RESUMO
BACKGROUND: Invariant chain (CD74, Ii) is a multifunctional protein expressed in antigen presenting cells. It assists the ER exit of various cargos and serves as a receptor for the macrophage migration inhibitory factor. The newly translated Ii chains trimerize, a structural feature that is not readily understood in the context of its MHCII chaperoning function. Two segments of Ii, the luminal C-terminal region (TRIM) and the transmembrane domain (TM), have been shown to participate in the trimerization process but their relative importance and impact on the assembly with MHCII molecules remains debated. Here, we addressed the requirement of these domains in the trimerization of human Ii as well as in the oligomerization with MHCII molecules. We used site-directed mutagenesis to generate series of Ii and DR mutants. These were transiently transfected in HEK293T cells to test their cell surface expression and analyse their interactions by co-immunoprecipitations. RESULTS: Our results showed that the TRIM domain is not essential for Ii trimerization nor for intracellular trafficking with MHCII molecules. We also gathered evidence that in the absence of TM, TRIM allows the formation of multi-subunit complexes with HLA-DR. Similarly, in the absence of TRIM, Ii can assemble into high-order structures with MHCII molecules. CONCLUSIONS: Altogether, our data show that trimerization of Ii through either TM or TRIM sustains nonameric complex formation with MHCII molecules.
Assuntos
Antígenos de Diferenciação de Linfócitos B/metabolismo , Membrana Celular/metabolismo , Antígenos HLA-DR/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Apresentação de Antígeno , Antígenos de Diferenciação de Linfócitos B/genética , Células HEK293 , Antígeno HLA-A24/metabolismo , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Mutagênese Sítio-Dirigida , Mutação/genética , Domínios Proteicos/genética , Multimerização ProteicaRESUMO
The immune system constantly monitors the emergence of cancerous cells and eliminates them. CD8+ cytotoxic T lymphocytes (CTLs), which kill tumor cells and provide antitumor immunity, select their targets by recognizing tumor antigenic peptides presented by MHC class-I (MHC-I) molecules. Cancer cells circumvent immune surveillance using diverse strategies. A key mechanism of cancer immune evasion is downregulation of MHC-I and key proteins of the antigen processing and presentation machinery (APM). Even though impaired MHC-I expression in cancers is well-known, reversing the MHC-I defects remains the least advanced area of tumor immunology. The discoveries that NLRC5 is the key transcriptional activator of MHC-I and APM genes, and genetic lesions and epigenetic modifications of NLRC5 are the most common cause of MHC-I defects in cancers, have raised the hopes for restoring MHC-I expression. Here, we provide an overview of cancer immunity mediated by CD8+ T cells and the functions of NLRC5 in MHC-I antigen presentation pathways. We describe the impressive advances made in understanding the regulation of NLRC5 expression, the data supporting the antitumor functions of NLRC5 and a few reports that argue for a pro-tumorigenic role. Finally, we explore the possible avenues of exploiting NLRC5 for cancer immunotherapy.
Assuntos
Genes MHC Classe I , Imunoterapia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias/imunologia , Neoplasias/terapia , Transativadores/metabolismo , Animais , Antígenos de Neoplasias/metabolismo , HumanosRESUMO
The COVID-19 pandemic has rapidly spread around the world with significant morbidity and mortality in a subset of patients including the elderly. The poorer outcomes are associated with 'cytokine storm-like' immune responses, otherwise referred to as 'hyperinflammation'. While most of the infected individuals show minimal or no symptoms and recover spontaneously, a small proportion of the patients exhibit severe symptoms characterized by extreme dyspnea and low tissue oxygen levels, with extensive damage to the lungs referred to as acute respiratory distress symptom (ARDS). The consensus is that the hyperinflammatory response of the host is akin to the cytokine storm observed during sepsis and is the major cause of death. Uncertainties remain on the factors that lead to hyperinflammatory response in some but not all individuals. Hyperinflammation is a common feature in different viral infections such as dengue where existing low-titer antibodies to the virus enhances the infection in immune cells through a process called antibody-dependent enhancement or ADE. ADE has been reported following vaccination or secondary infections with other corona, Ebola and dengue virus. Detailed analysis has shown that antibodies to any viral epitope can induce ADE when present in sub-optimal titers or is of low affinity. In this review we will discuss ADE in the context of dengue and coronavirus infections including Covid-19.
Assuntos
Anticorpos Facilitadores/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/veterinária , Peritonite Infecciosa Felina/imunologia , Inflamação/patologia , Pandemias/veterinária , Pneumonia Viral/imunologia , Pneumonia Viral/veterinária , Dengue Grave/imunologia , Animais , COVID-19 , Gatos , Citocinas/metabolismoRESUMO
Four different isoforms of the human invariant chain (Ii) have been described (p33, p35, p41 and p43). These heterotrimerize in the endoplasmic reticulum (ER) before associating with MHC class II molecules (MHCIIs). However, the final stoichiometry of the Ii/MHCII complex remains debated. This is particularly interesting as both p35 and p43 include a di-arginine motif that requires masking by MHCII to allow ER egress. Here, to functionally address the requirement for stoichiometric interactions, we used a recombinant DR heterodimer bearing its own cytoplasmic di-lysine ER-retention motif (DRKKAA). When coexpressed with p33 and a control myc-tagged DR (DRmyc), DRKKAA was retained in the ER but had little impact on surface expression of DRmyc. However, when coexpressed with p35, DRKKAA restricted the surface expression of DRmyc, indicating that Ii trimers can be loaded with more than one MHCII. Similar results were obtained using HLA-DQ instead of DRmyc, showing that a single trimeric Ii scaffold can include distinct MHCII isotypes. Altogether, these results demonstrate that the subunit stoichiometry of oligomeric Ii/MHCII complexes is influenced by p35.
Assuntos
Antígenos de Diferenciação de Linfócitos B/imunologia , Retículo Endoplasmático/imunologia , Regulação da Expressão Gênica/imunologia , Antígenos HLA-DQ/imunologia , Antígenos HLA-DR/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Motivos de Aminoácidos , Antígenos de Diferenciação de Linfócitos B/genética , Retículo Endoplasmático/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Antígenos HLA-DQ/genética , Antígenos HLA-DR/genética , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Isoformas de Proteínas/imunologiaRESUMO
Background: Inflammatory cytokines play key pathogenic roles in liver fibrosis. IL-15 is a proinflammatory cytokine produced by myeloid cells. IL-15 promotes pathogenesis of several chronic inflammatory diseases. However, increased liver fibrosis has been reported in mice lacking IL-15 receptor alpha chain (IL-15Rα), suggesting an anti-fibrogenic role for IL-15. As myeloid cells are key players in liver fibrosis and IL-15 signaling can occur independently of IL-15Rα, we investigated the requirement of IL-15 and IL-15Rα in liver fibrosis. Methods: We induced liver fibrosis in Il15-/- , Il15ra-/- and wildtype C57BL/6 mice by the administration of carbon tetrachloride (CCl4). Liver fibrosis was evaluated by Sirius red and Mason's trichrome staining and α-smooth muscle acting immunostaining of myofibroblasts. Gene expression of collagens, matrix modifying enzymes, cytokines and chemokines was quantified by RT-qPCR. The phenotype and the numbers of intrahepatic lymphoid and myeloid cell subsets were evaluated by flow cytometry. Results: Both Il15-/- and Il15ra-/- mice developed markedly reduced liver fibrosis compared to wildtype control mice, as revealed by reduced collagen deposition and myofibroblast content. Il15ra-/- mice showed further reduction in collagen deposition compared to Il15-/- mice. However, Col1a1 and Col1a3 genes were similarly induced in the fibrotic livers of wildtype, Il15-/- and Il15ra-/- mice, although notable variations were observed in the expression of matrix remodeling enzymes and chemokines. As expected, Il15-/- and Il15ra-/- mice showed markedly reduced numbers of NK cells compared to wildtype mice. They also showed markedly less staining of CD45+ immune cells and CD68+ macrophages, and significantly reduced inflammatory cell infiltration into the liver, with fewer pro-inflammatory and anti-inflammatory monocyte subsets compared to wildtype mice. Conclusion: Our findings indicate that IL-15 exerts its profibrogenic role in the liver by promoting macrophage activation and that this requires trans-presentation of IL-15 by IL-15Rα.
Assuntos
Tetracloreto de Carbono , Modelos Animais de Doenças , Subunidade alfa de Receptor de Interleucina-15 , Interleucina-15 , Cirrose Hepática , Camundongos Endogâmicos C57BL , Camundongos Knockout , Animais , Interleucina-15/metabolismo , Interleucina-15/genética , Camundongos , Subunidade alfa de Receptor de Interleucina-15/genética , Subunidade alfa de Receptor de Interleucina-15/metabolismo , Cirrose Hepática/imunologia , Cirrose Hepática/patologia , Cirrose Hepática/metabolismo , Cirrose Hepática/induzido quimicamente , Masculino , Fígado/patologia , Fígado/metabolismo , Fígado/imunologia , Citocinas/metabolismo , Receptores de Interleucina-15RESUMO
Monitoring hepatocyte proliferation in situ following partial hepatectomy is widely used to characterize cytokines, growth factors and signaling molecules and pathways as well as the regulatory mechanisms involved in liver regeneration. Periodic measurement of the liver/body mass ratio estimates the rate of liver regeneration, which is often supplemented by evaluating the proportion of proliferating hepatocytes using a synthetic nucleoside analog such as 5-bromo-2'-deoxyuridine (BrdU) or the nuclear accumulation of proliferating cell nuclear antigen (PCNA) in proliferating cells. The introduction of the thymidine analog 5-ethynyl-2'deoxyuridine (EdU) and its detection by "click chemistry" using fluorescently labeled reagents has simplified the evaluation of live cell proliferation as it eliminates certain limitations of antibody-mediated detection of BrdU. Here, we describe the EdU-based measurement of hepatocyte proliferation during liver regeneration and correlate the results with that of Ki67 and PCNA-based assays.
Assuntos
Regeneração Hepática , Nucleosídeos , Bromodesoxiuridina/metabolismo , Proliferação de Células , Citocinas , DNA/metabolismo , Hepatócitos/metabolismo , Antígeno Ki-67 , Antígeno Nuclear de Célula em Proliferação , TimidinaRESUMO
Overview: Long-term survival of T lymphocytes in quiescent state is essential to maintain their cell numbers in secondary lymphoid organs and in peripheral circulation. In the BioBreeding diabetes-prone strain of rats (BB-DP), loss of functional GIMAP5 (GTPase of the immune associated nucleotide binding protein 5) results in profound peripheral T lymphopenia. This discovery heralded the identification of a new family of proteins initially called Immune-associated nucleotide binding protein (IAN) family. In this review we will use 'GIMAP' to refer to this family of proteins. Recent studies suggest that GIMAP proteins may interact with each other and also be involved in the movement of the cellular cargo along the cytoskeletal network. Here we will summarize the current knowledge on the characteristics and functions of GIMAP family of proteins.
Assuntos
Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Família Multigênica , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Suscetibilidade a Doenças , Proteínas de Ligação ao GTP/química , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas , Humanos , Proteínas de Membrana/química , Mutação , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
The nucleotide-binding leucine-rich repeat-containing receptor (NLR) family protein-5 (NLRC5) controls NF-κB activation and production of inflammatory cytokines in certain cell types. NLRC5 is considered a potential regulator of hepatic fibrogenic response due to its ability to inhibit hepatic stellate activation in vitro. To test whether NLRC5 is critical to control liver fibrosis, we treated wildtype and NLRC5-deficient mice with carbon tetrachloride (CCl4) and assessed pathological changes in the liver. Serum alanine transaminase levels and histopathology examination of liver sections revealed that NLRC5 deficiency did not exacerbate CCl4-induced liver damage or inflammatory cell infiltration. Sirius red staining of collagen fibers and hydroxyproline content showed comparable levels of liver fibrosis in CCl4-treated NLRC5-deficient and control mice. Myofibroblast differentiation and induction of collagen genes were similarly increased in both groups. Strikingly, the fibrotic livers of NLRC5-deficient mice showed reduced expression of matrix metalloproteinase-3 (Mmp3) and tissue inhibitor of MMPs-1 (Timp1) but not Mmp2 or Timp2. Fibrotic livers of NLRC5-deficient mice had increased expression of TNF but similar induction of TGFß compared to wildtype mice. CCl4-treated control and NLRC5-deficient mice displayed similar upregulation of Cx3cr1, a monocyte chemoattractant receptor gene, and the Cd68 macrophage marker. However, the fibrotic livers of NLRC5-deficient mice showed increased expression of F4/80 (Adgre1), a marker of tissue-resident macrophages. NLRC5-deficient livers showed increased phosphorylation of the NF-κB subunit p65 that remained elevated following fibrosis induction. Taken together, NLRC5 deficiency deregulates hepatic inflammatory response following chemical injury but does not significantly aggravate the fibrogenic response, showing that NLRC5 is not a critical regulator of liver fibrosis pathogenesis.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Cirrose Hepática/imunologia , Alanina Transaminase/sangue , Animais , Tetracloreto de Carbono , Citocinas/sangue , Citocinas/genética , Citocinas/imunologia , Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fígado/imunologia , Fígado/patologia , Cirrose Hepática/sangue , Cirrose Hepática/genética , Cirrose Hepática/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Transcrição RelA/imunologiaRESUMO
Suppressor of Cytokine Signaling 1 (SOCS1) functions as a tumor suppressor in hepatocellular carcinoma and many other types of cancers. SOCS1 mediates its functions by inhibiting tyrosine kinases, promoting ubiquitination and proteasomal degradation of signal transducing proteins, and by modulating transcription factors. Here, we studied the impact of SOCS1 on the hepatocyte proteome using Stable Isotopic Labelling of Amino acids in Cell culture (SILAC)-based mass spectrometry on the Hepa1-6 murine HCC cell line stably expressing wildtype SOCS1 or a mutant SOCS1 with impaired SH2 domain. As SOCS1 regulates the hepatocyte growth factor (HGF) receptor, the MET receptor tyrosine kinase (RTK), the SILAC-labelled cells were stimulated or not with HGF. Following mass spectrometry analysis, differentially modulated proteins were identified, quantified and analyzed for pathway enrichment. Of the 3440 proteins identified in Hepa-SOCS1 cells at steady state, 181 proteins were significantly modulated compared to control cells. The SH2 domain mutation and HGF increased the number of differentially modulated proteins. Protein interaction network analysis revealed enrichment of SOCS1-modulated proteins within multiprotein complexes such as ubiquitin conjugating enzymes, proteasome, mRNA spliceosome, mRNA exosome and mitochondrial ribosome. Notably, the expression of UBE2D ubiquitin conjugating enzyme, which is implicated in the control of growth factor receptor tyrosine kinase signaling, was found to be regulated by SOCS1. These findings suggest that SOCS1, induced by cytokines, growth factors and diverse other stimuli, has the potential to dynamically modulate of large macromolecular regulatory complexes to help maintain cellular homeostasis.
Assuntos
Regulação para Baixo , Regulação Enzimológica da Expressão Gênica , Proteômica , Proteínas Proto-Oncogênicas c-met/biossíntese , Proteína 1 Supressora da Sinalização de Citocina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Animais , Linhagem Celular , Camundongos , Proteínas Proto-Oncogênicas c-met/genética , Proteína 1 Supressora da Sinalização de Citocina/genética , Enzimas de Conjugação de Ubiquitina/genética , Domínios de Homologia de srcRESUMO
As early as the beginning of the twentieth century some data indicated that macromolecules are able to cross the intestinal mucosa to reach the blood. Further evidence was added over the years; however, pathways for this transport still remain to be established. We report here the transfer of two pancreatic enzymes, amylase and lipase, from the intestinal lumen to the blood. Both are present in higher concentrations in the intestinal mucosa and in blood of fed rats. Upon cholinergic stimulation of pancreatic secretion, there was not only an increase in blood enzyme concentrations, but evidence for internalization by duodenal enterocytes was obtained. Following insertion of fluorochrome-tagged amylase and lipase into the duodenal lumen of fasting rats, blood and intestinal tissues were sampled at different time points. Serum activities for both enzymes clearly increased with time. Light microscopy established internalization of both proteins by duodenal enterocytes, and immunogold outlined the pathway taken by both proteins across the enterocytes. From the intestinal lumen, enzymes are channeled through the endosomal compartment to the Golgi apparatus and to the basolateral membrane reaching the interstitial space and blood circulation. Transcytosis through the intestinal mucosa thereby represents an access route for pancreatic enzymes to reach blood circulation.
Assuntos
Amilases/metabolismo , Mucosa Intestinal/enzimologia , Lipase/metabolismo , Pâncreas/enzimologia , Amilases/sangue , Animais , Duodeno/metabolismo , Duodeno/ultraestrutura , Enterócitos/metabolismo , Jejum , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Imuno-Histoquímica , Mucosa Intestinal/ultraestrutura , Lipase/sangue , Masculino , Transporte Proteico , Ratos , Ratos Sprague-DawleyRESUMO
Four invariant chain (Ii) isoforms assist the folding and trafficking of human MHC class II (MHCIIs). The main isoforms, Iip33 and Iip35, assemble in the ER into homo- and/or hetero-trimers. The sequential binding of up to three MHCII αß heterodimers to Ii trimers results in the formation of pentamers, heptamers and nonamers. MHCIIs are required to overcome the p35-encoded di-arginine (RxR) ER retention motif and to allow anterograde trafficking of the complex. Here, we show that inactivation of the RxR motif requires a direct cis interaction between p35 and the MHCII, precluding ER egress of some unsaturated Ii trimers. Interestingly, as opposed to MHCII/p33 complexes, those including p35 remained in the ER when co-expressed with the NleA protein of enterohaemorrhagic Escherichia coli. Taken together, our results demonstrate that p35 influences distinctively MHCII/Ii assembly and trafficking.
Assuntos
Antígenos de Diferenciação de Linfócitos B/metabolismo , Retículo Endoplasmático/metabolismo , Escherichia coli Êntero-Hemorrágica/imunologia , Proteínas de Escherichia coli/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Fatores de Virulência/metabolismo , Antígenos de Diferenciação de Linfócitos B/genética , Escherichia coli Êntero-Hemorrágica/patogenicidade , Proteínas de Escherichia coli/genética , Células HEK293 , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Sinais Direcionadores de Proteínas/genética , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Fatores de Virulência/genéticaRESUMO
The peptide repertoire (peptidome) associated with MHC class II molecules (MHCIIs) is influenced by the polymorphic nature of the peptide binding groove but also by cell-intrinsic factors. The invariant chain (Ii) chaperones MHCIIs, affecting their folding and trafficking. Recent discoveries relating to Ii functions have provided insights as to how it edits the MHCII peptidome. In humans, the Ii gene encodes four different isoforms for which structure-function analyses have highlighted common properties but also some non-redundant roles. Another layer of complexity arises from the fact that Ii heterotrimerizes, a characteristic that has the potential to affect the maturation of associated MHCIIs in many different ways, depending on the isoform combinations. Here, we emphasize the peptide editing properties of Ii and discuss the impact of the various isoforms on the MHCII peptidome.
RESUMO
IL-10 is a potent anti-inflammatory cytokine interfering with antigen presentation by inducing the intracellular sequestration of MHC class II (MHC-II) molecules. Here we studied the contribution of membrane-associated RING-CH (MARCH) ubiquitin ligase family members to the IL-10-induced down-regulation of MHC-II molecules. We found that MARCH1 and MARCH8 proteins are the most potent family members for the down-regulation of MHC-II surface expression in transfected cells, but only MARCH1 mRNA expression is strongly induced by IL-10 in human primary monocytes. We detected mono- and poly-ubiquitinated forms of MHC-II molecules both in IL-10-treated monocytes and in cells transfected with MARCH1. We also show direct interaction between MHC-II and MARCH1 molecules in co-immunoprecipitation assays. Finally, we found that siRNA-mediated knockdown of MARCH1 reverses IL-10-induced MHC-II down-regulation in primary monocytes. Thus, the immunosuppressive effect of IL-10 on antigen presentation is mediated through induced expression of MARCH1.