Anandamide modulation of monocyte-derived Langerhans cells: implications for immune homeostasis and skin inflammation.

Introduction: The endocannabinoid system (ECS), named after the chemical compounds found in the cannabis plant, is a regulatory network of neurotransmitters, receptors, and enzymes that plays crucial roles in skin health and disease. Endogenous ligands of the ECS, called endocannabinoids, have proven to be important regulators of immune responses. One of the most prevalent endocannabinoids, arachidonoylethanolamide (also known as anandamide), is known for its anti-inflammatory effects. Langerhans cells (LCs) are the sole antigen-presenting cells present in the human epidermis. They serve as the first line of defense against pathogens and are essential for the skin's specific immune responses and play a critical role in maintaining tissue homeostasis; however, little is known about the effect of endocannabinoids on these cells. Our research aimed to provide the connection between monocyte-derived Langerhans cells (moLCs) and the ECS, shedding light on their collaborative roles in immune homeostasis and inflammation. Methods: Human monocytes were differentiated into moLCs using established protocols. Anandamide was applied during the differentiation process to test its effect on the viability, marker expression, and cytokine production of the cells, as well as in short term treatments for intracellular calcium measurement. TLR ligands applied after the differentiation protocol were used to activate moLCs. The impact of anandamide on the functionality of moLCs was further assessed using differential gene expression analysis of bulk RNA-Seq data, moLC-T cell cocultures, while ELISpot was employed to determine polarization of T cells activated in the aforementioned cocultures. Results: Anandamide did not significantly affect the viability of moLCs up to 10 µM. When applied during the differentiation process it had only a negligible effect on CD207 expression, the prototypic marker of LCs; however, there was an observed reduction in CD1a expression by moLCs. Anandamide had no significant effects on the maturation status of moLCs, nor did it affect the maturation induced by TLR3 and TLR7/8 agonists. MoLCs differentiated in the presence of anandamide did however show decreased production of CXCL8, IL-6, IL-10 and IL-12 cytokines induced by TLR3 and TLR7/8 activation. Anandamide-treated moLCs showed an increased capability to activate naïve T cells; however, not to the level seen with combined TLR agonism. RNA sequencing analysis of moLCs differentiated with anandamide showed modest changes compared to control cells but did reveal an inhibitory effect on oxidative phosphorylation specifically in activated moLCs. Anandamide also promoted the polarization of naïve T cells towards a Th1 phenotype. Discussion: Our results show that anandamide has nuanced effects on the differentiation, maturation, cytokine secretion, metabolism and function of activated moLCs. Among these changes the decrease in CD1a expression on moLCs holds promise to selectively dampen inflammation induced by CD1a restricted T cells, which have been implicated as drivers of inflammation in common inflammatory skin conditions such as psoriasis, atopic dermatitis and contact dermatitis.

The interferon-rich skin environment regulates Langerhans cell ADAM17 to promote photosensitivity in lupus.

The autoimmune disease lupus erythematosus (lupus) is characterized by photosensitivity, where even ambient ultraviolet radiation (UVR) exposure can lead to development of inflammatory skin lesions. We have previously shown that Langerhans cells (LCs) limit keratinocyte apoptosis and photosensitivity via a disintegrin and metalloprotease 17 (ADAM17)-mediated release of epidermal growth factor receptor (EGFR) ligands and that LC ADAM17 sheddase activity is reduced in lupus. Here, we sought to understand how the lupus skin environment contributes to LC ADAM17 dysfunction and, in the process, differentiate between effects on LC ADAM17 sheddase function, LC ADAM17 expression, and LC numbers. We show through transcriptomic analysis a shared IFN-rich environment in non-lesional skin across human lupus and three murine models: MRL/lpr, B6.Sle1yaa, and imiquimod (IMQ) mice. IFN-I inhibits LC ADAM17 sheddase activity in murine and human LCs, and IFNAR blockade in lupus model mice restores LC ADAM17 sheddase activity, all without consistent effects on LC ADAM17 protein expression or LC numbers. Anti-IFNAR-mediated LC ADAM17 sheddase function restoration is associated with reduced photosensitive responses that are dependent on EGFR signaling and LC ADAM17. Reactive oxygen species (ROS) is a known mediator of ADAM17 activity; we show that UVR-induced LC ROS production is reduced in lupus model mice, restored by anti-IFNAR, and is cytoplasmic in origin. Our findings suggest that IFN-I promotes photosensitivity at least in part by inhibiting UVR-induced LC ADAM17 sheddase function and raise the possibility that anifrolumab ameliorates lupus skin disease in part by restoring this function. This work provides insight into IFN-I-mediated disease mechanisms, LC regulation, and a potential mechanism of action for anifrolumab in lupus.

IL-38 Aggravates Atopic Dermatitis via Facilitating Migration of Langerhans cells.

Atopic dermatitis (AD) is a common inflammation skin disease that involves dysregulated interplay between immune cells and keratinocytes. Interleukin-38 (IL-38), a poorly characterized IL-1 family cytokine, its role and mechanism in the pathogenesis of AD is elusive. Here, we show that IL-38 is mainly secreted by epidermal keratinocytes and highly expressed in the skin and downregulated in AD lesions. We generated IL-38 keratinocyte-specific knockout mice (K14Cre/+-IL-38f/f ) and induced AD models by 2,4-dinitrofluorobenzene (DNFB). Unexpectedly, after treatment with DNFB, K14Cre/+-IL-38f/f mice were less susceptible to cutaneous inflammation of AD. Moreover, keratinocyte-specific deletion of IL-38 suppressed the migration of Langerhans cells (LCs) into lymph nodes which results in disturbed differentiation of CD4+T cells and decreased the infiltration of immune cells into AD lesions. LCs are a type of dendritic cell that reside specifically in the epidermis and regulate immune responses. We developed LC-like cells in vitro from mouse bone marrow (BM) and treated with recombined IL-38. The results show that IL-38 depended on IL-36R, activated the phosphorylated expression of IRAK4 and NF-κB P65 and upregulated the expression of CCR7 to promoting the migration of LCs, nevertheless, the upregulation disappeared with the addition of IL-36 receptor antagonist (IL-36RA), IRAK4 or NF-κB P65 inhibitor. Furthermore, after treatment with IRAK4 inhibitors, the experimental AD phenotypes were alleviated and so IRAK4 is considered a promising target for the treatment of inflammatory diseases. Overall, our findings indicated a potential pathway that IL-38 depends on IL-36R, leading to LCs migration to promote AD by upregulating CCR7 via IRAK4/NF-κB and implied the prevention and treatment of AD, supporting potential clinical utilization of IRAK4 inhibitors in AD treatment.

FLT3L governs the development of partially overlapping hematopoietic lineages in humans and mice.

FMS-related tyrosine kinase 3 ligand (FLT3L), encoded by FLT3LG, is a hematopoietic factor essential for the development of natural killer (NK) cells, B cells, and dendritic cells (DCs) in mice. We describe three humans homozygous for a loss-of-function FLT3LG variant with a history of various recurrent infections, including severe cutaneous warts. The patients' bone marrow (BM) was hypoplastic, with low levels of hematopoietic progenitors, particularly myeloid and B cell precursors. Counts of B cells, monocytes, and DCs were low in the patients' blood, whereas the other blood subsets, including NK cells, were affected only moderately, if at all. The patients had normal counts of Langerhans cells (LCs) and dermal macrophages in the skin but lacked dermal DCs. Thus, FLT3L is required for B cell and DC development in mice and humans. However, unlike its murine counterpart, human FLT3L is required for the development of monocytes but not NK cells.

Rho-associated kinase regulates Langerhans cell morphology and responsiveness to tissue damage.

Skin damage requires efficient immune cell responses to restore organ function. Epidermal-resident immune cells known as Langerhans cells use dendritic protrusions to surveil the skin microenvironment, which contains keratinocytes and peripheral axons. The mechanisms governing Langerhans cell dendrite dynamics and responses to tissue damage are poorly understood. Using skin explants from adult zebrafish, we show that Langerhans cells maintain normal surveillance following axonal degeneration and use their dendrites to engulf small axonal debris. By contrast, a ramified-to-rounded shape transition accommodates the engulfment of larger keratinocyte debris. We find that Langerhans cell dendrites are populated with actin and sensitive to a broad-spectrum actin inhibitor. We show that Rho-associated kinase (ROCK) inhibition leads to elongated dendrites, perturbed clearance of large debris, and reduced Langerhans cell migration to epidermal wounds. Our work describes the dynamics of Langerhans cells and involvement of the ROCK pathway in immune cell responses.

Chronic consumption of imbalance diets high in sucrose or fat induces abdominal obesity with different pattern of metabolic disturbances and lost in Langerhans cells population.

AIM: Obesity is a worldwide health issue, associated with development of type 2 Diabetes Mellitus. The aim of this study is to analyze the effect of consumption of two hypercaloric diets on metabolic disturbance and beta cells damage. MAIN METHODS: Male Wistar rats were subjected to twelve months consumption of three diets: a Control balanced diet (CTD, carbohydrates 58 %, proteins 29 %, lipids 13 %) and two hypercaloric diets, high in sucrose (HSD, carbohydrates 68 %, proteins 22 %, lipids 10 %) or high in fat (HFD, carbohydrates 31 %, proteins 14 %, lipids 55 %). Serum levels of glucose, triglycerides and free fatty acids were measured after zoometric parameters determination. Antioxidant enzymes activity and oxidative stress-marker were measured in pancreas tissue among histological analysis of Langerhans islets. KEY FINDINGS: Although diets were hypercaloric, the amount of food consumed by rats decreased, resulting in an equal caloric consumption. The HSD induced hypertriglyceridemia and hyperglycemia with higher levels in free fatty acids (FFA, lipotoxicity); whereas HFD did not increased neither the triglycerides nor FFA, nevertheless the loss of islets' cell was larger. Both diets induced obesity with hyperglycemia and significant reduction in Langerhans islets size. SIGNIFICANCE: Our results demonstrate that consumption of HSD induces more significant metabolic disturbances that HFD, although both generated pancreas damage; as well hypercaloric diet consumption is not indispensable to becoming obese; the chronic consumption of unbalanced diets (rich in carbohydrates or lipids) may lead to abdominal obesity with metabolic and functional disturbances, although the total amount of calories are similar.

TRPV1 activation in human Langerhans cells and T cells inhibits mucosal HIV-1 infection via CGRP-dependent and independent mechanisms.

Upon its mucosal transmission, HIV type 1 (HIV-1) rapidly targets genital antigen-presenting Langerhans cells (LCs), which subsequently transfer infectious virus to CD4+ T cells. We previously described an inhibitory neuroimmune cross talk, whereby calcitonin gene-related peptide (CGRP), a neuropeptide secreted by peripheral pain-sensing nociceptor neurons innervating all mucosal epithelia and associating with LCs, strongly inhibits HIV-1 transfer. As nociceptors secret CGRP following the activation of their Ca2+ ion channel transient receptor potential vanilloid 1 (TRPV1), and as we reported that LCs secret low levels of CGRP, we investigated whether LCs express functional TRPV1. We found that human LCs expressed mRNA and protein of TRPV1, which was functional and induced Ca2+ influx following activation with TRPV1 agonists, including capsaicin (CP). The treatment of LCs with TRPV1 agonists also increased CGRP secretion, reaching its anti-HIV-1 inhibitory concentrations. Accordingly, CP pretreatment significantly inhibited LCs-mediated HIV-1 transfer to CD4+ T cells, which was abrogated by both TRPV1 and CGRP receptor antagonists. Like CGRP, CP-induced inhibition of HIV-1 transfer was mediated via increased CCL3 secretion and HIV-1 degradation. CP also inhibited direct CD4+ T cells HIV-1 infection, but in CGRP-independent manners. Finally, pretreatment of inner foreskin tissue explants with CP markedly increased CGRP and CCL3 secretion, and upon subsequent polarized exposure to HIV-1, inhibited an increase in LC-T cell conjugate formation and consequently T cell infection. Our results reveal that TRPV1 activation in human LCs and CD4+ T cells inhibits mucosal HIV-1 infection, via CGRP-dependent/independent mechanisms. Formulations containing TRPV1 agonists, already approved for pain relief, could hence be useful against HIV-1.

Localized immune surveillance of primary melanoma in the skin deciphered through executable modeling.

While skin is a site of active immune surveillance, primary melanomas often escape detection. Here, we have developed an in silico model to determine the local cross-talk between melanomas and Langerhans cells (LCs), the primary antigen-presenting cells at the site of melanoma development. The model predicts that melanomas fail to activate LC migration to lymph nodes until tumors reach a critical size, which is determined by a positive TNF-α feedback loop within melanomas, in line with our observations of murine tumors. In silico drug screening, supported by subsequent experimental testing, shows that treatment of primary tumors with MAPK pathway inhibitors may further prevent LC migration. In addition, our in silico model predicts treatment combinations that bypass LC dysfunction. In conclusion, our combined approach of in silico and in vivo studies suggests a molecular mechanism that explains how early melanomas develop under the radar of immune surveillance by LC.

Met-Signaling Controls Dendritic Cell Migration in Skin by Regulating Podosome Formation and Function.

Signaling through the HGF receptor/Met in skin-resident Langerhans cells (LCs) and dermal dendritic cells (DCs) is essential for their emigration toward draining lymph nodes upon inflammation-induced activation. In this study, we addressed the role of Met signaling in distinct steps of LC/dermal DC emigration from the skin by employing a conditionally Met-deficient mouse model (Metflox/flox). We found that Met deficiency severely impaired podosome formation in DCs and concomitantly decreased the proteolytic degradation of gelatin. Accordingly, Met-deficient LCs failed to efficiently cross the extracellular matrix-rich basement membrane between the epidermis and the dermis. We further observed that HGF-dependent Met activation reduced the adhesion of bone marrow-derived LCs to various extracellular matrix factors and enhanced the motility of DCs in three-dimensional collagen matrices, which was not the case for Met-deficient LCs/DCs. We found no impact of Met signaling on the integrin-independent amoeboid migration of DCs in response to the CCR7 ligand CCL19. Collectively, our data show that the Met-signaling pathway regulates the migratory properties of DC in HGF-dependent and HGF-independent manners.

Topical Application of Adenosine A2-Type Receptor Agonists Prevents Contact Hypersensitivity Reactions in Mice by Affecting Skin Dendritic Cells.

Adenosine (Ado) produced by skin and skin migratory CD73+ dendritic cells is critically involved in tolerance to haptens. We therefore investigated the use of Ado receptor agonists for the treatment of contact hypersensitivity reactions. A2A- 4-[2-[[6-Amino-9-(N-ethyl-ß-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino] ethyl]benzenepropanoic acid hydrochloride (CGS) and A2B- 2-[[6-Amino-3,5-dicyano-4-[4-[cyclopropylmethoxy]phenyl]-2-pyridinyl]thio]-acetamide (BAY) specific Ado receptor agonists were epicutaneously applied to the skin before sensitization and challenge with DNFB. Both agonists reduced ear swelling compared with solvent controls. This was accompanied by fewer activated T cells in the skin after the challenge and by higher numbers of T cells expressing anergic markers such as LAG-3, CD137, PD-1, CD272, and TIM-3 in the lymph nodes of CGS-treated groups. In ear tissue, Ado receptor agonist treatment reduced the production of proinflammatory cytokines and chemokines as well as the infiltration by neutrophils after sensitization. Moreover, reduced numbers of skin migratory dendritic cells producing less IL-12 and exhibiting lower expression of CD86 were recorded in lymph nodes after sensitization. In cocultures of skin migratory dendritic cells from CGS-treated mice with T cells, reduced proliferation of T cells and decreased secretion of proinflammatory cytokines compared with that of solvent controls were apparent. In conclusion, topical application of Ado receptor agonists to the skin prevents sensitization of T cells against haptens by reducing the migration and activation of skin migratory dendritic cells.

mTORC1 signaling in antigen-presenting cells of the skin restrains CD8+ T cell priming.

How mechanistic target of rapamycin complex 1 (mTORC1), a key regulator of cellular metabolism, affects dendritic cell (DC) metabolism and T cell-priming capacity has primarily been investigated in vitro, but how mTORC1 regulates this in vivo remains poorly defined. Here, using mice deficient for mTORC1 component raptor in DCs, we find that loss of mTORC1 negatively affects glycolytic and fatty acid metabolism and maturation of conventional DCs, particularly cDC1s. Nonetheless, antigen-specific CD8+ T cell responses to infection are not compromised and are even enhanced following skin immunization. This is associated with increased activation of Langerhans cells and a subpopulation of EpCAM-expressing cDC1s, of which the latter show an increased physical interaction with CD8+ T cells in situ. Together, this work reveals that mTORC1 limits CD8+ T cell priming in vivo by differentially orchestrating the metabolism and immunogenicity of distinct antigen-presenting cell subsets, which may have implications for clinical use of mTOR inhibitors.

Release of Matrix Metalloproteinases by Macrophages in Radicular Cysts and Residual Radicular Cysts.

INTRODUCTION: Radicular cysts (RCs) and residual radicular cysts (RRCs) are the sequelae of dental caries and that leads to proliferation of epithelial rests of Malassez in periapical tissues. OBJECTIVES: The aim was to evaluate the relationship between Langerhans cells, macrophages, matrix metalloproteinases (MMP-9, MMP-13), and tumor necrosis factor-alpha (TNF-α) in the capsule and lining epithelium of cystic lesions. MATERIALS AND METHODS: Twenty RCs and 20 RRCs were submitted to immunohistochemical analysis with anti-CD68, anti-CD1a, anti-MMP-9, anti-MMP-13, and anti-TNF-α antibodies. The Mann-Whitney test and the Spearman correlation test were used for analysis of the data (P<0.05). RESULTS: The immunoexpression of MMP-13 and CD68 was significantly higher in RCs when compared with RRCs (P=0.011 and 0.012, respectively). The presence of an intense inflammatory infiltrate was significantly correlated with the immunoexpression of CD68 in RCs (P=0.025). Expression of CD68 showed a significant positive correlation with MMP-13 (P=0.015). A moderate correlation was observed between MMP-9 and MMP-13 (P=0.010). TNF-α expression was more common in RCs (P=0.001). CD1a was more frequently expressed in atrophic epithelium (P=0.041) and was significantly correlated with TNF-α (P=0.014). CONCLUSION: Langerhans cells induce a greater release of TNF-α which, in turn, is responsible for the stimulation of M1 macrophages. Higher immunoexpression of MMP-13 and MMP-9 is observed in the early stages of RCs compared with RRCs. Therefore, the toxins of microorganisms present in highly inflamed RCs are the main factors triggering a proinflammatory immune response and greater cystic expansion in the early stages of these lesions.

BMPR1a Is Required for the Optimal TGFß1-Dependent CD207+ Langerhans Cell Differentiation and Limits Skin Inflammation through CD11c+ Cells.

The cytokine TGFß1 induces epidermal Langerhans cell (LC) differentiation from human precursors, an effect mediated through BMPR1a/ALK3 signaling, as revealed from ectopic expression and receptor inhibition studies. Whether TGFß1‒BMPR1a signaling is required for LC differentiation in vivo remained incompletely understood. We found that TGFß1-deficient mice show defective perinatal expansion and differentiation of LCs. LCs can be identified within the normal healthy human epidermis by anti-BMPR1a immunohistology staining. Deletion of BMPR1a in all (vav+) hematopoietic cells revealed that BMPR1a is required for the efficient TGFß1-dependent generation of CD207+ LC-like cells from CD11c+ intermediates in vitro. Similarly, BMPR1a was required for the optimal induction of CD207 by preformed major histocompatibility complex II‒positive epidermal resident LC precursors in the steady state. BMPR1a expression is strongly upregulated in epidermal cells in psoriatic lesions, and BMPR1aΔCD11c mice showed a defect in the resolution phase of allergic and psoriatic skin inflammation. Moreover, whereas LCs from these mice expressed CD207, BMPR1a counteracted LC activation and migration from skin explant cultures. Therefore, TGFß1‒BMPR1a signaling seems to be required for the efficient induction of CD207 during LC differentiation in the steady state, and bone marrow‒derived lesional CD11c+ cells may limit established skin inflammation through enhanced BMPR1a signaling.

Calcitonin gene-related peptide upregulates IL-17A and IL-22 in γδ-T cells through the paracrine effect of langerhans cells on LC/γδ-T co-culture model.

Intense mental stimulation and stress often directly induce or exacerbate psoriasis. On the contrary, patients with nerve injury and nervous system dysfunction have psoriasis remission. The nervous system plays an important role in the inflammatory process of psoriasis, and neuropeptides are considered as local mediators of disease maintenance. To examine the molecular mechanism involved in this, first we analyzed calcitonin gene-related peptide (CGRP)-treated langerhans Cells and γδ-T cells separately. CGRP induced IL-23 mRNA and protein expression via PDK1-Rsk signaling pathway. However, CGRP had no effect on secretion of IL-17A and IL-22 in γδ-T cells. Then we treated LCs/γδ-T cells Co-culture Model with CGRP. CGRP upregulated IL-17A and IL-22 expression in co-culture model through the paracrine effect of LCs. IL-17A and IL-22 are key cytokines of psoriasis. These findings provide a potential mechanism by which nerve factors affect the development of psoriasis.

Skin-resident dendritic cells mediate postoperative pain via CCR4 on sensory neurons.

Inflammatory pain, such as hypersensitivity resulting from surgical tissue injury, occurs as a result of interactions between the immune and nervous systems with the orchestrated recruitment and activation of tissue-resident and circulating immune cells to the site of injury. Our previous studies identified a central role for Ly6Clow myeloid cells in the pathogenesis of postoperative pain. We now show that the chemokines CCL17 and CCL22, with their cognate receptor CCR4, are key mediators of this response. Both chemokines are up-regulated early after tissue injury by skin-resident dendritic and Langerhans cells to act on peripheral sensory neurons that express CCR4. CCL22, and to a lesser extent CCL17, elicit acute mechanical and thermal hypersensitivity when administered subcutaneously; this response abrogated by pharmacological blockade or genetic silencing of CCR4. Electrophysiological assessment of dissociated sensory neurons from naïve and postoperative mice showed that CCL22 was able to directly activate neurons and enhance their excitability after injury. These responses were blocked using C 021 and small interfering RNA (siRNA)-targeting CCR4. Finally, our data show that acute postoperative pain is significantly reduced in mice lacking CCR4, wild-type animals treated with CCR4 antagonist/siRNA, as well as transgenic mice depleted of dendritic cells. Together, these results suggest an essential role for the peripheral CCL17/22:CCR4 axis in the genesis of inflammatory pain via direct communication between skin-resident dendritic cells and sensory neurons, opening therapeutic avenues for its control.

Early antitumor activity of oral Langerhans cells is compromised by a carcinogen.

Early diagnosis of oral squamous cell carcinoma (OSCC) remains an unmet clinical need. Therefore, elucidating the initial events of OSCC preceding tumor development could benefit OSCC prognosis. Here, we define the Langerhans cells (LCs) of the tongue and demonstrate that LCs protect the epithelium from carcinogen-induced OSCC by rapidly priming αßT cells capable of eliminating γH2AX+ epithelial cells, whereas γδT and natural killer cells are dispensable. The carcinogen, however, dysregulates the epithelial resident mononuclear phagocytes, reducing LC frequencies, while dendritic cells (DCs), macrophages, and plasmacytoid DCs (pDCs) populate the epithelium. Single-cell RNA-sequencing analysis indicates that these newly differentiated cells display an immunosuppressive phenotype accompanied by an expansion of T regulatory (Treg) cells. Accumulation of the Treg cells was regulated, in part, by pDCs and precedes the formation of visible tumors. This suggests LCs play an early protective role during OSCC, yet the capacity of the carcinogen to dysregulate the differentiation of mononuclear phagocytes facilitates oral carcinogenesis.

Epicutaneous Administration of 17ß-Estradiol Induces Langerhans Cells Depletion.

BACKGROUND: Langerhans cells (LC) number and function in mouse vaginal mucosa are affected by 17ß-estradiol (E2) application; nonetheless, its effect on epidermal LC has not been studied. The purpose of this study was to evaluate the effect of topical administration of E2 on the number, phenotype, and migratory ability of LC in mouse skin. METHODS: Ears of adult CD1 male mice were topically treated once with several doses. Immunohistochemical staining for CD207 and TUNEL staining were performed. LC migration to lymph nodes and the effect on the expression of costimulatory molecules on cultured dendritic cells (DC) were also evaluated. RESULTS: E2 decreased the number of CD207+ LC in a dose-dependent manner. One hour after treatment, 1 and 10 µg/mL E2 significantly reduced the LC number by 21% and 26%, respectively, after two hours, the reduction was 23% and 41%, respectively. After 48 hours, LC recovered, and after 96 hours of treatment, the CD207+/MHCII+ DC numbers were increased in regional lymph nodes. However, CD86 and CD40 molecules were expressed at lower levels than in positive control. The TUNEL assay did not show apoptotic cells. Furthermore, in cultured DC, E2 promoted a decrease in CD40 and CD86 expression and an increase in CD273, CD274, MHCII, and CCR7. CONCLUSIONS: The topical administration of E2 induced a transitory local diminution of LC population and a tolerogenic phenotype. This decrease in epidermal LC suggests that E2 may affect skin immune responses, inducing an inhibitory response, which should be considered when prescribing topical E2 medications.

Targeted delivery of a vaccine protein to Langerhans cells in the human skin via the C-type lectin receptor Langerin.

Human skin is a preferred vaccination site as it harbors multiple dendritic cell (DC) subsets, which display distinct C-type lectin receptors (CLR) that recognize pathogens. Antigens can be delivered to CLR by antibodies or ligands to boost antigen-specific immune responses. This concept has been established in mouse models but detailed insights into the functional consequences of antigen delivery to human skin DC in situ are sparse. In this study, we cloned and produced an anti-human Langerin antibody conjugated to the EBV nuclear antigen 1 (EBNA1). We confirmed specific binding of anti-Langerin-EBNA1 to Langerhans cells (LC). This novel LC-based vaccine was then compared to an existing anti-DEC-205-EBNA1 fusion protein by loading LC in epidermal cell suspensions before coculturing them with autologous T cells. After restimulation with EBNA1-peptides, we detected elevated levels of IFN-γ- and TNF-α-positive CD4+ T cells with both vaccines. When we injected the fusion proteins intradermally into human skin explants, emigrated skin DC targeted via DEC-205-induced cytokine production by T cells, whereas the Langerin-based vaccine failed to do so. In summary, we demonstrate that antibody-targeting approaches via the skin are promising vaccination strategies, however, further optimizations of vaccines are required to induce potent immune responses.

GAG Multivalent Systems to Interact with Langerin.

Langerin is a C-type Lectin expressed at the surface of Langerhans cells, which play a pivotal role protecting organisms against pathogen infections. To address this aim, Langerin presents at least two recognition sites, one Ca2+-dependent and another one independent, which are capable to recognize a variety of carbohydrate ligands. In contrast to other lectins, Langerin recognizes sulfated glycosaminoglycans (GAGs), a family of complex and heterogeneous polysaccharides present in the cell membrane and the extracellular matrix, at the interphase generated in the trimeric form of Langerin but absent in the monomeric form. The complexity of these oligosaccharides has impeded the development of welldefined monodisperse structures to study these interaction processes. However, in the last few decades, an improvement of synthetic developments to achieve the preparation of carbohydrate multivalent systems mimicking the GAGs has been described. Despite all these contributions, very few examples are reported where the GAG multivalent structures are used to evaluate the interaction with Langerin. These molecules should pave the way to explore these GAG-Langerin interactions.