Abstract Basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (SCC) are the most common keratinocyte-derived malignancies, yet they differ markedly in invasiveness, metastatic potential, and immune contexture. Although cancer-associated fibroblasts (CAFs) are increasingly recognized as key regulators of tumor architecture and tumor immunity, the spatial organization of distinct CAF subtypes in cutaneous carcinomas and their functional relationship with immune cells remains incompletely understood. Using a 33-plex imaging mass cytometry (IMC) panel, we profiled 28 regions of interest (ROIs) from 17 human BCC and SCC specimens, encompassing more than 739,000 single cells, and integrated these data with RNA fluorescence in situ hybridization (RNA-FISH), immunohistochemistry (IHC), multiplex immunofluorescence, and in vitro functional assays. We identified four fibroblast populations, including immunomodulatory CAFs (iCAFs), matrix CAFs (mCAFs), myofibroblast-like CAFs (myoCAFs), and reticular fibroblasts (retFIBs), and found that aggressive tumor subtypes were characterized by increased stromal area, extracellular matrix deposition, and altered CAF composition. CAF composition differed most prominently across BCC subtypes, with nodular BCC enriched for mCAFs and infiltrative BCC showing increased myoCAF density, consistent with a shift toward a contractile stromal program. Spatial analyses revealed distinct CAF-immune niches: iCAFs localized to immune-cell-rich, inflamed niches enriched for activated and/or exhaustion-associated immune-cell marker programs, whereas myoCAFs occupied fibroblast-dense, immune-poor niches with globally reduced immune activation. mCAFs were preferentially associated with immune cell accumulation in the stroma and spatial immune compartmentalization, with limited immune cell presence within tumor nests. At the invasive front, CAF-immune coupling was highly subset-dependent, with iCAFs linked to antigen-experienced T-cell states and myoCAFs linked to immune exclusion. In vitro, patient-derived CAF cultures from myoCAF-rich biopsies showed enhanced collagen-gel contraction, with cultures enriched for MCAM + CAFs displaying increased contractile capacity. Aggressive tumor variants displayed increased stromal nuclear YAP/TAZ, while complementary single-cell pathway analysis supported a mechanically remodeled stromal microenvironment in which mCAFs contribute ECM/matrix-remodeling programs and RGS5⁺/myoCAF-like populations show enhanced mechanotransduction-associated signaling, rather than a uniform CAF-wide increase in canonical YAP/TAZ transcriptional output. Together, these findings define spatially organized CAF programs in cutaneous carcinomas and identify myoCAF-rich stromal niches as a recurrent feature of aggressive, immune-repressed tumor architecture. These results nominate CAF composition as a biomarker of immune architecture and a potential determinant of therapeutic response.

Cancer-associated fibroblasts (CAFs) play a key role in cancer progression and treatment outcome. This study dissects the intra-tumoral diversity of CAFs in basal cell carcinoma, squamous cell carcinoma, and melanoma using molecular and spatial single-cell analysis. We identify three distinct CAF subtypes: myofibroblast-like RGS5+ CAFs, matrix CAFs (mCAFs), and immunomodulatory CAFs (iCAFs). Large-cohort tissue analysis reveals significant shifts in CAF subtype patterns with increasing malignancy. Two CAF subtypes exhibit immunomodulatory properties via different mechanisms. mCAFs sythesize extracellular matrix and may restrict T cell invasion in low-grade tumors via ensheathing tumor nests, while iCAFs are enriched in late-stage tumors, and express high levels of cytokines and chemokines to aid immune cell recruitment and activation. This is supported by the induction of an iCAF-like phenotype with immunomodulatory functions in primary healthy fibroblasts exposed to skin cancer cell secretomes. Thus, targeting CAF variants holds promise to enhance immunotherapy efficacy in skin cancers. Fibroblast heterogeneity in the tumor microenvironment can explain their multifaceted role in cancer. Here by single-cell transcriptomic analysis of basal cell carcinoma, squamous cell carcinoma and melanoma samples, the authors explore fibroblast heterogeneity in skin cancer and their potential to modulate the tumor-immune microenvironment.

Negative expectations regarding nerve reconstruction in the elderly prevail in the literature, but little is known about the effectiveness of nerve transfers in patients with brachial plexus injuries aged over 60 years. We present a series of five patients (1 female, 4 male) aged between 60 and 81 years (median 62.0 years) who underwent nerve reconstruction using multiple nerve transfers in brachial plexopathies. The etiology of brachial plexus injury was trauma (n = 2), or iatrogenic, secondary to spinal surgical laminectomy, tumor excision and radiation for breast cancer (n = 3). All but one patient underwent a one-stage reconstruction including neurolysis and extra-anatomical nerve transfer alone (n = 2) or combined with anatomical reconstruction by sural nerve grafts (n = 2). One patient underwent a two-stage reconstruction, which involved a first stage anatomical brachial plexus reconstruction followed by a second stage nerve transfer. Neurotizations were performed as double (n = 3), triple (n = 1) or quadruple (n = 1) nerve or fascicular transfers. Overall, at least one year postoperatively, successful results, characterized by a muscle strength of M3 or more, were restored in all cases, two patients even achieving M4 grading in the elbow flexion. This patient series challenges the widely held dogma that brachial plexus reconstruction in older patients will produce poor outcomes. Distal nerve transfers are advantageous as they shorten the reinnervation distance. Healthy, more elderly patients should be judiciously offered the whole spectrum of reconstructive methods and postoperative rehabilitation concepts to regain useful arm and hand function and thus preserve independence after a traumatic or nontraumatic brachial plexus injury.

Introduction: Autologous nerve grafts are accompanied by morbidity of the donor site, leading to loss of sensation and possible complications such as end neuroma formation or persistent pain. Moreover, the indications are narrow mostly limited to defects in larger motor nerves with good prognosis. One alternative to autografts might be the commercially available allograft (Avance® Nerve Graft). The aim of this study was to assess the safety and outcome of the Avance® Nerve Graft for the reconstruction of peripheral nerves after neuromas, and tumor resection. Materials and Methods: For this purpose, ten patients with eleven implanted grafts were recruited for the prospective study. Five patients suffered from a neuroma-in-continuity, four from an terminal neuroma formation and two from Schwannomas. Seven patients were symptomatic for pain. After the resection, the peripheral nerves were reconstructed with the Avance® Nerve Graft. The patients were followed up for at least six months, by assessing recovery of motor, and sensibility and pain reduction. Results: A significant decrease in pain levels was observed after reconstruction. All patients achieved or remained a meaningful sensory function with levels of S3 or above. Regarding motor function, 85.7% (6 of 7 patients) demonstrated M3 or higher. One patient received a revisional reconstruction upon recurrent neuroma formation. In summary, all patients demonstrated signs of recovery and improvement of their paint situation. Nevertheless, one patient missed a meaningful motor recovery and a second one underwent an additional intervention. Conclusion: The reconstruction with Avance® Nerve Graft demonstrated good outcomes for motor, and sensory function, by achieving a pain reduction in all patients. Of particular interest, was the reconstruction of lesioned purely sensible nerves, for those currently no reasonable reconstruction option exists. Declaration of potential conflicts of interest The financial resources for the conduct of the RANGER study at the Medical University of Vienna are provided by Axogen Inc.

Cancer-associated fibroblasts (CAFs) play a key role in cancer progression and treatment outcome. Here, we elucidate the yet unresolved intra-tumoral CAF variety in three skin cancer types at molecular and spatial single-cell resolution in a large cohort. We show that two out of three CAF subtypes contribute to tumor immune surveillance with distinct mechanisms. Matrix CAFs (mCAFs), a previously unknown subtype present in early-stage tumors, ensheath tumor nests and synthesize extracellular-matrix to prevent T cell invasion. Immuno CAFs (iCAFs), which express proinflammatory and immunomodulatory factors, are only detected in high abundance in aggressive tumors. Strikingly, iCAFs but not tumor cells are the exclusive celltype producing chemokines and, thus, play a key role in immune cell recruitment and activation. Mechanistically, we show that cancer cells transform adjacent healthy fibroblasts into cytokine-expressing iCAFs, which subsequently recruit immune cells and modulate the immune response. In conclusion, targeting CAF variants holds promise for improved efficacy of immunotherapy. Statement of significance: While it is accepted that fibroblasts affect cancer progression, the underlying molecular programs remain unclear. We unravel a multi-step cascade demonstrating that tumor cells transform healthy fibroblasts into CAFs, which critically impact immune surveillance via iCAFs being the exclusive source of chemokines and mCAFs promoting T cell exclusion.

Immunohistochemistry is a powerful tool for studying neuronal tissue from humans at the molecular level. Obtaining fresh neuronal tissue from human organ donors is difficult and sometimes impossible. In anatomical body donations, neuronal tissue is dedicated to research purposes and because of its easier availability, it may be an alternative source for research. In this study, we harvested spinal cord from a single organ donor 2 h (h) postmortem and spinal cord from body donors 24, 48, and 72 h postmortem and tested how long after death, valid multi-color immunofluorescence or horseradish peroxidase (HRP) immunohistochemistry is possible. We used general and specific neuronal markers and glial markers for immunolabeling experiments. Here we showed that it is possible to visualize molecularly different neuronal elements with high precision in the body donor spinal cord 24 h postmortem and the quality of the image data was comparable to those from the fresh organ donor spinal cord. High-contrast multicolor images of the 24-h spinal cords allowed accurate automated quantification of different neuronal elements in the same sample. Although there was antibody-specific signal reduction over postmortem intervals, the signal quality for most antibodies was acceptable at 48 h but no longer at 72 h postmortem. In conclusion, our study has defined a postmortem time window of more than 24 h during which valid immunohistochemical information can be obtained from the body donor spinal cord. Due to the easier availability, neuronal tissue from body donors is an alternative source for basic and clinical research.

Objective The objective of this surgery is to achieve early reinnervation of the intrinsic hand muscles through axons of the median nerve, preventing irreversible atrophy of the muscle tissue. The nerve transfer is achieved via a babysitter graft, which is sutured end-to-side to the donor as well as the recipient nerve. The procedure is carried out in combination with a proximal reconstruction of the ulnar nerve. Indications High-grade lesions of the ulnar nerve without spontaneous regeneration, particularly when lesions are located proximally and/or when patients present late. Contraindications Irreversible denervation of the intrinsic muscles; weakness or palsy of the thenar branch. Surgical technique The approach is taken through a longitudinal incision over the volar wrist. The deep branch of the ulnar nerve as well as the thenar branch of the median nerve are visualized after transection of the flexor retinaculum. An autologous graft is then placed between the two nerves, sutured to the donor (thenar branch) as well as the recipient nerve (ulnar deep branch) via an epineural window in an end-to-side manner. This facilitates timely regeneration of motor axons from the median nerve into the intrinsic muscles, thereby preventing irreversible degeneration. Through the end-to-side nerve coaptation, damage to the donor nerve is reduced to a minimum. At the same time reconstruction of the ulnar nerve is performed proximally to the lesion, facilitating original reinnervation of the intrinsic muscles at a later time. Postoperative management Postoperatively, Penrose drains are placed and a sterile hand dressing is applied. Drain removal and dressing change are performed on the first day, suture removal after 2 weeks. Physical therapy for mobility of the joints can be started as early as 1 week after surgery. After the first signs of motor and/or sensory reinnervation, a targeted retraining of daily skills should be initiated. Results This procedure has so far been reported on three patients with high-grade ulnar nerve injury. After a follow-up duration of 6 years, each achieved muscle strength of >= M3, with good to excellent overall regeneration according to the modified Bishop rating scale.

The innervation of the long head of the biceps tendon (LHBT) is not sufficiently documented. This is a drawback since pathologies of the LHBT are a major source of shoulder pain. Thus, the study aimed to characterize structurally and molecularly nervous elements of the LHBT. The proximal part of 11 LHBTs was harvested intraoperatively. There were 8 female and 3 male specimens. Age ranged from 66 to 86 years. For structural analyses, nervous elements were viewed in the transmission electron microscope. For molecular characterization, we used general neuronal markers including antibodies against neurofilament and protein gene product 9.5 (PGP9.5) as well as specific neuronal markers including antibodies against myelin basic protein (MBP), calcitonin gene-related product (CGRP), substance P (SP), tyrosine hydroxylase (TH), and growth-associated protein 43 (GAP43). Anti-neurofilament and anti-PGP9.5 visualized the overall innervation. Anti-MBP visualized myelination, anti-CGRP and anti-SP nociceptive fibers, anti-TH sympathetic nerve fibers, and anti-GAP43 nerve fibers during development and regeneration. Immunolabeled sections were analyzed in the confocal laser scanning microscope. We show that the LHBT contains unmyelinated as well as myelinated nerve fibers which group in nerve fascicles and follow blood vessels. Manny myelinated and unmyelinated axons exhibit molecular features of nociceptive nerve fibers. Another subpopulation of unmyelinated axons exhibits molecular characteristics of sympathetic nerve fibers. Unmyelinated sympathetic fibers and unmyelinated nociceptive fibers express proteins that are found during development and regeneration. Present findings support the hypothesis that ingrowth of nociceptive fibers are the source of chronic tendon pain.

BACKGROUND:Tendon pathologies are common and several data suggests that the peripheral nervous system is involved in this disorder. Immunohistochemistry (IHC) is one of the pillars to characterize nervous structures and their implication in the pathogenesis of chronic tendon pain. Most commonly, formalin-fixed, paraffin-embedded (FFPE) tendons are used for immunohistochemical characterization of the innervation. However, FFPE specimens exhibit major disadvantages: First, antigens (proteins) are masked and antigen retrieval is necessary to restore antigenicity. Second, FFPE specimens involve immunolabeling with enzyme-conjugated antibodies but this approach has limitations when multiple antigens are of interest simultaneously. Consequently, there is a demand in the orthopedic community for an alternative immunohistochemical approach to visualize tendon innervations. RESULTS:Here, we present a guide how to visualize tendon innervation. This guide couples paraformaldehyde fixation, cryo-embedding, immunofluorescence, and confocal laser scanning microscopy. We demonstrate the utility of our approach in the long head of the biceps tendon. For nerve fiber characterization, we used different neuronal markers including antibodies against neurofilament, protein gene product 9.5, calcitonin gene related peptide, and substance P. We show that it is possible to collect high quality, multicolor images of the innervation pattern of tendons. To map immunolabeled structures and the anatomical structures of the tendon fluorescence images and bright field images were merged. CONCLUSION:For the orthopedic community our approach might be a convenient research tool to simultaneously utilize multiple neuronal markers on the same tissue section and to define with greater accuracy the heterogeneity of tendon innervation.

The type II superior labrum anterior to posterior (SLAP) repair is a viable option in young and demanding patients, although a prolonged period of pain after surgery is described in the literature. The reason for this fact remains unknown. Thus, the purpose of this study was to investigate the molecular pattern of the biceps tendon anchor, where the sutures for repair are placed. The long head of the biceps tendon (LHBT), including the superior labrum, was dissected in the setting of reverse total shoulder arthroplasty. Immunohistochemical staining was performed using neurofilament (NF) and protein gene product (PGP) 9.5 as general markers for axons and calcitonin gene-related peptide (CGRP) and substance P for nociceptive transmission. A quantitative assessment was performed according to the two regions of interest (ROIs), i.e., the anterosuperior (ROI I) and the posterosuperior labrum (ROI II). Eleven LHBTs with a mean age of 73 years (range: 66-87 years) were harvested intraoperatively. Six LHBTs were gained in osteoarthrosis and five in fractures. We found an inhomogeneous distribution of axons in the anterosuperior and posterosuperior parts of the labrum in all the specimens irrespective of the age, gender, and baseline situation. There was a significantly higher number (p < 0.01) as well as density (p < 0.001) of NF-positive axons in ROI I compared to ROI II. Nociceptive fibers were always found along the NF-positive axons. Thus, our results indicate that the biceps tendon anchor itself is a highly innervated region comprising different nerve qualities. The anterosuperior labrum contains a higher absolute number and density of axons compared to the posterosuperior parts. Furthermore, we were able to prove the presence of nociceptive fibers in the superior labrum. The results obtained in this study could contribute to the variability of pain after SLAP repair.

INTRODUCTION: Any surgical nerve reconstruction must take into account the amount of individual nerve fibres at any given level of injury. To date, however, literature on qualitative and quantitative assessment of motor axons of the peripheral nerves of the upper extremity is scarce. The aim of the present study is to present the total number of motor fibres of the brachial plexus from each root down to the level of its corresponding branches. MATERIAL AND METHODS: Nerve samples have been harvested from 9 organ donors immediately after death. From 8 incisions ranging from the neck to the wrist a total of 36 nerve samples were gained per organ donor. A special immunohistochemical protocol was applied to visualize the specific structure of interest within the nerve cross section. Antibody against neurofilament served to determine the total amount of myelinated and unmyelinated axons. Antibody against choline acetyltranferase (ChAT) was used to detect cholinergic/motor fibres. Peripheral nerve cross sections were then scanned and evaluated with a digital software program to allow quantification of each cross section. These numbers were cross checked in an animal model with standard retrograde tracing methods. Finally, the quality of this method was also cross checked with staining ventral and dorsal roots of organ donors at spinal cord level. RESULTS: As expected any given peripheral nerve contains afferent fibers. To our surprise, however, only around 10% of all axons in a mixed peripheral nerve are efferent fibers. In a “pure” peripheral motor nerve (thoracodorsal nerve) one fifth of the axons are cholinergic. In a pure cranial motor nerve the motor portion rises to about 25%(accessory nerve). The control experiments in a rodent animal model show good correlation between retrogradely labelled motor neurons with ChAT positive labels in the peripheral nerve section. CONCLUSION: Here we present for the first time a quantitative analysis of all afferent and efferent fibres of the brachial plexus and its consecutive nerves. The surprising finding is that even “pure” motor nerves with a suspected high number of motor fibres (thoracodorsal nerve) only have a relatively small number of efferents. Since this ratio is relatively constant for motor nerves at different levels of the extremity these results challenge the traditional view of fiber distribution and innervation density in man.

Newts and salamanders, both urodele amphibians, are the only vertebrates with tremendous regenerative potency throughout their lifetime. In contrast to the limited regenerative potential of most mammals, including humans, they can regenerate an entire limb after amputation and many other structures of their bodies, whereas humans mainly respond to injury by the formation of a scar. The intention of plastic surgery is to restore function of injured body parts, with the highest principle to replace "like with like." Despite tremendous improvements in surgical techniques over the last century, the remaining drawbacks include the availability of autologous tissue for transfer to restore extensive tissue loss. Here, some regenerative features of the urodeles are reviewed, in particular wound healing, nerve and limb regeneration, and their potential impact for reconstructive surgery are discussed. With a detailed molecular and cellular understanding of the urodele regeneration processes in combination with recent advances in tissue engineering, new perspectives for plastic surgery and especially improvements in regards to tissue regeneration are opened.

Human skin dermis is composed of the superficial papillary dermis and the reticular dermis in the lower layers, which can easily be distinguished histologically. In vitro analyses of fibroblasts from explant cultures from superficial and lower dermal layers suggest that human skin comprises at least two fibroblast lineages with distinct morphology, expression profiles, and functions. However, while for mouse skin cell surface markers have been identified, allowing the isolation of pure populations of one lineage or the other via FACS, this has not been achieved for human skin fibroblasts. We have now discovered two cell surface markers that discriminate between papillary and reticular fibroblasts. While FAP+CD90- cells display increased proliferative potential, express PDPN and NTN1, and cannot be differentiated into adipocytes, FAP-CD90+ fibroblasts express high levels of ACTA2, MGP, PPARγ, and CD36 and readily undergo adipogenic differentiation, a hallmark of reticular fibroblasts. Flow cytometric analysis of fibroblasts isolated from superficial and lower layers of human dermis showed that FAP+CD90- cells are enriched in the papillary dermis. Altogether, functional analysis and expression profiling confirms that FAP+CD90- cells represent papillary fibroblasts, whereas FAP-CD90+ fibroblasts derive from the reticular lineage. Although papillary and reticular fibroblasts are enriched in the upper or lower dermis, respectively, they are not spatially restricted, and the microenvironment seems to affect their function.

Newts and salamanders, both urodele amphibians, are the only vertebrates with tremendous regenerative potency throughout their lifetime. In contrast to the limited regenerative potential of most mammals, including humans, they can regenerate an entire limb after amputation and many other structures of their bodies while humans mainly respond to injury by the formation of a scar. The intention of plastic surgery is to restore function of injured body parts, with the highest principle to replace “like with like”. Despite tremendous improvements in surgical techniques over the last century, the remaining drawbacks include the availability of autologous tissue for transfer to restore extensive tissue loss. We want to present some regenerative features of the urodeles, in particular wound healing, nerveand limb regeneration and to discuss their potential impact for reconstructive surgery. With a detailed molecular and cellular understanding of the urodele regeneration processes in combination with recent advances in tissue engineering, new perspectives for plastic surgery and especially improvements in regards to tissue regeneration are opened.

OBJECTIVE:Axons traveling within the brachial plexus are responsible for the dexterous control of human arm and hand movements. Despite comprehensive knowledge on the topographical anatomy of nerves innervating the human upper limbs, the definite quantity of sensory and motor axons within this neural network remains elusive. Our aim was to perform a quantitative analysis of the axonal components of human upper limb nerves based on highly specific molecular features from spinal cord level to the terminal nerves at wrist level.METHODS:Nerve specimen harvest at predefined harvesting sites (plexus roots and cords as well as major nerves originating from the brachial plexus innervating the arm and hand) was performed in 9 human heart-beating organ donors. Double immunofluorescence staining using antibodies against choline-acetyltransferase and neurofilament was performed to differentiate motor and sensory axons on nerve cross sections.RESULTS:Three hundred fifty thousand axons emerge from the spinal cord to innervate the human upper limb, of which 10% are motor neurons. In all nerves studied, sensory axons outnumber motor axons by a ratio of at least 9:1. The sensory axon contribution increases when moving distally, whereas only 1,700 motor axons reach the hand to innervate the intrinsic musculature.INTERPRETATION:Our results suggest that upper limb motor execution, and particularly dexterous coordination of hand movement, require an unexpectedly low number of motor neurons, with a large convergence of afferent input for feedback control. Ann Neurol 2017;82:396-408.

Purpose: To test whether palisade endings are a general feature of mammalian extraocular muscles (EOMs). Methods: Thirteen species, some frontal-eyed (human, monkey, cat, and ferret), and others lateral-eyed (pig, sheep, calf, horse, rabbit, rat, mouse, gerbil, and guinea pig) were analyzed. Palisade endings were labeled by using different combinations of immunofluorescence techniques. Three-dimensional reconstructions of immunolabeled palisade endings were done. Results: In all frontal-eyed species, palisade endings were a consistent feature in the rectus EOMs. Their total number was high and they exhibited an EOM-specific distribution. In particular, the number of palisade endings in the medial recti was significantly higher than in the other rectus muscles. In the lateral-eyed animals, palisade endings were infrequent and, when present, their total number was rather low. They were only found in ungulates (sheep, calf, pig, and horse) and in rabbit. In rodents (rat, guinea pig, mouse, and gerbil) palisade endings were found infrequently (e.g., rat) or were completely absent. Palisade endings in frontal-eyed species and in some lateral-eyed species (pig, sheep, calf, and horse) had a uniform morphology. They generally lacked α-bungarotoxin staining, with a few exceptions in primates. Palisade endings in other lateral-eyed species (rabbit and rat) exhibited a simplified morphology and bound α-bungarotoxin. Conclusions: Palisade endings are not a universal feature of mammalian EOMs. So, if they are proprioceptors, not all species require them. Because in frontal-eyed species, the medial rectus muscle has the highest number of palisade endings, they likely play a special role in convergence.

During inflammation, neutrophils are rapidly mobilized from the bone marrow storage pool into peripheral blood (PB) to enter lesional sites, where most rapidly undergo apoptosis. Monocytes constitute a second wave of inflammatory immigrates, giving rise to long-lived macrophages and dendritic cell subsets. According to descriptive immunophenotypic and cell culture studies, neutrophils may directly "transdifferentiate" into monocytes/macrophages. We provide mechanistic data in human and murine models supporting the existence of this cellular pathway. First, the inflammatory signal-induced MKK6-p38MAPK cascade activates a monocyte differentiation program in human granulocyte colony-stimulating factor-dependent neutrophils. Second, adoptively transferred neutrophils isolated from G-CSF-pretreated mice rapidly acquired monocyte characteristics in response to inflammatory signals in vivo. Consistently, inflammatory signals led to the recruitment of osteoclast progenitor cell potential from ex vivo-isolated G-CSF-mobilized human blood neutrophils. Monocytic cell differentiation potential was retained in left-shifted band-stage neutrophils but lost in neutrophils from steady-state PB. MKK6-p38MAPK signaling in HL60 model cells led to diminishment of the transcription factor C/EBPα, which enabled the induction of a monocytic cell differentiation program. Gene profiling confirmed lineage conversion from band-stage neutrophils to monocytic cells. Therefore, inflammatory signals relayed by the MKK6-p38MAPK cascade induce monocytic cell differentiation from band-stage neutrophils.

Langerhans cells (LCs) are a unique subset of dendritic cells (DCs) that express epithelial adhesion molecules, allowing them to form contacts with epithelial cells and reside in epidermal/epithelial tissues. The dynamic regulation of epithelial adhesion plays a decisive role in the life cycle of LCs. It controls whether LCs remain immature and sessile within the epidermis or mature and egress to initiate immune responses. So far, the molecular machinery regulating epithelial adhesion molecules during LC maturation remains elusive. Here, we generated pure populations of immature human LCs in vitro to systematically probe for gene‐expression changes during LC maturation. LCs down‐regulate a set of epithelial genes including E‐cadherin, while they upregulate the mesenchymal marker N‐cadherin known to facilitate cell migration. In addition, N‐cadherin is constitutively expressed by monocyte‐derived DCs known to exhibit characteristics of both inflammatory‐type and interstitial/dermal DCs. Moreover, the transcription factors ZEB1 and ZEB2 (ZEB is zinc‐finger E‐box‐binding homeobox) are upregulated in migratory LCs. ZEB1 and ZEB2 have been shown to induce epithelial‐to‐mesenchymal transition (EMT) and invasive behavior in cancer cells undergoing metastasis. Our results provide the first hint that the molecular EMT machinery might facilitate LC mobilization. Moreover, our study suggests that N‐cadherin plays a role during DC migration.

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.