RESUMO
OBJECTIVES: This study aims to investigate the effects of type 17 immune response on the proliferation of oral epithelial cells in periodontitis. DESIGN: A time-dependent ligature induced periodontitis mouse model was utilized to explore gingival hyperplasia and the infiltration of interleukin 17A (IL-17A) positive cells. Immunohistochemistry and flow cytometry were employed to determine the localization and expression of IL-17A in the ligature induced periodontitis model. A pre-existing single-cell RNA sequencing dataset, comparing individuals affected by periodontitis with healthy counterparts, was reanalyzed to evaluate IL-17A expression levels. We examined proliferation markers, including proliferating cell nuclear antigen (PCNA), signal transducer and activator of transcription (STAT3), Yes-associated protein (YAP), and c-JUN, in the gingival and tongue epithelium of the periodontitis model. An anti-IL-17A agent was administered daily to observe proliferative changes in the oral mucosa within the periodontitis model. Cell number quantification, immunofluorescence, and western blot analyses were performed to assess the proliferative responses of human normal oral keratinocytes to IL-17A treatment in vitro. RESULTS: The ligature induced periodontitis model exhibited a marked infiltration of IL-17A-positive cells, alongside significant increase in thickness of the gingival and tongue epithelium. IL-17A triggers the proliferation of human normal oral keratinocytes, accompanied by upregulation of PCNA, STAT3, YAP, and c-JUN. The administration of an anti-IL-17A agent attenuated the proliferation in oral mucosa. CONCLUSIONS: These findings indicate that type 17 immune response, in response to periodontitis, facilitates the proliferation of oral epithelial cells, thus highlighting its crucial role in maintaining the oral epithelial barrier.
Assuntos
Imunidade Adaptativa , Proliferação de Células , Células Epiteliais , Interleucina-17 , Periodontite , Periodontite/imunologia , Células Epiteliais/citologia , Células Epiteliais/imunologia , Proliferação de Células/genética , Animais , Camundongos , Modelos Animais de Doenças , Interleucina-17/genética , Interleucina-17/imunologia , Transporte Proteico/imunologia , Queratinócitos/citologia , Queratinócitos/imunologia , Humanos , Linhagem Celular , Perda do Osso Alveolar/imunologia , Imunidade Adaptativa/imunologiaRESUMO
Histone H2A is a nuclear molecule tightly associated in the form of the nucleosome. Our previous studies have demonstrated the antibacterial property of piscine H2A variants against gram-negative bacteria Edwardsiella piscicida and Gram-positive bacteria Streptococcus agalactiae. In this study, we show the function and mechanism of piscine H2A in the negative regulation of RLR signaling pathway and host innate immune response against spring viremia of carp virus (SVCV) infection. SVCV infection significantly inhibits the expression of histone H2A during an early stage of infection, but induces the expression of histone H2A during the late stage of infection such as at 48 and 72 hpi. Under normal physiological conditions, histone H2A is nuclear-localized. However, SVCV infection promotes the migration of histone H2A from the nucleus to the cytoplasm. The in vivo studies revealed that histone H2A overexpression led to the increased expression of SVCV gene and decreased survival rate. The overexpression of histone H2A also significantly impaired the expression levels of those genes involved in RLR antiviral signaling pathway. Furthermore, histone H2A targeted TBK1 and IRF3 to promote their protein degradation via the lysosomal pathway and impair the formation of TBK1-IRF3 functional complex. Importantly, histone H2A completely abolished TBK1-mediated antiviral activity and enormously impaired the protein expression of IRF3, especially nuclear IRF3. Further analysis demonstrated that the inhibition of histone H2A nuclear/cytoplasmic trafficking could relieve the protein degradation of TBK1 and IRF3, and blocked the negative regulation of histone H2A on the SVCV infection. Collectively, our results suggest that histone H2A nuclear/cytoplasmic trafficking is essential for negative regulation of RLR signaling pathway and antiviral immune response in response to SVCV infection.
Assuntos
Histonas/imunologia , Imunidade Inata/imunologia , Fator Regulador 3 de Interferon/imunologia , Lisossomos/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Rhabdoviridae/imunologia , Proteínas de Peixe-Zebra/imunologia , Peixe-Zebra/imunologia , Animais , Linhagem Celular , Núcleo Celular/imunologia , Núcleo Celular/metabolismo , Citoplasma/imunologia , Citoplasma/metabolismo , Regulação da Expressão Gênica/imunologia , Histonas/genética , Histonas/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Larva/imunologia , Larva/metabolismo , Larva/virologia , Lisossomos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico/imunologia , Proteólise , Rhabdoviridae/fisiologia , Peixe-Zebra/metabolismo , Peixe-Zebra/virologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Mucosal Associated Invariant T (MAIT) cells can sense intracellular infection by a broad array of pathogens. These cells are activated upon encountering microbial antigen(s) displayed by MR1 on the surface of an infected cell. Human MR1 undergoes alternative splicing. The full-length isoform, MR1A, can activate MAIT cells, while the function of the isoforms, MR1B and MR1C, are incompletely understood. In this report, we sought to characterize the expression and function of these splice variants. Using a transcriptomic analysis in conjunction with qPCR, we find that that MR1A and MR1B transcripts are widely expressed. However only MR1A can present mycobacterial antigen to MAIT cells. Coexpression of MR1B with MR1A decreases MAIT cell activation following bacterial infection. Additionally, expression of MR1B prior to MR1A lowers total MR1A abundance, suggesting competition between MR1A and MR1B for either ligands or chaperones required for folding and/or trafficking. Finally, we evaluated CD4/CD8 double positive thymocytes expressing surface MR1. Here, we find that relative expression of MR1A/MR1B transcript is associated with the prevalence of MR1 + CD4/CD8 cells in the thymus. Our results suggest alternative splicing of MR1 represents a means of regulating MAIT activation in response to microbial ligand(s).
Assuntos
Processamento Alternativo/genética , Processamento Alternativo/imunologia , Apresentação de Antígeno/genética , Apresentação de Antígeno/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Menor/genética , Células T Invariantes Associadas à Mucosa/imunologia , Células A549 , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Ligantes , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Antígenos de Histocompatibilidade Menor/imunologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Transporte Proteico/genética , Transporte Proteico/imunologia , Timócitos/imunologia , Transcriptoma/genética , Transcriptoma/imunologiaRESUMO
MHC class I molecules on the cellular surface display peptides that either derive from endogenous proteins (self or viral), or from endocytosis of molecules, dying cells or pathogens. The conventional antigen-processing pathway for MHC class I presentation depends on proteasome-mediated degradation of the protein followed by transporter associated with antigen-processing (TAP)-mediated transport of the generated peptides into the endoplasmic reticulum (ER). Here, peptides are loaded onto MHC I molecules before transportation to the cell surface. However, several alternative mechanisms have emerged. These include TAP-independent mechanisms, the vacuolar pathway and involvement of autophagy. Autophagy is a cell intrinsic recycling system. It also functions as a defence mechanism that removes pathogens and damaged endocytic compartments from the cytosol. Therefore, it appears likely that autophagy would intersect with the MHC class I presentation pathway to alarm CD8+ T cells of an ongoing intracellular infection. However, the importance of autophagy as a source of antigen for presentation on MHC I molecules remains to be defined. Here, original research papers which suggest involvement of autophagy in MHC I antigen presentation are reviewed. The antigens are from herpesvirus, cytomegalovirus and chlamydia. The studies point towards autophagy as important in MHC class I presentation of endogenous proteins during conditions of immune evasion. Because autophagy is a regulated process which is induced upon activation of, for example, pattern recognition receptors (PRRs), it will be crucial to use relevant stimulatory conditions together with primary cells when aiming to confirm the importance of autophagy in MHC class I antigen presentation in future studies.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos/imunologia , Autofagia/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Animais , Antígenos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Complexo de Endopeptidases do Proteassoma/imunologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Transporte Proteico/imunologiaRESUMO
Trafficking of toll-like receptor 3 (TLR3) from the endoplasmic reticulum (ER) to endolysosomes and its subsequent proteolytic cleavage are required for it to sense viral double-stranded RNA (dsRNA) and trigger antiviral response, yet the underlying mechanisms remain enigmatic. We show that the E3 ubiquitin ligase TRIM3 is mainly located in the Golgi apparatus and transported to the early endosomes upon stimulation with the dsRNA analog poly(I:C). TRIM3 mediates K63-linked polyubiquitination of TLR3 at K831, which is enhanced following poly(I:C) stimulation. The polyubiquitinated TLR3 is recognized and sorted by the ESCRT (endosomal sorting complex required for transport) complexes to endolysosomes. Deficiency of TRIM3 impairs TLR3 trafficking from the Golgi apparatus to endosomes and its subsequent activation. Trim3-/- cells and mice express lower levels of antiviral genes and show lower levels of inflammatory response following poly(I:C) but not lipopolysaccharide (LPS) stimulation. These findings suggest that TRIM3-mediated polyubiquitination of TLR3 represents a feedback-positive regulatory mechanism for TLR3-mediated innate immune and inflammatory responses.
Assuntos
Proteínas de Transporte/imunologia , Complexos Endossomais de Distribuição Requeridos para Transporte/imunologia , Imunidade Inata/imunologia , Receptor 3 Toll-Like/imunologia , Ubiquitinação/imunologia , Animais , Antivirais/imunologia , Células HEK293 , Humanos , Lisossomos/imunologia , Camundongos , Transporte Proteico/imunologia , RNA Viral/imunologia , Transdução de Sinais/imunologiaRESUMO
The identification of novel peptide hormones by functional screening is challenging because posttranslational processing is frequently required to generate biologically active hormones from inactive precursors. We developed an approach for functional screening of novel potential hormones by expressing them in endocrine host cells competent for posttranslational processing. Candidate preprohormones were selected by bioinformatics analysis, and stable endocrine host cell lines were engineered to express the preprohormones. The production of mature hormones was demonstrated by including the preprohormones insulin and glucagon, which require the regulated secretory pathway for production of the active forms. As proof of concept, we screened a set of G-protein-coupled receptors (GPCRs) and identified protein FAM237A as a specific activator of GPR83, a GPCR implicated in central nervous system and regulatory T-cell function. We identified the active form of FAM237A as a C-terminally cleaved, amidated 9 kDa secreted protein. The related protein FAM237B, which is 64% homologous to FAM237A, demonstrated similar posttranslational modification and activation of GPR83, albeit with reduced potency. These results demonstrate that our approach is capable of identifying and characterizing novel hormones that require processing for activity.
Assuntos
Hormônios Peptídicos/isolamento & purificação , Biblioteca de Peptídeos , Transporte Proteico/genética , Receptores Acoplados a Proteínas G/genética , Humanos , Ligantes , Hormônios Peptídicos/genética , Hormônios Peptídicos/imunologia , Ligação Proteica/genética , Transporte Proteico/imunologia , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/imunologia , Transdução de Sinais/genética , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
In humans, maternal IgGs are transferred to the fetus from the second trimester of pregnancy onwards. The transplacental delivery of maternal IgG is mediated by its binding to the neonatal Fc receptor (FcRn) after endocytosis by the syncytiotrophoblast. IgGs present in the maternal milk are also transferred to the newborn through the digestive epithelium upon binding to the FcRn. Importantly, the binding of IgGs to the FcRn is also responsible for the recycling of circulating IgGs that confers them with a long half-life. Maternally delivered IgG provides passive immunity to the newborn, for instance by conferring protective anti-flu or anti-pertussis toxin IgGs. It may, however, lead to the development of autoimmune manifestations when pathological autoantibodies from the mother cross the placenta and reach the circulation of the fetus. In recent years, strategies that exploit the transplacental delivery of antigen/IgG complexes or of Fc-fused proteins have been validated in mouse models of human diseases to impose antigen-specific tolerance, particularly in the case of Fc-fused factor VIII (FVIII) domains in hemophilia A mice or pre-pro-insulin (PPI) in the case of preclinical models of type 1 diabetes (T1D). The present review summarizes the mechanisms underlying the FcRn-mediated transcytosis of IgGs, the physiopathological relevance of this phenomenon, and the repercussion for drug delivery and shaping of the immune system during its ontogeny.
Assuntos
Antígenos/imunologia , Tolerância Imunológica , Troca Materno-Fetal/imunologia , Animais , Autoanticorpos/metabolismo , Feminino , Feto/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Sistema Imunitário/embriologia , Sistema Imunitário/metabolismo , Imunoglobulina G/metabolismo , Camundongos , Placenta/imunologia , Gravidez , Transporte Proteico/imunologia , Receptores Fc/metabolismo , Transcitose/imunologiaRESUMO
Effector CD4+ T cells can be classified by the cytokines they secrete, with T helper 1 (Th1) cells generating interferon (IFN)γ and Th17 cells secreting interleukin (IL)-17. Both Th1 and Th17 cells are strongly implicated in the initiation and chronicity of autoimmune diseases such as multiple sclerosis. The endoplasmic reticulum (ER) has been implicated as a potentially crucial site in regulating CD4+ T cell function. Secretory and transmembrane proteins are shuttled into the ER via the Sec61 translocon, where they undergo appropriate folding; misfolded proteins are retro-translocated from the ER in a p97-dependent manner. Here, we provide evidence that both processes are crucial to the secretion of inflammatory cytokines from effector CD4+ T cells. The pan-ER inhibitor eeeyarestatin-1 (ESI), which interferes with both Sec61 translocation and p97 retro-translocation, inhibited secretion of interferon (IFN)γ, interleukin (IL)-2 and tumor necrosis factor (TNF)α from Th1 cells in a dose-dependent manner. Selective inhibition of Sec61 by Apratoxin A (ApraA) revealed that ER translocation is crucial for Th1 cytokine secretion, while inhibition of p97 by NMS-873 also inhibited Th1 function, albeit to a lesser degree. By contrast, none of ESI, ApraA or NMS-873 could significantly reduce IL-17 secretion from Th17 cells. ApraA, but not NMS-873, reduced phosphorylation of Stat1 in Th1 cells, indicating the involvement of ER translocation in Th1 differentiation pathways. ApraA had modest effects on activation of the Th17 transcription factor Stat3, while NMS-873 had no effect. Interestingly, NMS-873 was able to reduce disease severity in CD4+ T cell-driven experimental autoimmune encephalomyelitis (EAE). Together, our data indicate that CD4+ T cell function, and Th1 cell function in particular, is dependent on protein translocation and dislocation across the ER.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Retículo Endoplasmático/imunologia , Inflamação/imunologia , Transporte Proteico/imunologia , Animais , Diferenciação Celular/imunologia , Sistema Nervoso Central/imunologia , Citocinas/imunologia , Encefalomielite Autoimune Experimental/imunologia , Interferon gama/imunologia , Interleucina-17/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/imunologia , Células Th1/imunologia , Células Th17/imunologiaRESUMO
All protective and pathogenic immune and inflammatory responses rely heavily on leukocyte migration and localization. Chemokines are secreted chemoattractants that orchestrate the positioning and migration of leukocytes through concentration gradients. The mechanisms underlying chemokine gradient establishment and control include physical as well as biological phenomena. Mathematical models offer the potential to both understand this complexity and suggest interventions to modulate immune function. Constructing models that have powerful predictive capability relies on experimental data to estimate model parameters accurately, but even with a reductionist approach most experiments include multiple cell types, competing interdependent processes and considerable uncertainty. Therefore, we propose the use of reduced modeling and experimental frameworks in complement, to minimize the number of parameters to be estimated. We present a Bayesian optimization framework that accounts for advection and diffusion of a chemokine surrogate and the chemokine CCL19, transport processes that are known to contribute to the establishment of spatio-temporal chemokine gradients. Three examples are provided that demonstrate the estimation of the governing parameters as well as the underlying uncertainty. This study demonstrates how a synergistic approach between experimental and computational modeling benefits from the Bayesian approach to provide a robust analysis of chemokine transport. It provides a building block for a larger research effort to gain holistic insight and generate novel and testable hypotheses in chemokine biology and leukocyte trafficking.
Assuntos
Movimento Celular/imunologia , Quimiocina CCL19/imunologia , Simulação por Computador , Leucócitos/imunologia , Modelos Imunológicos , Teorema de Bayes , Humanos , Leucócitos/citologia , Transporte Proteico/imunologiaRESUMO
Alzheimer's disease (AD) is a primary neurological disease with no effective cure. A hallmark of AD is the presence of intracellular tangles and extracellular plaques derived from the aberrant aggregation of tau- and beta-amyloid (Aß). Aß presents in the brain as well as in cerebrospinal fluid and the circulation, and Aß toxicity has been attributed to amyloidosis and inflammation, among other causes. In this study, the effects of the plasma protein corona have been investigated with regard to the blood cell association and cytokine secretion of oligomeric (Aßo) and fibrillar Aß1-42(Aßf), two major forms of the peptide aggregates. Aßo displayed little change in membrane association in whole blood or washed blood (i.e., cells in the absence of plasma proteins) at 37 °C, while Aßf showed a clear preference for binding with all cell types sans plasma proteins. Immune cells exposed to Aßo, but not to Aßf, resulted in significant expression of cytokines IL-6 and TNF measured in real-time by a localized surface plasmon resonance sensor. These observations indicate greater immune cell association and cytokine stimulation of Aßo than Aßf and shed new light on the contrasting toxicities of Aßo and Aßf resulting from their differential capacities in acquiring a plasma protein corona. These results further implicate a close connection between Aß amyloidosis and immunopathology in AD.
Assuntos
Doença de Alzheimer/imunologia , Amiloide/imunologia , Fragmentos de Peptídeos/química , Placa Amiloide/imunologia , Coroa de Proteína/química , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Amiloide/química , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/imunologia , Encéfalo/imunologia , Encéfalo/patologia , Citocinas/biossíntese , Citocinas/química , Humanos , Microglia/imunologia , Neurônios/imunologia , Neurônios/patologia , Fragmentos de Peptídeos/imunologia , Placa Amiloide/tratamento farmacológico , Placa Amiloide/patologia , Coroa de Proteína/imunologia , Transporte Proteico/imunologiaRESUMO
STING plays central roles in the innate immune response to pathogens that contain DNA. Sensing cytoplasmic DNA by cyclic GMP-AMP synthase produces cyclic GMP-AMP, which binds to and activates STING and induces STING translocation from the endoplasmic reticulum to the perinuclear microsome. However, this trafficking process has not been fully elucidated yet. In this study, we identified YIPF5 as a positive regulator of STING trafficking. YIPF5 is essential for DNA virus- or intracellular DNA-triggered production of type I IFNs. Consistently, knockdown of YIPF5 impairs cellular antiviral responses to DNA virus. Mechanistically, YIPF5 interacts with both STING and components of COPII, facilitating STING recruitment to COPII in the presence of cytoplasmic dsDNA. Furthermore, knockdown of components of COPII inhibits DNA virus-triggered production of type I IFNs, suggesting that COPII is involved in innate immune responses to DNA viruses. Collectively, our findings demonstrate that YIPF5 positively regulates STING-mediated innate immune responses by recruiting STING to COPII-coated vesicles and facilitating STING trafficking from the endoplasmic reticulum to Golgi, providing important insights into the molecular mechanisms of intracellular DNA-stimulated STING trafficking and activation.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/imunologia , Vírus de DNA/imunologia , Imunidade Inata/imunologia , Proteínas de Membrana/imunologia , Transporte Proteico/imunologia , Transdução de Sinais/imunologia , Proteínas de Transporte Vesicular/imunologia , Animais , DNA Viral/imunologia , Retículo Endoplasmático/imunologia , Complexo de Golgi/imunologia , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Recent studies have demonstrated that CD4+ T cells can efficiently reject MHC-II-negative tumors. This requires indirect presentation of tumor-associated antigens on surrounding antigen-presenting cells. We hypothesized that intercellular transfer of proteins is not the sole consequence of cell death-mediated protein release, but depends on heat-shock cognate protein 70 (HSC70) and its KFERQ-like binding motif on substrate proteins. Using human Y chromosome antigen DBY, we showed that mutation of one of its 2 putative binding motifs markedly diminished T cell activation after indirect presentation and reduced protein-protein interaction with HSC70. Intercellular antigen transfer was shown to be independent of cell-cell contact, but relied on engulfment within secreted microvesicles. In vivo, alterations of the homologous KFERQ-like motif in murine DBY hampered tumor rejection, T cell activation, and migration into the tumor and substantially impaired survival. Collectively, we show that intercellular antigen transfer of DBY is tightly regulated via binding to HSC70 and that this mechanism influences recognition and rejection of MHC-II-negative tumors in vivo.
Assuntos
RNA Helicases DEAD-box/imunologia , Proteínas de Choque Térmico HSC70/imunologia , Antígenos de Histocompatibilidade Menor/imunologia , Proteínas de Neoplasias/imunologia , Neoplasias/imunologia , Vesículas Secretórias/imunologia , Motivos de Aminoácidos , Animais , RNA Helicases DEAD-box/genética , Proteínas de Choque Térmico HSC70/genética , Células HeLa , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Ativação Linfocitária , Células MCF-7 , Camundongos , Antígenos de Histocompatibilidade Menor/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Transporte Proteico/genética , Transporte Proteico/imunologia , Vesículas Secretórias/genética , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
Astrocytes constantly adapt their ramified morphology in order to support brain cell assemblies. Such plasticity is partly mediated by ion and water fluxes, which rely on the water channel aquaporin-4 (AQP4). The mechanism by which this channel locally contributes to process dynamics has remained elusive. Using a combination of single-molecule and calcium imaging approaches, we here investigated in hippocampal astrocytes the dynamic distribution of the AQP4 isoforms M1 and M23. Surface AQP4-M1 formed small aggregates that contrast with the large AQP4-M23 clusters that are enriched near glutamatergic synapses. Strikingly, stabilizing surface AQP4-M23 tuned the motility of astrocyte processes and favors glutamate synapse activity. Furthermore, human autoantibodies directed against AQP4 from neuromyelitis optica (NMO) patients impaired AQP4-M23 dynamic distribution and, consequently, astrocyte process and synaptic activity. Collectively, it emerges that the membrane dynamics of AQP4 isoform regulate brain cell assemblies in health and autoimmune brain disease targeting AQP4.
Assuntos
Aquaporina 4/imunologia , Astrócitos/imunologia , Hipocampo/imunologia , Neuromielite Óptica/imunologia , Sinapses/imunologia , Animais , Astrócitos/patologia , Autoanticorpos/imunologia , Cálcio/imunologia , Hipocampo/patologia , Humanos , Neuromielite Óptica/patologia , Transporte Proteico/imunologia , Ratos , Ratos Sprague-Dawley , Sinapses/patologiaRESUMO
Lymph nodes (LNs) filter lymph to mount effective immune responses. Small soluble lymph-borne molecules from the periphery enter the draining LNs via a reticular conduit system. Intact antibodies and other larger molecules, in contrast, are physically unable to enter the conduits, and they are thought to be transported to the LNs only within migratory DCs after proteolytic degradation. Here, we discovered that lymph-borne antibodies and other large biomolecules enter within seconds into the parenchyma of the draining LN in an intact form. Mechanistically, we found that the uptake of large molecules is a receptor-independent, fluid-phase process that takes place by dynamin-dependent vesicular transcytosis through the lymphatic endothelial cells in the subcapsular sinus of the LN. Physiologically, this pathway mediates a very fast transfer of large protein antigens from the periphery to LN-resident DCs and macrophages. We show that exploitation of the transcytosis system allows enhanced whole-organ imaging and spatially controlled lymphocyte activation by s.c. administered antibodies in vivo. Transcytosis through the floor of the subcapsular sinus thus represents what we believe to be a new physiological and targetable mode of lymph filtering.
Assuntos
Anticorpos/imunologia , Células Endoteliais/imunologia , Linfonodos/imunologia , Transcitose/imunologia , Animais , Células Dendríticas/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Transporte Proteico/imunologiaRESUMO
The innate immune system plays an essential role in initial recognition of pathogen infection by producing inflammatory cytokines and type I interferons. cGAS is a cytoplasmic sensor for DNA derived from DNA viruses. cGAS binding with DNA induces the production of cGAMP, a second messenger that associates with STING in endoplasmic reticulum (ER). STING changes its cellular distribution from ER to perinuclear Golgi, where it activates the protein kinase TBK1 that catalyzes the phosphorylation of IRF3. Here we found that STING trafficking is regulated by myotubularin-related protein (MTMR) 3 and MTMR4, members of protein tyrosine phosphatases that dephosphorylate 3' position in phosphatidylinositol (PtdIns) and generate PtdIns5P from PtdIns3,5P2 and PtdIns from PtdIns3P. We established MTMR3 and MTMR4 double knockout (DKO) RAW264.7 macrophage cells and found that they exhibited increased type I interferon production after interferon-stimulatory DNA (ISD) stimulation and herpes simplex virus 1 infection concomitant with enhanced IRF3 phosphorylation. In DKO cells, STING rapidly trafficked from ER to Golgi after ISD stimulation. Notably, DKO cells exhibited enlarged cytosolic puncta positive for PtdIns3P and STING was aberrantly accumulated in this puncta. Taken together, these results suggest that MTMR3 and MTMR4 regulate the production of PtdIns3P, which plays a critical role in suppressing DNA-mediated innate immune responses via modulating STING trafficking.
Assuntos
DNA Viral/imunologia , Herpesvirus Humano 1/imunologia , Imunidade Inata , Macrófagos/imunologia , Proteínas de Membrana/imunologia , Fosfatos de Fosfatidilinositol/imunologia , Proteínas Tirosina Fosfatases não Receptoras/imunologia , Animais , DNA Viral/genética , Herpesvirus Humano 1/genética , Proteínas de Membrana/genética , Camundongos , Fosfatos de Fosfatidilinositol/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Transporte Proteico/genética , Transporte Proteico/imunologia , Proteínas Tirosina Fosfatases não Receptoras/genética , Células RAW 264.7RESUMO
Single-domain antibodies, also known as nanobodies, are small antigen-binding fragments (~15kDa) that are derived from heavy chain only antibodies present in camelids (VHH, from camels and llamas), and cartilaginous fishes (VNAR, from sharks). Nanobody V-like domains are useful alternatives to conventional antibodies due to their small size, and high solubility and stability across many applications. In addition, phage display, ribosome display, and mRNA/cDNA display methods can be used for the efficient generation and optimization of binders in vitro. The resulting nanobodies can be genetically encoded, tagged, and expressed in cells for in vivo localization and functional studies of target proteins. Collectively, these properties make nanobodies ideal for use within echinoderm embryos. This chapter describes the optimization and imaging of genetically encoded nanobodies in the sea urchin embryo. Examples of live-cell antigen tagging (LCAT) and the manipulation of green fluorescent protein (GFP) are shown. We discuss the potentially transformative applications of nanobody technology for probing membrane protein trafficking, cytoskeleton re-organization, receptor signaling events, and gene regulation during echinoderm development.
Assuntos
Biologia Molecular/métodos , Proteínas/isolamento & purificação , Ouriços-do-Mar/ultraestrutura , Anticorpos de Domínio Único/biossíntese , Animais , Camelídeos Americanos/imunologia , Camelus/imunologia , Embrião não Mamífero/ultraestrutura , Desenvolvimento Embrionário/genética , Cadeias Pesadas de Imunoglobulinas/genética , Transporte Proteico/genética , Transporte Proteico/imunologia , Proteínas/genética , Ouriços-do-Mar/crescimento & desenvolvimento , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologiaRESUMO
Mast cells (MCs) contribute to the control of local inflammatory reactions and become hyporesponsive after prolonged TLR4 activation by bacterial LPS. The molecular mechanisms involved in endotoxin tolerance (ET) induction in MCs are not fully understood. In this study, we demonstrate that the endocannabinoid 2-arachidonoylglycerol (2-AG) and its receptor, cannabinoid receptor 2 (CB2), play a role in the establishment of ET in bone marrow-derived MCs from C57BL/6J mice. We found that CB2 antagonism prevented the development of ET and that bone marrow-derived MCs produce 2-AG in a TLR4-dependent fashion. Exogenous 2-AG induced ET similarly to LPS, blocking the phosphorylation of IKK and the p65 subunit of NF-κB and inducing the synthesis of molecular markers of ET. LPS caused CB2 receptor trafficking in Rab11-, Rab7-, and Lamp2-positive vesicles, indicating recycling and degradation of the receptor. 2-AG also prevented LPS-induced TNF secretion in vivo, in a MC-dependent model of endotoxemia, demonstrating that TLR4 engagement leads to 2-AG secretion, which contributes to the negative control of MCs activation. Our study uncovers a functional role for the endocannabinoid system in the inhibition of MC-dependent innate immune responses in vivo.
Assuntos
Ácidos Araquidônicos/farmacologia , Endocanabinoides/farmacologia , Glicerídeos/farmacologia , Tolerância Imunológica/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , Mastócitos/imunologia , Receptor CB2 de Canabinoide/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Tolerância Imunológica/imunologia , Proteína 2 de Membrana Associada ao Lisossomo/genética , Proteína 2 de Membrana Associada ao Lisossomo/imunologia , Camundongos , Camundongos Knockout , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Transporte Proteico/imunologia , Receptor CB2 de Canabinoide/genética , Receptor 4 Toll-Like/genética , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/imunologia , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/imunologia , proteínas de unión al GTP Rab7RESUMO
Dendritic cells (DCs) trigger CD8â¯+â¯T cell responses after the internalization of exogenous antigens in a process called cross-presentation. Multiple intracellular transport events within the endocytic and secretory routes take place in order to accomplish this fundamental immunological process. The endomembrane system can be envisioned as a complex network of membrane domains coordinately working in the fusion of organelles, the budding of vesicles and tubules, and modifying the molecular composition of the limiting membranes. In this context of tightly regulated and dynamic endomembrane transport, small GTPases of the Rab family display a pivotal role by organizing membrane microdomains and defining specific identities to the different intracellular compartments. In this review, we synthesize and update the current knowledge about Rab22a, which has been involved in several immune functions. In this way, we analyze the intracellular localization of Rab22a and its important role in the endocytic recycling, including its relevance during MHC-I trafficking, antigen cross-presentation by DCs and the formation of T cell conjugates. We also describe how different pathogenic microorganisms hijack Rab22a functions to achieve efficient infection and intracellular survival strategies. Furthermore, we examine the oncogenic properties of Rab22a and how its expression determines the progression of many tumors. In summary, we highlight the role of Rab22a as a key effector of the intracellular trafficking that could be exploited in future therapies to modulate the immune system.
Assuntos
Proteínas rab de Ligação ao GTP/imunologia , Animais , Apresentação de Antígeno/imunologia , Apresentação Cruzada/imunologia , Células Dendríticas/imunologia , Endocitose/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Transporte Proteico/imunologiaRESUMO
Cross-presentation is thought to require transport of proteasome-generated peptides by the TAP transporters into MHC class I loading compartments for most antigens. However, a proteasome-dependent but TAP-independent pathway has also been described. Depletion of the pool of recycling cell surface MHC class I molecules available for loading with cross-presented peptides might partly or largely account for the critical role of TAP in cross-presentation of phagocytosed antigens. Here we examined a potential role of the homodimeric lysosomal TAP-like transporter in cross-presentation and in presentation of endogenous peptides by MHC class II molecules. We find that TAP-L is strongly recruited to dendritic cell phagosomes at a late stage, when internalized antigen and MHC class I molecules have been degraded or sorted away from phagosomes. Cross-presentation of a receptor-targeted antigen in vitro and of a phagocytosed antigen in vivo, as well as presentation of a cytosolic antigen by MHC class II molecules, is not affected by TAP-L deficiency. However, accumulation in vitro of a peptide optimally adapted to TAP-L selectivity in purified phagosomes is abolished by TAP-L deficiency. Unexpectedly, we find that TAP-L deficiency accelerates phagosome maturation, as reflected in increased Lamp2b recruitment and enhanced proteolytic degradation of phagocytosed antigen and in vitro transported peptides. Although additional experimentation will be required to definitely conclude on the role of TAP-L in transport of peptides presented by MHC class I and class II molecules, our data suggest that the principal role of TAP-L in dendritic cells may be related to regulation of phagosome maturation.
Assuntos
Transportadores de Cassetes de Ligação de ATP/imunologia , Apresentação de Antígeno/imunologia , Fagossomos/imunologia , Animais , Linhagem Celular Tumoral , Apresentação Cruzada/imunologia , Células Dendríticas/imunologia , Células HeLa , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Proteínas de Membrana Transportadoras/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptídeos/imunologia , Fagocitose/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Transporte Proteico/imunologia , ProteóliseRESUMO
In our previous studies, we have identified several in vivo-induced antigens and evaluated their potential as subunit vaccine candidates in a murine model, in which the recombinant protein GalT showed the most potent immunogenicity and immunoprotective efficacy against Actinobacillus pleuropneumoniae. To exploit a more efficient way of delivering GalT proteins, in this study, we employed the widely studied E. coli outer membrane vesicles (OMVs) as a platform to deliver GalT protein and performed the vaccine trial using the recombinant GalT-OMVs in the murine model. Results revealed that GalT-OMVs could elicit a highly-specific, IgG antibody titer that was comparable with the adjuvant GalT group. Significantly higher lymphocyte proliferation and cytokines secretion levels were observed in the GalT-OMVs group. 87.5% and 50% of mice were protected from a lethal dose challenge using A. pleuropneumoniae in active or passive immunization, respectively. Histopathologic and immunohistochemical analyses showed remarkably reduced pathological changes and infiltration of neutrophils in the lungs of mice immunized with GalT-OMVs after the challenge. Taken together, these findings confirm that OMVs can be used as a platform to deliver GalT protein and enhance its immunogenicity to induce both humoral and cellular immune responses in mice.