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Autoimmune diseases are characterized by dendritic cell (DC)-driven activation of pro-inflammatory T cell responses. Therapeutic options for these severe diseases comprise small molecules such as dimethyl fumarate, or "gasotransmitters" such as CO. Herein we describe the synthesis of bifunctional enzyme-triggered CO-releasing molecules (ET-CORMs) that allow the simultaneous intracellular release of both CO and methyl fumarate. Using bone-marrow-derived DCs the impressive therapeutic potential of these methyl fumarate-derived compounds (FumET-CORMs) is demonstrated by strong inhibition of lipopolysaccharide-induced pro-inflammatory signaling pathways and blockade of downstream interleukin-12 or -23 production. The data also show that FumET-CORMs are able to transform DCs into an anti-inflammatory phenotype. Thus, these novel compounds have great clinical potential, for example, for the treatment of psoriasis or other inflammatory conditions of the skin., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Brucella abortus is the causative agent of brucellosis, which causes abortion in domestic animals and undulant fever in humans. This bacterium infects and proliferates mainly in macrophages and dendritic cells, where it is recognized by pattern recognition receptors (PRRs) including Nod-like receptors (NLRs). Our group recently demonstrated the role of AIM2 and NLRP3 in Brucella recognition. Here, we investigated the participation of NLRP12 in innate immune response to B. abortus. We show that NLRP12 inhibits the early production of IL-12 by bone marrow-derived macrophages upon B. abortus infection. We also observed that NLRP12 suppresses in vitro NF-κB and MAPK signaling in response to Brucella. Moreover, we show that NLRP12 modulates caspase-1 activation and IL-1β secretion in B. abortus infected-macrophages. Furthermore, we show that mice lacking NLRP12 are more resistant in the early stages of B. abortus infection: NLRP12 -/- infected-mice have reduced bacterial burdens in the spleens and increased production of IFN-γ and IL-1β compared with wild-type controls. In addition, NLRP12 deficiency leads to reduction in granuloma number and size in mouse livers. Altogether, our findings suggest that NLRP12 plays an important role in negatively regulating the early inflammatory responses against B. abortus., Competing Interests: disclosure The authors have no financial or commercial conflicts of interest., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

IL-35 is a cytokine of the IL-12 family, existing as a heterodimer of IL-12p35 and Ebi3. IL-35 has anti-inflammatory properties and is produced by regulatory T cells in humans and mice, where it is required for optimal suppression of immune responses. Distinct from other IL-12 cytokines, the expression of IL-35 has not been described in antigen-presenting cells. In view of the immune-regulatory properties of IL-35, we investigated the expression, regulation, and function of IL-12p35 and Ebi3 in human monocyte-derived dendritic cells and tolerogenic DCs (tolDCs). These tolDCs do not produce IL-12p70 or the homodimer IL-12p40. We demonstrate that tolDCs completely lack transcriptional expression of IL-12p40. However, tolDCs maintain mRNA expression of IL-12p35 and Ebi3. Using intracellular flow cytometry and Western blot analysis, we show that tolDCs produce Ebi3 and IL-12p35, and both can be enhanced upon stimulation with IFN-γ, LPS, or CD40L. tolDCs supernatants have the capacity to suppress T-cell activation. Using IL12A silencing, we demonstrate that IL-12p35 is required for tolDCs to reach their full suppressive potential. Taken together, our results indicate that tolDCs produce IL-35, providing an additional novel mechanism by which tolDCs elicit their tolerogenic potential., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

With increasing interest in alternative options to interferon-alpha-based treatments, IFN-λ has shown therapeutic promise in a variety of diseases. Although the antiviral activity of IFN-λ has been extensively studied, there is limited knowledge regarding the immunological functions of IFN-λ and how these differ from those of other classes of IFNs. In this study, we investigated the effects of IFN-λ on primary human NK cells, both in a direct and indirect capacity. We demonstrate that in contrast to interferon-alpha, IFN-λ is unable to directly stimulate NK cells, due to the absence of IFN-λ receptor chain 1 (IFN-λR1) on NK cells. However, IFN-λ, in combination with TLR4 challenge, is able to induce the production of select members of the IL-12 family of cytokines in monocyte-derived macrophages. We further show that through macrophage-mediated IL-12 production, IFN-λ is able to indirectly affect NK cells and ultimately induce IFN-γ production., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Understanding the signaling that governs the immunogenicity of human dendritic cells (DCs) is a prerequisite for improving DC-based therapeutic vaccination strategies, in which the ability of DCs to induce robust and lasting Ag-specific CTL responses is of critical importance. Cytokine-matured DCs are regularly used, but to induce memory-type CTLs, they require additional activation stimuli, such as CD4+ T-cell help or TLR activation. One common denominator of these stimuli is the activation of NF-κB. Here, we show that human monocyte-derived, cytokine cocktail-matured DCs transfected with constitutively active mutants of IκB kinases (caIKKs) by mRNA electroporation, further upregulated maturation markers, and secreted enhanced amounts of cytokines, including IL-12p70, which was produced for more than 48 h after transfection. Most importantly, cytotoxic T cells induced by caIKK-transfected DCs combined high CD27 expression, indicating a more memory-like phenotype, and a markedly enhanced secondary expandability with a high lytic capacity. In contrast, CTLs primed and expanded with unmodified cytokine cocktail-matured DCs did not maintain their proliferative capacity upon repetitive stimulations. We hypothesize that "designer" DCs expressing constitutively active IκB kinases will prove highly immunogenic also in vivo and possibly emerge as a new strategy to improve the clinical efficacy of therapeutic vaccinations against cancer and other chronic diseases., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mycobacterium avium has been reported to signal through both Toll-like receptor (TLR2) and TLR9. To investigate the role of TLR6 in innate immune responses to M. avium, TLR6, MyD88, TLR2, and TLR2/6 KO mice were infected with this pathogen. Bacterial burdens were higher in the lungs and livers of infected TLR6, TLR2, TLR2/6, and MyD88 KO mice compared with those in C57BL/6 mice, which indicates that TLR6 is required for the efficient control of M. avium infection. However, TLR6 KO spleen cells presented with normal M. avium induced IFN-γ responses as measured by ELISA and flow cytometry. In contrast, the production of IFN-γ in lung tissue was diminished in all studied KO mice. Furthermore, only MyD88 deficiency reduced granuloma areas in mouse livers. Moreover, we determined that TLR6 plays an important role in controlling bacterial growth within macrophages and in the production of TNF-α, IL-12, and IL-6 by M. avium infected DCs. Finally, the lack of TLR6 reduced activation of MAPKs and NF-κB in DCs. In summary, TLR6 is required for full resistance to M. avium and for the activation of DCs to produce proinflammatory cytokines., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Early growth response gene 2 (Egr2), which encodes a zinc finger transcription factor, is rapidly and transiently induced in various cell types independently of de novo protein synthesis. Although a role for Egr2 is well established in T-cell development, Egr2 expression and its biological function in dendritic cells (DCs) have not yet been described. Here, we demonstrate Egr2 expression during DC development, and its role in DC-mediated immune responses. Egr2 is expressed in the later stage of DC development from BM precursor cells. Even at steady state, Egr2 is highly expressed in mouse splenic DCs. Egr2-knockdown (Egr2-KD) DCs showed increased levels of major histocompatability complex (MHC) class I and II and co-stimulatory molecules, and enhanced antigen uptake and migratory capacities. Furthermore, Egr2-KD abolished SOCS1 expression and signal transducer and activator of transcription 5 (STAT5) activation during DC development, probably resulting in the enhancement of IL-12 expression and Th1 immunogenicity of a DC vaccine. DC-mediated cytotoxic T lymphocyte (CTL) activation and antitumor immunity were significantly enhanced by Egr2-KD, and impaired by Egr2 overexpression in antigen-pulsed DC vaccines. These data suggest that Egr2 acts as an intrinsic negative regulator of DC immunogenicity and can be an attractive molecular target for DC vaccine development., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Plasmodium spp. parasites, the causative agents of malaria, survive and replicate in human hosts by modulating host protective immune responses. In a rodent model, malaria manifests as a severe splenomegaly, with infiltration of cells and lympho-proliferation as major contributing factors of the immunopathology. However, the cellular contents and the functions of these cells have not been well studied. Here, we report that Plasmodium berghei infection of mice leads to massive recruitment of mesenchymal stem cells (MSCs) in secondary lymphoid organs. Infusion of these cells into naïve mice was able to confer host resistance against malaria. Furthermore, MSCs augmented interleukin (IL)-12 production but suppressed IL-10 production in recipient animals. In addition, we observed dramatic reductions of regulatory T (Treg) cells in animals that received MSCs. Taken together, our findings have identified recruitment of MSCs as a novel host protective mechanism adopted by the host to combat malaria by modulating Treg-cell responses., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

DX5(+) CD4(+) T cells have been shown to dampen collagen-induced arthritis and delayed-type hypersensitivity reactions in mice. These cells are also potent modulators of T-helper cell responses through direct effects on CD4(+) T cells in an IL-4 dependent manner. To further characterize this T-cell population, we studied their effect on DCs and the potential consequences on T-cell activation. Here, we show that mouse DX5(+) CD4(+) T cells modulate DCs by robustly inhibiting IL-12 production. This modulation is IL-10 dependent and does not require cell contact. Furthermore, DX5(+) CD4(+) T cells modulate the surface phenotype of LPS-matured DCs. DCs modulated by DX5(+) CD4(+) T-cell supernatant express high levels of the co-inhibitor molecules PDL-1 and PDL-2. OVA-specific CD4(+) T cells primed with DCs exposed to DX5(+) CD4(+) T-cell supernatant produce less IFN-γ than CD4(+) T cells primed by DCs exposed to either medium or DX5(-) CD4(+) T-cell supernatant. The addition of IL-12 to the co-culture with DX5(+) DCs restores IFN-γ production. When IL-10 present in the DX5(+) CD4(+) T-cell supernatant is blocked, DCs re-establish their ability to produce IL-12 and to efficiently prime CD4(+) T cells. These data show that DX5(+) CD4(+) T cells can indirectly affect the outcome of the T-cell response by inducing DCs that have poor Th1 stimulatory function., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Resistance to murine visceral leishmaniasis (VL) correlates with the development of an IFN-γ predominant immune response. Beta1,4-galactose terminal glycans are potent inducers of IFN-γ. Here, we demonstrate the efficacy of a 29 kDa β1,4-galactose terminal glycoprotein (GP29) of Leishmania donovani (LD) in an in vitro macrophage model and an in vivo mouse model of VL. GP29 induced splenic macrophages to release NO and ROS in appreciable amounts that resulted in effective parasite clearance from macrophages. This was associated with the toll-like receptor (TLR)-4 mediated IL-12 induction and inhibition of TLR2-mediated IL-10 production. Two subcutaneous injections of GP29 at fortnightly intervals resulted in dominant IL-12-mediated IFN-γ production and 100% animals were protected against a subsequent challenge with virulent LD parasites. Vaccinated mice showed a reversal of T-cell anergy, significantly elevated expression of iNOS and a type-1 IgG subclass response. Moreover, vaccinated mice downregulated arginase1 and IL-10 expression but did not alter IL-4 expression. The IFN-γ/IL-10 ratio regulated the intensity of the protective immune response. Experiments with IFN-γ and IL-10 knockout mice reiterated the role IL-10 and IFN-γ play in disease progression or resolution in the murine model of VL., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Peptidoglycan is a widespread bacterial PAMP molecule and a powerful initiator of innate immune responses. It consists of repeating units of MDP, which as a monomer is only weakly immunostimulatory. Here, MDP-coupled dendrimers were prepared and investigated for stimulation of pig blood mononuclear cells. Compared to monomeric MDP, MDP-dendrimers induced a markedly enhanced production of IL-12 p40, IL-1β and IL-6 and completely down-regulated surface expression of B7 and MHC class II. These results suggest a possible novel strategy based on controlled multimerization of minimal PAMP motifs on dendrimers for preparing molecularly defined immunostimulators with predictable bioactivities., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Autosomal-dominant hyper-IgE syndrome (AD-HIES) is a primary immunodeficiency caused by STAT3 mutations. This inherited condition is characterized by eczema, staphylococcal cold abscesses and recurrent pulmonary infections. Given that STAT3 is involved in IL-10 signaling, we examined the immunoregulatory role of IL-10 in inflammation by studying the effects of IL-10 on monocytes, neutrophils and monocyte-derived DCs from HIES subjects. Analysis of gene expression in PBMCs and neutrophils isolated from HIES patients and stimulated with LPS in the presence of IL-10 showed reduced expression of IL1RN, which encodes IL-1 receptor antagonist (IL-1ra), and SOCS3 mRNA but increased CXCL8 mRNA expression. Moreover, secretion of the anti-inflammatory protein IL-1ra was reduced in AD-HIES patients. DCs from HIES patients secreted higher levels of TNF-α, IL-6 and, to a lesser extent, IL-12 when these cells were cultured in the presence of IL-10. These results suggest that IL-10 activity is affected in myeloid cells (e.g. monocytes, DCs) of HIES patients. Impairment of IL-10 signaling in patients with AD-HIES might result in an altered balance between pro-inflammatory and anti-inflammatory signals and might lead to persistent inflammation and delayed healing after infections., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The protein kinase C (PKC) family is involved in the regulation of many intracellular signalling pathways. Here, we report that the PKCδ isoform regulates IL-12p40/p70 production in macrophages and DC and that PKCδ deficiency in mice transforms the 129/Sv healer to a non-healer strain during cutaneous leishmaniasis. Leishmania major-infected PKCδ(-/-) 129/Sv mice developed a rapid increase in footpad swelling and parasite burden with disease progression, leading to necrosis and ulceration similar to non-healer BALB/c mice. Moreover, PKCδ(-/-) mice failed to develop delayed-type hypersensitivity responses against Leishmania antigen. PKCδ(-/-) macrophages were fully functional with normal MHC class II surface expression and GM-CSF production, recruitment to the draining lymph node and killing effector functions by NO production. In contrast, macrophages and DC produced significantly reduced IL-12p40 and IL-12p70 compared to the WT cells. Decreased IL-12 production resulted in diminished Th1 differentiation, as determined by a striking reduction in IFN-γ by antigen-specific stimulated CD4(+) T cells isolated from popliteal lymph nodes of L. major-infected PKCδ(-/-) mice, explaining the "non-healer" phenotype. We conclude from these data that PKCδ is a regulator of IL-12p40/p70 production by DC and macrophages, driving the healer phenotype during cutaneous leishmaniasis., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

DC apoptosis has been observed in patients with cancer and sepsis, and defects in DC apoptosis have been implicated in the development of autoimmune diseases. However, the mechanisms of how DC apoptosis affects immune responses, are unclear. In this study, we showed that immature viable DC have the ability to uptake apoptotic DC as well as necrotic DC without it being recognized as an inflammatory event by immature viable DC. However, the specific uptake of apoptotic DC converted immature viable DC into tolerogenic DC, which were resistant to LPS-induced maturation. These tolerogenic DC secreted increased levels of TGF-beta1, which induced differentiation of naïve T cells into Foxp3(+) Treg. Furthermore, induction of Treg differentiation only occurred upon uptake of apoptotic DC and not apoptotic splenocytes by viable DC, indicating that it is specifically the uptake of apoptotic DC that gives viable immature DC the potential to induce Foxp3(+) Treg. Taken together, these findings identify uptake of apoptotic DC by viable immature DC as an immunologically tolerogenic event.

To develop cytolytic effector functions, CD8(+) T lymphocytes need to recognize specific Ag/MHC class I complexes in the context of costimuli on Ag-presenting DC. Thereafter they differentiate into effector and memory CTL able to confer protection against pathogen infection. Using transgenic mice with DC-selective MHC class I expression and DC-specific versus ubiquitous vaccination regimen, we found that DC are sufficient to prime CTL responses. However, Ag recognition on parenchymal non-professional APC negatively affected CD8(+) T-cell responses in mice by inducing expression of the pro-apoptotic bcl2-family member bim in CTL. This unexpected induction of apoptosis in the early phase of effector CTL accumulation lead to suboptimal clonal burst size and diminished long-term memory. Thus, our data demonstrate that effector CTL differentiation and apoptosis are regulated independently. Moreover, Ag distribution on cells other than DC critically reduces CTL responses.

Recognition of microbial products by germ-line-encoded PRR initiates immune responses, but how PRR mediate specific host responses to infectious agents is poorly understood. We and others have proposed that specificity is achieved by collaborative responses mediated between different PRR. One such example comprises the fungal beta-glucan receptor Dectin-1, which collaborates with TLR to induce TNF production. We show here that collaborative responses mediated by Dectin-1 and TLR2 are more extensive than first appreciated, and result in enhanced IL-23, IL-6 and IL-10 production in DC, while down-regulating IL-12 relative to the levels produced by TLR ligation alone. Such down-regulation occurred with multiple MyD88-coupled TLR, was dependent on signaling through Dectin-1 and also occurred in macrophages. These findings explain how fungi can induce IL-23 and IL-6, while suppressing IL-12, a combination which has previously been shown to contribute to the development of Th17 responses found during fungal infections. Furthermore, these data reveal how the collaboration of different PRR can tailor specific responses to infectious agents.

IL-4 is expressed at high levels in allergic diseases and dominates the early phases of multiple acquired immune responses. However, the precise role of IL-4 during early inflammation and its impact on the differentiation of newly recruited DC precursors remains elusive. In order to characterize the impact of IL-4 on the differentiation of human DC, we investigated the role of IL-4 on the differentiation of monocytes into DC. Human DC were differentiated from peripheral blood precursors under either low or high concentrations of IL-4. We analyzed their cytokine profile and capacity to polarize T-cell differentiation. Concentrations of 5 (low) and 50 (high) ng/mL IL-4 induced two distinct types of DC. DC differentiated under low-dose IL-4 (5 ng/mL) produced almost no IL-12p70, and primed naïve CD4+ T cells allowing IL-4 secretion and Th2 induction. In contrast, DC generated under high concentrations of IL-4 (50 ng/mL) produced large amounts of IL-12p70, low IL-10 and primed naïve CD4+ T cells to become Th1 cells. Thus, we demonstrate that the Th2 cell cytokine IL-4 decisively determines the phenotype of ongoing immune responses by orchestrating the functional phenotype of newly immigrating DC precursors.

Galectin-3 is a beta-galactoside-binding lectin implicated in the fine-tuning of innate immunity. Rhodococcus equi, a facultative intracellular bacterium of macrophages, causes severe granulomatous bronchopneumonia in young horses and immunocompromised humans. The aim of this study is to investigate the role of galectin-3 in the innate resistance mechanism against R. equi infection. The bacterial challenge of galectin-3-deficient mice (gal3-/-) and their wild-type counterpart (gal3+/+) revealed that the LD50 for the gal3(-/-) mice was about seven times higher than that for the gal3+/+ mice. When challenged with a sublethal dose, gal3(-/-) mice showed lower bacteria counts and higher production of IL-12 and IFN-gamma production, besides exhibiting a delayed although increased inflammatory reaction. Gal3(-/-) macrophages exhibited a decreased frequency of bacterial replication and survival, and higher transcript levels of IL-1beta, IL-6, IL-10, TLR2 and MyD88. R. equi-infected gal3+/+ macrophages showed decreased expression of TLR2, whereas R. equi-infected gal3(-/-) macrophages showed enhanced expression of this receptor. Furthermore, galectin-3 deficiency in macrophages may be responsible for the higher IL-1beta serum levels detected in infected gal3(-/-) mice. Therefore galectin-3 may exert a regulatory role in innate immunity by diminishing IL-1beta production and thus affecting resistance to R. equi infection.

Cancer immunotherapy aims at inducing immune responses against tumour-associated antigens that mediate the eradication of tumour cells. For successful vaccination against antigens expressed by the tumour, the immune system has to be provided with sufficient amounts of these antigens in connection with strong immunostimulatory signals such as toll-like receptor (TLR) ligands. Tumour cells represent a convenient source of relevant tumour-associated antigens but can have suppressive properties. In this study, we explored how different forms of tumour cell material influence the activation of dendritic cells (DC), which play a crucial role in the induction of anti-tumour immune responses. We show that freeze-and-thaw-disrupted tumour cells inhibit DC activation in response to TLR stimulation, a phenomenon that is only partially seen with non-disrupted control cells. This suppression of DC stimulation is independent of tumour cell- and species-specific factors. We tested the hypothesis that phosphatidylserine on cells with disrupted membrane integrity mediates inhibition of TLR-induced DC activation. Our experimental evidence indicates that phosphatidylserine is not involved in the inhibition of TLR-mediated DC activation by freeze-and-thaw-disrupted cells. The inhibitory activity associated with disrupted tumour cells could explain why such preparations are less effective tumour vaccines than apoptotic tumour cells.

CD8alpha(+) DC are implicated as the principle DC subset for cross-presentation and cross-priming of cytotoxic CD8 T cell responses. In this study, we demonstrate another unique facet of the CD8alpha(+) DC and CD8 T cell relationship, by showing that CD8 T cells reciprocally activate CD8alpha(+) DC, but not CD8alpha(-) DC, for IL-12p70 production, the key Th1-promoting cytokine. This effect was observed during an antigen-specific interaction between DC and activated CD8 T cells, along with secondary TLR stimulation of DC by LPS. Activated CD8 T cells use a combination of IFN-gamma and CD40L, which is rapidly up-regulated post-stimulation, to prime DC for IL-12p70 production during an antigen-specific response. Our results suggest that the interaction between CD8alpha(+) DC and antigen-primed CD8 T cells may form an important component of Th1-mediated immunity through the induction of IL-12p70.

The present study addressed the modulatory role of CC chemokine receptor 4 (CCR4) in Toll-like receptor (TLR) 9-mediated innate immunity and explored the underlying molecular mechanisms. Our results demonstrated that CCR4-deficient mice were resistant to both septic peritonitis induced by cecal ligation and puncture (CLP) and CpG DNA/D-galactosamine-induced shock. In bone marrow-derived macrophages (BMMPhi) from CLP-treated CCR4-deficient mice, TLR9-mediated pathways of MAPK/AP-1, PI3K/Akt, and IkappaB kinase (IKK)/NF-kappaB were impaired compared to wild-type (WT) cells. While TLR9 expression was not altered, the intensity of internalized CpG DNA was increased in CCR4-deficient macrophages when compared to WT macrophages. Pharmacological inhibitor studies revealed that impaired activation of JNK, PI3K/Akt, and/or IKK/NF-kappaB could be responsible for decreased proinflammatory cytokine expression in CCR4-deficient macrophages. Interestingly, the CCR4-deficient BMMPhi exhibited an alternatively activated (M2) phenotype and the impaired TLR9-mediated signal transduction responses in CCR4-deficient cells were similar to the signaling responses observed in WT BMMPhi skewed to an alternatively activated phenotype. These results indicate that macrophages deficient in CCR4 impart a regulatory influence on TLR9-mediated innate immunity.

Mice deficient for the TLR adaptor molecule MyD88 succumb to a local infection with Leishmania (L.) major. However, the TLR(s) that contribute to the control of this intracellular parasite remain to be defined. Here, we show that TLR9 was required for the induction of IL-12 in bone marrow-derived DC by intact L. major parasites or L. major DNA and for the early IFN-gamma expression and cytotoxicity of NK cells following infection with L. major in vivo. During the acute phase of infection TLR9-/- mice exhibited more severe skin lesions and higher parasite burdens than C57BL/6 wild-type controls. Although TLR9 deficiency led to a transient increase of IL-4, IL-13 and arginase 1 mRNA and a reduced expression of iNOS at the site of infection and in the draining lymph nodes, it did not prevent the development of Th1 cells and the ultimate resolution of the infection. We conclude that TLR9 signaling is essential for NK cell activation, but dispensable for a protective T cell response to L. major in vivo.

Type I IFN are cytokines which play a central role in host resistance to viral or microbial infections and are important components linking innate and adaptive immunity. We and others have previously demonstrated that the production of IFN-beta by DC following bacterial infections or TLR triggering influences, in an autocrine manner, their maturation. In this study, we investigated whether IFN-beta release modulates the phenotype of the immature DC and their response to a subsequent TLR stimulation. The induction of CD86, HLA-DR, CD38 and B7H1 and the absence of CCR7 and CD83 expression upon IFN-beta treatment suggest that IFN-beta-primed DC remain at the site of infection acquiring an activated phenotype. These results prompted us to investigate the response of IFN-beta-primed DC to TLR stimulation. While IFN-beta pretreatment increases slightly the expression of maturation markers in TLR2- or TLR4-stimulated DC, it is able to modulate selectively the secretion of inflammatory and immuno-regulating cytokines. Interestingly, IL-27p28 subunit was induced by IFN-beta alone or during LPS-induced maturation of DC in a type I IFN-dependent manner through IFN regulatory factor-1 (IRF-1) activation. Taken together, our results shed light on the capacity of IFN-beta to finely tune DC response to invading pathogens.

Amebiasis is a human disease caused by the protozoan intestinal parasite Entamoeba histolytica. Vaccine development has focused on the parasite's surface galactose-N-acetyl-D-galactosamine inhibitable lectin (Gal-lectin) as a protective antigen. The Gal-lectin is immunogenic and has been shown to induce Th1 cytokines in vitro and in vivo. The immunological basis of the protective immune response elicited by the Gal-lectin is unknown. In this study, we investigated the response of BALB/c bone marrow-derived DC to E. histolytica Gal-lectin. Incubation of immature DC with Gal-lectin resulted in activation and maturation after 24 h. FACS analysis demonstrated an up-regulation of DC maturation markers CD80, CD86, CD40 and MHC class II upon exposure to Gal-lectin. The Gal-lectin also induced DC production of IL-12, indicating a Th1 response. Gal-lectin-activated DC were able to stimulate T cell proliferation in an allogeneic mixed leukocyte reaction and adoptive transfer of Gal-lectin-treated DC into naïve mice resulted in IFN-gamma-producing Gal-lectin-sensitized T cells. The activation of DC by Gal-lectin was mediated by MAPK and NF-kappaB. These findings indicate that E. histolytica Gal-lectin is a potent vaccine antigen capable of directly initiating DC maturation and activation characterized by Th1 cytokine production.

DC are believed to play important roles in the induction and regulation of immune responses in the liver, an organ implicated in peripheral tolerance. Since the liver is located downstream of the gut, it is constantly exposed to bacterial LPS. Our recent observations indicate that prior exposure to endotoxin modulates subsequent liver DC responses to this TLR4 ligand. In this study, we demonstrate that endotoxin modifies the capacity of mouse liver myeloid DC (MDC) activated by CpG (TLR9 ligand) to direct Th1-type responses. IL-12 production by liver MDC was significantly lower than that of spleen MDC following CpG or Imiquimod (R837; TLR7 ligand) activation in vitro. In addition, allogeneic T cells stimulated by CpG-activated liver MDC secreted significantly lower levels of IFN-gamma than T cells stimulated with CpG-activated spleen MDC. A similar effect on liver DC was observed in response to in vivo CpG administration. This effect may be explained by exposure of the DC to endotoxin, because LPS attenuated IL-12 production by CpG-stimulated liver MDC, both in vitro and in vivo. Moreover, attenuation of the response to CpG was not observed in liver MDC from TLR4-mutant (C3H/HeJ) mice, in which TLR4 signaling is impaired. These data suggest that endotoxin-induced 'cross-tolerance' to TLR ligands in liver DC may contribute to down-regulation of hepatic immune responses.

Adoptive dendritic cell (DC) immunotherapy provides a useful experimental tool to evaluate immunoregulation in vivo and has previously been successfully used to enhance host resistance in a variety of experimental models of leishmaniasis. Here, we used this approach to identify IL-6 and IL-12p40 as critical cytokines that cooperate to mediate host protection to Leishmania donovani but which act independently to regulate expansion of IL-10(+) CD4(+) T cells, shown here for the first time to be associated with this infection. Adoptive transfer of LPS-activated bone marrow-derived DC (BMDC) from wild-type mice was therapeutically beneficial and led to enhanced resistance as measured by spleen parasite burden. In contrast, IL-6- or IL-12p40-deficient BMDC had no protective benefit, indicating that production of both cytokines was essential for the therapeutic efficacy of DC. IL-10 production by CD25(-) FoxP3(-) IL-10(+) CD4(+) T cells is a strong correlate of disease progression, and BMDC from wild-type mice inhibited expansion of these cells. Strikingly, IL-12-deficient BMDC could also inhibit the expansion of this T cell population whereas IL-6-deficient BMDC could not, indicating that IL-6 played a key role in this aspect of DC function in vivo. Breadth of cytokine production is thus an important factor when considering strategies for DC-based interventions.

To investigate the role of interferon regulatory factor-1 (IRF-1) in the development of lupus nephritis, IRF-1(-/-) genotype mice were bred onto the MRL/lpJfas(lpr) (MRL/lpr) background. We examined kidney mesangial cell function and disease progression. Endpoints evaluated included inflammatory mediators, autoantibody production, immune complex deposition, renal pathology, T cell subset analysis, and duration of survival. Mesangial cells cultured from IRF-1(-/-) mice produced significantly lower levels of nitric oxide and IL-12 but not TNF-alpha when stimulated with LPS + IFN-gamma. IRF-1(-/-) mice showed less aggravated dermatitis compared to the wild-type mice. Anti-double-stranded DNA production and proteinuria were significantly decreased in IRF-1(-/-) mice compared to IRF-1(+/+) mice. IgG and C3 deposition as well as glomerulonephritis were decreased in IRF-1(-/-) mice at 26 wk of age compared to the IRF-1(+/+) mice. Splenic CD4- CD8- CD44+ T cells were decreased while CD4+ CD25+ T cells were increased in the IRF-1(-/-) mice when compared to IRF-1(+/+) mice. Survival rates (ED50) were 22 wk for IRF-1(+/+) mice and 45 wk for IRF-1(-/-) mice. These findings suggest an important role of IRF-1 in mediating renal disease in MRL/lpr mice.

Human newborns present impaired T helper type 1 cell responses, associated with a defect in the synthesis of IL-12 by dendritic cells (DC). IL-23 is a heterodimeric cytokine structurally related to IL-12, implicated in protective and autoimmune responses. We recently showed that upon activation neonatal T cells up-regulate a functional IL-23 receptor and that this cytokine polarizes the differentiation of naive T cells. We therefore investigated the capacity of neonatal DC to secrete IL-23. Lipopolysaccharide (LPS) stimulation induced the transcription of IL-23(p19) mRNA in both adult and neonatal DC, in sharp contrast to the repressed IL-12(p35) gene expression observed in neonatal cells. In comparison to adult DC, neonatal DC produced similar levels of IL-23 protein, in reponse to Toll-like receptor (TLR)-2- and TLR-3 ligands, and higher levels in response to TLR-4- or TLR-8 ligands. The same profile was observed in neonatal mononuclear cells. The supernatant of LPS-stimulated DC induced the secretion of IL-17 by polyclonally activated neonatal CD8(+) T cells, confirming the IL-23 bioactivity. Altogether, these observations strongly suggest that IL-23 could play a role in the immune system of human newborns. In particular, a functional IL-23/IL-17 axis might compensate a suboptimal IL-12/IFN-gamma pathway in early life.

T helper type 1 (Th1) cells perform a critical role in fighting intracellular organisms, and interleukin-12 (IL-12) is known to promote a Thl response. This study was conducted to identify whether an IL-12-independent Th1 reaction is induced by the varicella-zoster virus (VZV) in human beings. It was found that different intracellular microorganisms could induce IFNgamma but not IL-12 production. Induction of IFNgamma production by VZV was associated with IFNalpha production and phosphorylation of both the signal transducer and activator of transcription-1 (STAT-1) and STAT-4 in lymphocytes. In contrast, Bacillus Calmette-Guerin (BCG) induced IL-12 production in association with STAT-4 but not STAT-1 activation. Anti-IFNalpha but not anti-IL-12 antibodies blocked the VZV-induced Th1 polarization. A patient with an IL-12 receptor beta1 chain deficiency showed a normal VZV- but not a normal BCG-induced Th1 reaction, further supporting the concept of an IFNalpha-mediated, IL-12-independent Th1 reaction in response to certain intracellular infections. Identification of the early Th1 polarization induced by IFNalpha versus IL-12 in response to specific viruses may enable the development of better therapeutic strategies tailored to different infections.

We investigated here the potential role of Toll-like receptors (TLR) and the adaptor protein MyD88 in innate immunity responses to Cryptococcus neoformans, a pathogenic encapsulated yeast. Peritoneal macrophages from MyD88(-/-) or TLR2(-/-) mice released significantly less TNF-alpha, compared with wild-type controls, after in vitro stimulation with whole yeasts. In contrast, no differences in TNF-alpha release were noted between macrophages from C3H/HeJ mice, which have a loss of function mutation in TLR4, relative to C3H/HeN controls. When MyD88- or TLR2-deficient mice were infected with low doses of the H99 serotype A strain, all of the control animals, but none of MyD88(-/-) and only 38% of the TLR2(-/-) animals survived, in association with higher fungal burden in the mutant mice. Both MyD88(-/-) and TLR2(-/-) animals showed decreased TNF-alpha, IL-12p40 and/or IFN-gamma expression in various organs during infection. No difference in susceptibility to experimental cryptococcosis was found between C3H/HeJ mice and C3H/HeN controls. In conclusion, our data indicate that TLR2 and MyD88, but not TLR4, critically contribute to anti-cryptococcal defenses through the induction of increased TNF-alpha, IL-12 and IFN-gamma expression.

The IL-12/IFN-gamma axis is crucial for protective immunity to Mycobacterium in humans and mice. Our goal was to analyze the relative contribution of various human blood cell subsets and molecules to the production of, or response to IL-12 and IFN-gamma. We designed an assay for the stimulation of whole blood by live M. bovis Bacillus Calmette-Guerin (BCG) alone, or BCG plus IL-12 or IFN-gamma, measuring IFN-gamma and IL-12 levels. We studied patients with a variety of specific inherited immunodeficiencies resulting in a lack of leukocytes, or T, B, and/or NK lymphocytes, or polymorphonuclear cells, or a lack of expression of key molecules such as HLA class II, CD40L, NF-kappaB essential modulator (NEMO), and IL-1 receptor-associated kinase-4 (IRAK-4). Patients with deficiencies in IL-12p40, IL-12 receptor beta1 chain (IL-12Rbeta1), IFN-gammaR1, IFN-gammaR2, and STAT-1 were used as internal controls. We showed that monocytes were probably the main producers of IL-12, and that NK and T cells produced similar amounts of IFN-gamma. NEMO and IRAK-4 were found to be important for IL-12 production and subsequent IFN-gamma production, while a lack of CD40L or HLA class II had no major impact on the IL-12/IFN-gamma axis. The stimulation of whole blood by live BCG thus triggers the IL-12/IFN-gamma axis by an IRAK-4- and NEMO-dependent, non-cognate interaction between monocytes, NK, and T lymphocytes.

IFN-gamma plays a critical role in protection against Bordetella pertussis, but Th1 cells are only detectable after the infection has started to resolve, suggesting a protective role for innate IFN-gamma early in infection. Here, we demonstrate significant recruitment of NK cells and NKT cells into the lungs following respiratory challenge with B. pertussis. Furthermore, NK cells are the primary source of IFN-gamma in the lungs during the acute stage of infection. Stimulation of IFN-gamma production by NK cells was indirect through B. pertussis-activated IL-12 or IL-23 production by dendritic cells. Depletion of NK cells with anti-asialo ganglio-N-tetraosylceramide antibody resulted in a lethal infection, with enhancement of bacterial load in the lungs and dissemination of the bacteria to the liver via the blood. NK cell-depleted mice had significantly reduced B. pertussis-specific IFN-gamma and enhanced IgG1 and IL-5, but not IL-10 production, suggesting that regulatory T cells are induced simultaneously with Th1 cells, but the absence of NK cells resulted in enhancement of Th2-type responses. These findings suggest that NK cells confer resistance to B. pertussis by activating IL-12-mediated production of IFN-gamma, which enhances the anti-bacterial activity of macrophages, but also promotes the differentiation of Th1 cells., (Copyright 2004 Wiley-VCH Verlag GmbH & Co.)

Treatment with the hematopoietic growth factor Flt3 ligand (FL) increases DC numbers in neonatal mice and enhances their resistance against intracellular pathogens. Flow cytometric analysis showed the presence of conventional DC (cDC) and plasmacytoid pre-DC (pDC) in neonatal spleens from untreated and FL-treated mice. CD8alpha and MHC class II expression on cDC and pDC was higher on DC from FL-treated mice than on DC from control littermates. After FL treatment, two additional subpopulations of DC-lineage cells were found that were able to produce IL-12 and IFN-alpha. The IL-12 production of cDC from FL-treated animals was more than 50-fold increased and their ability to stimulate T cell proliferation was also increased. We conclude that the enhanced resistance against intracellular pathogens was due to increased numbers of DC-lineage cells and their increased ability to produce the essential cytokines.

RNA interference technology has been used to modulate dendritic cell (DC) function by targeting the expression of genes such as IL-12 and NF-kappa B. In this paper, we demonstrate that transfection of DC with IL-10-specific double strands of small interference RNA (siRNA) resulted in potent suppression of IL-10 gene expression without inducing DC apoptosis or blocking DC maturation. Inhibition of IL-10 by siRNA was accompanied by increased CD40 expression and IL-12 production after maturation, which endowed DC with the ability to significantly enhance allogeneic T cell proliferation. IL-10 siRNA transfection did not affect MHC class II, CD86, CD83, or CD54 expression in mature DC. To further test the ability of IL-10 siRNA-treated DC to induce a T cell response, naive CD4 T cells were stimulated by autologous DC pulsed with KLH. The results indicated that IL-10 siRNA-transfected DC enhanced Th1 responses by increasing IFN-gamma and decreasing IL-4 production. These findings suggest the potential for a novel immunotherapeutic strategy of using IL-10 siRNA-transfected antigen-presenting cells as vaccine delivery agents to boost the Th1 response against pathogens and tumors that are controlled by Th1 immunity.

Dendritic cells (DC) can induce Th1 cell differentiation by producing IL-12. In experimental infection with Leishmania major, DC could differently respond to infection and induce Th1 cells in C57BL/6 but not BALB/c mice, and thus determine the resistance or susceptibility of these mice. We characterized L. major antigen-containing DC in vivo in draining lymph nodes of both strains. Conventional experimental infection is shown to result in two waves of these DC and our data argue against a relevant genetic difference in the DC initiating the anti-parasite Th cell response in these mice. In both strains the first wave of DC presented L. major antigens but was not infected, produced IL-12 but induced disease-mediating Th2 cells upon adoptive transfer. In contrast to current belief, this response was therefore not initiated by infected DC, which were only detected in the second wave. The kinetics of the two waves suggests that DC turnover has an important impact on antigen presentation during infections with complex pathogens.

We show here that C1q suppresses IL-12p40 production in LPS-stimulated murine bone marrow-derived dendritic cells (BMDC). Serum IL-12p40 concentration of C1q-deficient mice was higher than that of wild-type mice after intraperitoneal LPS-injection. Because neither globular head of C1q (gC1q) nor collagen-like region of C1q (cC1q) failed to suppress LPS-induced IL-12p40 production, both gC1q and cC1q, and/or some specialized conformation of native C1q may be required for the inhibition. While C1q did not affect mRNA expression of Toll-like receptor 4 (TLR4), MD-2, and myeloid differentiation factor 88 (MyD88), BMDC treated with C1q showed the reduced activity of NF-kappaB and the delayed phosphorylation of p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinase after LPS-stimulation. CpG oligodeoxynucleotide-induced IL-12p40 and TNF-alpha production, another MyD88-dependent TLR-mediated signal, was also suppressed by C1q treatment. Therefore, C1q is likely to suppress MyD88-dependent pathway in TLR-mediated signals. In contrast, C1q failed to suppress colony formation of B cells responding to LPS or LPS-induced CD40 and CD86 expression on BMDC in MyD88-deficient mice, indicating that inhibitory effects of C1q on MyD88-independent pathways may be limited. Taken together, C1q may regulate innate and adaptive immune systems via modification of signals mediated by interactions between invading pathogens and TLR.

The majority of hepatitis C virus (HCV) infections become chronic, despite the presence of HCV-specific cellular and humoral immune responses. We have previously suggested that IL-10-secreting antigen-specific regulatory T cells may contribute to viral persistence, and demonstrate here that peripheral blood mononuclear cells (PBMC) from chronically HCV-infected patients secrete IL-10, but not IFN-gamma, in response to HCV nonstructural protein 4 (NS4). A neutralizing anti-IL-10 antibody restored this defective antigen-specific IFN-gamma production in vitro. Furthermore, PBMC from normal individuals secreted IL-10 in response to NS4, suggesting that cells of the innate immune system, in addition to T cells, produced IL-10 in the HCV-infected patients. Cell separation experiments revealed that the innate IL-10 was produced by blood monocytes, but not dendritic cells (DC). In addition, NS4 inhibited IL-12 production by PBMC in response to LPS and IFN-gamma, and Th1 responses to recall antigens in normal individuals. Furthermore, supernatants from NS4-stimulated monocytes inhibited LPS-induced maturation of DC and suppressed their capacity to stimulate proliferation and IFN-gamma production by allospecific T cells. Our data suggest that HCV subverts cellular immunity by inducing IL-10 and inhibiting IL-12 production by monocytes, which in turn inhibits the activation of DC that drive the differentiation of Th1 cells.

In allergy and asthma, the fine balance between the T helper (Th) 1, Th2 and T regulatory cytokine responses appears to be shifted towards Th2. Here, we report that synthetic lipopeptides which contain the typical lipid part of the lipoprotein of gram-negative bacteria stimulate a distinct regulatory cytokine pattern and inhibit several Th2 cell-related phenomena. The most potent analogue of synthetic lipopeptides, lipopeptide CGP 40774 (LP40) was not active in MyD88-deficient mice and stimulated Toll-like receptor (TLR)-2, but not TLR-4. LP40 potentiated the production of IFN-gamma and IL-10, but not IL-4 and IL-5 by human T cells. In addition, triggering of TLR-2 by lipopeptides promoted the in vitro differentiation of naive T cells towards IL-10- and IFN-gamma-producing T cells and suppressed IL-4 production by Th2 cells. Accordingly, LP40 inhibited IgE production induced by allergen, anti-IgD antibody, Nippostrongylus brasiliensis or murine acquired immunodeficiency virus. Furthermore, ovalbumin-induced lung eosinophilic inflammation was abolished and Schistosoma mansoni egg-induced granuloma size and eosinophil counts were suppressed in mice by LP40. These results demonstrate that stimulation of TLR-2 by lipopeptides represents a novel way for possible treatment of allergy and asthma by regulating the disrupted cytokine balance.

Active targeting of CpG-containing oligodeoxynucleotide (CpG-ODN) to macrophages was studied by incorporating it in mannose-coated liposomes, using visceral leishmaniasis as the model macrophage disease. Mannosylated liposomal CpG-ODN was more effective than liposomal or free CpG-ODN in inhibiting amastigote multiplication within macrophages. Moreover, in a 60-day mouse model of visceral leishmaniasis, complete elimination of spleen parasite burden was achieved by mannosylated liposomal CpG-ODN, compared to 62% and 81% parasite suppression by free and liposomal ODN, respectively, at a similar dose. Although in vitro exposure of CpG-ODN did not induce marked nitric oxide (NO) generation by macrophages, considerably enhanced amount of NO was generated by macrophages of CpG-ODN-treated animals. Their splenocytes secreted soluble factors required for the induction of NO generation, and the increased NO generation was paralleled by an increase in antileishmanial activity. Inducible NO generation was suppressed by treating splenocyte supernatants with anti-IFN-gamma or anti-IL-12 antibodies, whereas in vivo administration of these anti-cytokine Ab along with CpG-ODN reversed protection against infection. CpG-ODN treatment resulted in reduced levels of IL-4, but increased levels of IFN-gamma, IL-12 and inducible NO synthase in infected spleen cells, which was magnified by encapsulation in mannose-coated liposomes. This targeted treatment was not only curative, but it also imparted resistance to reinfection. These results represent a general approach for intracellular targeting of CpG-ODN, which effectively enhances its therapeutic potential in redirecting curative Th1 responses in Th2-driven disorders.

Neutrophils are generally considered to contribute to host defense through their potent microbicidal activity. However, there is accumulating evidence that neutrophils also have an important regulatory role in establishing the balance of Th1 and Th2 responses. This study investigated the role of neutrophils in defense against pulmonary Cryptococcus neoformans infection using neutrophil-depleted BALB/c mice generated by administering mAb RB6-8C5. Neutropenic mice with pulmonary infection survived significantly longer than control mice, but there was no difference between groups infected intravenously. On day 1 of infection, neutropenic mice had significantly smaller fungal burdens than control mice. On day 7, neutropenic mice had significantly higher lung concentrations of IL-10, TNF-alpha, IL-4, and IL-12 than control mice, but there was no difference in IFN-gamma and MCP-1 levels. Neutrophils influenced the outcome of cryptococcal infection in mice through mechanisms that did not involve a reduction in early fungal burden. The absence of neutrophils in lung tissue during the initial stages of infection appeared to alter the inflammatory response in a manner that was subsequently beneficial to the host. Higher levels of Th1- and Th2-associated cytokines in neutropenic mice could have simultaneously promoted a strong cellular response while reducing inflammatory damage to the lung. Our results support the emerging concept that neutrophils play an important function in modulating the development of the immune response.

CD11c(+) (F4/80(-) CD68(-)) dendritic cells (DC) in the colonic lamina propria (cLP) of normal and immunodeficient (RAG1(-/-)) C57BL/6 (B6) mice show high surface expression of MHC class I/II molecules and CD1d, and low surface expression of CD40, CD80, CD86 costimulator molecules. CD4(+) alpha beta T cells from normal or MHC class II-deficient B6 mice transferred into congenic RAG1(-/-) hosts induce a progressive, lethal colitis. Concomitant with colitis development, DC in the inflamed cLP increase in number and up-regulate surface expression of CD1d, MHC class II molecules and CD40, CD80, CD86 costimulator molecules. cLP DC from non-transplanted (healthy) and transplanted (diseased) mice produce similar amounts of IL-12 p70 and IL-10 in response to CD40 signaling, but the inducible IL-12 p40 release is 5-15-fold higher in mice with colitis than in non-transplanted mice. Binding of IL-12 p40 to p19 generates IL-23. Freshly isolated cLP lymphocytes (cLPL) from transplanted, diseased mice express 3-10-fold more p19 transcripts than cLPL from non-transplanted, healthy mice. p19 expression by cLPL is further up-regulated in response to CD40 ligation. Freshly isolated cLP DC from transplanted mice with colitis (but not from non-transplanted controls) stimulate IFN-gamma (but not IL-4 or IL-13) release by co-cultured NKT cells. Incolitis, DC accumulate in the cLP, show an activated surface phenotype, up-regulate IL-12 p40 and p19 expression, and 'spontaneously' stimulate NKT-like cells. cLP DC may be interesting targets for novel therapeutic approaches to modulate mucosal T cell responses in situ.

Prostaglandin (PG) D(2), and its metabolites, are known to be important mediators during acute and chronic inflammation. However, their functions during the early phases of the immune response are poorly documented. In the present study, we show that PGD(2 )inhibits, in a dose-dependent manner, the CD40- and LPS-induced secretion of the Th1-driving factor IL-12 by murine splenic dendritic cells (DC), the most potent antigen-presenting cells. The inhibition of IL-12 production is mediated only in part by the cell surface G alpha s protein-coupled D prostanoid receptor (termed DP1) but not by the G alpha i protein-coupled DP receptor, DP2. We show that recruitment of DP1 in DC results in the activation of a cyclic AMP/protein kinase A pathway that is partially responsible for the inhibition of IL-12 production. We also suggest that the DP1-independent effects exerted by PGD(2) on IL-12 production may be due to the action of ist PGJ(2), but not PGF(2)alpha, metabolites. Electrophoretic mobility shift assays demonstrated that PGD(2) affects NF-kappa B activation through (the) DP1-independent pathway(s). Together these data suggest that PGD(2), by interacting with DP1 and by binding to other target cellular proteins, may regulate immune responses by affecting IL-12 production in DC.

IL-12 and IL-4, respectively, dominate Th1 versus Th2 polarization. Additionally IL-4 can inhibit IL-12 p40 production in DC. Here we show that macrophages respond to bacterial CpG-DNA with IL-12 p40 production in an IL-4-sensitive manner. Analysis of the molecular mechanism of this inhibition shows that IL-4 acts by reducing the stability of IL-12 p40 mRNA as well as by affecting promoter activity. IL-4 did not affect early CpG-DNA-induced signal transduction. However, IL-4 reduced the activity of the IL-12 p40 promoter and when de novo transcription of IL-12 p40 mRNA was blocked, IL-4 led to acceleration of IL-12 p40 mRNA degradation. These data show that IL-4 regulates IL-12 p40 expression by influencing promoter activity and by interfering with mRNA stability.

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-gamma production by allogeneic CD4+ T cells. Furthermore, we found that PKC inhibition impaired LPS-induced I kappa B-alpha degradation and subsequent nuclear factor (NF)-kappa B activation in DC. LPS resulted in the phosphorylation of conventional alpha/beta and novel epsilon PKC isoforms in DC. Inhibition of LPS-induced PKC activity using pseudosubstrate peptides specific for PKC isoforms established that PKC epsilon but not PKC alpha/beta was involved in the production of IL-12 and TNF-alpha. Overall, these data provide evidence that PKC inhibition impairs LPS signaling in DC and identify PKC epsilon as a potential target for the inhibition of Toll-like receptor-4-mediated, IL-12-dependent Th1 type responses.

Bordetella pertussis toxin (PTX), a key component of acellular pertussis 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-alpha 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 that PTX directly stimulates MHC class II and costimulatory molecules up-regulation, cytokine synthesis and NF-kappa B 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.

Intracellular pathogens have developed strategies for evading elimination by the defenses of the host immune system. Here we describe an escape mechanism utilized by Mycobacterium tuberculosis that involves the interference with the generation of fully competent DC from monocytes. We show that monocytes infected with live M. tuberculosis differentiated into mature, CD83+ and CCR7+ DC (Mt-MoDC), but were characterized by a selective failure in the expression of the family of CD1 molecules. These cells also showed levels of MHC class II and CD80 (B7.1) that were reduced in comparison with LPS-matured DC. In addition, Mt-MoDC produced TNF-alpha and IL-10, but were unable to secrete IL-12. The generation of Mt-MoDC required the infection of monocytes with live M. tuberculosis, since infection with heat-killed bacteria partially abrogated the effects on monocyte differentiation. Interestingly, Mt-MoDC revealed an impaired antigen-presentation function as assessed by the reduced capability to induce proliferation of cord blood T lymphocytes. Further, naive T lymphocytes expanded by Mt-MoDC were unable to secrete cytokines, in particular IL-4 and IFN-gamma, suggesting that they could be ineffective in helping the macrophage-mediated killing of intracellular mycobacteria. Our results suggest that the interference with monocyte differentiation into fully competent DC is an evasion mechanism of M. tuberculosis that could contribute to its intracellular persistence by avoiding immune recognition.

Murine mature splenic DC with elevated intracellular glutathione, pretreated with IL-18, strikingly augmented the production of IFN-gamma in response to IL-12, whereas intracellular glutathione deprivation ablated this effect of IL-18. Likewise, macrophages with elevated intracellular glutathione augmented IFN-gamma production upon LPS or IL-12+IL-18 stimulation, whereas macrophages with reduced intracellular glutathione showed the reciprocal response. Under hypoxia, macrophages displayed a functional phenotype with decreased intracellular glutathione, i.e. decreased NO and IL-12, and elevated IL-10 production. However, mature DC and macrophages produced an elevated amount of IFN-gamma under hypoxia. Taken together, our results suggest that the intracellular redox status of DC and macrophages may play a pivotal role in local innate immunity, depending on local oxygen tension.

To clarify the functional consequences of adenine nucleotides action on human monocyte-derived dendritic cells (DC), we have systematically compared the effects of adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), an ATP analog active on the P2Y(11) receptor, on the responses to three DC stimuli, TNF-alpha, LPS, sCD40L, tested at various concentrations, using two different IL-12 assays. We observed that ATPgammaS potentiated the IL-12p40 release induced by TNF-alpha, but also by lipopolysaccharides (LPS) and soluble CD40 ligand (sCD40L). This potentiation was observed as long as the IL-12p40 concentration under agonist stimulation remained below a threshold value close to 10 ng/ml; inhibition was observed above this value. The combinations ATPgammaS-TNF-alpha and ATPgammaS-sCD40L were unable to induce detectable bioactive IL-12p70 production and at concentrations of LPS that induced a significant stimulation of IL-12p70, the effect of ATPgammaS was purely inhibitory. Our results also show that ATPgammaS synergized with LPS and sCD40L, but not TNF-alpha, to stimulate IL-10 production. In conclusion, we have clarified the discrepancies in the literature concerning the action of adenine nucleotides on DC and our study supports the concept that, like prostaglandin E(2) and other agents increasing cyclic AMP, they favor either a Th2 response or tolerance.

Like dendritic cells (DC), activated B cells are effective antigen-presenting cells (APC) for naïve CD4 cells due to their expression of MHC class II and multiple costimulatory molecules. We showed previously that CD4 cells primed in B cell-deficient micro MT) mice undergo more limited expansion than in normal animals after immunization with keyhole limpet hemocyanin. Here we report that in the absence of B cells, priming of effectors with the capacity to produce the Th2 cytokines, IL-4, IL-5 and IL-13, was profoundly reduced whereas the development of effectors that secrete the Th1 cytokine IFN-gamma was much less affected. A blockade of IL-12 reduced priming of IFN-gamma-secreting effectors but did not reverse the IL-4, IL-5, or IL-13 deficiency of the response. CD4 cell expansion and priming for Th2 cytokines in micro MT mice was reconstituted by adoptive transfer of activated splenic B cells, which were present throughout the primary response. However, transfer of splenic DC from either control or micro MT mice also supported development of Th2 cytokine responses, indicating that an APC deficit rather than a unique contribution of B cells accounted for diminished effector priming. We conclude that CD4 cell expansion must be sustained via APC for the development of Th2 cytokine-secreting effectors in vivo and that in responses to protein antigen, B cells can be a crucial population to serve in this role. The results suggest that the level of APC engagement can not only determine the extent of effector expansion, but also the overall Th1/Th2 cytokine balance.

The chemokine receptors CCR5 and CXCR3 have been implicated as playing a central role in directing a Th1 inflammatory response. Here, we investigated whether a synthetic CCR5 antagonist affects the process of T cell migration to sites of inflammation. Immunization of DBA/1 mice with type II collagen resulted in typical arthritis, which is associated with cellular infiltration. Treatment with a CCR5 antagonist strikingly affected the development of arthritis by reducing both incidence and severity of disease. There was no substantial difference between collagen-immunized mice with and without antagonist treatment in the induction of anti-collagen T cell responses and the capacity to produce IL-12. This endogenous IL-12 functioned to induce comparable levels of CCR5 in these two immunized groups of T cells. Whereas a massive infiltration of inflammatory cells including CCR5(+) T cells occurred in the joints of mice immunized without antagonist, cellular infiltration in the antagonist-treated group was only marginal. These results indicate that administration of a CCR5 antagonist inhibits the development of arthritis not by affecting the generation of collagen-sensitized T cells but by interfering with their migration to joint lesions.