IL-22 is rapidly induced by Pathogen Recognition Receptors Stimulation in Bone-Marrow-derived Dendritic Cells in the Absence of IL-23

www.nature.com/scientificreports OPEN received: 18 March 2016 accepted: 04 September 2016 Published: 22 September 2016 IL-22 is rapidly induced by Pathogen Recognition Receptors Stimulation in Bone-Marrowderived Dendritic Cells in the Absence of IL-23 Silvia Fumagalli1,*, Anna Torri1,*, Angela Papagna1, Stefania Citterio2, Federica Mainoldi1 & Maria Foti1 In vertebrates, microorganisms are recognized by pathogen recognition receptors (PRRs). Exposure of immune cells to the ligands of these receptors activates intracellular signaling cascades that rapidly induce the expression of a variety of genes. Within these genes, the cytokines family plays a crucial function because of its role in adaptive immunity induction and in tissue-specific functional regulation, such as tissue repair and tissue homeostasis during steady state conditions. Within the myeloid compartment, dendritic cells (DCs) release a variety of inflammatory cytokines in response to microbes. In this study, we show that BMDCs release IL-22 directly upon PRRs activation without the need of IL-23 signaling as reported for other IL22-producing cells. Moreover, we demonstrate that cytokine IL-22 is rapidly released in a cell-specific manner as macrophages are not able to produce IL-22 through the same PRRs system. In addition, we characterize the intracellular signaling cascade required for IL-22 release in BMDCs. Myd88, MEK1/2, NFkb and AhR, but not p38, NFAT, and RORgt, were found to be involved in IL-22 regulation in DCs. Our study suggests that BMDCs possess a unique intracellular molecular plasticity which, once activated, directs different BMDCs functions in a cell-specific manner. Pathogen recognition receptors (PRRs) stimulation induces the release of critical pro-inflammatory cytokines necessary to activate potent immune responses. The induction of specific cytokines by cells of the innate immunity early during infection influences the magnitude of the nascent inflammatory response and directs the type of adaptive response that provides best sterilizing immunity. Therefore, understanding the molecular intracellular pathways to PRRs stimulation will provide information about the basic mechanisms of how immunity is developed. Moreover, it will also provide an understanding of target-specific pathways and help to control the inflammatory process itself and the nascent adaptive response required. Immunity to microbes has provided an extensive understanding of the complexity operating within the immune system. Microbial insults induce both common and specific activatory pathways in cells of the immune system as a result of a steady adaptation of the mutual interaction between microbes and their host. The intracellular pathways induced by PRRs have been intensively studied. The downstream transcription factors induced, such as NF-KB, AP-1, IRFs, and MAPKs signaling pathways, play a major role in cytokine gene expression in a number of cellular systems. However, the precise mechanism governing cytokine gene regulation in a cell-specific context following PRR activation is still poorly understood. Dendritic Cells (DCs) reside in the peripheral tissues and the secondary lymphoid organs in an immature state and act as a “sensors” of a number of “danger” and/or “self/non self ” signals. Particular interest has been directed towards the molecular profiles between dendritic Cells (DCs) and live bacteria1–4. Specific receptors, signaling pathways and response types under specific microbial interactions are currently being studied intensively. DCs are located at the anatomical interface where first microbial contact occurs and are therefore supposed to interact with microbial pathogens in 1 School of Medicine and Surgery, University of Milano-Bicocca, Milan, 20126, Italy. 2Department of Biotechnology and Bioscience, University of Milano-Bicocca, Milan, 20126, Italy. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to M.F. (email: ) Scientific Reports | 6:33900 | DOI: 10.1038/srep33900 1 www.nature.com/scientificreports/ the early stages. They also actively participate in the regulation of the inflammatory process together with lymphoid and myeloid cells and in the induction of the most appropriate adaptive response5,6. Analysis of genes induced in DCs in response to microbes is used to predict the impact of such interactions on DCs activation1,7. Our study shows that a common genetic program is induced in DCs by live Gram+ and Gram- bacteria, including the induction of cytokines of the IL10 family such as IL-22. IL-22 is a recently discovered cytokine which regulates innate responses at the epithelial barriers interface. Immune cells from both the innate and the adaptive immunity are able to release the cytokine IL-22, although with different efficiency8,9. IL-22 expression is restricted to immune cell lineages, whereas the functional IL22R receptor seems to be restricted to the stromal cell compartments of different tissues and organs, such as skin, pancreas, intestines, liver, lung, and kidney10,11. IL-22 is important for the induction of genes involved in tissue inflammation, immunosurveillance, and homeostasis11–20. In mice, IL22 is both expressed by innate and adaptive immune system cells. The innate cellular source of the cytokine includes NK cells, NKT cells, γδT cells, innate lymphoid cells (ILCs), and CD11c+ DCs14,16,21–23. Induction of IL-22 is mainly initiated by IL-23 released by antigen-presenting cells (APC) following PRR stimulation24–26. In DCs, cytokines are produced following the binding of PRRs or endogenous danger signals to TLRs, NOD-like receptors, and C-type lectin receptors, such as dectin-127,28. However, the induction of specific cytokines (e.g. IL-12 or IL-23) is regulated by TLR and C-type lectin receptors in DCs29. NFkB, IFN regulatory factor (IRF)-1, and IRF-8 signaling pathways play important roles in the expression of IL-1229–32. In contrast, induction of IL6- and IL-23 in DCs by dectin-1 agonist is mediated by signaling via Syk and CARD933. In this study, we show that BMDCs directly release IL-22 upon PRRs activation without the need of IL-23 signaling, in contrast to previously described cellular systems (T Cell, NK cells, LTi cells). We further identify signaling requirements and mechanisms of IL-22 release in BMDCs. We examine the role of a number of signaling molecules downstream to PRR activation and show that the absence of Myd88 or inhibition of ERK-MEK1/ JNK suppresses IL-22 production by BMDCs stimulation with TLRs and dectin-1 agonists. Similar patterns of cytokine regulation by the Myd88 and ERK-MEK/JNK signaling pathways are observed in splenic CD11c+ cells isolated from wild type mice. In contrast, IL-23 production was not affected by inhibition of ERK-MEK1/JNK activation. We conclude that multiple intracellular signaling pathways not operating on macrophages control IL-22 cytokine release in BMDCs with (...truncated)