Uptake of synthetic naked RNA by skin-resident dendritic cells via macropinocytosis allows antigen expression and induction of T-cell responses in mice.

Intradermal administration of antigen-encoding RNA has entered clinical testing for cancer vaccination. However, insight into the underlying mechanism of RNA uptake, translation and antigen presentation is still limited. Utilizing pharmacologically optimized naked RNA, the dose-response kinetics revealed a rise in reporter signal with increasing RNA amounts and a prolonged RNA translation of reporter protein up to 30 days after intradermal injection. Dendritic cells (DCs) in the dermis were shown to engulf RNA, and the signal arising from the reporter RNA was significantly diminished after DC depletion. Macropinocytosis was relevant for intradermal RNA uptake and translation in vitro and in vivo. By combining intradermal RNA vaccination and inhibition of macropinocytosis, we show that effective priming of antigen-specific CD8(+) T-cells also relies on this uptake mechanism. This report demonstrates that direct antigen translation by dermal DCs after intradermal naked RNA vaccination is relevant for efficient priming of antigen-specific T-cells.

Animal model for cutaneous leishmaniasis.

Using cutaneous leishmaniasis of mice, the existence of so-called T helper (Th) cells type 1 and type 2 had been identified more than 20 years ago. Nowadays, it is well accepted that additional T cell populations as well as B cell-mediated immunity is required for immunity against Leishmania major. Finally, using inbred mouse strains, the relevance of genetical factors that influence anti-pathogen immunity as well as elements of the skin-immune system have been identified. This protocol describes a model for murine experimental leishmaniasis that tries to mimic natural parasite transmission by several means: (1) utilization of only infectious-stage parasites that are found in sand fly saliva, (2) intradermal inoculation, and (3) infection with only 1,000 parasites similar to the numbers inoculated by an infected sand fly.

Dendritic cells in Leishmania major infections: mechanisms of parasite uptake, cell activation and evidence for physiological relevance.

Leishmaniasis is one of the most important infectious diseases worldwide; a vaccine is still not available. Infected dendritic cells (DC) are critical for the initiation of protective Th1 immunity against Leishmania major. Phagocytosis of L. major by DC leads to cell activation, IL-12 release and (cross-) presentation of Leishmania antigens by DC. Here, we review the role of Fcγ receptor- and B cell-mediated processes for parasite internalization by DC. In addition, the early events after parasite inoculation that consist of mast cell activation, parasite uptake by skin-resident macrophages (MΦ), followed by neutrophil and monocyte immigration and DC activation are described. All these events contribute significantly to antigen processing in infected DC and influence resulting T cell priming in vivo. A detailed understanding of the role of DC for the development of efficient anti-Leishmania immunity will aid the development of potent anti-parasite drugs and/or vaccines.

Dendritic cells ameliorate autoimmunity in the CNS by controlling the homeostasis of PD-1 receptor(+) regulatory T cells.

Mature dendritic cells (DCs) are established as unrivaled antigen-presenting cells (APCs) in the initiation of immune responses, whereas steady-state DCs induce peripheral T cell tolerance. Using various genetic approaches, we depleted CD11c(+) DCs in mice and induced autoimmune CNS inflammation. Unexpectedly, mice lacking DCs developed aggravated disease compared to control mice. Furthermore, when we engineered DCs to present a CNS-associated autoantigen in an induced manner, we found robust tolerance that prevented disease, which coincided with an upregulation of the PD-1 receptor on antigen-specific T cells. Additionally, we showed that PD-1 was necessary for DC-mediated induction of regulatory T cells. Our results show that a reduction of DCs interferes with tolerance, resulting in a stronger inflammatory response, and that other APC populations could compensate for the loss of immunogenic APC function in DC-depleted mice.

Langerhans cells are negative regulators of the anti-Leishmania response.

Migratory skin dendritic cells (DCs) are thought to play an important role in priming T cell immune responses against Leishmania major, but DC subtypes responsible for the induction of protective immunity against this pathogen are still controversial. In this study, we analyzed the role of Langerin(+) skin-derived DCs in the Leishmania model using inducible in vivo cell ablation. After physiologically relevant low-dose infection with L. major (1,000 parasites), mice depleted of all Langerin(+) DCs developed significantly smaller ear lesions with decreased parasite loads and a reduced number of CD4(+) Foxp3(+) regulatory T cells (T reg cells) as compared with controls. This was accompanied by increased interferon γ production in lymph nodes in the absence of Langerin(+) DCs. Moreover, selective depletion of Langerhans cells (LCs) demonstrated that the absence of LCs, and not Langerin(+) dermal DC, was responsible for the reduced T reg cell immigration and the enhanced Th1 response, resulting in attenuated disease. Our data reveal a unique and novel suppressive role for epidermal LCs in L. major infection by driving the expansion of T reg cells. A better understanding of the various roles of different DC subsets in cutaneous leishmaniasis will improve the development of a potent therapeutic/prophylactic vaccine.

A role for leukocyte-derived IL-1RA in DC homeostasis revealed by increased susceptibility of IL-1RA-deficient mice to cutaneous leishmaniasis.

Dendritic cell (DC)-derived IL-1α/ß plays a critical role in the induction of T helper type 1 (Th1)-dependent immunity against Leishmania. DCs from susceptible BALB/c mice produce less IL-1α/ß when compared with resistant C57BL/6 mice, contributing to aberrant Th2 development and ultimate death of infected mice. We have extended our studies of the role of IL-1 in leishmaniasis using IL-1RA(-/-) BALB/c mice that are characterized by upregulated IL-1 receptor signaling. Unexpectedly, infection of IL-1RA(-/-) mice led to significantly worsened disease outcome with larger lesions, dramatically higher parasite burdens, and decreased IFN-γ production by antigen-specific T cells. We determined that IL-1RA(-/-) DCs were more mature already in the steady state, exhibited less phagocytotic capacity, and IL-12 production in response to various stimuli was impaired. Our data suggest that in addition to effects on Th education, IL-1α/ß signaling also modulates DC homeostasis with increased signaling, leading to downmodulation of IL-12 synthesis and worsened disease outcome after infection with Leishmania major. Thus, the complex regulation of various members of the IL-1 cytokine family mediated through effects on both DCs and T cells critically contributes to disease outcome against this important human pathogen.

IL-1 signalling is dispensable for protective immunity in Leishmania-resistant mice.

Leishmaniasis is a parasitic disease affecting ∼12 million people. Control of infection (e.g. in C57BL/6 mice) results from IL-12-dependent production of IFNγ by Th1/Tc1 cells. In contrast, BALB/c mice succumb to infection because of preferential Th2-type cytokine induction. Infected dendritic cells (DC) represent important sources of IL-12. Genetically determined differences in DC IL-1α/ß production contribute to disease outcome. Whereas the course of disease was not dramatically altered in IL-1RI(-/-) mice, local administration of IL-1α to infected C57BL/6 mice improved disease outcome. To definitively elucidate the involvement of IL-1 in immunity against leishmaniasis, we now utilized IL-1α/ß-double-deficient C57BL/6 mice. C57BL/6 mice are believed to be a good surrogate model for human, self limited cutaneous leishmaniasis (CL). Leishmania major-infected IL-1α/ß(-/-) mice were resistant to experimental CL comparable to controls. In addition, DC-based vaccination against leishmaniasis in C57BL/6 mice was independent of IL-1. Thus, in Leishmania-resistant C57BL/6 mice, IL-1 signalling is dispensable for protection.

Leishmaniasis, contact hypersensitivity and graft-versus-host disease: understanding the role of dendritic cell subsets in balancing skin immunity and tolerance.

Dendritic cells (DC) are key elements of the immune system. In peripheral tissues, they function as sentinels taking up and processing antigens. After migration to the draining lymph nodes, the DC either present antigenic peptides by themselves or transfer them to lymph node-resident DC. The skin is the primary interface between the body and the environment and host's various DC subsets, including dermal DC (dDC) and Langerhans cells (LC). Because of their anatomical position in the epidermis, LC are believed to be responsible for induction of adaptive cutaneous immune responses. The functions of LC and dDC in the skin immune system in vivo are manifold, and it is still discussed controversially whether the differentiation of T-cell subtypes (e.g. effector T cells and regulatory T cells) may be initiated by distinct DC subtypes. As skin DC are able to promote or downmodulate immune responses, we chose different skin diseases (cutaneous leishmaniasis, contact hypersensitivity, UV radiation-induced suppression, and graft-versus-host disease) to describe the biological interactions between different DC subtypes and T cells that lead to the development of efficient or unwanted immune responses. A detailed knowledge about the immune modulatory capacity of different cutaneous DC subsets might be helpful to specifically target these cells through the skin during therapeutic interventions.