Ezra Aksoy

Monophosphoryl Lipid A Activates Both Human Dendritic Cells and T Cells

The induction of dendritic cell (DC) maturation is critical for the induction of Ag-specific T lymphocyte responses and may be essential for the development of human vaccines relying on T cell immunity. In this study, we have investigated the effects of monophosphoryl lipid A (MPL) on human monocyte-derived DC as well as peripheral blood T cells. Calcium mobilization, mitogen-activated protein kinase activation, and the NF-κB transcription factor were induced after MPL stimulation of DC and required high doses of MPL (100 μg/ml). Maturation parameters such as production of IL-12 and increases in cell surface expression of HLA-DR, CD80, CD86, CD40, and CD83 were observed following DC treatment with MPL. However, lower levels of IL-12 were induced by MPL when compared with lipopolysaccharide. This is likely to be related to differences in the kinetics of extracellular signal-related kinase 1/2 and p-38 phosphorylation induced by both molecules. Although maturation induced by MPL was weaker when compared with lipopolysaccharide, it appeared to be sufficient to support optimal activation of allogeneic naive CD45RA+ T cell and anti-tetanus toxoid CD4 T cells. MPL at low doses (5 μg/ml) had no impact on DC maturation, while its addition to DC-T cell cocultures induced full T cell activation. The observed effect was related to the fact that MPL also acts directly on T cells, likely through their Toll-like receptors, by increasing their intracellular calcium and up-regulating their CD40 ligand expression. Together, these data support a model where MPL enhances T cell responses by having an impact on DC and T cells.

Inhibition of phosphoinositide 3-kinase enhances TRIF-dependent NF-kappa B activation and IFN-beta synthesis downstream of Toll-like receptor 3 and 4.

Phosphoinositide 3‐kinases (PI3K) are known to regulate Toll‐like receptor (TLR)‐mediated inflammatory responses, but their impact on the different pathways of TLR signaling remains to be clarified. Here, we investigated the consequences of pharmacological inhibition of PI3K on Toll‐IL‐1 receptor domain‐containing adapter‐inducing IFN‐β (TRIF)‐dependent signaling, which induces IFN‐β gene expression downstream of TLR3 and TLR4. First, treatment of monocyte‐derived dendritic cells (DC) with wortmannin or LY294002 was found to enhance IFN‐β expression upon TLR3 or TLR4 engagement. In the same models of DC activation, PI3K inhibition increased DNA‐binding activity of NF‐κB, but not interferon response factor (IRF)‐3, the key transcription factors required for TLR‐mediated IFN‐β synthesis. In parallel, wortmannin‐treated DC exhibited enhanced levels of IκB kinase (IKK)‐α/β phosphorylation and IκB‐α degradation with a concomitant increase in NF‐κB nuclear translocation. Experiments carried out in HEK 293T cells stably expressing TLR3 or TLR4 confirmed that inhibition of PI3K activity enhances NF‐κB‐dependent promoters as well as IFN‐β promoter activities without interfering with transcription at the positive regulatory domain III‐I. Furthermore, wortmannin enhanced NF‐κB activity induced by TRIF overexpression in HEK 293T cells, while overexpression of catalytically active PI3K selectively attenuated TRIF‐mediated NF‐κB transcriptional activity. Finally, in co‐immunoprecipitation experiments, we showed that PI3K physically interacted with TRIF. We conclude that inhibition of PI3K activity enhances TRIF‐dependent NF‐κB activity, and thereby increases IFN‐β synthesis elicited by TLR3 or TLR4 ligands.

Protein kinase C epsilon: a new target to control inflammation and immune-mediated disorders

Recent advances in understanding the molecular basis for mammalian host immune responses to microbial invasion suggest that the first line of defense against microbes is the recognition of pathogen-associated molecular patterns by a set of germline-encoded receptors: the Toll-like receptors (TLRs). TLRs have been identified as being part of a large family of pathogen-recognition receptors that play a decisive role in the induction of both innate and adaptive immunity. Indeed, activation of T lymphocytes depends on their interaction with dendritic cells previously stimulated by TLR agonists such as bacterial lipopolysaccharide (LPS), a TLR-4 ligand. A novel PKC epsilon (epsilon) was recently found to be a critical component of TLR-4 signaling pathway and thereby to play a key role in macrophage and dendritic cell (DC) activation in response to LPS. Thus, controlling the kinase activity of PKC epsilon might represent an efficient strategy to prevent or treat certain inflammatory disorders of microbial origin.

Bordetella pertussis toxin induces the release of inflammatory cytokines and dendritic cell activation in whole blood: impaired responses in human newborns

Bordetella pertussis toxin (PTX), a key component of acellular petussis vaccines, is known to be endowed with adjuvant properties. In experiments designed to get insights into the interactions between PTX and circulating immune cells, we first observed that addition of PTX to adult whole blood induced the release of IL‐12 and TNF‐α as well as maturation of myeloid dendritic cells (DC). These effects were still present with a toxin mutant devoid of ADP‐ribosyltransferase activity but not with a formaldehyde‐inactivated toxin. These findings indicate that cytokine production and DC maturation require the intact structure of PTX but not its enzymatic activity. Secondly, studies on DC generated in vitro by culturing monocytes with IL‐4 and GM‐CSF showed thatPTX directly stimulates MHC class II and costimulatory molecules up‐regulation, cytokine synthesis and NF‐κB activation. Finally, comparison of data obtained in adult vs. cord blood revealed deficient responses of neonatal DC to PTX. These data suggest new applications of PTX and PTX mutants as vaccine adjuvants.

Tumoricidal activity of monocyte-derived dendritic cells: Evidence for a caspase-8-dependent, Fas-associated death domain-independent mechanism.

Monocyte-derived dendritic cells (DC) were found to be cytotoxic for several tumor cell lines including Jurkat cells, which were killed through a calcium-independent pathway. K562 cells were resistant, excluding a NK cell-like activity. DC-mediated apoptosis did not involve classical death receptors because it was not reversed by blocking TNF/TNFR, CD95/CD95 ligand, or TNF-related apoptosis-inducing ligand/TNF-related apoptosis-inducing ligand receptor interactions. Fas-associated death domain-deficient, but not caspase-8 deficient, Jurkat cells were killed by DC. Indeed, caspase-8 cleavage was demonstrated in Jurkat cells cocultured with DC, and the use of specific caspase inhibitors confirmed that apoptosis triggered by DC was caspase-8 dependent. Furthermore, the involvement of Bcl-2 family members in the control of DC-mediated apoptosis was demonstrated by Bid cleavage in Jurkat cells cocultured with DC and resistance of Jurkat cells overexpressing Bcl-2 to DC-mediated cytotoxicity. Overall, these data indicate that monocyte-derived DC exert a caspase-8-dependent, Fas associated death domain-independent tumoricidal activity, a finding that could be relevant to their therapeutic use in cancer.

Critical role of protein kinase C epsilon for lipopolysaccharide-induced IL-12 synthesis in monocyte-derived dendritic cells.

In the present study we have investigated the potential involvement of protein kinase C (PKC) in the maturation of human dendritic cells (DC) by bacterial lipopolysaccharide (LPS). LPS stimulation of DC derived from human monocytes resulted in PKC phosphorylation. Inhibition of PKC activation using bisindolylmaleimide (Bis), a pan‐PKC inhibitor, was associated with a dose‐dependent decrease of LPS‐induced IL‐12 production. In contrast, up‐regulation of MHC class II, CD80 and CD86 was not altered. Consistent with the diminished IL‐12 synthesis, DC stimulated with LPS in presence of Bis were deficient in the induction of IFN‐γ production by allogeneic CD4+ T cells. Furthermore, we found that PKC inhibition impaired LPS‐induced IκB‐α degradation and subsequent nuclear factor (NF)‐κB activation in DC. LPS resulted in the phosphorylation of conventional α/β and novel ϵ PKC isoforms in DC. Inhibition of LPS‐induced PKC activity using pseudosubstrate peptides specific for PKC isoforms established that PKC ϵ but not PKC α/β was involved in the production of IL‐12 and TNF‐α. Overall, these data provide evidence thatPKC inhibition impairs LPS signaling in DC and identify PKC ϵ as a potential target for the inhibition of Toll‐like receptor‐4‐mediated, IL‐12‐dependent Th1 type responses.

PKC‐α controls MYD88‐dependent TLR/IL‐1R signaling and cytokine production in mouse and human dendritic cells

Conventional PKC (cPKC)‐α regulates TRIF‐dependent IFN response factor 3 (IRF3)‐mediated gene transcription, but its role in MyD88‐dependent TLR signaling remains unknown. Herein, we demonstrate that PKC‐α is induced by several MyD88‐dependent TLR/IL‐1R ligands and regulates cytokine expression in human and murine DC. First, inhibition of cPKC activity in human DC by cPKC‐specific inhibitors, Gö6976 or HBDDe, downregulated the production of classical inflammatory/immunomodulatory cytokines induced by TLR2, TLR5 or IL‐1R but not by TLR3 stimulation. Similarly, dominant negative PKC‐α repressed Pam3CSK4 induced NF‐κB‐ and AP‐1‐driven promoter activities in TLR2‐expressing human embryonic kidney 293 T cells. Dominant negative PKC‐α inhibited NF‐κB reporter activity mediated by overexpression of MyD88 but not TRIF. Unexpectedly, BM‐derived DC from PKC‐α−/− mice exhibited decreased TNF‐α and IL‐12p40 production induced by both MyD88‐ and TRIF‐dependent ligands. Furthermore, PKC‐α is coupled to TLR2 signaling proximal to MyD88 since MAPK and IκB kinase‐α/β phosphorylations and IκBα degradation were inhibited in PKC‐α−/− BM‐derived DC. Finally, co‐immunoprecipitation assays revealed that PKC‐α physically interacts with Pam3CSK4 activated TLR2 in WT but not in MyD88−/− DC. Collectively this study identifies a species‐specific role of PKC‐α as a key component that controls MyD88‐dependent cytokine gene expression in human and mouse but differentially regulates production of TRIF‐dependent cytokines.

CD40 expression on human pancreatic duct cells: role in nuclear factor-kappa B activation and production of pro-inflammatory cytokines.

Aims/hypothesisHuman pancreatic duct cells are closely associated with islet beta cells, and contaminate islet suspensions transplanted in Type 1 diabetes mellitus patients. Activated duct cells produce cytotoxic mediators and possibly contribute to the pathogenesis of Type 1 diabetes mellitus or islet graft rejection. As CD40 transduces activation signals involved in inflammatory and immune disorders, we investigated CD40 expression on duct cells and their response to CD40 engagement.MethodsCD40 expression on human pancreatic duct cells was analysed by flow cytometry and immunohistochemical analyses. To assess the function of CD40 expression on duct cells, activation of the transcription factor nuclear factor-kappa B was determined using electrophoretic mobility shift assay and ELISA. Cytokine mRNA levels were quantified by real-time RT-PCR, and protein levels by Luminex technology.ResultsIsolated human pancreatic duct cells and Capan-2 cell lines were found to express constitutively CD40. The expression of CD40 on duct cells was confirmed in vivo on human normal and pathological pancreatic specimens. CD40 ligation on Capan-2 cells induced rapid nuclear factor-kappa B activation, and supershift assays demonstrated that p50/p65 heterodimers and p50/p50 homodimers were present in the activated complexes in the nucleus. This activation was accompanied by tumour necrosis factor-α and interleukin-1β mRNA accumulation. Tumour necrosis factor-α protein secretion was confirmed in CD40-activated Capan-2 cells and in isolated human pancreatic duct cells.Conclusions/interpretationInteraction between activated T lymphocytes expressing CD40 ligand and duct cells expressing CD40 may contribute to the immune responses involved in Type 1 diabetes mellitus and islet graft rejection. Interfering with CD40-mediated duct cell activation could alleviate beta cell damage of immune origin.

Protein kinase Calpha is involved in interferon regulatory factor 3 activation and type I interferon-beta synthesis.

Protein kinase C (PKC) isoforms are critically involved in the regulation of innate immune responses. Herein, we investigated the role of conventional PKCα in the regulation of IFN-β gene expression mediated by the Toll-like receptor 3 (TLR3) signaling pathway. Inhibition of conventional PKC (cPKC) activity in monocyte-derived dendritic cells or TLR3-expressing cells by an isoform-specific inhibitor, Gö6976, selectively inhibited IFN-β synthesis induced by double-stranded RNA polyinosine-polycytidylic acid. Furthermore, reporter gene assays confirmed that PKCα regulates IFN-β promoter activity, since overexpression of dominant negative PKCα but not PKCβI repressed interferon regulatory factor 3 (IRF-3)-dependent but not NF-κB-mediated promoter activity upon TLR3 engagement in HEK 293 cells. Dominant negative PKCα inhibited IRF-3 transcriptional activity mediated by overexpression of TIR domain-containing adapter inducing IFN-β and Tank-binding kinase-1. Additional biochemical analysis demonstrated that Gö6976-treated dendritic cells exhibited IRF-3 phosphorylation, dimerization, nuclear translocation, and DNA binding activity analogous to their control counterparts in response to polyinosine-polycytidylic acid. In contrast, co-immunoprecipitation experiments revealed that TLR3-induced cPKC activity is essential for mediating the interaction of IRF-3 but not p65/RelA with the co-activator CREB-binding protein. Furthermore, PKCα knock-down with specific small interfering RNA inhibited IFN-β expression and down-regulated IRF-3-dependent promoter activity, establishing PKCα as a component of TLR3 signaling that regulates IFN-β gene expression by targeting IRF-3-CREB-binding protein interaction. Finally, we analyzed the involvement of cPKCs in other signaling pathways leading to IFN-β synthesis. These experiments revealed that cPKCs play a role in the synthesis of IFN-β induced via both TLR-dependent and -independent pathways.

An Alternative Pathway of NF-κB Activation Results in Maturation and T Cell Priming Activity of Dendritic Cells Overexpressing a Mutated IκBα

Maturation of dendritic cells (DC) is a critical step in the induction of T cell responses and depends on the activation of NF-κB transcription factors. Therefore, inhibition of NF-κB activation has been proposed as a strategy to maintain DC in an immature stage and to promote immune tolerance. Herein, we generated murine myeloid DC expressing a mutated IκBα acting as a superrepressor of the classical NF-κB pathway (s-rIκB DC) to investigate the consequences of NF-κB inhibition on the ability of DC to prime T cell responses. Upon in vitro LPS activation, maturation of s-rIκB DC was profoundly impaired as indicated by defective up-regulation of MHC class II and costimulatory molecules and reduced secretion of IL-12 p70 and TNF-α. In contrast, after injection, s-rIκB DC had the same capacity as control DC to migrate to draining lymph node and to induce Th1- and Th2-type cytokine production in a MHC class II-incompatible host mice. Likewise, s-rIκB DC pulsed with OVA were as efficient as control DC to induce Ag-specific T cell responses in vivo. Indeed, further in vitro experiments established that s-rIκB DC undergo efficient maturation upon prolonged contact with activated T cells via the alternative pathway of NF-κB activation triggered at least partly by lymphotoxin β receptor ligation and involving processing of p100/RelB complexes.