PD-L1 expression on tolerogenic APCs is controlled by STAT-3.

During infection, TLR agonists are released and trigger mature as well as differentiating innate immune cells. Early encounter with TLR agonists (R848; LPS) blocks conventional differentiation of CD14(+) monocytes into immature dendritic cells (iDCs) resulting in a deviated phenotype. We and others characterized these APCs (TLR-APC) by a retained expression of CD14 and a lack of CD1a. Here, we show in addition, expression of programmed death ligand-1 (PD-L1). TLR-APCs failed to induce T-cell proliferation and furthermore were able to induce CD25(+) Foxp3(+) T regulatory cells (Tregs). Since PD-L1 is described as a key negative regulator and inducer of tolerance, we further analyzed its regulation. PD-L1 expression was regulated in a MAPK/cytokine/STAT-3-dependent manner: high levels of IL-6 and IL-10 that signal via STAT-3 were produced by TLR-APCs. Blocking of STAT-3 activation prevented PD-L1 expression. Moreover, chromatin immunoprecipitation revealed direct binding of STAT-3 to the PD-L1 promoter. Those findings indicate a pivotal role of STAT-3 in regulating PD-L1 expression. MAPKs were indirectly engaged, as blocking of p38 and p44/42 MAPKs decreased IL-6 and IL-10 thus reducing STAT-3 activation and subsequent PD-L1 expression. Hence, during DC differentiation TLR agonists induce a STAT-3-mediated expression of PD-L1 and favor the development of tolerogenic APCs.

Regulation of Toll-like receptor 4-mediated immune responses through Pasteurella multocida toxin-induced G protein signalling.

BACKGROUND: Lipopolysaccharide (LPS)-triggered Toll-like receptor (TLR) 4-signalling belongs to the key innate defence mechanisms upon infection with Gram-negative bacteria and triggers the subsequent activation of adaptive immunity. There is an active crosstalk between TLR4-mediated and other signalling cascades to secure an effective immune response, but also to prevent excessive inflammation. Many pathogens induce signalling cascades via secreted factors that interfere with TLR signalling to modify and presumably escape the host response. In this context heterotrimeric G proteins and their coupled receptors have been recognized as major cellular targets. Toxigenic strains of Gram-negative Pasteurella multocida produce a toxin (PMT) that constitutively activates the heterotrimeric G proteins Gαq, Gα13 and Gαi independently of G protein-coupled receptors through deamidation. PMT is known to induce signalling events involved in cell proliferation, cell survival and cytoskeleton rearrangement. RESULTS: Here we show that the activation of heterotrimeric G proteins through PMT suppresses LPS-stimulated IL-12p40 production and eventually impairs the T cell-activating ability of LPS-treated monocytes. This inhibition of TLR4-induced IL-12p40 expression is mediated by Gαi-triggered signalling as well as by Gßγ-dependent activation of PI3kinase and JNK.Taken together we propose the following model: LPS stimulates TLR4-mediated activation of the NFĸB-pathway and thereby the production of TNF-α, IL-6 and IL-12p40. PMT inhibits the production of IL-12p40 by Gαi-mediated inhibition of adenylate cyclase and cAMP accumulation and by Gßγ-mediated activation of PI3kinase and JNK activation. CONCLUSIONS: On the basis of the experiments with PMT this study gives an example of a pathogen-induced interaction between G protein-mediated and TLR4-triggered signalling and illustrates how a bacterial toxin is able to interfere with the host's immune response.

IL-1ß As Mediator of Resolution That Reprograms Human Peripheral Monocytes toward a Suppressive Phenotype.

During infection pathogen-associated molecular patterns activate immune cells to initiate a cascade of reactions leading to inflammation and the activation of the adaptive immune response culminating in the elimination of foreign pathogens. However, shortly after activation of the host defense machinery, a return to homeostasis is preferred to prevent inflammation-induced tissue damage. This switch from the initial immunogenic to the subsequent tolerogenic phase after clearance of the infection can be mediated through highly plastic peripheral monocytes. Our studies reveal that an early encounter with toll-like receptor 7/8-ligand R848 mediates a strong pro-inflammatory monocytic phenotype that primes its own reprogramming toward an immunosuppressive one. Previously, we showed that these R848-treated antigen-presenting cells (APCs) fail to activate allogeneic T cells and induce regulatory T cells (Tregs) through signal transducer and activator of transcription 3 (STAT3)-dependent PD-L1. Here, we further demonstrate that R848-treated APCs suppress CD3/CD28-mediated and dendritic cell-mediated T cell activation and that adenosine and indoleamine 2,3-dioxygenase/kynurenin pathways are involved in tolerance induction. Reprogramming of monocytes after R848 stimulation requires the pro-inflammatory cytokine IL-1ß and a boosted IL-6 release. The subsequent autocrine prolonged activation of STAT3 induces direct upregulation of tolerogenic factors which finally downregulate proliferation of activated T cells and mediate Tregs. Thereby our study suggests that inflammatory cytokines, such as IL-1ß and IL-6, should be considered as mediators of resolution of inflammation.

Bifunctional oligodeoxynucleotide/antagomiR constructs: evaluation of a new tool for microRNA silencing.

MicroRNAs (miRNAs) are fine-tuners in cellular processes, including those of the immune response. To study their functions and effects in immune cells, it is necessary to achieve specific silencing of individual miRNAs. To date, introduction of antisense microRNAs (antagomiRs) into primary cells is based on electroporation, lipofection, and viral vectors. However, these techniques often compromise viability, proliferative capacity, and differentiation. Furthermore, efficiency varies depending on the cell type and some are not suitable for in vivo approaches. To overcome these limitations we exploited the property of phosphorothioate (PTO)-modified DNA oligodeoxynucleotides (ODN) to enter cells with high efficacy: we developed and evaluated ODN/antagomiR constructs that consist of a PTO-ODN carrier covalently linked to a fully methylated antagomiR RNA sequence. Using these constructs, we achieved transfection efficiency of approximately 99% in leukocytes-in particular, in B lymphocytes that are hard to transfect with other methods. Our data demonstrate that miRNA silencing by the antagomiR portion of the constructs was specific and efficient, which could be further confirmed by an increase in target protein under silencing conditions. The constructs were successfully tested in human B cells, plasmacytoid dendritic cells, monocytes, and monocyte-derived dendritic cells, thus demonstrating their versatility. Moreover, introduction of stimulatory CpG sequences into the ODN portion conveys immune stimulatory quality when intended. Thus, bifunctional ODN/antagomiR constructs represent a highly efficient, versatile, and easy-to-handle tool to manipulate cellular miRNA expression levels and to allow the subsequent investigation of specific miRNA functions.