Adenylate cycalse toxin of Bordetella pertussis inhibits TLR-induced IRF-1 and IRF-8 activation and IL-12 production and enhances IL-10 through MAPK activation in dendritic cells

Adenylate cyclase toxin (CyaA) of Bordetella pertussis binds to CD11b/CD18 on macrophages and dendritic cells (DC) and confers virulence to the bacteria by subverting innate immune responses of the host. We have previously demonstrated that CyaA promotes the induction of IL-10-secreting regulatory T cells in vivo by modulating DC activation. Here, we examine the mechanism of immune subversion, specifically, the modulation of TLR signaling pathways in DC. We found that CyaA synergized with LPS to induce IL-10 mRNA and protein expression in DC but significantly inhibited IL-12p70 production. CyaA enhanced LPS-induced phosphorylation of p38 MAPK and ERK in DC, and inhibitors of p38 MAPK, MEK, or NF-kappaB suppressed IL-10 production in response to LPS and CyaA. However, inhibition of p38 MAPK, MEK, and NF-kappaB did not reverse the inhibitory effect of CyaA on TLR agonist-induced IL-12 production. Furthermore, CyaA suppression of IL-12 was independent of IL-10. In contrast, CyaA suppressed LPS- and IFN-gamma-induced IFN-regulatory factor-1 (IRF-1) and IRF-8 expression in DC. The modulatory effects of CyaA were dependent on adenylate cyclase activity and induction of intracellular cAMP, as an enzyme-inactive mutant of CyaA failed to modulate TLR-induced signaling in DC, whereas the effects of the wild-type toxin were mimicked by stimulation of the DC with PGE2. Our findings demonstrate that CyaA modulates TLR agonist-induced IL-10 and IL-12p70 production in DC by, respectively, enhancing MAPK phosphorylation and inhibiting IRF-1 and IRF-8 expression and that this is mediated by elevation of intercellular cAMP concentrations.     

Leukotriene B4 enhances tumour necrosis factor-α-induced CCL27 production in human keratinocytes

BACKGROUND: A chemokine CCL27 recruits skin-homing T cells. CCL27 production by epidermal keratinocytes is dependent on nuclear factor-kappaB (NF-kappaB) activity and is enhanced in lesions with atopic dermatitis or allergic contact dermatitis. A lipid mediator leukotriene B(4) (LTB(4)) may be involved in the development of these allergic dermatoses. LTB(4) acts on cell surface G-protein-coupled receptors, BLT1 and BLT2. OBJECTIVE: The aim of this study was to investigate the in vitro effects of LTB(4) on CCL27 production in human keratinocytes. METHODS: Keratinocytes were incubated with TNF-alpha and LTB(4). CCL27 secretion and mRNA levels were analysed by ELISA and RT-PCR, respectively. NF-kappaB activities were analysed by luciferase assays. Protein levels or phosphorylation status were analysed by cell-based ELISA. RESULTS: LTB(4) alone did not enhance CCL27 production and modestly enhanced NF-kappaB activity in human keratinocytes. However, LTB(4) potently enhanced TNF-alpha-induced CCL27 secretion and mRNA expression and NF-kappaB activity. LTB(4) alone or together with TNF-alpha, induced phosphorylation and degradation of inhibitory NF-kappaB alpha (IkappaBalpha) and phosphorylation of NF-kappaB p65. These effects of LTB(4) were suppressed by BLT1 antagonist U75302, pertussis toxin, phosphoinositide-3 kinase (PI3K) inhibitor LY294002 and extracellular signal-regulated kinase (ERK) kinase inhibitor U0126, but not by BLT2 antagonist LY255283. LTB(4) induced phosphorylation of ERK and Akt, downstream kinase of PI3K; LY294002 suppressed phosphorylation of both kinases while U0126 suppressed only the former. CONCLUSION: These results suggest that LTB(4) may enhance TNF-alpha-induced CCL27 production by activating NF-kappaB via the BLT1/G(i/o)/PI3K/ERK pathway in human keratinocytes. LTB(4) may contribute to the enhanced CCL27 production of keratinocytes in lesions with atopic dermatitis or allergic contact dermatitis.     

IL-17 suppresses TNF-alpha-induced CCL27 production through induction of COX-2 in human keratinocytes

BACKGROUND: The chemokine CCL27 attracts skin-homing T cells. CCL27 production by keratinocytes is dependent on nuclear factor kappaB (NF-kappaB) activity and enhanced in lesions of patients with atopic dermatitis, psoriasis vulgaris, or allergic contact dermatitis. IL-17 is released from activated memory T cells and modulates skin inflammation. OBJECTIVE: We examined the in vitro effects of IL-17 on TNF-alpha-induced CCL27 production in human keratinocytes. METHODS: Keratinocytes were incubated with TNF-alpha, IL-17, or both. CCL27 secretion and mRNA levels were analyzed by means of ELISA and RT-PCR, respectively. COX-2 promoter and NF-kappaB activities were analyzed by using luciferase assays. COX-2 protein levels were analyzed by means of Western blotting. RESULTS: IL-17 suppressed TNF-alpha-induced CCL27 secretion and mRNA expression and NF-kappaB activity in keratinocytes. The COX-2 inhibitor NS398 counteracted the effects of IL-17, and prostaglandin E(2) prevented counteraction by NS398. IL-17 alone or synergistically with TNF-alpha increased prostaglandin E(2) release from keratinocytes, and the increase was suppressed by NS398. IL-17 alone or synergistically with TNF-alpha increased COX-2 mRNA and protein levels, promoter activity, and mRNA stability. The stimulatory effects of IL-17 on COX-2 expression were suppressed by inhibitors of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) kinase. IL-17 alone or synergistically with TNF-alpha induced dual phosphorylation of p38 MAPK and ERK. CONCLUSION: IL-17 might suppress TNF-alpha-induced CCL27 production by inhibiting NF-kappaB through induction of COX-2. The induction of COX-2 might be mediated by activation of p38 MAPK and ERK. T cell-derived IL-17 might alleviate T-cell skin infiltration through inhibition of CCL27 production.     

Signaling events involved in cytokine and chemokine production induced by secretory phospholipase A2 in human lung macrophages

Secretory phospholipases A(2) (sPLA(2)) are enzymes released during inflammatory reactions. These molecules activate immune cells by mechanisms either related or unrelated to their enzymatic activity. We examined the signaling events activated by group IA (GIA) and group IB (GIB) sPLA(2) in human lung macrophages leading to cytokine/chemokine production. sPLA(2) induced the production of cytokines (TNF-alpha, IL-6 and IL-10) and chemokines (CCL2, CCL3, CCL4 and CXCL8), whereas no effect was observed on IL-12, CCL1, CCL5 and CCL22. sPLA(2) induced the phosphorylation of the MAPK p38 and ERK1/2, and inhibition of these kinases by SB203580 and PD98059, respectively, reduced TNF-alpha and CXCL8 release. Suppression of sPLA(2) enzymatic activity by a site-directed inhibitor influenced neither cytokine/chemokine production nor activation of MAPK, whereas alteration of sPLA(2) secondary structure suppressed both responses. GIA activated the phosphatidylinositol 3-kinase (PI3 K)/Akt system and a specific inhibitor of PI3 K (LY294002) reduced sPLA(2)-induced release of TNF-alpha and CXCL8. GIA promoted phosphorylation and degradation of IkappaB and inhibition of NF-kappaB by MG-132 and 6-amino-4-phenoxyphenylethylamino-quinazoline suppressed the production of TNF-alpha and CXCL8. These results indicate that sPLA(2) induce the production of cytokines and chemokines in human macrophages by a non-enzymatic mechanism involving the PI3 K/Akt system, the MAPK p38 and ERK1/2 and NF-kappaB.     

Human β-defensin-2 enhances IFN-γ and IL-10 production and suppresses IL-17 production in T cells

Psoriasis is an inflammatory dermatosis with enhanced expression of hBD-2 in keratinocytes and infiltration of cytokine-producing T cells, which in turn, up- or down-regulate hBD-2 expression. We determined the serum levels of hBD-2 and cytokines in psoriasis patients and analyzed the effects of hBD-2 on cytokine production in human peripheral blood T cells. Serum hBD-2 levels in patients were higher than those in controls and correlated with PASI, serum IFN-γ, and IL-10 levels and correlated inversely with serum IL-17 levels. IFN-γ, IL-17, IL-22, TNF-α, IL-1β, and IL-6 enhanced, and IL-10, IL-4, and IL-13 suppressed hBD-2 secretion from keratinocytes. hBD-2 enhanced secretion and mRNA levels of IFN-γ, TNF-α, IL-10, IL-1β, IL-6, and IL-22 and reduced those of IL-17 in CD3/28-stimulated T cells. These effects of hBD-2 were counteracted by PTX. hBD-2 induced phosphorylation of JNK, ERK, and Akt in T cells. Inhibitors of these signals attenuated hBD-2-induced production of IFN-γ, TNF-α, IL-10, IL-1β, IL-6, and IL-22. hBD-2 suppressed phosphorylation of STAT3 and enhanced expression of SOCS3 in CD3/28-stimulated T cells. siRNA against SOCS3 reversed hBD-2-induced suppression of IL-17 production and STAT3 phosphorylation. JNK and MEK inhibitors suppressed hBD-2-induced expression of SOCS3. In conclusion, hBD-2 may bind PTX-sensitive GPCR(s) on T cells and act as a stimulator by enhancing IFN-γ, TNF-α, IL-1β, IL-6, and IL-22 production via JNK, MEK/ERK, and PI3K/Akt and as a regulator by suppressing IL-17 production via SOCS3 or by stimulating IL-10 production.     

Treatment of monocytes with interleukin (IL)-12 plus IL-18 stimulates survival, differentiation and the production of CXC chemokine ligands (CXCL)8, CXCL9 and CXCL10

During inflammation, interleukin (IL)-12 and IL-18 are produced by macrophages and other cell types such as neutrophils (IL-12), keratinocytes and damaged endothelial cells (IL-18). To explore the role of IL-12 and IL-18 in inflammatory innate immune responses we investigated their impact on human peripheral blood monocytes and mature bronchoalveolar lavage (BAL) macrophages. IL-12 and IL-18 together, but not alone, prevented spontaneous apoptosis of cultured monocytes, promoted monocyte clustering and subsequent differentiation into macrophages. These morphological changes were accompanied by increased secretion of CXC chemokine ligands (CXCL)9, CXCL10 (up to 100-fold, P < 0.001) and CXCL8 (up to 10-fold, P < 0.001) but not CCL3, CCL4 or CCL5. Mature macrophages (from BALs) expressed high basal levels of CXCL8, that were no modified upon stimulation with IL-12 and IL-18. In contrast, the basal production of CXCL9 and CXCL10 by BALs was increased by 10-fold (P < 0.001) in the presence of either IL-12 or IL-18 alone and by 50-fold in the presence of both cytokines. In conclusion, our results indicate a relevant role for IL-12 and IL-18 in the activation and resolution of inflammatory immune responses, by increasing the survival of monocytes and by inducing the production of chemokines. In particular, those that may regulate angiogenesis and promote the recruitment of monocytes, activated T cells (CXCL9 and CXCL10) and granulocytes (CXCL8).     

Interferon regulatory factor modulation underlies the bystander suppression of malaria antigen-driven IL-12 and IFN-γ in filaria-malaria co-infection

In areas where polyparasitism is highly prevalent, the impact of multiple parasites on the host response is underestimated. In particular, the presence of helminth infection coincident with malaria profoundly alters the production of malaria-specific IFN-γ, IL-12p70, CXCL9, CXCL10 and CXCL11, cytokines/chemokines known to be critical in mediating malaria-specific immunity. In order to elucidate the mechanisms underlying the suppression of malaria-specific cytokines/chemokines, we assessed the expression of malaria-specific IL-12Rβ1, IL-12Rβ2 and interferon regulatory factor (IRF)-1 in blood obtained from 18 filaria-infected (Fil(+)) and 17 filaria-uninfected (Fil(-)) individuals in a filaria-malaria co-endemic region of Mali. We found that Fil(+) individuals had significantly lower RNA expression of IRF-1 but not IL-12Rβ1 or IL-12Rβ2 in response to malaria antigen stimulation. We also measured the frequency of IL-12-producing DCs from these subjects and found that Fil(+) subjects had lower frequencies of IL-12(+) mDCs after malaria antigen stimulation than did the Fil(-) subjects. Modeling these data in vitro, we found that mDCs pre-exposed to live microfilariae not only produced significantly lower levels of CXCL-9, CXCL-10, IL-12p35, IL-12p40, IL-12p19 and CXCL-11 following stimulation with malaria antigen but also markedly downregulated the expression of IRF-1, IRF-2 and IRF-3 compared with microfilaria-unexposed mDCs. siRNA-inhibition of irf-1 in mDCs downregulated the production of IL-12p70 through repression of IL-12p35. Our data demonstrate that the modulation of IRFs seen in filarial (and presumably other tissue-invasive helminths) infection underlies the suppression of malaria-specific cytokines/chemokines that play a crucial role in immunity to malaria.     

CXCR3-mediated opposite effects of CXCL10 and CXCL4 on TH1 or TH2 cytokine production

BACKGROUND: Two variants of the CXCR3 receptor exist, one (CXCR3-A) reactive with CXCL9, CXCL10, and CXCL11 and the other (CXCR3-B) also reactive with CXCL4. Both variants are contemporarily expressed by human T cells. OBJECTIVE: We sought to investigate the in vitro effects of CXCL10 and CXCL4 on the production of TH1 or TH2 cytokines. METHODS: The cytokine profile of antigen-specific human CD4+ T-cell lines obtained in the absence or presence of CXCL10 or CXCL4 was evaluated by means of quantitative RT-PCR, flow cytometry, and ELISA. RESULTS: CXCL10 upregulated IFN-gamma and downregulated IL-4, IL-5, and IL-13 production, whereas CXCL4 downregulated IFN-gamma and upregulated TH2 cytokines. Similar effects were also observed on polyclonally activated pure naive CD4+ T cells. The opposite effects of CXCL10 and CXCL4 on TH1 and TH2 cytokine production were inhibited by an anti-CXCR3 antibody able to neutralize both CXCR3-A and CXCR3-B and were apparently related to the activation of distinct signal transduction pathways. Moreover, CXCL10 upregulated mRNA levels of T-box expressed in T cells and downregulated GATA-3 expression, whereas CXCL4 downregulated T-box expressed in T cells and upregulated GATA-3. Finally, CXCL4, but not CXCL10, induced direct activation of IL-5 and IL-13 promoters. CONCLUSION: CXCL10 and CXCL4 exert opposite effects on the production of human TH1 and TH2 cytokines, likely through their respective interaction with CXCR3-A or CXCR3-B and the consequent activation of different signal transduction pathways. This might represent an internal regulatory pathway of TH cell responses and might contribute to the modulation of chronic inflammatory reactions, including allergy.     

CXCL1 confers a survival advantage in Kaposi's sarcoma-associated herpesvirus-infected human endothelial cells through STAT3 phosphorylation

Many cytokines produced by Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells have been shown to participate in the pathogenesis of KSHV. Determination of the exact role of cytokines in Kaposi's sarcoma (KS) pathogenesis is limited, however, by the difficulty to manipulate the target genes in human endothelial cells. In this study, we sought to elucidate the role of cytokines in KSHV-infected human immortalized endothelial cell line (HuARLT cells) by knockout (KO) of the corresponding target genes using the CRISPR/Cas9 system. The cytokine production profile of KSHV-infected HuARLT cells was analyzed using a protein array, and several cytokines were found to be highly upregulated following KSHV infection. This study focused on CXCL1, which was investigated by knocked out in HuARLT cells. KSHV-infected CXCL1 KO cells underwent increased cell death compared to KSHV-infected wild-type (WT) cells and mock-infected CXCL1 KO cells. Lytic replication was not observed in KSHV-infected WT nor CXCL1 KO cells. Phosphorylation of STAT3 was significantly suppressed in KSHV-infected CXCL1 KO cells. Additionally, inhibitors of STAT3 and CXCL1 induced cell death in KSHV-infected endothelial cells. Our results show that CXCL1 production is required for the survival of KSHV-infected endothelial cells, and the CXCL1 to STAT3 phosphorylation signaling pathway may be a therapeutic target for KS.     

Evaluation of CXCL10, CXCL11, CXCL12 and CXCL13 chemokines in serum and cerebrospinal fluid in patients with tick borne encephalitis (TBE)

PurposeThe aim of the study was to assess the concentration of chemokines: CXCL10, XCL11, CXCL12, CXCL13 in serum and cerebrospinal fluid (CSF) in patients with tick-borne encephalitis (TBE) before and after treatment. We evaluated also the usefulness of these molecules in diagnosis and monitoring of inflammation in TBE.MethodsTwenty three patients hospitalized in The Department of Infectious Diseases and Neuroinfections of Medical University in Bia?ystok, Poland were included in the study. Patients were divided into 2 groups: TBE group-patients with confirmed TBE and control group (CG): patients with excluded TBE and other inflammatory diseases of CNS. Concentration of CXCL10/IP-10, CXCL11/I-TAC, CXCL12/SDF-1α, CXCL13/BLC/BCA-1 in serum and CSF were measured with ELISA kits (R&;D Systems, USA) according to the protocols.ResultsThe analysis of chemokines concentration in TBE patients before treatment and control group using ROC showed that serum CXCL10 and CXCL13 and CSF CXCL10, CXCL11, CXCL12 and CXCL13 differentiate both groups (p<0.05). The analysis of CXCL10, CXCL11, CXCL12 and CXCL13 before and after treatment showed that CXCL10 and CXCL11 in CSF and CXCL13 in serum differentiates both groups with p<0.05.ConclusionsConcentration of CSF CXCL10, CXCL11, CXCL12, CXCL13 and serum CXCL10, CXCL13 may be good biomarkers of CNS inflammation caused by TBEV. Moreover concentration of CXCL10 in CSF and CXCL13 in serum may be used as indicators of patients recovery.     

Aberrant activation profile of cytokines and mitogen-activated protein kinases in type 2 diabetic patients with nephropathy

Cytokine-induced inflammation is involved in the pathogenesis of type 2 diabetes mellitus (DM). We investigated plasma concentrations and ex vivo production of cytokines and chemokines, and intracellular signalling molecules, mitogen-activated protein kinases (MAPK) in T helper (Th) cells and monocytes in 94 type 2 diabetic patients with or without nephropathy and 20 healthy controls. Plasma concentrations of inflammatory cytokines tumour necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-18 and chemokine CCL2 in patients with diabetic nephropathy (DN) were significantly higher than control subjects, while IL-10, CXCL8, CXCL9, CXCL10 and adiponectin concentrations of DN were significantly higher than patients without diabetic nephropathy (NDN) and control subjects (all P < 0.05). Plasma concentrations of TNF-alpha, IL-6, IL-10, IL-18, CCL2, CXCL8, CXCL9, CXCL10 and adiponectin exhibited significant positive correlation with urine albumin : creatinine ratio in DN patients. The percentage increases of ex vivo production of IL-6, CXCL8, CXCL10, CCL2 and CCL5 upon TNF-alpha activation were significantly higher in both NDN and DN patients than controls (all P < 0.05). The percentage increases in IL-18-induced phosphorylation of extracellular signal-regulated kinase (ERK) in Th cells of NDN and DN were significantly higher than controls (P < 0.05), while the percentage increase in TNF-alpha-induced phosphorylation of p38 MAPK in monocytes and IL-18-induced phosphorylation of p38 MAPK in Th cells and monocytes were significantly higher in NDN patients than controls. These results confirmed that the aberrant production of inflammatory cytokines and chemokines and differential activation of MAPK in different leucocytes are the underlying immunopathological mechanisms of type 2 DM patients with DN.     

Apoptotic cells inhibit LPS-induced cytokine and chemokine production and IFN responses in macrophages

Apoptosis is a critical process in tissue homeostasis and results in immediate removal of the dying cell by professional phagocytes such as macrophages and dendritic cells. Phagocytosis of apoptotic cells actively suppresses production of proinflammatory growth factors and cytokines. Impaired phagocytosis of apoptotic cells has been implicated in the pathogenesis of chronic inflammatory and autoimmune diseases. In this study we found that, in addition to suppressing lipopolysaccharide (LPS)-induced production of TNF-alpha and IL-6, phagocytosis of apoptotic cells by macrophages suppressed production of the chemokine CXCL10 that is activated by LPS-induced autocrine-acting type I IFNs. Inhibition of cytokine and chemokine production was not universally affected because LPS-induced production of IL-10 and IL-8 was not significantly affected. Apoptotic cells had minimal effects on LPS-induced activation of NF-kappaB and MAPKs, but induced expression of SOCS proteins and substantially suppressed induction of CXCL10 expression by IFN-alpha. In addition to suppressing LPS responses, apoptotic cells inhibited macrophage responses to another major macrophage activator IFN-gamma by attenuating IFN-gamma-induced STAT1 activation and downstream gene expression. These results identify suppressive effects of apoptotic cells on signal transduction, and extend our understanding of the anti-inflammatory effects of apoptotic cells to include suppression of Jak-STAT signaling.     

Cbl and Akt regulate CXCL8-induced and CXCR1- and CXCR2-mediated chemotaxis

CXCL8 (IL-8) plays an important role in the pathogenesis of a variety of inflammatory diseases. However, little is known about the signaling pathways that regulate CXCL8-induced chemotaxis. Here, we found that CXCL8 treatment of CXCR1- and CXCR2-over-expressing L1.2 cells (CXCR1-L1.2 and CXCR2-L1.2, respectively) induced the phosphorylation of Cbl and Akt. The tyrosine kinase inhibitor Tyrphostin A9, phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002 as well as proteasome inhibitors significantly blocked the CXCL8-induced chemotaxis of L1.2 cells and human neutrophils. We further found that stimulation with CXCL8 enhanced the association of the PI3K subunit p85 with Cbl. Additionally, over-expression of wild-type Cbl and G306E-Cbl (mutation in the tyrosine kinase-binding domain) inhibited chemotaxis by approximately 50% as compared with the vector control, whereas the 70Z mutant (deletion in the RING finger domain) did not reduce migration. However, wild-type Cbl or its mutants had no effect on the CXCL8-induced activation of MAPK, indicating that Cbl specifically modulated CXCL8-induced chemotaxis. Furthermore, over-expression of the kinase-dead Akt mutant decreased CXCL8-induced chemotaxis by 60% and diminished Cbl phosphorylation as compared with the vector control. The CXCL8-induced phosphorylation of Cbl was also reduced when cells were pre-treated with the PI3K inhibitor LY294002. Lastly, we have shown that pre-treatment of L1.2 cells with the proteasome inhibitor Lactacystin blocks CXCL8-induced internalization of the CXCR1 and CXCR2 receptors. These studies provide new information regarding CXCL8-induced signaling pathways that may regulate chemotaxis and receptor internalization.     

Differential regulation of cytokine genes in gingival epithelial cells challenged by Fusobacterium nucleatum and Porphyromonas gingivalis

IL-8 mRNA in human gingival epithelial cells (HGECs) is up-regulated by Fusobacterium nucleatum, and up-/down-regulated by Porphyromonas gingivalis in a complex interaction in the early stages (< or = 4 h) after infection. The mechanisms involved in this regulation in response to F. nucleatum and/or P. gingivalis infection, and identification of co-regulated cytokine genes, are the focus of this investigation. Heat, formalin or protease treatment of F. nucleatum cells attenuated the IL-8 mRNA up-regulation. NF-kappaB, mitogen-activated protein kinase (MAPK) p38 and MAPK kinase/extracellular signal-regulated kinase (MEK/ERK) pathways were involved in IL-8 mRNA induction by F. nucleatum. Pretreatment of P. gingivalis with heat, formalin or protease enhanced IL-8 mRNA induction. NF-kappaB, MARK p38, and MEK/ERK pathways were also involved in this induction. In contrast, down-regulation of IL-8 mRNA by P. gingivalis involved MEK/ERK, but not NF-kappaB or MAPK p38 pathways. cDNA arrays analysis revealed that mRNA down-regulation by P. gingivalis is a specific reaction that only a number of genes, e.g. IL-1beta, IL-8, macrophage inflammatory protein-2alpha, and migration inhibitory factor-related protein-14, are affected based on examination of 278 cytokine/receptor genes. These data indicate that F. nucleatum and P. gingivalis trigger specific and differential gene regulation pathways in HGECs.