Progress in Biomechanics and Mechanobiology of Dendritic Cells / 医用生物力学

Dendritic cells (DCs) are now known as the most powerful antigen-presenting cells in vivo, with efficient antigen uptaking, and processing capabilities. They can present antigens to naïve T cells in secondary lymphoid tissues, thereby induce immune response or tolerance, and play a key role in initiating and amplifying innate and adaptive immunity. DCs experience complex chemical and mechanical microenvironment changes and show different mechanophenotypes and immunophenotypes in the process of exerting their physiological functions. Deeply understanding the chemical and mechanical factors that regulate the mechanophenotypes and immunophenotypes of DCs is a prerequisite for using DCs to treat immune related diseases. In this review, the progress in the biomechanics and mechanobiology research of DCs was mainly introduced, and their potential applications and future development directions in the treatment of immune related diseases were explored.

Wuhutang Alleviates Respiratory Syncytial Virus-induced Asthma in Mice by Inhibiting STAT3 Protein and Regulating Dendritic Cell Autophagy / 中国实验方剂学杂志

Objective:To investigate the therapeutic mechanism of Wuhutang on respiratory syncytial virus （RSV）-induced asthma in mice and its influence on the expression of signal transducer and activator of transcription 3 （STAT3） in lung tissue. Method:One hundred female BALB/c mice of SPF grade were randomly divided into a normal group and an experimental group. After successful modeling via aerosol inhalation of RSV and ovalbumin （OAV）， the mice in the experimental group were further randomized into the following seven groups： model， positive control （dexamethasone， 1.82 mg·kg^-1）， STAT3 inhibitor （STATTIC， 3.75 mg·kg^-1）， STAT3 inducer （colivelin， 1.0 mg·kg^-1）， and low-， medium-， and high-dose （1.6， 3.2， and 6.4 g·kg^-1， respectively） Wuhutang groups. The corresponding drugs were administered for two weeks， followed by the detection of airway reactivity using a small animal ventilator， the pathological changes in lung tissue， mucus secretion by goblet cells and collagen deposition in airway were observed by hematoxylin-eosin （HE）， periodic acid-Schiff （PAS） and Masson staining， the serum levels of interleukin-6 （IL-6）， IL-10， and IL-17 were detected by enzyme-linked immunosorbent assay （ELISA）. The mRNA expression levels of TGF-<italic>β</italic>₁ and <italic>α</italic>-SMA in lung tissue were detected by fluorescence-based real-time polymerase chain reaction （Real-time PCR）， autophagosomes present in lung tissue were examined by transmission electron microscopy， the protein expression levels of ATG5 and SQSTM1 in dendritic cells （DCs） and STAT3 and p-STAT3 in lung tissue were detected by Western blot. Result:The airway reactivity of the model group was enhanced in contrast to that in the model group （<italic>P<</italic>0.01）， manifested as inflammatory cell infiltration around the lung tissue， excessive metaplasia of goblet cells， and extensive deposition of airway collagen， the expression levels of serum IL-6 and IL-17 were increased （<italic>P<</italic>0.01）， while that of IL-10 declined （<italic>P<</italic>0.01）， the mRNA expression levels of TGF-<italic>β</italic>₁ and <italic>α</italic>-SMA were elevated （<italic>P</italic><0.01）， the number of autophagosomes in the lung tissue increased. The protein expression levels of ATG5， STAT3， and p-STAT were up-regulated， while that of SQSTM1 was down-regulated （<italic>P<</italic>0.01）. Compared with the model group， Wuhutang and STATTIC significantly reduced the airway hyperresponsiveness of asthmatic mice （<italic>P<</italic>0.05， <italic>P<</italic>0.01）， alleviated RSV-induced pathological changes in lung tissue， reduced the contents of serum IL-6 and IL-17 （<italic>P<</italic>0.01）， increased serum IL-10 and ATG5 in DCs （<italic>P<</italic>0.01）， down-regulated the mRNA expression levels of TGF-<italic>β</italic>₁ and <italic>α</italic>-SMA as well as the protein expression levels of SQSTM1， STAT3 and p-STAT3 （<italic>P<</italic>0.05，<italic>P<</italic>0.01）， and elevated the number of autophagosomes. Conclusion:Wuhutang relieves airway inflammation， improves airway remodeling and reduces airway hyperresponsiveness in RSV-induced asthmatic mice by inhibiting STAT3 protein and up-regulating DC autophagy in lung tissue.

Salidroside regulates DC through TLR4 to increase the lethality of T cells to lung cancer 3LLcells / 中国肿瘤生物治疗杂志

@#Objective： ：To investigatetheeffectofsalidroside(SAL)onthephenotype of dendritic cells (DCs) and the antitumor ability of cytotoxic T lymphocytes (CTL). Methods： ：Lewis lung cancer cell line 3LL, wild type (WT) C57BL/6 mice and TLR4-/- C57BL/6 mice were chosen for this study. Mice bone marrow derived DC precursor cells were obtained to differentiate into immature DCs, which were harvested on the sixth day of culture. CD11c+ DCs were obtained by magnetic beads screening, and further divided into PBS group, SAL group and lipopolysaccharide (LPS) group.After being cultured for 48 h, the effects of SAL on surface molecules and phagocytosis of DCs as well as the efffect of TLR4 pathway on the killing effect of T cells were detected by Fow cytometry. Results： ： Compared with PBS group, expressions of DC surface molecules CD80, CD86 and MHC Ⅱ significantly increased (all P<0.05), phagocytosis significantly decreased (P<0.05), and TLR4 expression level significantly increased (P<0.01) in SAL group; Compared with WT group, after being treated with SAL or LPS, the expressions of DC surface molecules CD80, CD86 and MHC Ⅱ decreased significantly in TLR4-/- group (all P<0.05); ComparedwithPBSgroup,theactivatedCTLinSALgroupexhibited a significantly elevated killing effect against lung cancer 3LLcells (P<0.05). Conclusion：SAL can induce DC maturation by regulating TLR4, thus improving the killing ability of T cells.

Effects of miR-30 a on cell cycle and dendritic cell-suppressive function of MSCs / 中国免疫学杂志

Objective:Mesenchymal stem cells( MSCs) have self-renewal capacity and potential to differentiate into the cells.It was reported that the expression of miR-30a changed in some immune diseases.But it remains unclear the effect of miR-30a on the im-munoregulatory functions of MSCs.Here we studied the impact of miR-30a on the phenotype,cell viability,apoptosis,cell cycle and im-munoregulatory functions of MSCs.Methods: The mixed enzyme methods were used for the isolation of human umbilical cord MSCs.Flow cytometry(FCM)was used to investigate the effect of overexpressed miR-30a on the phenotype of MSCs.CCK-8 was used to examine the cell viability of miR-30a-overexpressed MSCs.Annexin V/PI was used for the detection of apoptosis of MSCs.Q-PCR and Western blot were used to investigate the effect of miR-30a on the expression of Cyclin E2( CCNE2).CCNE2 was one putative target of miR-30a predicted by Targetscan database.The effects of miR-30a-overexpressed MSCs on the maturation of dendritic cells(DCs)were determined.Results:Overexpression of miR-30a blocked the cell cycle of MSCs in the G0/G1 phase by inhibiting the expression of CCNE2,but did not affect the phenotype, cell viability and apoptosis of MSCs.When co-cultured with DCs, although MSCs down-regulated the expression of CD40 and CD86 on DCs,overexpression of miR-30a more significantly enhanced the suppressive impact of MSCs on the maturation of DCs.Conclusion: miR-30a affects the cell cycle of MSCs and enhances its immunosuppressive effect on DCs.

Effect of Kidney-tonification, Spleen-invigoration and Detoxification Methods on T lymphocytes Mediated by Peripheral Blood DCs in Chronic HBV Infection Patients / 世界科学技术-中医药现代化

This study was aimed to compare the effect of traditional Chinese medicine ( TCM ) treatment method of kidney-tonification, spleen-invigoration and detoxification on T lymphocytes of patients with chronic HBV infec-tion, which were mediated by peripheral blood dendritic cells (DCs). Ten patients with chronic HBV infection and eight healthy controls were included in this study. DCs were differentiated from PBMCs which were isolated from their peripheral blood, incubated and induced. Then, the DCs were interfered by plasma of medicine through the treatment methods of kidney-tonification, spleen-invigoration and detoxification. Models of interaction were estab-lished. Expressions of CD3+, CD4+, CD8+, CD8+CD28+, CD8+PD-1 of DCs and T cells were detected by FACS; and the levels of IFN-γ in supernatants were detected by ELISA. The results showed that the kidney-tonification plasma, spleen-invigoration plasma, detoxification plasma deal with HBcAg in load DCs incubated T lymphocytes. The CD3+ and CD4+ expression rate were increased; the CD8+ and CD8+PD-1 expression were reduced; IFN-γ level was increased; but only the Liuweidihuang Tang group was with significant difference compared to the model group (P < 0.05); the CD8+CD28+ expression rate was increased in the Sijunzi Tang and Liuweidihuang Tang group compared to the model group ( P < 0 . 05 ) . It was concluded that to a certain extent , the treatment method of kidney-tonification, spleen-invigoration and detoxification can restore the function of peripheral blood DCs in chronic HBV infection patients in order to restore its downstream T lymphocyte function. Among these treatment methods, the optimal method is kidney-tonification.

The role of NF-κB in the differentiation process of dendritic cells induced by astragalus polysaccharides from human cord blood monocyte / 中华微生物学和免疫学杂志

Objective To investigatethe role of NF-κB played in the process of the cord blood monocytes differentiating into dendritic cells(DCs)induced by astragalus polysaccharide(APS)and to explore the signal transduction pathway involved in this process.Methods Umbilica]cord blood was collected in aseptic conditions.The cord blood monocytes were obtained by density gradient centrifugation and were divided into three groups afterwards.In the control group.cells were cultivated in the RPMI 1640 complete medium.In the APS group.cells were cultivated in the RPMI 1640 complete medium containing 100 mg/L APS.In the PDTC group:cells were treated with 10 μmol/L disulfide carbamate(PDTC).NF-κB inhibitor in 30 min followed by cultivalion in the RPMI 1640 complete medium containing 100 mg/L APS.,The morphological changes were observed during the process of cultivation by the optical microscope and transmission electron microscopy.Cells were collected 12 d later and the cellular immunophenotyping was assayed by FCM.,The activation and migration of NF-κB fluorescence in the cells was examined by the immunoflouresce microscopy.Results (1)Cells in the control group grown up without cluster forformation and were found fusiform and macrophage-like in 12 d.Cells in the APS group grown up in clnstem,and morphological changes were found from the circular shape to a typical dendritic cells-like shape.Cells in the inhibitor group grown up slowly and without cluster formation,and cell morphdogy had no significant change.(2)The expression of DCs-specific antigen CD80,CD83 and CD86 in the APS group was higher than that in the control group and inhibitom group(P0.05).(3)NF-κB fluorescence in the nuclei was examined by the immunoflourescence microscopy and was much higher in the APS group than that in khe other groups,especially in 72 h with the activation rate of NF-κB (75.20±7.37)%,while(13.20±3.46)% of PDTC group and(8.20 ±1.92)%,respectively(P<0.01).Conclusion Astragalus polysaccharide can induce the differentiation of umbilical cord blood cells into DCs,and NF-κB is the key component of the signal transduction pathway involved in this process.

Dendritic Cells-based Vaccine and Immune Monitoring for Hepatocellular Carcinoma

Human tumors, including those of the hepatobiliary system, express a number of specific antigens that can be recognized by T cells, and may provide potential targets for cancer immunotherapy. Dendritic cells (DCs) are rare leucocytes that are uniquely potent in their ability to capture, process and present antigens to T cells. The ability to culture sufficient numbers of DCs from human bone marrow or blood progenitors has attracted a great deal of interest in their potential utilization in human tumor vaccination. CD34+ peripheral blood stem cells (PBSCs) were obtained from a patient with a hepatocellular carcinoma. The PBSCs were cultured in the X-VIVO 20 medium supplemented with the Flt-3 Ligand (FL), GM-CSF, IL-4 and TNF-alpha for 12 days. The morphology and functions of the cells were examined. The generated cells had the typical morphology of DCs. When the DCs were reinjected into the same patient, an augmentation of the cytotoxic T lymphocyte (CTL) activity was observed. Concomitantly, an increase in the natural killer (NK) cell activity was also detected in the patient. These results suggest that DCs-based cancer immunotherapy may become an important treatment option for cancer patients in the future.

APS on Inducing the Effect of Differentiating Cord Blood Monocyte into the Dendritic Cells Induced by Astragalus Polysaccharides In Vitro and its Immunological Characteristic / 医学研究杂志

Objectives To elucidate the effect of APS replacing cytokine on inducing the cord blood monocyte in vitro into the dendritic cells (DCs) and its cellar immunological characteristic. Methods The cord blood monocytes were isolated and obtained by lymphocyte isolation. three groups were divided: ②Cultured in the RPMI-1640 culture with GM-CSF/IL-4/TNF-?,as the positive control group, with APS in concentration (100mg/L) as the experimental group,and without GM-CSF/IL-4/ TNF-?and APS,as the negative control group, respectively. The morphotype of DCs was identified by inverted optical microscope or scanning electron microscope. The phenotype of cultured 12 days DCs (CD1a, CD80, CD86, and CD83) was identified by flowcytometry. Results Cultured for 72 hours , the morphous of cell of the experimental group grew clustering and began to change from round to irregularity, appearing rough cell face and barb pustute. The longer cell cultured, the more obvious the dendritic structure is. The experimental group cell cultured for 12 days had the most typical dendritic structure. the negative control group cell had no dendritic structure and became the macrophage when cultured for 12 days. The experimental group cell cultured for 10 days showed typical dendritic morphotype by SEM. The experiment group cell and the positive group cell cultured for 12 days significantly expressed the high level phenotype of DCs((CD1a, CD80, CD86, and CD83))by flowcytometry. Conclusions APS and cytokine both could induce the cord blood monocyte to direofive differentiate into functional DC.

The studies on the properties of dendritic cells from human cord blood and malignant pleural effusions / 肿瘤研究与临床

Objective To investigate the culture and proliferation of dendritic cells from human cord blood and malignant pleural effusions and identify the phenotype and the function of them in vitro. Methods Existing DCs were obtained from cord blood and malignant pleural effusions. The DCs were cultured with IL-4, GM-CSF and TNF-?. The morphological properties of DCs were observed by invert optical microscope. The phenotypes of DCs were analysed by flow cytometry. The proliferative bioactivities of T cells activated by DCs were observed with mixed lymphocyte reaction (MLR). Results No remarkable differences were found in the morphology and cellular phenotype of the DC, derived from two different sources, but there was remarkable difference between their culture time. The DCs from two different sources could activate homologous lymphocytes to proliferate mostly. But the capacity to stimulate the proliferation of homologous lymphocytes had remarkable difference. Conclusion Mature DCs could be induced from cord blood and malignant pleural effusions.

Promotion of Angelica polysaccharide on inducing chronic myelocytic leukemia cells into dendritic cells / 中草药

Object To investigate the effect of Angelica polysaccharide (APS) on the induction of chronic myelocytic leukemia cells into chronic myelocytic leukaemia dendritic cells (CML-DCs). Methods Bone marrow monocytes from CML patients were cultured in GM-CSF/IL-4 or in GM-CSF/IL-4 combined with APS in each concentration (50, 100, 200 mg/L), respectively. The morphotype of CML-DCs was identified by optical microscope or electron microscope, CML-DCs viability was calculated by Trypan Blue exclusion. The phenotype of CML-DCs (CD 80, CD 86, and CD 83) was identified by flow cytometry. The capability of stimulating auto-lymphocyte or allo-lymphocyte proliferation was tested with mixed leukocyte reaction (MLR). Results Bone marrow monocytes from CML patients, which were cultured in GM-CSF/IL-4 or in GM-CSF/IL-4/APS showed typical morphotype and expressed the high level phenotype of CML-DCs. The capability of proliferation and the survival rate of CML-DCs were enhanced markedly and the expression of CD 83, CD 80, and CD 86 on CML-DCs were significantly increased when CML-DCs were cultured in GM-CSF/IL-4/APS. The capability of stimulating lymphocyte proliferation was more competent in 100 mg/L APS group. Conclusion The expression of CD 83, CD 80, and CD 86 on CML-DCs cultured in GM-CSF/IL-4/APS is significantly higher than those in GM-CSF/IL-4. The capability of CML-DCs of stimulating lymphocyte proliferation is more potential in GM-CSF/IL-4/APS than in GM-CSF/IL-4. APS can promote the induction and mature of CML-DCs cultured in IL-4 and GM-CSF.

Astragalus polysaccharides(APS) induces the cord blood monocytes into mature dendritic cells(DCs) directly and its impact on T cells proliferation / 中国免疫学杂志

Objective:To observe the effect of Astragalus polysaccharides(APS) on inducing the cord blood monocytes into mature dendritic cells(DCs) in vitro and to investigate their morphous,cellar immunological characteristics,and contribution to T cell proliferation.Methods:①The cord blood monocytes were isolated by lymphocyte isolating solution under axenic condition,and three groups were divided.②Cells cultured with APS in concentration of 100 mg/L as the experiment group, that with the cytokines of GM-CSF/IL-4/TNF-? as the positive control, and another without either GM-CSF/IL-4/TNF-? or APS as the negative control, respectively. The morphology of DCs was identified by inverted optical microscope or scanning electron microscope. The phenotype of 12 days cultures of DCs(CD1a, CD80, CD86 and CD83) were identified by flowcytometry. The DCs preparations from the experiment group were treated with mitomycin for 45 min to remove their proliferative activity as incentive cells in the mixed cultures with allogenic peripheral blood mononuclear cells from healthy volunteers as responders. T cell proliferation induced with the DCs preparations was detected by MTT chromatometry.Results:After cultured for 72 hours, the cell of both the experiment group and the positive control grew dusteringly and began to change from round to irregularin shapes. The longer the cells were cultured, the more obvious the dendritic structure is. The cells of experiment group and the positive control group when cultured for 12 days had the most typical dendritic structure. The negative control group cells had no dendritic structure and became macrophages when cultured for 12 days. The experiment group cells cultured for 10 days showed typical dendritic morphology by SEM. The experiment group cells and the positive group cells cultured for 12 days significantly expressed high level of the phenotypes of DCs(CD1a, CD80, CD86 and CD83) by flowcytometry.And the difference exhibitied statistical significance when compared with the negative control group(P0.05).The mixed lymphocyte reaction showed that the DCs induced by APS trigerred proliferation of allogenic T cells obviously.Conclusion:Both APS and cytokine could induce the cord blood monocytes to differentiate into functional DCs committedly. DCs reduced by APS stimualate proliferation of the allogenic T cells obviously.