Summary The skin is a site of constant dialogue between the immune system and commensal bacteria. However, the molecular mechanisms that allow us to tolerate the presence of skin commensals without eliciting destructive inflammation are unknown. Using a model system to study the antigen-specific response to S. epidermidis, we demonstrated that skin colonization during a defined period of neonatal life was required to establish immune tolerance to commensal microbes. This crucial window was characterized by an abrupt influx of highly activated regulatory T (Treg) cells into neonatal skin. Selective inhibition of this Treg cell wave completely abrogated tolerance. Thus, the host-commensal relationship in the skin relied on a unique Treg cell population that mediated tolerance to bacterial antigens during a defined developmental window. This suggests that the cutaneous microbiome composition in neonatal life is crucial in shaping adaptive immune responses to commensals, and disrupting these interactions may have enduring health implications.

T cell activation and function require a structured engagement of antigen-presenting cells. These cell contacts are characterized by two distinct dynamics in vivo: transient contacts resulting from promigratory junctions called immunological kinapses or prolonged contacts from stable junctions called immunological synapses. Kinapses operate in the steady state to allow referencing to selfpeptide-MHC (pMHC) and searching for pathogen-derived pMHC. Synapses are induced by T cell receptor (TCR) interactions with agonist pMHC under specific conditions and correlate with robust immune responses that generate effector and memory T cells. High-resolution imaging has revealed that the synapse is highly coordinated, integrating cell adhesion, TCR recognition of pMHC complexes, and an array of activating and inhibitory ligands to promote or prevent T cell signaling. In this review, we examine the molecular components, geometry, and timing underlying kinapses and synapses. We integrate recent molecular and physiological data to provide a synthesis and suggest ways forward.

Natural killer (NK) cells are sentinel components of the innate response to pathogens, but the cell types, pathogen recognition receptors, and cytokines required for their activation in vivo are poorly defined. Here, we investigated the role of plasmacytoid dendritic cells (pDCs), myeloid DCs (mDCs), Toll-like receptors (TLRs), and of NK cell stimulatory cytokines for the induction of an NK cell response to the protozoan parasite Leishmania infantum. In vitro, pDCs did not endocytose Leishmania promastigotes but nevertheless released interferon (IFN)-α/β and interleukin (IL)-12 in a TLR9-dependent manner. mDCs rapidly internalized Leishmania and, in the presence of TLR9, produced IL-12, but not IFN-α/β. Depletion of pDCs did not impair the activation of NK cells in L. infantum–infected mice. In contrast, L. infantum–induced NK cell cytotoxicity and IFN-γ production were abolished in mDC-depleted mice. The same phenotype was observed in TLR9−/− mice, which lacked IL-12 expression by mDCs, and in IL-12−/− mice, whereas IFN-α/β receptor−/− mice showed only a minor reduction of NK cell IFN-γ expression. This study provides the first direct evidence that mDCs are essential for eliciting NK cell cytotoxicity and IFN-γ release in vivo and demonstrates that TLR9, mDCs, and IL-12 are functionally linked to the activation of NK cells in visceral leishmaniasis.

Mice deficient for the TLR adaptor molecule MyD88 succumb to a local infection with Leishmania (L.) major. However, the TLR(s) that contribute to the control of this intracellular parasite remain to be defined. Here, we show that TLR9 was required for the induction of IL-12 in bone marrow-derived DC by intact L. major parasites or L. major DNA and for the early IFN-c expression and cytotoxicity of NK cells following infection with L. major in vivo. During the acute phase of infection TLR9 -/-mice exhibited more severe skin lesions and higher parasite burdens than C57BL/6 wild-type controls. Although TLR9 deficiency led to a transient increase of IL-4, IL-13 and arginase 1 mRNA and a reduced expression of iNOS at the site of infection and in the draining lymph nodes, it did not prevent the development of Th1 cells and the ultimate resolution of the infection. We conclude that TLR9 signaling is essential for NK cell activation, but dispensable for a protective T cell response to L. major in vivo.

The early systemic production of interferon (IFN)-αβ is an essential component of the antiviral host defense mechanisms, but is also thought to contribute to the toxic side effects accompanying gene therapy with adenoviral vectors. Here we investigated the IFN-αβ response to human adenoviruses (Ads) in mice. By comparing the responses of normal, myeloid (m)DC- and plasmacytoid (p)DC-depleted mice and by measuring IFN-αβ mRNA expression in different organs and cells types, we show that in vivo, Ads elicit strong and rapid IFN-αβ production, almost exclusively in splenic mDCs. Using knockout mice, various strains of Ads (wild type, mutant and UV-inactivated) and MAP kinase inhibitors, we demonstrate that the Ad-induced IFN-αβ response does not require Toll-like receptors (TLR), known cytosolic sensors of RNA (RIG-I/MDA-5) and DNA (DAI) recognition and interferon regulatory factor (IRF)-3, but is dependent on viral endosomal escape, signaling via the MAP kinase SAPK/JNK and IRF-7. Furthermore, we show that Ads induce IFN-αβ and IL-6 in vivo by distinct pathways and confirm that IFN-αβ positively regulates the IL-6 response. Finally, by measuring TNF-α responses to LPS in Ad-infected wild type and IFN-αβR−/− mice, we show that IFN-αβ is the key mediator of Ad-induced hypersensitivity to LPS. These findings indicate that, like endosomal TLR signaling in pDCs, TLR-independent virus recognition in splenic mDCs can also produce a robust early IFN-αβ response, which is responsible for the bulk of IFN-αβ production induced by adenovirus in vivo. The signaling requirements are different from known TLR-dependent or cytosolic IFN-αβ induction mechanisms and suggest a novel cytosolic viral induction pathway. The hypersensitivity to components of the microbial flora and invading pathogens may in part explain the toxic side effects of adenoviral gene therapy and contribute to the pathogenesis of adenoviral disease.

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Certain strains ofClostridium difficile is the causative pathogen of antibioticassociated diarrhea and pseudomembranous colitis (10, 14). It produces two major protein toxins (toxins A and B), which are the prototypes of the family of large clostridial cytotoxins. Both toxins inhibit the functions of Rho GTPases by monoglucosylation. Beside toxins A and B, some strains of C. difficile produce a binary ADP-ribosylating toxin (CDT) which modifies actin (22). The pathogenic role of CDT in diseases induced by C. difficile is not clear, but about 6 to 12.5% of strains isolated from patients with enteritis contained CDT genes (20,26).The binary CDT is composed of the enzymatic component, CDTa (48 kDa), and the binding and translocation component, CDTb (94 kDa), which mediates cell entry of CDTa (19). CDT belongs to the group of binary actin ADP-ribosylating toxins (4), which can be divided into C2 toxin and iota toxin subfamilies (2, 5, 21). The subfamilies differ with respect to their actin substrate specificities (24). Clostridium botulinum C2 toxin ADP-ribosylates only ␤/␥-nonmuscle actin and ␥-smooth muscle actin, whereas iota-like toxins ADP-ribosylate all actin isoforms, including ␣-actin (16). Furthermore, whereas the binding components of the iota toxin subfamily, including those of iota toxin, Clostridium spiroforme toxin, and CDTa, are interchangeable, no functional complementation between the binding components and the enzymatic components of C2 toxin and the toxins of the iota subfamily was observed (12, 21).Here we studied the enzyme component of CDT and characterized its enzyme activity. We show that the N-terminal part of CDTa is responsible for interaction with the binding component, whereas the C-terminal part harbors transferase activity. For delivery of CDTa into cells we used the Ib binding and translocation component from iota toxin, because CDTb was not expressed as a recombinant protein in Escherichia coli. Recently, we characterized the catalytic center of the ADPribosyltransferase C2I and identified several amino acid residues essential for the transferase activity (8). Here we studied the functional roles of several amino acid residues of CDTa suggested to be conserved among the actin ADP-ribosylating toxins. Moreover, we compared the minimal structural requirements of CDTa with those of the enzyme components of iota toxin (Ia) and C2 toxin (C2I).

Nonetheless, INR should be less than 4, local hemostatic measures are of high importance and patients need to be instructed and closely monitored as minor bleedings might occur more often in OAT patients.