Eosinophil resistance to glucocorticoid-induced apoptosis is mediated by the transcription factor NFIL3

The mainstay of asthma therapy, glucocorticoids (GCs) exert their therapeutic effects through the inhibition of inflammatory signaling and induction of eosinophil apoptosis. However, laboratory and clinical observations of GC-resistant asthma suggest that GCs’ effects on eosinophil viability may depend on the state of eosinophil activation. In the present study we demonstrate that eosinophils stimulated with IL-5 show impaired pro-apoptotic response to GCs. We sought to determine the contribution of GC-mediated transactivating (TA) and transrepressing (TR) pathways in modulation of activated eosinophils’ response to GC by comparing their response to the selective GC receptor (GR) agonist Compound A (CpdA) devoid of TA activity to that upon treatment with Dexamethasone (Dex). IL-5-activated eosinophils showed contrasting responses to CpdA and Dex, as IL-5-treated eosinophils showed no increase in apoptosis compared to cells treated with Dex alone, while CpdA elicited an apoptotic response regardless of IL-5 stimulation. Proteomic analysis revealed that both Nuclear Factor IL-3 (NFIL3) and Map Kinase Phosphatase 1 (MKP1) were inducible by IL-5 and enhanced by Dex; however, CpdA had no effect on NFIL3 and MKP1 expression. We found that inhibiting NFIL3 with specific siRNA or by blocking the IL-5-inducible Pim-1 kinase abrogated the protective effect of IL-5 on Dex-induced apoptosis, indicating crosstalk between IL-5 anti-apoptotic pathways and GR-mediated TA signaling occurring via the NFIL3 molecule. Collectively, these results indicate that (1) GCs’ TA pathway may support eosinophil viability in IL-5-stimulated cells through synergistic upregulation of NFIL3; and (2) functional inhibition of IL-5 signaling (anti-Pim1) or the use of selective GR agonists that don’t upregulate NFIL3 may be effective strategies for the restoring pro-apoptotic effect of GCs on IL-5-activated eosinophils.     

Glucocorticoids reduce inflammation in cystic fibrosis bronchial epithelial cells

Reduction of lung inflammation is one of the goals of cystic fibrosis (CF) therapy. Among anti-inflammatory molecules, glucocorticoids (GC) are one of the most prescribed. However, CF patients seem to be resistant to glucocorticoid treatment. Several molecular mechanisms that contribute to decrease anti-inflammatory effects of glucocorticoids have been identified in pulmonary diseases, but the molecular actions of glucocorticoids have never been studied in CF. In the cytoplasm, glucocorticoids bind to glucocorticoid receptor (GR) and then, control NF-κB and MAPK pathways through direct interaction with AP-1 and NF-κB in the nucleus. Conversely, MAPK can regulate glucocorticoid activation by targeting GR phosphorylation. Together these pathways regulate IL-8 release in the lung. Using bronchial epithelial cell lines derived from non CF and CF patients, we analyzed GR-based effects of glucocorticoids on NF-κB and MAPK pathways, after stimulation with TNF-α. We demonstrate that the synthetic glucocorticoid dexamethasone (Dex) significantly decreases IL-8 secretion, AP-1 and NF-κB activity in CF cells in a pro-inflammatory context. Moreover, we show that p38 MAPK controls IL-8 release by determining GR activation through specific phosphorylation on serine 211. Finally, we demonstrate a synergistic effect of dexamethasone treatment and inhibition of p38 MAPK inducing more than 90% inhibition of IL-8 production in CF cells. All together, these results demonstrate the good responsiveness to glucocorticoids of CF bronchial epithelial cells and the reciprocal link between glucocorticoids and p38 MAPK in the control of CF lung inflammation.     

Adiponectin induces dendritic cell activation via PLCγ/JNK/NF-κB pathways, leading to Th1 and Th17 polarization

Adiponectin (APN) is a crucial regulator for many inflammatory processes, but its effect on Th cell-mediated responses has not been fully understood. Thus, we investigated the immune-modulatory effects of APN on dendritic cells (DCs) controlling Th cell polarization. APN induced maturation and activation of DCs, as demonstrated by the increased expression of MHC class II, costimulatory molecules in both mouse and human DCs, and it significantly enhanced production of proinflammatory cytokines. APN triggered degradation of IκB proteins, nuclear translocation of NF-κB p65 subunit, and phosphorylation of MAPKs in DCs. Pretreatment with a phospholipase C (PLC)γ inhibitor and a JNK inhibitor suppressed IL-12 production and NF-κB binding activity. Additionally, PLCγ inhibitor downregulated phosphorylation of JNK, indicating that PLCγ and JNK may be upstream molecules of NF-κB. Importantly, APN-treated DCs significantly induced both Th1 and Th17 responses in allogeneic CD4(+) T cells. The addition of a neutralizing anti-IL-12 mAb to the cocultures abolished the secretion of IFN-γ, whereas the blockage of IL-23 and IL-1β suppressed APN-induced IL-17 production. Immunization of mice with OVA-pulsed, APN-treated DCs efficiently led to Ag-specific Th1 and Th17 cell responses. Taken together, these results demonstrated that APN effectively induced activation of DCs through PLCγ/JNK/NF-κB-signaling pathways, leading to enhanced Th1 and Th17 responses.     

Oncostatin M induces dendritic cell maturation and Th1 polarization

Oncostatin M (OSM) is a pleiotropic cytokine and a member of the gp130/IL-6 cytokine family that has been found to be involved in both pro- and anti-inflammatory responses in cell-mediated immunity. Maturation of dendritic cells (DCs) is crucial for initiation of primary immune responses and is regulated by several stimuli. In this study, the role of OSM in the phenotypic and functional maturation of DCs was evaluated in vitro. Stimulation with OSM upregulated the expression of CD80, CD86, MHC class I and MHC class II and reduced the endocytic capacity of immature DCs. Moreover, OSM induced the allogeneic immunostimulatory capacity of DCs by stimulating the production of the Th1-promoting cytokine IL-12. OSM also increased the production of IFN-γ by T cells in mixed-lymphocyte reactions, which would be expected to contribute to the Th1 polarization of the immune response. The expression of surface markers and cytokine production in DCs was mediated by both the MAPK and NF-κB pathways. Taken together, these results indicate that OSM may play a role in innate immunity and in acquired immunity by enhancing DCs maturation and promoting Th1 immune responses.     

Treponema pallidum recombinant protein Tp47 enhanced interleukin-6 secretion in human dermal fibroblasts through the toll-like receptor 2 via the p38, PI3K/Akt,and NF-κB signalling pathways

Interleukin-6 (IL-6) is a multi-effective cytokine involved in multiple immune responses. Whether fibroblasts also turn out to be a cytokine IL-6 factory during interaction with Treponema pallidum is not yet understood. To explore whether fibroblasts participate in inflammation due to syphilis, a series of experiments were performed to explore the role of T. pallidum lipoprotein Tp47 in IL-6 production in human dermal fibroblasts. The Toll-like receptor 2 (TLR2) and participating signalling pathways in this process were also evaluated. The results showed that the expressions of IL-6 and the protein levels of TLR2 in fibroblasts were upregulated after stimulation with Tp47, and this effect was impeded by the TLR2 inhibitor C29. In addition, Tp47 promoted the phosphorylation of p38, PI3K/Akt, and nuclear factor-kappaB (NF-κB), and the translocation of NF-κB in fibroblasts. Moreover, p38, PI3K, and NF-κB inhibitors significantly reduced IL-6 production in fibroblasts stimulated with Tp47. Furthermore, the TLR2 inhibitor C29 inhibited the phosphorylation of p38, Akt, and NF-κB, and the translocation of NF-κB in fibroblasts. In conclusion, our results showed that Tp47 enhanced IL-6 secretion in human dermal fibroblasts through TLR2 via p38, PI3K/Akt, and NF-κB signalling pathways. These findings contribute to our understanding of syphilis inflammation.     

IL-32γ induces chemotaxis of activated T cells via dendritic cell-derived CCL5

Interleukin (IL)-32 has been associated with a variety of inflammatory diseases including rheumatoid arthritis, vasculitis and Crohn’s disease. We have previously reported that IL-32γ, the IL-32 isoform with the highest biological activity, could act as an immune modulator through regulation of dendritic cell (DC) functions in immune responses. Cell locomotion is crucial for induction of an effective immune response. In this study, we investigated the effect and underlying mechanisms of IL-32γ on recruitment of T cells. IL-32γ upregulated the expression of several chemokines including CCL2, CCL4, and CCL5 in the DCs. In particular, IL-32γ significantly increased CCL5 expression in a dose-dependent manner. Treatment with JNK and NF-κB inhibitors suppressed IL-32γ-induced CCL5 expression in DCs, indicating that IL-32γ induced CCL5 production through the JNK and NF-κB pathways. Furthermore, supernatants from IL-32γ-treated DCs showed chemotactic activities controlling migration of activated CD4⁺ and CD8⁺ T cells, and these activities were suppressed by addition of neutralizing anti-CCL5 antibody. These results show that IL-32γ effectively promotes migration of activated T cells via CCL5 production in DCs. The chemotactic potential of IL-32γ may explain the pro-inflammatory effects of IL-32 and the pathologic role of IL-32 in immune disorders such as rheumatoid arthritis.     

Distinct control of MyD88 adapter-dependent and Akt kinase-regulated responses by the interleukin (IL)-1RI co-receptor, TILRR

Inflammatory responses are controlled through members of the interleukin-1 receptor (IL-1R)/Toll-like receptor superfamily. Our earlier work demonstrates that the IL-1 receptor type 1 (IL-1RI) co-receptor, Toll-like and IL-1 receptor regulator (TILRR), amplifies IL-1 activation of NF-κB and inflammatory genes. Here we show that TILRR similarly promotes IL-1-induced anti-apoptotic signals and reduces caspase-3 activity. Further, the TILRR-induced effects on cell survival and inflammatory responses are controlled through distinct parts of the IL-1RI regulatory Toll IL-1 receptor (TIR) domain. Alanine-scanning mutagenesis identified a functional TILRR mutant (R425A), which blocked increases in cell survival and upstream activation of Akt but had no effect on amplification of MyD88-dependent inflammatory responses. A second mutant (D448A) blocked TILRR potentiation of MyD88-dependent signals and inflammatory activation but had no impact on cell survival. Secondary structure predictions suggested that the mutations induce distinct alterations in the α-helical structure of the TILRR core protein. The results indicate a role for TILRR in selective amplification of NF-κB responses through IL-1RI and suggest that the specificity is determined by changes in receptor conformation and adapter protein recruitment.     

Glucocorticoids inhibit lipopolysaccharide-mediated inflammatory response by downregulating microRNA-155: a novel anti-inflammation mechanism

Glucocorticoids (GCs) are among the most widely used and effective therapies for many chronic inflammatory diseases. Although attempts have been made to identify important protein-coding genes and pathways involved in the anti-inflammatory effect of GCs, knowledge of genomic aberrations associated with noncoding genes, such as micro-RNAs (miRNAs), and their contributions is relatively limited. In this study, a systematic screening of the miRNA expression profile by microarray showed that GCs inhibited the expression of miR-155 in lipopolysaccharide (LPS)-induced macrophage inflammatory responses. Overexpression of miR-155 markedly reversed the suppressive action of GCs, whereas inhibition of miR-155 exhibited an effect similar to that of GCs on LPS-treated RAW264.7 cells, indicating miR-155 to be a functional regulator in the anti-inflammatory effect of GCs. Furthermore, GCs inhibited miR-155 expression in a GC receptor- and NF-κB-dependent manner. Bioinformatics analysis and luciferase assay revealed that the NF-κB binding site located in the promoter region of the B-cell integration cluster was important in mediating the GC-driven suppression of miR-155 in response to LPS stimulation. In addition, the combination of treatment with GCs and inhibition of miR-155 enhanced the anti-inflammatory effect of GCs on LPS-stimulated RAW264.7 cells. Therefore, we identify miR-155 to be a novel target through which GCs exert their anti-inflammatory effect on the LPS-induced macrophage inflammatory response. These findings may provide a basic rationale for new approaches in the effort to develop anti-inflammatory therapeutics.     

Ginsenoside Rg1, a novel glucocorticoid receptor agonist of plant origin, maintains glucocorticoid efficacy with reduced side effects

Glucocorticoids (GCs) are widely used to treat inflammatory diseases. However, they cause debilitating side effects, which limit the use of these compounds. In the past decade, many researchers have attempted to find so-called dissociated GCs that have separate distinct transactivation and transrepression activities. Anti-inflammation of GCs is a result of glucocorticoid receptor (GR)-mediated transactivation and transrepression in some tissues, similar to their side effects; therefore, the goal to discover a compound that has anti-inflammatory properties, but lacks the negative side effects seen with GCs, has yet to be achieved. In the present study, we introduce a plant-derived compound, ginsenoside Rg1, which possesses GC and estrogen-like activities. In this study, we show that Rg1 downmodulates LPS-induced proinflammatory cytokine release and inhibits NF-κB nuclear translocation and DNA binding activity. The negative effects on NF-κB activation are due to a decrease in IκB phosphorylation and protein stabilization. Furthermore, the inhibitory effect of Rg1 on NF-κB is GR-dependent, as small interfering RNA knockdown of GR abrogated this function. Rg1 also displayed profound inhibitory effects on LPS-induced MAPK activation. Importantly, Rg1 did not impair proliferation or differentiation of mouse osteoblasts. Finally, we show that Rg1 can effectively inhibit acute and chronic inflammation in vivo, but it does not cause hyperglycemia or osteoporosis as seen with dexamethasone. These results suggest that ginsenoside Rg1 may serve as a novel anti-inflammatory agent and may exhibit a potential profile for therapeutic intervention in inflammatory diseases.     

Both adiponectin and interleukin-10 inhibit LPS-induced activation of the NF-κB pathway in 3T3-L1 adipocytes

Adiponectin and interleukin 10 (IL-10) are adipokines that are predominantly secreted by differentiated adipocytes and are involved in energy homeostasis, insulin sensitivity, and the anti-inflammatory response. These two adipokines are reduced in obese subjects, which favors increased activation of nuclear factor kappa B (NF-κB) and leads to elevation of pro-inflammatory adipokines. However, the effects of adiponectin and IL-10 on NF-κB DNA binding activity (NF-κBp50 and NF-κBp65) and proteins involved with the toll-like receptor (TLR-2 and TLR-4) pathway, such as MYD88 and TRAF6 expression, in lipopolysaccharide-treated 3T3-L1 adipocytes are unknown. Stimulation of lipopolysaccharide-treated 3T3-L1 adipocytes for 24 h elevated IL-6 levels; activated the NF-κB pathway cascade; increased protein expression of IL-6R, TLR-4, MYD88, and TRAF6; and increased the nuclear activity of NF-κB (p50 and p65) DNA binding. Adiponectin and IL-10 inhibited the elevation of IL-6 levels and activated NF-κB (p50 and p65) DNA binding. Taken together, the present results provide evidence that adiponectin and IL-10 have an important role in the anti-inflammatory response in adipocytes. In addition, inhibition of NF-κB signaling pathways may be an excellent strategy for the treatment of inflammation in obese individuals.     

IL-32gamma induces the maturation of dendritic cells with Th1- and Th17-polarizing ability through enhanced IL-12 and IL-6 production

IL-32, a newly described multifunctional cytokine, has been associated with a variety of inflammatory diseases, including rheumatoid arthritis, vasculitis, and Crohn's disease. In this study, we investigated the immunomodulatory effects of IL-32γ on bone marrow-derived dendritic cell (DC)-driven Th responses and analyzed the underlying signaling events. IL-32γ-treated DCs exhibited upregulated expression of cell-surface molecules and proinflammatory cytokines associated with DC maturation and activation. In particular, IL-32γ treatment significantly increased production of IL-12 and IL-6 in DCs, which are known as Th1- and Th17-polarizing cytokines, respectively. This increased production was inhibited by the addition of specific inhibitors of the activities of phospholipase C (PLC), JNK, and NF-κB. IL-32γ treatment increased the phosphorylation of JNK and the degradation of both IκBα and IκBβ in DCs, as well as NF-κB binding activity to the κB site. The PLC inhibitor suppressed NF-κB DNA binding activity and JNK phosphorylation increased by IL-32γ treatment, thereby indicating that IL-32γ induced IL-12 and IL-6 production in DCs via a PLC/JNK/NF-κB signaling pathway. Importantly, IL-32γ-stimulated DCs significantly induced both Th1 and Th17 responses when cocultured with CD4(+) T cells. The addition of a neutralizing anti-IL-12 mAb abolished the secretion of IFN-γ in a dose-dependent manner; additionally, the blockage of IL-1β and IL-6, but not of IL-21 or IL-23p19, profoundly inhibited IL-32γ-induced IL-17 production. These results demonstrated that IL-32γ could effectively induce the maturation and activation of immature DCs, leading to enhanced Th1 and Th17 responses as the result of increased IL-12 and IL-6 production in DCs.     

Syk negatively regulates TLR4-mediated IFNβ and IL-10 production and promotes inflammatory responses in dendritic cells

BackgroundWhile Syk has been shown to associate with TLR4, the immune consequences of Syk–TLR interactions and related molecular mechanisms are unclear.MethodsGain- and loss-of-function approaches were utilized to determine the regulatory function of Syk and elucidate the related molecular mechanisms in TLR4-mediated inflammatory responses. Cytokine production was measured by ELISA and phosphorylation of signaling molecules determined by Western blotting.ResultsSyk deficiency in murine dendritic cells resulted in the enhancement of LPS-induced IFNβ and IL-10 but suppression of pro-inflammatory cytokines (TNFα, IL-6). Deficiency of Syk enhanced the activity of PI3K and elevated the phosphorylation of PI3K and Akt, which in turn, lead to the phospho-inactivation of the downstream, central gatekeeper of the innate response, GSK3β. Inhibition of PI3K or Akt abrogated the ability of Syk deficiency to enhance IFNβ and IL-10 in Syk deficient cells, confirmed by the overexpression of Akt (Myr–Akt) or constitutively active GSK3β (GSK3 S9A). Moreover, neither inhibition of PI3K–Akt signaling nor neutralization of de novo synthesized IFNβ could rescue TNFα and IL-6 production in LPS-stimulated Syk deficient cells. Syk deficiency resulted in decreased phosphorylation of IKKβ and the NF-κB p65 subunit, further suggesting a divergent influence of Syk on pro- and anti-inflammatory TLR responses.ConclusionsSyk negatively regulates TLR4-mediated production of IFNβ and IL-10 and promotes inflammatory responses in dendritic cells through divergent regulation of downstream PI3K–Akt and NF-κB signaling pathways.General significanceSyk may represent a novel target for manipulating the direction or intensity of the innate response, depending on clinical necessity.     

TRAF6 mediates IL-1β/LPS-induced suppression of TGF-β signaling through its interaction with the type III TGF-β receptor

Transforming growth factor-β1 (TGF-β1) is an important anti-inflammatory cytokine that modulates and resolves inflammatory responses. Recent studies have demonstrated that inflammation enhances neoplastic risk and potentiates tumor progression. In the evolution of cancer, pro-inflammatory cytokines such as IL-1β must overcome the anti-inflammatory effects of TGF-β to boost pro-inflammatory responses in epithelial cells. Here we show that IL-1β or Lipopolysaccharide (LPS) suppresses TGF-β-induced anti-inflammatory signaling in a NF-κB-independent manner. TRAF6, a key molecule in IL-1β signaling, mediates this suppressive effect through interaction with the type III TGF-β receptor (TβRIII), which is TGF-β-dependent and requires type I TGF-β receptor (TβRI) kinase activity. TβRI phosphorylates TβRIII at residue S829, which promotes the TRAF6/TβRIII interaction and consequent sequestration of TβRIII from the TβRII/TβRI complex. Our data indicate that IL-1β enhances the pro-inflammatory response by suppressing TGF-β signaling through TRAF6-mediated sequestration of TβRIII, which may be an important contributor to the early stages of tumor progression.     

Expression of HAb18G in non-small lung cancer and characterization of activation, migration, proliferation, and apoptosis in A549 cells following siRNA-induced downregulation of HAb18G

VSL#3 probiotics can be effective on induction and maintenance of the remission of clinical ulcerative colitis. However, the mechanisms are not fully understood. The aim of this study was to examine the effects of VSL#3 probiotics on dextran sulfate sodium (DSS)-induced colitis in rats. Acute colitis was induced by administration of DSS 3.5 % for 7 days in rats. Rats in two groups were treated with either 15 mg VSL#3 or placebo via gastric tube once daily after induction of colitis; rats in other two groups were treated with either the wortmannin (1 mg/kg) via intraperitoneal injection or the wortmannin + VSL#3 after induction of colitis. Anti-inflammatory activity was assessed by myeloperoxidase (MPO) activity. Expression of inflammatory related mediators (iNOS, COX-2, NF-κB, Akt, and p-Akt) and cytokines (TNF-α, IL-6, and IL-10) in colonic tissue were assessed. TNF-α, IL-6, and IL-10 serum levels were also measured. Our results demonstrated that VSL#3 and wortmannin have anti-inflammatory properties by the reduced disease activity index and MPO activity. In addition, administration of VSL#3 and wortmannin for 7 days resulted in a decrease of iNOS, COX-2, NF-κB, TNF-α, IL-6, and p-Akt and an increase of IL-10 expression in colonic tissue. At the same time, administration of VSL#3 and wortmannin resulted in a decrease of TNF-α and IL-6 and an increase of IL-10 serum levels. VSL#3 probiotics therapy exerts the anti-inflammatory activity in rat model of DSS-induced colitis by inhibiting PI3K/Akt and NF-κB pathway.