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

Receptor-interacting serine/threonine kinase 2(RIPK2) is an adaptor molecule involved in the signal pathway of TLRs. However, there is no report on association between RIPK2 expression and infectious disease including mycobacterial disease in which TLRs play main role on interaction of infection. We evaluated relationship between Mycobacterium leprae and RIPK 2 by real-time RT-PCR. This study revealed that RIPK2 expression was down-regulated in the footpads and skin but was up-regulated in the liver, lymph node, and spleen of Mycobacterium leprae-infected nu/nu mice compared with those of non-infected nu/nu mice. It was observed that the IL-12p40, IFN-gamma, and IL-18 involved in the susceptibility of Mycobacterium leprae were down-regulated in the skin and footpad but up-regulated in the liver. These results suggest that regulation of RIPK2 expression is tissue-specific and is associated with M. leprae infection.
Animals ; Communicable Diseases ; Interleukin-12 ; Interleukin-12 Subunit p40 ; Interleukin-18 ; Liver ; Lymph Nodes ; Mice ; Mycobacterium leprae* ; Mycobacterium* ; Phosphotransferases ; Signal Transduction ; Skin ; Spleen

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

OBJECTIVE: To assess the capability of phosphodiesterase type IV inhibitor (rolipram) to suppress IL-12 in human decidua and the subsequent changes of Th-2 cytokine (IL-10) and Th-1 cytokine (TNF-alpha). METHODS: Decidual tissues of 10 first-trimester pregnant women and 10 first-trimester pregnant women diagnosed as missed abortion were collected by dilatation and currettage. The decidual tissues were treated with rolipram for 6 hours. Protein and mRNA expression in the tissues were analysed by western blotting, immunohistochemistry and reverse transcription-polymerase chain reaction. RESULTS: Rolipram, in the concentration above 1 microgram/ml, could decrease the expression of IL-12p35 (control: 46.37+/-7.38, rolipram: 24.34+/-8.46) and IL-12p40 mRNA (control: 31.7+/-5.8, rolipram: 14.9+/-4.6) and protein (control: 52.4+/-8.9, rolipram: 40.9+/-12.1). However, the expression of IL-10 and TNF-alpha mRNA and protein did not changed by rolipram. There was no difference in the cytokine expression pattern between the decidual tissues of normal pregnancy and missed abortion. CONCLUSION: Rolipram, the phosphodiesterase type IV inhibitor, could induce the decrease of IL-12 in human decidua. In human decidual tissue, unlike other human tissues, the decrease of IL-12 by rolipram did not modulate other Th-1/Th-2 cytokines. Inability of IL-12 to modulate other Th-1/Th-2 cytokines might be related with unique cytokine network in human decidua rather than its small extent of decrease.
Abortion, Missed ; Blotting, Western ; Cyclic Nucleotide Phosphodiesterases, Type 4* ; Cytokines* ; Decidua ; Dilatation ; Female ; Humans* ; Immunohistochemistry ; Interleukin-10 ; Interleukin-12 Subunit p35 ; Interleukin-12 Subunit p40 ; Interleukin-12* ; Pregnancy ; Pregnant Women ; RNA, Messenger ; Rolipram ; Tumor Necrosis Factor-alpha

Cell Survival ; drug effects ; Cells, Cultured ; Dendritic Cells ; drug effects ; immunology ; physiology ; Humans ; Immunophenotyping ; Interleukin-10 ; pharmacology ; Interleukin-12 ; genetics ; secretion ; Interleukin-12 Subunit p35 ; Interleukin-12 Subunit p40 ; Protein Subunits ; genetics ; secretion ; RNA, Messenger ; analysis

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: Glycogen synthase kinase 3beta (GSK3beta) is a ubiquitous serine/threonine kinase that is regulated by serine phosphorylation at 9. Recent studies have reported the beneficial effects of a number of the pharmacological GSK3beta inhibitors in rodent models of septic shock. Since most of the GSK3beta inhibitors are targeted at the ATP-binding site, which is highly conserved among diverse protein kinases, the development of novel non-ATP competitive GSK3beta inhibitors is needed. METHODS: Based on the unique phosphorylation motif of GSK3beta, we designed and generated a novel class of GSK3beta inhibitor (GSK3i) peptides. In addition, we investigated the effects of a GSK3i peptide on lipopolysaccharide (LPS)-stimulated cytokine production and septic shock. Mice were intraperitoneally injected with GSK3i peptide and monitored over a 7-day period for survival. RESULTS: We first demonstrate its effects on LPS-stimulated pro-inflammatory cytokine production including interleukin (IL)-6 and IL-12p40. LPS-induced IL-6 and IL-12p40 production in macrophages was suppressed when macrophages were treated with the GSKi peptide. Administration of the GSK3i peptide potently suppressed LPS-mediated endotoxin shock. CONCLUSION: Collectively, we present a rational strategy for the development of a therapeutic GSK3i peptide. This peptide may serve as a novel template for the design of non-ATP competitive GSK3 inhibitors.
Animals ; Cytokines ; Glycogen Synthase Kinase 3 ; Glycogen Synthase Kinases ; Interleukin-12 Subunit p40 ; Interleukin-6 ; Interleukins ; Macrophages ; Mice ; Peptides ; Phosphorylation ; Phosphotransferases ; Protein Kinases ; Rodentia ; Serine ; Shock ; Shock, Septic

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

Humans ; Interferon-gamma ; metabolism ; Interleukin-12 Subunit p40 ; metabolism ; Meningitis, Bacterial ; blood ; cerebrospinal fluid ; Multiple Sclerosis, Relapsing-Remitting ; blood ; cerebrospinal fluid ; Staphylococcal Infections ; blood ; cerebrospinal fluid

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