Monocyte-derived Langerhans Cells Research Articles

336 BNP supports monocyte-derived Langerhans cells’ anti-inflammatory properties during TLR stimulus

336 BNP supports monocyte-derived Langerhans cells’ anti-inflammatory properties during TLR stimulus

Monocytes give rise to Langerhans cells that preferentially migrate to lymph nodes at steady state

Current evidence suggests that ontogeny may account for the functional heterogeneity of some tissue macrophages, but not others. Here, we asked whether developmental origin drives different functions of skin Langerhans cells (LCs), an embryo-derived mononuclear phagocyte with features of both tissue macrophages and dendritic cells. Using time-course analyses, bone marrow chimeras, and fate tracing models, we found that the complete elimination of embryo-derived LCs at steady state results in their repopulation from circulating monocytes. However, monocyte-derived LCs inefficiently replenished the epidermal niche. Instead, these cells preferentially migrated to skin-draining lymph nodes. Mechanistically, we show that the enhanced migratory capability of monocyte-derived LCs is associated with higher expression of CD207/Langerin, a C-type lectin involved in the capture of skin microbes. Our data demonstrate that ontogeny plays a role in the migratory behavior of epidermal LCs.

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

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. 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. 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. 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.

Integrin α5 regulates motility of human monocyte-derived Langerhans cells during immune response.

Langerhans cells (LCs) are mainly present in the epidermis and mucosa, and have important roles during skin infection. Migration of LCs to lymph nodes is essential for antigen presentation. However, due to the difficulties in isolating and culturing human LCs, it is not fully understood how LCs move and interact with the extracellular matrix (ECM) through their adhesion molecules such as integrin, during the immune responses. In this study, we aimed to investigate LC motility, cell shape and the role of integrin under inflammatory conditions using monocyte-derived Langerhans cells (moLCs) as a model. As a result, lipopolysaccharide (LPS) stimulation increased adhesion on fibronectin coated substrate and integrin α5 expression in moLCs. Time-lapse imaging of moLCs revealed that stimulation with LPS elongated cell shape, whilst decreasing their motility. Additionally, this decrease in motility was not observed when pre-treated with a neutralising antibody targeting integrin α5. Together, our data suggested that activation of LCs decreases their motility by promoting integrin α5 expression to enhance their affinity to the fibronectin, which may contribute to their migration during inflammation.

1026 NPR1 receptor expression in monocyte-derived langerhans cells

1026 NPR1 receptor expression in monocyte-derived langerhans cells

932 Integrin α5 regulates dynamics and motility of human monocyte-derived langerhans cells during immune response

932 Integrin α5 regulates dynamics and motility of human monocyte-derived langerhans cells during immune response

TRPV4 Activation Increases the Expression of CD207 (Langerin) of Monocyte-Derived Langerhans Cells without Affecting their Maturation

Langerhans cells (LCs) are the sole professional antigen-presenting cell normally found in the human epidermal compartment. Research into their physiological role is hindered by the fact that they are invariably activated during isolation from the skin. To overcome this challenge, we turned to a monocyte-derived LC (moLC) model, which we characterized with RNA sequencing, and compared the transcriptome of moLCs with that of donor-matched immature dendritic cells. We found that moLCs express markers characteristic of LC2 cells as well as TRPV4. TRPV4 is especially important in the skin because it has been linked to the conservation of the skin barrier, immunological responses, as well as acute and chronic itch, but we know little about its function on LCs. Our results show that TRPV4 activation increased the expression of Langerin and led to increased intracellular calcium concentration in moLCs. Regarding the functionality of moLCs, we found that TRPV4 agonism had a mitigating effect on their inflammatory responses because it decreased their cytokine production and T-cell activating capability. Because TRPV4 has emerged as a potential therapeutic target in dermatological conditions, it is important to highlight LCs as, to our knowledge, a previously unreported target of these therapies.

CGRP inhibits human Langerhans cells infection with HSV by differentially modulating specific HSV-1 and HSV-2 entry mechanisms.

Herpes simplex virus (HSV) is widespread globally, with both HSV-1 and HSV-2 responsible for genital herpes. During sexual transmission, HSV targets epithelial cells, sensory peripheral pain neurons secreting the mucosal neuropeptide calcitonin gene-related peptide (CGRP), and mucosal immune cells including Langerhans cells (LCs). We previously described a neuro-immune crosstalk, whereby CGRP inhibits LCs-mediated human immunodeficiency virus type 1 (HIV-1) transmission. Herein, to further explore CGRP-mediated anti-viral function, we investigated whether CGRP affects LCs infection with HSV. We found that both HSV-1 and HSV-2 primary isolates productively infect monocyte-derived LCs (MDLCs) and inner foreskin LCs. Moreover, CGRP significantly inhibits infection with both HSV subtypes of MDLCs and langerinhigh, but not langerinlow, inner foreskin LCs. For HSV-1, infection is mediated via the HSV-1-specific entry receptor 3-O sulfated heparan sulfate (3-OS HS) in a pH-depended manner, and CGRP down-regulates 3-OS HS surface expression, as well as abrogates pH dependency. For HSV-2, infection involves langerin-mediated endocytosis in a pH-independent manner, and CGRP up-regulates surface expression of atypical langerin double-trimer oligomers. Our results show that CGRP inhibits mucosal HSV infection by differentially modulating subtype-specific entry receptors and mechanisms in human LCs. CGRP could turn out useful for prevention of LCs-mediated HSV infection and HSV/HIV-1 co-infection.

Usutu Virus escapes langerin-induced restriction to productively infect human Langerhans cells, unlike West Nile virus

ABSTRACT Usutu virus (USUV) and West Nile virus (WNV) are phylogenetically close emerging arboviruses and constitute a global public health threat. Since USUV and WNV are transmitted by mosquitoes, the first immune cells they encounter are skin-resident dendritic cells, the most peripheral outpost of immune defense. This unique network is composed of Langerhans cells (LCs) and dermal DCs, which reside in the epidermis and the dermis, respectively. Using human skin explants, we show that while both viruses can replicate in keratinocytes, they can also infect resident DCs with distinct tropism: WNV preferentially infects DCs in the dermis, whereas USUV has a greater propensity to infect LCs. Using both purified human epidermal LCs (eLCs) and monocyte derived LCs (MoLCs), we confirm that LCs sustain a faster and more efficient replication of USUV than WNV and that this correlates with a more intense innate immune response to USUV compared with WNV. Next, we show that ectopic expression of the LC-specific C-type lectin receptor (CLR), langerin, in HEK293T cells allows WNV and USUV to bind and enter, but supports the subsequent replication of USUV only. Conversely, blocking or silencing langerin in MoLCs or eLCs made them resistant to USUV infection, thus demonstrating that USUV uses langerin to enter and replicate in LCs. Altogether, our results demonstrate that LCs constitute privileged target cells for USUV in human skin, because langerin favours its entry and replication. Intriguingly, this suggests that USUV efficiently escapes the antiviral functions of langerin, which normally safeguards LCs from most viral infections.

233 Langerhans cells as targets of neuroimmune Communication in the Epidermal Compartment of Human Skin

The immune system is strongly linked to the nervous system, to the point that in many diseases they cannot be discussed as separate entities. Specifically, inflammatory skin diseases such as atopic dermatitis and psoriasis have been reported to be intensified by stress, and, interestingly the skin lesions in these diseases sometimes resolve after nerve injury. The putative cause underlying this empirical observation is that sensory nerves secrete neuropeptides (NPs) and other mediators that are important members of neuro-immune communication. Inside the epidermis, the only dedicated resident immune cells under steady-state are the antigen-presenting cells called Langerhans cells (LCs). LCs have been shown to be anatomically associated with neurons that produce Calcitonin Gene-Related Peptide (CGRP), supporting the hypothesis that NPs possibly play a role in cutaneous inflammation. Previous reports have shown that CGRP reduces LC antigen-presentation to a Th1 clone while simultaneously, enhancing antigen-presentation for Th2 clones in mice, but little is known in humans. As a first, exploratory step we utilized RNA sequencing from monocyte-derived LCs samples from five donors and we detected the expression of neuropeptide receptor genes. We found high expression of CGRP receptor and its coreceptor RAMP1 and also one of the neurotensin receptors (SORT1, also known as NTR3) and the Brain Natriuretic peptide receptor NPR1, and validated these with RT-qPCR. We also determined the effect of all three neuropeptides on the differentiation of monocyte-derived LCs with flow cytometry and found that NPR1 activation significantly increased the differentiation of LCs, while the others had no effect. Interestingly, BNP had only minimal effect on the T cell stimulatory capability of LCs, or on their cytokine (IL-6, IL-8, IL-10 and IL-12) production. These data suggest that neuroimmune communication might be an important prerequisite of LC differentiation.

194 Female hormones enhance HIV-1 acquisition in female Langerhans cells, but not in male Langerhans cells

Hormonal contraceptives, which are composed of progesterone with or without estrogen, are used world-widely. Several published epidemiologic reports have elucidated that usage of hormonal contraceptives increased the risk of HIV acquisition about two-fold in women of heterosexual HIV-1-serodiscordant couples. However, the underlying mechanism is not determined. Langerhans cells (LCs) distributed in vaginal epithelium and skin epidermis are the initial cellular target of HIV-1. HIV-1 productively infects LCs through CD4 and CCR5 on LCs, followed by migration of HIV-1-infected LCs to draining lymph nodes. Dendritic cells (DCs) distributed in vaginal lamina propria and skin dermis could be a target of HIV-1 in the case that epithelium is ablated. To examine whether 17β-estradiol and progesterone modulate HIV-1 infection in LCs and DCs, we generated monocyte-derived LCs and DCs (mLCs and mDCs, respectively). Pretreatment of female mLCs, but not male mLCs, with 17β-estradiol or progesterone increased HIV-1 infection about two-fold. Pretreatment of female and male mDCs with 17β-estradiol or progesterone did not modulate HIV-1 infection. Both 17β-estradiol and progesterone increased HIV-1 infection in mLCs from both premenopausal and postmenopausal female, suggesting that female hormones enhance the risk of HIV-1 acquisition in female mLCs. Interference of receptors for estrogen and progesterone did not inhibit the increased risk of HIV-1 acquisition by female hormones in female mLCs. Collectively, female hormones enhance risk of HIV-1 acquisition in female LCs thought unknown putative signaling pathway.

Extracellular vesicles produced by primary human keratinocytes in response to TLR agonists induce stimulus-specific responses in antigen-presenting cells

Cells can communicate through the extracellular vesicles (EVs) they secrete. Pathogen associated molecular patterns (PAMPs), alter the biophysical and communicative properties of EVs released from cells, but the functional consequences of these changes are unknown. Characterization of keratinocyte-derived EVs after poly(I:C) treatment (poly(I:C)-EVs) showed slight differences in levels of EV markers TSG101 and Alix, a loss of CD63 and were positive for autophagosome marker LC3b-II and the cytokine IL36γ compared to EVs from unstimulated keratinocytes (control-EVs). Flagellin treatment (flagellin-EVs) led to an EV marker profile like control-EVs but lacked LC3b-II. Flagellin-EVs also lacked IL-36γ despite nearly identical intracellular levels. While poly(I:C) treatment led to the clear emergence of a > 200 nm diameter EV sub-population, these were not found in flagellin-EVs. EV associated IL-36γ colocalized with LC3b-II in density gradient analysis, equilibrating to 1.10 g/mL, indicating a common EV species. Poly(I:C), but not flagellin, induced intracellular vesicles positive for IL-36γ, LC3b-II, Alix and TSG101, consistent with fusion of autophagosomes and multivesicular bodies. Simultaneous rapamycin and flagellin treatment induced similar intracellular vesicles but was insufficient for the release of IL-36γ+/LC3b-II+ EVs. Finally, a qRT-PCR array screen showed eight cytokine/chemokine transcripts were altered (p < 0.05) in monocyte-derived Langerhans cells (LCs) when stimulated with poly(I:C)-EVs while three were altered when LCs were stimulated with flagellin-EVs compared to control-EVs. After independent confirmation, poly(I:C)-EVs upregulated BMP6 (p = 0.035) and flagellin-EVs upregulated CXCL8 (p = 0.005), VEGFA (p = 0.018) and PTGS2 (p = 0.020) compared to control-EVs. We conclude that exogenous signals derived from pathogens can alter keratinocyte-mediated modulation of the local immune responses by inducing changes in the types of EVs secreted and responses in antigen presenting cells.

Notch-Mediated Generation of Monocyte-Derived Langerhans Cells: Phenotype and Function

Langerhans cells (LCs) in the skin are a first line of defense against pathogens but also play an essential role in skin homeostasis. Their exclusive expression of the C-type lectin receptor Langerin makes them prominent candidates for immunotherapy. For vaccine testing, an easily accessible cell platform would be desirable as an alternative to the time-consuming purification of LCs from human skin. Here, we present such a model and demonstrate that monocytes in the presence of GM-CSF, TGF-β1, and the Notch ligand DLL4 differentiate within 3 days into CD1a+Langerin+cells containing Birbeck granules. RNA sequencing of these monocyte-derived LCs (moLCs) confirmed gene expression of LC-related molecules, pattern recognition receptors, and enhanced expression of genes involved in the antigen-presenting machinery. On the protein level, moLCs showed low expression of costimulatory molecules but prominent expression of C-type lectin receptors. MoLCs can be matured, secrete IL-12p70 and TNF-α, and stimulate proliferation and cytokine production in allogeneic CD4+ and CD8+ T cells. In regard to vaccine testing, a recently characterized glycomimetic Langerin ligand conjugated to liposomes demonstrated specific and fast internalization into moLCs. Hence, these short-term in vitro‒generated moLCs represent an interesting tool to screen LC-based vaccines in the future.

Dendritic Cell Subsets in Intestinal Immunity and Inflammation.

The mammalian intestine is a complex environment that is constantly exposed to Ags derived from food, microbiota, and metabolites. Intestinal dendritic cells (DC) have the responsibility of establishing oral tolerance against these Ags while initiating immune responses against mucosal pathogens. We now know that DC are a heterogeneous population of innate immune cells composed of classical and monocyte-derived DC, Langerhans cells, and plasmacytoid DC. In the intestine, DC are found in organized lymphoid tissues, such as the mesenteric lymph nodes and Peyer's patches, as well as in the lamina propria. In this Brief Review, we review recent work that describes a division of labor between and collaboration among gut DC subsets in the context of intestinal homeostasis and inflammation. Understanding relationships between DC subtypes and their biological functions will rationalize oral vaccine design and will provide insights into treatments that quiet pathological intestinal inflammation.

406 TRPV4 stimulates differentiation of monocyte derived Langerhans cells without influencing their maturation

Transient receptor potential (TRP) ion channels, originally described mostly in neuronal cells, are now known to be expressed in numerous tissues, including immune cells. They affect many cellular functions, such as migration, cytokine secretion, and phagocytosis. TRP vanilloid (TRPV) channels were shown to be expressed on dendritic cells, where they decreased their differentiation and maturation. We have no information however, about their putative role on Langerhans cells (LCs). Our previous work highlighted the role of TRPV4 on monocyte-derived LCs (moLCs), where we showed that TRPV4 activation increased their differentiation, and led to an increase in intracellular calcium concentration, which effects could be abrogated by the specific antagonist of TRPV4. In our current work we further investigated the role of TRPV4 on moLCs. To support our functional data we showed that TRPV4 is expressed on the protein level. We next wanted to investigate the role of TRPV4 on the maturation of moLCs, which we induced by the addition of peptidoglycan (PGN). The TRPV4 agonist GSK1016790A when applied with PGN did not further stimulate the maturation of LCs, showing that TPRV4 influences the differentiation, but not the activation of moLCs. To determine what role LCs might play in the epidermal intracellular communication network we investigated the proteins secreted by our moLCs using a cytokine array. Our results showed that these cells produce CXCL8 and MIF among others, and that the transcription of these genes was not influenced by TRPV4 activation. Our results show that while TRPV4 stimulates the differentiation of moLCs, it does not affect the maturation of the cells.

Tissue microenvironment initiates an immune response to structural components of Staphylococcus aureus.

Cell-to-cell communication in skin participates to the maintenance of homeostatic responses to foreign substances. Certain strains of Staphylococcus (S) aureus are vicious pathogens that cause deleterious effects in host cells and tissues. Both secreted toxins and structural components of S.aureus trigger an immune response, though how S.aureus stimulates host immune responses is poorly understood. We explored here how keratinocytes and fibroblasts initiate the first steps of an immune response by activating dendritic cells (DCs) through recognition of structural components of S.aureus. We treated monocyte-derived Langerhans cells (moLCs) and monocyte-derived DCs (moDCs) with conditioned media from keratinocytes (K-CM) and fibroblasts (F-CM) treated with heat-killed S.aureus (HKSA) respectively, or directly with HKSA. Immune and inflammatory responses from keratinocytes, fibroblasts, moLCs and moDCs were assessed by analysis of cell surface markers and cytokine production using flow cytometry, real-time PCR and ELISA assays. K-CM and F-CM increased the expression of CD86 and HLA-DR on moLCs and moDCs, in association with a specific cytokine profile. K-CM upregulated TNFA, IL-1B and GM-CSF mRNA expression in moLCs, while F-CM upregulated IL-12 and downregulated TNFA and TGFB mRNA expression in moDCs. Additionally, F-CM attenuated the induction of an inflammatory profile in monocytes. The recognition of structural components from S.aureus by cutaneous microenvironment induces the activation and the expression of specific cytokines from LCs and DCs.

Differentiation of Langerhans Cells from Monocytes and Their Specific Function in Inducing IL-22-Specific Th Cells.

Human mucosal tissues and skin contain two distinct types of dendritic cell (DC) subsets, epidermal Langerhans cells (LCs) and dermal DCs, which can be distinguished by the expression of C-type lectin receptors, Langerin and DC-SIGN, respectively. Although peripheral blood monocytes differentiate into these distinct subsets, monocyte-derived LCs (moLCs) induced by coculture with GM-CSF, IL-4, and TGF-β1 coexpress both Langerin and DC-SIGN, suggesting that the environmental cues remain unclear. In this study, we show that LC differentiation is TGF-β1 dependent and that cofactors such as IL-4 and TNF-α promote TGF-β1-dependent LC differentiation into Langerin+DC-SIGN- moLCs but continuous exposure to IL-4 blocks differentiation. Steroids such as dexamethasone greatly enhanced TNF-α-induced moLC differentiation and blocked DC-SIGN expression. Consistent with primary LCs, dexamethasone-treated moLCs express CD1a, whereas monocyte-derived DCs (moDCs) express CD1b, CD1c, and CD1d. moDCs but not moLCs produced inflammatory cytokines after stimulation with CD1b and CD1d ligands mycolic acid and α-galactosylceramide, respectively. Strikingly, CD1a triggering with squalene on moLCs but not moDCs induced strong IL-22-producing CD4+ helper T cell responses. As IL-22 is an important cytokine in the maintenance of skin homeostasis, these data suggest that CD1a on LCs is involved in maintaining the immune barrier in the skin.

965 Expression and role pruritic receptors on monocyte-derived Langerhans cells

965 Expression and role pruritic receptors on monocyte-derived Langerhans cells

Resident and monocyte-derived Langerhans cells are required for imiquimod-induced psoriasis-like dermatitis model

BackgroundLangerhans cells (LCs) are dendritic cells that reside in the epidermis and local inflammation results in an increased differentiation of monocyte-derived LCs. Only few studies have investigated on the role of LCs in psoriasis-like dermatitis model, but the results are variable and the exact role of LCs in psoriasis model remains to be elucidated. ObjectiveTo explore the functional role of resident (rLCs) and monocyte-derived LCs (mLCs) in imiquimod (IMQ)-induced psoriasis-like inflammation using human Langerin-diphtheria toxin subunit A (huLang-DTA) mice. Methods5% IMQ cream was topically applied on the skins. Clinical and histopathological features were evaluated. Psoriasis-related gene expression was analyzed by quantitative polymerase chain reaction. The production of psoriasis-related cytokines including IL-17A and IL-22 by T cells were assessed by flow cytometry from the lesional skins. ResultshuLang-DTA mice showed a common depletion of both rLCs and mLCs in the IMQ-treated skins. huLang-DTA mice had a reduced IMQ-induced psoriasis-like inflammation featuring erythema, scale, and thickness compared with wild-type mice. Psoriatic lesions from huLang-DTA mice had a decreased level of Il23a and accordingly demonstrated an attenuated cytokine production of IL-17A and IL-22 from γδlow T cells. mLCs revealed a significantly greater level of IL-23 expression compared to rLCs in response to topical IMQ treatment. ConclusionAlthough both rLCs and mLCs are involved in the development of IMQ-induced psoriasis-like dermatitis, inflammation-induced mLCs present a superior capacity for producing IL-23 in this murine experimental model of psoriasis.

GM-CSF Monocyte-Derived Cells and Langerhans Cells As Part of the Dendritic Cell Family.

Dendritic cells (DCs) and macrophages (Mph) share many characteristics as components of the innate immune system. The criteria to classify the multitude of subsets within the mononuclear phagocyte system are currently phenotype, ontogeny, transcription patterns, epigenetic adaptations, and function. More recently, ontogenetic, transcriptional, and proteomic research approaches uncovered major developmental differences between Flt3L-dependent conventional DCs as compared with Mphs and monocyte-derived DCs (MoDCs), the latter mainly generated in vitro from murine bone marrow-derived DCs (BM-DCs) or human CD14+ peripheral blood monocytes. Conversely, in vitro GM-CSF-dependent monocyte-derived Mphs largely resemble MoDCs whereas tissue-resident Mphs show a common embryonic origin from yolk sac and fetal liver with Langerhans cells (LCs). The novel ontogenetic findings opened discussions on the terminology of DCs versus Mphs. Here, we bring forward arguments to facilitate definitions of BM-DCs, MoDCs, and LCs. We propose a group model of terminology for all DC subsets that attempts to encompass both ontogeny and function.