ABSTRACT
The involvement of Group 3 innate lymphoid cells (ILC3s) and dendritic cells (DCs) in chronic lung inflammation has been increasingly regarded as the key to understand the inflammatory mechanisms of smoke-related chronic obstructive pulmonary disease (COPD). However, the mechanism underlying the engagement of both remains unclear. Our study aimed to explore NCR-ILC3 differentiation in the lungs of mice exposed to cigarette smoke (CS) and to further investigate whether DCs activated by CS exposure contribute to the differentiation of ILCs into NCR-ILC3s. The study involved both in vivo and in vitro experiments. In the former, the frequencies of lung NCR-ILC3s and NKp46-IL-17A+ ILCs and the expression of DCs, CD40, CD86, IL-23, and IL-1ß quantified by flow cytometry were compared between CS-exposed mice and air-exposed mice. In the latter, NKp46-IL-17A+ ILC frequencies quantified by flow cytometry were compared after two cocultures, one involving lung CD45+Lin-CD127+ ILCs sorted from air-exposed mice and DCs sifted by CD11c magnetic beads from CS-exposed mice and another including identical CD45+Lin-CD127+ ILCs and DCs from air-exposed mice. The results indicated significant increases in the frequencies of NCR-ILC3s and NKp46-IL-17A+ ILCs; in the expression of DCs, CD40, CD86, IL-23, and IL-1ß in CS-exposed mice; and in the frequency of NKp46-IL-17A+ ILCs after the coculture with DCs from CS-exposed mice. In conclusion, CS exposure increases the frequency of lung ILCs and NCR-ILC3s. CS-induced DC activation enhances the differentiation of ILCs into NCR-ILC3s, which likely acts as a mediating step in the involvement of NCR-ILC3s in chronic lung inflammation.
Subject(s)
Cell Differentiation , Dendritic Cells , Interleukin-17 , Interleukin-1beta , Lung , Natural Cytotoxicity Triggering Receptor 1 , Animals , Dendritic Cells/immunology , Mice , Lung/immunology , Lung/metabolism , Natural Cytotoxicity Triggering Receptor 1/metabolism , Interleukin-17/metabolism , Interleukin-1beta/metabolism , Lymphocytes/immunology , Lymphocytes/metabolism , Interleukin-23/metabolism , B7-2 Antigen/metabolism , Mice, Inbred C57BL , Smoke/adverse effects , Pulmonary Disease, Chronic Obstructive/immunology , CD40 Antigens/metabolism , Cigarette Smoking/adverse effects , Immunity, Innate , Antigens, Ly/metabolism , Coculture Techniques , MaleABSTRACT
Individuals with type 2 diabetes (T2D) show signs of low-grade inflammation, which is related to the development of insulin resistance and beta-cell dysfunction. However, the underlying triggers remain unknown. The gut microbiota is a plausible source as it comprises pro-inflammatory bacteria, bacterial metabolites and viruses, including (bacterio)phages. These prokaryotic viruses have been shown to mediate inflammatory responses in gastrointestinal disease. Given the differences in phage populations in healthy individuals versus those with cardiometabolic diseases such as T2D, we here questioned whether phages from T2D individuals would have increased immunogenic potential. To address this, we isolated intestinal phages from a fresh stool sample of healthy controls and individuals with newly diagnosed, treatment-naive T2D. Phages were purified using cesium chloride ultracentrifugation and incubated with healthy donor dendritic cells (DCs) and autologous T cells. Donors with T2D had slightly higher free viral particle numbers compared to healthy controls (p = .1972), which has been previously associated with disease states. Further, phages from T2D induced a higher inflammatory response in DCs and T cells than phages from HC. For example, the expression of the co-stimulatory molecule CD86 on DCs (p < .001) and interferon-y secretion from T cells (p < .01) were increased when comparing the two groups. These results suggest that phages might play a role in low-grade inflammation in T2D individuals.
Subject(s)
Bacteriophages , Coculture Techniques , Dendritic Cells , Diabetes Mellitus, Type 2 , Inflammation , Humans , Diabetes Mellitus, Type 2/immunology , Dendritic Cells/immunology , Bacteriophages/isolation & purification , Bacteriophages/physiology , Male , Middle Aged , Inflammation/immunology , Inflammation/virology , Female , Feces/virology , Feces/microbiology , Adult , Gastrointestinal Microbiome , T-Lymphocytes/immunology , Aged , B7-2 Antigen/metabolismABSTRACT
This study investigates the mechanism through which paeoniflorin inhibits TSLP expression to regulate dendritic cell activation in corticosteroid-dependent dermatitis treatment. Utilizing databases like TCMSP, we identified paeoniflorin's components, targets, and constructed networks. Molecular docking and gene enrichment analysis helped pinpoint key targets and pathways affected by paeoniflorin. In vitro and in vivo models were used to study CD80, CD86, cytokines, T-cell activation, skin lesions, histopathological changes, TSLP, CD80, and CD86 expression. Our study revealed paeoniflorin's active constituent targeting IL-6 in corticosteroid-dependent dermatitis. In vitro experiments demonstrated reduced TSLP expression, CD80, CD86, and cytokine secretion post-paeoniflorin treatment. In vivo, paeoniflorin significantly decreased skin lesion severity, cytokine levels, TSLP, CD80, and CD86 expression. The study highlights paeoniflorin's efficacy in inhibiting TSLP expression and suppressing dendritic cell activation in corticosteroid-dependent dermatitis, suggesting its potential as a therapeutic intervention. Additionally, it offers insights into the complex molecular mechanisms underlying paeoniflorin's anti-inflammatory properties in treating corticosteroid-dependent dermatitis.
Subject(s)
Cytokines , Dendritic Cells , Glucosides , Monoterpenes , Dendritic Cells/drug effects , Dendritic Cells/immunology , Dendritic Cells/metabolism , Glucosides/pharmacology , Glucosides/therapeutic use , Animals , Cytokines/metabolism , Monoterpenes/pharmacology , Monoterpenes/therapeutic use , Humans , Mice , Dermatitis/drug therapy , Dermatitis/immunology , Dermatitis/metabolism , Interleukin-6/metabolism , Molecular Docking Simulation , Skin/pathology , Skin/drug effects , Skin/immunology , Skin/metabolism , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Disease Models, Animal , B7-1 Antigen/metabolism , B7-2 Antigen/metabolism , Male , Thymic Stromal Lymphopoietin , Lymphocyte Activation/drug effectsABSTRACT
Mixed lymphocyte culture under the blockade of CD80/CD86-CD28 co-stimulation induces anergic (completely hyporesponsive) T cells with immune suppressive function (inducible suppressing T cells: iTS cells). Previously, iTS cell therapy has demonstrated outstanding benefits in clinical trials for organ transplantation. Here, we examined whether peptide antigen-specific iTS cells are inducible. DO 11.10 iTS cells were obtained from splenocytes of BALB/c DO 11.10 mice by stimulation with OVA peptide and antagonistic anti-CD80/CD86 mAbs. When DO 11.10 iTS or Foxp3- DO 11.10 iTS cells were stimulated with OVA, these cells produced IL-13, but not IL-4. DO 11.10 iTS cells decreased IL-4 and increased IL-13 production from OVA-stimulated naïve DO 11.10 splenocytes. When Foxp3+ DO 11.10 iTS cells were prepared, these cells significantly inhibited the production of IL-4 and IL-13 compared with freshly isolated Foxp3+ DO 11.10 T cells. Moreover, an increase in the population expressing OX40, ICOS, and 4-1BB suggested activation of Foxp3+ DO 11.10 iTS cells. Thus, blockade of CD80/CD86-CD28 co-stimulation during peptide antigen stimulation augments the inhibitory function of Foxp3+ regulatory T cells, and does not induce anergic Foxp3- conventional T cells. Peptide-specific Foxp3+ regulatory iTS cells could be useful for the treatment of allergic and autoimmune diseases without adverse effects.
Subject(s)
B7-1 Antigen , B7-2 Antigen , CD28 Antigens , T-Lymphocytes, Regulatory , Animals , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , CD28 Antigens/immunology , CD28 Antigens/metabolism , Mice , B7-1 Antigen/metabolism , B7-1 Antigen/immunology , B7-2 Antigen/metabolism , B7-2 Antigen/immunology , Mice, Inbred BALB C , Forkhead Transcription Factors/metabolism , Peptides/pharmacology , Peptides/immunology , Lymphocyte Activation/immunology , Interleukin-4/metabolism , Interleukin-4/immunology , Interleukin-13/metabolism , Interleukin-13/immunology , Ovalbumin/immunology , Spleen/immunology , Spleen/cytology , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/immunologyABSTRACT
Dendritic cells (DCs) can initiate immune response through the presenting antigens to naïve T lymphocytes. Esculeoside A (EsA), a spirosolane glycoside, is reported as a major component in the ripe fruit of tomato. Little is known about the effect of tomato saponin on mice bone marrow-derived DCs. This study revealed that EsA and its aglycon, esculeogenin A (Esg-A), attenuated the phenotypic and functional maturation of murine DCs stimulated by lipopolysaccharide (LPS). We found that EsA/Esg-A down-regulated the expression of major histocompatibility complex type II molecules and costimulatory molecule CD86 after LPS stimulation. It was also determined that EsA-/Esg-A-treated DCs were poor stimulators of allogeneic T-cell proliferation and exhibited impaired interleukin-12 and TNF-α production. Additionally, EsA/Esg-A was able to inhibit TLR4-related and p-NFκB signaling pathways. This study shows new insights into the immunopharmacology of EsA/Esg-A, and represents a novel approach to controlling DCs for therapeutic application.
Subject(s)
Dendritic Cells , Saponins , Signal Transduction , Solanum lycopersicum , Toll-Like Receptor 4 , Animals , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Dendritic Cells/immunology , Toll-Like Receptor 4/metabolism , Signal Transduction/drug effects , Saponins/pharmacology , Mice , NF-kappa B/metabolism , Lipopolysaccharides/pharmacology , Mice, Inbred C57BL , Interleukin-12/metabolism , Cell Proliferation/drug effects , Mice, Inbred BALB C , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , T-Lymphocytes/immunology , Tumor Necrosis Factor-alpha/metabolism , Fruit/chemistry , B7-2 Antigen/metabolism , SapogeninsABSTRACT
When developing a program of preclinical studies of human cell-based drugs intended for adoptive immunotherapy of cancer patients, the biological effect should be substantiated by data describing their immunological action. Administration and study of human autologous dendritic cell vaccine to immunocompetent animals are not adequate in terms of immunological compatibility. It is possible to use immunocompromised, knockout, or transgenic animals or to obtain a homologous cellular product, namely, a preparation based on animal cells using a technology similar to obtaining the original preparation for clinical practice in humans. Within the framework of this study, we have developed a protocol for obtaining a homologous cell product based on animal dendritic cells (mice, rats) according to a similar technology for obtaining human vaccine dendritic cells, and demonstrated the comparability of morphological characteristics and expression of differentiation antigens of dendritic cells (CD11c, CD80, CD86, and CD83) of animals (mice) and humans.
Subject(s)
Cancer Vaccines , Dendritic Cells , Immunotherapy, Adoptive , Animals , Dendritic Cells/immunology , Dendritic Cells/drug effects , Cancer Vaccines/immunology , Mice , Humans , Rats , Immunotherapy, Adoptive/methods , B7-1 Antigen/immunology , B7-1 Antigen/metabolism , B7-1 Antigen/genetics , CD11c Antigen/metabolism , CD11c Antigen/immunology , B7-2 Antigen/metabolism , B7-2 Antigen/immunology , B7-2 Antigen/geneticsABSTRACT
BACKGROUND: The surplus cytokines remaining after use in the early stages of the inflammatory response stimulate immune cells even after the response is over, causing a secondary inflammatory response and ultimately damaging the host, which is called a cytokine storm. Inhibiting heat shock protein 90 (Hsp90), which has recently been shown to play an important role in regulating inflammation in various cell types, may help control excessive inflammatory responses and cytokine storms. METHODS: We discovered an anti-inflammatory compound by measuring the inhibitory effect of CD86 expression on spleen DCs (sDCs) using the chemical compounds library of Hsp90 inhibitors. Subsequently, to select the hit compound, the production of cytokines and expression of surface molecules were measured on the bone marrow-derived DCs (BMDCs) and peritoneal macrophages. Then, we analyzed the response of antigen-specific Th1 cells. Finally, we confirmed the effect of the compound using acute lung injury (ALI) and delayed-type hypersensitivity (DTH) models. RESULTS: We identified Be01 as the hit compound, which reduced CD86 expression the most in sDCs. Treatment with Be01 decreased the production of pro-inflammatory cytokines (IL-6, TNF-α, and IL-1ß) in BMDC and peritoneal macrophages stimulated by LPS. Under the DTH model, Be01 treatment reduced ear swelling and pro-inflammatory cytokines in the spleen. Similarly, Be01 treatment in the ALI model decreased neutrophil infiltration and lower levels of secreted cytokines (IL-6, TNF-α). CONCLUSIONS: Reduction of CD80 and CD86 expression on DCs by Be01 indicates reduced secondary inflammatory response by Th1 cells, and reduced release of pro-inflammatory cytokines by peritoneal macrophages may initially control the cytokine storm.
Subject(s)
Anti-Inflammatory Agents , Cytokines , Dendritic Cells , HSP90 Heat-Shock Proteins , Macrophages, Peritoneal , Mice, Inbred C57BL , Animals , Dendritic Cells/drug effects , Dendritic Cells/immunology , HSP90 Heat-Shock Proteins/antagonists & inhibitors , HSP90 Heat-Shock Proteins/metabolism , Cytokines/metabolism , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Mice , Macrophages, Peritoneal/drug effects , Macrophages, Peritoneal/immunology , Macrophages, Peritoneal/metabolism , Hypersensitivity, Delayed/drug therapy , Hypersensitivity, Delayed/immunology , B7-2 Antigen/metabolism , Acute Lung Injury/drug therapy , Acute Lung Injury/immunology , Cells, Cultured , Cytokine Release Syndrome/drug therapy , Cytokine Release Syndrome/immunology , Th1 Cells/immunology , Th1 Cells/drug effects , Inflammation/drug therapy , Inflammation/immunology , Female , Disease Models, Animal , Spleen/immunology , Spleen/drug effectsABSTRACT
Obesity is a leading risk factor for progression and metastasis of many cancers1,2, yet can in some cases enhance survival3-5 and responses to immune checkpoint blockade therapies, including anti-PD-1, which targets PD-1 (encoded by PDCD1), an inhibitory receptor expressed on immune cells6-8. Although obesity promotes chronic inflammation, the role of the immune system in the obesity-cancer connection and immunotherapy remains unclear. It has been shown that in addition to T cells, macrophages can express PD-19-12. Here we found that obesity selectively induced PD-1 expression on tumour-associated macrophages (TAMs). Type I inflammatory cytokines and molecules linked to obesity, including interferon-γ, tumour necrosis factor, leptin, insulin and palmitate, induced macrophage PD-1 expression in an mTORC1- and glycolysis-dependent manner. PD-1 then provided negative feedback to TAMs that suppressed glycolysis, phagocytosis and T cell stimulatory potential. Conversely, PD-1 blockade increased the level of macrophage glycolysis, which was essential for PD-1 inhibition to augment TAM expression of CD86 and major histocompatibility complex I and II molecules and ability to activate T cells. Myeloid-specific PD-1 deficiency slowed tumour growth, enhanced TAM glycolysis and antigen-presentation capability, and led to increased CD8+ T cell activity with a reduced level of markers of exhaustion. These findings show that obesity-associated metabolic signalling and inflammatory cues cause TAMs to induce PD-1 expression, which then drives a TAM-specific feedback mechanism that impairs tumour immune surveillance. This may contribute to increased cancer risk yet improved response to PD-1 immunotherapy in obesity.
Subject(s)
Neoplasms , Obesity , Programmed Cell Death 1 Receptor , Tumor-Associated Macrophages , Animals , Female , Humans , Male , Mice , Antigen Presentation/drug effects , B7-2 Antigen/antagonists & inhibitors , B7-2 Antigen/immunology , B7-2 Antigen/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Line, Tumor , Glycolysis/drug effects , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class II/immunology , Immune Checkpoint Inhibitors/pharmacology , Immune Checkpoint Inhibitors/therapeutic use , Inflammation Mediators/immunology , Inflammation Mediators/metabolism , Lymphocyte Activation , Mechanistic Target of Rapamycin Complex 1/metabolism , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Mice, Inbred C57BL , Neoplasms/drug therapy , Neoplasms/immunology , Neoplasms/metabolism , Neoplasms/pathology , Obesity/immunology , Obesity/metabolism , Phagocytosis/drug effects , Programmed Cell Death 1 Receptor/metabolism , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Tumor-Associated Macrophages/immunology , Tumor-Associated Macrophages/metabolism , Tumor-Associated Macrophages/drug effectsABSTRACT
To explore the effect of K33 only mutant ubiquitin (K33O) on bone marrow-derived dendritic cells' (BMDCs') maturity, antigen uptake capability, surface molecule expressions and BMDC-mediated CTL priming, and further investigate the role of PI3K-Akt engaged in K33O-increased BMDC maturation, antigen uptake and presentation, surface molecule expressions and BMDC-based CTL priming. BMDCs were conferred K33O and other ubiquitin mutants (K33R, K48R, K63R-mutant ubiquitin) incubation or LY294002 and wortmannin pretreatment. PI3K-Akt phosphorylation, antigen uptake, antigenic presentation and CD86/MHC class I expression in BMDC were determined by western blot or flow cytometry. BMDC-based CTL proliferation and priming were determined by in vitro mixed lymphocyte reaction (MLR), ex vivo enzyme-linked immunospot assay (Elispot) and flow cytometry with intracellular staining, respectively. The treatment with K33O effectively augmented PI3K-Akt phosphorylation, BMDCs' antigen uptake, antigenic presentation, CD86/MHC class I and CD11c expressions. MLR, Elispot and flow cytometry revealed that K33O treatment obviously enhanced CTL proliferation, CTL priming and perforin/granzyme B expression. The pretreatment with PI3K-Akt inhibitors efficiently abrogated K33O's effects on BMDC. The replenishment of K33 only mutant ubiquitin augments BMDC-mediated CTL priming in bone marrow-derived dendritic cells via PI3K-Akt signalling.
Subject(s)
Antigen Presentation , Bone Marrow Cells , Dendritic Cells , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins c-akt , Signal Transduction , T-Lymphocytes, Cytotoxic , Ubiquitin , Dendritic Cells/immunology , Dendritic Cells/metabolism , Animals , Mice , Proto-Oncogene Proteins c-akt/metabolism , Ubiquitin/metabolism , T-Lymphocytes, Cytotoxic/immunology , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Antigen Presentation/immunology , Mice, Inbred C57BL , Phosphorylation , Lymphocyte Activation , Cell Differentiation , Mutation , Morpholines/pharmacology , Lymphocyte Culture Test, Mixed , Cell Proliferation , B7-2 Antigen/metabolism , B7-2 Antigen/genetics , B7-2 Antigen/immunology , Cells, Cultured , Chromones/pharmacology , Wortmannin/pharmacology , Androstadienes/pharmacologyABSTRACT
Dendritic cells (DCs) play a pivotal role in maintaining immune tolerance. Using recombinant adenovirus (rAd) to deliver vectors to immature dendritic cells (imDCs) is an important method for studying the tolerogenic function of DCs. We found that using RPMI medium and a higher MOI during transduction increased the expression of CD80, CD86, and MHC-II on the surface of imDCs. Our data reveal a significant increase in the secretion of the pro-inflammatory cytokine IL-6 in the group showing the most pronounced phenotypic changes. In the mouse heart transplant model, imDCs with unstable phenotype and function due to adenoviral transduction resulted in an increased proportion of Th1 and Th17 cells in recipients. However, these effects can be managed, and our proposed optimized transduction strategy significantly minimizes these adverse effects. Our study holds significant implications for the development and optimization of immunotherapy utilizing tolerogenic dendritic cells.
Subject(s)
Adenoviridae , Dendritic Cells , Genetic Vectors , Immunotherapy , Transduction, Genetic , Dendritic Cells/immunology , Animals , Adenoviridae/genetics , Mice , Immunotherapy/methods , Genetic Vectors/genetics , Heart Transplantation , Mice, Inbred C57BL , Interleukin-6/metabolism , Immune Tolerance , B7-1 Antigen/genetics , B7-1 Antigen/metabolism , Th1 Cells/immunology , Th17 Cells/immunology , B7-2 Antigen/metabolism , B7-2 Antigen/geneticsABSTRACT
T-cells play a significant role in the development of autoimmune diseases. The CD28-B7 costimulatory pathway is crucial for activating T-cells, and blocking this pathway is essential for treating autoimmune diseases. Therapeutic antibodies and fusion proteins that target costimulatory molecules like CD80, CD86, CTLA-4, and CD28 have been developed to explore the costimulation process and as targeted treatments. To advance our understanding of costimulation in autoimmunity and the inhibition of the costimulatory pathway, it is crucial to have an accurate, precise, and direct method for detecting and quantifying the soluble form of these molecules in body fluids and various biological systems. Herein, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying the four costimulatory proteins depending on the signature peptides derived from the soluble isoform of these proteins in multiple reaction monitoring (MRM) mode. The method was validated using the US FDA guidelines. The LOQ was determined as â¼0.5â¯nM for the four analytes, with quantification extended to 20â¯nM with a correlation coefficient of R2>0.998. The developed MRM method was used to analyze on-bead digested protein mixtures to establish a competitive assay for the CD28-B7 costimulatory pathway using CTLA4-Ig (Abatacept ™) as an FDA-approved drug for rheumatoid arthritis. The IC50 was determined to be 2.99 and 159.8â¯nM for sCD80 and sCD86, respectively. A straightforward MRM-based competitive assay will advance the knowledge about the costimulatory role in autoimmunity and the autoimmune therapeutic drug discovery, with the need for broad application on different in vitro and in vivo models to discover new targeted inhibitors.
Subject(s)
Autoimmune Diseases , Immunoconjugates , Humans , CD28 Antigens/metabolism , Antigens, CD/metabolism , B7-2 Antigen , Chromatography, Liquid , Liquid Chromatography-Mass Spectrometry , Tandem Mass Spectrometry , B7-1 Antigen/metabolism , AbataceptABSTRACT
Autoimmune diseases are diseases in which the regulatory mechanisms of the immune response are disturbed. As a result, the body loses self-tolerance. Since one of the main regulatory mechanisms of the immune response is the CTLA4-CD80/86 axis, this hypothesis suggests that autoimmune diseases potentially share a similar molecular basis of pathogenesis. Hence, investigating the CTLA4-CD80/86 axis may be helpful in finding an appropriate treatment strategy. Therefore, this study aims to investigate the molecular basis of the CTLA4-CD80/86 axis in the regulation of the immune response, and then its role in developing some autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, and multiple sclerosis. As well, the main therapeutic strategies affecting the CTLA4-CD80/86 axis have been summarized to highlight the importance of this axis in management of autoimmune diseases.
Subject(s)
Autoimmune Diseases , Immunoconjugates , Humans , CTLA-4 Antigen , Antigens, CD , B7-2 Antigen , B7-1 Antigen/physiology , Autoimmune Diseases/therapy , Cell Adhesion MoleculesABSTRACT
BACKGROUND AND AIMS: Rheumatoid arthritis (RA) is a chronic autoimmune disease. RA-induced immunological responses are coordinated by T-cell stimulation. The costimulatory signal CD28-B7 is essential for T-cell activation by interacting CD28 with CD80 and CD86 costimulatory proteins. CTLA4 is another costimulatory protein that binds to CD80 and CD86 to inhibit T-cell activity. The soluble costimulatory proteins: sCD80, sCD86, sCD28, and sCTLA-4 were detected and quantified in human plasma and correlated with RA development. As potential diagnostic biomarkers for RA, developing a sensitive, specific, and reproducible method for quantifying these costimulatory molecules in human plasma and establishing quantitative ranges for each protein in healthy and RA patients' plasma is essential for advancing the clinical diagnostic and health outcomes. MATERIALS AND METHODS: A novel quantitative liquid chromatography-tandem spectrometry (LC-MS/MS) technique using multiple reaction monitoring (MRM) modes was developed and validated to measure soluble costimulatory molecules sCTLA4, sCD28, sCD80, and sCD86 in human plasma samples. Furthermore, the method was applied to determine sCTLA4, sCD28, sCD80, and sCD86 levels in plasma samples from RA patients (n = 23) and healthy controls (n = 21). RESULTS: The method was successfully developed and validated according to international inter- and intra-assay precision and accuracy guidelines. The linearity of the method was achieved between 0.5 nM and 100 nM for each protein with a correlation coefficient of > 0.998. The plasma level of sCTLA4, sCD80, and sCD86 in RA patients was significantly elevated compared to controls. RA patients had 63.32 ± 17.63 nM sCTLA4 and controls 36.05 ± 18.83 nM; p < 0.0001. The performance of the four proteins was determined using ROC curves, where sCTLA4 showed the highest diagnostic and clinical performance compared to the others. CONCLUSIONS: This study reports the first use of LC-MS/MS in MRM mode to accurately quantify soluble costimulatory molecules in plasma samples as potential RA diagnostic biomarkers. Determination of the reference range for each protein with high selectivity and sensitivity increases the potential for utilizing this method as a clinical diagnostic.
Subject(s)
Arthritis, Rheumatoid , CD28 Antigens , Humans , Antigens, CD , B7-2 Antigen , Chromatography, Liquid , Tandem Mass Spectrometry , B7-1 Antigen/metabolism , Transcription Factors , Arthritis, Rheumatoid/diagnosis , BiomarkersABSTRACT
One of the major challenges in chemical toxicity testing is the possibility to protect human health against adverse effects with non-animal methods. In this paper, 4-Octylphenol (OP) was tested for skin sensitization and immunomodulatory effects using an integrated in silico-in vitro test approach. In silico tools (QSAR TOOLBOX 4.5, ToxTree and VEGA) were used together with several in vitro tests including HaCaT cells (quantification of IL-6; IL-8; IL-1α and IL-18 by ELISA and expression of genes TNF, IL1A, IL6 and IL8 by RT- qPCR), RHE model (quantification of IL-6; IL-8; IL-1α and IL-18 by ELISA) and THP-1 activation assay (CD86/CD54 expression and IL-8 release). Additionally, the immunomodulatory effect of OP was investigated using lncRNAs MALAT1 and NEAT1 expression and LPS-induced THP-1 activation (CD86/CD54 expression and IL-8 release). The in silico tools predicted OP as a sensitizer. In vitro tests are also concordant with the in silico prediction. OP increased IL-6 expression (HaCaT cells); IL-18 and IL-8 expressions (RHE model). An irritant potential was also shown by a great expression of IL-1α (RHE model); and increased expression of CD54 marker and IL-8 in THP-1 cells. Immunomodulatory effects of OP were demonstrated by the downregulation of NEAT1, MALAT1 (epigenetic markers), IL6 and IL8; and an increase in LPS-induced CD54 and IL-8 expressions. Overall, results indicate that OP is a skin sensitizer, being positive in three key events of the AOP for skin sensitization, also showing immunomodulatory effects.
Subject(s)
Interleukin-8 , RNA, Long Noncoding , Humans , Interleukin-8/genetics , Interleukin-18/pharmacology , Interleukin-6 , Lipopolysaccharides/toxicity , B7-2 Antigen/metabolism , B7-2 Antigen/pharmacology , Skin , AllergensABSTRACT
B7 ligands (CD80 and CD86), expressed by professional antigen-presenting cells (APCs), activate the main co-stimulatory receptor CD28 on T cells in trans. However, in peripheral tissues, APCs expressing B7 ligands are relatively scarce. This raises the questions of whether and how CD28 co-stimulation occurs in peripheral tissues. Here, we report that CD8+ T cells displayed B7 ligands that interacted with CD28 in cis at membrane invaginations of the immunological synapse as a result of membrane remodeling driven by phosphoinositide-3-kinase (PI3K) and sorting-nexin-9 (SNX9). cis-B7:CD28 interactions triggered CD28 signaling through protein kinase C theta (PKCθ) and promoted CD8+ T cell survival, migration, and cytokine production. In mouse tumor models, loss of T cell-intrinsic cis-B7:CD28 interactions decreased intratumoral T cells and accelerated tumor growth. Thus, B7 ligands on CD8+ T cells can evoke cell-autonomous CD28 co-stimulation in cis in peripheral tissues, suggesting cis-signaling as a general mechanism for boosting T cell functionality.
Subject(s)
CD28 Antigens , CD8-Positive T-Lymphocytes , Mice , Animals , CD28 Antigens/metabolism , Antigens, CD/metabolism , Ligands , Synaptic Membranes/metabolism , B7-2 Antigen , Membrane Glycoproteins/metabolism , B7-1 Antigen/metabolism , Cell Adhesion Molecules , Lymphocyte ActivationABSTRACT
Increased mechanical endothelial cell stretch contributes to the development of numerous cardiovascular and renal pathologies. Recent studies have shone a light on the importance of sex-dependent inflammation in the pathogenesis of renal disease states. The endothelium plays an intimate and critical role in the orchestration of immune cell activation through upregulation of adhesion molecules and secretion of cytokines and chemokines. While endothelial cells are not recognized as professional antigen-presenting cells, in response to cytokine stimulation, endothelial cells can express both major histocompatibility complex (MHC) I and MHC II. MHCs are essential to forming a part of the immunological synapse interface during antigen presentation to adaptive immune cells. Whether MHC I and II are increased under increased mechanical stretch is unknown. Due to hypertension being multifactorial, we hypothesized that increased mechanical endothelial stretch promotes the regulation of MHCs and key costimulatory proteins on mouse renal endothelial cells (MRECs) in a stretch-dependent manner. MRECs derived from both sexes underwent 5%, 10%, or 15% uniaxial cyclical stretch, and immunological synapse interface proteins were determined by immunofluorescence microscopy, immunoblot analysis, and RNA sequencing. We found that increased endothelial mechanical stretch conditions promoted downregulation of MHC I in male MRECs but upregulation in female MRECs. Moreover, MHC II was upregulated by mechanical stretch in both male and female MRECs, whereas CD86 and CD70 were regulated in a sex-dependent manner. By bulk RNA sequencing, we found that increased mechanical endothelial cell stretch promoted differential gene expression of key antigen processing and presentation genes in female MRECs, demonstrating that females have upregulation of key antigen presentation pathways. Taken together, our data demonstrate that mechanical endothelial stretch regulates endothelial activation and immunological synapse interface formation in renal endothelial cells in a sex-dependent manner.NEW & NOTEWORTHY Endothelial cells contribute to the development of renal inflammation and have the unique ability to express antigen presentation proteins. Whether increased endothelial mechanical stretch regulates immunological synapse interface proteins remains unknown. We found that antigen presentation proteins and costimulatory proteins on renal endothelial cells are modulated by mechanical stretch in a sex-dependent manner. Our data provide novel insights into the sex-dependent ability of renal endothelial cells to present antigens in response to endothelial mechanical stimuli.
Subject(s)
Blood Vessels , Endothelial Cells , Immunological Synapses , Kidney , Endothelial Cells/physiology , Cells, Cultured , Male , Female , Animals , Mice , Kidney/blood supply , Mice, Inbred C57BL , Blood Vessels/cytology , Biomechanical Phenomena , Inflammation/metabolism , Secretome/metabolism , Sex Characteristics , Major Histocompatibility Complex , B7-2 Antigen/metabolism , Antigen PresentationABSTRACT
CD28 and CTLA4 are T cell coreceptors that competitively engage B7 ligands CD80 and CD86 to control adaptive immune responses. While the role of CTLA4 in restraining CD28 costimulatory signaling is well-established, the mechanism has remained unclear. Here, we report that human T cells acquire antigen-presenting-cell (APC)-derived B7 ligands and major histocompatibility complex (MHC) via trogocytosis through CD28:B7 binding. Acquired MHC and B7 enabled T cells to autostimulate, and this process was limited cell-intrinsically by CTLA4, which depletes B7 ligands trogocytosed or endogenously expressed by T cells through cis-endocytosis. Extending this model to the previously proposed extrinsic function of CTLA4 in human regulatory T cells (Treg), we show that blockade of either CD28 or CTLA4 attenuates Treg-mediated depletion of APC B7, indicating that trogocytosis and CTLA4-mediated cis-endocytosis work together to deplete B7 from APCs. Our study establishes CTLA4 as a cell-intrinsic molecular sink that limits B7 availability on the surface of T cells, with implications for CTLA4-targeted therapy.
Subject(s)
CD28 Antigens , Immunoconjugates , Humans , CTLA-4 Antigen/metabolism , CD28 Antigens/metabolism , Antigens, CD/metabolism , Ligands , Antigens, Differentiation , Abatacept/pharmacology , B7-2 Antigen , Membrane Glycoproteins/metabolism , B7-1 Antigen/metabolism , Cell Adhesion MoleculesABSTRACT
BACKGROUND: Fragrances are important contact allergens; however, investigation of their skin sensitization potency has been challenging in new approach methods (NAMs). Many fragrance chemicals are susceptible to autoxidation or can be metabolized by enzymes constitutively expressed in skin keratinocytes. Strong sensitizers can be formed in both of these processes. Further, keratinocytes can modulate the dendritic cell (DC) activation and maturation potential, a key event in the acquisition of contact allergy. OBJECTIVES: To evaluate the 2D coculture model consisting of keratinocytes and DCs using different weak to moderate sensitizing fragrance chemicals. Further, to investigate fragrances and related oxidation products in the in vitro model and compare to in vivo data. METHODS: Chemicals were tested in the coculture activation test (COCAT), consisting of HaCaT keratinocytes and THP-1 cells. THP-1 cell surface expression of costimulatory and adhesion molecules (CD86 and CD54) collected after 24 h incubation with the chemicals was analysed using flow cytometry. RESULTS: Twenty-four molecules were tested positive, three were negative (n = 27). Four pairs were evaluated, with aldehydes showing a 6- to 13-fold stronger responses compared to their corresponding alcohols. CONCLUSIONS: Results provide insight into the activation of DC in their natural environment of keratinocytes. α,ß-Unsaturated alcohols were classified as weaker sensitizers compared to their corresponding aldehydes. In sum, testing of fragrances retrieved results in good agreement with in vivo data.
Subject(s)
Dermatitis, Allergic Contact , Odorants , Humans , Coculture Techniques , Dermatitis, Allergic Contact/etiology , Dermatitis, Allergic Contact/metabolism , Dendritic Cells , B7-2 Antigen/metabolism , Keratinocytes/metabolism , Allergens , AldehydesABSTRACT
We previously showed that cell-surface CD86 expressed on multiple myeloma (MM) cells contributed to not only tumor growth but also antitumor cytotoxic T-lymphocyte responses mediated by induction of IL-10-producing CD4+ T cells. The soluble form of CD86 (sCD86) was also detected in serum from patients with MM. Thus, to determine whether sCD86 levels are a useful prognostic factor, we investigated the association of serum sCD86 levels with disease progression and prognosis in 103 newly diagnosed patients with MM. Serum sCD86 was detected in 71% of the patients with MM but was only rarely detected in patients with monoclonal gammopathy of undetermined significance and healthy controls, and the level was significantly increased in patients with advanced-stage MM. When we examined differences in clinical characteristics according to the level of serum sCD86, those in the high (≥2.18 ng/mL, n = 38) group exhibited more aggressive clinical characteristics, with shorter overall survival times compared with those in the low (<2.18 ng/mL, n = 65) group. On the other hand, it was difficult to stratify the patients with MM into different risk groups based on the expression levels of cell-surface CD86. The levels of serum sCD86 were significantly correlated with the expression levels of the messenger RNA (mRNA) transcripts of CD86 variant 3, which lack exon 6, resulting in a truncated transmembrane region, and its variant transcripts were upregulated in the high group. Thus, our findings suggest that sCD86 can be easily measured in peripheral blood samples and is a useful prognostic marker in patients with MM.
Subject(s)
B7-2 Antigen , Multiple Myeloma , Humans , B7-2 Antigen/blood , B7-2 Antigen/genetics , Disease Progression , Multiple Myeloma/diagnosis , Multiple Myeloma/genetics , PrognosisABSTRACT
CD80 or cluster of differentiation 80, also known as B7-1, is a member of the immunoglobulin super family, which binds to CTLA-4 and CD28 T cell receptors and induces inhibitory and inductive signals respectively. Although CTLA-4 and CD28 receptors belong to the same protein family, slight differences in their structures leads to CD80 having a higher binding affinity to CTLA-4 (-14.55 kcal/mol) compared with CD28(-12.51 kcal/mol). In this study, we constructed a variant of CD80 protein with increased binding affinity to CTLA-4 and decreased binding affinity to CD28. This variant has no signaling capability, and can act as a cap for these receptors to protect them from natural CD80 proteins existing in the body. The first step was the evolutionary and alanine scanning analysis of CD80 protein to determine conserved regions in this protein. Next, complex alanine scanning technique was employed to determine CD80 protein hotspots in CD80-CTLA-4 and CD80-CD28 protein complexes. This information was fed into a computational model developed in R for in silico mutagenesis and CD80 variant library construction. The 3D structures of variants were modeled using the Swiss model webserver. After modeling the 3D structures, HADDOCK server was employed to build all protein-protein complexes, which contain CTLA-4-CD80 variant complexes, Wild type CD80-CD28 complexes and CD28-CD80 variant complexes. Protein-protein binding free energy was determined using FoldX and the variant number 316 with mutations at 29, 31, 33 positions showed increased binding affinity to CTLA-4 (-21.43 kcal/mol) and decreased binding affinity to CD28 (- 9.54 kcal/mol). Finally, molecular dynamics (MD) simulations confirmed the stability of variant 316. In conclusion, we designed a new CD80 protein variant with potential immunotherapeutic applications.