Myeloid-derived suppressor cell cytokine secretion as prognostic factor in myelodysplastic syndromes

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that play a critical immunosuppressive role in the tumour micro-environment. Although biological research on MDSCs has made progress, the relationship between the secretion of cytokines by MDSCs and poor prognosis is not clear, and there are no criteria to measure the functional status of MDSCs. Here, we detected the mRNA expression of IL-10, IL-12, TGF-β and TNF-α in MDSCs and the levels of these cytokines in MDSC culture supernatants of patients with myelodysplastic syndromes, and quantified the functional status of MDSCs by IL-10/IL-12 ratio and TGF-β/TNF-α ratio. We found that the ratio of IL-10/IL-12 and TGF-β/TNF-α was significantly higher in higher-risk MDS than in lower-risk MDS and normal control groups. The TGF-β/TNF-α ratio in MDSCs was positively correlated with the percentage of blast cells and was negatively correlated with the percentage of CD3⁺CD8⁺ T lymphocytes. Meanwhile, the TGF-β/TNF-α ratio was higher in patients with a lower absolute neutrophil count. It suggested that MDSCs in higher-risk MDS have a stronger immunosuppressive effect and might be related to poor prognosis. Quantifying the functional status of MDSCs with IL-10/IL-12 and TGF-β/TNF-α ratio might help to evaluate the balance of cellular immunity of MDSCs in MDS.     

High Concentrations of TNF-α Induce Cell Death during Interactions between Human Umbilical Cord Mesenchymal Stem Cells and Peripheral Blood Mononuclear Cells

Human umbilical cord mesenchymal stromal cells (hUC-MSCs) are currently being used as novel therapeutic agents in numerous clinical trials. Previous works have shown that hUC-MSCs possess profound immunomodulatory capacities through IL-1 stimulation produced by peripheral blood mononuclear cells (PBMCs), their main cellular partner in most pathophysiological and therapeutic situations. The present study was designed to explore the role of TNF-α in these interactions. In these experiments, we demonstrated that TNF-α originated from PBMCs under the influence of IL-1. We also showed that TNF-α acted differently depending upon the concentrations reached. At low concentrations it clearly contributed to IL-6 and monocyte chemotactic protein 1 (MCP-1) production. At high concentrations, used alone or in association with the TNF-related apoptosis-inducing ligand, TNF-α also stimulated hUC-MSC IL-6 but, more intensely, MCP-1 production. This stimulation was associated but independent of apoptosis induction in a process involving Inhibitor of Apoptosis Proteins. Interferon gamma (IFN-γ), tested to stimulate PBMC and tissue activation, amplified IL-6 and MCP-1 production and cell death by, apparently, a different process involving necrosis. Our findings bring new insights into the complex interactions between hUC-MSCs and PBMCs, involving cytokines, chemokines and cell death, and are of fundamental importance for tissue homeostasis.     

Morin,a Bioflavonoid Suppresses Monosodium Urate Crystal-Induced Inflammatory Immune Response in RAW 264.7 Macrophages through the Inhibition of Inflammatory Mediators,Intracellular ROS Levels and NF-κB Activation

Our previous studies had reported that morin, a bioflavanoid exhibited potent anti-inflammatory effect against adjuvant-induced arthritic rats. In this current study, we investigated the anti-inflammatory mechanism of morin against monosodium urate crystal (MSU)-induced inflammation in RAW 264.7 macrophage cells, an in vitro model for acute gouty arthritis. For comparison purpose, colchicine was used as a reference drug. We have observed that morin (100–300 μM) treatment significantly suppressed the levels of inflammatory cytokines (TNF-α, IL-1β, IL-6, MCP-1 and VEGF), inflammatory mediators (NO and PEG₂), and lysosomal enzymes (acid phosphatase, β-galactosidase, N-acetyl glucosamindase and cathepsin D) in MSU-crystals stimulated macrophage cells. The mRNA expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and MCP-1), inflammatory enzymes (iNOS and COX-2), and NF-κBp65 was found downregulated in MSU crystal stimulated macrophage cells by morin treatment, however, the mRNA expression of hypoxanthine phospho ribosyl transferse (HPRT) was found to be increased. The flow cytometry analysis revealed that morin treatment decreased intracellular reactive oxygen species levels in MSU crystal stimulated macrophage cells. The western blot analysis clearly showed that morin mainly exerts its anti-inflammatory effects by inhibiting the MSU crystal-induced COX-2 and TNF-α protein expression through the inactivation of NF-κB signaling pathway in RAW 264.7 macrophage cells similar to that of BAY 11–7082 (IκB kinase inhibitor). Our results collectively suggest that morin can be a potential therapeutic agent for inflammatory disorders like acute gouty arthritis.     

Epratuzumab inhibits the production of the proinflammatory cytokines IL-6 and TNF-α, but not the regulatory cytokine IL-10, by B cells from healthy donors and SLE patients

IntroductionCytokines produced by B cells are believed to play important roles in autoimmune diseases. CD22 targeting by epratuzumab has been demonstrated to inhibit phosphorylation of B cell receptor (BCR) downstream signaling in B cells. It has been shown that other sialoadhesin molecules related to CD22 have immunoregulatory functions; therefore, in the present study, we addressed the role of epratuzumab on the production of key cytokines by B cells of patients with systemic lupus erythematosus (SLE) and of healthy donors (HD).MethodsPeripheral blood B cells were purified and activated by BCR with or without Toll-like receptor 9 (TLR9) stimulation in the presence or absence of epratuzumab. Cytokine production by B cells (interleukin [IL]-6, tumor necrosis factor [TNF]-α and IL-10) in the supernatant and the induction of IL-10⁺ B cells from patients with SLE and HD were analyzed.ResultsThe secretion of the proinflammatory cytokines TNF-α and IL-6 by anti-BCR and BCR- and/or TLR9-activated B cells from HD and patients with SLE was inhibited by epratuzumab. In contrast, the production of IL-10 by B cells was not affected by epratuzumab under either stimulation condition. Consistently, the induction of IL-10–producing B cells in culture was not affected by epratuzumab.ConclusionsEpratuzumab, by targeting CD22, was able to inhibit the production of the proinflammatory cytokines IL-6 and TNF-α by B cells, in contrast to IL-10, in vitro. These data suggest that targeting CD22 alters the balance between proinflammatory cytokines (TNF-α, IL-6) and the regulatory cytokine IL-10 as another B cell effector mechanism.     

Effects of the Commercial Flame Retardant Mixture DE-71 on Cytokine Production by Human Immune Cells

IntroductionAlthough production of polybrominated diphenyl ethers (PBDEs) is now banned, release from existing products will continue for many years. The PBDEs are assumed to be neurotoxic and toxic to endocrine organs at low concentrations. Their effect on the immune system has not been investigated thoroughly. We aimed to investigate the influence of DE-71 on cytokine production by peripheral blood mononuclear cells (PBMCs) stimulated with Escherichia Coli lipopolysaccharide (LPS) or phytohaemagglutinin-L (PHA-L).ResultsAt non-cytotoxic concentrations (0.01–10 μg/mL), DE-71 significantly enhanced secretion of IL-1β, IL-6, CXCL8, IL-10, and TNF-α (p<0.001–0.019; n = 6) from LPS-stimulated PBMCs. IFN-γ, TNF-α, IL-17A, and IL-17F (p = <0.001–0.043; n = 6) secretion were enhanced from PHA-L-stimulated PBMCs as well. Secretion of IL-1β, IL-2, IL-10, IL-8 and IL-6 was not significantly affected by DE-71.ConclusionsWe demonstrate an enhancing effect of DE-71 on cytokine production by normal human PBMCs stimulated with LPS or PHA-L ex vivo.     

Demethylation of MicroRNA-124a Genes Attenuated Proliferation of Rheumatoid Arthritis Derived Fibroblast-Like Synoviocytes and Synthesis of Tumor Necrosis Factor-α

ObjectiveTo examine the impact of 5-Aza-2ʹ-deoxycytidine (5-AzadC) on methylation status of miR-124a genes in rheumatoid arthritis (RA) associated fibroblast-like synoviocytes (FLS) and its effect on RA-FLS proliferation and TNF-α expression.ResultsAfter 5-AzadC treatment, the expression of miR-124a was significantly increased compared with the control group (1.545 ± 0.189 vs 0.836 ± 0.166, p = 0.001). On the other hand, 5-AzadC significantly reduced IL-1β-mediated cell proliferation by nearly 2.5 fold (p = 0.006). Also, the level of TNF-α secreted from the cells treated with IL-1β plus 5-AzadC was considerably less than that from the cells treated with IL-1β alone (324.99 ± 22.73 ng/L vs 387.91 ± 58.51 ng/L, p = 0.022). After transfection with miR-124a inhibitor in RA-FLS treated with IL-1β plus 5-AzadC, the cell proliferation was increased by 18.2% and the TNF-α expression was increased by 19.0% (p = 0.001 and 0.011, respectively).ConclusionMethylation of miR-124a genes contributed to IL-1β-mediated RA-FLS proliferation and TNF-α expression.     

Human Langerhans Cells Control Th Cells via Programmed Death-Ligand 1 in Response to Bacterial Stimuli and Nickel-Induced Contact Allergy

Langerhans cells (LCs) are suspected to initiate inflammatory immune responses to contact allergens and pathogenic bacteria. In chronic infectious diseases, programmed death ligand (PD-L) 1 exhibits both inhibitory and costimulatory functions on T cell-mediated activation and tolerance. Here, we investigated the effects of contact allergens and bacterial stimuli on PD-L1 expression in LCs and the effects of altered PD-L1 expression on cytokine release of subsequently cocultured T cells. Monocyte-derived LCs (MoLCs), LCs, and skin sections of patients suffering from allergic contact dermatitis were challenged with nickel and then analyzed for PD-L1 expression by confocal laser scanning microscopy and flow cytometry. In blocking experiments, we found that the release of Th cell specific cytokines was dependent on both stimulation of LCs and inhibition of PD-L1-PD-1 interactions. Stimulation with peptidoglycan (PGN) or lipopolysaccharide (LPS) and blockage of PD-L1 with a specific antibody triggered the release of high levels of IL-17, IL-22, TNF-α, and IFN-γ in CD4⁺T cells. If nickel was used as a stimulus, blockage of PD-L1 led to high amounts of TNF-α and IL-22. A closer look revealed PD-L1-dependent upregulation of IL-17 secretion in FACS-sorted CCR6⁺/CCR4⁺ T memory cells. In the presence of anti-PD-L1, PGN induced secretion of IFN-γ and IL-17 in total CCR6⁺ cells, while nickel triggered secretion of IFN-γ and IL-17 exclusively in CCR6⁺/CCR4⁺ cells. Our findings suggest that PD-L1 on LCs plays a crucial role in type IV allergic reactions and in response to bacterial stimuli by controlling the nature of inflammatory Th cell responses.     

ApoE Production in Human Monocytes and Its Regulation by Inflammatory Cytokines

The apoE production by tissue macrophages is crucial for the prevention of atherosclerosis and the aim of this study was to further elucidate how this apolipoprotein is regulated by cytokines present during inflammation. Here we studied apoE production in peripheral blood mononuclear cells (PBMC) and analysis was made with a newly developed apoE ELISpot assay. In PBMC, apoE secretion was restricted to monocytes with classical (CD14⁺⁺CD16⁻) and intermediate (CD14⁺CD16⁺) monocytes being the main producers. As earlier described for macrophages, production was strongly upregulated by TGF-β and downregulated by bacterial lipopolysaccharide (LPS) and the inflammatory cytokines IFN-γ, TNF-α and IL-1β. We could here show that a similar down-regulatory effect was also observed with the type I interferon, IFN-α, while IL-6, often regarded as one of the more prominent inflammatory cytokines, did not affect TGF-β-induced apoE production. The TNF-α inhibitor Enbrel could partly block the down-regulatory effect of IFN-γ, IFN-α and IL-1β, indicating that inhibition of apoE by these cytokines may be dependent on or synergize with TNF-α. Other cytokines tested, IL-2, IL-4, IL-12, IL-13, IL-17A and IL-23, had no inhibitory effect on apoE production. In contrast to the effect on monocytes, apoE production by primary hepatocytes and the hepatoma cell line HepG2 was more or less unaffected by treatment with cytokines or LPS.     

The Pelargonium sidoides Extract EPs 7630 Drives the Innate Immune Defense by Activating Selected MAP Kinase Pathways in Human Monocytes

Pelargonium sidoides is a medical herb and respective extracts are used very frequently for the treatment of respiratory tract infections. However, the effects of Pelargonium sidoides and a special extract prepared from its roots (EPs 7630) on human immune cells are not fully understood. Here we demonstrate that EPs 7630 induced a rapid and dose-dependent production of TNF-α, IL-6, and IL-10 by human blood immune cells. This EPs 7630-induced cytokine profile was more pro-inflammatory in comparison with the profile induced by viral or bacterial infection-mimicking agents. The search for EPs 7630 target cells revealed that T-cells did not respond to EPs 7630 stimulation by production of TNF-α, IL-6, or IL-10. Furthermore, pretreatment of T-cells with EPs 7630 did not modulate their TNF-α, IL-6, and IL-10 secretion during subsequent activation. In contrast to lymphocytes, monocytes showed clear intracellular TNF-α staining after EPs 7630 treatment. Accordingly, EPs 7630 predominantly provoked activation of MAP kinases and inhibition of p38 strongly reduced the monocyte TNF-α production. The pretreatment of blood immune cells with EPs 7630 lowered their secretion of TNF-α and IL-10 and caused an IL-6 dominant response during second stimulation with viral or bacterial infection-mimicking agents. In summary, we demonstrate that EPs 7630 activates human monocytes, induces MAP kinase-dependent pro-inflammatory cytokines in these cells, and specifically modulates their production capacity of mediators known to lead to an increase of acute phase protein production in the liver, neutrophil generation in the bone marrow, and the generation of adaptive Th17 and Th22 cells.     

HSP70 Enhances Immunosuppressive Function of CD4+CD25+FoxP3+ T Regulatory Cells and Cytotoxicity in CD4+CD25? T Cells

Human CD4⁺CD25⁺FoxP3⁺ T regulatory cells (Tregs) control effector T cells and play a central role in peripheral tolerance and immune homeostasis. Heat shock protein 70 (HSP70) is a major immunomodulatory molecule, but its effect on the functions of Tregs is not well understood. To investigate target-dependent and –independent Treg functions, we studied cytokine expression, regulation of proliferation and cytotoxicity after exposure of Tregs to HSP70. HSP70-treated Tregs significantly inhibited proliferation of CD4⁺CD25⁻ target cells and downregulated the secretion of the proinflammatory cytokines IFN-γ and TNF-α. By contrast, HSP70 increased the secretion of Treg suppressor cytokines IL-10 and TGF-β. Treatment with HSP70 enhanced the cytotoxic properties of Tregs only to a minor extent (4-fold), but led to stronger responses in CD4⁺CD25⁻ cells (42-fold). HSP70-induced modulation of T-cell responses was further enhanced by combined treatment with HSP70 plus IL-2. Treatment of Tregs with HSP70 led to phosphorylation of PI3K/AKT and the MAPKs JNK and p38, but not that of ERK1/2. Exposure of Tregs to specific inhibitors of PI3K/AKT and the MAPKs JNK and p38 reduced the immunosuppressive function of HSP70-treated Tregs as indicated by the modified secretion of specific target cell (IFN-γ, TNF-α) and suppressor cytokines (IL-10, TGF-β). Taken together, the data show that HSP70 enhances the suppressive capacity of Tregs to neutralize target immune cells. Thus HSP70-enhanced suppression of Tregs may prevent exaggerated immune responses and may play a major role in maintaining immune homeostasis.     

Vitamin D Regulates Cytokine Patterns Secreted by Dendritic Cells to Promote Differentiation of IL-22-Producing T Cells

One central mechanism, by which vitamin D regulates human immune responses, is the direct modulation of dendritic cells (DCs). However, the effect of vitamin D on several key DC functions, such as the secretion of central inflammatory cytokines, remains controversial. Moreover, whether vitamin D treatment of DCs regulates their ability to promote differentiation of IL-17-/IL-22-producing T cell subsets, such as Th17 and Th22 cell, is not known. Here, we report that vitamin D treatment during differentiation of monocytes into DCs markedly enhanced their ability to secrete TNF-α, IL-6, IL-1β and IL-23. Cytokines secreted by vitamin D-treated DC were significantly more potent in driving differentiation of IL-22-producing T cells, but not IL-17-producing T cells, as compared to secreted cytokines of not-vitamin D-treated DCs. Finally, we found that the differentiation of IL-22-producing T cells mediated by supernatants of vitamin D-treated DCs was dependent on TNF-α IL-6 and IL-23. In summary, our study suggests a novel role of vitamin D in regulating DC-mediated immune responses in humans.     

Regulation of retinoschisin secretion in Weri-Rb1 cells by the F-actin and microtubule cytoskeleton

Retinoschisin is encoded by the gene responsible for X-linked retinoschisis (XLRS), an early onset macular degeneration that results in a splitting of the inner layers of the retina and severe loss in vision. Retinoschisin is predominantly expressed and secreted from photoreceptor cells as a homo-oligomer protein; it then associates with the surface of retinal cells and maintains the retina cellular architecture. Many missense mutations in the XLRS1 gene are known to cause intracellular retention of retinoschisin, indicating that the secretion process of the protein is a critical step for its normal function in the retina. However, the molecular mechanisms underlying retinoschisin's secretion remain to be fully elucidated. In this study, we investigated the role of the F-actin cytoskeleton in the secretion of retinoschisin by treating Weri-Rb1 cells, which are known to secrete retinoschisin, with cytochalasin D, jasplakinolide, Y-27632, and dibutyryl cGMP. Our results show that cytochalasin D and jasplakinolide inhibit retinoschisin secretion, whereas Y-27632 and dibutyryl cGMP enhance secretion causing F-actin alterations. We also demonstrate that high concentrations of taxol, which hyperpolymerizes microtubules, inhibit retinoschisin secretion. Our data suggest that retinoschisin secretion is regulated by the F-actin cytoskeleton, that cGMP or inhibition of ROCK alters F-actin structure enhancing the secretion, and that the microtubule cytoskeleton is also involved in this process.     

S100-β aggravates spinal cord injury via activation of M1 macrophage phenotype

Objectives:S100-β has been identified as a sensitive biomarker in central nervous system injuries. However, the functions and mechanisms of S100-β are unknown in spinal cord injury.Methods:Spinal cord injury (SCI) mouse model was generated by surgical operation, microglia activation model was established by inducing BV-2 cells with LPS. The SCI model was evaluated by Basso-Beattie-Bresnahan (BBB) behavioral score, HE staining, and Nissl staining. The expression level of S100-β was detected by qRT-PCR, western blot, and immunofluorescence. qRT-PCR and western blot were used to detect the expression of iNOS and CD16. Pro-inflammatory cytokines TNF-α and IL-1β levels were detected by qRT-PCR and ELISA.Results:The expression of IL-1β, TNF-α, iNOS, and CD16 increased at 3^rd day after SCI. In BV2 microglia, LPS treatment promoted the expression of S100-β, IL-1β, TNF-α, iNOS, and CD16. Knockdown of S100-β reduced the expression of iNOS stimulated by LPS. Over-expression of S100-β increased IL-1β and TNF-α, and S100-β inhibition suppressed IL-1β and TNF-α. In SCI mice, knockdown of S100-β attenuated the spinal cord injury and inhibited the expression of iNOS, IL-1β, and TNF-α.Conclusions:Down-regulation of S100-β could inhibit the pathogenesis of SCI and inhibit the activation of M1 macrophages. S100-β may be a useful diagnostic biomarker or therapeutic target for SCI.     

Crosstalk between Immune Cells and Adipocytes Requires Both Paracrine Factors and Cell Contact to Modify Cytokine Secretion

Increased adiposity results in a heightened infiltration of immune cells into fat depots, which in turn generates a pro-inflammatory phenotype in obese individuals. To better understand the causal factors that establish this pro-inflammatory profile, we examined events leading to crosstalk between adipocytes and immune cells. Using isolated spleen-derived immune cells, stimulated with LPS, together with cultured adipocytes, we differentiated the effects of paracrine factors and cell-cell contact on TNFα, IL-6 and MCP-1 secretion levels and secretion profiles. When splenocytes and adipocytes were co-cultured without direct contact, permitting only paracrine communication, secretion of IL-6 and MCP-1 were increased by 3- and 2.5-fold, respectively, over what was secreted by individual cultures, whereas TNFα secretion was reduced by 55%. When cells were co-cultured with direct cell-cell contact, IL-6 and MCP-1 secretion were increased by an additional 36% and 38%, respectively, over that measured from just paracrine stimulation alone, indicating that cell contact provides a synergistic signal that amplifies elevated cytokine secretion stimulated by paracrine signals. Using splenocytes from TNFα^-/- mice showed that the absence of TNFα has little effect on paracrine stimulation of cytokine secretion, but attenuates cell contact-mediated enhancement of IL-6 and MCP-1 secretion. Furthermore, TNFα supports cell contact-mediated signaling in part, but not exclusively, through Nuclear Factor-κB activation. These findings indicate that engagement of cell contact between immune cells and adipocytes, in conjunction with locally secreted paracrine factors, activates a unique signaling pathway that mediates crosstalk between these cell types leading to marked effects on cytokine secretion and profile.     

Butein,a tetrahydroxychalcone,suppresses pro-inflammatory responses in HaCaT keratinocytes

Up-regulation of cell adhesion molecules and proinflammatory cytokines contributes to enhanced monocyte adhesiveness and infiltration into the skin, during the pathogenesis of various inflammatory skin diseases, including atopic dermatitis. In this study, we examined the anti-inflammatory effects of butein, a tetrahydroxychalcone, and its action mechanisms using TNF-α-stimulated keratinocytes. Butein significantly inhibited TNF-α-induced ICAM-I expression and monocyte adhesion in human keratinocyte cell line HaCaT. Butein also decreased TNF-α-induced pro-inflammatory mediators, such as IL-6, IP-10 and MCP-1, in HaCaT cells. Butein decreased TNF-α-induced ROS generation in a dose-dependent manner in HaCaT cells. In addition, treatment of HaCaT cells with butein suppressed TNF-α-induced MAPK activation. Furthermore, butein suppressed TNF-α-induced NF-kappaB activation. Overall, our results indicate that butein has immunomodulatory activities by inhibiting expression of proinflammatory mediators in keratinocytes. Therefore, butein may be used as a therapeutic agent for the treatment of inflammatory skin diseases. [BMB Reports 2015; 48(9): 495-500]     

A Trifluoromethyl Analogue of Celecoxib Exerts Beneficial Effects in Neuroinflammation

Celecoxib is a selective cyclooxygenase-2 (COX2) inhibitor. We have previously shown that celecoxib inhibits experimental autoimmune encephalomyelitis (EAE) in COX-2-deficient mice, suggestive for a mode of action involving COX2-independent pathways. In the present study, we tested the effect of a trifluoromethyl analogue of celecoxib (TFM-C) with 205-fold lower COX-2 inhibitory activity in two models of neuroinflammation, i.e. cerebellar organotypic cultures challenged with LPS and the EAE mouse model for multiple sclerosis. TFM-C inhibited secretion of IL-1β, IL-12 and IL-17, enhanced that of TNF-α and RANTES, reduced neuronal axonal damage and protected from oxidative stress in the organotypic model. TFM-C blocked TNF-α release in microglial cells through a process involving intracellular retention, but induced TNF-α secretion in primary astrocyte cultures. Finally, we demonstrate that TFM-C and celecoxib ameliorated EAE with equal potency. This coincided with reduced secretion of IL-17 and IFN-γ by MOG-reactive T-cells and of IL-23 and inflammatory cytokines by bone marrow-derived dendritic cells. Our study reveals that non-coxib analogues of celecoxib may have translational value in the treatment of neuro-inflammatory conditions.     

Tilapia Hepcidin 2-3 Peptide Modulates Lipopolysaccharide-induced Cytokines and Inhibits Tumor Necrosis Factor-α through Cyclooxygenase-2 and Phosphodiesterase 4D

The antimicrobial peptide, tilapia hepcidin (TH) 2-3, belongs to the hepcidin family, and its antibacterial function has been reported. Here, we examined the TH2-3-mediated regulation of proinflammatory cytokines in bacterial endotoxin lipopolysaccharide (LPS)-stimulated mouse macrophages. The presence of TH2-3 in LPS-stimulated cells reduced the amount of tumor necrosis factor (TNF)-α secretion. From a microarray, real-time polymerase chain reaction (PCR), and cytokine array studies, we showed down-regulation of the proinflammatory cytokines TNF-α, interleukin (IL)-1α, IL-1β, IL-6, and the prostaglandin synthesis gene, cyclooxygenase (COX)-2, by TH2-3. Studies with the COX-2-specific inhibitor, melaxicam, and with COX-2-overexpressing cells demonstrated the positive regulation of TNF-α and negative regulation of cAMP degradation-specific phosphodiesterase (PDE) 4D by COX-2. In LPS-stimulated cells, TH2-3 acts like melaxicam and down-regulates COX-2 and up-regulates PDE4D. The reduction in intracellular cAMP by TH2-3 or melaxicam in LPS-stimulated cells supports the negative regulation of PDE4D by COX-2 and TH2-3. This demonstrates that the inhibition of COX-2 is among the mechanisms through which TH2-3 controls TNF-α release. At 1 h after treatment, the presence of TH2-3 in LPS-stimulated cells had suppressed the induction of pERK1/2 and prevented the LPS-stimulated nuclear accumulation of NF-κB family proteins of p65, NF-κB2, and c-Rel. In conclusion, TH2-3 inhibits TNF-α and other proinflammatory cytokines through COX-2-, PDE4D-, and pERK1/2-dependent mechanisms.     

HBsAg Inhibits IFN-α Production in Plasmacytoid Dendritic Cells through TNF-α and IL-10 Induction in Monocytes

Type I Interferon (IFN) is one of the first lines of defense against viral infection. Plasmacytoid dendritic cells (pDCs) are professional IFN-α-producing cells that play an important role in the antiviral immune response. Previous studies have reported that IFN-α production is impaired in chronic hepatitis B (CHB) patients. However, the mechanisms underlying the impairment in IFN-α production are not fully understood. Here, we report that plasma-derived hepatitis B surface antigen (HBsAg) and HBsAg expressed in CHO cells can significantly inhibit toll like receptor (TLR) 9-mediated Interferon-α (IFN-α) production in peripheral blood mononuclear cells (PBMCs) from healthy donors. Further analysis indicated that monocytes participate in the inhibitory effect of HBsAg on pDCs through the secretion of TNF-α and IL-10. Furthermore, TLR9 expression on pDCs was down-regulated by TNF-α, IL-10 and HBsAg treatment. This down-regulation may partially explain the inhibition of IFN-α production in pDCs. In conclusion, we determined that HBsAg inhibited the production of IFN-α by pDCs through the induction of monocytes that secreted TNF-α and IL-10 and through the down-regulation of TLR9 expression on pDCs. These data may aid in the development of effective antiviral treatments and lead to the immune control of the viral infections.