Jutta Horejs-Hoeck

Rapamycin Promotes Expansion of Functional CD4+CD25+FOXP3+ Regulatory T Cells of Both Healthy Subjects and Type 1 Diabetic Patients

CD4+CD25+FOXP3+ T regulatory cells (Tregs) are pivotal for the induction and maintenance of peripheral tolerance in both mice and humans. Rapamycin has been shown to promote tolerance in experimental models and to favor CD4+CD25+ Treg-dependent suppression. We recently reported that rapamycin allows in vitro expansion of murine CD4+CD25+FoxP3+ Tregs, which preserve their suppressive function. In the current study, we show that activation of human CD4+ T cells from healthy subjects in the presence of rapamycin leads to growth of CD4+CD25+FOXP3+ Tregs and to selective depletion of CD4+CD25− T effector cells, which are highly sensitive to the antiproliferative effect of the compound. The rapamycin-expanded Tregs suppress proliferation of both syngeneic and allogeneic CD4+ and CD8+ T cells. Interestingly, rapamycin promotes expansion of functional CD4+CD25+FOXP3+ Tregs also in type 1 diabetic patients, in whom a defect in freshly isolated CD4+CD25+ Tregs has been reported. The capacity of rapamycin to allow growth of functional CD4+CD25+FOXP3+ Tregs, but also to deplete T effector cells, can be exploited for the design of novel and safe in vitro protocols for cellular immunotherapy in T cell-mediated diseases.

IL-4 and IL-13 Induce SOCS-1 Gene Expression in A549 Cells by Three Functional STAT6-Binding Motifs Located Upstream of the Transcription Initiation Site

Proteins of the suppressors of cytokine signaling (SOCS) family have important functions as negative regulators of cytokine signaling. We show here that SOCS-1 expression can be induced in the human epithelial lung cell line A549 by IL-4 and IL-13. Analysis of reporter gene constructs under control of the SOCS-1 promoter provides evidence that IL-4- and IL-13-induced up-regulation is dependent on three IFN-γ-activated sequence motifs of the sequence TTC(N)4GAA, which is known for binding STAT6. The three motifs are situated close to each other ∼600 bp upstream of the transcriptional initiation site. When mutations were inserted into all three IFN-γ-activated sequence motifs at the same time, IL-4-IL-13-induced luciferase activity was abrogated. With single and double mutants, promoter activity was diminished in comparison with the wild-type promoter. STAT6 is therefore required for IL-4-IL-13-dependent SOCS-1 expression in A549 cells, and the three identified binding motifs cooperate to induce maximal transcription. EMSAs conducted with nuclear extracts of IL-4- and IL-13-stimulated A549 cells showed that STAT6 was able to bind to each of the three binding motifs. Finally, cotransfection of a SOCS-1 expression vector inhibited activation of SOCS-1 promoter luciferase constructs. Thus, SOCS-1 is able to autoregulate its expression via a negative feedback loop.

IL-4 induces expression of TARC/CCL17 via two STAT6 binding sites

A cardinal feature of allergic disorders and immune responses is enhanced leukocyte trafficking. This is largely orchestrated by chemokines. The CC chemokine thymus‐ and activation‐regulated chemokine (TARC/CCL17) selectively attracts Th2 cells via the G protein‐coupled chemokine receptor CCR4. We show here that TARC/CCL17 is expressed by human T cells upon stimulation with IL‐4. Mapping of the transcriptional start site revealed the presence of two putative STAT6 binding motifs in proximity to the start position. EMSA and chromatin immunoprecipitation experiments demonstrated that STAT6 was able to bind to both motifs. A fragment of the TARC/CCL17 promoter containing both sites was tested in reporter gene assays for IL‐4 inducibility. The promoter was inducible in a STAT6‐deficient cell line only after introduction of functional STAT6. When mutations were inserted into one of the STAT6 motifs, IL‐4‐induced promoter activation was reduced. With both sites mutated, inducibility was completely abrogated. These data demonstrate collectively that T cells serve as a source of TARC/CCL17 when stimulated with IL‐4 and that STAT6 is essential for this.

STAT6-dependent and -independent mechanisms in Th2 polarization.

Th2 cells play a key role in directing immune responses against helminths. Additionally, Th2 cells are crucial for many types of allergic reactions. Whereas the molecular mechanisms underlying the differentiation of other types of Th cells are well understood, Th2 differentiation is still a controversial topic. IL‐4 and its downstream transcription factor signal transducer and activator of transcription (STAT)6 are well‐known key mediators in Th2 differentiation. The fact that Th2 cells themselves are the most potent source of IL‐4 suggests that additional mechanisms promoting the initiation of Th2 differentiation exist. This article gives an overview on STAT6‐dependent and ‐independent mechanisms involved in the process of Th2 polarization, including Notch, mTORC2, IL‐2/STAT5, and Wnt. Furthermore, we emphasize the role of STAT6 not only as a transcriptional activator promoting Th2 development, but also in fine‐tuning alternative signaling pathways which are involved in the initiation of Th2 polarization.

Residual Endotoxin Contaminations in Recombinant Proteins Are Sufficient to Activate Human CD1c + Dendritic Cells

Many commercially available recombinant proteins are produced in Escherichia coli, and most suppliers guarantee contamination levels of less than 1 endotoxin unit (EU). When we analysed commercially available proteins for their endotoxin content, we found contamination levels in the same range as generally stated in the data sheets, but also some that were higher. To analyse whether these low levels of contamination have an effect on immune cells, we stimulated the monocytic cell line THP-1, primary human monocytes, in vitro differentiated human monocyte-derived dendritic cells, and primary human CD1c+ dendritic cells (DCs) with very low concentrations of lipopolysaccharide (LPS; ranging from 0.002–2 ng/ml). We show that CD1c+ DCs especially can be activated by minimal amounts of LPS, equivalent to the levels of endotoxin contamination we detected in some commercially available proteins. Notably, the enhanced endotoxin sensitivity of CD1c+ DCs was closely correlated with high CD14 expression levels observed in CD1c+ DCs that had been maintained in cell culture medium for 24 hours. When working with cells that are particularly sensitive to LPS, even low endotoxin contamination may generate erroneous data. We therefore recommend that recombinant proteins be thoroughly screened for endotoxin contamination using the limulus amebocyte lysate test, fluorescence-based assays, or a luciferase based NF-κB reporter assay involving highly LPS-sensitive cells overexpressing TLR4, MD-2 and CD14.

Suppressor of Cytokine Signaling 2 Is a Feedback Inhibitor of TLR-Induced Activation in Human Monocyte-Derived Dendritic Cells

Dendritic cells (DCs) are key players in initiating and directing the immune response. Therefore, their activation state and functional differentiation need to be tightly controlled. The activating stimuli and their signaling networks have long been an area of focus in DC research. Recent investigations have also shed light on the mechanisms of counterregulation and fine-tuning of DC functions. One class of proteins involved in these processes is the family of suppressors of cytokine signaling (SOCS), whose members were originally described as feedback inhibitors of cytokine-induced JAK/STAT signaling. Essential roles in DC function have been assigned to SOCS1 and SOCS3. In this article, we show that SOCS2 also is involved in DC regulation. In human and in murine DCs, SOCS2 is a highly TLR-responsive gene, which is expressed in a time-delayed fashion beginning 8 h after TLR ligation. Functionally, silencing of SOCS2 in DCs results in hyperphosphorylation of STAT3 at later time points. As a consequence, SOCS2-deficient DCs secrete increased amounts of the cytokines IL-1β and IL-10, both being transcriptional targets of STAT3. We propose a model in which SOCS2 acts as a negative regulator of TLR-induced DC activation. The delayed expression of SOCS2 provides a mechanism of late-phase counterregulation and limitation of inflammation-driving DC activity.

The diesel exhaust component pyrene induces expression of IL-8 but not of eotaxin

Environmental pollutants can influence the expression of immunoregulatory molecules and, in this way, promote allergies. The local synthesis of proinflammatory chemokines is an important aspect in the development of allergic airway inflammation. We have characterized the influence of pyrene, a polycyclic aromatic hydrocarbon (PAH) contained, for example, in diesel exhaust particles (DEP), on transcription and secretion of the chemokines interleukin-8 (IL-8) and eotaxin. Reporter genes under control of the respective promoters were tested in the human cell lines A549 and HeLa, mRNA production was assayed in A549 cells and protein production was measured by ELISA in cell supernatants from primary human fibroblasts. Pyrene content of cell supernatants was measured by analytical HPLC. Promoter activity, mRNA production and protein expression of IL-8 were increased by pyrene. The activating effect in reporter gene studies was abolished by mutating either an NF-kappaB or an AP-1 binding site in the IL-8 promoter. In contrast, pyrene showed no effect on transcription from the eotaxin promoter, despite the important role of this chemokine in asthma. Our data show that pyrene has specific effects on chemokine synthesis, which are not restricted to mediators primarily associated with atopic diseases. Pyrene also affected cells not derived from lung tissue, which suggests a broader immunoregulatory influence for this pollutant.

Modulation of T Lymphocyte Function by the Pregnane X Receptor

The pregnane X receptor (PXR) is a ligand-activated transcription factor regulating genes central to drug and hormone metabolism in the liver. Previous reports indicated that PXR is expressed in PBMC, but the role of PXR in immune cells remains unknown. In this paper, we report increased PXR expression in mouse and human T lymphocytes upon immune activation. Furthermore, pharmacologic activation of PXR inhibits T lymphocyte proliferation and anergizes T lymphocytes by decreasing the expression of CD25 and IFN-γ and decreasing phosphorylated NF-κB and MEK1/2. Although these effects are preceded by an increase of suppressor of cytokine signaling 1, a master switch for IFN-γ expression, in a PXR-dependent manner, T-bet expression remains unchanged. Conversely, PXR-deficient mice exhibit an exaggerated T lymphocyte proliferation and increased CD25 expression. Furthermore, PXR-deficient lymphocytes produce more IFN-γ and less of the anti-inflammatory cytokine IL-10. In summary, these results reveal a novel immune-regulatory role of PXR in T lymphocytes and identify suppressor of cytokine signaling 1 as an early signal in PXR-mediated T lymphocyte suppression.

Patulin influences the expression of Th1/Th2 cytokines by activated peripheral blood mononuclear cells and T cells through depletion of intracellular glutathione.

Patulin is a mold toxin secreted mainly by fungi of the Penicillium species. Exposure generally results from consumption of moldy fruits and fruit products. Since recent studies identified mold exposure as a risk factor for allergic diseases, we examined the effects of patulin on human peripheral blood mononuclear cells (PBMC) prepared from buffy coats of healthy donors. Cells were stimulated with CD3‐ and CD28‐specific antibodies in the presence or absence of patulin. Effects of patulin on PBMCs were evaluated by proliferation, viability assays, and cytokine ELISAs. The presence of 50 ng/mL patulin strongly decreased the amounts of several cytokines in the supernatant of stimulated PBMCs. This decrease in cytokine secretion was not due to cytotoxic effects of patulin. Moreover, the extent of the reduction of cytokine amounts was cytokine specific, affecting some (IL‐4, IL‐13, IFNγ, and IL‐10), but not others (IL‐8, IL‐5). We show that all effects could be abolished by adding thiol containing compounds. A depletion of intracellular GSH could be measured after incubation of cells with patulin. Taken together, our data indicate that patulin modulates the functional activation of PBMCs with respect to proliferation and cytokine secretion patterns by depletion of intracellular GSH. The depletion of intracellular glutathione may influence the balance between Th1 and Th2 cells and have implications for allergic diseases.

Dendritic Cells Activated by IFN-γ/STAT1 Express IL-31 Receptor and Release Proinflammatory Mediators upon IL-31 Treatment

IL-31 is a T cell-derived cytokine that signals via a heterodimeric receptor composed of IL-31Rα and oncostatin M receptor β. Although several studies have aimed to investigate IL-31–mediated effects, the biological functions of this cytokine are currently not well understood. IL-31 expression correlates with the expression of IL-4 and IL-13 and is associated with atopic dermatitis in humans, indicating that IL-31 is involved in Th2-mediated skin inflammation. Because dendritic cells are the main activators of Th cell responses, we posed the question of whether dendritic cells express the IL-31R complex and govern immune responses triggered by IL-31. In the current study, we report that primary human CD1c+ as well as monocyte-derived dendritic cells significantly upregulate the IL-31Rα receptor chain upon stimulation with IFN-γ. EMSAs, chromatin immunoprecipitation assays, and small interfering RNA-based silencing assays revealed that STAT1 is the main transcription factor involved in IFN-γ–dependent IL-31Rα expression. Subsequent IL-31 stimulation resulted in a dose-dependent release of proinflammatory mediators, including TNF-α, IL-6, CXCL8, CCL2, CCL5, and CCL22. Because these cytokines are crucially involved in skin inflammation, we hypothesize that IL-31–specific activation of dendritic cells may be part of a positive feedback loop driving the progression of inflammatory skin diseases.