Christopher G. Mueller

Selection of a synthetic glycan oligomer from a library of DNA-templated fragments against DC-SIGN and inhibition of HIV gp120 binding to dendritic cells

We report the synthesis of a nucleic acid-encoded carbohydrate library, its combinatorial self-assembly into 37,485 pairs and a screen against DC-SIGN leading to the identification of consensus ligand motifs. A prototypical example from the selected pairs was shown to have enhanced binding. A dendrimer incorporating the selected motifs inhibited gp120s binding to dendritic cells with higher efficiency than mannan.

Receptor activator of NF-kappaB (RANK) stimulates the proliferation of epithelial cells of the epidermo-pilosebaceous unit.

Receptor activator of NF-κB (RANK), known for controlling bone mass, has been recognized for its role in epithelial cell activation of the mammary gland. Because bone and the epidermo-pilosebaceous unit of the skin share a lifelong renewal activity where similar molecular players operate, and because mammary glands and hair follicles are both skin appendages, we have addressed the function of RANK in the hair follicle and the epidermis. Here, we show that mice deficient in RANK ligand (RANKL) are unable to initiate a new growth phase of the hair cycle and display arrested epidermal homeostasis. However, transgenic mice overexpressing RANK in the hair follicle or administration of recombinant RANKL both activate the hair cycle and epidermal growth. RANK is expressed by the hair follicle germ and bulge stem cells and the epidermal basal cells, cell types implicated in the renewal of the epidermo-pilosebaceous unit. RANK signaling is dispensable for the formation of the stem cell compartment and the inductive hair follicle mesenchyme, and the hair cycle can be rescued by Rankl knockout skin transplantation onto nude mice. RANKL is actively transcribed by the hair follicle at initiation of its growth phase, providing a mechanism for stem cell RANK engagement and hair-cycle entry. Thus, RANK–RANKL regulates hair renewal and epidermal homeostasis and provides a link between these two activities.

Dermal-Type Macrophages Expressing CD209/DC-SIGN Show Inherent Resistance to Dengue Virus Growth

Background An important question in dengue pathogenesis is the identity of immune cells involved in the control of dengue virus infection at the site of the mosquito bite. There is evidence that infection of immature myeloid dendritic cells plays a crucial role in dengue pathogenesis and that the interaction of the viral envelope E glycoprotein with CD209/DC-SIGN is a key element for their productive infection. Dermal macrophages express CD209, yet little is known about their role in dengue virus infection. Methods and Findings Here, we showed that dermal macrophages bound recombinant envelope E glycoprotein fused to green fluorescent protein. Because dermal macrophages stain for IL-10 in situ, we generated dermal-type macrophages from monocytes in the presence of IL-10 to study their infection by dengue virus. The macrophages were able to internalize the virus, but progeny virus production was undetectable in the infected cells. In addition, no IFN-α was produced in response to the virus. The inability of dengue virus to grow in the macrophages was attributable to accumulation of internalized virus particles into poorly-acidified phagosomes. Conclusions Aborting infection by viral sequestration in early phagosomes would present a novel means to curb infection of enveloped virus and may constitute a prime defense system to prevent dengue virus spread shortly after the bite of the infected mosquito.

Bone resorption control of tooth eruption and root morphogenesis: Involvement of the receptor activator of NF-κB (RANK).

Activation of the receptor activator of NF‐κB (RANK) is a crucial step in osteoclastogenesis. Loss‐ and gain‐of‐function mutations in the Rank gene cause, respectively, osteopetrosis and several forms of extensive osteolysis. Tooth and alveolar bone alterations are associated with these pathologies but remain to be better characterized. The aim of the present study was to establish the tooth and alveolar bone phenotype of a transgenic mouse model of RANK over‐expression in osteoclast precursors. Early tooth eruption and accelerated tooth root elongation were observed subsequent to an increase in osteoclast numbers surrounding the tooth. The final root length appeared not to be affected by RANK over‐expression, but a significant reduction in root diameter occurred in both control and root‐morphogenesis‐defective Msx2 null mutant mice. These results indicate that root length is independent of the surrounding bone resorption activity. In contrast, root diameter is sensitive to the activity of alveolar bone osteoclasts. These data suggest that early eruption and thin root are phenotypic features that could be associated with extensive osteolytic pathologies. J. Cell. Physiol. 226: 74–85, 2010.

Epidermal Receptor Activator of NF-κB Ligand Controls Langerhans Cells Numbers and Proliferation

Langerhans cells (LC) are the dendritic APC population of the epidermis, where they reside for long periods and are self-replicating. The molecular signals underlying these characteristics are unknown. The TNF superfamily member receptor activator of NF-κB ligand (RANKL, TNFSF11) has been shown to sustain viability of blood dendritic cells in addition to its role in promoting proliferation and differentiation of several cell types, notably osteoclasts. In this study, we have studied expression of the RANKL system in skin and have defined a key role for this molecule in LC homeostasis. In vitro and in vivo, human KC expressed RANKL and epidermal LC expressed cell surface RANK. In vitro, RANKL sustained CD34+ progenitor-derived LC viability following 72-h cultures in cytokine-free medium (79.5 ± 1% vs 55.2 ± 5.7% live cells, respectively; n = 4; p < 0.05). In vivo, RANKL-deficient mice displayed a marked reduction in epidermal LC density (507.1 ± 77.2 vs 873.6 ± 41.6 LC per mm2; n = 9; p < 0.05) and their proliferation was impaired without a detectable effect on apoptosis. These data indicate a key role for the RANKL system in the regulation of LC survival within the skin and suggest a regulatory role for KC in the maintenance of epidermal LC homeostasis.

RANKL Induces Organized Lymph Node Growth by Stromal Cell Proliferation

RANK and its ligand RANKL play important roles in the development and regulation of the immune system. We show that mice transgenic for Rank in hair follicles display massive postnatal growth of skin-draining lymph nodes. The proportions of hematopoietic and nonhematopoietic stromal cells and their organization are maintained, with the exception of an increase in B cell follicles. The hematopoietic cells are not activated and respond to immunization by foreign Ag and adjuvant. We demonstrate that soluble RANKL is overproduced from the transgenic hair follicles and that its neutralization normalizes lymph node size, inclusive area, and numbers of B cell follicles. Reticular fibroblastic and vascular stromal cells, important for secondary lymphoid organ formation and organization, express RANK and undergo hyperproliferation, which is abrogated by RANKL neutralization. In addition, they express higher levels of CXCL13 and CCL19 chemokines, as well as MAdCAM-1 and VCAM-1 cell-adhesion molecules. These findings highlight the importance of tissue-derived cues for secondary lymphoid organ homeostasis and identify RANKL as a key molecule for controlling the plasticity of the immune system.

Emerging Functions of RANKL in Lymphoid Tissues

The tumor necrosis factor superfamily (TNFSF) members play pivotal roles in embryonic development of lymphoid tissue and their homeostasis. RANKL (Receptor activator of NF-κB ligand, also called TRANCE, TNFSF11) is recognized as an important player in bone homeostasis and lymphoid tissue formation. In its absence bone mass control is deregulated and lymph nodes fail to develop. While its function in bone is well described, there is still little functional insight into the action of RANKL in lymphoid tissue development and homeostasis. Here we provide an overview of the known functions of RANKL, its signaling receptor RANK and its decoy receptor OPG from the perspective of lymphoid tissue development and immune activation in the mouse. Expressed by the hematopoietic lymphoid tissue inducing (LTi) cells and the mesenchymal lymphoid tissue organizer (LTo) cells, RANKL was shown to stimulate Lymphotoxin (LT) expression and to be implicated in LTi cell accumulation. Our recent finding that RANKL also triggers proliferation of adult lymph node stroma suggests that RANKL may furthermore directly activate LTo cells. Beyond bone, the RANKL-RANK-OPG triad plays important roles in immunobiology that are waiting to be unraveled.

Dermal CD14+ Dendritic Cell and Macrophage Infection by Dengue Virus Is Stimulated by Interleukin-4

Dengue virus (DENV) is responsible for the most prevalent arthropod-borne viral infection in humans. Events decisive for disease development occur in the skin after virus inoculation by the mosquito. Yet, the role of human dermis-resident immune cells in dengue infection and disease remains elusive. Here we investigated how dermal dendritic cells (dDCs) and macrophages (dMs) react to DENV and impact on immunopathology. We show that both CD1c(+) and CD14(+) dDC subsets were infected, but viral load greatly increased in CD14(+) dDCs upon IL-4 stimulation, which correlated with upregulation of virus-binding lectins Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Nonintegrin (DC-SIGN/CD209) and mannose receptor (CD206). IL-4 also enhanced T-cell activation by dDCs, which was further increased upon dengue infection. dMs purified from digested dermis were initially poorly infected but actively replicated the virus and produced TNF-α upon lectin upregulation in response to IL-4. DC-SIGN(+) cells are abundant in inflammatory skin with scabies infection or Th2-type dermatitis, suggesting that skin reactions to mosquito bites heighten the risk of infection and subsequent immunopathology. Our data identify dDCs and dMs as primary arbovirus target cells in humans and suggest that dDCs initiate a potent virus-directed T-cell response, whereas dMs fuel the inflammatory cascade characteristic of dengue fever.

Lymphatic Endothelial Cells Control Initiation of Lymph Node Organogenesis

Summary Lymph nodes (LNs) are strategically situated throughout the body at junctures of the blood vascular and lymphatic systems to direct immune responses against antigens draining from peripheral tissues. The current paradigm describes LN development as a programmed process that is governed through the interaction between mesenchymal lymphoid tissue organizer (LTo) cells and hematopoietic lymphoid tissue inducer (LTi) cells. Using cell‐type‐specific ablation of key molecules involved in lymphoid organogenesis, we found that initiation of LN development is dependent on LTi‐cell‐mediated activation of lymphatic endothelial cells (LECs) and that engagement of mesenchymal stromal cells is a succeeding event. LEC activation was mediated mainly by signaling through receptor activator of NF‐&kgr;B (RANK) and the non‐canonical NF‐&kgr;B pathway and was steered by sphingosine‐1‐phosphate‐receptor‐dependent retention of LTi cells in the LN anlage. Finally, the finding that pharmacologically enforced interaction between LTi cells and LECs promotes ectopic LN formation underscores the central LTo function of LECs. Graphical Abstract Figure. No Caption available. HighlightsLT&bgr;R signals in mesenchymal LTo cells are not essential for LN organogenesisRANK‐mediated signaling in lymphatic endothelial cells (LECs) drives LN developmentLECs control retention of LTi cells in embryonic LN anlagenEnforced LTi cell retention in lymphatics induces formation of ectopic LNs &NA; Lymph node (LN) formation is thought to rely mainly on interactions between mesenchymal lymphoid tissue organizer cells and lymphoid tissue inducer cells. Onder et al. now show that LN formation is governed by RANK‐dependent activation of lymphatic endothelial cells that control retention of lymphoid tissue inducer cells in embryonic LN anlagen.

A new organotypic model containing dermal-type macrophages

Abstract:  Human skin equivalents (SEs) are popular three‐dimensional (D) cell culture systems in fundamental and applied dermatology. They have been made to contain dendritic cells, but so far no study on the incorporation of potentially anti‐inflammatory dermal macrophages has been performed. Here, we show that monocyte‐derived dermal‐type macrophages can be introduced into a rigid scaffold with dermal fibroblasts. They maintain their cell surface markers CD163, DC‐SIGN/CD209 and HLA‐DR, which discriminate them from monocytes and dendritic cells. They retain the ability to produce the anti‐inflammatory cytokine IL‐10 in response to lipopolysaccharide (LPS) and to phagocytose latex beads. We thus demonstrate the feasibility of creating macrophage–fibroblast 3D cultures as a first step towards generating SEs with dermal macrophages.