Talin critically controls integrin-dependent cell migration, but its regulatory role in skin dendritic cells (DCs) during inflammatory responses has not been investigated. via direct interaction with MyD88 and PIP5K. Local production of PIP2 by PIP5K then recruited TIRAP to the preassembled complexes, which were required for TLR signalosome assembly during DC activation. Thus, talin1 regulates MyD88-dependent TLR signaling pathways in DCs through a novel mechanism with implications for antimicrobial and inflammatory immune responses. Graphical Abstract Open in a separate window Introduction Phagocytes, such as dendritic cells (DCs) and GCN5L macrophages, sense pathogens through various pattern recognition receptors (PRRs) but exert distinct effector functions (Eisenbarth, 2019; Kaplan, 2017; Malissen et al., 2014; Merad et al., 2008). In contrast to the direct destruction of phagocytosed microbes by macrophages, pathogens phagocytosed by DCs are processed and effectively used for T cell priming (Eisenbarth, 2019; Malissen et al., 2014; Merad et al., 2008). The ability of DCs to regulate immunity is strictly dependent on their maturation and activation state (Eisenbarth, 2019; Malissen et al., 2014; Worbs et al., 2017). Microbe recognition via PRRs activates and induces the maturation of DCs, which not only promotes their migratory ability to deliver pathogen-derived antigens to immune cells in nearby lymphoid organs, but also greatly enhances their T cell stimulatory capacity by up-regulating MHC and costimulatory molecules (Malissen et al., 2014). TLRs are perhaps the best characterized PRRs (Gay et al., 2014; Kawasaki and Kawai, 2014; ONeill and Bowie, 2007). Ligand binding to various TLRs either induces receptor dimerization or alters the conformation of preexisting dimers (Gay et al., 2014; Park et al., 2009), thereby triggering distinct signaling pathways involving two adaptor molecules, myeloid differentiation primary response protein 88 (MyD88) and TIR domainCcontaining adapter-inducing interferon- (TRIF), to promote NFB or IRF3/7 activation and proinflammatory cytokine production (Gay et al., 2014; Kawasaki and Kawai, 2014; ONeill and Bowie, 2007). Notably, all TLRs require the engagement of MyD88 to initiate downstream signaling pathways with the exception of TLR3, which only uses only TRIF (Gay et al., 2014; Kawasaki and Kawai, 2014; ONeill and Bowie, 2007). Mature DCs are fast migrating cells that are able to move through various tissues by switching between different migration modes (L?mmermann et al., 2008; PhiKan 083 hydrochloride Renkawitz et al., 2009; Worbs et al., 2017). For instance, following cutaneous insults, epidermal DCs called Langerhans cells (LCs) up-regulate 6 integrin (Price et al., 1997) to enable passage across the basement membrane into the dermis while concomitantly up-regulating chemokine receptors CXCR4 and CCR7 to promote migration to the skin-draining lymph nodes (sdLNs; Kabashima et al., 2007; Ohl et al., 2004). PhiKan 083 hydrochloride Whereas transmigration across the basement membrane is an integrin-dependent process (Gunawan et al., 2015; Loh et al., 2018; Worbs et al., 2017), the movement of migratory LCs (mLCs) or dermal DCs (mdDCs) from the dermis to sdLNs via interstitial tissues and lymphatic vessels is mainly achieved by integrin-independent actomyosin contractions (Loh et al., 2018; Worbs et al., 2017). Talin, a key adaptor molecule of integrin signaling, is required for integrin-dependent cell adhesion and migration, but not actomyosin contractility-driven cell migration (Calderwood et al., 2013; L?mmermann et al., 2008). Consistently, talin1-deficient B cells and T cells are defective in homing to LNs because of the failure of integrin-dependent extravasation from blood vessels (Manevich-Mendelson et al., 2010; Wernimont et al., 2011). A similar inability to migrate contributes to the defective infiltration of talin1-deficient neutrophils into the peritoneal cavity during peritonitis (Lim and Su, 2018; Yago et PhiKan 083 hydrochloride al., 2015). Integrins are likely to have broader physiological roles in addition to their well-described functions in cell adhesion/migration and cellCcell contacts. Several atypical functions of integrins have been reported. For example, v6 and v8 integrins bind to and activate pro-TGF- in a cytoskeletal force-dependent manner (Shi et al., 2011; Wang et al., 2017). However, alternative functions of talin, besides cell adhesion, have not been investigated. Here, we demonstrate for the first time that talin1 critically regulates DC activation by forming a preassembled TLR complex that is required for ligand-induced PhiKan 083 hydrochloride MyD88-dependent TLR signaling pathways. Talin1-deficient DCs showed compromised integrin-dependent and -independent migration as well as inflammatory cytokine production. Consequently, antimicrobial immune responses and tolerance induction against innocuous cutaneous allergen were compromised in mice with talin1-deficient DCs. Further molecular studies revealed that Talin1 links active integrins to TLRs via direct interaction with PhiKan 083 hydrochloride MyD88 while promoting TIR-domain containing adapter protein (TIRAP) recruitment by increasing local PIP2 concentration through interaction with PIP5K. In conclusion, we provide mechanistic insight into the novel mechanism of talin1-regulated TLR-mediated DC activation and migration both in vitro and in vivo. Results Talin1 regulates skin DC migration to sdLNs Because the migration of.