Dendritic Cells Promote the Differentiation of ILCs into NCR-ILC3s in the Lungs of Mice Exposed to Cigarette Smoke.

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.

Bacteriophages from treatment-naïve type 2 diabetes individuals drive an inflammatory response in human co-cultures of dendritic cells and T cells.

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.

Targeting dendritic cell activation: the therapeutic impact of paeoniflorin in cortosteroid-dependent dermatitis management.

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.

Obesity induces PD-1 on macrophages to suppress anti-tumour immunity.

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.

Development of a Protocol for Obtaining a Homologous Cell Product of Animal Origin Intended for Preclinical Studies of Antitumor Vaccine CaTeVac.

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.

Blockade of CD80/CD86-CD28 co-stimulation augments the inhibitory function of peptide antigen-specific regulatory T cells.

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.

Development of Hsp90 inhibitor to regulate cytokine storms in excessive delayed- and acute inflammation.

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.

Saponin Esculeoside A and Aglycon Esculeogenin A from Ripe Tomatoes Inhibit Dendritic Cell Function by Attenuation of Toll-like Receptor 4 Signaling.

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.

Optimal conditions for adenoviral transduction of immature dendritic cells without affecting the tolerogenic activity of DC-based immunotherapy.

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.

K33 only mutant ubiquitin augments bone marrow-derived dendritic cell-mediated CTL priming via PI3K-Akt pathway.

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.

Co-stimulatory pathway competitive assay development using Liquid chromatography-tandem mass spectrometry (LC-MS/MS).

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 ∼0.5 nM for the four analytes, with quantification extended to 20 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 159.8 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.

TRAM deletion attenuates monocyte exhaustion and alleviates sepsis severity.

Monocyte exhaustion characterized by immune-suppressive features can develop during sepsis and contribute to adverse patient outcomes. However, molecular mechanisms responsible for the establishment of immune-suppressive monocytes with reduced expression of immune-enhancing mediators such as CD86 during sepsis are not well understood. In this study, we identified that the TLR4 intracellular adaptor TRAM plays a key role in mediating the sustained reduction of CD86 expression on exhausted monocytes and generating an immune-suppressive monocyte state. TRAM contributes to the prolonged suppression of CD86 through inducing TAX1BP1 as well as SARM1, collectively inhibiting Akt and NFκB. TRAM deficient mice are protected from cecal slurry-induced experimental sepsis and retain immune-competent monocytes with CD86 expression. Our data reveal a key molecular circuitry responsible for monocyte exhaustion and provide a viable target for rejuvenating functional monocytes and treating sepsis.

Long-chain non-coding RNA n337374 relieves symptoms of respiratory syncytial virus-induced asthma by inhibiting dendritic cell maturation via the CD86 and the ERK pathway

Background: In this study, we investigated the relationship between long-chain non-coding RNAs (lncRNAs) and respiratory syncytial virus (RSV)-exacerbated asthma. Methods: Transcriptome microarray was used to detect differentially expressed lncRNAs in dendritic cells (DCs) co-cultured with RSV-infected human airway epithelial cells and DCs infected with RSV. The identified downregulation of lncRNA n337374 was validated using fluorescence RT-qPCR. LncRNA n337374-overexpressing DCs and RSV-exacerbated asthmatic mouse models were established. Airway hyper-reactivity and bronchoalveolar lavage fluid (BALF) were examined, and pathological changes in lung tissues were observed in mice. Surface molecules in DCs were detected by flow cytometry and RT-qPCR and the expression of CD86 and mitogen-activated protein kinases was determined by western blot. Results: In an RSV-exacerbated asthmatic mouse model, the airway wall was thickened, luminal stenosis was observed, a large number of inflammatory cells were infiltrated in the lung tissue, lung function was impaired, and counts of inflammatory cells in the BALF were increased. The overexpression of lncRNA n337374 ameliorated these pathological changes and improved impaired lung function and inflammation in an asthmatic mouse model. In DCs co-cultured with RSV-infected human airway epithelial cells, CD86 expression was promoted and ERK was markedly phosphorylated. When lncRNA n337374-overexpressing DCs were used in the co-cultures, the expression of CD86 and phosphorylated ERK was decreased. Conclusion: The results suggest that lncRNA n337374 overexpression may suppress DC maturation by downregulating the CD86 and ERK pathway, subsequently relieving the symptoms of RSV-induced asthma. LncRNA n337374 may be a promising target in the treatment of RSV infection-induced asthma (AU)

Switch to belatacept in kidney graft recipients / Cambio a belatacept en trasplante renal

No disponible

Study of Trichostation A-Induced Expression of Costimulatory Molecules CD80 and CD86 in Acute Myelocytic Leukemia Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the trichostain A (TSA)-induced expression of costinmulatory molecules CD80 and CD86 in HL-60, K562 and mononuclear cells (MNC) of bone marrow in AML patients and its clinical significance.</p><p><b>METHODS</b>The TSA-induced expression of costimulatory molecules CD80, CD86 in HL-60, K562 and BMMNC, and the cell viability were detected by flow cytometry; the mRNA expression of CD80 and CD86 was detected by RT-PCR; after the TSA-induced HL-60 cells and K562 cells were irradiated with 75 Gy, the effect of these cells on proliferation of PBMNC from healthy volunteers was determined with CCK-8 method.</p><p><b>RESULTS</b>The HL-60 cells and BMMNC in AML patients expressed CD86, not expressed CD80, while the K562 cells not expressed CD86 and CD80. TSA could up-regulate the expression of CD86 in HL-60 cells and BMMNC of AML patients. The TSA-induced HL-60 cells expressing costimulatory molecule CD86 showed the proliferative effect on BMMNC from healthy volunteers.</p><p><b>CONCLUSION</b>The TSA can induce the expression of costimulatory molecule CD86 in HL-60 cells and BMMNC in AML patients, and can improve the proliferation of PBMNC in healthy volunteers.</p>

Role of glycogen synthase kinase 3β in maturation and function of murine myeloid dendritic cells in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the role of glycogen synthase kinase 3β (GSK-3β) in the maturation and function of murine bone marrow-derived dendritic cells (BMDCs).</p><p><b>METHODS</b>Mature DCs (mDCs) induced by LPS were examined for GSK-3β phosphorylation level with Western blotting before and after LPS exposure. To explore the role of GSK-3β in maturation and function of DCs, we added SB216763, a selective inhibitor of GSK-3β, in the cell culture of immature DCs (iDCs), and examined CD40 and CD86 expressions in the cells by flow cytometry and the expression of IL-6, IL-12 and IL-10 mRNA by real-time PCR; the changes of the immunogenicity of the cells was evaluated by mixed lymphocyte reaction. The expression of GSK-3β and RelB was examined by Western blotting in DC2.4 cells transfected with a lentiviral vector over-expressing murine GSK-3β gene.</p><p><b>RESULTS</b>LPS exposure significantly lowered GSK-3β activity in iDCs as demonstrated by increased Ser9 phosphorylation and reduced Tyr216 phosphorylation. GSK-3β inhibition induced DC maturation by increasing the expression of surface costimulatory molecules CD40 and CD86, lowered the expressions of IL-6 and IL-12 while enhanced the expression of IL-10 in iDCs, and impaired mixed lymphocyte reaction of the cells. In DC2.4 cells, lentivirus-mediated over-expression of GSK-3β obviously down-regulated the expression of RelB.</p><p><b>CONCLUSIONS</b>GSK-3β is a crucial enzyme involved in the differentiation and maintenance of an immature phenotype of DCs. GSK-3β is constitutively active in iDCs to inhibit their spontaneous maturation. DCs become phenotypically mature after inhibition of GSK-3β, which also executes a proinflammatory task in DC activation. The reduction of RelB protein levels as a result of GSK-3β overexpression supports GSK-3β as a new target for inducing tolerogenic DCs.</p>

Functional disruption of human leukocyte antigen II in human embryonic stem cell

BACKGROUND: Theoretically human embryonic stem cells (hESCs) have the capacity to self-renew and differentiate into all human cell types. Therefore, the greatest promise of hESCs-based therapy is to replace the damaged tissues of patients suffering from traumatic or degenerative diseases by the exact same type of cells derived from hESCs. Allo-graft immune rejection is one of the obstacles for hESCs-based clinical applications. Human leukocyte antigen (HLA) II leads to CD4+ T cells-mediated allograft rejection. Hence, we focus on optimizing hESCs for clinic application through gene modification. RESULTS: Transcription activator-like effector nucleases (TALENs) were used to target MHC class II transactivator (CIITA) in hESCs efficiently. CIITA(-/-)hESCs did not show any difference in the differentiation potential and self-renewal capacity. Dendritic cells (DCs) derived from CIITA(-/-)hESCs expressed CD83 and CD86 but without the constitutive HLA II. Fibroblasts derived from CIITA(-/-)hESCs were powerless in IFN-γ inducible expression of HLA II. CONCLUSION: We generated HLA II defected hESCs via deleting CIITA, a master regulator of constitutive and IFN-γ inducible expression of HLA II genes. CIITA(-/-)hESCs can differentiate into tissue cells with non-HLA II expression. It's promising that CIITA(-/-)hESCs-derived cells could be used in cell therapy (e.g., T cells and DCs) and escape the attack of receptors' CD4+ T cells, which are the main effector cells of cellular immunity in allograft.

Tanshinone II A inhibits dendritic cell-mediated adaptive immunity: potential role in anti-atherosclerotic activity / 中国结合医学杂志

<p><b>OBJECTIVE</b>Antigen-presenting cells such as monocytes and dendritic cells (DCs) stimulate T-cell proliferation and activation during adaptive immunity. This cellular interaction plays a role in the growth of atherosclerotic plaques. Tanshinone II A (TSN) had been shown to decrease the growth of atherosclerotic lesions. We therefore investigated the ability of TSN to inhibit human monocyte-derived DCs and their T-cellstimulatory capacity.</p><p><b>METHODS</b>DCs derived from human monocytes cultured with recombinant human interleukin (IL)-4 and recombinant human granulocyte-macrophage colony-stimulating factor were co-cultured with TSN and lipopolysaccharide for 48 h. Phosphate-buffered saline was used as a negative control. Activation markers and the capacity of DCs for endocytosis were measured by flow cytometry, and proinflammatory cytokines were measured by enzyme-linked immunosorbent assays. DCs were co-cultured with lymphocytes to measure T-cell proliferation and IL-2 secretion by mixed lymphocyte reactions.</p><p><b>RESULTS</b>TSN dose-dependently attenuated DC expression of costimulatory molecules (CD86), and decreased expression of major histocompatibility complex class II (human loukocyte antigen-DR) and adhesion molecules (CD54). Moreover, TSN reduced secretion of the proinflammatory cytokines IL-12 and IL-1 by human DCs, and restored the capacity for endocytosis. Finally, TSN-preincubated DCs showed a reduced capacity to stimulate T-cell proliferation and cytokine secretion.</p><p><b>CONCLUSIONS</b>TSN inhibits DC maturation and decreases the expression of proinflammatory cytokines, while impairing their capacity to stimulate T-cell proliferation and cytokine secretion. These effects may contribute to the influence of TSN on the progression of atherosclerotic lesions.</p>

Role of Toll-like receptor 2/4-nuclear factor-κB signaling pathway in invasion of Mycobacterium tuberculosis to mouse dendritic cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the mechanism of Mycobacterium tuberculosis invasion to mouse dendritic cells (DC).</p><p><b>METHODS</b>Mycobacterium tuberculosis strain H37Rv was co-cultured with mouse DC2.4 cells.The mRNA expression of Toll-like receptor 2/4(TLR2/4) in DC2.4 cells was detected by fluorescent quantitative real-time PCR and the protein expression of nuclear factor κB(NF-κB) was assessed by Western blotting.The extracellular concentration of tumor necrosis factor α(TNF-α) was measured by ELISA methods during Mycobacterium Tuberculosis invasion.Indirect immunofluorescent staining and flow cytometry assay were used to detect the expression of CD80 and CD86 on DC2.4 cells before and after invasion.</p><p><b>RESULTS</b>The invasion of Mycobacterium tuberculosis in DC2.4 cells was observed after 2 h of co-incubation.The rates of invasion were (37.9±5.6)%,(51.2±7.6)%,(57.2±8.9)% and(63.9±6.8)% at 6,8,10 and 12 h after co-incubation,respectively.The mRNA expression level of TLR2 /4 was significantly increased at 6 h but decreased at 10 h after co-incubation.The expressions of NF-κB p65 and TNF-α were higher in DC2.4 cells after being invaded by 6,8,and 10 h and then gradually decreased.CD80 and CD86 expression were increased on DC2.4 at 6 h after co-incubation.</p><p><b>CONCLUSION</b>Invasion of Mycobacterium tuberculosis strain H37Rv to DC might enhance its antigen-presenting function through activation of TLR2/4-NF-kB signaling pathway.</p>

Expression of costimulatory molecule CD86 in HL-60 cells induced by MG132 and its effect on allogeneic mixed lymphocyte reaction / 中国实验血液学杂志

This study was aimed to elucidate the expression of costimulatory molecule CD80 and CD86 in HL-60 cells induced by proteasome inhibitor MG132 and its effect on allogeneic mixed lymphocyte reaction. Acute myelocytic leukemia cell line HL-60 and chronic myelocytic leukemia cell line K562 were cultured. The viability of the cells was measured by flow cytometry. Proteasome inhibitor MG132 at the concentrations of 2 or 3 µmol/L was used to stimulate the HL-60 cell cultured for 24 h and 48 h respectively, and the Annexin V/7-AAD staining and flow cytomotry were used to detect the apoptosis of the HL-60 cells. HL-60 and K562 cells were treated with 1 µmol/L MG132 for 24 h and 48 h respectively, then CD80 and CD86 antibodies were added, finally the expression of CD80 and CD86 was analysed by flow cytomery. The mRNA expression of CD86 in the HL-60 cells treated with 1 µmol/L MG132 was detected by RT-PCR. HL-60 and K562 cells were treated by 1 µmol/L MG132 and then underwent irradiation of 75 Gy (60)Co to kill the cells with their antigenicity preserved. Peripheral blood mononuclear cells (PBMNCs) of healthy volunteers, as reactive cells, were isolated and inoculated into the (60)Co irradiated HL-60 cells of different concentrations, as stimulating cells, CCK-8 was added and then the A value of absorbance was measured at the wave length of 450 nm in an enzyme labeling instrument. The results showed that the cell viability of the HL-60 cells treated with 1 µmol/L MG132 for 24 h an d 48 h was 92.95% and 85.87% respectively. The apoptotic rates of the HL-60 cells treated with MG132 increased in dose-and time-dependent manner. High-concentration of MG132 directly killed HL-60 cells. Before MG132 treatment K562 cells did not express CD86, but the CD86 expression of the HL-60 cells was up-regulated time-dependently after MG132 treatment (P < 0.01). The mRNA expression of CD86 in the HL-60 treated with MG132 was up-regulated time-dependently (P < 0.01). CCK-8 test showed that the proliferation level of PBMNC gradually increased along with the concentration of HL-60 cells treated with MG132 and reached its peak when the concentration of the HL-60 cells was 1×10(5) (P < 0.01). No remarkable proliferation of PBMNC was observed in the K562 groups no matter if the HL-60 cells had been treated with MG132. It is concluded that the high concentration of MG132 can directly kill HL-60 cells, low-concentration of MG132 can induce the expression of costimulatory molecule CD86 in HL-60 cells, also can improve the proliferation of PBMNC.