Human Epidermal Langerhans Cells Research Articles

216 Human epidermal Langerhans cells emigrate and induce a tolerogenic immune response in response to tick feeding and tick-borne pathogens

216 Human epidermal Langerhans cells emigrate and induce a tolerogenic immune response in response to tick feeding and tick-borne pathogens

Transcriptional programming of immunoregulatory responses in human Langerhans cells.

Human epidermal Langerhans cells (LCs) maintain immune homeostasis in the skin. To examine transcriptional programming of human primary LCs during homeostasis, we performed scRNA-seq analysis of LCs before and after migration from the epidermis, coupled with functional assessment of their regulatory T cell priming capabilities. The analysis revealed that steady-state LCs exist in a continuum of maturation states and upregulate antigen presentation genes along with an immunoregulatory module including the genes IDO1, LGALS1, LAMTOR1, IL4I, upon their migration. The migration-induced transition in genomic state is accompanied by the ability of LCs to more efficiently prime regulatory T cell responses in co-culture assays. Computational analyses of the scRNAseq datasets using SCENIC and Partial Information Decomposition in Context identified a set of migration-induced transcription factors including IRF4, KLF6 and RelB as key nodes within a immunoregulatory gene regulatory network. These findings support a model in which efficient priming of immunoregulatory responses by LCs is dependent on coordinated upregulation of a migration-coupled maturation program with a immunoregulation-promoting genomic module.

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.

Pioneers in Dermatology and Venereology: an interview with Prof. Lasse R. Braathen

Pioneers in Dermatology and Venereology: an interview with Prof. Lasse R. Braathen

Reduction in Human Epidermal Langerhans Cells with Age Is Associated with Decline in CXCL14-Mediated Recruitment of CD14+ Monocytes

The skin provides the first line of physical and immunological defense against environmental insults. However, the age-related changes in the immune function of human skin are unclear. Here, we investigated the age-related changes in epidermal Langerhans cells (LCs), which play a sentinel role in the initiation of the immune responses in the skin. We found a significant reduction in the number of epidermal LCs in sun-protected skin with age. Among the possible explanations for this reduction, the number of CD14+ CD207+ CCR6+ dermal-resident monocytes that can differentiate into epidermal LCs was markedly reduced with age (P=0.0057). Among the chemokines that can recruit these cells into the skin, the expression of CXCL14 was significantly down-regulated in epidermal keratinocytes with age. In addition, we discovered that young skin recruited a significantly higher number of monocytic THP-1 cells compared with old skin exvivo. This recruitment was blocked by CXCL14 neutralizing antibody and conversely promoted by CXCL14 treatment. Collectively, our findings indicate that decreased CXCL14-mediated recruitment of CD14+ monocytes in human skin results in the reduction of epidermal LCs with age, and CXCL14 may provide a therapeutic target for the prevention of age-related reduction in LCs.

Tacrolimus Reverses UVB Irradiation-Induced Epidermal Langerhans Cell Reduction by Inhibiting TNF-α Secretion in Keratinocytes via Regulation of NF-κB/p65.

Background: Topical calcineurin inhibitors including tacrolimus and pimecrolimus are used in the treatment of many inflammatory skin diseases mainly via blocking T-cell proliferation. Our previous studies found that pimecrolimus 1% cream could reverse high-dose ultraviolet B (UVB) irradiation-induced epidermal Langerhans cell (LC) reduction via inhibition of LC migration. We conducted this study to investigate the effects of topical tacrolimus 0.03% ointment on high-dose UVB-irradiated human epidermal LCs.Methods: Twenty fresh human foreskin tissues were randomly divided into four groups as follows: Control, Tacrolimus (0.03%), UVB (180 mJ/cm2), and UVB (180 mJ/cm2) + Tacrolimus (0.03%). Four time points were set as follows: 0, 18, 24, and 48 h. We collected culture medium and tissues at each time point. The percentage of CD1a+ cells in the medium was detected by means of flow cytometry. Each tissue was prepared for immunohistochemistry, real-time quantitative PCR, and western blot. HaCaT cells were cultured and divided into four groups: Control, Tacrolimus (1 μg/ml), UVB (30 mJ/cm2), and UVB (30 mJ/cm2) + Tacrolimus (1 μg/ml). The cells were incubated for 24 h and prepared for real-time quantitative PCR and western blot.Results: Topical tacrolimus significantly reversed high-dose UVB irradiation-induced epidermal LC reduction and CD1a+ cell increment in culture medium. Tacrolimus significantly inhibited UVB irradiation-induced tumor necrosis factor-α (TNF-α) and nuclear factor kappa B (NF-κB)/p65 mRNA and protein expression in HaCaT cells. Tacrolimus also significantly inhibited high-dose UVB irradiation-induced TNF-α expression in cultured tissues. Finally, TNF-α antagonist (recombinant human TNF-α receptor II: IgG Fc fusion protein) could significantly reverse UVB irradiation-induced epidermal LC reduction.Conclusion: Topical tacrolimus 0.03% could reverse UVB irradiation-induced epidermal LC reduction by inhibiting TNF-α secretion in keratinocytes via regulation of NF-κB/p65.

A type of human skin dendritic cell marked by CD5 is associated with the development of inflammatory skin disease.

Dendritic cells (DCs) are important in regulating immunity and tolerance and consist of functionally distinct subsets that differentially regulate T lymphocyte function. The underlying basis for this subset specificity is lacking, particularly in humans, where the classification of tissue DCs is currently incomplete. Examination of healthy human epidermal Langerhans cells and dermal skin cells revealed a tissue CD5-expressing DC subtype. The CD5+ DCs were potent inducers of cytotoxic T cells and Th22 cells. The products of these T cells, IL-22 and IFN-γ, play a key role in the pathogenesis of psoriasis. Remarkably, CD5+ DCs were significantly enriched in lesional psoriatic skin compared with distal tissues, suggesting their involvement in the disease. We show that CD5+ DCs can be differentiated from hematopoietic progenitor cells independently of the CD5- DCs. A progenitor population found in human cord blood and in the dermal skin layer, marked as CD34-CD123+CD117dimCD45RA+, was an immediate precursor of these CD11c+CD1c+CD5+ DCs. Overall, our discovery of the CD5-expressing DC subtype suggests that strategies to regulate their composition or function in the skin will represent an innovative approach for the treatment of immune-mediated disorders in and beyond the skin.

Identification of bone morphogenetic protein 7 (BMP7) as an instructive factor for human epidermal Langerhans cell differentiation

Human Langerhans cell (LC) precursors populate the epidermis early during prenatal development and thereafter undergo massive proliferation. The prototypic antiproliferative cytokine TGF-β1 is required for LC differentiation from human CD34(+) hematopoietic progenitor cells and blood monocytes in vitro. Similarly, TGF-β1 deficiency results in LC loss in vivo. However, immunohistology studies revealed that human LC niches in early prenatal epidermis and adult basal (germinal) keratinocyte layers lack detectable TGF-β1. Here we demonstrated that these LC niches express high levels of bone morphogenetic protein 7 (BMP7) and that Bmp7-deficient mice exhibit substantially diminished LC numbers, with the remaining cells appearing less dendritic. BMP7 induces LC differentiation and proliferation by activating the BMP type-I receptor ALK3 in the absence of canonical TGF-β1-ALK5 signaling. Conversely, TGF-β1-induced in vitro LC differentiation is mediated via ALK3; however, co-induction of ALK5 diminished TGF-β1-driven LC generation. Therefore, selective ALK3 signaling by BMP7 promotes high LC yields. Within epidermis, BMP7 shows an inverse expression pattern relative to TGF-β1, the latter induced in suprabasal layers and up-regulated in outer layers. We observed that TGF-β1 inhibits microbial activation of BMP7-generated LCs. Therefore, TGF-β1 in suprabasal/outer epidermal layers might inhibit LC activation, resulting in LC network maintenance.

Transcriptional and functional conservation between human Langerhans cells and mouse thymic DCs (P5067)

Abstract The characterization of human DC subsets is essential for the design of new vaccines and for better translation of mouse immunological data to the clinic. Previously, we showed that human epidermal Langerhans cells (LCs) were highly efficient at priming effector CD8+ T cell responses. However, the ability of mouse LCs to cross-present antigens and drive potent CTL responses is controversial. We used expression profiling to find the human LC equivalent in the mouse. Our studies suggest that the mouse equivalent to the human LC are the two mouse thymic DC subsets. Our studies also reveal a distinct genetic signature of over 25 genes, that could identify and predict which DC subsets can cross-present. The cross-presentation signature was present in the human blood CD141+ DCs and correctly identified the ability of other distinct human and mouse DC subsets to cross present. We confirmed these predictions with the functional ability of DCs to efficiently induce antigen-specific effector CD8+ T cells. Our studies may help bridge differences between mouse and human DCs and facilitate the application of these data to the clinic.

Identification of Axl as a downstream effector of TGF-β1 during Langerhans cell differentiation and epidermal homeostasis

Transforming growth factor-β1 (TGF-β1) is a fundamental regulator of immune cell development and function. In this study, we investigated the effects of TGF-β1 on the differentiation of human Langerhans cells (LCs) and identified Axl as a key TGF-β1 effector. Axl belongs to the TAM (Tyro3, Axl, and Mer) receptor tyrosine kinase family, whose members function as inhibitors of innate inflammatory responses in dendritic cells and are essential to the prevention of lupus-like autoimmunity. We found that Axl expression is induced by TGF-β1 during LC differentiation and that LC precursors acquire Axl early during differentiation. We also describe prominent steady-state expression as well as inflammation-induced activation of Axl in human epidermal keratinocytes and LCs. TGF-β1-induced Axl enhances apoptotic cell (AC) uptake and blocks proinflammatory cytokine production. The antiinflammatory role of Axl in the skin is reflected in a marked impairment of the LC network preceding spontaneous skin inflammation in mutant mice that lack all three TAM receptors. Our findings highlight the importance of constitutive Axl expression to tolerogenic barrier immunity in the epidermis and define a mechanism by which TGF-β1 enables silent homeostatic clearing of ACs to maintain long-term self-tolerance.

The differential production of cytokines by human Langerhans cells and dermal CD14+ DCs controls CTL priming

AbstractWe recently reported that human epidermal Langerhans cells (LCs) are more efficient than dermal CD14+ DCs at priming naive CD8+ T cells into potent CTLs. We hypothesized that distinctive dendritic cell (DC) cytokine expression profiles (ie, IL-15 produced by LCs and IL-10 expressed by dermal CD14+ DCs) might explain the observed functional difference. Blocking IL-15 during CD8+ T-cell priming reduced T-cell proliferation by ∼ 50%. These IL-15–deprived CD8+ T cells did not acquire the phenotype of effector memory cells. They secreted less IL-2 and IFN-γ and expressed only low amounts of CD107a, granzymes and perforin, and reduced levels of the antiapoptotic protein Bcl-2. Confocal microscopy analysis showed that IL-15 is localized at the immunologic synapse of LCs and naive CD8+ T cells. Conversely, blocking IL-10 during cocultures of dermal CD14+ DCs and naive CD8+ T cells enhanced the generation of effector CTLs, whereas addition of IL-10 to cultures of LCs and naive CD8+ T cells inhibited their induction. TGF-β1 that is transcribed by dermal CD14+ DCs further enhanced the inhibitory effect of IL-10. Thus, the respective production of IL-15 and IL-10 explains the contrasting effects of LCs and dermal CD14+ DCs on CD8+ T-cell priming.

Human Epidermal Langerhans Cells Maintain Immune Homeostasis in Skin by Activating Skin Resident Regulatory T Cells

Recent studies have demonstrated that the skin of a normal adult human contains 10-20 billion resident memory Tcells, including various helper, cytotoxic, and regulatory T cell subsets, that are poised to respond to environmental antigens. Using only autologous human tissues, we report that both invitro and invivo, resting epidermal Langerhan cells (LCs) selectively and specifically induced the activation and proliferation of skin resident regulatory T (Treg) cells, a minor subset of skin resident memory Tcells. In the presence of foreign pathogen, however, the same LCs activated and induced proliferation of effector memory T (Tem) cells and limited Treg cells' activation. These underappreciated properties of LCs, namely maintenance of tolerance in normal skin, and activation of protective skin resident memory Tcells upon infectious challenge, help clarify the role of LCs in skin.

HLA-DQA2 and HLA-DQB2 Genes Are Specifically Expressed in Human Langerhans Cells and Encode a New HLA Class II Molecule

The precise role of human epidermal Langerhans cells (LCs) in immune response is highly controversial. While studying the gene expression profile of these cells, we were intrigued to identify the HLA-DQB2 gene as potentially expressed in LCs. Despite a strong evolutionary conservation of their sequences, the concomitant expression of the poorly polymorphic HLA-DQA2/HLA-DQB2 genes, paralogous to the HLA-DQA1/HLA-DQB1 genes, has never been detected in any cell type. We confirmed by RT-PCR that the HLA-DQA2 and -DQB2 genes are both expressed in LCs, but not in monocyte-derived dendritic cells, or in blood CD1c(+) or plasmacytoid dendritic cells. The presence of the HLA-DQβ2 chain in LCs could be demonstrated by Western blotting, whereas immunofluorescence revealed its localization in early endosomes. As in the case of other HLA class II molecules, the HLA-DQα2 and -DQβ2 chains formed heterodimers that had to associate with the invariant chain to reach endosomal compartments. HLA-DQα2/β2 heterodimers were expressed at the cell surface, where they could mediate staphylococcal superantigen stimulation of T cells. Interestingly, HLA-DQα2 and HLA-DQβ1 chains formed mixed heterodimers which efficiently left the endoplasmic reticulum. These observations strongly suggest that the poorly polymorphic HLA-DQA2 and -DQB2 genes should be considered to be of immunological importance. The HLA-DQα2/β2 molecules could influence the complexity of the repertoire of Ags presented by LCs.

Inhibition of Transcription Factor Specificity Protein 1 Alters the Gene Expression Profile of Keratinocytes Leading to Upregulation of Kallikrein-Related Peptidases and Thymic Stromal Lymphopoietin

Transcription factor specificity protein 1 (Sp1) is involved in diverse cellular functions. We recently found that Sp1 was significantly decreased in skin biopsies from patients with atopic dermatitis (AD) and had an even greater reduction in AD patients with a history of eczema herpeticum. In the current study, we sought to better understand the role of Sp1 in skin biological processes by using a small interfering RNA (siRNA) technique to knock down Sp1 gene expression in normal human keratinocytes (NHK) and investigated the genome-wide gene expression profiling of Sp1- silenced NHK. The gene arrays revealed that 53 genes had more than three-fold changes in expression in Sp1-silenced NHK as compared to scrambled siRNA silenced cells. Strikingly, six kallikrein-related peptidase genes, KLK5, KLK6, KLK7, KLK8, KLK10, and KLK12 were up-regulated in NHK following Sp1 silencing. Functionally, protease activity was significantly enhanced in Sp1-silenced keratinocytes as compared to scrambled siRNA silenced keratinocytes. Moreover, thymic stromal lymphopoietin (TSLP), an epithelial derived TH2 promoting cytokine, was induced in Sp1-silenced keratinocytes due to elevated kallikrein activity. These results indicate that Sp1 expression deficiency leads to abnormally increased kallikrein protease activity in keratinocytes and may contribute to TH2 immune responses in the skin by inducing TSLP.

Identification of TROP2 (TACSTD2), an EpCAM-Like Molecule, as a Specific Marker for TGF-β1-Dependent Human Epidermal Langerhans Cells

Langerin (CD207) expression is a hallmark of epidermal Langerhans cells (LCs); however, CD207(+) cells comprise several functional subsets. Murine studies showed that epidermal, but not dermal, CD207(+) cells require transforming growth factor-β 1 (TGF-β1) for development, whereas human data are lacking. Using gene profiling, we found that the surface molecule TROP2 (TACSTD2) is strongly and rapidly induced during TGF-β1-dependent LC commitment of human CD34(+) hematopoietic progenitor cells or monocytes. TROP2 is conserved between mouse and human, and shares substantial amino-acid identity with EpCAM, a marker for murine epidermal LCs. To our knowledge, neither TROP2 nor EpCAM expression has been analyzed in human dendritic cell (DC) subsets. We found that (i) all human epidermal LCs are TROP2(+)EpCAM(+); (ii) human dermis lacks CD207(+)EpCAM(-) or CD207(+)TROP2(-) DCs, i.e., equivalents of murine dermal CD207(+) DCs; and (iii) pulmonary CD207(+) cells are TROP2(-)EpCAM(-). Moreover, although EpCAM was broadly expressed by pulmonary and intestinal epithelial cells, as well as by bone marrow erythroid progenitor cells, these cells lacked TROP2. However, although TROP2 is expressed by human LCs as well as by human and murine keratinocytes, most murine LCs, except of a small subset, lacked TROP2. Therefore, TROP2 is a marker for human TGF-β1-dependent epidermal LCs.

Human Epidermal Langerhans Cells Replenish Skin Xenografts and Are Depleted by Alloreactive T Cells In Vivo

Epidermal Langerhans cells (LC) are potent APCs surveying the skin. They are crucial regulators of T cell activation in the context of inflammatory skin disease and graft-versus-host disease (GVHD). In contrast to other dendritic cell subtypes, murine LC are able to reconstitute after local depletion without the need of peripheral blood-derived precursors. In this study, we introduce an experimental model of human skin grafted to NOD-SCID IL2Rγ(null) mice. In this model, we demonstrate that xenografting leads to the transient loss of LC from the human skin grafts. Despite the lack of a human hematopoietic system, human LC repopulated the xenografts 6 to 9 wk after transplantation. By staining of LC with the proliferation marker Ki67, we show that one third of the replenishing LC exhibit proliferative activity in vivo. We further used the skin xenograft as an in vivo model for human GVHD. HLA-disparate third-party T cells stimulated with skin donor-derived dendritic cells were injected intravenously into NOD-SCID IL2Rγ(null) mice that had been transplanted with human skin. The application of alloreactive T cells led to erythema and was associated with histological signs of GVHD limited to the transplanted human skin. The inflammation also led to the depletion of LC from the epidermis. In summary, we provide evidence that human LC are able to repopulate the skin independent of blood-derived precursor cells and that this at least partly relates to their proliferative capacity. Our data also propose xeno-transplantation of human skin as a model system for studying the role of skin dendritic cells in the efferent arm of GVHD.

Increased MAPK and NF-κB expression of Langerhans cells is dependent on TLR2 and TLR4, and increased IRF-3 expression is partially dependent on TLR4 following UV exposure

Toll-like receptors (TLRs) and epidermal Langerhans cells (LCs) play a crucial role in innate and adaptive immunity. To date, the pattern of TLR expression has not been fully analyzed. The effects of ultraviolet (UV) light on TLR expression and the downstream signaling cascades of human LC have not been examined. In this study, we purified human epidermal LCs using a density gradient centrifugation method and an immunomagnetic microbead method. We found that cultured purified LCs from human skin express mRNAs encoding TLR2, TLR4, TLR5 and TLR7-9. The expression of TLR2 and TLR4 protein was confirmed by Western blot analysis. The results showed for the first time that UV exposure up-regulated the mRNA and protein expression of TLR2 and TLR4 in human LCs. We also found that UV exposure-induced up-regulated MAPK and NF-κB/p65 expression was dependent on TLR2 and TLR4, and up-regulated IRF-3 expression was partially dependent on TLR4. In conclusion, UV light up-regulates the expression of TLR2, TLR4 and downstream signaling molecules MAPK, NF-κB/p65 and IRF-3 in human LCs. This suggests that UV light has a significant effect on skin immune responses.

Self‐renewal capacity of human epidermal Langerhans cells: observations made on a composite tissue allograft

Epidermal Langerhans cells (LC) are dendritic, antigen-presenting cells residing within mammalian epidermis and mucosal epithelia. When massively depleted, they are replaced by cells of bone-marrow origin. However, their renewal within normal skin under steady-state conditions is not precisely known. We observed that epidermal LC within a human hand allograft remain stable in the long term (10 years) and are not replaced by cells of recipient's origin; furthermore, we observed a Langerhans cell in mitosis within the epidermis 8 years postgraft. These results show that under almost physiological conditions, human LC renew in the epidermis by local mitoses of preexisting cells.

New insights into mechanisms of immunoregulation in 2007

Substantial progress in understanding the mechanisms of immune regulation in allergic diseases and asthma has been made during the last year. In asthma, rhinitis, and atopic dermatitis the immune system is activated by allergens, autoantigens, and components of superimposed infectious agents. Immune regulation in the lymphatic organs and in the tissue has an important role in the control and suppression of allergic disease in all stages of the inflammatory process, such as cell migration to tissues, cells gaining an inflammatory and tissue-destructive phenotype in the tissues, and their interaction with resident tissue cells to augment the inflammation. After the discovery of regulatory T cells, the importance of their unique suppressive capacity was strongly emphasized for the suppression of effector T-cell responses. However, it seems that all 3 subsets of effector T(H)1, T(H)2, and T(H)17 cells, as well as regulatory T cells, regulate each other at the level of transcription, major cytokines, and surface molecules. This review highlights key advances in immune regulation that were published in the Journal of Allergy and Clinical Immunology.

Functional Specializations of Human Epidermal Langerhans Cells and CD14 + Dermal Dendritic Cells

Little is known about the functional differences between the human skin myeloid dendritic cell (DC) subsets, epidermal CD207(+) Langerhans cells (LCs) and dermal CD14(+) DCs. We showed that CD14(+) DCs primed CD4(+) T cells into cells that induce naive B cells to switch isotype and become plasma cells. In contrast, LCs preferentially induced the differentiation of CD4(+) T cells secreting T helper 2 (Th2) cell cytokines and were efficient at priming and crosspriming naive CD8(+) T cells. A third DC population, CD14(-)CD207(-)CD1a(+) DC, which resides in the dermis, could activate CD8(+) T cells better than CD14(+) DCs but less efficiently than LCs. Thus, the human skin displays three DC subsets, two of which, i.e., CD14(+) DCs and LCs, display functional specializations, the preferential activation of humoral and cellular immunity, respectively.