ABSTRACT
CLEC12A is a myeloid inhibitory receptor that negatively regulates inflammation in mouse models of autoimmune and autoinflammatory arthritis. Reduced CLEC12A expression enhances myeloid cell activation and inflammation in CLEC12A knock-out mice with collagen antibody-induced or gout-like arthritis. Similarly to other C-type lectin receptors, CLEC12A harbours a stalk domain between its ligand binding and transmembrane domains. While it is presumed that the cysteines in the stalk domain have multimerisation properties, their role in CLEC12A expression and/or signaling remain unknown. We thus used site-directed mutagenesis to determine whether the stalk domain cysteines play a role in CLEC12A expression, internalisation, oligomerisation, and/or signaling. Mutation of C118 blocks CLEC12A transport through the secretory pathway diminishing its cell-surface expression. In contrast, mutating C130 does not affect CLEC12A cell-surface expression but increases its oligomerisation, inducing ligand-independent phosphorylation of the receptor. Moreover, we provide evidence that CLEC12A dimerisation is regulated in a redox-dependent manner. We also show that antibody-induced CLEC12A cross-linking induces flotillin oligomerisation in insoluble membrane domains in which CLEC12A signals. Taken together, these data indicate that the stalk cysteines in CLEC12A differentially modulate this inhibitory receptor's expression, oligomerisation and signaling, suggestive of the regulation of CLEC12A in a redox-dependent manner during inflammation.
Subject(s)
Lectins, C-Type/genetics , Lectins, C-Type/metabolism , Membrane Proteins/metabolism , Myeloid Cells/metabolism , Protein Multimerization/genetics , Receptors, Mitogen/genetics , Receptors, Mitogen/metabolism , Cell Line, Tumor , Cysteine/metabolism , HEK293 Cells , HeLa Cells , Humans , Inflammation/genetics , Lectins, C-Type/biosynthesis , Membrane Proteins/genetics , Mutagenesis, Site-Directed , Phosphorylation , Protein Domains/genetics , Protein Transport/genetics , Receptors, Mitogen/biosynthesis , Signal Transduction/immunologyABSTRACT
BACKGROUND: CD8+ T-lymphocyte subsets defined by killer lectin-like receptor G1 (KLRG1) and CD127 expression have been reported to have an important role in infection, but their role in the setting of lymphoid malignancies, specifically follicular lymphoma (FL), has not been studied. METHODS: To characterize the phenotype of KLRG1/CD127-defined CD8+ subsets, surface and intracellular markers were measured by flow cytometry and Cytometry by time of flight (CyTOF), and the transcriptional profile of these cells was determined by CITE-Seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing). The functional capacity of each subset was determined, as was their impact on overall survival (OS) and event-free survival (EFS) of patients with FL. RESULTS: We found that intratumoral CD8+ cells in FL are skewed toward effector cell subsets, particularly KLRG+CD127- and KLRG1-CD127- cells over memory cell subsets, such as KLRG1-CD127+ and KLRG1+CD127+ cells. While effector subsets exhibited increased capacity to produce cytokines/granules when compared with memory subsets, their proliferative capacity and viability were found to be substantially inferior. Clinically, a skewed distribution of intratumoral CD8+ T cells favoring effector subtypes was associated with an inferior outcome in patients with FL. Increased numbers of CD127+KLRG1- and CD127+KLRG1+ were significantly associated with a favorable OS and EFS, while CD127-KLRG1- correlated with a poor EFS and OS in patients with FL. Furthermore, we demonstrated that interleukin (IL)-15 promotes CD127-KLRG1+ cell development in the presence of dendritic cells via a phosphoinositide 3-kinase (PI3K)-dependent mechanism, and treatment of CD8+ T cells with a PI3K inhibitor downregulated the transcription factors responsible for CD127-KLRG1+ differentiation. CONCLUSIONS: Taken together, these results reveal not only a biological and prognostic role for KLRG1/CD127-defined CD8+ subsets in FL but also a potential role for PI3K inhibitors to manipulate the differentiation of CD8+ T cells, thereby promoting a more effective antitumor immune response.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Interleukin-7 Receptor alpha Subunit/metabolism , Lectins, C-Type/biosynthesis , Lymphoma, Follicular/metabolism , Receptors, Immunologic/biosynthesis , Cell Differentiation/immunology , Female , Humans , Interleukin-7 Receptor alpha Subunit/biosynthesis , Interleukin-7 Receptor alpha Subunit/genetics , Interleukin-7 Receptor alpha Subunit/immunology , Lectins, C-Type/genetics , Lectins, C-Type/immunology , Lymphoma, Follicular/genetics , Lymphoma, Follicular/immunology , Receptors, Immunologic/genetics , Receptors, Immunologic/immunology , Treatment Outcome , Tumor MicroenvironmentABSTRACT
In breast cancer, humoral immune responses may contribute to clinical outcomes, especially in more immunogenic subtypes. Here, we investigated B lymphocyte subsets, immunoglobulin expression, and clonal features in breast tumors, focusing on aggressive triple-negative breast cancers (TNBC). In samples from patients with TNBC and healthy volunteers, circulating and tumor-infiltrating B lymphocytes (TIL-B) were evaluated. CD20+CD27+IgD- isotype-switched B lymphocytes were increased in tumors, compared with matched blood. TIL-B frequently formed stromal clusters with T lymphocytes and engaged in bidirectional functional cross-talk, consistent with gene signatures associated with lymphoid assembly, costimulation, cytokine-cytokine receptor interactions, cytotoxic T-cell activation, and T-cell-dependent B-cell activation. TIL-B-upregulated B-cell receptor (BCR) pathway molecules FOS and JUN, germinal center chemokine regulator RGS1, activation marker CD69, and TNFα signal transduction via NFκB, suggesting BCR-immune complex formation. Expression of genes associated with B lymphocyte recruitment and lymphoid assembly, including CXCL13, CXCR4, and DC-LAMP, was elevated in TNBC compared with other subtypes and normal breast. TIL-B-rich tumors showed expansion of IgG but not IgA isotypes, and IgG isotype switching positively associated with survival outcomes in TNBC. Clonal expansion was biased toward IgG, showing expansive clonal families with specific variable region gene combinations and narrow repertoires. Stronger positive selection pressure was present in the complementarity determining regions of IgG compared with their clonally related IgA in tumor samples. Overall, class-switched B lymphocyte lineage traits were conspicuous in TNBC, associated with improved clinical outcomes, and conferred IgG-biased, clonally expanded, and likely antigen-driven humoral responses. SIGNIFICANCE: Tumor-infiltrating B lymphocytes assemble in clusters, undergoing B-cell receptor-driven activation, proliferation, and isotype switching. Clonally expanded, IgG isotype-biased humoral immunity associates with favorable prognosis primarily in triple-negative breast cancers.
Subject(s)
B-Lymphocytes/metabolism , Immunoglobulin G/immunology , Triple Negative Breast Neoplasms/metabolism , Antigens, CD/biosynthesis , Antigens, CD20/biosynthesis , Antigens, Differentiation, T-Lymphocyte/biosynthesis , B-Lymphocytes/pathology , Base Sequence , Cell Line, Tumor , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Immunoglobulin D/biosynthesis , Immunohistochemistry , Lectins, C-Type/biosynthesis , Lymphocytes/cytology , Models, Statistical , Phenotype , Prognosis , RNA-Seq , Receptors, Antigen, B-Cell/metabolism , Single-Cell Analysis , Transcriptome , Triple Negative Breast Neoplasms/immunology , Tumor Necrosis Factor Receptor Superfamily, Member 7/biosynthesis , Tumor Necrosis Factor-alpha/biosynthesis , User-Computer InterfaceABSTRACT
BACKGROUND: Tissue resident memory T cells (TRMs) persist long-term in peripheral tissues without recirculation, triggering an immediate protective inflammatory state upon the re-recognition of the antigen. Despite evidence incriminating the dysregulation of TRMs in autoimmune diseases, few studies have examined their expression in cutaneous lupus erythematosus (CLE). OBJECTIVES: We aimed to examine whether there are differences among TRM populations in CLE depending on different clinical conditions, such as the CLE subtype or association with systemic lupus erythematosus, and to determine the effect of type I interferon (IFN) on the development of TRMs in CLE. METHODS: CLE disease activity was evaluated using the Cutaneous Lupus Erythematosus Disease Area and Severity Index. The expression of the TRM markers CD69 and CD103 in CLE lesions was evaluated by immunofluorescence. Flow cytometry was performed on peripheral blood mononuclear cells after IFNα treatment. RESULTS: The number of TRMs expressing either CD69 or CD103 was significantly higher in CLE lesions than in control skin; however, it was not significantly different between discoid lupus erythematosus and subacute CLE, or dependent on the presence of concomitant systemic lupus. Lesional severity was not correlated with an increase in TRMs in CLE. IFNα treatment induced a conspicuous increase in CD69 expression in skin-homing T cells, more profoundly in CD4+ T cells than in CD8+ T cells. CONCLUSIONS: Skin TRMs, either CD69 or CD103-positive cells, showed increased levels in the lesional skin of CLE, and IFNα increased the expression of CD69 in T cells.
Subject(s)
Interferon-alpha/immunology , Lupus Erythematosus, Cutaneous/immunology , Memory T Cells/immunology , Skin/immunology , Adult , Antigens, CD/biosynthesis , Antigens, CD/immunology , Antigens, Differentiation, T-Lymphocyte/biosynthesis , Antigens, Differentiation, T-Lymphocyte/immunology , Female , Humans , Integrin alpha Chains/biosynthesis , Integrin alpha Chains/immunology , Interferon-alpha/pharmacology , Lectins, C-Type/biosynthesis , Lectins, C-Type/immunology , Lupus Erythematosus, Discoid/immunology , Lupus Erythematosus, Systemic/immunology , Male , Middle Aged , Young AdultABSTRACT
Background: Extracellular vesicles (EVs) are secreted by cells from their membrane within circulation and body fluids. Knowledge of the involvement of EVs in pathogenesis of lung diseases is increasing. The present study aimed to evaluate the expression of exosomal surface epitopes in a cohort of idiopathic pulmonary fibrosis (IPF) patients followed in two Italian Referral Centres for Interstitial Lung Diseases, comparing them with a group of healthy volunteers. Materials and Methods: Ninety IPF patients (median age and interquartile range (IQR) 71 (66-75) years; 69 males) were selected retrospectively. Blood samples were obtained from patients before starting antifibrotic therapy. A MACSPlex Exosome Kit, human, (Miltenyi Biotec, Bergisch-Gladbach, Germany), to detect 37 exosomal surface epitopes, was used. Results: CD19, CD69, CD8, and CD86 were significantly higher in IPF patients than in controls (p = 0.0023, p = 0.0471, p = 0.0082, and p = 0.0143, respectively). CD42a was lower in IPF subjects than in controls (p = 0.0153), while CD209, Cd133/1, MCSP, and ROR1 were higher in IPF patients than in controls (p = 0.0007, p = 0.0050, p = 0.0139, and p = 0.0335, respectively). Kaplan-Meier survival analysis for IPF patients: for median values and a cut-off of 0.48 for CD25, the two subgroups showed a significant difference in survival rate (p = 0.0243, hazard ratio: 0.52 (95%CI 0.29-0.92); the same was true for CD8 (cut-off 1.53, p = 0.0309, hazard ratio: 1.39 (95%CI 0.75-2.53). Conclusion: Our multicenter study showed for the first time the expression of surface epitopes on EVs from IPF patients, providing interesting data on the communication signatures/exosomal profile in serum from IPF patients and new insights into the pathogenesis of the disease and a promising reliability in predicting mid-term survival of IPF patients.
Subject(s)
Extracellular Vesicles/metabolism , Idiopathic Pulmonary Fibrosis/immunology , Idiopathic Pulmonary Fibrosis/physiopathology , Aged , Antigens, CD/biosynthesis , Antigens, CD19/biosynthesis , Antigens, Differentiation, T-Lymphocyte/biosynthesis , B7-2 Antigen/biosynthesis , CD8 Antigens/biosynthesis , Cell Adhesion Molecules/metabolism , Cell Membrane/metabolism , Epitopes/chemistry , Exosomes/metabolism , Female , Flow Cytometry , Humans , Idiopathic Pulmonary Fibrosis/blood , Italy , Lectins, C-Type/biosynthesis , Lectins, C-Type/metabolism , Male , Middle Aged , Prognosis , Proportional Hazards Models , Receptors, Cell Surface/metabolism , Retrospective Studies , Treatment OutcomeABSTRACT
Purpose: The purpose of this study was to investigate the therapeutic effect of perillaldehyde (PAE) on Aspergillus fumigatus (A. fumigatus) keratitis. Methods: Human corneal epithelial cells (HCECs) were pretreated with PAE and stimulated with A. fumigatus mycelium. C57BL/6 mice were infected with A. fumigatus and treated with or without PAE 1 day after infection. Clinical scores, PCR, ELISA, and Western blotting were used to detect the expression of pro-inflammatory mediators, dendritic cell-associated c-type lectin-1 (Dectin-1), nuclear factor (erythroid-derived 2) like 2 (Nrf2), and heme oxygenase (HO-1). Nrf2 expression in HCECs pretreated with PAE was observed by immunofluorescence. NIMP-R14 protein expression and localization in mouse corneas were observed by immunofluorescence staining after treatment with PAE. Corneal colony counting, time-kill tests, and mycelial transformation inhibition tests were used to evaluate the antifungal effect of PAE. Results: C57BL/6 mice treated with PAE at 1 day after infection had a lower clinical score and decreased IL-1ß, TNF-α, IL-6, Dectin-1, and MPO levels. PAE treatment significantly reduced neutrophil recruitments to the corneal stroma. Compared with the DMSO-treated group, PAE treatment significantly decreased mRNA and protein levels of pro-inflammatory cytokines and Dectin-1 in HCECs. PAE pretreatment before A. fumigatus stimulation obviously elevated the mRNA and protein levels of components of the Nrf2/HO-1 axis. HCECs pretreated with PAE before infection showed a weakened ability to inhibit inflammation in the presence of brusatol (BT; an Nrf2 inhibitor) or ZnPP (an HO-1 inhibitor). PAE treatment significantly reduced the fungal load of C57BL/6 mouse corneas and inhibited fungal growth in vitro. Conclusions: These data proved that PAE may ameliorate A. fumigatus keratitis by activating the Nrf2/HO-1 signaling pathway and inhibiting the Dectin-1 mediated inflammatory response and neutrophil recruitment. Furthermore, PAE exerts direct fungicidal activity on A. fumigatus.
Subject(s)
Eye Infections, Fungal/drug therapy , Gene Expression Regulation , Keratitis/drug therapy , Lectins, C-Type/antagonists & inhibitors , Lectins, C-Type/genetics , Monoterpenes/pharmacology , NF-E2-Related Factor 2/metabolism , Animals , Aspergillosis/drug therapy , Aspergillosis/metabolism , Aspergillosis/microbiology , Aspergillus fumigatus/isolation & purification , Disease Models, Animal , Epithelium, Corneal/metabolism , Epithelium, Corneal/microbiology , Epithelium, Corneal/pathology , Eye Infections, Fungal/metabolism , Eye Infections, Fungal/microbiology , Female , Humans , Keratitis/metabolism , Keratitis/microbiology , Lectins, C-Type/biosynthesis , Mice , Mice, Inbred C57BL , NF-E2-Related Factor 2/drug effects , RNA/genetics , Signal TransductionABSTRACT
BACKGROUND: Docetaxel is an effective first-line chemotherapy agent used in the treatment of castration-resistant prostate cancer (CRPC) patients. However, most times chemotherapy with docetaxel eventually fails due to the development of docetaxel resistance. Natural killer (NK) cells are the first line of defense against cancer and infections. NK cell function is determined by a delicate balance between signals received via activating and inhibitory receptors. The aim of this study is to explore whether the potential docetaxel-resistant mechanism is associated with impaired NK cell cytotoxicity toward CRPC cells. METHODS: By performing MTT assay, we explored the role of docetaxel in regulating NK cells' cytotoxicity. Western blot and quantitative real-time polymerase chain reaction analysis were used to measure messenger RNA and protein levels separately. Luciferase reporter assay and chromatin immunoprecipitation assay were performed to analyze the mechanism. RESULTS: We found that docetaxel could suppress the immunotherapy efficacy of NK cells toward CRPC cells via the androgen receptor (AR)-lectin-like transcript 1 (LLT1) signals in vitro. Analysis of the mechanism revealed that docetaxel functioned through increasing AR to upregulate LLT1 expression in CRPC cells. AR transcriptionally activated LLT1 expression by binding to its promoter region. Furthermore, targeting AR with ASC-J9 or blocking LL1 by anti-human LLT1 monoclonal antibody could reverse the suppressive effect of docetaxel on the immunotherapy efficacy of NK cells toward CRPC cells. CONCLUSIONS: We concluded that chemotherapy agent docetaxel could increase AR that transcriptionally regulated the expression of NK inhibitory ligand LLT1 on CRPC cells. An increase of LL1 may further suppress the immunological efficacy of NK cells to kill CRPC cells. Additionally, targeting AR or blocking LL1 could enhance the immunotherapy efficacy of NK cells toward CRPC cells which might be considered as a new therapeutic option for the prevention or treatment of docetaxel resistance.
Subject(s)
Docetaxel/adverse effects , Killer Cells, Natural/drug effects , Lectins, C-Type/immunology , Prostatic Neoplasms, Castration-Resistant/immunology , Prostatic Neoplasms, Castration-Resistant/therapy , Receptors, Androgen/immunology , Receptors, Cell Surface/immunology , Androgen Receptor Antagonists/pharmacology , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/adverse effects , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Coculture Techniques , Combined Modality Therapy , Curcumin/analogs & derivatives , Curcumin/pharmacology , Docetaxel/therapeutic use , HEK293 Cells , Humans , Immunotherapy, Adoptive/methods , Killer Cells, Natural/immunology , Killer Cells, Natural/transplantation , Lectins, C-Type/antagonists & inhibitors , Lectins, C-Type/biosynthesis , Male , Prostatic Neoplasms, Castration-Resistant/drug therapy , Receptors, Androgen/biosynthesis , Receptors, Androgen/genetics , Receptors, Cell Surface/antagonists & inhibitors , Receptors, Cell Surface/biosynthesis , Up-Regulation/drug effectsABSTRACT
PURPOSE: To investigate the clinical implications of CD3+CD69+ T-cells and CD8+CD28+ T-cells in the peripheral blood of patients prior to radical prostatectomy. MATERIALS AND METHODS: A total of 91 prostate cancer (PCa) patients and 50 benign prostatic hyperplasia (BPH) patients were enrolled from January 2016 to December 2017. The proportions of CD3+CD69+ T-cells and CD8+CD28+ T-cells in the peripheral blood of PCa and BPH patients were detected by flow cytometry, and the association of these T-cell populations with pathological Grade Group and pathological TNM classification was evaluated. Data analysis was performed with SAS version 9.4 software. RESULTS: The proportions of CD3+CD69+ and CD8+CD28+ T-cells in peripheral blood were higher in PCa patients than those in BPH patients. Multivariate analysis identified a higher CD3+CD69+ T-cell proportion as a risk factor for PCa (odds ratio (OR) = 4.783, P = 0.0013), but the diagnostic efficacy of the CD3+CD69+ T-cell proportion (area under the curve (AUC)=0.6833, P = 0.0003) for PCa was still inferior to that of the tPSA level (AUC=0.7531, P < 0.0001). The AUCs for CD3+CD69+ T-cell and CD8+CD28+ T-cell proportions for PCa were 0.6959 (P = 0.0372) and 0.6935 (P = 0.0395), respectively, among men with tPSA levels of 4.0-10.0 ng/mL. A lower CD3+CD69+ T-cell proportion was associated with higher pathological Grade Group (P=0.0074). CONCLUSION: The proportions of CD3+CD69+ T-cells and CD8+CD28+ T-cells in peripheral blood are potential diagnostic indicators for PCa. The preoperative proportion of CD3+CD69+ T-cells in peripheral blood may have prognostic value in terms of the pathological Grade Group in PCa.
Subject(s)
Antigens, CD/biosynthesis , Antigens, Differentiation, T-Lymphocyte/biosynthesis , CD28 Antigens/biosynthesis , CD3 Complex/biosynthesis , CD8-Positive T-Lymphocytes , Lectins, C-Type/biosynthesis , Prostatectomy , Prostatic Hyperplasia/blood , Prostatic Hyperplasia/surgery , Prostatic Neoplasms/blood , Prostatic Neoplasms/surgery , T-Lymphocytes/metabolism , Aged , Humans , Lymphocyte Count , Male , Middle Aged , Neoplasm Staging , Preoperative Period , Prostatic Hyperplasia/pathology , Prostatic Neoplasms/pathologyABSTRACT
OBJECTIVE: Delayed thrombolytic therapy with recombinant tissue plasminogen activator (tPA) may exacerbate blood-brain barrier (BBB) breakdown after ischemic stroke and lead to catastrophic hemorrhagic transformation (HT). Rosiglitazone(RSG), a widely used antidiabetic drug that activates peroxisome proliferator-activated receptor-γ (PPAR-γ), has been shown to protect against cerebral ischemia through promoting poststroke microglial polarization toward the beneficial anti-inflammatory phenotype. However, whether RSG can alleviate HT after delayed tPA treatment remains unknown. In this study, we sort to examine the role of RSG on tPA-induced HT after stroke. METHODS AND RESULTS: We used the murine suture middle cerebral artery occlusion (MCAO) models of stroke followed by delayed administration of tPA (10 mg/kg, 2 hours after suture occlusion) to investigate the therapeutic potential of RSG against tPA-induced HT. When RSG(6 mg/kg) was intraperitoneally administered 1 hour before MCAO in tPA-treated MCAO mice, HT in the ischemic territory was significantly attenuated 1 day after stroke. In the tPA-treated MCAO mice, we found RSG significantly mitigated BBB disruption and hemorrhage development compared to tPA-alone-treated stroke mice. Using flow cytometry and immunostaining, we confirmed that the expression of CD206 was significantly upregulated while the expression of iNOS was down-regulated in microglia of the RSG-treated mice. We further found that the expression of Arg-1 was also upregulated in those tPA and RSG-treated stroke mice and the protection against tPA-induced HT and BBB disruption in these mice were abolished in the presence of PPAR-γ antagonist GW9662 (4 mg/kg, 1 hour before dMCAO through intraperitoneal injection). CONCLUSIONS: RSG treatment protects against BBB damage and ameliorates HT in delayed tPA-treated stroke mice by activating PPAR-γ and favoring microglial polarization toward anti-inflammatory phenotype.
Subject(s)
Hypoglycemic Agents/therapeutic use , Intracranial Hemorrhages/chemically induced , Intracranial Hemorrhages/prevention & control , Plasminogen Activators/adverse effects , Rosiglitazone/therapeutic use , Stroke/complications , Tissue Plasminogen Activator/adverse effects , Anilides/pharmacology , Animals , Anti-Inflammatory Agents/pharmacology , Blood-Brain Barrier/drug effects , Hypoglycemic Agents/administration & dosage , Hypoglycemic Agents/antagonists & inhibitors , Infarction, Middle Cerebral Artery/drug therapy , Infarction, Middle Cerebral Artery/pathology , Injections, Intraperitoneal , Lectins, C-Type/biosynthesis , Lectins, C-Type/genetics , Male , Mannose Receptor , Mannose-Binding Lectins/biosynthesis , Mannose-Binding Lectins/genetics , Mice , Mice, Inbred C57BL , Nitric Oxide Synthase Type II/biosynthesis , Nitric Oxide Synthase Type II/genetics , PPAR gamma/antagonists & inhibitors , Plasminogen Activators/therapeutic use , Receptors, Cell Surface/biosynthesis , Receptors, Cell Surface/genetics , Rosiglitazone/administration & dosage , Rosiglitazone/antagonists & inhibitors , Stroke/drug therapy , Tissue Plasminogen Activator/therapeutic useABSTRACT
Inclusion body myositis is a late onset treatment-refractory autoimmune disease of skeletal muscle associated with a blood autoantibody (anti-cN1A), an HLA autoimmune haplotype, and muscle pathology characterized by cytotoxic CD8+ T cell destruction of myofibres. Here, we report on translational studies of inclusion body myositis patient muscle compared with a diverse set of other muscle disease samples. Using available microarray data on 411 muscle samples from patients with inclusion body myositis (n = 40), other muscle diseases (n = 265), and without neuromuscular disease (normal, n = 106), we identified a signature of T-cell cytotoxicity in inclusion body myositis muscle coupled with a signature of highly differentiated CD8 T-cell effector memory and terminally differentiated effector cells. Further, we examined killer cell lectin-like receptor G1 (KLRG1) as a marker of this population of cells, demonstrated the correlation of KLRG1 gene expression with lymphocyte cytotoxicity across 28 870 human tissue samples, and identified the presence of KLRG1 on pathogenic inclusion body myositis muscle invading T cells and an increase in KLRG1 expressing T cells in inclusion body myositis blood. We examined inclusion body myositis muscle T-cell proliferation by Ki67 immunohistochemistry demonstrating that diseased muscle-invading T cells are minimally or non-proliferative, in accordance with known properties of highly differentiated or terminally differentiated T cells. We found low expression of KLRG1 on infection-protective human lymphoid tissue central memory T cells and autoimmune-protective human blood regulatory T cells. Targeting highly differentiated cytotoxic T cells could be a favourable approach to treatment of inclusion body myositis.
Subject(s)
CD8-Positive T-Lymphocytes/metabolism , Cell Differentiation/physiology , Lectins, C-Type/biosynthesis , Muscle, Skeletal/metabolism , Myositis, Inclusion Body/metabolism , Receptors, Immunologic/biosynthesis , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/pathology , Humans , Lectins, C-Type/immunology , Muscle, Skeletal/immunology , Muscle, Skeletal/pathology , Myositis, Inclusion Body/immunology , Myositis, Inclusion Body/pathology , Receptors, Immunologic/immunologyABSTRACT
BACKGROUND: Multiple sclerosis (MS) involves a misdirected immune attack against myelin in the brain and spinal cord, leading to profound neuroinflammation and neurodegeneration. While the mechanisms of disease pathogenesis have been widely studied, the suppression mechanisms that lead to the resolution of the autoimmune response are still poorly understood. Here, we investigated the role of the C-type lectin receptor macrophage galactose-type lectin (MGL), usually expressed on tolerogenic antigen-presenting cells (APCs), as a negative regulator of autoimmune-driven neuroinflammation. METHODS: We used in silico, immunohistochemical, immunofluorescence, quantitative real-time polymerase chain reaction (qRT-PCR) and flow cytometry analysis to explore the expression and functionality of MGL in human macrophages and microglia, as well as in MS post-mortem tissue. In vitro, we studied the capacity of MGL to mediate apoptosis of experimental autoimmune encephalomyelitis (EAE)-derived T cells and mouse CD4+ T cells. Finally, we evaluated in vivo and ex vivo the immunomodulatory potential of MGL in EAE. RESULTS: MGL plays a critical role in the resolution phase of EAE as MGL1-deficient (Clec10a-/-) mice showed a similar day of onset but experienced a higher clinical score to that of WT littermates. We demonstrate that the mouse ortholog MGL1 induces apoptosis of autoreactive T cells and diminishes the expression of pro-inflammatory cytokines and inflammatory autoantibodies. Moreover, we show that MGL1 but not MGL2 induces apoptosis of activated mouse CD4+ T cells in vitro. In human settings, we show that MGL expression is increased in active MS lesions and on alternatively activated microglia and macrophages which, in turn, induces the secretion of the immunoregulatory cytokine IL-10, underscoring the clinical relevance of this lectin. CONCLUSIONS: Our results show a new role of MGL-expressing APCs as an anti-inflammatory mechanism in autoimmune neuroinflammation by dampening pathogenic T and B cell responses, uncovering a novel clue for neuroprotective therapeutic strategies with relevance for in MS clinical applications.
Subject(s)
Asialoglycoproteins/biosynthesis , Encephalomyelitis, Autoimmune, Experimental/metabolism , Lectins, C-Type/biosynthesis , Membrane Proteins/biosynthesis , Microglia/metabolism , Animals , Cells, Cultured , Encephalomyelitis, Autoimmune, Experimental/immunology , Female , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Microglia/immunology , RatsABSTRACT
Nontumour cells in the tumour microenvironment, especially fibroblasts, contribute to tumour progression and metastasis. The occurrence and evolution of colorectal cancer (CRC) is closely related to cancer-associated fibroblasts (CAFs). The aim of this work was to evaluate the effects of the growth factors and cytokines secreted by CAFs on CRC progression. The secreted cytokines were examined in CAFs by Human Cytokine Antibody array. We screened 37 differentially secreted cytokines in the culture supernatants of CAFs and NFs. CLEC3B, attractin, kallikrein 5 and legumain were selected for further verification. CLEC3B was more highly expressed in the stroma of CRC tissues than the other 3 cytokines. Immunohistochemistry revealed that CLEC3B expression was associated with serosal invasion by CRC. Patients with co-expression of CLEC3B and α-SMA had worse survival outcomes than those with only CLEC3B or α-SMA expression. CLEC3B secreted from CAFs may promote tumour migration. Knockdown of endogenous CLEC3B in CAFs markedly decreased CRC cell migration, while recombinant human CLEC3B clearly promoted CRC cell migration and actin remodelling. In conclusion, our findings suggest that CAFs promote the CRC cell migration and skeletal reorganization by secreting CLEC3B. CLEC3B might be a potential therapeutic molecule for CRC treatment.
Subject(s)
Cancer-Associated Fibroblasts/metabolism , Cancer-Associated Fibroblasts/pathology , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Lectins, C-Type/biosynthesis , Actins/metabolism , Adult , Aged , Biomarkers , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Colorectal Neoplasms/genetics , Disease Progression , Female , Gene Expression Profiling , Humans , Immunohistochemistry , Male , Middle Aged , Neoplasm Grading , Neoplasm Staging , Tumor Microenvironment/geneticsABSTRACT
Although littoral cell angiomas (LCAs) are phenotypically well characterized, the antibodies used to support the diagnosis identify many other cells in the normal spleen, and some may be found in other angiomatous lesions. Based on a langerin/CD207+ LCA index case, langerin and other selected immunohistochemical staining was performed on 10 LCAs, 20 other splenic angiomatous lesions, and 7 reactive lymph nodes to further investigate the role of langerin as a diagnostic tool. Ninety percent (9/10) of LCAs were langerin positive, whereas only 1 (5%) of 20 other splenic vascular lesions was partially positive (P < .00001). All LCAs were CD1a-, CD68+, CD34-, and CD8-; 20% were S100+, 70% CD21+, and 90% cyclin D1+. Ultrastructural studies of one LCA did not show Birbeck-type granules in definite lining cells. Sinus lining cells in 7 of 7 reactive lymph nodes showed partial langerin positivity, and 4 of 4 showed partial cyclin D1 positivity. In conclusion, langerin staining is an easily interpreted and highly sensitive and specific (sensitivity [0.90], specificity [0.95]) ancillary study to help distinguish LCA from other vascular tumors of the spleen. Whether this represents cross-reactivity or true CD207 expression is uncertain, as other immunohistochemical and ultrastructural studies do not support a Langerhans cell origin. The cyclin D1 staining seen in most LCA would be consistent with their expression of other selected vascular and histiocytic markers. The similar staining pattern in some lymph node sinus lining cells suggests a possible similar cell of origin, although LCA of lymph nodes is not described.
Subject(s)
Antigens, CD/biosynthesis , Biomarkers, Tumor/analysis , Hemangioma/diagnosis , Lectins, C-Type/biosynthesis , Mannose-Binding Lectins/biosynthesis , Splenic Neoplasms/diagnosis , Adolescent , Adult , Aged , Aged, 80 and over , Antigens, CD/analysis , Female , Humans , Lectins, C-Type/analysis , Male , Mannose-Binding Lectins/analysis , Middle Aged , Sensitivity and Specificity , Young AdultABSTRACT
OBJECTIVE: The aim of the study was to investigate the role of dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) in intestinal epithelial cells (IECs) in regulating sepsis-induced acute intestinal injury and systemic inflammatory response. METHODS: To induce sepsis condition, Male C57BL/6 mice were exposed to cecal ligation and puncture (CLP) in vivo, whereas a normal human IECs line (FHs74Int) was stimulated with lipopolysaccharide (LPS) in vitro. DC-SIGN siRNA pretreatment was used to knock down DC-SIGN expression both in vivo and in vitro. The expression of DC-SIGN was detected by western blot and immunohistochemistry. The expression of total and phosphorylation of ERK1/2 and NF-κB/p65 was examined by western blot. The levels of cytokines in serum and culture supernatant were measured by ELISA. The survival rate and organ injures of septic mice were also assessed. RESULTS: In vivo, DC-SIGN expression in mouse IECs was time-dependently upregulated by CLP. CLP-induced phosphorylation of ERK1/2 and NF-κB/p65 was effectively inhibited by DC-SIGN siRNA pretreatment, leading to the decrease of systemic inflammatory cytokines (TNF-α, IL-1ß, IL-6, IL-10, and IFN-γ), which alleviated multiple organ injuries and increased the survival rate of septic mice. In vitro, DC-SIGN expression in FHs74Int was significantly upregulated by LPS stimulation in a time- and dose-dependent manner. DC-SIGN knockdown abolished LPS-induced ERK1/2 and NF-κB/p65 phosphorylation, resulting in the decrease of cytokines release by FHs74Int. CONCLUSIONS: Sepsis-induced DC-SIGN expression in IECs plays a significant role in regulating acute intestinal injury and systemic inflammatory response. The inhibition of DC-SIGN exhibited protective effects on sepsis-associated organ injury and systemic inflammation.
Subject(s)
Cell Adhesion Molecules/biosynthesis , Epithelial Cells , Gene Expression Regulation , Intestinal Diseases , Intestinal Mucosa , Lectins, C-Type/biosynthesis , MAP Kinase Signaling System , Receptors, Cell Surface/biosynthesis , Sepsis , Animals , Cell Line , Epithelial Cells/metabolism , Epithelial Cells/pathology , Humans , Intestinal Diseases/etiology , Intestinal Diseases/metabolism , Intestinal Diseases/pathology , Intestinal Mucosa/injuries , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Male , Mice , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Sepsis/complications , Sepsis/metabolism , Sepsis/pathology , Transcription Factor RelA/metabolismABSTRACT
There is a need for more detailed elucidation of T-cell immunity in chikungunya infection. CD8 T cells are one of main actors against viruses. Here, we analysed CD8+ T lymphocytes from patients in the acute and chronic phases of chikungunya disease (CHIKD). Our results demonstrate that CD8+ T cells expressed higher ex vivo granzyme B, perforin and CD107A expression in patients in the acute phase of CHIKD compared with healthy individuals and higher ex vivo expression of CD69, interleukin-17A, interleukin-10 and CD95 ligand, and co-expression of CD95/CD95 ligand. These results elucidate the importance of these lymphocytes, demonstrating immune mechanisms mediated in human chikungunya infection.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Chikungunya Fever/immunology , Chikungunya virus/immunology , Cytokines/biosynthesis , Lymphocyte Activation/immunology , Antigens, CD/biosynthesis , Antigens, CD/immunology , Antigens, Differentiation, T-Lymphocyte/biosynthesis , Antigens, Differentiation, T-Lymphocyte/immunology , Chikungunya Fever/pathology , Chikungunya Fever/virology , Cytokines/immunology , Cytotoxicity, Immunologic/immunology , Fas Ligand Protein/biosynthesis , Fas Ligand Protein/immunology , Granzymes/biosynthesis , Granzymes/immunology , Humans , Interleukin-10/biosynthesis , Interleukin-10/immunology , Interleukin-17/biosynthesis , Interleukin-17/immunology , Lectins, C-Type/biosynthesis , Lectins, C-Type/immunology , Lysosomal-Associated Membrane Protein 1/biosynthesis , Lysosomal-Associated Membrane Protein 1/immunology , Perforin/biosynthesis , Perforin/immunology , fas Receptor/biosynthesis , fas Receptor/immunologyABSTRACT
BACKGROUND: MicroRNA-126 (miR-126), a distinct miRNA family member, has been reported to be involved in the development and function of some types of immune cells. However, the potential role of miR-126 in the development of CD4+ T cells remains to be elucidated. OBJECTIVES: To investigate the potential role of miR-126 in the development of CD4+ T cells in the thymus and explore its significance. METHODS: The relative expression level of miR-126 in thymus CD4+ single-positive (SP) cells was detected by Real-Time PCR assay. The possible change in thymus tissue was assessed by histopathology. The total cell number of thymocytes and the expression of activation-associated molecules including CD62L, CD69, and CD44, as well as proliferation-associated nuclear antigen Ki-67, in CD4+ SP cells were assessed by flow cytometric analysis. The expression of IRS-1 and related signaling pathways including Akt and Erk were determined by flow cytometric analysis. RESULTS: Compared with that in wild-type (WT) mice, the total cell number of thymocytes in miR-126 knockdown (KD) mice increased significantly. Moreover, the proportion and absolute cell number of thymic CD4+ SP cells decreased significantly in miR-126 KD mice. Further analysis showed that the frequencies of activation-associated molecules including CD62L, CD69, and CD44, as well as proliferation-associated nuclear antigen Ki-67 in CD4+ SP cells also changed significantly, respectively. Mechanism aspect, the expression level of IRS-1, a putative target of miR-126, increased significantly in CD4+ SP cells in miR-126 KD mice. Moreover, the expression levels of the signaling molecules phosphorylated (p)-Akt and p-Erk also changed significantly. CONCLUSIONS: Our work is the first to reveal a previously unknown role of miR-126 in the development of CD4+ SP cells in the thymus, which might ultimately benefit studies on development of thymocytes.
Subject(s)
CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/immunology , Cell Differentiation/genetics , Lymphocyte Activation/genetics , MicroRNAs/genetics , Animals , Antigens, CD/biosynthesis , Antigens, Differentiation, T-Lymphocyte/biosynthesis , Cell Differentiation/immunology , Flow Cytometry , Hyaluronan Receptors/biosynthesis , Insulin Receptor Substrate Proteins/metabolism , Ki-67 Antigen/biosynthesis , L-Selectin/biosynthesis , Lectins, C-Type/biosynthesis , Lymphocyte Activation/immunology , Mice , Mice, Knockout , Thymocytes/cytology , Thymocytes/immunology , Thymus Gland/cytologyABSTRACT
BACKGROUND: Chronic lymphocytic leukemia (CLL) is a condition characterized by the accumulation of morphologically mature monoclonal lymphocytes B with the CD19+/CD5+/CD23+ phenotype in lymphoid tissue, peripheral blood and bone marrow. The clinical course of patients with CLL is heterogeneous, ranging from indolent to aggressive. The role of lymphocyte activation in the natural history of CLL is still a matter of discussion. OBJECTIVES: The aim of this study was to determine the percentages and absolute numbers of lymphocytes B and T in peripheral blood and bone marrow of CLL patients. Moreover, we analyzed the relationship between the number of CD25-positive and CD69-positive lymphocytes and the established prognostic factors in CLL. MATERIAL AND METHODS: The study included 80 untreated patients with CLL and 20 healthy subjects. The immunophenotype of peripheral blood mononuclear cells (in both groups) and bone marrow cells (solely in the CLL group) was determined by means of flow cytometry. RESULTS: Patients with CLL showed a higher absolute number of activated lymphocytes B with phenotypes CD19+CD25+ and CD19+CD69+, as well as a higher absolute number of CD3+CD25+ lymphocytes T than the controls. The enhanced activation of peripheral blood and bone marrow lymphocytes was associated with higher Rai stages, an increased concentration of lactate dehydrogenase and beta-2 microglobulin and the progression of the disease. The number of lymphocytes B CD19+ZAP-70+ correlated positively with the number of CD19+CD25+ B cells and CD3+CD69+ T cells. CONCLUSIONS: The study confirmed the association between an unfavorable prognosis and a high expression of activation markers in CLL patients. The determination of CD25+ and CD69+ lymphocytes T and B constitutes a valuable diagnostic tool, completing the cytometric evaluation of CLL.
Subject(s)
B-Lymphocytes/pathology , Leukemia, Lymphocytic, Chronic, B-Cell/blood , Lymphocyte Subsets/immunology , T-Lymphocytes/pathology , Adult , Aged , Aged, 80 and over , Antigens, CD/biosynthesis , Antigens, Differentiation, T-Lymphocyte/biosynthesis , B-Lymphocytes/immunology , Female , Humans , Interleukin-2 Receptor alpha Subunit/biosynthesis , Lectins, C-Type/biosynthesis , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Lymphocyte Activation/immunology , Lymphocyte Count , Male , Middle Aged , Prognosis , T-Lymphocytes/immunologyABSTRACT
The absence of tumor necrosis factor (TNF) causes lethal infection by Leishmania major in normally resistant C57BL/6J (B6.WT) mice. The underlying pathogenic mechanism of this fatal disease has so far remained elusive. We found that B6.WT mice deficient for the tnf gene (B6.TNF-/-) displayed not only a non-healing cutaneous lesion but also a serious infection of the liver upon L. major inoculation. Infected B6.TNF-/- mice developed an enlarged liver that showed increased inflammation. Furthermore, we detected an accumulating monocyte-derived macrophage population (CD45+F4/80+CD11bhiLy6Clow) that displayed a M2 macrophage phenotype with high expression of CD206, arginase-1, and IL-6, supporting the notion that IL-6 could be involved in M2 differentiation. In in vitro experiments, we demonstrated that IL-6 upregulated M-CSF receptor expression and skewed monocyte differentiation from dendritic cells to macrophages. This was countered by the addition of TNF. Furthermore, TNF interfered with the activation of IL-6-induced gp130-signal transducer and activator of transcription (STAT) 3 and IL-4-STAT6 signaling, thereby abrogating IL-6-facilitated M2 macrophage polarization. Therefore, our results support the notion of a general role of TNF in the inflammatory activation of macrophages and define a new role of IL-6 signaling in macrophage polarization downstream of TNF.
Subject(s)
Interleukin-6/immunology , Liver/immunology , Macrophage Activation/immunology , Macrophages/immunology , Tumor Necrosis Factor-alpha/genetics , Animals , Arginase/biosynthesis , Cell Differentiation/genetics , Cell Differentiation/immunology , Cells, Cultured , Cytokine Receptor gp130/metabolism , Inflammation/immunology , Interleukin-4/metabolism , Interleukin-6/biosynthesis , Lectins, C-Type/biosynthesis , Leishmania major/immunology , Leishmaniasis, Cutaneous/immunology , Leishmaniasis, Cutaneous/parasitology , Liver/parasitology , Liver/pathology , Macrophage Activation/genetics , Macrophages/cytology , Mannose Receptor , Mannose-Binding Lectins/biosynthesis , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Monocytes/cytology , Parasite Load , Receptor, Macrophage Colony-Stimulating Factor/biosynthesis , Receptors, Cell Surface/biosynthesis , STAT3 Transcription Factor/metabolism , STAT6 Transcription Factor/metabolismABSTRACT
PURPOSE: Tumor cell resistance to platinum-based chemotherapeutic agents is one of the major hurdles to successful cancer treatment with these drugs, and is associated with alterations in tumor cell immune evasion and immunomodulatory properties. Immunocyte targeting is considered as a relevant approach to fight drug-resistant cancer. In this study, immunological hallmarks of cis-DDP-resistant Lewis lung carcinoma cells (LLC/R9) were investigated. METHODS: Immunological features of LLC/R9 cells cultured in vitro in normoxic and hypoxic conditions as well as of those that were grown in vivo were examined. The expression of immunologically relevant genes was evaluated by RT-PCR. Tumor cell susceptibility to the macrophage contact tumoricidal activity and NK-mediated cytolysis was investigated in MTT test. TNF-α-mediated tumor cell apoptosis as well as macrophage phagocytosis, oxidative metabolism, and CD206 expression after the treatment with conditioned media from normoxic and hypoxic tumor cells were studied by flow cytometry. Flow cytometry was also used to characterize dendritic cell maturity. RESULTS: When growing in vitro, LLC/R9 were characterized by slightly increased immunosuppressive cytokine gene expression. Transition to in vivo growth was associated with the enhancement of transcription of these genes in tumor cells. LLC/R9 cells had lowered sensitivity to contact-dependent macrophage-mediated cytotoxicity and to the TNFα-mediated apoptosis in vitro. Conditioned media from hypoxic LLC/R9 cells stimulated reactive oxygen species generation and CD206 expression in non-sensitized macrophages. Acquisition of drug resistance by LLC/R9 cells was associated with their increased sensitivity to NK-cell-mediated cytolysis. Meanwhile, the treatment of LLCR/9-bearing animals with generated ex vivo and loaded with LLC/R9 cell-lysate dendritic cells (DCs) resulted in profoundly enhanced tumor metastasizing. CONCLUSION: Decreased sensitivity to macrophage cytolysis, polarizing effect on DCs maturation along with increased susceptibility to NK-cell cytotoxic action promote extensive local growth of chemoresistant LLC/R9 tumors in vivo, but hamper their metastasizing.
Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Lewis Lung/drug therapy , Carcinoma, Lewis Lung/immunology , Cisplatin/pharmacology , Drug Resistance, Neoplasm/immunology , Algorithms , Animals , Apoptosis/immunology , Cell Line, Tumor , Dendritic Cells/immunology , Humans , Killer Cells, Natural/immunology , Lectins, C-Type/biosynthesis , Macrophages, Peritoneal/immunology , Mannose Receptor , Mannose-Binding Lectins/biosynthesis , Mice , Mice, Inbred C57BL , Phagocytosis , Reactive Oxygen Species , Receptors, Cell Surface/biosynthesis , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolismABSTRACT
C-type lectin domain family 3 member A (CLEC3A) is a poorly characterized protein belonging to the superfamily of C-type lectins. Its closest homologue tetranectin binds to the kringle 4 domain of plasminogen and enhances its association with tissue plasminogen activator (tPA) thereby enhancing plasmin production, but whether CLEC3A contributes to plasminogen activation is unknown. Here, we recombinantly expressed murine and human full-length CLEC3As as well as truncated forms of CLEC3A in HEK-293 Epstein-Barr nuclear antigen (EBNA) cells. We analyzed the structure of recombinant CLEC3A by SDS-PAGE and immunoblot, glycan analysis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, size-exclusion chromatography, circular dichroism spectroscopy, and electron microscopy; compared the properties of the recombinant protein with those of CLEC3A extracted from cartilage; and investigated its tissue distribution and extracellular assembly by immunohistochemistry and immunofluorescence microscopy. We found that CLEC3A mainly occurs as a monomer, but also forms dimers and trimers, potentially via a coiled-coil α-helix. We also noted that CLEC3A can be modified with chondroitin/dermatan sulfate side chains and tends to oligomerize to form higher aggregates. We show that CLEC3A is present in resting, proliferating, and hypertrophic growth-plate cartilage and assembles into an extended extracellular network in cultures of rat chondrosarcoma cells. Further, we found that CLEC3A specifically binds to plasminogen and enhances tPA-mediated plasminogen activation. In summary, we have determined the structure, tissue distribution, and molecular function of the cartilage-specific lectin CLEC3A and show that CLEC3A binds to plasminogen and participates in tPA-mediated plasminogen activation.