6-kDa early secretory antigenic target (ESAT6), a virulent factor of Mycobacterium tuberculosis, is involved in immune regulation. However, the underlying mechanism behind the activation and maturation of dendritic cells (DCs) by ESAT6 remains unclear. In this study, we investigated the effect on TLRs signaling on the regulation of ESAT6-induced activation and maturation of DCs. ESAT6 induced production of IL-6, TNF-α, and IL-12p40 in bone marrow-derived dendritic cells (BMDCs) from wild-type and TLR2-deficient mice, with this induction abolished in TLR4-deficient cells. NF-κB is essential for the ESAT6-induced production of the cytokines in BMDCs. TLR4 was also required for ESAT6-induced activation of NF-κB and MAPKs in BMDCs. ESAT6 additionally upregulated the expression of surface molecules CD80, CD86, and MHC-II, and also promoted the ability of CD4⁺ T cells to secrete IFN-γ via the TLR4-dependent pathway. Our findings suggest that TLR4 is critical in the activation and maturation of DCs in response to ESAT6.
Animals ; Cytokines ; Dendritic Cells ; Interleukin-12 Subunit p40 ; Interleukin-6 ; Mice ; Mycobacterium tuberculosis ; Mycobacterium ; T-Lymphocytes ; Toll-Like Receptor 4

BACKGROUND: Although phenotypic heterogeneity of psoriasis is suggested by the alternate activation of either T-helper (Th)1-related or Th17-related cytokines, little is known about the mRNA levels of inflammatory cytokines. OBJECTIVE: To investigate whether there is differential expression of Th1-related and Th17-related inflammatory cytokine genes 1) between psoriatic patients and healthy controls, and 2) between patients with different psoriasis phenotypes. METHODS: Twenty-five patients with psoriasis (10 with guttate psoriasis and 15 with plaque psoriasis) and 5 healthy volunteers were enrolled in this study. The mRNA levels of circulating cytokines (interleukin [IL]-2, IL-12p40, interferon-γ, IL-17A, IL-22, and IL-23R) were measured by real-time reverse transcription polymerase chain reaction. RESULTS: The comparison between psoriatic and healthy control samples revealed that IL-12p40, IL-17A, and IL-22 mRNA levels were significantly higher (approximately 4∼6 folds) in the patients with psoriasis. The mRNA levels of these six cytokines in the blood did not differ between the guttate and plaque psoriasis groups. CONCLUSION: We found that the mRNA levels of blood inflammatory cytokines (IL-12p40, IL-17A, and IL-22) were significantly elevated in patients with psoriasis compared to the levels in healthy controls, but they did not significantly differ between patients with guttate and plaque type psoriasis.
Cytokines* ; Gene Expression* ; Healthy Volunteers ; Humans ; Interleukin-12 Subunit p40 ; Interleukin-17 ; Phenotype ; Polymerase Chain Reaction ; Population Characteristics ; Psoriasis ; Reverse Transcription ; RNA, Messenger

OBJECTIVEThis paper aimed to determine the mRNA expression of osteoclast-related factors interleukin-6 (IL-6), interleukin-12 (IL-12) p35, IL-12p40, matrix metalloproteinase-9 (MMP-9), nuclear factor of activated T-cells cytoplasmic 1 (NFATcl), receptor activator of nuclear factor-KB (RANK), and tumor necrosis factor-α (TNF-α) mRNA in murine macrophages infected by a periodontitis patient's own tissue nucleic acid. Another aim was to investigate the effects of a periodontitis patient's own tissue nucleic acid on the differentiation of macrophages into osteoclasts.

METHODSInflammatory periodontal tissue samples of chronic periodontitis patients were taken during periodontal flap surgery, and healthy gingival tissue samples were taken from orthodontic patients during tooth extractions. Total RNA from periodontal tissue was extracted and reversely transcribed into cDNA and then cryo-preserved until further use. First, specific sequence oligodeoxynucleotide MT0I at a concentration of 1 µg · mL⁻¹ was added in murine macrophage RAW264.7, and the cells were incubated for 3 hours. Cells with PBS (1 µg · mL⁻¹) were used as negative controls. The inflammatory periodontal tissue cDNA and healthy periodontal tissue cDNA (1 µg · mL⁻¹) was added subsequently. There were four experimental groups: healthy periodontal tissue cDNA+ RAW264.7, inflammatory periodontal tissue cDNA+RAW264.7, MT01+healthy periodontal tissue cDNA+RAW264.7, and MT01+inflammatory periodontal tissue cDNA+RAW264.7. Real-time quantitative polymerase chain reaction was used to detect the mRNA expression of osteoclast-related factors IL-6, IL-12p35, IL-12p4O, MMP-9, NFATcl, RANK, and TNF-α mRNA after 3, 6, 12, and 24-hours.

RESULTSThe mRNA levels of osteoclast-related factors NFATc1, MMP-9, TNF-a, IL-6, IL-12p40, IL-12p35, and RANK in RAW264.7 were markedly upregulated with the treatment of periodontitis patient's own tissue nucleic acid. However, the mRNA expression of osteoclast-related factors was inhibited by use of an immunosuppressant MT01.

CONCLUSIONThe periodontitis patient's own tissue nucleic acid could promote the differentiation of murine macrophage into osteoclasts.

Animals ; Cell Differentiation ; Cytokines ; metabolism ; Gene Expression ; Gingiva ; Humans ; Interleukin-12 Subunit p40 ; Interleukin-6 ; Macrophages ; Matrix Metalloproteinase 9 ; Mice ; Osteoclasts ; metabolism ; Periodontitis ; RNA, Messenger ; Tumor Necrosis Factor-alpha

BACKGROUND: The phenotypic heterogeneity of psoriasis could be explained by the alternate activation of either T-helper (Th)-1- or Th-17-related cytokines. However, evidence directly supporting this hypothesis is scarce. OBJECTIVE: To characterize the expression of Th-1- and Th-17-related cytokines according to the morphological psoriasis phenotype: guttate vs. plaque. METHODS: In this study, we enrolled 68 patients exhibiting either guttate or plaque psoriasis, and 10 healthy controls. To avoid age-related bias, age matching was performed for each group. Circulating levels of interferon (IFN)-gamma, interleukin (IL)-1RA, IL-2, IL-12p40, IL-17A, IL-22, and IL-23 were measured with an enzyme-linked immunosorbent assay (ELISA). Psoriasis-affected tissue was obtained through biopsy sampling from the eight patients who exhibited the most typical morphology. Levels of IL-1RA, IL-12p40, IL-17, IL-22, and IL-23 in the psoriasis tissue samples were measured with western blot analysis. RESULTS: ELISAs of the serum samples showed higher levels of inflammatory cytokines such as IL-1RA, IL-2, IL-23, and IFN-gamma in patients with psoriasis than in healthy controls. However, the inflammatory cytokine levels did not differ significantly between guttate and plaque psoriasis patients. Western blot analysis of psoriatic tissue revealed higher protein levels of Th-1- and Th-17-related cytokines in patients than in healthy controls. The levels of IL-12p40 and IL-23 were unexpectedly higher in plaque tissue than in guttate tissue. CONCLUSION: The morphological phenotype of psoriasis does not appear to be determined by a specific activation of either the Th-1 or Th-17 pathway. Rather, the cytokine profile influences disease activity and is altered according to the status of the lesion (early or chronic).
Bias (Epidemiology) ; Biopsy ; Blotting, Western ; Cytokines ; Enzyme-Linked Immunosorbent Assay ; Humans ; Interferons ; Interleukin 1 Receptor Antagonist Protein ; Interleukin-12 Subunit p40 ; Interleukin-17 ; Interleukin-2 ; Interleukin-23 ; Interleukins ; Phenotype ; Population Characteristics ; Psoriasis*

The purpose of this study was to explore the effect of NOD2 signalling pathway activated by muramyl dipeptide (MDP) on the immunomodulation effect of human monocyte-derived dendritic cells (DC) loaded with leukemia cell lysates. Peripheral blood mononuclear cells (PBMNC) were isolated by density gradient centrifugation, These cells were cultured with three cytokines for 7 days to induce their maturation. On the 5th day, cells were loaded with leukemia cell HL-60 lysates. NOD2 expression was detected by RT-PCR and Western blot. The phenotype of DC were analyzed by flow cytometry, and ELISA was used to assay levels of IL-12 (p40) . The results showed that MDP could trigger NOD2 mRNA and protein expression in different groups of DC, especially in sensitized DC+MDP group, which was significantly higher than that in the DC+MDP group and sensitized DC without MDP stimulation, the difference was statistically significant (P < 0.05). Besides, the expression of surface molecules (HLA-DR, CD80, CD83, CD86, CD40) in the group of DC loaded with leukemia cell lysate and stimulated by MDP (sensitized DC+MDP) reached the highest level, followed by the group of DC loaded with leukemia cell lysate without MDP and DC only stimulated by MDP, non-treated DC were the lowest (P < 0.05). Similarly, compared with untreated unstimulated DC, after loading with HL-60 lysates or only stimulating with MDP, the secretion of IL-12p40 increased, but IL-12p40 level (573.86 ± 32.09 pg/ml) in DC+MDP group was higher than that in group of sensitized DC (365.03 ± 28.86 pg/ml) (P < 0.05), and it in sensitized DC+MDP group reached the highest (898.30 ± 61.08) pg/ml, compared to other groups (P < 0.05). It is concluded that MDP can significantly enhance the NOD2 mRNA and protein expression in sensitized DC, promote the expression of HLA-DR, synergistic costimulatory molecules and adhesion molecules of DC, at the same time, MDP can increase secretion of inflammatory factors IL-12p40. This study will provide a new ideas for DC application in leukemia immunotherapy.
Acetylmuramyl-Alanyl-Isoglutamine ; pharmacology ; Cells, Cultured ; Dendritic Cells ; immunology ; metabolism ; HL-60 Cells ; Humans ; Interleukin-12 Subunit p40 ; secretion ; Leukemia ; immunology ; metabolism ; Leukocytes, Mononuclear ; metabolism ; Membrane Proteins ; metabolism ; Nod2 Signaling Adaptor Protein ; metabolism

IL-12 is a secretory heterodimeric cytokine composed of p35 and p40 subunits. IL-12 p35 and p40 subunits are sometimes produced as monomers or homodimers. IL-12 is also produced as a membrane-bound form in some cases. In this study, we hypothesized that the membrane-bound form of IL-12 subunits may function as a costimulatory signal for selective activation of TAA-specific CTL through direct priming without involving antigen presenting cells and helper T cells. MethA fibrosarcoma cells were transfected with expression vectors of membrane-bound form of IL-12p35 (mbIL-12p35) or IL-12p40 subunit (mbIL-12p40) and were selected under G418-containing medium. The tumor cell clones were analyzed for the expression of mbIL-12p35 or p40 subunit and for their stimulatory effects on macrophages. The responsible T-cell subpopulation for antitumor activity of mbIL-12p35 expressing tumor clone was also analyzed in T cell subset-depleted mice. Expression of transfected membrane-bound form of IL-12 subunits was stable during more than 3 months of in vitro culture, and the chimeric molecules were not released into culture supernatants. Neither the mbIL-12p35-expressing tumor clones nor mbIL-12p40-expressing tumor clones activated macrophages to secrete TNF-alpha. Growth of mbIL-12p35-expressing tumor clones was more accelerated in the CD8+ T cell-depleted mice than in CD4+ T cell-depleted or normal mice. These results suggest that CD8+ T cells could be responsible for the rejection of mbIL-12p35-expressing tumor clone, which may bypass activation of antigen presenting cells and CD4+ helper T cells.
Animals ; Antigen-Presenting Cells ; Clone Cells ; Corynebacterium ; Fibrosarcoma ; Interleukin-12 ; Interleukin-12 Subunit p35 ; Interleukin-12 Subunit p40 ; Macrophages ; Mice ; Rejection (Psychology) ; T-Lymphocytes ; T-Lymphocytes, Helper-Inducer ; Tumor Necrosis Factor-alpha

BACKGROUND/AIMS: Epigallocatechin-3-gallate (EGCG), the primary catechin in green tea, has anti-inflammatory and anti-oxidative properties. The aim of the current study was to characterize the impact of EGCG on lipopolysaccharide (LPS)-induced innate signaling in bone marrow-derived macrophages (BMMs) isolated from ICR mice. METHODS: The effect of EGCG on LPS-induced pro-inflammatory gene expression and nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein kinase (MAPK) signaling was examined using reverse transcription-polymerase chain reaction, Western blotting, immunofluorescence, and the electrophoretic mobility shift assay. RESULTS: EGCG inhibited accumulation of LPS-induced IL-12p40, IL-6, MCP-1, ICAM-1, and VCAM-1 mRNA in BMMs. EGCG blocked LPS-induced IkappaBalpha degradation and RelA nuclear translocation. EGCG blocked the DNA-binding activity of NF-kappaB. LPS-induced phosphorylation of ERK1/2, JNK, and p38 was inhibited by EGCG. U0126 (an inhibitor of MEK-1/2) suppressed the LPS-induced IL-12p40, IL-6, MCP-1, ICAM-1, and VCAM-1 mRNA accumulation in BMMs. CONCLUSIONS: These results indicate that EGCG may prevent LPS-induced pro-inflammatory gene expression through blocking NF-kappaB and MAPK signaling pathways in BMMs.
Blotting, Western ; Butadienes ; Catechin ; Fluorescent Antibody Technique ; Gene Expression ; I-kappa B Proteins ; Intercellular Adhesion Molecule-1 ; Interleukin-12 Subunit p40 ; Interleukin-6 ; Macrophages ; NF-kappa B ; Nitriles ; Phosphorylation ; Protein Kinases ; RNA, Messenger ; Tea ; Vascular Cell Adhesion Molecule-1

The aim of this study was to investigate the expression and immunologic regulation function of interleukin-23 and its related cytokines in chronic idiopathic thrombocytopenic purpura (ITP) patients. Levels of cytokines in peripheral blood mononuclear cells (PBMNC) were detected by reverse-transcription real-time polymerase chain reaction in 30 patients with chronic ITP and 15 healthy volunteers. The quantity of IL-23, IL-12, IL-17 in serum was detected by enzyme-linked immunosorbent assay (ELISA). The results showed that low detectable mRNA levels of IL-23p19, IL-12p35, IL-27 and IL-12p40 were found in all patients and healthy persons. Trace of IL-17 mRNA were expressed in PBMNC of part of patients and normal controls. Levels of IL-12p35, IL-27, IL-17 mRNA between healthy persons and chronic ITP patients were not statistically different. Compared with normal controls, patients showed the lower expression levels of IL-23p19 and IL-12p40 mRNA (p < 0.01). The IL-12 levels of chronic ITP patients were significantly higher than that of normal controls (p < 0.01). The IL-23 and IL-17 levels of chronic ITP patients were same to that of normal controls. It is concluded that the imbalance of T cell subsets in ITP patients mainly associated with IL-12, but not with IL-23 and IL-17.
Adolescent ; Adult ; Case-Control Studies ; Chronic Disease ; Female ; Humans ; Interleukin-12 ; metabolism ; Interleukin-12 Subunit p35 ; metabolism ; Interleukin-12 Subunit p40 ; metabolism ; Interleukin-17 ; metabolism ; Interleukin-23 ; metabolism ; Interleukin-23 Subunit p19 ; metabolism ; Male ; Middle Aged ; Purpura, Thrombocytopenic, Idiopathic ; metabolism ; Young Adult

Genetic Predisposition to Disease ; Humans ; Interleukin-12 Subunit p40 ; physiology ; Receptors, Interferon ; physiology ; Receptors, Interleukin-12 ; physiology ; Toll-Like Receptors ; physiology ; Tuberculosis ; genetics ; immunology

OBJECTIVETo study the ex vivo effect of sirolimus on capacity of splenic dendritic cells (DC) from traumatized mice in inducing T cell responses.

METHODSTwenty-four BALB/c mice were divided into control group and trauma group according to the random number table, with 12 mice in each group. Mice in trauma group were bled followed by closed femur fracture after anaesthesia, while mice in control group were only anaesthetized without injury. Twenty-four hours later DC were isolated from spleens and divided into 4 subgroups: sirolimus devoid control (trauma) groups [consisted of cells from control (trauma) groups, without sirolimus treatment] and sirolimus treated control (trauma) groups [consisted of cells from control (trauma) groups, treated with 10 microg/L sirolimus for 6 hours]. Then their autophagic activity, DC-induced mixed lymphocyte reaction (MLR) were measured and recorded as fluorescence intensity (FI) value and absorbance value respectively. The expression of major histocompatibility complex class (MHC) II and costimulatory molecules CD40, CD80, and CD86 on DC surface were measured with flow cytometry. IL-12p40, IL-12p70 and IL-10 levels in lipopolysaccharide-stimulated DC supernatants were determined by ELISA. Data were processed with one-way analysis of variance.

RESULTS(1) Compared with those of sirolimus devoid control group (FI value = 22 +/- 6), DC autophagic activity (FI value = 13 +/- 2) and DC-induced MLR in mice from sirolimus devoid trauma group were significantly weakened (F = 212.836, P < 0.05). Compared with those of sirolimus devoid control (trauma) groups, DC autophagic activity in mice from sirolimus treated control (trauma) groups (FI = 45 +/- 8, 44 +/- 8 respectively) were significantly strengthened (F = 212.836, P < 0.05 or P < 0.01). MLR in mice from sirolimus treated trauma group was stronger than that from sirolimus devoid trauma group (with F value respectively 101.426, 86.533, P values all below 0.05). (2) Compared with those of sirolimus devoid control group [MHC II (85 +/- 6)%, CD40 (8 +/- 1)%], the expressions of MHCII [(60 +/- 9)%] and CD40 [(4 +/- 1)%] on DC surface from sirolimus devoid trauma group were significantly reduced (with F value respectively 37.918, 40.426, P values all below 0.05). The expression of MHCII from sirolimus treated trauma group [(78 +/- 7)%] was higher than that from sirolimus devoid trauma group (F = 37.918, P < 0.05). (3) IL-12p40, IL-12p70 secretion by DC from sirolimus devoid trauma group [(120 +/- 13), (10 +/- 3) pg/mL] were significantly reduced as compared with those from sirolimus devoid control group [(200 +/- 25), (20 +/- 6) pg/mL, with F value respectively 218.646, 310.253, P values all below 0.05]. Compared with those from sirolimus devoid control (trauma) groups, IL-12p40 [(560 +/- 34), (540 +/- 29) pg/mL], IL-12p70 [(55 +/- 8), (60 +/- 11) pg/mL] secretion by DC from sirolimus treated control (trauma) groups were obviously enhanced (with F value respectively 218.646, 310.253, P values all below 0.01), while IL-10 secretion levels were significantly decreased (F = 246.108, P < 0.01).

CONCLUSIONSSirolimus can partially ameliorate DC functions ex vivo in traumatized mice, and further enhance the capacity of DC in inducing T cell responses.

Animals ; B7-1 Antigen ; metabolism ; B7-2 Antigen ; metabolism ; CD40 Antigens ; metabolism ; Dendritic Cells ; drug effects ; immunology ; metabolism ; Histocompatibility Antigens Class II ; immunology ; Interleukin-12 Subunit p40 ; metabolism ; Lymphocyte Culture Test, Mixed ; Male ; Mice ; Mice, Inbred BALB C ; Sirolimus ; pharmacology ; Spleen ; cytology ; immunology ; T-Lymphocytes ; immunology ; Wounds and Injuries ; immunology