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Near-infrared (NIR) can penetrate the dermis. NIR is able to regulate cutaneous component cells and immune cells and shows significant anti-inflammatory therapeutic effects. However, the mechanisms of these effects are largely unknown. The purpose of this study is to elucidate NIR-induced molecular mechanisms on macrophages because macrophages play initial roles in directing immune responses by their M1 or M2 polarizations. Proteomic analysis revealed that NIR radiation enhanced the expression of mitochondrial respiratory gene citrate synthase. This increased citrate synthase expression was triggered by NIR-induced H3K4 hypermethylation on the citrate synthase gene promoter but not by heat, which led to macrophage M2 polarization and finally resulted in TGFβ1 release from CD4 + cells. These cellular effects were validated in human primary macrophages and abdominal NIR-irradiated mouse experiments. In a phorbol 12-myristate 13-acetate‒induced inflammatory model on mouse ear, we confirmed that NIR irradiation induced significant anti-inflammatory effects through decreased M1 counts, reduced TNF-α, and increased CCL22 and/or TGFβ1 levels., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The extracts of Schisandra chinensis (Turcz.) Baill. (Schisandraceae) have various therapeutic effects, including inflammation and allergy. In this study, gomisin M2 (GM2) was isolated from S. chinensis and its beneficial effects were assessed against atopic dermatitis (AD). We evaluated the therapeutic effects of GM2 on 2,4-dinitrochlorobenzene (DNCB) and Dermatophagoides farinae extract (DFE)-induced AD-like skin lesions with BALB/c mice ears and within the tumor necrosis factor (TNF)-α and interferon (IFN)-γ-stimulated keratinocytes. The oral administration of GM2 resulted in reduced epidermal and dermal thickness, infiltration of tissue eosinophils, mast cells, and helper T cells in AD-like lesions. GM2 suppressed the expression of IL-1β, IL-4, IL-5, IL-6, IL-12a, and TSLP in ear tissue and the expression of IFN-γ, IL-4, and IL-17A in auricular lymph nodes. GM2 also inhibited STAT1 and NF-κB phosphorylation in DNCB/DFE-induced AD-like lesions. The oral administration of GM2 reduced levels of IgE (DFE-specific and total) and IgG2a in the mice sera, as well as protein levels of IL-4, IL-6, and TSLP in ear tissues. In TNF-α/IFN-γ-stimulated keratinocytes, GM2 significantly inhibited IL-1β, IL-6, CXCL8, and CCL22 through the suppression of STAT1 phosphorylation and the nuclear translocation of NF-κB. Taken together, these results indicate that GM2 is a biologically active compound that exhibits inhibitory effects on skin inflammation and suggests that GM2 might serve as a remedy in inflammatory skin diseases, specifically on AD.

During aging, skin accumulates senescent cells. The transient presence of senescent cells, followed by their clearance by the immune system, is important in tissue repair and homeostasis. The persistence of senescent cells that evade clearance contributes to the age-related deterioration of the skin. The senescence-associated secretory phenotype of these cells contains immunomodulatory molecules that facilitate clearance but also promote chronic damage. Here, we investigated the epilipidome-the oxidative modifications of phospholipids-of senescent dermal fibroblasts, because these molecules are among the bioactive lipids that were recently identified as senescence-associated secretory phenotype factors. Using replicative- and stress- induced senescence protocols, we identified lysophosphatidylcholines as universally elevated in senescent fibroblasts, whereas other oxidized lipids displayed a pattern that was characteristic for the used senescence protocol. When we tested the lysophosphatidylcholines for senescence-associated secretory phenotype activity, we found that they elicit chemokine release in nonsenescent fibroblasts but also interfere with toll-like receptor 2 and 6/CD36 signaling and phagocytic capacity in macrophages. Using matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry imaging, we localized two lysophosphatidylcholine species in aged skin. This suggests that lysophospholipids may facilitate immune evasion and low-grade chronic inflammation in skin aging., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Gradients of chemokines and growth factors guide migrating cells and morphogenetic processes. Migration of antigen-presenting dendritic cells from the interstitium into the lymphatic system is dependent on chemokine CCL21, which is secreted by endothelial cells of the lymphatic capillary, binds heparan sulfates and forms gradients decaying into the interstitium. Despite the importance of CCL21 gradients, and chemokine gradients in general, the mechanisms of gradient formation are unclear. Studies on fibroblast growth factors have shown that limited diffusion is crucial for gradient formation. Here, we used the mouse dermis as a model tissue to address the necessity of CCL21 anchoring to lymphatic capillary heparan sulfates in the formation of interstitial CCL21 gradients. Surprisingly, the absence of lymphatic endothelial heparan sulfates resulted only in a modest decrease of CCL21 levels at the lymphatic capillaries and did neither affect interstitial CCL21 gradient shape nor dendritic cell migration toward lymphatic capillaries. Thus, heparan sulfates at the level of the lymphatic endothelium are dispensable for the formation of a functional CCL21 gradient., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Vaahtomeri, Moussion, Hauschild and Sixt.)

There is substantial experimental evidence to indicate that Leishmania infections that are transmitted naturally by the bites of infected sand flies differ in fundamental ways from those initiated by needle inocula. We have used flow cytometry and intravital microscopy (IVM) to reveal the heterogeneity of sand fly transmission sites with respect to the subsets of phagocytes in the skin that harbor L. major within the first hours and days after infection. By flow cytometry analysis, dermis resident macrophages (TRMs) were on average the predominant infected cell type at 1 hr and 24 hr. By confocal IVM, the co-localization of L. major and neutrophils varied depending on the proximity of deposited parasites to the presumed site of vascular damage, defined by the highly localized swarming of neutrophils. Some of the dermal TRMs could be visualized acquiring their infections via transfer from or efferocytosis of parasitized neutrophils, providing direct evidence for the "Trojan Horse" model. The role of neutrophil engulfment by dermal TRMs and the involvement of the Tyro3/Axl/Mertk family of receptor tyrosine kinases in these interactions and in sustaining the anti-inflammatory program of dermal TRMs was supported by the effects observed in neutrophil depleted and in Axl-/-Mertk-/- mice. The Axl-/-Mertk-/- mice also displayed reduced parasite burdens but more severe pathology following L. major infection transmitted by sand fly bite., Competing Interests: The authors have declared that no competing interests exist.

Dengue virus infection (DENV-2) is transmitted by infected mosquitoes via the skin, where many dermal and epidermal cells are potentially susceptible to infection. Most of the cells in an area of infection will establish an antiviral microenvironment to control viral replication. Although cumulative studies report permissive DENV-2 infection in dendritic cells, keratinocytes, and fibroblasts, among other cells also infected, little information is available regarding cell-to-cell crosstalk and the effect of this on the outcome of the infection. Therefore, our study focused on understanding the contribution of fibroblast and dendritic cell crosstalk to the control or promotion of dengue. Our results suggest that dendritic cells promote an antiviral state over fibroblasts by enhancing the production of type I interferon, but not proinflammatory cytokines. Infected and non-infected fibroblasts promoted partial dendritic cell maturation, and the fibroblast-matured cells were less permissive to infection and showed enhanced type I interferon production. We also observed that the soluble mediators produced by non-infected or Poly (I:C) transfected fibroblasts induced allogenic T cell proliferation, but mediators produced by DENV-2 infected fibroblasts inhibited this phenomenon. Additionally, the effects of fibroblast soluble mediators on CD14 + monocytes were analyzed to assess whether they affected the differentiation of monocyte derived dendritic cells (moDC). Our data showed that mediators produced by infected fibroblasts induced variable levels of monocyte differentiation into dendritic cells, even in the presence of recombinant GM-CSF and IL-4. Cells with dendritic cell-like morphology appeared in the culture; however, flow cytometry analysis showed that the mediators did not fully downregulate CD14 nor did they upregulate CD1a. Our data revealed that fibroblast-dendritic cell crosstalk promoted an antiviral response mediated manly by type I interferons over fibroblasts. Furthermore, the maturation of dendritic cells and T cell proliferation were promoted, which was inhibited by DENV-2-induced mediators. Together, our results suggest that activation of the adaptive immune response is influenced by the crosstalk of skin resident cells and the intensity of innate immune responses established in the microenvironment of the infected skin., (Copyright © 2020 Montes-Gómez, García-Cordero, Marcial-Juárez, Shrivastava, Visoso-Carvajal, Juárez-Delgado, Flores-Romo, Sanchez-Torres, Santos-Argumedo, Bustos-Arriaga and Cedillo-Barrón.)

Nickel (Ni) is the most frequent metal allergen and induces Th1-dependent type-IV allergies. In local skin, epidermal Langerhans cells (LCs) and/or dermal dendritic cells (DCs) uptake antigens and migrate to draining lymph nodes (LNs). However, the subsets of antigen-presenting cells that contribute to Ni presentation have not yet been identified. In this study, we analyzed the Ni-binding capabilities of murine DCs using fluorescent metal indicator Newport Green. Elicitation of Ni allergy was assessed after intradermal (i.d.) injection of Ni-treated DCs into ear pinnae of Ni-sensitized mice. The Ni-binding capabilities of MHC class II hi CD11c int migratory DCs were significantly stronger than those of MHC class II int CD11c hi resident DCs and CD11c int PDCA1 + MHC class II int B220 + plasmacytoid DCs. Migratory DCs in skin-draining and mandibular LNs showed significantly stronger Ni-binding capabilities than those in mesenteric and medial iliac LNs. An i.d. injection of IL-1β induced the activation of LCs and dermal DCs with strong Ni-binding capabilities. Ni-binding LCs were detected in draining LNs after i.d. challenge with IL-1β and Ni. Moreover, an i.d. injection of Ni-treated DCs purified from skin-draining LNs elicited Ni-allergic inflammation. These results demonstrated that migratory DCs in skin-draining LNs have strong Ni-binding capabilities and elicit Ni allergy.

Transcutaneous immunization (TCI) via needle-free and non-invasive drug delivery systems is a promising approach for overcoming the current limitations of conventional parenteral vaccination methods. The targeted access to professional antigen-presenting cell (APC) populations within the skin, such as Langerhans cells (LCs), various dermal dendritic cells (dDCs), macrophages, and others makes the skin an ideal vaccination site to specifically shape immune responses as required. The stratum corneum (SC) of the skin is the main penetration barrier that needs to be overcome by the vaccine components in a coordinated way to achieve optimal access to dermal APC populations that induce priming of T-cell or B-cell responses for protective immunity. While there are numerous approaches to penetrating the SC, such as electroporation, sono- or iontophoresis, barrier and ablative methods, jet and powder injectors, and microneedle-mediated transport, we will focus this review on the recent progress made in particle-based systems for TCI. This particular approach delivers vaccine antigens together with adjuvants to perifollicular APCs by diffusion and deposition in hair follicles. Different delivery systems including nanoparticles and lipid-based systems, for example, solid nano-emulsions, and their impact on immune cells and generation of a memory effect are discussed. Moreover, challenges for TCI are addressed, including timely and targeted delivery of antigens and adjuvants to APCs within the skin as well as a deeper understanding of the ill-defined mechanisms leading to the induction of effective memory responses., (Copyright © 2020 Pielenhofer, Sohl, Windbergs, Langguth and Radsak.)

Staphylococcus aureus is a common skin commensal but is also associated with various skin and soft tissue pathologies. Upon invasion, S. aureus is detected by resident innate immune cells through pattern-recognition receptors (PRRs), although a comprehensive understanding of the specific molecular interactions is lacking. Recently, we demonstrated that the PRR langerin (CD207) on epidermal Langerhans cells senses the conserved β-1,4-linked N-acetylglucosamine (GlcNAc) modification on S. aureus wall teichoic acid (WTA), thereby increasing skin inflammation. Interestingly, the S. aureus ST395 lineage as well as certain species of coagulase-negative staphylococci (CoNS) produce a structurally different WTA molecule, consisting of poly-glycerolphosphate with α-O-N-acetylgalactosamine (GalNAc) residues, which are attached by the glycosyltransferase TagN. Here, we demonstrate that S. aureus ST395 strains interact with the human Macrophage galactose-type lectin (MGL; CD301) receptor, which is expressed by dendritic cells and macrophages in the dermis. MGL bound S. aureus ST395 in a tagN- and GalNAc-dependent manner but did not interact with different tagN-positive CoNS species. However, heterologous expression of Staphylococcus lugdunensis tagN in S. aureus conferred phage infection and MGL binding, confirming the role of this CoNS enzyme as GalNAc-transferase. Functionally, the detection of GalNAc on S. aureus ST395 WTA by human monocyte-derived dendritic cells significantly enhanced cytokine production. Together, our findings highlight differential recognition of S. aureus glycoprofiles by specific human innate receptors, which may affect downstream adaptive immune responses and pathogen clearance., (© 2019 The Authors Cellular Microbiology Published by John Wiley & Sons Ltd.)

Pemphigoid diseases are a subgroup of autoimmune skin diseases characterized by widespread tense blisters. Standard of care typically involves immunosuppressive treatments, which may be insufficient and are often associated with significant adverse events. As such, a deeper understanding of the pathomechanism(s) of pemphigoid diseases is necessary in order to identify improved therapeutic approaches. A major initiator of pemphigoid diseases is the accumulation of autoantibodies against proteins at the dermal-epidermal junction (DEJ), followed by protease activation at the lesion. The contribution of proteases to pemphigoid disease pathogenesis has been investigated using a combination of in vitro and in vivo models. These studies suggest proteolytic degradation of anchoring proteins proximal to the DEJ is crucial for dermal-epidermal separation and blister formation. In addition, proteases can also augment inflammation, expose autoantigenic cryptic epitopes, and/or provoke autoantigen spreading, which are all important in pemphigoid disease pathology. The present review summarizes and critically evaluates the current understanding with respect to the role of proteases in pemphigoid diseases.

Psoriasis is a chronic autoimmune skin disease that can often be triggered upon skin injury, known as Koebner phenomenon. Type 1 interferons (IFNα and IFNβ), key cytokines that activate autoimmunity during viral infection, have been suggested to play an indispensable role in initiating psoriasis during skin injury. Type 1 IFN-inducible gene signature has been identified as one of the major upregulated gene signatures in psoriatic skin. Type 1 IFNs treatments often directly induce or exacerbate psoriasis, whereas blocking type 1 IFNs signaling pathway in animal models effectively inhibits the development of T cell-mediated skin inflammation and psoriasis-like inflammatory diseases. Epidermal keratinocytes (KCs) occupy the outermost position in the skin and are the first responder to skin injury. Skin injury rapidly induces IFNβ from KCs and IFNα from dermal plasmacytoid dendritic cells (pDCs) through distinct mechanisms. Host antimicrobial peptide LL37 potentiates double-stranded RNA (dsRNA) immune pathways in keratinocytes and single-stranded RNA or DNA pathways in pDCs, leading to production of distinct type 1 IFN genes. IFNβ from KC promotes dendritic cell maturation and the subsequent T cell proliferation, contributing to autoimmune activation during skin injury and psoriasis pathogenesis. Accumulating evidences have indicated an important role of this dsRNA immune pathway in psoriasis pathogenesis. Together, this review describes how skin injury induces type 1 IFNs from skin cells and how this may initiate autoimmune cascades that trigger psoriasis. Targeting keratinocytes or type 1 IFNs in combination with T cell therapy may result in more sustainable effect to treat auto-inflammatory skin diseases such as psoriasis.

Juvenile-onset systemic sclerosis (jSSc) is a rare and severe autoimmune disease with associated life-threatening organ inflammation and evidence of fibrosis. The organ manifestations of jSSc resemble adult SSc, but with better outcomes and survival. The etiology of jSSc appears to reflect adult-onset SSc, with similar inflammatory mediators and autoantibodies, but with a significant population of children with uncharacterized anti-nuclear antibodies. The genetics of patients with jSSc differ from women with SSc, resembling instead the genes of adult males with SSc, with additional HLA genes uniquely associated with childhood-onset disease. Current treatments are aimed at inhibiting the inflammatory aspect of disease, but important mechanisms of fibrosis regulated by dermal white adipose tissue dendritic cells may provide an avenue for targeting and potentially reversing the fibrotic stage.

The skin comprises tissue macrophages as the most abundant resident immune cell type. Their diverse tasks including resistance against invading pathogens, attraction of bypassing immune cells from vessels, and tissue repair require dynamic specification. Here, we delineated the postnatal development of dermal macrophages and their differentiation into subsets by adapting single-cell transcriptomics, fate mapping, and imaging. Thereby we identified a phenotypically and transcriptionally distinct subset of prenatally seeded dermal macrophages that self-maintained with very low postnatal exchange by hematopoietic stem cells. These macrophages specifically interacted with sensory nerves and surveilled and trimmed the myelin sheath. Overall, resident dermal macrophages contributed to axon sprouting after mechanical injury. In summary, our data show long-lasting functional specification of macrophages in the dermis that is driven by stepwise adaptation to guiding structures and ensures codevelopment of ontogenetically distinct cells within the same compartment., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Atopic dermatitis is a chronic inflammatory skin disease with persistent pruritus. To clarify its molecular mechanism, it is important to establish a mouse model similar to the phenotypes of atopic dermatitis patients, particularly in exhibiting scratching behavior. Ikk2, a component of the IκB kinase complex, exerts pro-inflammatory responses, whereas its deficiency in keratinocytes paradoxically causes skin inflammation. In this study, we sought to generate a mouse model exhibiting skin inflammation by which dermal fibroblasts lack Ikk2 expression and evaluate whether cutaneous inflammatory phenotypes are similar to those of atopic dermatitis patients. To generate Ikk2-deficient mice (Nestin cre ;Ikk2 FL/FL ) in which Ikk2 is deleted in dermal fibroblasts, we crossed female Ikk2 FL/FL mice to male Nestin cre ;Ikk2 FL/+ mice. These mice spontaneously developed skin inflammation limited to the face, with the appearance of Ikk2-deficient fibroblasts in the facial skin. These mice showed phenotypes similar to those of atopic dermatitis patients, including scratching behaviors, which are resistant to immunosuppressive or molecularly targeted drugs. These findings suggest that the Nestin cre ;Ikk2 FL/FL mouse is an atopic dermatitis model that will be useful in clarifying atopic dermatitis pathogenesis and in developing a novel therapeutic agent for atopic dermatitis symptoms., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Dermal white adipose tissue is a unique layer of adipocytes within the reticular dermis of the skin. Recently, several nonmetabolic activities have been discovered for dWAT and its fibroblast precursors. These functions include antimicrobial defense and roles in hair cycling, wound healing, and thermogenesis. In this review, we discuss recent progress in understanding the role of dermal white adipose tissue in immunity, both as an innate antimicrobial cell type and as an indirect communicator with other cutaneous immunocytes to enhance defense and potentially contribute to inflammatory disease., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Psoriasis is an autoimmune chronic dermatosis that is T cell-mediated, characterized by epidermal thickening, aberrant epidermal differentiation and inflammatory infiltrates, with a dominant Th1 and Th17 profile. Additional in vitro models are required to study the complex interactions between activated T cells and skin cells, and to develop new, more effective treatments. We have therefore sought to model this psoriatic inflammation by the generation of tissue-engineered immunocompetent tissues, and we have investigated the response of activated T-cell infiltration in models produced with lesional psoriatic skin cells on major hallmarks of psoriasis. The immunocompetent lesional skin model displayed a delayed onset of epidermal differentiation, an hyperproliferation of the basal keratinocytes, a drastic increase in the secretion of proinflammatory cytokines, and a disturbed expression of key transcription factors, as observed in lesional plaques, suggesting a crucial importance of combining the pathological phenotype of cutaneous cells to T cells in order to generate a relevant model for psoriasis. Finally, we found this skin model to be responsive to methotrexate treatment, making it a valuable tool for drug development.

Lesion features in cutaneous leishmaniasis (CL) depend on the infecting Leishmania species as well as on host immune reponse. In this study, we evaluated the histological and immunological differences between two forms of CL described in Tunisia: zoonotic cutaneous leishmaniasis (ZCL) caused by L. major and sporadic cutaneous leishmaniasis (SCL) caused by L. infantum. Histological analysis showed a mild to moderate infiltrate within ZCL lesions. In contrast, massive infiltration of the dermis was observed within SCL lesions. Contrary to ZCL, infiltrates within SCL lesions were organized and showed granuloma composed of macrophages and lymphocytes. In addition, immunohistochemical analysis showed a predominance of CD4 + T cells within both CL forms. Furthermore, expression of interferon-γ, interleukin (IL)-10, IL-8, IL-13 and monocyte chemotactic protein (MCP)-1 was evaluated using real-time quantitative polymerase chain reaction (RT-qPCR). MCP-1 and IL-10 were expressed at comparable levels in ZCL and SCL lesions. Interestingly, IL-8 mRNA levels were significantly higher in ZCL lesions compared to SCL lesions, but interferon-γ was significantly higher in SCL lesions than in ZCL lesions., (© T. Boussoffara et al., published by EDP Sciences, 2019.)

The skin immune system must discriminate between innocuous antigens and pathogens. Antigen applied topically using a Viaskin® patch elicits immune tolerance that can suppress colitis and food allergy. Here we show how topical antigen is acquired and presented by dendritic cells in the skin. Topical antigen is acquired by Langerhans cells (LC) and CD11b + cDC2s but not cDC1s, and both  LCs and CD11b + cDC2s reaching the lymph node can prime T cells and expand LAP + Tregs. However, LCs are neither required nor sufficient for T cell priming, and have no role in tolerance induction. Conversely, IRF-4-dependent cDC2s are required for T cell priming. Acquisition of antigen in the dermis, delivery to the draining lymph node, and generation of tolerance are all absent in hairless mice. These results indicate an important function for hair follicle niche and CD11b + cDC2s in antigen acquisition, and in generation of primary immune tolerance to topical antigens.

Mechanisms of immune suppression in peripheral tissues counteract protective immunity to prevent immunopathology and are coopted by tumors for immune evasion. While lymphatic vessels facilitate T cell priming, they also exert immune suppressive effects in lymph nodes at steady-state. Therefore, we hypothesized that peripheral lymphatic vessels acquire suppressive mechanisms to limit local effector CD8 + T cell accumulation in murine skin. We demonstrate that nonhematopoietic PD-L1 is largely expressed by lymphatic and blood endothelial cells and limits CD8 + T cell accumulation in tumor microenvironments. IFNγ produced by tissue-infiltrating, antigen-specific CD8 + T cells, which are in close proximity to tumor-associated lymphatic vessels, is sufficient to induce lymphatic vessel PD-L1 expression. Disruption of IFNγ-dependent crosstalk through lymphatic-specific loss of IFNγR boosts T cell accumulation in infected and malignant skin leading to increased viral pathology and tumor control, respectively. Consequently, we identify IFNγR as an immunological switch in lymphatic vessels that balances protective immunity and immunopathology leading to adaptive immune resistance in melanoma., (© 2018 Lane et al.)

Antimicrobial resistance needs to be tackled from new angles, and antimicrobial peptides could be future candidates for combating bacterial infections. This study aims to investigate in vitro the bactericidal effects of the lantibiotic gallidermin on Staphylococcus epidermidis and Staphylococcus aureus, possible cytotoxic effects and its impact on host-microbe interactions. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of gallidermin were determined, and cytotoxicity and proinflammatory effects of gallidermin on fibroblasts, red blood cells (RBCs) and in whole blood were investigated. Both MIC and MBC for all four tested strains of S. epidermidis was 6.25 μg/ml. Both MIC and MBC for methicillin-sensitive S. aureus was 12.5 μg/ml and for methicillin-resistant S. aureus (MRSA) 1.56 μg/ml. Gallidermin displayed no cytotoxic effects on fibroblasts, only a high dose of gallidermin induced low levels of CXCL8 and interleukin-6. Gallidermin hemolyzed less than 1% of human RBCs, and did not induce reactive oxygen species production or cell aggregation in whole blood. In cell culture, gallidermin inhibited the cytotoxic effects of the bacteria and totally suppressed the bacteria-induced release of CXCL8 and interleukin-6 from fibroblasts. We demonstrate that gallidermin, expressing low cell cytotoxicity, is a promising candidate for treating bacterial infections caused by S. epidermidis and S. aureus, especially MRSA., (© 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Plasmodium sporozoites are injected into the skin as mosquitoes probe for blood. From here, they migrate through the dermis to find blood vessels which they enter in order to be rapidly carried to the liver, where they invade hepatocytes and develop into the next life cycle stage, the exoerythrocytic stage. Once sporozoites enter the blood circulation, they are found in hepatocytes within minutes. In contrast, sporozoite exit from the inoculation site resembles a slow trickle and occurs over several hours. Thus, sporozoites spend the majority of their extracellular time at the inoculation site, raising the hypothesis that this is when the malarial parasite is most vulnerable to antibody-mediated destruction. Here, we investigate this hypothesis and demonstrate that the neutralizing capacity of circulating antibodies is greater at the inoculation site than in the blood circulation. Furthermore, these antibodies are working, at least in part, by impacting sporozoite motility at the inoculation site. Using actively and passively immunized mice, we found that most parasites are either immobilized at the site of injection or display reduced motility, particularly in their net displacement. We also found that antibodies severely impair the entry of sporozoites into the bloodstream. Overall, our data suggest that antibodies targeting the migratory sporozoite exert a large proportion of their protective effect at the inoculation site. IMPORTANCE Studies in experimental animal models and humans have shown that antibodies against Plasmodium sporozoites abolish parasite infectivity and provide sterile immunity. While it is well documented that these antibodies can be induced after immunization with attenuated parasites or subunit vaccines, the mechanisms by and location in which they neutralize parasites have not been fully elucidated. Here, we report studies indicating that these antibodies display a significant portion of their protective effect in the skin after injection of sporozoites and that one mechanism by which they work is by impairing sporozoite motility, thus diminishing their ability to reach blood vessels. These results suggest that immune protection against malaria begins at the earliest stages of parasite infection and emphasize the need of performing parasite challenge in the skin for the evaluation of protective immunity., (Copyright © 2018 Flores-Garcia et al.)

Objective: Graft rejection, with the possibility of a violent immune response, may be severe and life threatening. Our aim was to thoroughly investigate the biocompatibility and immunotoxicology of collagen-based dermal matrix (DM) before assessment in clinical trials., Methods: DM was subcutaneously implanted in BALB/c mice in two doses to induce a potential immune response. The spleen and lymph nodes were assessed for shape, cell number, cell phenotype via flow cytometry, cell activation via CCK8 kit, Annexin V kit, and Ki67 immunostaining. Serum samples were used to measure antibody concentration by enzyme-linked immunosorbent assay. Local inflammation was analyzed by histology and immunohistochemistry staining. Data analysis was performed by one-way ANOVA and non-parametric tests., Results: Our data illustrate that the spleen and lymph node sizes were similar between the negative control mice and mice implanted with DM. However, in the high-dose DM (DM-H) group, the total cell populations in the spleen and lymph nodes, T cells and B cells in the spleen had slight increases in prophase, and the low-dose DM (DM-L) group did not display gross abnormities. Moreover, DM-H initiated moderate cell activation and proliferation in the early phase post-immunization, whereas DM-L did not. Neither DM-H nor DM-L implantation noticeably increased IgM and IgG serum concentrations. Examination of the local cellular response revealed only benign cell infiltration and TNF-α expression in slides of DM in the early phase., Conclusion: Overall, DM-H may have induced a benign temporary acute immune response post-implantation, whereas DM-L had quite low immunogenicity. Thus, this DM can be regarded as a safe product., (Copyright © 2018 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

Background and Objectives: Surgery provokes inflammatory and immune responses, so efforts have been made to reduce host response by using less invasive techniques. The purpose of this experimental study was to investigate the surgical stress induced by skin incision and the role of liver response in this process., Methods: Seventy male anesthetized Wistar rats were subjected to a midline incision confined strictly to the skin (dermis) of either 1 cm long (n = 20), 10 cm long (n = 20), or no incision (n = 20) or served as controls (n = 10). Skin trauma was left open for a 20-minutes period, and then was meticulously sutured. At 3 and 24 hours later, laparotomy was performed on half the rats of each group, for blood and liver sampling. In serum and liver homogenates, cytokine-induced neutrophil chemoattractant (CINC)1/interleukin (IL)-8 and tumor necrosis factor (TNF)-α levels were measured with enzyme-linked immunosorbent assays and nitric oxide (NO) using a Griess reaction., Results: Skin trauma was found to significantly ( P < .01) increase all inflammatory mediators tested (CINC1/IL-8, TNF-α, NO) in serum of operated rats versus controls, the increase being proportionally dependent on the length of skin incision. In liver homogenates, CINC1/IL-8 was significantly ( P < .01) increased in operated animals versus controls, similarly to serum levels. In contrast, liver TNF-α levels were inversely related to serum levels, and a significant ( P < .01) decrease in TNF-α was observed in liver homogenates of operated animals compared with the controls, indicating that the increased TNF-α in blood reflects liver TNF-α secretion., Conclusion: Our findings suggest that inflammatory and immune reactions induced by skin-only surgical trauma are closely correlated to the length of skin incision., Competing Interests: Conflicts of Interest: All authors declare no conflict of interest regarding the publication of this article.

Infectious diseases propagated by arthropod vectors, such as tularemia, are commonly initiated via dermal infection of the skin. However, due to the technical difficulties in achieving accurate and reproducible dermal deposition, intradermal models are less commonly used. To overcome these limitations, we used microneedle arrays (MNAs), which are micron-scale polymeric structures, to temporarily disrupt the barrier function of the skin and deliver a bacterial inoculum directly to the dermis of an animal. MNAs increase reliability by eliminating leakage of the inoculum or blood from the injection site, thereby providing a biologically relevant model for arthropod-initiated disease. Here, we validate the use of MNAs as a means to induce intradermal infection using a murine model of tularemia initiated by Francisella novicida We demonstrate targeted delivery of the MNA bolus to the dermal layer of the skin, which subsequently led to innate immune cell infiltration. Additionally, F. novicida -coated MNAs were used to achieve lethality in a dose-dependent manner in C57BL/6 mice. The immune profile of infected mice mirrored that of established F. novicida infection models, consisting of markedly increased serum levels of interleukin-6 and keratinocyte chemoattractant, splenic T-cell depletion, and an increase in splenic granulocytes, together confirming that MNAs can be used to reproducibly induce tularemia-like pathogenesis in mice. When MNAs were used to immunize mice using an attenuated F. novicida mutant ( F. novicida Δ lpxD1 ), all immunized mice survived a lethal subcutaneous challenge. Thus, MNAs can be used to effectively deliver viable bacteria in vivo and provide a novel avenue to study intradermally induced microbial diseases in animal models., (Copyright © 2018 American Society for Microbiology.)

Multidrug-resistant bacterial strains are a rapidly emerging healthcare threat; therefore it is critical to develop new therapies to combat these organisms. Prior antibacterial strategies directly target pathogen growth or viability. Host-directed strategies to increase antimicrobial defenses may be an effective alternative to antibiotics and reduce development of resistant strains. In this study, we demonstrated the efficacy of a pyrimidine synthesis inhibitor, N-phosphonacetyl-L-aspartate (PALA), to enhance clearance of methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Acinetobacter baumannii strains by primary human dermal fibroblasts in vitro. PALA did not have a direct bactericidal effect, but enhanced cellular secretion of the antimicrobial peptides human β-defensin 2 (HBD2) and HBD3 from fibroblasts. When tested in porcine and human skin explant models, a topical PALA formulation was efficacious to enhance MRSA, P. aeruginosa, and A. baumannii clearance. Topical PALA treatment of human skin explants also resulted in increased HBD2 and cathelicidin (LL-37) production. The antimicrobial actions of PALA required expression of nucleotide-binding, oligomerization domain 2 (NOD2), receptor-interacting serine/threonine-protein kinase 2 (RIP2), and carbamoyl phosphatase synthase II/aspartate transcarbamylase/dihydroorotase (CAD). Our results indicate that PALA may be a new option to combat multidrug-resistant bacterial infections of the skin through enhancement of an integral pathway of the cutaneous innate immune defense system.

RTN1 is an endoplasmic reticulum-associated protein that was initially identified in neuronal tissues. Here we show that the main isoform RTN1A is a marker for dendritic cells. In the skin, HLA-DR + CD1a high CD207 + CD11c weak Langerhans cells were the only cells in the epidermis, and HLA-DR + CD11c + dendritic cells were the main cells in the dermis, expressing this protein. RTN1A + dendritic cells were also found in gingiva, trachea, tonsil, thymus, and peripheral blood. During differentiation of MUTZ-3 cells into Langerhans cells, expression of RTN1A mRNA and protein preceded established Langerhans cell markers CD1a and CD207, and RTN1A protein partially co-localized with the endoplasmic reticulum marker protein disulfide isomerase. In line with this observation, we found that RTN1A was expressed by around 80% of Langerhans cell precursors in human embryonic skin. Our findings show that RTN1A is a marker for cells of the dendritic lineage, including Langerhans cells and dermal dendritic cells. This unexpected finding will serve as a starting point for the elucidation of the, until now, elusive functional roles of RTN1A in both the immune and the nervous system., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Conventional dendritic cells (cDCs) are composed of heterogeneous subsets commonly arising from dendritic cell (DC)-committed progenitors. A population of CD301b-expressing DCs has recently been identified in non-lymphoid barrier tissues such as skin. However, whether CD301b + DCs in the skin represent an ontogenetically unique subpopulation of migratory cDCs has not been fully addressed. Here, we demonstrated that CD301b + dermal DCs were distinct subpopulation of FMS-like tyrosine kinase 3 ligand (FLT3L)-dependent CD11b + cDC2 lineage, which required an additional GM-CSF cue for the adequate development. Although the majority of lymphoid-resident cDC2 lacked CD301b expression, dermal migratory cDC2 contained a substantial fraction of CD301b + subset. Similar to CD301b - population, CD301b + dermal DC development was closely regulated by FLT3 signaling, suggesting their common origin from FLT3L-responsive cDC progenitors. However, FLT3L-driven cDC progenitor culture was not sufficient, but additional GM-CSF treatment was required to produce CD301b + cDC2. In vivo development of CD301b + cDC2 was significantly augmented by exogenous GM-CSF, while the repopulation of CD301b + dermal cDC2 was abrogated by GM-CSF neutralization. Functionally, CD301b + cDC2 was capable of producing a high level of IL-23, and the depletion of CD301b + cDC2 effectively prevented IL-17-mediated psoriasiform dermatitis. Therefore, our findings highlight the differentiation program of a distinct CD301b + dermal cDC2 subset in the skin and its involvement in psoriatic inflammation., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Conditioning regimens preceding allogeneic stem cell transplantation (alloSCT) can cause tissue damage and acceleration of the development of graft-versus-host disease (GVHD). T-cell-depleted alloSCT with postponed donor lymphocyte infusion (DLI) may reduce GVHD, because tissue injury can be restored at the time of DLI. In this study, we investigated the presence of tissue injury and inflammation in skin during the period of hematologic recovery and immune reconstitution after alloSCT., Methods: Skin biopsies were immunohistochemically stained for HLA class II, CD1a, CD11c, CD40, CD54, CD68, CD86, CD206, CD3, and CD8. HLA class II-expressing cells were characterized as activated T-cells, antigen-presenting cells (APCs), or tissue repairing macrophages. In sex-mismatched patient and donor couples, origin of cells was determined by multiplex analysis combining XY-FISH and fluorescent immunohistochemistry., Results: No inflammatory environment due to pretransplant conditioning was detected at the time of alloSCT, irrespective of the conditioning regimen. An increase in HLA class II-positive macrophages and CD3 T-cells was observed 12-24 weeks after myeloablative alloSCT, but these macrophages did not show signs of interaction with the co-localized T-cells. In contrast, during GVHD, an increase in HLA class II-expressing cells coinciding with T-cell interaction was observed, resulting in an overt inflammatory reaction with the presence of activated APC, activated donor T-cells, and localized upregulation of HLA class II expression on epidermal cells. In the absence of GVHD, patient derived macrophages were gradually replaced by donor-derived macrophages although patient-derived macrophages were detectable even 24 weeks after alloSCT., Conclusion: Conditioning regimens cause tissue damage in the skin, but this does not result in a local increase of activated APC. In contrast to the inflamed situation in GVHD, when interaction takes place between activated APC and donor T-cells, the tissue damage caused by myeloablative alloSCT results in dermal recruitment of HLA class II-positive tissue repairing macrophages co-existing with increased numbers of patient- and donor-derived T-cells, but without signs of specific interaction and initiation of an immune response. Thus, the local skin damage caused by the conditioning regimen appears to be insufficient as single factor to provoke GVHD induction.

Therapeutic strategy in late 20th century to prevent allergic diseases was derived from a conceptual framework of allergens elimination which was as same as that of coping with them after their onset. Manifold trials were implemented; however, most of them failed to verify the effectiveness of their preventive measures. Recent advancement of epidemiological studies and cutaneous biology revealed epidermal barrier dysfunction plays a major role of allergen sensitization and development of atopic dermatitis which ignites the inception of allergy march. For this decade, therapeutic strategy to prevent the development of food allergy has been confronted with a paradigm shift from avoidance and delayed introduction of allergenic foods based on the theoretical concept to early introduction of them based on the clinical and epidemiological evidences. Especially, prevention of peanut allergy and egg allergy has been established with the highest evidence verified by randomized controlled trials, although application in clinical practice should be done with attention. This paradigm shift concerning food allergy was also due to the discovery of cutaneous sensitization risk of food allergens for an infant with eczema revealed by prospective studies. Here we have recognized the increased importance of prevention of eczema/atopic dermatitis in infancy. Two randomized controlled trials using emollients showed successful results in prevention of atopic dermatitis in infancy; however, longer term safety and prognosis including allergy march should be pursued. To establish more fundamental strategy for prevention of the development of allergy, further studies clarifying the mechanisms of interaction between barrier dysfunction and microbial milieu are needed with macroscope to understand the relationship between allergic diseases and a diversity of environmental influences., (Copyright © 2017 Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.)

Programmed death ligand-1 (PD-L1) is a critical regulator of T cell function contributing to peripheral immune tolerance. Although it has been shown that posttranscriptional regulatory mechanisms control PD-L1 expression in cancer, it remains unknown whether such regulatory loops operate also in non-transformed cells. Here we studied PD-L1 expression in human dermal lymphatic endothelial cells (HDLECs), which play key roles in immunity and cancer. Treatment of HDLECs with the pro-inflammatory cytokines IFN-γ and TNF-α synergistically up-regulated PD-L1 expression. IFN-γ and TNF-α also affected expression of several microRNAs (miRNAs) that have the potential to suppress PD-L1 expression. The most highly up-regulated miRNA following IFN-γ and TNF-α treatment in HDLECs was miR-155, which has a central role in the immune system and cancer. Induction of miR-155 was driven by TNF-α, the effect of which was significantly enhanced by IFN-γ. The PD-L1 3'-UTR contains two functional miR-155-binding sites. Endogenous miR-155 controlled the kinetics and maximal levels of PD-L1 induction upon IFN-γ and TNF-α treatments. We obtained similar findings in dermal fibroblasts, demonstrating that the IFN-γ/TNF-α/miR-155/PD-L1 pathway is not restricted to HDLECs. These results reveal miR-155 as a critical component of an inflammation-induced regulatory loop controlling PD-L1 expression in primary cells., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Plasma leakage is the main pathophysiological feature in severe dengue, resulting from altered vascular barrier function associated with an inappropriate immune response triggered upon infection. The present study investigated functional changes using an electric cell-substrate impedance sensing system in four (brain, dermal, pulmonary and retinal) human microvascular endothelial cell (MEC) lines infected with purified dengue virus, followed by assessment of cytokine profiles and the expression of inter-endothelial junctional proteins. Modelling of changes in electrical impedance suggests that vascular leakage in dengue-infected MECs is mostly due to the modulation of cell-to-cell interactions, while this loss of vascular barrier function observed in the infected MECs varied between cell lines and DENV serotypes. High levels of inflammatory cytokines (IL-6 and TNF-α), chemokines (CXCL1, CXCL5, CXCL11, CX3CL1, CCL2 and CCL20) and adhesion molecules (VCAM-1) were differentially produced in the four infected MECs. Further, the tight junctional protein, ZO-1, was down-regulated in both the DENV-1-infected brain and pulmonary MECs, while claudin-1, PECAM-1 and VE-cadherin were differentially expressed in these two MECs after infection. Non-purified virus stock was also studied to investigate the impact of virus stock purity on dengue-specific immune responses, and the results suggest that virus stock propagated through cell culture may include factors that mask or alter the DENV-specific immune responses of the MECs. The findings of the present study show that high DENV load differentially modulates human microvascular endothelial barrier function and disrupts the function of inter-endothelial junctional proteins during early infection with organ-specific cytokine production.

The role of mouse dermal γδ T cells in inflammatory skin disorders and host defense has been studied extensively. It is known that dendritic epidermal T cells (DETC) have a monomorphic γδ T cell receptor (TCR) and reside in murine epidermis from birth. We asked if dermal γδ cells freely re-circulated out of skin, or behaved more like dermal resident memory T cells (TRM) in mice. We found that, unlike epidermal γδ T cells (DETC), dermal γδ cells are not homogeneous with regard to TCR, express the tissue resident T cell markers CD69 and CD103, bear skin homing receptors, and produce IL-17 and IL-22. We created GFP+: GFP- parabiotic mice and found that dermal γδ T cells re-circulate very slowly-more rapidly than authentic αβ TCR TRM, but more slowly than the recently described dermal αβ TCR T migratory memory cells (TMM). Mice lacking the TCR δ gene (δ-/-) had a significant reduction of 2,4-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity (CHS). We created mice deficient in dermal γδ T cells but not DETC, and these mice also showed a markedly reduced CHS response after DNFB challenge. The infiltration of effector T cells during CHS was not reduced in dermal γδ T cell-deficient mice; however, infiltration of Gr-1+CD11b+ neutrophils, as well as ear swelling, was reduced significantly. We next depleted Gr-1+ neutrophils in vivo, and demonstrated that neutrophils are required for ear swelling, the accepted metric for a CHS response. Depletion of IL-17-producing dermal Vγ4+ cells and neutralization of IL-17 in vivo, respectively, also led to a significantly reduced CHS response and diminished neutrophil infiltration. Our findings here suggest that dermal γδ T cells have an intermediate phenotype of T cell residence, and play an important role in primary CHS through producing IL-17 to promote neutrophil infiltration., Competing Interests: The authors have declared that no competing interests exist.

Tissue injury triggers the activation and differentiation of multiple cell types to minimize damage and initiate repair processes. In systemic sclerosis, these repair processes appear to run unchecked, leading to aberrant remodeling and fibrosis of the skin and multiple internal organs, yet the fundamental pathological defect remains unknown. We describe herein a transition wherein the abundant CD34(+) dermal fibroblasts present in healthy human skin disappear in the skin of systemic sclerosis patients, and CD34(-), podoplanin(+), and CD90(+) fibroblasts appear. This transition is limited to the upper dermis in several inflammatory skin diseases, yet in systemic sclerosis, it can occur in all regions of the dermis. In vitro, primary dermal fibroblasts readily express podoplanin in response to the inflammatory stimuli tumor necrosis factor and IL-1β. Furthermore, we show that on acute skin injury in both human and murine settings, this transition occurs quickly, consistent with a response to inflammatory signaling. Transitioned fibroblasts partially resemble the cells that form the reticular networks in organized lymphoid tissues, potentially linking two areas of fibroblast research. These results allow for the visualization and quantification of a basic stage of fibroblast differentiation in inflammatory and fibrotic diseases in the skin., (Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Lymphocyte- and leukocyte-mediated lymph node (LN) lymphatic sinus growth (lymphangiogenesis) is involved in immune responses and in diseases including cancer and arthritis. We previously discovered a 10.1.1 Ab that recognizes the lymphatic endothelial cell (LEC) surface protein mCLCA1, which is an interacting partner for LFA1 and Mac-1 that mediates lymphocyte adhesion to LECs. Here, we show that 10.1.1 Ab treatment specifically induces LEC proliferation, and influences migration and adhesion in vitro. Functional testing by injection of mice with 10.1.1 Ab but not control hamster Abs identified rapid induction of LN LEC proliferation and extensive lymphangiogenesis within 23 h. BrdU pulse-chase analysis demonstrated incorporation of proliferating LYVE-1-positive LEC into the growing medullary lymphatic sinuses. The 10.1.1 Ab-induced LN remodeling involved coordinate increases in LECs and also blood endothelial cells, fibroblastic reticular cells, and double negative stroma, as is observed during the LN response to inflammation. 10.1.1 Ab-induced lymphangiogenesis was restricted to LNs, as mCLCA1-expressing lymphatic vessels of the jejunum and dermis were unaffected by 23 h 10.1.1 Ab treatment. These findings demonstrate that 10.1.1 Ab rapidly and specifically induces proliferation and growth of LN lymphatic sinuses and stroma, suggesting a key role of mCLCA1 in coordinating LN remodeling during immune responses.

Regulatory T-cell (Treg) selection in the thymus is essential to prevent autoimmune diseases. Although important rules for Treg selection have been established, there is controversy regarding the degree of self-reactivity displayed by T-cell receptors expressed by Treg cells. In this study we have developed a model of autoimmune skin inflammation, to determine key parameters in the generation of skin-reactive Treg cells in the thymus (tTreg). tTreg development is predominantly AIRE dependent, with an AIRE-independent component. Without the knowledge of antigen recognized by skin-reactive Treg cells, we are able to enhance skin-specific tTreg cell generation using three approaches. First, we increase medullary thymic epithelial cells by using mice lacking osteoprotegerin or by adding TRANCE (RANKL, Tnfsf11). Second, we inject intrathymically peripheral dendritic cells from skin-draining sites. Finally, we inject skin tissue lysates intrathymically. These findings have implications for enhancing the generation of organ-specific Treg cells in autoimmune diseases.

Introduction: Proto-oncogene survivin is a member of the inhibitor of apoptosis (IAP) family of proteins. The presence of serous antibodies against survivin in patients with systemic sclerosis has been previously reported; however, there are few reports regarding the pathophysiological relationship between survivin and systemic sclerosis. We herein investigated the expression and function of survivin in SSc patients., Methods: We performed immunohistochemistry analyses to determine the expression of XIAP, cIAP and survivin in skin lesions from patients with SSc and non-SSc. The expression levels of survivin in peripheral blood mononuclear cells (PBMCs) obtained from SSc patients and healthy controls were evaluated using RT-PCR and flow cytometry. Additionally, the function of survivin was verified with overexpression experiments using monocyte-derived dendritic cells (Mo-DCs)., Results: The expression patterns of both XIAP and cIAP were similar, while only the survivin expression differed between the SSc and non-SSc skin lesions. Survivin-overexpressing cells were detected in the SSc dermis frequently. The positive rate of survivin in SSc dermis (64.3%, 9/14) was higher than that in non-SSc dermis (11.2%, 1/9). Furthermore, survivin+ cells expressed CD1a, one of the DC markers. Real-time PCR and FACS analyses revealed that the survivin-WT (wild type) expression levels in PBMCs, in particular CD14+ monocytes, from SSc patients were higher than that from healthy controls. Additionally, the overexpression experiments showed that survivin-WT-overexpressing CD1a+ Mo-DCs have the characteristics of promoting cell cycle progression and decreasing apoptotic cells., Conclusions: These findings suggest that dermal survivin+ CD1a+ cell infiltration may be a potential biomarker of SSc skin lesions. PBMCs and monocytes from SSc patients also overexpressed survivin; therefore, dermal survivin+ DC may be derived from peripheral blood monocytes. Additionally, survivin may be involved in dermal CD1a+ DC proliferation through cell cycle activation and resistance to apoptosis. Survivin may be an important molecule for the pathogenesis of SSc.

The series of events that occurs immediately after pathogen entrance into the body is largely speculative. Key aspects of these events are pathogen dissemination and pathogen interactions with the immune response as the invader moves into deeper tissues. We sought to define major events that occur early during infection of a highly virulent pathogen. To this end, we tracked early dissemination of Yersinia pestis, a highly pathogenic bacterium that causes bubonic plague in mammals. Specifically, we addressed two fundamental questions: (1) do the bacteria encounter barriers in disseminating to draining lymph nodes (LN), and (2) what mechanism does this nonmotile bacterium use to reach the LN compartment, as the prevailing model predicts trafficking in association with host cells. Infection was followed through microscopy imaging in addition to assessing bacterial population dynamics during dissemination from the skin. We found and characterized an unexpected bottleneck that severely restricts bacterial dissemination to LNs. The bacteria that do not pass through this bottleneck are confined to the skin, where large numbers of neutrophils arrive and efficiently control bacterial proliferation. Notably, bottleneck formation is route dependent, as it is abrogated after subcutaneous inoculation. Using a combination of approaches, including microscopy imaging, we tested the prevailing model of bacterial dissemination from the skin into LNs and found no evidence of involvement of migrating phagocytes in dissemination. Thus, early stages of infection are defined by a bottleneck that restricts bacterial dissemination and by neutrophil-dependent control of bacterial proliferation in the skin. Furthermore, and as opposed to current models, our data indicate an intracellular stage is not required by Y. pestis to disseminate from the skin to draining LNs. Because our findings address events that occur during early encounters of pathogen with the immune response, this work can inform efforts to prevent or control infection.

The skin has to effectively combat external attacks, while maintaining skin immune homeostasis under steady-state conditions. To fulfill these challenging tasks, the dermis harbors a variety of heterogeneous cell types that are able to suppress T-cell proliferation similar to bone marrow mesenchymal stromal cells. Here we show that plastic-adherent, human dermal cells induce FoxP3 expression in TCR-complex-stimulated CD25(-)CD4(+)CD45RA(+) T cells in the absence of CD28 co-ligation in a cell-contact-dependent manner. These FoxP3(+) T cells reveal an effective suppressive capacity in vitro. Moreover, we found that the vast majority of CD90(+) dermal cells are perivascularly located and generate a significantly higher percentage of regulatory T cells compared with cells expressing markers such as CD271 in vitro. Importantly, we further demonstrate that plastic-adherent dermal cells are also able to differentiate toward the endothelial lineage. Our data show that human skin harbors specific cell types with immunosuppressive potential, which are located in close vicinity to their likely operational area and provide evidence for a CD28-independent regulatory mechanism. Further, the differentiation potential into endothelial cells suggests the existence of a tissue-resident cell pool for vessel regeneration. These findings might have important implications for the clinical use of allogeneic dermal cells to rebuild an imbalanced human skin immune homeostasis.

Dengue virus (DENV) causes the most prevalent arthropod-borne viral disease in humans. Although Aedes mosquitoes transmit DENV when probing for blood in the skin, no information exists on DENV infection and immune response in the dermis, where the blood vessels are found. DENV suppresses the interferon response, replicates, and causes disease in humans but not wild-type mice. Here, we used mice lacking the interferon-α/β receptor (Ifnar(-/-)), which had normal cell populations in the skin and were susceptible to intradermal DENV infection, to investigate the dynamics of early DENV infection of immune cells in the skin. CD103(+) classical dendritic cells (cDCs), Ly6C(-) CD11b(+) cDCs, and macrophages in the steady-state dermis were initial targets of DENV infection 12-24 hours post-inoculation but then decreased in frequency. We demonstrated recruitment of adoptively-transferred Ly6C(high) monocytes from wild-type and Ifnar(-/-) origin to the DENV-infected dermis and differentiation to Ly6C(+) CD11b(+) monocyte-derived DCs (moDCs), which became DENV-infected after 48 hours, and were then the major targets for virus replication. Ly6C(high) monocytes that entered the DENV-infected dermis expressed chemokine receptor CCR2, likely mediating recruitment. Further, we show that ∼ 100-fold more hematopoietic cells in the dermis were DENV-infected compared to Langerhans cells in the epidermis. Overall, these results identify the dermis as the main site of early DENV replication and show that DENV infection in the skin occurs in two waves: initial infection of resident cDCs and macrophages, followed by infection of monocytes and moDCs that are recruited to the dermis. Our study reveals a novel viral strategy of exploiting monocyte recruitment to increase the number of targets for infection at the site of invasion in the skin and highlights the skin as a potential site for therapeutic action or intradermal vaccination.

Innate lymphoid cells (ILCs) are increasingly appreciated as important regulators of tissue homeostasis and inflammation. However, their role in human skin remains obscure. We found that healthy peripheral blood CD117(+) ILC3, lacking the natural cytotoxicity receptor (NCR) NKp44 (NCR(-) ILC3), CD117(-)NCR(-)CRTH2(-)CD161(+) ILC1, and CRTH2(+) ILC2, express the skin-homing receptor cutaneous lymphocyte antigen (CLA). NCR(+) ILC3 were scarce in peripheral blood. Consistently, we identified in normal skin ILC2 and NCR(-) ILC3, a small proportion of CD161(+) ILC1, and hardly any NCR(+) ILC3, whereas NCR(+) ILC3 were present in cultured dermal explants. The skin ILC2 and NCR(+) ILC3 subsets produced IL-13 and IL-22, respectively, upon cytokine stimulation. Remarkably, dermal NCR(-) ILC3 converted to NCR(+) ILC3 upon culture in IL-1β plus IL-23, cytokines known to be involved in psoriatic inflammation. In line with this observation, significantly increased proportions of NCR(+) ILC3 were present in lesional skin and peripheral blood of psoriasis patients as compared with skin and blood of healthy individuals, respectively, whereas the proportions of ILC2 and CD161(+) ILC1 remained unchanged. NCR(+) ILC3 from skin and blood of psoriasis patients produced IL-22, which is regarded as a key driver of epidermal thickening, suggesting that NCR(+) ILC3 may participate in psoriasis pathology.

Innate lymphoid cells (ILCs) are a recently discovered family of innate immune cells belonging to the lymphoid lineage, yet lacking antigen-specific receptors. ILCs were first identified in the intestinal tract, where they contribute to epithelial barrier integrity and host responses to commensal microbes. Teunissen et al. (in the current issue) and Villanova et al. (2014) now suggest an important role for type 3 ILCs (ILC3s) in the skin, particularly in psoriasis. Both groups found an increased frequency of IL-22- and/or IL-17A-producing ILCs in psoriatic skin and blood. These cells are activated in response to IL-1β and IL-23, correlate with disease severity, and are decreased following antitumor necrosis factor-α (anti-TNFα) treatment. The presence of a novel ILC population in psoriatic skin, one that responds to biologic therapeutics, suggests that dysregulation of ILCs is a contributing factor to psoriasis pathogenesis.

Objective: To explore the effects of SANPAOCAO (SPC), a compound traditional Chinese folk medicine, on chronic dermatitis/eczema in mice induced by 2, 4-dinitrochlorobenzene (DNCB)., Methods: Thirty-three Balb/c mice were randomly divided into a negative control group, a positive control group, a prednisolone treatment group, an SPC ethanol extract treatment group, a Cardiospermum halicacabum ethanol extract treatment group, a Physalis minima ethanol extract treatment group, and a Jussiaea repens ethanol extract treatment group. Mice in the six treatment groups had twenty-five microliters of 0.1% DNCB in acetone/olive oil (3: 1) applied to each side of their right ears and dorsal skin three times a week, over a 5 week period. They were treated with prednisolone or the various kinds of ethanol extract after each challenge. The weight difference between the two ears, pathological changes in the right ears, dermal inflammatory cell numbers, and total serum Ig E levels were used to assess the effects of the drugs., Results: after the 5 weeks of challenges, the weight differences of the ears in the SPC group and the prednisolone group were significantly less than those in the other groups. There was evidence of significant suppression of the development of dermatitis, as determined by a histological examination and the serum Ig E levels., Conclusion: SPC has beneficial effects when used in the treatment of chronic dermatitis-eczema in mice.

Lipid mediators derived from 5-lipoxygenase (5-LO) metabolism can activate both pro- and anti-inflammatory pathways, but their role in wound healing remains largely unexplored. In this study we show that 5-LO knockout (5-LO(-/-)) mice exhibited faster wound healing than wild-type (WT) animals, and exhibited upregulation of heme oxygenase-1 (HO-1). Furthermore, HO-1 inhibition in 5-LO(-/-) mice abolished the beneficial effect observed. Despite the fact that 5-LO(-/-) mice exhibited faster healing, in in vitro assays both migration and proliferation of human dermal fibroblasts (HDFs) were inhibited by the 5-LO pharmacologic inhibitor AA861. No changes were observed in the expression of fibronectin, transforming growth factor (I and III), and α-smooth muscle actin (α-SMA). Interestingly, AA861 treatment significantly decreased ROS formation by stimulated fibroblasts. Similar to 5-LO(-/-) mice, induction of HO-1, but not superoxide dismutase-2 (SOD-2), was also observed in response to 5-LO (AA861) or 5-LO activating protein (MK886) inhibitors. HO-1 induction was independent of nuclear factor (erythroid derived-2) like2 (Nrf-2), cyclooxygenase 2 (COX-2) products, or lipoxin action. Taken together, our results show that 5-LO disruption improves wound healing and alters fibroblast function by an antioxidant mechanism based on HO-1 induction. Overexpression of HO-1 in wounds may facilitate early wound resolution.

Langerhans cells (LCs) are professional antigen-presenting cells (APCs) residing in the epidermis. Despite their high potential to activate T lymphocytes, current understanding of human LC biology is limited. Genome-wide comparison of the transcriptional profiles of human skin migratory CD1a+ LCs and CD11c+ dermal dendritic cells (DDCs) demonstrated significant differences between these "dendritic cell (DC)" types, including preferential expression of 625 genes (P<0.05) in LC and 914 genes (P<0.05) in DDC. Analysis of the temporal regulation of molecular networks activated after stimulation with tumor necrosis factor-α (TNF-α) confirmed the unique molecular signature of LCs. Although LCs conformed to the phenotype of professional APC, inflammatory signaling activated primarily genes associated with cellular metabolism and mitochondrial activation (e.g., CYB561 and MRPS35), cell membrane re-organization, and antigen acquisition and degradation (CAV1 and PSMD14; P<0.05-P<0.0001). Conversely, TNF-α induced classical activation in DDCs with early downregulation of surface receptors (mannose receptor-1 (MRC1) and C-type lectins), and subsequent upregulation of cytokines, chemokines (IL1a, IL1b, and CCL18), and matrix metalloproteinases (MMP1, MMP3, and MMP9; P<0.05-P<0.0001). Functional interference of caveolin abrogated LCs superior ability to cross-present antigens to CD8+ T lymphocytes, highlighting the importance of these networks to biological function. Taken together, these observations support the idea of distinct biological roles of cutaneous DC types.

The immune privilege (IP) of hair follicles (HFs) has been well established in previous studies. However, whether cultured HF cells still exhibit IP properties, the individual factors involved in this process, and the detailed mechanisms that drive and maintain IP, are largely unidentified. We found preferential expression of IP-associated genes in cultured HF dermal papilla and dermal sheath cup cells (DSCCs) compared with non-follicular fibroblasts (FBs) at passage 4, suggesting a potential for functional IP. Notably, programmed cell death 1 ligand 1 (PD-L1) was significantly upregulated in DSCCs and dermal papilla cells relative to FBs. IFNγ secretion from peripheral blood mononuclear cells (PBMCs) co-cultured with histoincompatible DSCCs was significantly lower than with FB and higher percentages of early apoptotic, Annexin V+ cells were observed in PBMC co-cultured with DSCCs. Knockdown of PD-L1 translation by silencing interfering RNA in DSCCs enabled increased IFNγ secretion by PBMCs, whereas transfection of pCMV6-XL4/hPD-L1 in FB significantly reduced IFNγ secretion and increased apoptosis in co-cultured PBMCs. We also found that apoptosis in allogeneic T cells induced by DSCCs was largely dependent on the mitochondrial pathway. Our study suggests IP properties are exhibited in cultured DSCCs in part through expression of negative co-signaling molecule PD-L1.

The interaction between keratinocytes and immune cells plays a major role in the development of inflammatory skin diseases like psoriasis and atopic dermatitis. Pharmacological intervention to inhibit T cell-derived proinflammatory mediators is an effective therapy in the treatment of psoriasis. Here, we present a model to study the interaction between keratinocytes and T cells in a three-dimensional (3D) microenvironment, based on human skin equivalents populated with CD4+ T cells. T cell migration into the dermis initiated keratinocyte activation within 2 days, with hallmarks of a psoriasiform inflammation after 4 days. Expression of epidermal psoriasis marker genes was upregulated, and proinflammatory cytokines and chemokines were highly expressed. Disturbed epidermal differentiation was shown by downregulated filaggrin expression and involucrin expression in the spinous layer. These effects were mediated via soluble factors produced by the T cells. The psoriasiform inflammation was also observed using T helper type 1 (Th1)- and Th17-polarized CD4+ T cells. We validated our model by treatment with anti-inflammatory drugs that reduced the expression of proinflammatory cytokines and chemokines and suppressed the psoriasiform inflammation. We propose that our T cell-driven inflammatory skin equivalent model has potential to study the pathogenesis of inflammatory skin diseases and may serve as a preclinical screening tool for anti-inflammatory drugs.

Skin scarification (s.s.) with vaccinia virus (VACV) is essential for generation of an optimal protective T-cell memory immune response. Dendritic cells (DCs), which are professional antigen-presenting cells, are required for naive T-cell priming and activation. At least three subsets of skin-resident DC have been identified: Langerhans cells (LCs), dermal Langerin+ DC (Lang+ dDC), and dermal Langerin- DC (Lang- dDC). Using Langerin-diphtheria toxin receptor mice and established mouse model of VACV delivered by s.s., we demonstrated that Lang+ dDC, but not LC, are absolutely required for the induction of a rapid and robust antigen-specific CD8+ T-cell response after s.s. with VACV. The depletion of Lang+ dDC led to a significant delay in the priming and proliferation of antigen-specific CD8+ T cells. Moreover, CD8+ T cells generated after VACV s.s. in the absence of Lang+ dDC lacked effector cytotoxic functions both in vitro and in vivo. While s.s.-immunized wild-type and LC-depleted mice controlled the progression of OVA257-264 expressing T-cell lymphoma EG7 (injected intradermally), the depletion of Lang+ dDC led to rapid lymphoma progression and mortality. These data indicate that of all skin DC subsets, Lang+ dDC is the most critical for the generation of robust CD8+ T-cell immunity after s.s. with VACV.