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 1 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 1 Foxp3 1 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.Keywords: DC . PD-L1 . STAT-3 . Tolerance . TLR See accompanying Commentary by Sumpter and ThomsonSupporting Information available online IntroductionDC are initiators and modulators of the adaptive immune response [1]. They are able to induce T-cell activation as well as T-cell tolerance. During infection, DCs are confronted with pathogen-associated molecular patterns (PAMP), which in turn trigger effector functions in innate immune cells. For example, immature DCs (iDCs) generated from monocytes by in vitro culture with GM-CSF and IL-4 (G4) mature and become fully activated upon stimulation with TLR agonists. Mature DCs (mDCs) in turn activate most efficiently naïve T cells [2]. However, during infection induction of inhibitory immune pathways can also be observed [3,4]. Here, we investigate an alternative TLR-induced APC phenotype, which inhibits immune reactivity. It has been shown that encounter of monocytes with LPS during the very beginning of the differentiation process blocks conventional differentiation to iDCs. A phenotypically distinct APC type (TLR-APC) is generated, characterized by a [5][6][7]. Activation of p38 MAPK, the secretion of IL-10 and the inactivation of ERK and NF-kB [7] have been correlated with the generation of TLR-APCs. LPS-treated cells showed in addition an intense STAT-3 phosphorylation. Differentiation processes of DCs are plastic and can be influenced by various factors, e.g. cytokines. Many cytokines mediate their cellular response via the JAK/STAT signaling pathway thereby controlling the status of transcription and cellular differentiation. For instance, during the maturation of DCs, a switch occurs from constit...

Histone deacetylase (HDAC) inhibitors (HDACi) are clinically approved anticancer drugs that have important immune-modulatory properties. We report the surprising finding that HDACi promote LPS-induced IL-1β processing and secretion in human and murine dendritic cells and murine macrophages. HDACi/LPS-induced IL-1β maturation and secretion kinetics differed completely from those observed upon inflammasome activation. Moreover, this pathway of IL-1β secretion was dependent on caspase-8 but was independent of the inflammasome components NACHT, LRR, and PYD domains-containing protein 3, apoptosis-associated speck-like protein containing a carboxyl-terminal caspase-recruitment domain, and caspase-1. Genetic studies excluded HDAC6 and HDAC10 as relevant HDAC targets in this pathway, whereas pharmacological inhibitor studies implicated the involvement of HDAC11. Treatment of mice with HDACi in a dextran sodium sulfate–induced colitis model resulted in a strong increase in intestinal IL-1β, confirming that this pathway is also operative in vivo. Thus, in addition to the conventional inflammasome-dependent IL-1β cleavage pathway, dendritic cells and macrophages are capable of generating, secreting, and processing bioactive IL-1β by a novel, caspase-8–dependent mechanism. Given the widespread interest in the therapeutic targeting of IL-1β, as well as the use of HDACi for anti-inflammatory applications, these findings have substantial clinical implications.

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-1β 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-1β and IL-6, should be considered as mediators of resolution of inflammation.