Circadian clocks guide dendritic cells into skin lymphatics.

Migration of leukocytes from the skin to lymph nodes (LNs) via afferent lymphatic vessels (LVs) is pivotal for adaptive immune responses1,2. Circadian rhythms have emerged as important regulators of leukocyte trafficking to LNs via the blood3,4. Here, we demonstrate that dendritic cells (DCs) have a circadian migration pattern into LVs, which peaks during the rest phase in mice. This migration pattern is determined by rhythmic gradients in the expression of the chemokine CCL21 and of adhesion molecules in both mice and humans. Chronopharmacological targeting of the involved factors abrogates circadian migration of DCs. We identify cell-intrinsic circadian oscillations in skin lymphatic endothelial cells (LECs) and DCs that cogovern these rhythms, as their genetic disruption in either cell type ablates circadian trafficking. These observations indicate that circadian clocks control the infiltration of DCs into skin lymphatics, a process that is essential for many adaptive immune responses and relevant for vaccination and immunotherapies.

Expression of the membrane tetraspanin claudin 18 on cancer cells promotes T lymphocyte infiltration and antitumor immunity in pancreatic cancer.

Tumors weakly infiltrated by T lymphocytes poorly respond to immunotherapy. We aimed to unveil malignancy-associated programs regulating T cell entrance, arrest, and activation in the tumor environment. Differential expression of cell adhesion and tissue architecture programs, particularly the presence of the membrane tetraspanin claudin (CLDN)18 as a signature gene, demarcated immune-infiltrated from immune-depleted mouse pancreatic tumors. In human pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer, CLDN18 expression positively correlated with more differentiated histology and favorable prognosis. CLDN18 on the cell surface promoted accrual of cytotoxic T lymphocytes (CTLs), facilitating direct CTL contacts with tumor cells by driving the mobilization of the adhesion protein ALCAM to the lipid rafts of the tumor cell membrane through actin. This process favored the formation of robust immunological synapses (ISs) between CTLs and CLDN18-positive cancer cells, resulting in increased T cell activation. Our data reveal an immune role for CLDN18 in orchestrating T cell infiltration and shaping the tumor immune contexture.

Imaging leukocyte migration through afferent lymphatics.

Afferent lymphatics mediate the transport of antigen and leukocytes, especially of dendritic cells (DCs) and T cells, from peripheral tissues to draining lymph nodes (dLNs). As such they play important roles in the induction and regulation of adaptive immunity. Over the past 15 years, great advances in our understanding of leukocyte trafficking through afferent lymphatics have been made through time-lapse imaging studies performed in tissue explants and in vivo, allowing to visualize this process with cellular resolution. Intravital imaging has revealed that intralymphatic leukocytes continue to actively migrate once they have entered into lymphatic capillaries, as a consequence of the low flow conditions present in this compartment. In fact, leukocytes spend considerable time migrating, patrolling and interacting with the lymphatic endothelium or with other intralymphatic leukocytes within lymphatic capillaries. Cells typically only start to detach once they arrive in downstream-located collecting vessels, where vessel contractions contribute to enhanced lymph flow. In this review, we will introduce the biology of afferent lymphatic vessels and report on the presumed significance of DC and T cell migration via this route. We will specifically highlight how time-lapse imaging has contributed to the current model of lymphatic trafficking and the emerging notion that - besides transport - lymphatic capillaries exert additional roles in immune modulation.

Transcription factor FOXP2 is a flow-induced regulator of collecting lymphatic vessels.

The lymphatic system is composed of a hierarchical network of fluid absorbing lymphatic capillaries and transporting collecting vessels. Despite distinct functions and morphologies, molecular mechanisms that regulate the identity of the different vessel types are poorly understood. Through transcriptional analysis of murine dermal lymphatic endothelial cells (LECs), we identified Foxp2, a member of the FOXP family of transcription factors implicated in speech development, as a collecting vessel signature gene. FOXP2 expression was induced after initiation of lymph flow in vivo and upon shear stress on primary LECs in vitro. Loss of FOXC2, the major flow-responsive transcriptional regulator of lymphatic valve formation, abolished FOXP2 induction in vitro and in vivo. Genetic deletion of Foxp2 in mice using the endothelial-specific Tie2-Cre or the tamoxifen-inducible LEC-specific Prox1-CreERT2 line resulted in enlarged collecting vessels and defective valves characterized by loss of NFATc1 activity. Our results identify FOXP2 as a new flow-induced transcriptional regulator of collecting lymphatic vessel morphogenesis and highlight the existence of unique transcription factor codes in the establishment of vessel-type-specific endothelial cell identities.

Lymph Vessels Associate with Cancer Stem Cells from Initiation to Malignant Stages of Squamous Cell Carcinoma.

Tumor-associated lymph vessels and lymph node involvement are critical staging criteria in several cancers. In skin squamous cell carcinoma, lymph vessels play a role in cancer development and metastatic spread. However, their relationship with the cancer stem cell niche at early tumor stages remains unclear. To address this gap, we studied the lymph vessel localization at the cancer stem cell niche and observed an association from benign skin lesions to malignant stages of skin squamous cell carcinoma. By co-culturing lymphatic endothelial cells with cancer cell lines representing the initiation and promotion stages, and conducting RNA profiling, we observed a reciprocal induction of cell adhesion, immunity regulation, and vessel remodeling genes, suggesting dynamic interactions between lymphatic and cancer cells. Additionally, imaging analyses of the cultured cells revealed the establishment of heterotypic contacts between cancer cells and lymph endothelial cells, potentially contributing to the observed distribution and maintenance at the cancer stem cell niche, inducing downstream cellular responses. Our data provide evidence for an association of lymph vessels from the early stages of skin squamous cell carcinoma development, opening new avenues for better comprehending their involvement in cancer progression.

The lymphatic vasculature: An active and dynamic player in cancer progression.

The lymphatic vasculature has been widely described and explored for its key functions in fluid homeostasis and in the organization and modulation of the immune response. Besides transporting immune cells, lymphatic vessels play relevant roles in tumor growth and tumor cell dissemination. Cancer cells that have invaded into afferent lymphatics are propagated to tumor-draining lymph nodes (LNs), which represent an important hub for metastatic cell arrest and growth, immune modulation, and secondary dissemination to distant sites. In recent years many studies have reported new mechanisms by which the lymphatic vasculature affects cancer progression, ranging from induction of lymphangiogenesis to metastatic niche preconditioning or immune modulation. In this review, we provide an up-to-date description of lymphatic organization and function in peripheral tissues and in LNs and the changes induced to this system by tumor growth and progression. We will specifically focus on the reported interactions that occur between tumor cells and lymphatic endothelial cells (LECs), as well as on interactions between immune cells and LECs, both in the tumor microenvironment and in tumor-draining LNs. Moreover, the most recent prognostic and therapeutic implications of lymphatics in cancer will be reported and discussed in light of the new immune-modulatory roles that have been ascribed to LECs.

Sialoglycans on lymphatic endothelial cells augment interactions with Siglec-1 (CD169) of lymph node macrophages.

Cellular interactions between endothelial cells and macrophages regulate macrophage localization and phenotype, but the mechanisms underlying these interactions are poorly understood. Here we explored the role of sialoglycans on lymphatic endothelial cells (LEC) in interactions with macrophage-expressed Siglec-1 (CD169). Lectin-binding assays and mass spectrometric analyses revealed that LEC from human skin express more sialylated glycans than the corresponding blood endothelial cells. Higher amounts of sialylated and/or sulfated glycans on LEC than BEC were consistently observed in murine skin, lung and lymph nodes. The floor LEC of the subcapsular sinus (SCS) in murine lymph nodes (LN) displayed sialylated glycans at particularly high densities. The sialoglycans of LN LEC were strongly bound by Siglec-1. Such binding plays an important role in the localization of Siglec-1+ LN-SCS macrophages, as their numbers are strongly reduced in mice expressing a Siglec-1 mutant that is defective in sialoglycan binding. The residual Siglec-1+ macrophages are less proliferative and have a more anti-inflammatory phenotype. We propose that the densely clustered, sialylated glycans on the SCS floor LEC are a key component of the macrophage niche, providing anchorage for the Siglec-1+ LN-SCS macrophages.

AutoTube: a novel software for the automated morphometric analysis of vascular networks in tissues.

Due to their involvement in many physiologic and pathologic processes, there is a great interest in identifying new molecular pathways that mediate the formation and function of blood and lymphatic vessels. Vascular research increasingly involves the image-based analysis and quantification of vessel networks in tissue whole-mounts or of tube-like structures formed by cultured endothelial cells in vitro. While both types of experiments deliver important mechanistic insights into (lymph)angiogenic processes, the manual analysis and quantification of such experiments are typically labour-intensive and affected by inter-experimenter variability. To bypass these problems, we developed AutoTube, a new software that quantifies parameters like the area covered by vessels, vessel width, skeleton length and branching or crossing points of vascular networks in tissues and in in vitro assays. AutoTube is freely downloadable, comprises an intuitive graphical user interface and helps to perform otherwise highly time-consuming image analyses in a rapid, automated and reproducible manner. By analysing lymphatic and blood vascular networks in whole-mounts prepared from different tissues or from gene-targeted mice with known vascular abnormalities, we demonstrate the ability of AutoTube to determine vascular parameters in close agreement to the manual analyses and to identify statistically significant differences in vascular morphology in tissues and in vascular networks formed in in vitro assays.

IL-1 Coordinates the Neutrophil Response to C. albicans in the Oral Mucosa.

Mucosal infections with Candida albicans belong to the most frequent forms of fungal diseases. Host protection is conferred by cellular immunity; however, the induction of antifungal immunity is not well understood. Using a mouse model of oropharyngeal candidiasis (OPC) we show that interleukin-1 receptor (IL-1R) signaling is critical for fungal control at the onset of infection through its impact on neutrophils at two levels. We demonstrate that both the recruitment of circulating neutrophils to the site of infection and the mobilization of newly generated neutrophils from the bone marrow depended on IL-1R. Consistently, IL-1R-deficient mice displayed impaired chemokine production at the site of infection and defective secretion of granulocyte colony-stimulating factor (G-CSF) in the circulation in response to C. albicans. Strikingly, endothelial cells were identified as the primary cellular source of G-CSF during OPC, which responded to IL-1α that was released from keratinocytes in the infected tissue. The IL-1-dependent crosstalk between two different cellular subsets of the nonhematopoietic compartment was confirmed in vitro using a novel murine tongue-derived keratinocyte cell line and an established endothelial cell line. These data establish a new link between IL-1 and granulopoiesis in the context of fungal infection. Together, we identified two complementary mechanisms coordinating the neutrophil response in the oral mucosa, which is critical for preventing fungal growth and dissemination, and thus protects the host from disease.

Stromal Expression of Activated Leukocyte Cell Adhesion Molecule Promotes Lung Tumor Growth and Metastasis.

Activated leukocyte cell adhesion molecule (ALCAM) is expressed on various cell types, including leukocytes, endothelial cells, and certain tumor cells. Although ALCAM expression on tumor cells has been linked to tumor invasion and metastatic spread, the contribution of ALCAM expressed in cells forming the tumor stroma to cancer progression has not been investigated. In this study, ALCAM-deficient (ALCAM-/-) mice were used to evaluate the role of ALCAM in lung tumor growth and metastasis. ALCAM-/- mice displayed an altered blood vascular network in the lung and the diaphragm, indicative of an angiogenetic defect. The absence of ALCAM expression in cells forming the stromal tumor microenvironment profoundly affected lung tumor growth in three different i.v. metastasis models. In the case of Lewis lung carcinoma (LLC), an additional defect in tumor cell homing to the lungs and a resulting reduction in the number of lung tumor nodules were observed. Similarly, when LLC cells were implanted subcutaneously for the study of spontaneous tumor cell metastasis, the rate of LLC metastasis to the lungs was profoundly reduced in ALCAM-/- mice. Taken together, our work demonstrates for the first time the in vivo contribution of ALCAM to angiogenesis and reveals a novel role of stromally expressed ALCAM in supporting tumor growth and metastatic spread.

Postnatal Deletion of Podoplanin in Lymphatic Endothelium Results in Blood Filling of the Lymphatic System and Impairs Dendritic Cell Migration to Lymph Nodes.

OBJECTIVE: The lymphatic vascular system exerts major physiological functions in the transport of interstitial fluid from peripheral tissues back to the blood circulation and in the trafficking of immune cells to lymph nodes. Previous studies in global constitutive knockout mice for the lymphatic transmembrane molecule podoplanin reported perinatal lethality and a complex phenotype with lung abnormalities, cardiac defects, lymphedema, blood-filled lymphatic vessels, and lack of lymph node organization, reflecting the importance of podoplanin expression not only by the lymphatic endothelium but also by a variety of nonendothelial cell types. Therefore, we aimed to dissect the specific role of podoplanin expressed by adult lymphatic vessels. APPROACH AND RESULTS: We generated an inducible, lymphatic-specific podoplanin knockout mouse model (PdpnΔLEC) and induced gene deletion postnatally. PdpnΔLEC mice were viable, and their lymphatic vessels appeared morphologically normal with unaltered fluid drainage function. Intriguingly, PdpnΔLEC mice had blood-filled lymph nodes and vessels, most frequently in the neck and axillary region, and displayed a blood-filled thoracic duct, suggestive of retrograde filling of blood from the blood circulation into the lymphatic system. Histological and fluorescence-activated cell sorter analyses revealed normal lymph node organization with the presence of erythrocytes within lymph node lymphatic vessels but not surrounding high endothelial venules. Moreover, fluorescein isothiocyanate painting experiments revealed reduced dendritic cell migration to lymph nodes in PdpnΔLEC mice. CONCLUSIONS: These results reveal an important role of podoplanin expressed by lymphatic vessels in preventing postnatal blood filling of the lymphatic vascular system and in contributing to efficient dendritic cell migration to the lymph nodes.

A translocated effector required for Bartonella dissemination from derma to blood safeguards migratory host cells from damage by co-translocated effectors.

Numerous bacterial pathogens secrete multiple effectors to modulate host cellular functions. These effectors may interfere with each other to efficiently control the infection process. Bartonellae are Gram-negative, facultative intracellular bacteria using a VirB type IV secretion system to translocate a cocktail of Bartonella effector proteins (Beps) into host cells. Based on in vitro infection models we demonstrate here that BepE protects infected migratory cells from injurious effects triggered by BepC and is required for in vivo dissemination of bacteria from the dermal site of inoculation to blood. Human endothelial cells (HUVECs) infected with a ΔbepE mutant of B. henselae (Bhe) displayed a cell fragmentation phenotype resulting from Bep-dependent disturbance of rear edge detachment during migration. A ΔbepCE mutant did not show cell fragmentation, indicating that BepC is critical for triggering this deleterious phenotype. Complementation of ΔbepE with BepEBhe or its homologues from other Bartonella species abolished cell fragmentation. This cyto-protective activity is confined to the C-terminal Bartonella intracellular delivery (BID) domain of BepEBhe (BID2.EBhe). Ectopic expression of BID2.EBhe impeded the disruption of actin stress fibers by Rho Inhibitor 1, indicating that BepE restores normal cell migration via the RhoA signaling pathway, a major regulator of rear edge retraction. An intradermal (i.d.) model for B. tribocorum (Btr) infection in the rat reservoir host mimicking the natural route of infection by blood sucking arthropods allowed demonstrating a vital role for BepE in bacterial dissemination from derma to blood. While the Btr mutant ΔbepDE was abacteremic following i.d. inoculation, complementation with BepEBtr, BepEBhe or BIDs.EBhe restored bacteremia. Given that we observed a similar protective effect of BepEBhe on infected bone marrow-derived dendritic cells migrating through a monolayer of lymphatic endothelial cells we propose that infected dermal dendritic cells may be involved in disseminating Bartonella towards the blood stream in a BepE-dependent manner.

Interleukin-7 is produced by afferent lymphatic vessels and supports lymphatic drainage.

The cytokine interleukin (IL)-7 exerts essential roles in lymph node (LN) organogenesis and lymphocyte development and homeostasis. Recent studies have identified lymphatic endothelial cells (LECs) as a major source of IL-7 in LNs. Here, we report that LECs not only produce IL-7, but also express the IL-7 receptor chains IL-7Rα and CD132. Stimulation with recombinant IL-7 enhanced LEC in vitro activity and induced lymphangiogenesis in the cornea of wild-type (WT) mice. Whereas in IL-7Rα(-/-) mice, dermal lymphatic vessels (LVs) were abnormally organized and lymphatic drainage was compromised, transgenic overexpression of IL-7 in mice resulted in an expanded dermal LV network with increased drainage function. Moreover, systemic treatment with recombinant IL-7 enhanced lymphatic drainage in the skin of WT mice and of mice devoid of lymphocytes. Experiments in IL-7Rα(-/-) bone marrow chimeras demonstrated that the drainage-enhancing activity of IL-7 was exclusively dependent on IL-7Rα expression in stromal but not in hematopoietic cells. Finally, near-infrared in vivo imaging performed in IL-7Rα(-/-) mice revealed that the pumping activity of collecting vessels was normal but fluid uptake into lymphatic capillaries was defective. Overall, our data point toward an unexpected new role for IL-7 as a potential autocrine mediator of lymphatic drainage.

Multidimensional Design of Anticancer Peptides.

The computer-assisted design and optimization of peptides with selective cancer cell killing activity was achieved through merging the features of anticancer peptides, cell-penetrating peptides, and tumor-homing peptides. Machine-learning classifiers identified candidate peptides that possess the predicted properties. Starting from a template amino acid sequence, peptide cytotoxicity against a range of cancer cell lines was systematically optimized while minimizing the effects on primary human endothelial cells. The computer-generated sequences featured improved cancer-cell penetration, induced cancer-cell apoptosis, and were enabled a decrease in the cytotoxic concentration of co-administered chemotherapeutic agents in vitro. This study demonstrates the potential of multidimensional machine-learning methods for rapidly obtaining peptides with the desired cellular activities.

The inflammatory response of lymphatic endothelium.

Lymphatic vessels have traditionally been regarded as a rather inert drainage system, which just passively transports fluids, leukocytes and antigen. However, it is becoming increasingly clear that the lymphatic vasculature is highly dynamic and plays a much more active role in inflammatory and immune processes. Tissue inflammation induces a rapid, stimulus-specific upregulation of chemokines and adhesion molecules in lymphatic endothelial cells and a proliferative expansion of the lymphatic network in the inflamed tissue and in draining lymph nodes. Moreover, increasing evidence suggests that inflammation-induced changes in the lymphatic vasculature have a profound impact on the course of inflammatory and immune responses, by modulating fluid drainage, leukocyte migration or the removal of inflammatory mediators from tissues. In this review we will summarize and discuss current knowledge of the inflammatory response of lymphatic endothelium and of inflammation-induced lymphangiogenesis and the current perspective on the overall functional significance of these processes.

Piloting the membranolytic activities of peptides with a self-organizing map.

Antimicrobial peptides (AMPs) show remarkable selectivity toward lipid membranes and possess promising antibiotic potential. Their modes of action are diverse and not fully understood, and innovative peptide design strategies are needed to generate AMPs with improved properties. We present a de novo peptide design approach that resulted in new AMPs possessing low-nanomolar membranolytic activities. Thermal analysis revealed an entropy-driven mechanism of action. The study demonstrates sustained potential of advanced computational methods for designing peptides with the desired activity.

Differential requirement for ROCK in dendritic cell migration within lymphatic capillaries in steady-state and inflammation.

Dendritic cell (DC) migration via lymphatic vessels to draining lymph nodes (dLNs) is crucial for the initiation of adaptive immunity. We imaged this process by intravital microscopy (IVM) in the ear skin of transgenic mice bearing red-fluorescent vasculature and yellow-fluorescent DCs. DCs within lymphatic capillaries were rarely transported by flow, but actively migrated within lymphatics and were significantly faster than in the interstitium. Pharmacologic blockade of the Rho-associated protein kinase (ROCK), which mediates nuclear contraction and de-adhesion from integrin ligands, significantly reduced DC migration from skin to dLNs in steady-state. IVM revealed that ROCK blockade strongly reduced the velocity of interstitial DC migration, but only marginally affected intralymphatic DC migration. By contrast, during tissue inflammation, ROCK blockade profoundly decreased both interstitial and intralymphatic DC migration. Inhibition of intralymphatic migration was paralleled by a strong up-regulation of ICAM-1 in lymphatic endothelium, suggesting that during inflammation ROCK mediates de-adhesion of DC-expressed integrins from lymphatic-expressed ICAM-1. Flow chamber assays confirmed an involvement of lymphatic-expressed ICAM-1 and DC-expressed ROCK in DC crawling on lymphatic endothelium. Overall, our findings further define the role of ROCK in DC migration to dLNs and reveal a differential requirement for ROCK in intralymphatic DC crawling during steady-state and inflammation.

IL-7-producing stromal cells are critical for lymph node remodeling.

Nonhematopoietic stromal cells of secondary lymphoid organs form important scaffold and fluid transport structures, such as lymph node (LN) trabeculae, lymph vessels, and conduits. Furthermore, through the production of chemokines and cytokines, these cells generate a particular microenvironment that determines lymphocyte positioning and supports lymphocyte homeostasis. IL-7 is an important stromal cell-derived cytokine that has been considered to be derived mainly from T-cell zone fibroblastic reticular cells. We show here that lymphatic endothelial cells (LECs) are a prominent source of IL-7 both in human and murine LNs. Using bacterial artificial chromosome transgenic IL-7-Cre mice, we found that fibroblastic reticular cells and LECs strongly up-regulated IL-7 expression during LN remodeling after viral infection and LN reconstruction after avascular transplantation. Furthermore, IL-7-producing stromal cells contributed to de novo formation of LyveI-positive lymphatic structures connecting reconstructed LNs with the surrounding tissue. Importantly, diphtheria toxin-mediated depletion of IL-7-producing stromal cells completely abolished LN reconstruction. Taken together, this study identifies LN LECs as a major source of IL-7 and shows that IL-7-producing stromal cells are critical for reconstruction and remodeling of the distinct LN microenvironment.

Novel role for ALCAM in lymphatic network formation and function.

Adhesion molecules play an important role in vascular biology because they mediate vascular stability, permeability, and leukocyte trafficking to and from tissues. Performing microarray analyses, we have recently identified activated leukocyte cell adhesion molecule (ALCAM) as an inflammation-induced gene in lymphatic endothelial cells (LECs). ALCAM belongs to the immunoglobulin superfamily and engages in homophilic as well as heterophilic interactions. In this study, we found ALCAM to be expressed at the protein level in human and murine lymphatic and blood vascular endothelial cells in vitro and in the vasculature of human and murine tissues in vivo. Functional in vitro experiments revealed that ALCAM mediates adhesive interactions, migration, and tube formation in LECs, suggesting a role for ALCAM in lymphatic vessel (LV) stability and in lymphangiogenesis. Furthermore, ALCAM supported dendritic cell (DC) adhesion to lymphatic endothelium. In agreement with these findings, experiments performed in ALCAM mice detected reduced LEC numbers in various tissues and defects in the formation of an organized LV network. Moreover, DC migration from lung to draining lymph nodes was compromised in ALCAM mice. Collectively, our data reveal a novel role for ALCAM in stabilizing LEC-LEC interactions and in the organization and function of the LV network.

Single-B cell analysis correlates high-lactate secretion with stress and increased apoptosis.

While cellular metabolism was proposed to be a driving factor of the activation and differentiation of B cells and the function of the resulting antibody-secreting cells (ASCs), the study of correlations between cellular metabolism and functionalities has been difficult due to the absence of technologies enabling the parallel measurement. Herein, we performed single-cell transcriptomics and introduced a direct concurrent functional and metabolic flux quantitation of individual murine B cells. Our transcriptomic data identified lactate metabolism as dynamic in ASCs, but antibody secretion did not correlate with lactate secretion rates (LSRs). Instead, our study of all splenic B cells during an immune response linked increased lactate metabolism with acidic intracellular pH and the upregulation of apoptosis. T cell-dependent responses increased LSRs, and added TLR4 agonists affected the magnitude and boosted LSRhigh B cells in vivo, while resulting in only a few immunoglobulin-G secreting cells (IgG-SCs). Therefore, our observations indicated that LSRhigh cells were not differentiating into IgG-SCs, and were rather removed due to apoptosis.