BACKGROUNDDendritic cells are professional antigen-presenting cells found in an immature state in epithelia and interstitial space, where they capture antigens such as pathogens or damaged tissue. Matrix metallopeptidase 13 (MMP-13), a member of the collagenase subfamily, is involved in many different cellular processes and is expressed in murine bone marrow-derived dendritic cells (DCs). The function of MMP-13 in DCs is not well understood. Here, we investigated the effect of MMP-13 on DC maturation, apoptosis, and phagocytosis.

METHODSBone marrow-derived dendritic cells were obtained from C57BL/6 mice. One short-interfering RNA specific for MMP-13 was used to transfect DCs. MMP-13-silenced DCs and control DCs were prepared, and apoptosis was measured using real-time polymerase chain reaction and Western blotting. MMP-13-silenced DCs and control DCs were analyzed for surface expression of CD80 and CD86 and phagocytosis capability using flow cytometry.

RESULTSCompared to the control DCs, MMP-13-silenced DCs increased expression of anti-apoptosis-related genes, BAG1 (control group vs. MMP-13-silenced group: 4.08 ± 0.60 vs. 6.11 ± 0.87, P = 0.008), BCL-2 (control group vs. MMP-13-silenced group: 7.54 ± 0.76 vs. 9.54 ± 1.29, P = 0.036), and TP73 (control group vs. MMP-13-silenced group: 4.33 ± 0.29 vs. 5.60 ± 0.32, P = 0.001) and decreased apoptosis-related genes, CASP1 (control group vs. MMP-13-silenced group: 3.79 ± 0.67 vs. 2.54 ± 0.39, P = 0.019), LTBR (control group vs. MMP-13-silenced group: 9.23 ± 1.25 vs. 6.24 ± 1.15, P = 0.012), and CASP4 (control group vs. MMP-13-silenced group: 2.07 ± 0.56 vs. 0.35 ± 0.35, P = 0.002). Protein levels confirmed the same expression pattern. MMP-13-silenced groups decreased expression of CD86 on DCs; however, there was no statistical difference in CD80 surface expression. Furthermore, MMP-13-silenced groups exhibited weaker phagocytosis capability.

CONCLUSIONThese results indicate that MMP-13 inhibition dampens DC maturation, apoptosis, and phagocytosis.

Animals ; Apoptosis ; drug effects ; physiology ; Bone Marrow Cells ; cytology ; Dendritic Cells ; cytology ; drug effects ; metabolism ; Female ; Lipopolysaccharides ; pharmacology ; Matrix Metalloproteinase 13 ; metabolism ; physiology ; Mice ; Mice, Inbred C57BL ; RNA, Small Interfering

Interleukin-21 (IL-21) is a new member of the interleukin-2 family. It is mainly synthesized and secreted by the activated of CD4(+) T cells and natural killer T cells. IL-21 receptor (IL-21R) is mainly expressed in T cells, B cells, and natural killer (NK) cells. After binding to its receptor, IL-21 can regulate the activation and proliferation of T cells, B cells, and NK cells through activating JAKs-STATs signaling pathways. As a new immunoregulatory factor, IL-21 and its receptor play important roles in the development and progression of various autoimmune diseases. Regulation of the expression levels of IL-21 and IL-21R and blocking of their signal transduction pathways with blockers may be new treatment options for autoimmune diseases.
Animals ; Autoimmune Diseases ; etiology ; immunology ; Dendritic Cells ; immunology ; Humans ; Interleukins ; physiology ; Killer Cells, Natural ; immunology ; Lymphocytes ; immunology ; Receptors, Interleukin-21 ; physiology

OBJECTIVETo compare the CC-chemokine ligand 18 (CCL18) expression in the serum and malignant pleural effusion (MPE) of NSCLC patients and explore its regulatory effect on differentiation of monocyte-derived dendritic cells (Mo-DC).

METHODSCCL18 levels in the serum and MPE from 62 NSCLC patients were quantitated by immunoassay. CCL18 in sera from 26 healthy individuals, 28 exudative pleural effusions from inflammatory pulmonary diseases and 17 transudative pleural effusions from non-inflammatory diseases were used as control. Mo-DC was generated by culturing NSCLC-derived monocytes with GM-CSF and IL-4 in the presence or absence of CCL18. The mean fluorescent intensity (MFI) of CD14, CD80, CD83, CD86 and HLA-DR were analyzed by flow cytometry (FCM). Mo-DC was then co-cultured with purified T cells and the percence of CD25(+)FoxP3(+) cells was assayed by FCM.

RESULTSCCL18 levels in the sera of NSCLC patients and healthy individuals were (132.70 ± 15.52) ng/ml and (18.44 ± 0.99) ng/ml, respectively (P < 0.001). The levels of CCL18 in MPE, exudative PE and transudative PE were (155.6 ± 13.58) ng/ml, (190.4 ± 22.33) ng/ml and (20.89 ± 3.03) ng/ml, respectively. CCL18 in the MPE was significantly higher than that in transudates (P < 0.001), however, no significant difference was observed between CCL18 expression in exudative PE and MPE (P = 0.172). Of note, a moderate positive correlation (r = 0.421, P < 0.01) was observed between CCL18 levels in the paired MPE and serum of NSCLC. In the healthy control group, Mo-DC cultured in the presence of CCL18 showed 31.4 ± 15.8 (MFI) of CD14 expression, which was significantly higher than that in Mo-DC cultured in the absence of CCL18 (18.5 ± 8.9, P < 0.05). In contrast, the expressions of MFI of CD80, CD83, CD86 and HLA-DR were significantly decreased upon CCL18 induction (P < 0.05). In the NSCLC group, GM-CSF+IL-4+CCL18 induced a MFI of 45.2 ± 13.8 of CD14 expression in Mo-DC, which was also significantly higher than that of GM-CSF+ IL-4 induction (22.6 ± 10.5, P < 0.01). Similarly, the expressions of MFI of CD80, CD83, CD86 and HLA-DR were significantly decreased in the presence of CCL18 (P < 0.05). Furthermore, the MFI of CD14, CD83, CD86 and HLA-DR had significant differences between GM-CSF/IL-4/CCL18-induced Mo-DC derived from NSCLC patients and healthy control (P < 0.05). Finally, CD4(+) T cells co-cultured with NSCLC-derived, GM-CSF/IL-4/CCL18-treated Mo-DC had significantly higher percent of CD25(+)FoxP3(+) cells compared with that of CD4(+) T cells stimulated with Mo-DC induced by GM-CSF/IL-4(P < 0.01).

CONCLUSIONSCCL18 is present at a high level in MPE and serum of NSCLC patients complicated with pleural effusion and a moderate positive correlation exists between CCL18 levels in the two fluids. CCL18 inhibits maturation of Mo-DC, which consequently stimulates T cells to differentiate into CD25(+)FoxP3(+) regulatory T cells.

Carcinoma, Non-Small-Cell Lung ; metabolism ; Cell Differentiation ; Chemokines ; Chemokines, CC ; metabolism ; Coculture Techniques ; Dendritic Cells ; metabolism ; Flow Cytometry ; Granulocyte-Macrophage Colony-Stimulating Factor ; metabolism ; Humans ; Interleukin-4 ; metabolism ; Ligands ; Lung Neoplasms ; Monocytes ; physiology ; Pleural Effusion ; T-Lymphocytes, Regulatory

BACKGROUNDDespite extensive research, the mechanism of immature dendritic cells (DCs) induced immune hyporesponsiveness remains incompletely understood.

METHODSRecipient DCs from C3H mouse bone marrow cells were incubated with donor antigen from splenic lymphocytes of C57BL/6 mouse; these DCs were transfected with CD80/86 specific siRNA using lentiviral vectors. Flow cytometry was used to evaluate expression of CD80/86 on the antigen-pulsed recipient DCs. Immune regulatory activity was examined by mixed lymphocyte reaction, in which irradiated DCs were cultured with C3H spleen T cells. After the reaction, interleukin (IL)-2, IL-4, IL-10, and interferon (INF)-γ levels of mixed lymphocyte reaction culture supernatant were measured by enzyme-linked immunosorbent assay. The apoptotic T lymphocytes were identified by Annexin V and CD3 staining.

RESULTSThere was a significant inhibition of CD80/86 expression in DCs transfected with CD80/86 lentiviral vectors compared with the control groups (P < 0.05), indicating the specificity of RNA interference. Enzyme-linked immunosorbent assay results showed a significant reduction of INF-γ, IL-2 and IL-10 in the CD80/86 lentivirus transfected group compared to the control groups (P < 0.05). There was no significant difference in IL-4 levels between the groups (P > 0.05). We also showed that CD80/86 low DCs loaded with alloantigen (1) stimulated low T cell proliferative responses via the indirect recognition pathway and (2) enhanced apoptotic activity (P < 0.05) in co-cultured T cells.

CONCLUSIONSLentiviral vector transfection can effectively and specifically knock down target genes in DCs. The CD80/86 low DCs may show tolerogenic activity via induction of T-cell apoptosis, thereby modulating the activity of recipient-derived DCs. The use of this approach may potentially be clinically applicable.

Animals ; Apoptosis ; B7-1 Antigen ; genetics ; physiology ; B7-2 Antigen ; genetics ; physiology ; Dendritic Cells ; immunology ; Lentivirus ; genetics ; Lymphocyte Activation ; Mice ; Mice, Inbred C3H ; Mice, Inbred C57BL ; RNA Interference ; T-Lymphocytes ; cytology ; immunology

OBJECTIVETo investigate the anti-tumor activity of CCL21-exCD40L eukaryotic expression vector.

METHODSCCL21-exCD40L fusion gene were constructed by overlap PCR connecting CCL21 and exCD40L through a flexible linker (Gly3Ser)4, and then was cloned into expression vector pcDNA3.1(+). pcDNA3.1(+)/CCL21 and pcDNA3.1(+)/exCD were constructed as negative control. Wsestern blot was used to identify the fusion protein. CHO cells was transfected with pcDNA3.1(+)/CCL21-exCD, pcDNA3.1(+)/CCL21 and pcDNA3.1(+), respectively. The chemotatic function of the expressed product was detected by Transwell method and its anti-tumor activity was tested with vivo transfection.

RESULTSGene sequencing and restrictive digestion proved the successful construction of pcDNA3.1(+)/CCL21-exCD40L,and its expression was conformed by western blot. The transfectant supernantes of pcDNA3.1(+)/CCL21-exCD40 group had a significant chmotactic function to DCs, of which the cell numbers passing through the film was 14.95 times of blank control every high power microscope visual field. After tumor orthotoic injection of plasmid carrying fusion gene in Balb/c mouse, the tumor mass reduced remarkablely, and all the mouse in fusion gene group survived after 4 weeks.

CONCLUSIONCCL21-exCD40L fusion protein had a remarkable function to DCs and it can inhibit tumor growth and prolong the mouse survival time, which is more effective than all control group.

Animals ; CD40 Ligand ; genetics ; pharmacology ; CHO Cells ; Cell Line, Tumor ; Chemokine CCL21 ; genetics ; pharmacology ; Colonic Neoplasms ; therapy ; Cricetulus ; Dendritic Cells ; drug effects ; physiology ; Genetic Therapy ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; pharmacology

The fungus Trichophyton schoenleinii (T. schoenleinii) is the causative agent of Trichophytosis and Tinea favosa of the scalp in certain regions of Eurasia and Africa. Human innate immune system plays an important role in combating with various pathogens including fungi. The inflammasome is one of the most critical arms of host innate immunity, which is a protein complex controlling maturation of IL-1β. To clarify whether T. schoenleinii is able to activate the inflammasome, we analyzed human monocytic cell line THP-1 for IL-1β production upon infection with T. schoenleinii strain isolated from Tinea favosa patients, and rapid IL-1β secretion from THP-1 cells was observed. Moreover, applying competitive inhibitors and gene specific silencing with shRNA, we found that T. schoenleinii induced IL-1β secretion, ASC pyroptosome formation as well as caspase-1 activation were all dependent on NLRP3. Cathepsin B activity, ROS production and K⁺ efflux were required for the inflammasome activation by T. schoenleinii. Our data thus reveal that the NLRP3 inflammasome plays an important role in host defense against T. schoenleinii, and suggest that manipulating NLRP3 signaling can be a novel approach for control of diseases caused by T. schoenleinii infection.
Animals ; Bone Marrow Cells ; cytology ; Carrier Proteins ; metabolism ; Caspase 1 ; metabolism ; Cell Line ; Dendritic Cells ; cytology ; metabolism ; microbiology ; Enzyme Activation ; Hot Temperature ; Humans ; Inflammasomes ; metabolism ; Interleukin-1beta ; biosynthesis ; metabolism ; Lysosomes ; metabolism ; Mice ; Monocytes ; cytology ; metabolism ; microbiology ; NLR Family, Pyrin Domain-Containing 3 Protein ; Potassium ; metabolism ; Reactive Oxygen Species ; metabolism ; Signal Transduction ; Trichophyton ; physiology

OBJECTIVEThis paper aims to investigate the anti-tumor mechanism of inactivated Sendai virus (Hemagglutinating virus of Japan envelope, HVJ-E) for murine melanoma (B16F10).

METHODSThe murine dendritic cells (DCs) were treated with HVJ-E, and then the cytokines secreted from DCs and costimulation-related molecules on DCs were measured. Meanwhile, the expression of β-catenin in HVJ-E treated murine melanoma cells was detected. In addition, HVJ-E was intratumorally injected into the melanoma on C57BL/6 mice, and the immune cells, CTL response and tumor volume were analyzed.

RESULTSHVJ-E injected into B16F10 melanoma obviously inhibited the growth of the tumor and prolonged the survival time of the tumor-bearing mice. Profiles of cytokines secreted by dendritic cells (DCs) after HVJ-E stimulation showed that the number of cytokines released was significantly higher than that elicited by PBS (1P<0.05). The co-stimulation-related molecules on DCs were comparable to those stimulated by LPS. Immunohistochemical examinations demonstrated the repression of β-catenin in B16F10 melanoma cells after HVJ-E treatment. Meanwhile, real-time reverse transcription PCR revealed that HVJ-E induced a remarkable infiltration of CD11c positive cells, chemokine ligand 10 (CXCL10) molecules, interleukin-2 (IL-2) molecule, CD4(+) and CD8(+) T cells into HVJ-E injected tumors. Furthermore, the mRNA expression level of β-catenin in the HVJ-E injected tumors was also down-regulated. In addition, B16F10-specific CTLs were induced significantly after HVJ-E was injected into the tumor-bearing mice.

CONCLUSIONThis is the first report to show the effective inhibition of melanoma tumors by HVJ-E alone and the mechanism through which it induces antitumor immune responses and regulates important signal pathways for melanoma invasion. Therefore, HVJ-E shows its prospect as a novel therapeutic for melanoma therapy.

Animals ; Cell Line, Tumor ; Cytokines ; genetics ; metabolism ; Dendritic Cells ; immunology ; physiology ; virology ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; Melanoma ; immunology ; pathology ; virology ; Mice ; Mice, Inbred C57BL ; Neoplasms, Experimental ; Real-Time Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; Sendai virus ; physiology ; Virus Inactivation ; Virus Replication ; beta Catenin ; genetics ; metabolism

BACKGROUNDAlthough traumatic brain injury can lead to opening the blood-brain barrier and leaking of blood substances (including water) into brain tissue, few studies of brain antigens leaking into the blood and the pathways have been reported. Brain antigens result in damage to brain tissues by stimulating the immune system to produce anti-brain antibodies, but no treatment has been reported to reduce the production of anti-brain antibodies and protect the brain tissue. The aim of the study is to confirm the relationship between immune injury and arachnoid granulations following traumatic brain injury, and provide some new methods to inhibit the immune injury.

METHODSIn part one, methylene blue was injected into the rabbits' cisterna magna after traumatic brain injury, and concentrations of methylene blue and tumor necrosis factor (TNF)-α in blood were detected to determine the permeability of arachnoid granulations. In part two, umbilical cord mesenchymal stem cells and immature dendritic cells were injected into veins, and concentrations of interleukin 1 (IL-1), IL-10, interferon (IFN)-γ, transforming growth factor (TGF)-β, anti-brain antibodies (ABAb), and IL-12 were measured by ELISA on days 1, 3, 7, 14 and 21 after injury, and the numbers of leukocytes in the blood were counted. Twenty-one days after injury, expression of glutamate in brain tissue was determined by immunohistochemical staining, and neuronal degeneration was detected by H&E staining.

RESULTSIn part one, blood concentrations of methylene blue and TNF-α in the traumatic brain injury group were higher than in the control group (P < 0.05). Concentrations of methylene blue and TNF-α in the trauma cerebrospinal fluid (CSF) injected group were higher than in the control cerebrospinal fluid injected group (P < 0.05). In part two, concentrations of IL-1, IFN-γ, ABAb, IL-12, expression of glutamate (Glu), neuronal degeneration and number of peripheral blood leukocytes were lower in the group with cell treatment compared to the control group. IL-10 and TGF-β were elevated compared to the control group.

CONCLUSIONSTraumatic brain injury can lead to stronger arachnoid granulations (AGs) permeability; umbilical cord mesenchymal stem cells and immature dendritic cells can induce immune tolerance and reduce inflammation and anti-brain antibodies to protect the brain tissue.

Adipocytes ; cytology ; Animals ; Antigens ; blood ; metabolism ; Brain Injuries ; blood ; cerebrospinal fluid ; metabolism ; Cell Differentiation ; physiology ; Cells, Cultured ; Dendritic Cells ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Interleukin-1 ; blood ; cerebrospinal fluid ; Interleukin-10 ; blood ; cerebrospinal fluid ; Interleukin-12 ; blood ; cerebrospinal fluid ; Mesenchymal Stromal Cells ; cytology ; Methylene Blue ; metabolism ; Osteoblasts ; cytology ; Rabbits ; Transforming Growth Factor beta ; blood ; cerebrospinal fluid ; Tumor Necrosis Factor-alpha ; blood ; cerebrospinal fluid

BACKGROUNDVibrio vulnificus (Vv) is an estuarine bacterium that can cause primary septicemia as well as serious wound infections. However, little is known about the mechanisms by which Vv infects dendritic cells (DCs) and its effects on cytoskeleton. In this study, we aimed to investigate the invasion, internalization, and the organelles damage of the cultured dendritic cells (a DC 2.4 strain) during Vv infection.

METHODSThe study model was the cultured DCs infected by a Vv 1.758 strain. Electron microscopy was used to observe the localization of bacteria at the different time points of infection, cell morphology, and the process of organelles changes. The cytoskeleton structure including the microfilaments and the microtubules rearrangement was examined under a fluorescence microscope.

RESULTSThe Vv were pinocytosised into the DC cells through double-sides, and localized at 1 - 2 mm of the inner side membrane. It took 1.3, 1.9, and 3.4 hours to reach the infection ratio of 25%, 50%, and 75%, respectively. Using electron microscopy, the DCs had been observed to have developed chromatin aggregation within 4.0 hours, and significant cytoskeleton structure disruption was noted within 6.0 hours.

CONCLUSIONThe high lethality of Vv infection may be associated with the direct disruption of the DCs cytoskeleton structure.

Animals ; Apoptosis ; physiology ; Cells, Cultured ; Cytoskeleton ; metabolism ; ultrastructure ; DNA Fragmentation ; Dendritic Cells ; metabolism ; microbiology ; ultrastructure ; Mice ; Microscopy, Electron ; Microscopy, Electron, Transmission ; Vibrio Infections ; metabolism ; Vibrio vulnificus ; pathogenicity

Dendritic cells (DCs) as a rare type of leukocytes play an important role in bridging the innate and adaptive immune system. A subset of DCs, monocyte-derived dendritic cells (moDCs), exists in very low numbers at steady state but become abundant in inflammatory states. These inflammation-associated DCs are potent producers of pro-inflammatory cytokines and potent inducers of T helper differentiation. They behave as a "double-edge" sword so that they not only mediate protective immunity but also immuno-pathology. It is still incompletely understood how their function is regulated. Emerging evidence indicates that microRNAs (miRNAs), as a new class of gene regulators, potently regulate the function of moDCs. Here we summarize recent progress in this area.
Animals ; Antigen Presentation ; genetics ; Cell Differentiation ; Cytokines ; genetics ; metabolism ; Dendritic Cells ; metabolism ; physiology ; Humans ; Inflammation ; immunology ; pathology ; MicroRNAs ; metabolism ; physiology ; RNA Interference