ß-Catenin in dendritic cells exerts opposite functions in cross-priming and maintenance of CD8+ T cells through regulation of IL-10.

Recent studies have demonstrated that ß-catenin in DCs serves as a key mediator in promoting both CD4(+) and CD8(+) T-cell tolerance, although how ß-catenin exerts its functions remains incompletely understood. Here we report that activation of ß-catenin in DCs inhibits cross-priming of CD8(+) T cells by up-regulating mTOR-dependent IL-10, suggesting blocking ß-catenin/mTOR/IL-10 signaling as a viable approach to augment CD8(+) T-cell immunity. However, vaccination of DC-ß-catenin(-/-) (CD11c-specific deletion of ß-catenin) mice surprisingly failed to protect them against tumor challenge. Further studies revealed that DC-ß-catenin(-/-) mice were deficient in generating CD8(+) T-cell immunity despite normal clonal expansion, likely due to impaired IL-10 production by ß-catenin(-/-) DCs. Deletion of ß-catenin in DCs or blocking IL-10 after clonal expansion similarly led to reduced CD8(+) T cells, suggesting that ß-catenin in DCs plays a positive role in CD8(+) T-cell maintenance postclonal expansion through IL-10. Thus, our study has not only identified mTOR/IL-10 as a previously unidentified mechanism for ß-catenin-dependent inhibition of cross-priming, but also uncovered an unexpected positive role that ß-catenin plays in maintenance of CD8(+) T cells. Despite ß-catenin's opposite functions in regulating CD8(+) T-cell responses, selectively blocking ß-catenin with a pharmacological inhibitor during priming phase augmented DC vaccine-induced CD8(+) T-cell immunity and improved antitumor efficacy, suggesting manipulating ß-catenin signaling as a feasible therapeutic strategy to improve DC vaccine efficacy.

Langerin(neg) conventional dendritic cells produce IL-23 to drive psoriatic plaque formation in mice.

Psoriasis is an autoinflammatory skin disease of unknown etiology. Topical application of Aldara cream containing the Toll-like receptor (TLR)7 agonist Imiquimod (IMQ) onto patients induces flares of psoriasis. Likewise, in mice IMQ triggers pathological changes closely resembling psoriatic plaque formation. Key cytokines like IL-23 and type-I IFN (IFN-I), both being produced mainly by dendritic cells (DCs), have been implicated in psoriasis. Although plasmacytoid DCs (pDCs) are the main source of IFNα and thought to initiate disease, conventional DCs (cDCs) appear to maintain the psoriatic lesions. Any role of cDCs during lesion formation remains elusive. Here, we report that selective activation of TLR7 signaling specifically in CD11c(+) DCs was sufficient to induce psoriasiform skin disease in mice. Intriguingly, both pDCs and the IFN-I pathway were dispensable for the development of local skin inflammation. Selective TLR7 triggering of Langerin(+) DCs resulted in attenuated disease, whereas their depletion did not alter the severity of skin lesions. Moreover, after IMQ-painting, IL-23 was exclusively produced by Langerin(neg) DCs in vivo. In conclusion, TLR7-activated Langerin(neg) cDCs trigger psoriatic plaque formation via IL-23-mediated activation of innate IL-17/IL-22-producing lymphocytes, independently of pDCs or IFN-I. These results suggest therapeutic targeting of IL-23 production by cDCs to refine current treatment strategies for psoriasis.

Zinc signals promote IL-2-dependent proliferation of T cells.

Zinc signals, i.e. a change of the intracellular concentration of free zinc ions in response to receptor stimulation, are involved in signal transduction in several immune cells. Here, the role of zinc signals in T-cell activation by IL-2 was investigated in the murine cytotoxic T-cell line CTLL-2 and in primary human T cells. Measurements with the fluorescent dyes FluoZin-3 and Zinquin showed that zinc is released from lysosomes into the cytosol in response to stimulation of the IL-2-receptor. Activation of the ERK-pathway was blocked by chelation of free zinc with N,N,N',N'-tetrakis-2(pyridyl-methyl)ethylenediamine, whereas zinc was not required for STAT5 phosphorylation. In addition, the key signaling molecules MEK and ERK were activated in response to elevated free intracellular zinc, induced by incubation with zinc and the ionophore pyrithione. Downstream of ERK activation, ERK-specific gene expression of c-fos and IL-2-induced proliferation was found to depend on zinc. Further experiments indicated that inhibition of MEK and ERK-dephosphorylating protein phosphatases is the molecular mechanism for the influence of zinc on this pathway. In conclusion, an increase of cytoplasmic free zinc is required for IL-2-induced ERK signaling and proliferation of T cells.

Cadmium ions promote monocytic differentiation of human leukemia HL-60 cells treated with 1α,25-dihydroxyvitamin D3.

Cadmium exposure has multiple effects on the immune system. These can be stimulating, leading to improved clearance of infections, or inhibiting, increasing susceptibility toward infectious agents. One in vivo observation in cadmium-exposed individuals is increased monocyte numbers. Therefore, the objective of this study is to investigate the impact of cadmium on monocyte differentiation in the HL-60 model cell line. Administered alone, cadmium had no effect. However, cadmium amplified the expression of monocyte surface markers CD11b and CD14 when differentiation was induced by 1α,25-dihydroxyvitamin D3 (VD3). Furthermore, differentiation with VD3 in the presence of cadmium augmented key monocyte functions: the capacities to perform phagocytosis and generate an oxidative burst. One important signaling pathway required for monocyte differentiation involves extracellular signal-regulated kinase (ERK)1/2. Notably, cadmium induced ERK1/2 phosphorylation in HL-60 cells. Furthermore, U0126, which inhibits ERK1/2 phosphorylation by upstream MAPK/ERK kinases (MEK)1/2, reduced VD3-mediated differentiation and abrogated the effects of cadmium. In conclusion, cadmium can augment monocytic differentiation by activating ERK1/2 signaling, leading to increased generation of functional monocytes. These increased monocyte numbers could contribute to the impact of cadmium on the immune system owing to their role in the production of pro-inflammatory cytokines and activation of T-cells by antigen presentation.

Zinc signals are essential for lipopolysaccharide-induced signal transduction in monocytes.

Cytosolic alterations of calcium ion concentrations are an integral part of signal transduction. Similar functions have been hypothesized for other metal ions, in particular zinc (Zn(2+)), but this still awaits experimental verification. Zn(2+) is important for multiple cellular functions, especially in the immune system. Among other effects, it influences formation and secretion of pro-inflammatory cytokines, including TNF-alpha. Here we demonstrate that these effects are due to a physiological signaling system involving intracellular Zn(2+) signals. An increase of the intracellular zinc ion concentration occurs upon stimulation of human leukocytes with Escherichia coli, LPS, Pam(3)CSK(4), TNF-alpha, or insulin, predominantly in monocytes. Chelating this zinc signal with the membrane permeable zinc-specific chelator TPEN (N,N,N',N'-tetrakis-(2-pyridyl-methyl)ethylenediamine) completely blocks activation of LPS-induced signaling pathways involving p38 MAPK, ERK1/2, and NF-kappaB, and abrogates the release of proinflammatory cytokines, including TNF-alpha. This function of Zn(2+) is not limited to monocytes or even the immune system, but seems to be another generalized signaling system based on intracellular fluctuations of metal ion concentrations, acting parallel to Ca(2+).

Monitoring Skin Dendritic Cells in Steady State and Inflammation by Immunofluorescence Microscopy and Flow Cytometry.

Skin dendritic cells (DC) are strategically positioned at the body's second largest epithelial border to the environment. Hence they are the first antigen presenting cells that encounter invading pathogens and environmental antigens, including contact sensitizers and carcinogens penetrating the skin. Moreover, DC have the unique ability to induce immunity or tolerance and thus take center stage in regulating innate and adaptive immune responses. Skin DC can be divided into several phenotypically and functionally distinct subtypes. The three main subsets are Langerin+ epidermal Langerhans cells (LC) and Langerin+ as well as Langerinneg dermal DC. In the steady state skin DC form a dense network to survey the periphery for pathogens and harmful substances breaching the cutaneous barrier. During inflammation DC become rapidly activated and start their migration to skin-draining lymph nodes where they initiate antigen-specific T cell responses. The homeostasis and mobilization of DC in the skin can be visualized by immunofluorescent staining of epidermal and dermal sheet preparations or skin sections. Here, we describe in detail how inflammation can be induced in the skin with tape stripping or FITC painting and how the skin DC network can be monitored using immunofluorescence microscopy and flow cytometry.

Dendritic Cell-Specific Deletion of ß-Catenin Results in Fewer Regulatory T-Cells without Exacerbating Autoimmune Collagen-Induced Arthritis.

Dendritic cells (DCs) are professional antigen presenting cells that have the dual ability to stimulate immunity and maintain tolerance. However, the signalling pathways mediating tolerogenic DC function in vivo remain largely unknown. The ß-catenin pathway has been suggested to promote a regulatory DC phenotype. The aim of this study was to unravel the role of ß-catenin signalling to control DC function in the autoimmune collagen-induced arthritis model (CIA). Deletion of ß-catenin specifically in DCs was achieved by crossing conditional knockout mice with a CD11c-Cre transgenic mouse line. Bone marrow-derived DCs (BMDCs) were generated and used to study the maturation profile of these cells in response to a TLR2 or TLR4 ligand stimulation. CIA was induced by intra-dermal immunization with 100 µg chicken type II collagen in complete Freund's adjuvant on days 0 and 21. CIA incidence and severity was monitored macroscopically and by histology. The T cell profile as well as their cytokine production were analysed by flow cytometry. Lack of ß-catenin specifically in DCs did not affect the spontaneous, TLR2- or TLR4-induced maturation and activation of BMDCs or their cytokine production. Moreover, no effect on the incidence and severity of CIA was observed in mice lacking ß-catenin in CD11c+ cells. A decreased frequency of splenic CD3+CD8+ T cells and of regulatory T cells (Tregs) (CD4+CD25highFoxP3+), but no changes in the frequency of splenic Th17 (CCR6+CXCR3-CCR4+), Th2 (CCR6-CXCR3-CCR4+) and Th1 (CCR6-CXCR3+CCR4-) cells were observed in these mice under CIA condition. Furthermore, the expression of IL-17A, IL-17F, IL-22, IL-4 or IFNγ was also not affected. Our data indicate that ablation of ß-catenin expression in DCs did not alter the course and severity of CIA. We conclude that although deletion of ß-catenin resulted in a lower frequency of Tregs, this decrease was not sufficient to aggravate the onset and severity of CIA.

ß-catenin mediates tumor-induced immunosuppression by inhibiting cross-priming of CD8⁺ T cells.

Whereas CD8⁺ T cells are essential for anti-tumor immunity, tumors often evade CD8⁺ T cell surveillance by immunosuppression. As the initiators of antigen-specific immune responses, DCs are likely to play a central role in regulating the balance between immunity and tolerance to tumor antigens and are specialized in their ability to cross-present exogenous tumor antigens on MHC class I molecules to initiate CD8⁺ T cell immunity. However, it remains unclear whether and how tumors modulate DC functions to suppress CD8⁺ T cell responses. We have shown previously that ß-catenin signaling in DCs promotes DC-mediated CD8⁺ T cell tolerance. Here, we tested the hypothesis that ß-catenin in DCs mediates tumor-induced suppression of CD8⁺ T cell immunity by inhibiting the ability of DCs in cross-priming. ß-Catenin was activated in DCs by multiple tumors in vivo and in vitro. B16 melanoma-bearing mice, when vaccinated with DC-targeting anti-DEC-205 mAb fused with tumor antigens, exhibited dampened CD8⁺ immunity, similar to DC-ß-catenin(active) mice. DCs from DC-ß-catenin(active) and tumor-bearing mice were deficient in cross-priming, and antigen-specific CD8⁺ T cells primed in these mice resulted in dampened CD8⁺ memory responses. Importantly, DC-ß-catenin⁻/⁻ mice completely abrogate tumor-mediated inhibition of cross-priming, suggesting that tumor-induced inhibition of cross-priming is dependent on ß-catenin. Finally, enhancing cross-priming at the priming or recall phase rescued ß-catenin-suppressed CD8⁺ immunity in DC-ß-catenin(active) and tumor-bearing mice. Thus, ß-catenin-mediated inhibition of cross-priming represents a new and potentially general mechanism that tumors employ to achieve immunosuppression.

Cadmium ions induce monocytic production of tumor necrosis factor-alpha by inhibiting mitogen activated protein kinase dephosphorylation.

Cadmium ions (Cd(2+)) are carcinogenic and have cytotoxic effects in a variety of organisms. In addition to its direct cytotoxicity, Cd(2+) acts as an immunomodulator at sub-toxic concentrations. Among other influences Cd(2+) can induce inflammation, but the molecular basis for this effect is not well investigated. In this manuscript, we analyze the impact of Cd(2+) on monocytes/macrophages, which are potent producers of pro-inflammatory cytokines, finding that Cd(2+) treatment induced tumor necrosis factor (TNF)-alpha secretion. Based on the observation that another group IIb metal, zinc (Zn(2+)), has a physiological role in these events, we investigated if Cd(2+) acts on the same molecular targets. Like Zn(2+), Cd(2+) inhibits phosphatases, and hereby dephosphorylation of mitogen activated protein kinases (MAPK). Consequently, treatment of cells with Cd(2+) resulted in stimulation of ERK 1/2 and p38 MAPK phosphorylation. Furthermore, Cd(2+)-induced release of TNF-alpha from primary human monocytes was blocked by inhibitors for ERK 1/2 (U0126) and p38 MAPK (SB202190), demonstrating that MAPKs are involved in the induction of TNF-alpha by Cd(2+).