT for genital warts on local immunity and to investigate the recruitment and significance of immune cells in tissues. METHODS: Local immune changes in T lymphocytes (CD3+, CD4+, CD8+), plasmacytoid dendritic cells (pDCs) (CD123+), and myeloid dendritic cells (CD1a+) after PDT in patients were evaluated by immunohistochemistry staining. Changes in mRNA levels of IFN-γ, IFN-α, IFN-ß, interferon-stimulated gene 15 kDa (ISG-15), Mx2, Toll-like receptor 9 (TLR9), and interferon regulatory factor 7 (IRF7) were analyzed by real-time quantitative polymerase chain reaction. RESULTS: At 4 hours after PDT, CD4+ increased, accompanied by increased levels of mRNA expression of IFN-γ, but CD4+ and mRNA expression levels of IFN-γ were decreased at 24 hours after PDT. CD123+ pDCs showed an increasing trend. CD1a+ LCs in the epidermis gradually decreased, and DCs in the epidermis gradually increased. CD3+ infiltrated and migrated to the superficial dermis, but CD8+ did not change significantly after PDT. The mRNA expression levels of IFN-α, IFN-ß, ISG-15, Mx2, TLR9, and IRF7 showed an increasing trend after PDT. As compared with the patients without significantly increased IFN-α and IFN-ß after PDT sessions, patients with significant increases needed fewer sessions of PDT for remission. CONCLUSIONS: PDT for genital warts can activate T lymphocyte-mediated, DC-related, and pDC-related immunity. The clinical efficacy of PDT for genital warts may be related to the increased levels of IFN-α and IFN-ß after treatment.


Assuntos
Ácido Aminolevulínico/farmacologia , Condiloma Acuminado/tratamento farmacológico , Epiderme/imunologia , Células de Langerhans/imunologia , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Adulto , Ácido Aminolevulínico/uso terapêutico , Antígenos CD1/metabolismo , Complexo CD3/metabolismo , Contagem de Linfócito CD4 , Linfócitos T CD8-Positivos/imunologia , Citocinas/genética , Epiderme/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Fator Regulador 7 de Interferon/genética , Interferon-alfa/genética , Interferon beta/genética , Interferon gama/genética , Subunidade alfa de Receptor de Interleucina-3/metabolismo , Células de Langerhans/efeitos dos fármacos , Células de Langerhans/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas de Resistência a Myxovirus/genética , Fármacos Fotossensibilizantes/uso terapêutico , RNA Mensageiro/metabolismo , Receptor Toll-Like 9/genética , Ubiquitinas/genética , Adulto Jovem
14.
J Immunol ; 202(9): 2529-2534, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30936294

RESUMO

Systemic lupus erythematosus severity correlates with elevated serum levels of type I IFNs, cytokines produced in large quantities by plasmacytoid dendritic cells (pDC) in response to engagement of TLR7 and TLR9 with endocytosed nucleic acids. B cell adaptor for PI3K (BCAP) promoted many aspects of TLR7-driven lupus-like disease, including Isg15 and Ifit1 expression in blood and an immature pDC phenotype associated with higher IFN production. BCAP-/- mice produced significantly less serum IFN-α than wild-type mice after injection of TLR9 agonist, and BCAP promoted TLR7 and TLR9-induced IFN-α production specifically in pDC. TLR-induced IFN-α production in pDC requires DOCK2-mediated activation of Rac1 leading to activation of IKKα, a mechanism we show was dependent on BCAP. BCAP-/- pDC had decreased actin polymerization and Rac1 activation and reduced IKKα phosphorylation upon TLR9 stimulation. We show a novel role for BCAP in promoting TLR-induced IFN-α production in pDC and in systemic lupus erythematosus pathogenesis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Células Dendríticas/imunologia , Interferon-alfa/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Glicoproteínas de Membrana/imunologia , Plasmócitos/imunologia , Receptor 7 Toll-Like/imunologia , Receptor Toll-Like 9/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Citocinas/genética , Citocinas/imunologia , Células Dendríticas/patologia , Feminino , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/imunologia , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/imunologia , Interferon-alfa/genética , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/patologia , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Neuropeptídeos/genética , Neuropeptídeos/imunologia , Plasmócitos/patologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Receptor 7 Toll-Like/genética , Receptor Toll-Like 9/genética , Ubiquitinas/genética , Ubiquitinas/imunologia , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/imunologia
15.
PLoS Negl Trop Dis ; 13(2): e0007146, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30802247

RESUMO

Leishmania (L.) amazonensis is one of the etiological agents of cutaneous leishmaniasis (CL) in Brazil. Currently, there is no vaccine approved for human use against leishmaniasis, although several vaccine preparations are in experimental stages. One of them is Leishvacin, or LaAg, a first-generation vaccine composed of total L. amazonensis antigens that has consistently shown an increase of mouse resistance against CL when administered intranasally (i.n.). Since Toll-like receptor 9 (TLR9) is highly expressed in the nasal mucosa and LaAg is composed of TLR9-binding DNA CpG motifs, in this study we proposed to investigate the role of TLR9 in both L. amazonensis infection and in LaAg vaccine efficacy in C57BL/6 (WT) mice and TLR9-/- mice. First, we evaluated, the infection of macrophages by L. amazonensis in vitro, showing no significant difference between macrophages from WT and TLR9-/- mice in terms of both infection percentage and total number of intracellular amastigotes, as well as NO production. In addition, neutrophils from WT and TLR9-/- mice had similar capacity to produce neutrophil extracellular traps (NETs) in response to L. amazonensis. L. amazonensis did not activate dendritic cells from WT and TLR9-/- mice, analysed by MHCII and CD86 expression. However, in vivo, TLR9-/- mice were slightly more susceptible to L. amazonensis infection than WT mice, presenting a larger lesion and an increased parasite load at the peak of infection and in the chronic phase. The increased TLR9-/- mice susceptibility was accompanied by an increased IgG and IgG1 production; a decrease of IFN-γ in infected tissue, but not IL-4 and IL-10; and a decreased number of IFN-γ producing CD8+ T cells, but not CD4+ T cells in the lesion-draining lymph nodes. Also, TLR9-/- mice could not control parasite growth following i.n. LaAg vaccination unlike the WT mice. This protection failure was associated with a reduction of the hypersensitivity response induced by immunization. The TLR9-/- vaccinated mice failed to respond to antigen stimulation and to produce IFN-γ by lymph node cells. Together, these results suggest that TLR9 contributes to C57BL/6 mouse resistance against L. amazonensis, and that the TLR9-binding LaAg comprising CpG motifs may be important for intranasal vaccine efficacy against CL.


Assuntos
Leishmania mexicana/imunologia , Leishmaniose Cutânea/imunologia , Vacinas Protozoárias/imunologia , Receptor Toll-Like 9/imunologia , Administração Intranasal , Animais , Antígenos de Protozoários/imunologia , Ilhas de CpG , Células Dendríticas/imunologia , Células Dendríticas/parasitologia , Armadilhas Extracelulares , Interferon gama/imunologia , Macrófagos/imunologia , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologia , Neutrófilos/parasitologia , Óxido Nítrico/biossíntese , Carga Parasitária , Receptor Toll-Like 9/genética , Vacinação
16.
Kidney Int ; 95(4): 859-879, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30777286

RESUMO

Intestinal Paneth cells play a critical role in ischemic acute kidney injury (AKI) by releasing interleukin 17A (IL-17A). Because Toll-like receptor 9 (TLR9) activation degranulates Paneth cells and necrotic tubular epithelial cells release several damage associated molecular patterns that target TLR9, we tested the hypothesis that intestinal TLR9 deficiency would protect against ischemic AKI and associated remote intestinal and hepatic dysfunction by decreasing Paneth cell degranulation. We generated mice lacking TLR9 in intestinal epithelia (TLR9fl/fl Villin Cre mice) and compared them to wild type (TLR9fl/fl) mice following right nephrectomy and left ischemia/reperfusion. To our surprise, mice lacking intestinal TLR9 had exacerbated kidney, liver, and small intestine injury after ischemia/reperfusion compared to wild type mice, characterized by increased kidney and intestinal inflammation, apoptosis, and necrosis as well as increased hepatic inflammation and apoptosis. Mice lacking intestinal TLR9 had larger Paneth cell granule size, pronounced intestinal macrophage infiltration, and higher intestinal crypt IL-17A expression. Administration of IL-17A neutralizing antibody prevented the exacerbation of ischemic AKI in mice lacking intestinal TLR9. These studies suggest that intestinal TLR9 activation protects against ischemic AKI and associated remote multi-organ dysfunction syndrome by regulating Paneth cell IL-17A synthesis.


Assuntos
Lesão Renal Aguda/imunologia , Interleucina-17/metabolismo , Insuficiência de Múltiplos Órgãos/imunologia , Celulas de Paneth/patologia , Receptor Toll-Like 9/metabolismo , Lesão Renal Aguda/patologia , Animais , Apoptose , Modelos Animais de Doenças , Progressão da Doença , Humanos , Hiperplasia/imunologia , Hiperplasia/patologia , Interleucina-17/antagonistas & inibidores , Interleucina-17/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Intestinos/imunologia , Intestinos/patologia , Rim/imunologia , Rim/patologia , Fígado/imunologia , Fígado/patologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Transgênicos , Insuficiência de Múltiplos Órgãos/patologia , Celulas de Paneth/imunologia , Celulas de Paneth/metabolismo , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/patologia , Receptor Toll-Like 9/genética
17.
Fish Shellfish Immunol ; 87: 879-885, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30794932

RESUMO

Toll-like receptor 9 (TLR9) is activated by bacterial DNA and induces the production of inflammatory cytokines. In this study, the darkbarbel catfish Pelteobagrus vachellii TLR9 cDNA was cloned and sequenced. The daily expression pattern of TLR9 mRNA was investigated in various tissues. Furthermore, its expression was analyzed following exposure to the pathogen Aeromonas hydrophila. The 4249 bp cDNA includes a 3201 bp open reading frame (ORF) encoding 1067 amino acids. The predicted amino acid sequence comprises a leucine-rich domain (LRD), a toll/interleukin-1 receptor (TIR), and a transmembrane domain. P. vachellii TLR9 showed 42-87% amino acid sequence identity with TLR9 sequences of Ictalurus punctatus, Rhincodon typus, and Miichthys miiuy. The P. vachellii TLR9 mRNA was highly expressed in intestines, head kidney, and spleen in an apparently healthy fish. Following pathogen challenge, TLR9 expression increased significantly (P < 0.05) and peaked at 48 h post-exposure in the liver, at 24 in the head kidney, and at 12 h in the spleen. In addition, the pattern of TLR9 expression over a 24-h period showed a circadian rhythm in the head kidney, spleen, and intestine, with the acrophase at 20:34, 18:45, and 3:50, respectively. This result provided the basis for further study of the rhythm of innate immunity against bacteria in catfish.


Assuntos
Peixes-Gato/genética , Peixes-Gato/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/imunologia , Aeromonas hydrophila/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Filogenia , Alinhamento de Sequência/veterinária , Receptor Toll-Like 9/química
18.
PLoS Pathog ; 15(1): e1007560, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30682165

RESUMO

Bacterial lung infections, particularly with methicillin-resistant Staphylococcus aureus (MRSA), increase mortality following influenza infection, but the mechanisms remain unclear. Here we show that expression of TLR9, a microbial DNA sensor, is increased in murine lung macrophages, dendritic cells, CD8+ T cells and epithelial cells post-influenza infection. TLR9-/- mice did not show differences in handling influenza nor MRSA infection alone. However, TLR9-/- mice have improved survival and bacterial clearance in the lung post-influenza and MRSA dual infection, with no difference in viral load during dual infection. We demonstrate that TLR9 is upregulated on macrophages even when they are not themselves infected, suggesting that TLR9 upregulation is related to soluble mediators. We rule out a role for elevations in interferon-γ (IFNγ) in mediating the beneficial MRSA clearance in TLR9-/- mice. While macrophages from WT and TLR9-/- mice show similar phagocytosis and bacterial killing to MRSA alone, following influenza infection, there is a marked upregulation of scavenger receptor A and MRSA phagocytosis as well as inducible nitric oxide synthase (Inos) and improved bacterial killing that is specific to TLR9-deficient cells. Bone marrow transplant chimera experiments and in vitro experiments using TLR9 antagonists suggest TLR9 expression on non-hematopoietic cells, rather than the macrophages themselves, is important for regulating myeloid cell function. Interestingly, improved bacterial clearance post-dual infection was restricted to MRSA, as there was no difference in the clearance of Streptococcus pneumoniae. Taken together these data show a surprising inhibitory role for TLR9 signaling in mediating clearance of MRSA that manifests following influenza infection.


Assuntos
Staphylococcus aureus Resistente à Meticilina/imunologia , Staphylococcus aureus Resistente à Meticilina/metabolismo , Receptor Toll-Like 9/metabolismo , Animais , Humanos , Influenza Humana/imunologia , Pulmão/imunologia , Macrófagos , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/patologia , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Infecções por Orthomyxoviridae/imunologia , Fagocitose , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/imunologia , Receptor Toll-Like 9/genética
19.
BMC Infect Dis ; 19(1): 56, 2019 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-30651082

RESUMO

BACKGROUND: The mechanism behind HIV mediated immune activation remains debated, although the role of virus replication in this process is increasingly evident. Toll like Receptor 9 (TLR9) has been implicated in HIV mediated immune activation via sensing of viral CpG DNA. Polymorphisms in the TLR9 gene and promoter region including TLR9 1635A/G and 1486C/T have been found to be associated with multiple infectious diseases and cancers. METHODS: In the current study, we looked at the correlation of TLR9 polymorphisms 1635A/G and 1486C/T with key hallmarks of HIV disease in a cohort of 50 HIV infected patients. We analyzed CD4 counts, T cell immune activation characterized by upregulation of CD38 and HLA-DR and upregulation of plasma biomarkers of inflammation like LPS, sCD14, IL-6 and IP10 in the HIV patient cohort and compared it to healthy controls. RESULTS: We found that TLR9 1635AA genotype was associated with lower CD4 counts and significantly higher immune activation in both CD4+ and CD8+ T cells. Analysis of HIV associated plasma biomarkers including LPS, sCD14, IL-6 and IP10 revealed a strong correlation between IP10 and immune activation. Interestingly, IP10 levels were also found to be higher in HIV patients with the 1635AA genotype. Furthermore, the TLR9 1486C/T polymorphism that is in linkage disequilibrium with 1635A/G was weakly associated with lower CD4 counts, higher CD8 immune activation and higher IP10 levels. CONCLUSIONS: As TLR9 stimulation is known to induce IP10 production by dendritic cells, our findings provide new insights into HIV mediated immune activation and CD4 loss. TLR9 stimulation by viral CpG DNA may be important to HIV immunopathogenesis and the TLR9 polymorphisms 1635A/G and 1486C/T may be associated with disease progression.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , Polimorfismo Genético , Receptores de Citocinas/sangue , Receptor Toll-Like 9/genética , Adulto , Linfócitos T CD8-Positivos/imunologia , Estudos de Coortes , Estudos Transversais , DNA Viral , Feminino , Infecções por HIV/genética , Infecções por HIV/virologia , Antígenos HLA-DR/genética , Antígenos HLA-DR/imunologia , Humanos , Interleucina-6/sangue , Ativação Linfocitária/imunologia , Masculino , Receptores de Citocinas/genética , Replicação Viral
20.
Immunol Invest ; 48(1): 79-95, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30239236

RESUMO

PURPOSE: Toll like receptor (TLR) engagement is primarily a function of the innate immune cells. The purpose of the study was to assess direct uptake of ODN 2216 in T helper cells and effects on cell proliferation and cytokine expression. METHODS: We isolated CD4+ CD25- T helper cells by magnetic sorting and studied the uptake of ODN 2216 using flow cytometry and confocal microscopy. We then studied the effect of ODN 2216 engagement on cell proliferation and cytokine expression using flow cytometry and gene expression of TLR9 signaling genes using real time RT-PCR. RESULTS: We made a chance observation that purified T helper cells from healthy individuals consistently bind to the TLR9 ligand ODN 2216. In PBMCs, on the other hand, 98% of monocytes preferentially bound to ODN 2216 FITC, indicating that they competed with the lymphocytes. We confirmed intracellular localization of ODN 2216 FITC as well as intracellular expression of TLR9 in Thelper cells. Furthermore, ODN 2216 FITC was also co-localized with the lysosomal membrane associated protein 1. The uptake of TLR9 ligand culminated in cellular proliferation, up-regulation of cytokines and increased mRNA expression of TLR9 and IRF7 in T helper cells, in the absence of antigen presenting cells. ODN 2216 uptake was inhibited by promethazine as well as by TLR9 antagonist. CONCLUSIONS: Our results show a direct engagement of TLR9 ligand in T helper cells and suggest involvement of TLR9 signalling in CD4+T cells, which may envisage novel targets for TLR inhibitors.


Assuntos
Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Oligodesoxirribonucleotídeos/genética , Linfócitos T Auxiliares-Indutores/fisiologia , Receptor Toll-Like 9/metabolismo , Proliferação de Células , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Citometria de Fluxo , Humanos , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/metabolismo , Ativação Linfocitária , Microscopia Confocal , Ligação Proteica , Transporte Proteico , Transdução de Sinais/genética , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/genética
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