This study was aimed to investigate the influence of TLR2 and TLR4 agonists on the migration and adhesion activity of umbilical cord blood (UCB) CD34(+) cells and to explore the underlying mechanism. The expression of TLR2 and TLR4 on UCB CD34(+) cells was detected with flow cytometry. The effect of TLR2 agonist (PAM3CSK4) and TLR2 agonist (LPS) on the migration and adhesion ability of UCB CD34(+) cells was evaluated with chemotaxis and adhesion assays. The results indicated that expression levels of TLR2 and TLR4 were (14.2 ± 3.8)%, (19.6 ± 4.1)% respectively. Compared with the control group, the migration activity of UCB CD34(+) cells toward SDF-1 decreased significantly in LPS group (p < 0.01). The adhesion activity was not altered significantly in LPS group. However, both the migration activity towards SDF-1 and the adhesion activity of UCB CD34(+) cells were not changed significantly in PAM3CSK4 group. Further study found that LPS did not affect the expression level of CXCR4 on CD34(+) cells, but could inhibit the spontaneous migration ability of CD34(+) cells. It is concluded that TLR4 activation can decrease the chemotaxis function of CD34(+) cells towards SDF-1, which may associate with the decreased spontaneous migration ability of CD34(+) cells.
Antigens, CD34 ; blood ; Cell Movement ; drug effects ; Cells, Cultured ; Chemokine CXCL12 ; Fetal Blood ; cytology ; immunology ; Humans ; Lipopeptides ; pharmacology ; Lipopolysaccharides ; pharmacology ; Toll-Like Receptor 2 ; agonists ; Toll-Like Receptor 4 ; agonists

This study was aimed to investigate the influence of TLR2 and TLR4 agonists on the migration and adhesion activity of human bone marrow-derived mesenchymal stem cells (MSC) and to clarify the underlying mechanisms. The expression of TLR2 and TLR4 on MSC was detected by flow cytometry. The effects of TLR2 agonist (PAM3CSK4) and TLR2 agonist (LPS) on MSC migration and adhesion ability were evaluated with chemotaxis and adhesion test. The results indicated that expressive levels of TLR2 and TLR4 on surface of human bone marrow MSC were (24.5 ± 3.2)% and (91.3 ± 5.2)% respectively. Compared with the control group, the migration activity of MSC toward SDF-1 was decreased significantly in PAM3CSK4 group, while the adhesion activity of MSC was promoted by PAM3CSK4 exposure. However, both the migration activity toward SDF-1 and the adhesion activity of MSC were not changed significantly in LPS-treated group. Further, it was found that PAM3CSK4 did not affect the expressive level of CXCR4 on MSC, however, it could inhibit the spontaneous migration of MSC in dose dependent manner. It is concluded that activation of TLR2 can decrease the migration ability of MSC, which may associate with the decreased spontaneous migration ability and the increased adhesion activity of MSC.
Bone Marrow Cells ; cytology ; drug effects ; Cell Movement ; drug effects ; Cells, Cultured ; Humans ; Lipopeptides ; pharmacology ; Lipopolysaccharides ; pharmacology ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Toll-Like Receptor 2 ; agonists ; Toll-Like Receptor 4 ; agonists

Dendritic cells (DCs) play a role in natural killer (NK) cell activation, while NK cells are also able to activate and mature DCs. Toll-like receptors (TLRs) on the surface of DCs and NK cells induce the maturation and activation of these cells when engaged with their cognate ligand. We investigated to generate potent DCs by maturation with NK cells in the presence of TLR agonist in vitro and tested the efficacy of these DC vaccinations in mouse colon cancer model. The optimal ratios of DCs versus NK cells were 1:1 to 1:2. Immature DCs were mature with NK cells in the presence of lipopolysaccharide, which is TLR4 agonist, and further addition of IL-2 induced phenotypically and functionally mature bone marrow-derived DCs. These potent DCs exhibited not only high expression of several costimulatory molecules and high production of IL-12p40 and IL-12p70, but also high allogeneic T cells stimulatory capacity, and the induction of the high activities to generate tumor-specific CTLs. Consistently, vaccination with these DCs efficiently inhibited CT-26 tumor growth in mouse colon cancer model when compared to other vaccination strategies. Interestingly, combination therapy of these DC-based vaccines and with low-dose cyclophosphamide showed dramatic inhibition effects of tumor growth. These results suggest that the DCs maturated with NK cells in the presence of TLR agonist are potent inducer of antitumor immune responses in mouse model and may provide a new source of DC-based vaccines for the development of immunotherapy against colon cancer.
Animals ; Cancer Vaccines/immunology/metabolism ; Carcinoma/immunology/pathology/*therapy ; Cell Line, Tumor ; Cells, Cultured ; Colonic Neoplasms/immunology/pathology/*therapy ; Dendritic Cells/*drug effects/*immunology/transplantation ; Female ; Immunotherapy, Adoptive/*methods ; Killer Cells, Natural/*immunology/physiology ; Lipopolysaccharides/pharmacology ; Mice ; Mice, Inbred BALB C ; Toll-Like Receptor 4/agonists ; Toll-Like Receptors/*agonists

In this study, we showed the direct interaction between Mycobacterium avium subsp. paratuberculosis fibronectin attachment protein (FAP) and toll-like receptor4 (TLR4) via co-localization and binding by using confocal microscopy and co-immunoprecipitation assays. FAP triggered the expression of pro- and anti-inflammatory cytokines in a TLR4-dependent manner. In addition, FAP-induced cytokine expression in bone marrow-derived dendritic cells (BMDCs) was modulated in part by glycogen synthase kinase-3 (GSK-3). FAP-induced expression of CD80, CD86, major histocompatibility complex (MHC) class I, and MHC class II in TLR4+/+ BMDCs was not observed in TLR4-/- BMDCs. Furthermore, FAP induced DC-mediated CD8+ T cell proliferation and cytotoxic T lymphocyte (CTL) activity, and suppressed tumor growth with DC-based tumor vaccination in EG7 thymoma murine model. Taken together, these results indicate that the TLR4 agonist, FAP, a potential immunoadjuvant for DC-based cancer vaccination, improves the DC-based immune response via the TLR4 signaling pathway.
*Adhesins, Bacterial/genetics/metabolism ; Animals ; CD8-Positive T-Lymphocytes/metabolism ; *Cancer Vaccines/therapeutic use ; Cell Proliferation ; Cytokines/metabolism ; Dendritic Cells/*cytology ; Disease Models, Animal ; Gene Expression Regulation ; Glycogen Synthase Kinase 3/metabolism ; Humans ; Mice ; Mice, Inbred C57BL ; Mycobacterium avium/genetics/metabolism ; Paratuberculosis/metabolism ; Protein Binding ; Signal Transduction ; T-Lymphocytes, Cytotoxic/metabolism ; *Thymoma/genetics/metabolism ; *Toll-Like Receptor 4/agonists/genetics/metabolism

BACKGROUND/AIMS: Asthma is characterized by airway hyperresponsiveness, inflammation, and remodeling. Peroxisome proliferator-activated receptors have been reported to regulate inflammatory responses in many cells. In this study, we examined the effects of intranasal rosiglitazone on airway remodeling in a chronic asthma model. METHODS: We developed a mouse model of airway remodeling, including smooth muscle thickening, in which ovalbumin (OVA)-sensitized mice were repeatedly exposed to intranasal OVA administration twice per week for 3 months. Mice were treated intranasally with rosiglitazone with or without an antagonist during OVA challenge. We determined airway inflammation and the degree of airway remodeling by smooth muscle actin area and collagen deposition. RESULTS: Mice chronically exposed to OVA developed sustained eosinophilic airway inflammation, compared with control mice. Additionally, the mice developed features of airway remodeling, including thickening of the peribronchial smooth muscle layer. Administration of rosiglitazone intranasally inhibited the eosinophilic inflammation significantly, and, importantly, airway smooth muscle remodeling in mice chronically exposed to OVA. Expression of Toll-like receptor (TLR)-4 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) was increased in the OVA group and decreased in the rosiglitazone group. Co-treatment with GW9660 (a rosiglitazone antagonist) and rosiglitazone increased the expression of TLR-4 and NF-kappaB. CONCLUSIONS: These results suggest that intranasal administration of rosiglitazone can prevent not only air way inf lammation but also air way remodeling associated with chronic allergen challenge. This beneficial effect is mediated by inhibition of TLR-4 and NF-kappaB pathways.
Actins/metabolism ; Administration, Inhalation ; Airway Remodeling/*drug effects ; Animals ; Anti-Asthmatic Agents/*administration & dosage ; Asthma/chemically induced/*drug therapy/metabolism/physiopathology ; Chronic Disease ; Collagen/metabolism ; Disease Models, Animal ; Female ; Lung/*drug effects/metabolism/physiopathology ; Mice, Inbred BALB C ; NF-kappa B/metabolism ; Ovalbumin ; PPAR gamma/agonists/metabolism ; Pneumonia/chemically induced/physiopathology ; Pulmonary Eosinophilia/chemically induced/prevention & control ; Signal Transduction/drug effects ; Thiazolidinediones/*administration & dosage ; Toll-Like Receptor 4/metabolism

We evaluated the effectiveness of rhamnogalacturonan II (RG-II)-stimulated bone marrow-derived dendritic cells (BMDCs) vaccination on the induction of antitumor immunity in a mouse lymphoma model using EG7-lymphoma cells expressing ovalbumin (OVA). BMDCs treated with RG-II had an activated phenotype. RG-II induced interleukin (IL)-12, IL-1beta, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) production during dendritic cell (DC) maturation. BMDCs stimulated with RG-II facilitate the proliferation of CD8+ T cells. Using BMDCs from the mice deficient in Toll-like receptors (TLRs), we revealed that RG-II activity is dependent on TLR4. RG-II showed a preventive effect of immunization with OVA-pulsed BMDCs against EG7 lymphoma. These results suggested that RG-II expedites the DC-based immune response through the TLR4 signaling pathway.
Acute-Phase Proteins/metabolism ; Adaptor Proteins, Vesicular Transport/metabolism ; Animals ; Antigens, CD14/metabolism ; Bone Marrow Cells/cytology/drug effects ; CD8-Positive T-Lymphocytes/*immunology ; Carrier Proteins/metabolism ; Cell Differentiation/drug effects ; Cell Nucleus/drug effects/metabolism ; Cell Proliferation/drug effects ; Cytokines/biosynthesis ; Dendritic Cells/cytology/drug effects/enzymology/*immunology ; Enzyme Activation/drug effects ; Lymphocyte Activation/*drug effects ; Membrane Glycoproteins/metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mitogen-Activated Protein Kinases/metabolism ; Myeloid Differentiation Factor 88/metabolism ; NF-kappa B/metabolism ; Neoplasms/immunology/*pathology ; Pectins/*pharmacology ; Phenotype ; Protein Transport/drug effects ; Receptors, Chemokine/metabolism ; Signal Transduction/drug effects ; T-Lymphocytes, Cytotoxic/cytology/drug effects ; Toll-Like Receptor 4/*agonists/metabolism