ABSTRACT
Adoptive transfer of genetically engineered chimeric antigen receptor (CAR) T cells is becoming a promising treatment option for hematological malignancies. However, T cell immunotherapies have mostly failed in individuals with solid tumors. Here, with a CRISPR-Cas9 pooled library, we performed an in vivo targeted loss-of-function screen and identified ST3 ß-galactoside α-2,3-sialyltransferase 1 (ST3GAL1) as a negative regulator of the cancer-specific migration of CAR T cells. Analysis of glycosylated proteins revealed that CD18 is a major effector of ST3GAL1 in activated CD8+ T cells. ST3GAL1-mediated glycosylation induces the spontaneous nonspecific tissue sequestration of T cells by altering lymphocyte function-associated antigen-1 (LFA-1) endocytic recycling. Engineered CAR T cells with enhanced expression of ßII-spectrin, a central LFA-1-associated cytoskeleton molecule, reversed ST3GAL1-mediated nonspecific T cell migration and reduced tumor growth in mice by improving tumor-specific homing of CAR T cells. These findings identify the ST3GAL1-ßII-spectrin axis as a major cell-intrinsic program for cancer-targeting CAR T cell migration and as a promising strategy for effective T cell immunotherapy.
Subject(s)
Receptors, Chimeric Antigen , Animals , Mice , CD8-Positive T-Lymphocytes , Cell Line, Tumor , Cell Movement , Immunotherapy, Adoptive , Lymphocyte Function-Associated Antigen-1 , Spectrin , Humans , FemaleABSTRACT
Early recruitment of neutrophils from the blood to sites of tissue infection is a hallmark of innate immune responses. However, little is known about the mechanisms by which apoptotic neutrophils are cleared in infected tissues during resolution and the immunological consequences of in situ efferocytosis. Using intravital multiphoton microscopy, we show previously unrecognized motility patterns of interactions between neutrophils and tissue-resident phagocytes within the influenza-infected mouse airway. Newly infiltrated inflammatory monocytes become a chief pool of phagocytes and play a key role in the clearance of highly motile apoptotic neutrophils during the resolution phase. Apoptotic neutrophils further release epidermal growth factor and promote the differentiation of monocytes into tissue-resident antigen-presenting cells for activation of antiviral T cell effector functions. Collectively, these results suggest that the presence of in situ neutrophil resolution at the infected tissue is critical for optimal CD8+ T cell-mediated immune protection.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Influenza A virus/physiology , Influenza, Human/immunology , Neutrophils/immunology , Orthomyxoviridae Infections/immunology , Phagocytes/immunology , Receptors, CCR2/metabolism , Animals , Antigen Presentation , Apoptosis , Cell Movement , Cells, Cultured , Humans , Immunity, Innate , Intravital Microscopy , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Fluorescence, Multiphoton , Receptors, CCR2/geneticsABSTRACT
Protein kinase D (PKD) is a serine/threonine kinase family with three isoforms (PKD1-3) that are expressed in most cells and implicated in a wide array of signaling pathways, including cell growth, differentiation, transcription, secretion, polarization and actin turnover. Despite growing interest in PKD, relatively little is known about the role of PKD in immune responses. We recently published that inhibiting PKD limits proinflammatory cytokine secretion and leukocyte accumulation in mouse models of viral infection, and that PKD3 is highly expressed in the murine lung and immune cell populations. Here we focus on the immune-related phenotypes of PKD3 knockout mice. We report that PKD3 is necessary for maximal neutrophil accumulation in the lung following challenge with inhaled polyinosinic:polycytidylic acid, a double-stranded RNA, as well as following influenza A virus infection. Using reciprocal bone marrow chimeras, we found that PKD3 is required in the hematopoietic compartment for optimal neutrophil migration to the lung. Ex vivo transwell and chemokinesis assays confirmed that PKD3-/- neutrophils possess an intrinsic motility defect, partly because of reduced surface expression of CD18, which is critical for leukocyte migration. Finally, the peak of neutrophilia was significantly reduced in PKD3-/- mice after lethal influenza A virus infection. Together, these results demonstrate that PKD3 has an essential, and nonredundant, role in promoting neutrophil recruitment to the lung. A better understanding of the isoform-specific and cell type-specific activities of PKD has the potential to lead to novel therapeutics for respiratory illnesses.
Subject(s)
Neutrophils , Protein Kinase C , Virus Diseases , Animals , Mice , Neutrophils/metabolism , Protein Isoforms , Signal Transduction , Protein Kinase C/metabolismABSTRACT
BACKGROUND AND OBJECTIVES: Studies implicate the lung in moderating systemic immune activation via effects on circulating leukocytes. In this study, we investigated whether targeted expression of the antioxidant extracellular superoxide dismutase (SOD3) within the lung would influence post-ischemic peripheral neutrophil activation and CNS reperfusion injury. METHODS: Adult, male mice expressing human SOD3 within type II pneumocytes were subjected to 15 min of transient global cerebral ischemia. Three days post-reperfusion, lung and brain tissue was collected and analyzed by immunohistochemistry for inflammation and injury markers. In vitro motility and neurotoxicity assays were conducted to ascertain the direct effects of hSOD3 on PMN activation. Results were compared against C57BL/6 age and sex-matched controls. RESULTS: Relative to wild-type controls, hSOD3 heterozygous mice exhibited a reduction in lung inflammation, blood-brain barrier damage, and post-ischemic neuronal injury within the hippocampus and cortex. PMNs harvested from hSOD3 mice were also resistant to LPS priming, slower-moving, and less toxic to primary neuronal cultures. CONCLUSIONS: Constitutive, focal expression of hSOD3 is neuroprotective in a model of global cerebral ischemia-reperfusion injury. The underlying mechanism of SOD3-dependent protection is attributable in part to effects on the activation state and toxic potential of circulating neutrophils. These results implicate lung-brain coupling as a determinant of cerebral ischemia-reperfusion injury and highlight post-stroke lung inflammation as a potential therapeutic target in acute ischemic cerebrovascular injuries.
Subject(s)
Alveolar Epithelial Cells/enzymology , Brain Ischemia/enzymology , Brain/metabolism , Neurons/metabolism , Neutrophil Activation , Neutrophils/metabolism , Pneumonia/prevention & control , Reperfusion Injury/prevention & control , Superoxide Dismutase/metabolism , Alveolar Epithelial Cells/pathology , Animals , Brain/pathology , Brain Ischemia/genetics , Brain Ischemia/immunology , Cells, Cultured , Disease Models, Animal , Humans , Immunity, Innate , Male , Mice, Inbred C57BL , Mice, Transgenic , Neurons/pathology , Neutrophils/immunology , Pneumonia/enzymology , Pneumonia/genetics , Pneumonia/immunology , Reperfusion Injury/enzymology , Reperfusion Injury/genetics , Reperfusion Injury/immunology , Signal Transduction , Superoxide Dismutase/geneticsABSTRACT
Adoptive cell transfer of ex vivo-generated immune-promoting or tolerogenic T cells to either enhance immunity or promote tolerance in patients has been used with some success. However, effective trafficking of the transferred cells to the target tissue sites is the main barrier to achieving successful clinical outcomes. Here we developed a strategy for optically controlling T-cell trafficking using a photoactivatable (PA) chemokine receptor. Photoactivatable-chemokine C-X-C motif receptor 4 (PA-CXCR4) transmitted intracellular CXCR4 signals in response to 505-nm light. Localized activation of PA-CXCR4 induced T-cell polarization and directional migration (phototaxis) both in vitro and in vivo. Directing light onto the melanoma was sufficient to recruit PA-CXCR4-expressing tumor-targeting cytotoxic T cells and improved the efficacy of adoptive T-cell transfer immunotherapy, with a significant reduction in tumor growth in mice. These findings suggest that the use of photoactivatable chemokine receptors allows remotely controlled leukocyte trafficking with outstanding spatial resolution in tissues and may be feasible in other cell transfer therapies.
Subject(s)
Cell Movement , Optogenetics , Receptors, CXCR4/metabolism , T-Lymphocytes/cytology , Amino Acid Sequence , Animals , Cell Adhesion/radiation effects , Cell Movement/radiation effects , Cell Polarity/immunology , Cell Polarity/radiation effects , Immunotherapy , Light , Mice , Molecular Sequence Data , Neoplasms/immunology , Neoplasms/therapy , Protein Engineering , Receptors, CXCR4/chemistry , Signal Transduction/radiation effects , T-Lymphocytes/immunologyABSTRACT
Integrin-mediated migration of neutrophils to infected tissue sites is vital for pathogen clearance and therefore host survival. Although ß2 integrins have been shown to mediate neutrophil transendothelial migration during systemic and local inflammation, relatively little information is available regarding neutrophil migration in sepsis beyond the endothelial cell layer. In this study, we report that integrin α3ß1 (VLA-3; CD49c/CD29) is dramatically upregulated on neutrophils isolated from both human septic patients and in mouse models of sepsis. Compared with the α3ß1 (low) granulocytes, α3ß1 (high) cells from septic animals displayed hyperinflammatory phenotypes. Administration of a α3ß1 blocking peptide and conditional deletion of α3 in granulocytes significantly reduced the number of extravasating neutrophils and improved survival in septic mice. In addition, expression of α3ß1 on neutrophils was associated with Toll-like receptor-induced inflammatory responses and cytokine productions. Thus, our results show that α3ß1 is a novel marker of tissue homing and hyperresponsive neutrophil subtypes in sepsis, and blocking of α3ß1 may represent a new therapeutic approach in sepsis treatment.
Subject(s)
Cytokines/immunology , Integrin alpha3beta1/immunology , Neutrophil Infiltration/immunology , Neutrophils/immunology , Sepsis/immunology , Toll-Like Receptors/immunology , Animals , Cytokines/genetics , Disease Models, Animal , Humans , Integrin alpha3beta1/antagonists & inhibitors , Integrin alpha3beta1/genetics , Male , Mice , Mice, Knockout , Neutrophil Infiltration/drug effects , Neutrophil Infiltration/genetics , Neutrophils/pathology , Peptides/pharmacology , Sepsis/chemically induced , Sepsis/drug therapy , Sepsis/genetics , Sepsis/pathology , Toll-Like Receptors/geneticsABSTRACT
The addition of O-linked N-acetylglucosamine (O-GlcNAc) on serine or threonine modifies a myriad of proteins and regulates their function, stability and localization. O-GlcNAc modification is common among chromosome-associated proteins, such as transcription factors, suggesting its extensive involvement in gene expression regulation. In this study, we demonstrate the O-GlcNAc status of the Sp family members of transcription factors and the functional impact on their transcriptional activities. We highlight the presence of O-GlcNAc residues in Sp3 and Sp4, but not Sp2, as demonstrated by their enrichment in GlcNAc positive protein fractions and by detection of O-GlcNAc residues on Sp3 and Sp4 co-expressed in Escherichia coli together with O-GlcNAc transferase (OGT) using an O-GlcNAc-specific antibody. Deletion mutants of Sp3 and Sp4 indicate that the majority of O-GlcNAc sites reside in their N-terminal transactivation domain. Overall, using reporter gene assays and co-immunoprecipitations, we demonstrate a functional inhibitory role of O-GlcNAc modifications in Sp3 and Sp4 transcription factors. Thereby, our study strengthens the current notion that O-GlcNAc modification is an important regulator of protein interactome.
Subject(s)
Acetylglucosamine/metabolism , Protein Processing, Post-Translational , Sp3 Transcription Factor/metabolism , Sp4 Transcription Factor/metabolism , Transcription, Genetic , Escherichia coli , Genes, Reporter , HEK293 Cells , HeLa Cells , Humans , Luciferases/genetics , Luciferases/metabolism , N-Acetylglucosaminyltransferases/genetics , N-Acetylglucosaminyltransferases/metabolism , Plasmids/chemistry , Plasmids/metabolism , Protein Structure, Tertiary , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Serine/metabolism , Signal Transduction , Sp2 Transcription Factor/genetics , Sp2 Transcription Factor/metabolism , Sp3 Transcription Factor/genetics , Sp4 Transcription Factor/genetics , Threonine/metabolismABSTRACT
Glycolysis, the primary pathway metabolizing glucose for energy production, is connected to the hexosamine biosynthetic pathway (HBP) which produces UDP-N-acetylglucosamine (UDP-GlcNAc), a GlcNAc donor for O-linked GlcNAc modification (O-GlcNAc), as well as for traditional elongated glycosylation. Thus, glycolysis and O-GlcNAc are intimately associated. The present study reports the transcriptional activation of glycolytic genes by the transcription factor Sp1 and the O-GlcNAc-mediated suppression of Sp1-dependent activation of glycolytic genes. O-GlcNAc-deficient mutant Sp1 stimulated the transcription of nine glycolytic genes and cellular production of pyruvate, the final product of glycolysis, to a greater extent than wild-type Sp1. Consistently, this mutant Sp1 increased the protein levels of the two key glycolytic enzymes, phosphofructokinase (PFK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), to a greater extent than wild-type Sp1. Finally, the mutant Sp1 occupied GC-rich elements on PFK and GAPDH promoters more efficiently than wild-type Sp1. These results suggest that O-GlcNAcylation of Sp1 suppresses Sp1-mediated activation of glycolytic gene transcription.
Subject(s)
Glycolysis , Sp1 Transcription Factor/metabolism , Transcriptional Activation , Uridine Diphosphate N-Acetylglucosamine/metabolism , Base Sequence , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , HEK293 Cells , Humans , Molecular Sequence Data , Mutation , Phosphofructokinases/genetics , Promoter Regions, Genetic , Sp1 Transcription Factor/genetics , Uridine Diphosphate N-Acetylglucosamine/geneticsABSTRACT
T cells spend the majority of their time perusing lymphoid organs in search of cognate antigen presented by antigen presenting cells (APCs) and then quickly recirculate through the bloodstream to another lymph node. Therefore, regulation of a T-cell response is dependent upon the ability of cells to arrive in the correct location following chemokine gradients ("go" signal) as well as to receive appropriate T-cell receptor (TCR) activation signals upon cognate antigen recognition ("stop" signal). However, the mechanisms by which T cells regulate these go and stop signals remain unclear. We found that overexpression of the hematopoietic-specific RhoH protein in the presence of chemokine signals resulted in decreased Rap1-GTP and LFA-1 adhesiveness to ICAM-1, thus impairing T-cell chemotaxis; while in the presence of TCR signals, there were enhanced and sustained Rap1-GTP and LFA-1 activation as well as prolonged T:APC conjugates. RT-PCR analyses of activated CD4(+) T cells and live images of T-cell migration and immunological synapse (IS) formation revealed that functions of RhoH took place primarily at the levels of transcription and intracellular distribution. Thus, we conclude that RhoH expression provides a key molecular determinant that allows T cells to switch between sensing chemokine-mediated go signals and TCR-dependent stop signals.
Subject(s)
Lymphocyte Activation , T-Lymphocytes/cytology , Transcription Factors/physiology , rho GTP-Binding Proteins/physiology , Humans , Receptors, Antigen, T-Cell/physiology , Receptors, Chemokine/physiologyABSTRACT
Immune cell migration is required for the development of an effective and robust immune response. This elegant process is regulated by both cellular and environmental factors, with variables such as immune cell state, anatomical location, and disease state that govern differences in migration patterns. In all cases, a major factor is the expression of cell surface receptors and their cognate ligands. Rapid adaptation to environmental conditions partly depends on intrinsic cellular immune factors that affect a cell's ability to adjust to new environment. In this review, we discuss both myeloid and lymphoid cells and outline key determinants that govern immune cell migration, including molecules required for immune cell adhesion, modes of migration, chemotaxis, and specific chemokine signaling. Furthermore, we summarize tumor-specific elements that contribute to immune cell trafficking to cancer, while also exploring microenvironment factors that can alter these cellular dynamics within the tumor in both a pro and antitumor fashion. Specifically, we highlight the importance of the secretome in these later aspects. This review considers a myriad of factors that impact immune cell trajectory in cancer. We aim to highlight the immunotherapeutic targets that can be harnessed to achieve controlled immune trafficking to and within tumors.
Subject(s)
Cell Movement , Neoplasms , Tumor Microenvironment , Humans , Neoplasms/immunology , Neoplasms/pathology , Tumor Microenvironment/immunology , Animals , Lymphocytes/immunology , Lymphocytes/metabolism , Signal TransductionABSTRACT
The adaptive immune response is reliant on a T cell's ability to migrate through blood, lymph, and tissue in response to pathogens and foreign bodies. T cell migration is a complex process that requires the coordination of many signal inputs from the environment and local immune cells, including chemokines, chemokine receptors, and adhesion molecules. Furthermore, T cell motility is influenced by dynamic surrounding environmental cues, which can alter activation state, transcriptional landscape, adhesion molecule expression, and more. In vivo, the complexity of these seemingly intertwined factors makes it difficult to distinguish individual signals that contribute to T cell migration. This protocol provides a string of methods from T cell isolation to computer-aided analysis to assess T cell migration in real-time under highly specific environmental conditions. These conditions may help elucidate mechanisms that regulate migration, improving our understanding of T cell kinetics and providing strong mechanistic evidence that is difficult to attain through animal experiments. A deeper understanding of the molecular interactions that impact cell migration is important to develop improved therapeutics.
Subject(s)
CD8-Positive T-Lymphocytes , Cell Movement , Animals , Mice , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/cytology , Cell Movement/physiology , Cell Movement/immunology , Cell Migration Assays/methodsABSTRACT
Sepsis is a life-threatening systemic inflammatory condition causing approximately 11 million annual deaths worldwide. Although key hyperinflammation-based organ dysfunctions that drive disease pathology have been recognized, our understanding of the factors that predispose patients to septic mortality is limited. Due to the lack of reliable prognostic measures, the development of appropriate clinical management that improves patient survival remains challenging. Here, we discovered that a subpopulation of CD49chigh neutrophils with dramatic upregulation of the complement component 1q (C1q) gene expression arises during severe sepsis. We further found that deceased septic patients failed to maintain C1q protein expression in their neutrophils, whereas septic survivors expressed higher levels of C1q. In mouse sepsis models, blocking C1q with neutralizing antibodies or conditionally knocking out C1q in neutrophils led to a significant increase in septic mortality. Apoptotic neutrophils release C1q to control their own clearance in critically injured organs during sepsis; thus, treatment of septic mice with C1q drastically increased survival. These results suggest that neutrophil C1q is a reliable prognostic biomarker of septic mortality and a potential novel therapeutic target for the treatment of sepsis.
Subject(s)
Neutrophils , Sepsis , Animals , Complement C1q/genetics , Disease Models, Animal , Humans , Mice , Sepsis/mortality , Up-RegulationABSTRACT
Although cancer immunotherapy is effective against hematological malignancies, it is less effective against solid tumors due in part to significant metabolic challenges present in the tumor microenvironment (TME), where infiltrated CD8+ T cells face fierce competition with cancer cells for limited nutrients. Strong metabolic suppression in the TME is often associated with impaired T cell recruitment to the tumor site and hyporesponsive effector function via T cell exhaustion. Increasing evidence suggests that mitochondria play a key role in CD8+ T cell activation, effector function, and persistence in tumors. In this study, we showed that there was an increase in overall mitochondrial function, including mitochondrial mass and membrane potential, during both mouse and human CD8+ T cell activation. CD8+ T cell mitochondrial membrane potential was closely correlated with granzyme B and IFN-γ production, demonstrating the significance of mitochondria in effector T cell function. Additionally, activated CD8+ T cells that migrate on ICAM-1 and CXCL12 consumed significantly more oxygen than stationary CD8+ T cells. Inhibition of mitochondrial respiration decreased the velocity of CD8+ T cell migration, indicating the importance of mitochondrial metabolism in CD8+ T cell migration. Remote optical stimulation of CD8+ T cells that express our newly developed "OptoMito-On" successfully enhanced mitochondrial ATP production and improved overall CD8+ T cell migration and effector function. Our study provides new insight into the effect of the mitochondrial membrane potential on CD8+ T cell effector function and demonstrates the development of a novel optogenetic technique to remotely control T cell metabolism and effector function at the target tumor site with outstanding specificity and temporospatial resolution.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/radiation effects , Optogenetics/methods , Adenosine Triphosphate/metabolism , Animals , CD8-Positive T-Lymphocytes/metabolism , Cell Movement/radiation effects , Cytokines/metabolism , Cytoskeleton/metabolism , Cytoskeleton/radiation effects , Humans , Immunotherapy , Lymphocyte Activation/radiation effects , Membrane Potential, Mitochondrial/genetics , Membrane Potential, Mitochondrial/radiation effects , Mice , Mitochondria/metabolism , Mitochondria/radiation effects , Neoplasms/immunology , Neoplasms/therapyABSTRACT
O-linked N-acetylglucosamine (O-GlcNAc), a monosaccharide N-acetylglucosamine on the serine and threonine residues of nucleocytoplasmic proteins, is a novel protein modification that is ubiquitous among eukaryotes and implicated in cell regulation. Recent evidence indicates that O-GlcNAc regulates protein-protein interactions. Here we provide evidence that O-GlcNAc interrupts a known interaction between Sp1 and sterol regulatory element binding protein 2 (SREBP2), thereby inhibiting expression of the gene encoding acetyl-CoA synthetase 1, which is involved in lipid synthesis. This study suggests a novel mechanism in which lipid biosynthesis may be regulated by O-GlcNAc.
Subject(s)
Acetylglucosamine/metabolism , Coenzyme A Ligases/genetics , Gene Expression Regulation, Enzymologic , Lipids/biosynthesis , Sp1 Transcription Factor/antagonists & inhibitors , Sterol Regulatory Element Binding Protein 2/antagonists & inhibitors , Animals , Cell Line , Humans , Lipids/genetics , Mice , Sp1 Transcription Factor/metabolism , Sterol Regulatory Element Binding Protein 2/metabolism , Transcription, GeneticABSTRACT
O-Linked N-acetylglucosamine (O-GlcNAc), a single GlcNAc modification of proteins, is abundant in nucleocytoplasmic proteins of eukaryotes. Most nuclear transcriptional regulator proteins carry O-GlcNAc, implicating O-GlcNAc in gene regulation. This study suggested the possibility that O-GlcNAc regulates cooperative binding of Sp1 and its collaborating transcription factors, Oct1 and Elf-1, onto DNA templates in vivo. Chromatin immunoprecipitation assays on cells in which O-GlcNAc was modulated pharmacologically revealed that Sp1-Oct1- and Sp1-Elf-1-paired occupancies of previously known target promoter regions were suppressed by elevated O-GlcNAc modification. Since these pairs of transcription factors bind the target promoters cooperatively and DNA binding of Sp1 alone is not affected by O-GlcNAc, our results imply that O-GlcNAc weakens the DNA binding of Sp1 and its cooperative binding partners by inhibiting stable interaction on DNA templates.
Subject(s)
Acetylglucosamine/metabolism , DNA/metabolism , Oxygen/metabolism , Sp1 Transcription Factor/metabolism , Animals , Base Sequence , Cell Line, Tumor , Humans , Molecular Sequence Data , Protein Binding/drug effects , RNA, Small Nuclear/genetics , Rats , Streptozocin/pharmacology , Transcription, GeneticABSTRACT
The novel protein modification, O-linked N-acetylglucosamine (O-GlcNAc), plays an important role in various aspects of cell regulation. Although most of nuclear transcription regulatory factors are modified by O-GlcNAc, O-GlcNAc effects on transcription remain largely undefined yet. In this study, we show that O-GlcNAc inhibits a physical interaction between Sp1 and Elf-1 transcription factors, and negatively regulates transcription of placenta and embryonic expression oncofetal protein gene (Pem). These findings suggest that O-GlcNAc inhibits Sp1-mediated gene transcription possibly by interrupting Sp1 interaction with its cooperative factor.
Subject(s)
Ephrin-A2/antagonists & inhibitors , Gene Expression Regulation , Homeodomain Proteins/genetics , Sp1 Transcription Factor/antagonists & inhibitors , Transcription Factors/genetics , Acetylglucosamine , Animals , Cell Line, Tumor , Ephrin-A2/metabolism , Humans , Mice , Rats , Sp1 Transcription Factor/metabolism , Transcription, GeneticABSTRACT
O-linked N-acetylglucosamine (O-GlcNAc), a monosaccharide N-acetylglucosamine addition on nucleocytoplasmic proteins, is abundant in transcription regulators and has been implicated in gene regulation. Sp1 transcription factor is multiply modified by O-GlcNAc within its serine/threonine-rich region and glutamine-rich transactivation domain. In the present study, we show that O-GlcNAc of Sp1 serine/threonine-rich region interrupts a physical interaction between Sp1 and NF-YA, thus inhibiting Sp1-NF-Y cooperative activation of gene transcription. Our results strengthen the notion that O-GlcNAc regulates gene transcription by modulating the protein-protein interaction network among transcription regulatory proteins.
Subject(s)
Acetylglucosamine/metabolism , CCAAT-Binding Factor/metabolism , Sp1 Transcription Factor/metabolism , Transcriptional Activation , Acylation , Cell Line , Humans , Immunoprecipitation , Promoter Regions, Genetic , Protein Structure, Tertiary , Transcription, GeneticABSTRACT
The chemokine CCL7 (MCP3) is known to promote the recruitment of many innate immune cell types including monocytes and neutrophils to sites of bacterial and viral infection and eosinophils and basophils to sites of allergic inflammation. CCL7 upregulation has been associated with many inflammatory settings including infection, cardiovascular disease, and the tumor microenvironment. CCL7's pleotropic effects are due in part to its ability to bind numerous chemokine receptors, namely CCR1, CCR2, CCR3, CCR5, and CCR10. CCL7-blockade or CCL7-deficiency is often marked by decreased inflammation and poor pathogen control. In the context of Leishmania major infection, CCL7 is specifically upregulated in the skin one-2 weeks after infection but its role in L. major control is unclear. To determine CCL7's impact on the response to L. major we infected WT and CCL7-/- C57BL/6 mice. L. major infection of CCL7-deficient mice led to an unexpected increase in inflammation in the infected skin 2 weeks post-infection. A broad increase in immune cell subsets was observed but was dominated by enhanced neutrophilic infiltration. Increased neutrophil recruitment was associated with an enhanced IL-17 gene profile in the infected skin. CCL7 was shown to directly antagonize neutrophil migration in vitro and CCL7 add-back in vivo specifically reduced neutrophil influx into the infected skin revealing an unexpected role for CCL7 in limiting neutrophil recruitment during L. major infection. Enhanced neutrophilic infiltration in CCL7-deficient mice changed the balance of L. major infected host cells with an increase in the ratio of infected neutrophils over monocytes/macrophages. To determine the consequence of CCL7 deficiency on L. major control we analyzed parasite load cutaneously at the site of infection and viscerally in the draining LN and spleen. The CCL7-/- mice supported robust cutaneous parasite control similar to their WT C57BL/6 counterparts. In contrast, CCL7-deficiency led to greater parasite dissemination and poor parasite control in the spleen. Our studies reveal a novel role for CCL7 in negatively regulating cutaneous inflammation, specifically neutrophils, early during L. major infection. We propose that CCL7-mediated dampening of the early immune response in the skin may limit the ability of the parasite to disseminate without compromising cutaneous control.
Subject(s)
Chemokine CCL7/immunology , Chemokine CCL7/metabolism , Leishmania major/immunology , Leishmaniasis, Cutaneous/immunology , Analysis of Variance , Animals , Cell Movement , Chemokine CCL7/genetics , Chemokine CXCL2/metabolism , Gene Expression , Inflammation/genetics , Inflammation/immunology , Interleukin-17/genetics , Leishmaniasis, Cutaneous/pathology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Neutrophil Infiltration/immunology , Neutrophils/physiology , Statistics, NonparametricABSTRACT
Bacteriophage phiFC1 integrase (MJ1) was previously shown to perform a site-specific recombination between a phage attachment site (attP) and a host attachment site (attB) in its host, Enterococcus faecalis, and also in a non-host bacterium, Escherichia coli. Here, we investigated biochemical features of MJ1 integrase. First, MJ1 integrase could perform in vitro recombination between attP and attB in the absence of additional factors. Second, MJ1 integrase interacted with att sites. Electrophoretic mobility shift assays and DNase I footprinting revealed that MJ1 integrase could efficiently bind to all the att sites and that MJ1 integrase recognized relatively short sequences (approximately 50 bp) containing an overlapping region within attB and attP. These results demonstrate that MJ1 integrase indeed catalyzes an integrative recombination between attP and attB, the mechanism of which might be simple and unidirectional, as found in serine integrases.
Subject(s)
Attachment Sites, Microbiological/genetics , Bacteriophages/enzymology , Enterococcus faecalis/virology , Integrases/physiology , Recombination, Genetic , Amino Acid Sequence , Base Sequence , Molecular Sequence DataABSTRACT
We previously identified the origin of replication of p703/5, a small cryptic plasmid from the KBL703 strain of Enterococcus faecalis. The origin of replication contains putative regulatory cis-elements required for replication and a replication initiator (RepA) gene. The replicon of p703/5 is similar in its structural organization to theta-type plasmids, and RepA is homologous to a family of Rep proteins identified in several plasmids from Gram-positive bacteria. Here, we report molecular interactions between RepA and the replication origin of p703/5. DNase I footprinting using recombinant RepA together with electrophoretic mobility shift assays confirmed the binding of RepA to the replication origin of p703/5 via iterons and an inverted repeat. We also demonstrated the formation of RepA dimers and the different binding of RepA to the iteron and the inverted repeat using gel filtration chromatographic analysis, a chemical crosslinking assay, and electrophoretic mobility shift assays in the presence of guanidine hydrochloride. Our results suggest that RepA plays a regulatory role in the replication of the enterococcal plasmid p703/5 via mechanisms similar to those of typical iteron-carrying theta-type plasmids.