The lipopolysaccharide core of Brucella abortus acts as a shield against innate immunity recognition

Innate immunity recognizes bacterial molecules bearing pathogen-associated molecular patterns to launch inflammatory responses leading to the activation of adaptive immunity. However, the lipopolysaccharide (LPS) of the gram-negative bacterium Brucella lacks a marked pathogen-associated molecular pattern, and it has been postulated that this delays the development of immunity, creating a gap that is critical for the bacterium to reach the intracellular replicative niche. We found that a B. abortus mutant in the wadC gene displayed a disrupted LPS core while keeping both the LPS O-polysaccharide and lipid A. In mice, the wadC mutant induced proinflammatory responses and was attenuated. In addition, it was sensitive to killing by non-immune serum and bactericidal peptides and did not multiply in dendritic cells being targeted to lysosomal compartments. In contrast to wild type B. abortus, the wadC mutant induced dendritic cell maturation and secretion of pro-inflammatory cytokines. All these properties were reproduced by the wadC mutant purified LPS in a TLR4-dependent manner. Moreover, the core-mutated LPS displayed an increased binding to MD-2, the TLR4 co-receptor leading to subsequent increase in intracellular signaling. Here we show that Brucella escapes recognition in early stages of infection by expressing a shield against recognition by innate immunity in its LPS core and identify a novel virulence mechanism in intracellular pathogenic gram-negative bacteria. These results also encourage for an improvement in the generation of novel bacterial vaccines.

The Role of Direct Presentation by Donor Dendritic Cells in Rejection of Minor Histocompatibility Antigen-Mismatched Skin and Hematopoietic Cell Grafts

Background. The success of transplantation is hampered by rejection of the graft by alloreactive T cells. Donor dendritic cells (DC) have been shown to be required for direct priming of immune responses to antigens from major histocompatibility complex-mismatched grafts. However, for immune responses to major histocompatibility complex-matched, minor histocompatibility (H) antigen mismatched grafts, the magnitude of the T-cell response to directly presented antigens is reduced, and the indirect pathway is more important. Therefore, we aimed to investigate the requirement for donor DC to directly present antigen from minor H antigen mismatched skin and hematopoietic grafts.

Langerin(+) Dermal Dendritic Cells Are Critical for CD8(+) T Cell Activation and IgH gamma-1 Class Switching in Response to Gene Gun Vaccines

The C-type lectin langerin/CD207 was originally discovered as a specific marker for epidermal Langerhans cells (LC). Recently, additional and distinct subsets of langerin(+) dendritic cells (DC) have been identified in lymph nodes and peripheral tissues of mice. Although the role of LC for immune activation or modulation is now being discussed controversially, other langerin(+) DC appear crucial for protective immunity in a growing set of infection and vaccination models. In knock-in mice that express the human diphtheria toxin receptor under control of the langerin promoter, injection of diphtheria toxin ablates LC for several weeks whereas other langerin(+) DC subsets are replenished within just a few days. Thus, by careful timing of diphtheria toxin injections selective states of deficiency in either LC only or all langerin(+) cells can be established. Taking advantage of this system, we found that, unlike selective LC deficiency, ablation of all langerin(+) DC abrogated the activation of IFN-gamma producing and cytolytic CD8(+) T cells after gene gun vaccination. Moreover, we identified migratory langerin(+) dermal DC as the subset that directly activated CD8(+) T cells in lymph nodes. Langerin(+) DC were also critical for IgG1 but not IgG2a Ab induction, suggesting differential polarization of CD4(+) T helper cells by langerin(+) or langerin-negative DC, respectively. In contrast, protein vaccines administered with various adjuvants induced IgG1 independently of langerin(+) DC. Taken together, these findings reflect a highly specialized division of labor between different DC subsets both with respect to Ag encounter as well as downstream processes of immune activation. The Journal of Immunology, 2011, 186: 1377-1383.

Skin langerin+ dendritic cells transport intradermally injected anti-DEC-205 antibodies but are not essential for subsequent cytotoxic CD8+ T cell responses

Incorporation of Ags by dendritic cells (DCs) increases when Ags are targeted to endocytic receptors by mAbs. We have previously demonstrated in the mouse that mAbs against C-type lectins administered intradermally are taken up by epidermal Langerhans cells (LCs), dermal Langerin(neg) DCs, and dermal Langerin(+) DCs in situ. However, the relative contribution of these skin DC subsets to the induction of immune responses after Ag targeting has not been addressed in vivo. We show in this study that murine epidermal LCs and dermal DCs transport intradermally injected mAbs against the lectin receptor DEC-205/CD205 in vivo. Skin DCs targeted in situ with mAbs migrated through lymphatic vessels in steady state and inflammation. In the skin-draining lymph nodes, targeting mAbs were found in resident CD8a(+) DCs and in migrating skin DCs. More than 70% of targeted DCs expressed Langerin, including dermal Langerin(+) DCs and LCs. Numbers of targeted skin DCs in the nodes increased 2-3-fold when skin was topically inflamed by the TLR7 agonist imiquimod. Complete removal of the site where OVA-coupled anti-DEC-205 had been injected decreased endogenous cytotoxic responses against OVA peptide-loaded target cells by 40-50%. Surprisingly, selective ablation of all Langerin(+) skin DCs in Langerin-DTR knock-in mice did not affect such responses independently of the adjuvant chosen. Thus, in cutaneous immunization strategies where Ag is targeted to DCs, Langerin(+) skin DCs play a major role in transport of anti-DEC-205 mAb, although Langerin(neg) dermal DCs and CD8a(+) DCs are sufficient to subsequent CD8(+) T cell responses.

Dynamics and Function of Langerhans Cells In Vivo: Dermal Dendritic Cells Colonize Lymph Node Areas Distinct from Slower Migrating Langerhans Cells

Langerhans cells (LCs) are prominent dendritic cells (DCs) in epithelia, but their role in immunity is poorly defined. To track and discriminate LCs from dermal DCs in vivo, we developed knockin mice expressing enhanced green fluorescent protein (EGFP) under the control of the langerin (CD207) gene. By using vital imaging, we showed that most EGFP(+) LCs were sessile under steady-state conditions, whereas skin inflammation induced LC motility and emigration to lymph nodes (LNs). After skin immunization, dermal DCs arrived in LNs first and colonized areas distinct from slower migrating LCs. LCs reaching LNs under steady-state or inflammatory conditions expressed similar levels of costimulatory molecules. Langerin and EGFP were also expressed on thymic DCs and on blood-derived, CD8alpha(+) DCs from all secondary lymphoid organs. By using a similar knockin strategy involving a diphtheria toxin receptor (DTR) fused to EGFP, we demonstrated that LCs were dispensable for triggering hapten-specific T cell effectors through skin immunization.

Disruption of the langerin/CD207 Gene Abolishes Birbeck Granules without a Marked Loss of Langerhans Cell Function

Langerin is a C-type lectin expressed by a subset of dendritic leukocytes, the Langerhans cells (LC). Langerin is a cell surface receptor that induces the formation of an LC-specific organelle, the Birbeck granule (BG). We generated a langerin(-/-) mouse on a C57BL/6 background which did not display any macroscopic aberrant development. In the absence of langerin, LC were detected in normal numbers in the epidermis but the cells lacked BG. LC of langerin(-/-) mice did not present other phenotypic alterations compared to wild-type littermates. Functionally, the langerin(-/-) LC were able to capture antigen, to migrate towards skin draining lymph nodes, and to undergo phenotypic maturation. In addition, langerin(-/-) mice were not impaired in their capacity to process native OVA protein for I-A(b)-restricted presentation to CD4(+) T lymphocytes or for H-2K(b)-restricted cross-presentation to CD8(+) T lymphocytes. langerin(-/-) mice inoculated with mannosylated or skin-tropic microorganisms did not display an altered pathogen susceptibility. Finally, chemical mutagenesis resulted in a similar rate of skin tumor development in langerin(-/-) and wild-type mice. Overall, our data indicate that langerin and BG are dispensable for a number of LC functions. The langerin(-/-) C57BL/6 mouse should be a valuable model for further functional exploration of langerin and the role of BG.

Monocyte-derived dendritic cells: a potential target for therapy in multiple sclerosis (MS)

Monocytes can differentiate into dendritic cells (DC), cells with a pivotal role in both protective immunity and tolerance. Defects in the maturation or function of DC may be important in the development of autoimmune disease. We sought to establish if there were differences in the cytokine (granulocyte-macrophage colony-stimulating factor and IL-4)-driven maturation of monocytes to DC in patients with MS and whether drugs used to treat MS affected this process in vitro. We have demonstrated that there is no defect in the ability of magnetic activated cell sorting (MACS)-purified monocytes from patients with MS to differentiate to DC, but equally they show no tendency to acquire a DC phenotype without exogenous cytokines. Interferon-beta1a prevents the acquisition of a full DC phenotype as determined by light and electron microscopy and by flow cytometry. Methylprednisolone not only prevents the development of monocyte-derived DC but totally redirects monocyte differentiation towards a macrophage phenotype. Evidence is evolving for a role for DC in central nervous system immunity, either within the brain or in cervical lymph nodes. The demonstrated effect of both drugs on monocyte differentiation may represent an important site for immune therapy in MS.

Epidermal Langerhans Cells are Dispensable for Humoral and Cell-Mediated Immunity Elicited by Gene Gun Immunization.

Gene gun immunization, i.e., bombardment of skin with DNA-coated particles, is an efficient method for the administration of DNA vaccines. Direct transfection of APC or cross-presentation of exogenous Ag acquired from transfected nonimmune cells enables MHC-I-restricted activation of CD8(+) T cells. Additionally, MHC-II-restricted presentation of exogenous Ag activates CD4(+) Th cells. Being the principal APC in the epidermis, Langerhans cells (LC) seem ideal candidates to accomplish these functions. However, the dependence on LC of gene gun-induced immune reactions has not yet been demonstrated directly. This was primarily hampered by difficulties to discriminate the contributions of LC from those of other dermal dendritic cells. To address this problem, we have used Langerin-diphtheria toxin receptor knockin mice that allow for selective inducible ablation of LC. LC deficiency, even over the entire duration of experiments, did not affect any of the gene gun-induced immune functions examined, including proliferation of CD4(+) and CD8(+) T cells, IFN-gamma secretion by spleen cells, Ab production, CTL activity, and development of protective antitumor immunity.

Identification of a novel population of Langerin+ dendritic cells.

Langerhans cells (LCs) are antigen-presenting cells that reside in the epidermis of the skin and traffic to lymph nodes (LNs). The general role of these cells in skin immune responses is not clear because distinct models of LC depletion resulted in opposite conclusions about their role in contact hypersensitivity (CHS) responses. While comparing these models, we discovered a novel population of LCs that resides in the dermis and does not represent migrating epidermal LCs, as previously thought. Unlike epidermal LCs, dermal Langerin(+) dendritic cells (DCs) were radiosensitive and displayed a distinct cell surface phenotype. Dermal Langerin(+) DCs migrate from the skin to the LNs after inflammation and in the steady state, and represent the majority of Langerin(+) DCs in skin draining LNs. Both epidermal and dermal Langerin(+) DCs were depleted by treatment with diphtheria toxin in Lang-DTREGFP knock-in mice. In contrast, transgenic hLang-DTA mice lack epidermal LCs, but have normal numbers of dermal Langerin(+) DCs. CHS responses were abrogated upon depletion of both epidermal and dermal LCs, but were unaffected in the absence of only epidermal LCs. This suggests that dermal LCs can mediate CHS and provides an explanation for previous differences observed in the two-model systems.

Antigen-specific T-T interactions regulate CD4 T-cell expansion.

The regulation of CD4 T cell numbers during an immune response should take account of the amount of antigen (Ag), the initial frequency of Ag-specific T cells, the mix of naive versus experienced cells, and (ideally) the diversity of the repertoire. Here we describe a novel mechanism of T cell regulation that potentially deals with all of these parameters. We found that CD4 T cells establish a negative feedback loop by capturing their cognate MHC/peptide complexes from Ag-presenting cells and presenting them to Ag-experienced CD4 T cells, thereby inhibiting their recruitment into the response while allowing recruitment of naive T cells. The inhibition is Ag specific, begins at day 2 (long before Ag disappearance), and cannot be overcome by providing new Ag-loaded dendritic cells. In this way CD4 T cell proliferation is regulated in a functional relationship to the amount of Ag, while allowing naive T cells to generate repertoire variety.

Langerhans cell (LC) proliferation mediates neonatal development, homeostasis, and inflammation-associated expansion of the epidermal LC network.

Most tissues develop from stem cells and precursors that undergo differentiation as their proliferative potential decreases. Mature differentiated cells rarely proliferate and are replaced at the end of their life by new cells derived from precursors. Langerhans cells (LCs) of the epidermis, although of myeloid origin, were shown to renew in tissues independently from the bone marrow, suggesting the existence of a dermal or epidermal progenitor. We investigated the mechanisms involved in LC development and homeostasis. We observed that a single wave of LC precursors was recruited in the epidermis of mice around embryonic day 18 and acquired a dendritic morphology, major histocompatibility complex II, CD11c, and langerin expression immediately after birth. Langerin+ cells then undergo a massive burst of proliferation between postnatal day 2 (P2) and P7, expanding their numbers by 10–20-fold. After the first week of life, we observed low-level proliferation of langerin+ cells within the epidermis. However, in a mouse model of atopic dermatitis (AD), a keratinocyte signal triggered increased epidermal LC proliferation. Similar findings were observed in epidermis from human patients with AD. Therefore, proliferation of differentiated resident cells represents an alternative pathway for development in the newborn, homeostasis, and expansion in adults of selected myeloid cell populations such as LCs. This mechanism may be relevant in locations where leukocyte trafficking is limited.