Langerhans cells (LCs) are antigen-presenting cells that reside in the epidermis of the skin and traffic to lymph nodes (LNs). The general role of these cells in skin immune responses is not clear because distinct models of LC depletion resulted in opposite conclusions about their role in contact hypersensitivity (CHS) responses. While comparing these models, we discovered a novel population of LCs that resides in the dermis and does not represent migrating epidermal LCs, as previously thought. Unlike epidermal LCs, dermal Langerin+ dendritic cells (DCs) were radiosensitive and displayed a distinct cell surface phenotype. Dermal Langerin+ DCs migrate from the skin to the LNs after inflammation and in the steady state, and represent the majority of Langerin+ DCs in skin draining LNs. Both epidermal and dermal Langerin+ DCs were depleted by treatment with diphtheria toxin in Lang-DTREGFP knock-in mice. In contrast, transgenic hLang-DTA mice lack epidermal LCs, but have normal numbers of dermal Langerin+ DCs. CHS responses were abrogated upon depletion of both epidermal and dermal LCs, but were unaffected in the absence of only epidermal LCs. This suggests that dermal LCs can mediate CHS and provides an explanation for previous differences observed in the two-model systems.

There was an error published in Development 138, 3647-3656.The panel labels on the left indicating genotypes were misaligned in Fig. 5A. The corrected Fig. 5 appears in full below.The authors apologise to readers for this mistake.

Rationale: Acute respiratory distress syndrome (ARDS) remains a major cause of respiratory failure in critically ill patients. Mesenchymal stromal cells (MSCs) are a promising candidate for a cell-based therapy. However, the mechanisms of MSCs' effects in ARDS are not well understood. In this study, we focused on the paracrine effect of MSCs on macrophage polarization and the role of extracellular vesicle (EV)-mediated mitochondrial transfer.Objectives: To determine the effects of human MSCs on macrophage function in the ARDS environment and to elucidate the mechanisms of these effects.Methods: Human monocyte-derived macrophages (MDMs) were studied in noncontact coculture with human MSCs when stimulated with LPS or bronchoalveolar lavage fluid (BALF) from patients with ARDS. Murine alveolar macrophages (AMs) were cultured ex vivo with/without human MSC-derived EVs before adoptive transfer to LPS-injured mice.Measurements and Main Results: MSCs suppressed cytokine production, increased M2 macrophage marker expression, and augmented phagocytic capacity of human MDMs stimulated with LPS or ARDS BALF. These effects were partially mediated by CD44-expressing EVs. Adoptive transfer of AMs pretreated with MSCderived EVs reduced inflammation and lung injury in LPS-injured mice. Inhibition of oxidative phosphorylation in MDMs prevented the modulatory effects of MSCs. Generating dysfunctional mitochondria in MSCs using rhodamine 6G pretreatment also abrogated these effects.Conclusions: In the ARDS environment, MSCs promote an antiinflammatory and highly phagocytic macrophage phenotype through EV-mediated mitochondrial transfer. MSC-induced changes in macrophage phenotype critically depend on enhancement of macrophage oxidative phosphorylation. AMs treated with MSCderived EVs ameliorate lung injury in vivo.

Mesenchymal stromal cells (MSC) have been reported to improve bacterial clearance in preclinical models of Acute Respiratory Distress Syndrome (ARDS) and sepsis. The mechanism of this effect is not fully elucidated yet. The primary objective of this study was to investigate the hypothesis that the antimicrobial effect of MSC in vivo depends on their modulation of macrophage phagocytic activity which occurs through mitochondrial transfer. We established that selective depletion of alveolar macrophages (AM) with intranasal (IN) administration of liposomal clodronate resulted in complete abrogation of MSC antimicrobial effect in the in vivo model of Escherichia coli pneumonia. Furthermore, we showed that MSC administration was associated with enhanced AM phagocytosis in vivo. We showed that direct coculture of MSC with monocyte‐derived macrophages enhanced their phagocytic capacity. By fluorescent imaging and flow cytometry we demonstrated extensive mitochondrial transfer from MSC to macrophages which occurred at least partially through tunneling nanotubes (TNT)‐like structures. We also detected that lung macrophages readily acquire MSC mitochondria in vivo, and macrophages which are positive for MSC mitochondria display more pronounced phagocytic activity. Finally, partial inhibition of mitochondrial transfer through blockage of TNT formation by MSC resulted in failure to improve macrophage bioenergetics and complete abrogation of the MSC effect on macrophage phagocytosis in vitro and the antimicrobial effect of MSC in vivo. Collectively, this work for the first time demonstrates that mitochondrial transfer from MSC to innate immune cells leads to enhancement in phagocytic activity and reveals an important novel mechanism for the antimicrobial effect of MSC in ARDS. Stem Cells 2016;34:2210–2223

Langerhans cells (LCs) are prominent dendritic cells (DCs) in epithelia, but their role in immunity is poorly defined. To track and discriminate LCs from dermal DCs in vivo, we developed knockin mice expressing enhanced green fluorescent protein (EGFP) under the control of the langerin (CD207) gene. By using vital imaging, we showed that most EGFP(+) LCs were sessile under steady-state conditions, whereas skin inflammation induced LC motility and emigration to lymph nodes (LNs). After skin immunization, dermal DCs arrived in LNs first and colonized areas distinct from slower migrating LCs. LCs reaching LNs under steady-state or inflammatory conditions expressed similar levels of costimulatory molecules. Langerin and EGFP were also expressed on thymic DCs and on blood-derived, CD8alpha(+) DCs from all secondary lymphoid organs. By using a similar knockin strategy involving a diphtheria toxin receptor (DTR) fused to EGFP, we demonstrated that LCs were dispensable for triggering hapten-specific T cell effectors through skin immunization.

Most tissues develop from stem cells and precursors that undergo differentiation as their proliferative potential decreases. Mature differentiated cells rarely proliferate and are replaced at the end of their life by new cells derived from precursors. Langerhans cells (LCs) of the epidermis, although of myeloid origin, were shown to renew in tissues independently from the bone marrow, suggesting the existence of a dermal or epidermal progenitor. We investigated the mechanisms involved in LC development and homeostasis. We observed that a single wave of LC precursors was recruited in the epidermis of mice around embryonic day 18 and acquired a dendritic morphology, major histocompatibility complex II, CD11c, and langerin expression immediately after birth. Langerin+ cells then undergo a massive burst of proliferation between postnatal day 2 (P2) and P7, expanding their numbers by 10–20-fold. After the first week of life, we observed low-level proliferation of langerin+ cells within the epidermis. However, in a mouse model of atopic dermatitis (AD), a keratinocyte signal triggered increased epidermal LC proliferation. Similar findings were observed in epidermis from human patients with AD. Therefore, proliferation of differentiated resident cells represents an alternative pathway for development in the newborn, homeostasis, and expansion in adults of selected myeloid cell populations such as LCs. This mechanism may be relevant in locations where leukocyte trafficking is limited.

Langerhans cells (LCs) constitute a subset of dendritic cells (DCs) that express the lectin langerin and that reside in their immature state in epidermis. Paradoxically, in mice permitting diphtheria toxin (DT)–mediated ablation of LCs, epidermal LCs reappeared with kinetics that lagged behind that of their putative progeny found in lymph nodes (LNs). Using bone marrow (BM) chimeras, we showed that a major fraction of the langerin+, skin-derived DCs found in LNs originates from a developmental pathway that is independent from that of epidermal LCs. This pathway, the existence of which was unexpected, originates in the dermis and gives rise to langerin+ dermal DCs (DDCs) that should not be confused with epidermal LCs en route to LNs. It explains that after DT treatment, some langerin+, skin-derived DCs reappear in LNs long before LC-derived DCs. Using CD45 expression and BrdU-labeling kinetics, both LCs and langerin+ DDCs were found to coexist in wild-type mice. Moreover, DT-mediated ablation of epidermal LCs opened otherwise filled niches and permitted repopulation of adult noninflammatory epidermis with BM-derived LCs. Our results stress that the langerin+ DC network is more complex than originally thought and have implications for the development of transcutaneous vaccines and the improvement of humanized mouse models.

Langerin is a C-type lectin receptor that recognizes glycosylated patterns on pathogens. Langerin is used to identify human and mouse epidermal Langerhans cells (LCs), as well as migratory LCs in the dermis and the skin draining lymph nodes (DLNs). Using a mouse model that allows conditional ablation of langerin+ cells in vivo, together with congenic bone marrow chimeras and parabiotic mice as tools to differentiate LC- and blood-derived dendritic cells (DCs), we have revisited the origin of langerin+ DCs in the skin DLNs. Our results show that in contrast to the current view, langerin+CD8− DCs in the skin DLNs do not derive exclusively from migratory LCs, but also include blood-borne langerin+ DCs that transit through the dermis before reaching the DLN. The recruitment of circulating langerin+ DCs to the skin is dependent on endothelial selectins and CCR2, whereas their recruitment to the skin DLNs requires CCR7 and is independent of CD62L. We also show that circulating langerin+ DCs patrol the dermis in the steady state and migrate to the skin DLNs charged with skin antigens. We propose that this is an important and previously unappreciated element of immunosurveillance that needs to be taken into account in the design of novel vaccine strategies.