ABSTRACT
Dendritic cell (DC) migration from peripheral tissues via afferent lymphatic vessels to draining lymph nodes (dLNs) is important for the organism's immune regulation and immune protection. Several lymphatic endothelial cell (LEC)-expressed adhesion molecules have thus far been found to support transmigration and movement within the lymphatic vasculature. In this study, we investigated the contribution of CD112, an adhesion molecule that we recently found to be highly expressed in murine LECs, to this process. Performing in vitro assays in the murine system, we found that transmigration of bone marrow-derived dendritic cells (BM-DCs) across or adhesion to murine LEC monolayers was reduced when CD112 was absent on LECs, DCs, or both cell types, suggesting the involvement of homophilic CD112-CD112 interactions. While CD112 was highly expressed in murine dermal LECs, CD112 levels were low in endogenous murine dermal DCs and BM-DCs. This might explain why we observed no defect in the in vivo lymphatic migration of adoptively transferred BM-DCs or endogenous DCs from the skin to dLNs. Compared to murine DCs, human monocyte-derived DCs expressed higher CD112 levels, and their migration across human CD112-expressing LECs was significantly reduced upon CD112 blockade. CD112 expression was also readily detected in endogenous human dermal DCs and LECs by flow cytometry and immunofluorescence. Upon incubating human skin punch biopsies in the presence of CD112-blocking antibodies, DC emigration from the tissue into the culture medium was significantly reduced, indicating impaired lymphatic migration. Overall, our data reveal a contribution of CD112 to human DC migration.
Subject(s)
Langerhans Cells , Lymphatic Vessels , Nectins , Animals , Humans , Mice , Cell Movement/physiology , Endothelium, Lymphatic , Langerhans Cells/physiology , Nectins/metabolismABSTRACT
Anti-PD-1 therapy targets intratumoral CD8+ T cells to promote clinical responses in cancer patients. Recent evidence suggests an additional activity in the periphery, but the underlying mechanism is unclear. Here, we show that anti-PD-1 mAb enhances CD8+ T cell responses in tumor-draining lymph nodes by stimulating cytokine production in follicular helper T cells (Tfh). In two different models, anti-PD-1 mAb increased the activation and proliferation of tumor-specific T cells in lymph nodes. Surprisingly, anti-PD-1 mAb did not primarily target CD8+ T cells but instead stimulated IL-4 production by Tfh cells, the major population bound by anti-PD-1 mAb. Blocking IL-4 or inhibiting the Tfh master transcription factor BCL6 abrogated anti-PD-1 mAb activity in lymph nodes while injection of IL-4 complexes was sufficient to recapitulate anti-PD-1 mAb activity. A similar mechanism was observed in a vaccine model. Finally, nivolumab also boosted human Tfh cells in humanized mice. We propose that Tfh cells and IL-4 play a key role in the peripheral activity of anti-PD-1 mAb.
Subject(s)
Neoplasms , T Follicular Helper Cells , Humans , Mice , Animals , T-Lymphocytes, Helper-Inducer , Interleukin-4/metabolism , Lymph Nodes , Neoplasms/pathology , CD8-Positive T-LymphocytesABSTRACT
T cells become activated following one or multiple contacts with antigen-presenting cells. Calcium influx is a key signaling event elicited during these cellular interactions; however, it is unclear whether T cells recall and integrate calcium signals elicited during temporally separated contacts. To study the integration of calcium signals, we designed a programmable, multiplex illumination strategy for temporally patterned optogenetics (TEMPO). We found that a single round of calcium elevation was insufficient to promote nuclear factor of activated T cells (NFAT) activity and cytokine production in a T cell line. However, robust responses were detected after a second identical stimulation even when signals were separated by several hours. Our results suggest the existence of a biochemical memory of calcium signals in T cells that favors signal integration during temporally separated contacts and promote cytokine production. As illustrated here, TEMPO is a versatile approach for dissecting temporal integration in defined signaling pathways.
ABSTRACT
The regulation of cellular energy metabolism is central to most physiological and pathophysiological processes. However, most current methods have limited ability to functionally probe metabolic pathways in individual cells. Here, we describe SPICE-Met (Single-cell Profiling and Imaging of Cell Energy Metabolism), a method for profiling energy metabolism in single cells using flow cytometry or imaging. We generated a transgenic mouse expressing PercevalHR, a fluorescent reporter for cellular ATP:ADP ratio. Modulation of PercevalHR fluorescence with metabolic inhibitors was used to infer the dependence of energy metabolism on oxidative phosphorylation and glycolysis in defined cell populations identified by flow cytometry. We applied SPICE-Met to analyze T-cell memory development during vaccination. Finally, we used SPICE-Met in combination with real-time imaging to dissect the heterogeneity and plasticity of energy metabolism in single macrophages ex vivo and identify three distinct metabolic patterns. Functional probing of energy metabolism with single-cell resolution should greatly facilitate the study of immunometabolism at a steady state, during disease pathogenesis or in response to therapy.
Subject(s)
Energy Metabolism , Oxidative Phosphorylation , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Animals , Energy Metabolism/physiology , Glycolysis/physiology , Mice , Mice, TransgenicABSTRACT
Junctional adhesion proteins play important roles in controlling angiogenesis, vascular permeability and leukocyte trafficking. CD112 (nectin-2) belongs to the immunoglobulin superfamily and was shown to engage in homophilic and heterophilic interactions with a variety of binding partners expressed on endothelial cells and on leukocytes. Recent in vitro studies suggested that CD112 regulates human endothelial cell migration and proliferation as well as transendothelial migration of leukocytes. However, so far, the role of CD112 in endothelial cell biology and in leukocyte trafficking has not been elucidated in vivo. We found CD112 to be expressed by lymphatic and blood endothelial cells in different murine tissues. In CD112-deficient mice, the blood vessel coverage in the retina and spleen was significantly enhanced. In functional in vitro studies, a blockade of CD112 modulated endothelial cell migration and significantly enhanced endothelial tube formation. An antibody-based blockade of CD112 also significantly reduced T cell transmigration across endothelial monolayers in vitro. Moreover, T cell homing to the spleen was significantly reduced in CD112-deficient mice. Overall, our results identify CD112 as a regulator of angiogenic processes in vivo and demonstrate a novel role for CD112 in T cell entry into the spleen.
Subject(s)
Nectins/metabolism , Neovascularization, Pathologic , Spleen/metabolism , T-Lymphocytes/metabolism , Animals , Blood Vessels/metabolism , Blood Vessels/pathology , Capillary Permeability , Cell Movement , Endothelial Cells/metabolism , Gene Expression Regulation , Leukocytes/cytology , Lymphatic Vessels/cytology , Mice , Mice, Inbred C57BL , Microscopy, Fluorescence , Neutrophils/metabolism , Permeability , Protein Binding , T-Lymphocytes/cytology , Virus InternalizationABSTRACT
Premature infants in the neonatal intensive care unit (NICU) may be subjected to numerous painful procedures without analgesics. One necessary, though acutely painful, procedure is the use of heel lances to monitor blood composition. The current study examined the acute effects of neonatal pain on maternal behavior as well as amygdalar and hypothalamic activation, and the long-term effects of neonatal pain on later-life anxiety-like behavior, using a rodent model. Neonatal manipulations consisted of either painful needle pricks or non-painful tactile stimulation in subjects' left plantar paw surface which occurred four times daily during the first week of life [postnatal day (PND)1-PND7]. Additionally, maternal behaviors in manipulated litters were compared against undisturbed litters via scoring of videotaped interactions to examine the long-term effects of pain on dam-pup interactions. Select subjects underwent neonatal brain collection (PND6) and fluorescent in situ hybridization (FISH) for corticotropin-releasing hormone (CRH) and the immediate early gene c-fos. Other subjects were raised to juvenile age (PND24 and PND25) and underwent innate anxiety testing utilizing an elevated plus maze (EPM) protocol. FISH indicated that neonatal pain influenced amygdalar CRH and c-fos expression, predominately in males. No significant increase in c-fos or CRH expression was observed in the hypothalamus. Additionally, neonatal pain altered anxiety behaviors independent of sex, with neonatal pain subjects showing the highest frequency of exploratory behavior. Neonatal manipulations did not alter maternal behaviors. Overall, neonatal pain drives CRH expression and produces behavioral changes in anxiety that persist until the juvenile stage.
Subject(s)
Acute Pain/metabolism , Amygdala/metabolism , Anxiety/metabolism , Corticotropin-Releasing Hormone/metabolism , Hypothalamus/metabolism , Animals , Animals, Newborn , Behavior, Animal/physiology , Disease Models, Animal , Exploratory Behavior/physiology , Female , Male , Maternal Behavior , Neurons/metabolism , Proto-Oncogene Proteins c-fos/metabolism , Rats , Rats, Sprague-Dawley , Stress, Psychological/metabolismABSTRACT
Afferent lymphatic vessels contribute to immunity by transporting antigen and leukocytes to draining lymph nodes (LNs) and are emerging as new players in the regulation of peripheral tolerance. Performing intravital microscopy in inflamed murine ear skin we found that migrating dendritic cells (DCs) and antigen-experienced effector T cells spend considerable time arresting or clustering within afferent lymphatic capillaries. We also observed that intralymphatic T cells frequently interacted with DCs. When imaging polyclonal T cells during an ongoing contact-hypersensitivity response, most intralymphatic DC-T cell interactions were short-lived. Conversely, during a delayed-type-hypersensitivity response, cognate antigen-bearing DCs engaged in long-lived MHCII-(I-A/I-E)-dependent interactions with antigen-specific T cells. Long-lived intralymphatic DC-T cell interactions reduced the speed of DC crawling but did not delay overall DC migration to draining LNs. While further consequences of these intralymphatic interactions still need to be explored, our findings suggest that lymphatic capillaries represent a unique compartment in which adaptive immune interaction and modulation occur.
Subject(s)
Cell Communication/immunology , Cell Movement/immunology , Dendritic Cells/immunology , Lymphatic Vessels/immunology , T-Lymphocytes/immunology , Animals , Cell Communication/genetics , Cell Movement/genetics , Dendritic Cells/cytology , Lymphatic Vessels/cytology , Mice , Mice, Knockout , T-Lymphocytes/cytologyABSTRACT
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.
Subject(s)
Endothelial Cells/physiology , Image Processing, Computer-Assisted/methods , Lymphangiogenesis/physiology , Lymphatic Vessels/cytology , Neovascularization, Physiologic/physiology , Software , Animals , Cell Communication/physiology , Cell Count/methods , Cell Size , Cells, Cultured , Endothelial Cells/cytology , Humans , Lymphatic Vessels/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Microvessels/cytologyABSTRACT
Enlargement of the lymphatic vascular network in tumor-draining lymph nodes (LNs) often precedes LN metastasis, likely providing a lymphovascular niche for tumor cells. We investigated morphological and molecular changes associated with the lymphatic remodeling process, using the 4T1 breast cancer and B16F10 melanoma models. Lymphatic expansion in tumor-draining LNs is mediated by sprouting and proliferation of lymphatic endothelial cells (LECs) as early as 4 days after tumor implantation. RNA sequencing revealed an altered transcriptional profile of LECs from tumor-draining compared to naive LNs with similar changes in both tumor models. Integrin αIIb is upregulated in LECs of tumor-draining LNs and mediates LEC adhesion to fibrinogen in vitro. LEC-associated fibrinogen was also detected in LNs in vivo, suggesting a role of integrin αIIb in lymphatic remodeling. Together, our results identify specific responses of LN LECs to tumor stimuli and provide insights into the mechanisms of lymphovascular niche formation in tumor-draining LNs.
Subject(s)
Lymph Nodes/pathology , Lymphatic Vessels/pathology , Neoplasms/pathology , Animals , Cell Adhesion , Cell Adhesion Molecules/metabolism , Cell Line, Tumor , Cell Proliferation , Endothelial Cells/metabolism , Female , Fibrinogen/metabolism , Gene Expression Regulation, Neoplastic , Lymphatic Metastasis , Mice, Inbred BALB C , Mice, Inbred C57BL , Neoplasms/genetics , Neovascularization, Physiologic , Platelet Membrane Glycoprotein IIb/metabolism , Up-RegulationABSTRACT
Microinjection is extensively used across fields to deliver material intracellularly. Here we address the fundamental aspects of introducing exogenous organelles into cells to endow them with artificial functions. Nanocarriers encapsulating biologically active cargo or extreme intraluminal pH were injected directly into the cytosol of cells, where they bypassed subcellular processing pathways and remained intact for several days. Nanocarriers' size was found to dictate their intracellular distribution pattern upon injection, with larger vesicles adopting polarized agglomerated distributions and smaller colloids spreading evenly in the cytosol. This in turn determined the symmetry or asymmetry of their dilution following cell division, ultimately affecting the intracellular dose at a cell population level. As an example of microinjection's applicability, a cell type relevant for cell-based therapies (dendritic cells) was injected with vesicles, and its migratory properties were studied in a co-culture system mimicking lymphatic capillaries.
Subject(s)
Bioreactors , Liposomes/chemistry , Microinjections/methods , Animals , Cell Division/physiology , Cell Movement/physiology , Cell- and Tissue-Based Therapy , Drug Carriers/chemistry , Humans , Nanoparticles/chemistryABSTRACT
T cells are the most abundant cell type found in afferent lymph, but their migration through lymphatic vessels (LVs) remains poorly understood. Performing intravital microscopy in the murine skin, we imaged T cell migration through afferent LVs in vivo. T cells entered into and actively migrated within lymphatic capillaries but were passively transported in contractile collecting vessels. Intralymphatic T cell number and motility were increased during contact-hypersensitivity-induced inflammation and dependent on ICAM-1/LFA-1 interactions. In vitro, blockade of endothelial cell-expressed ICAM-1 reduced T cell adhesion, crawling, and transmigration across lymphatic endothelium and decreased T cell advancement from capillaries into lymphatic collectors in skin explants. In vivo, T cell migration to draining lymph nodes was significantly reduced upon ICAM-1 or LFA-1 blockade. Our findings indicate that T cell migration through LVs occurs in distinct steps and reveal a key role for ICAM-1/LFA-1 interactions in this process.
Subject(s)
Inflammation/immunology , Intercellular Adhesion Molecule-1/metabolism , Lymph Nodes/metabolism , Lymphocyte Function-Associated Antigen-1/metabolism , Skin/metabolism , T-Lymphocytes/physiology , Animals , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/physiology , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/physiology , Cell Adhesion/drug effects , Cell Movement/drug effects , Flow Cytometry , Inflammation/chemically induced , Inflammation/pathology , Intercellular Adhesion Molecule-1/chemistry , Interferon-gamma/pharmacology , Killer Cells, Natural/cytology , Killer Cells, Natural/immunology , Killer Cells, Natural/physiology , Lymphatic Vessels/metabolism , Lymphocyte Function-Associated Antigen-1/chemistry , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Confocal , Oxazolone/toxicity , Skin/pathology , T-Lymphocytes/cytology , T-Lymphocytes/immunology , Time-Lapse Imaging , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
PURPOSE: A shear stress-induced atherosclerosis mouse model was characterized for its expression of inflammation markers with focus on CD80. With this model, we evaluated two positron emission tomography (PET) radiotracers targeting CD80 as well as 2-deoxy-2-[18F]fluoro-D-mannose ([18F]FDM) in comparison with 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG). PROCEDURE: A flow constrictive cuff implanted around the common carotid artery in apolipoprotein E knockout mice resulted in plaque formation. CD80 expression levels and plaque histopathology were evaluated. Serial PET/X-ray computed tomography scans were performed to follow inflammation. RESULTS: Plaque formation with increased levels of CD80 was observed. Histologically, plaques presented macrophage-rich and large necrotic areas covered by a thin fibrous cap. Of the CD80-specific tracers, one displayed an increased uptake in plaques by PET. Both [18F]FDG and [18F]FDM accumulated in atherosclerotic plaques. CONCLUSION: This mouse model presented, similar to humans, an increased expression of CD80 which renders it suitable for non-invasively targeting CD80-positive immune cells and evaluating CD80-specific radiotracers.
Subject(s)
Atherosclerosis/diagnostic imaging , Atherosclerosis/metabolism , B7-1 Antigen/metabolism , Positron-Emission Tomography , Radiopharmaceuticals/chemistry , Stress, Mechanical , Up-Regulation , Animals , Antigens, CD/genetics , Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/genetics , Antigens, Differentiation, Myelomonocytic/metabolism , Apolipoproteins E/deficiency , Apolipoproteins E/metabolism , Arteries/metabolism , Arteries/pathology , Atherosclerosis/blood , Atherosclerosis/pathology , B7-1 Antigen/genetics , Body Weight , Disease Models, Animal , Gene Expression Regulation , Immunohistochemistry , Lipids/blood , Mice, Inbred C57BL , Mice, Knockout , Tomography, X-Ray ComputedABSTRACT
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.
Subject(s)
Blood Circulation , Cell Movement , Dendritic Cells/metabolism , Endothelial Cells/metabolism , Endothelium, Lymphatic/metabolism , Lymph Nodes/metabolism , Membrane Glycoproteins/deficiency , Thoracic Duct/metabolism , Animals , Body Patterning , Dendritic Cells/pathology , Endothelial Cells/pathology , Endothelium, Lymphatic/pathology , Erythrocytes/metabolism , Gene Expression Regulation, Developmental , Genotype , Lymph Nodes/pathology , Lymphangiogenesis , Membrane Glycoproteins/genetics , Mice, Inbred C57BL , Mice, Knockout , Phenotype , Signal Transduction , Thoracic Duct/pathologyABSTRACT
To induce adaptive immunity, dendritic cells (DCs) migrate through afferent lymphatic vessels (LVs) to draining lymph nodes (dLNs). This process occurs in several consecutive steps. Upon entry into lymphatic capillaries, DCs first actively crawl into downstream collecting vessels. From there, they are next passively and rapidly transported to the dLN by lymph flow. Here, we describe a role for the chemokine CCL21 in intralymphatic DC crawling. Performing time-lapse imaging in murine skin, we found that blockade of CCL21-but not the absence of lymph flow-completely abolished DC migration from capillaries toward collecting vessels and reduced the ability of intralymphatic DCs to emigrate from skin. Moreover, we found that in vitro low laminar flow established a CCL21 gradient along lymphatic endothelial monolayers, thereby inducing downstream-directed DC migration. These findings reveal a role for intralymphatic CCL21 in promoting DC trafficking to dLNs, through the formation of a flow-induced gradient.
Subject(s)
Bone Marrow Cells/cytology , Chemokine CCL21/immunology , Dendritic Cells/cytology , Endothelium, Lymphatic/immunology , Lymph Nodes/immunology , Lymphatic Vessels/immunology , Animals , Bone Marrow Cells/immunology , Cell Movement , Chemokine CCL21/genetics , Dendritic Cells/immunology , Ear , Endothelium, Lymphatic/ultrastructure , Gene Expression , Lymph Nodes/ultrastructure , Lymphatic Vessels/ultrastructure , Mice , Mice, Inbred C57BL , Mice, Transgenic , Rheology , Skin/cytology , Skin/immunology , Time-Lapse ImagingABSTRACT
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 inâ vitro. This study demonstrates the potential of multidimensional machine-learning methods for rapidly obtaining peptides with the desired cellular activities.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Cell-Penetrating Peptides/pharmacology , Computer-Aided Design , Dermis/drug effects , Apoptosis/drug effects , Breast Neoplasms/pathology , Cell Survival/drug effects , Cells, Cultured , Dermis/cytology , Drug Therapy, Combination , Female , HumansABSTRACT
In contrast to leukocyte migration through blood vessels, trafficking via lymphatic vessels (LVs) is much less well characterized. An important cell type migrating via this route is antigen-presenting dendritic cells (DCs), which are key for the induction of protective immunity as well as for the maintenance of immunological tolerance. In this review, we will summarize and discuss current knowledge of the cellular and molecular events that control DC migration from the skin towards, into, and within LVs, followed by DC arrival and migration in draining lymph nodes. Finally, we will discuss potential strategies to therapeutically target this migratory step to modulate immune responses.
Subject(s)
Cell Movement , Dendritic Cells/physiology , Lymph Nodes/physiology , Lymphatic Vessels/physiology , Animals , Endothelium, Lymphatic/metabolism , Humans , Inflammation/genetics , Inflammation/immunology , Inflammation/metabolism , Inflammation/therapy , Transendothelial and Transepithelial MigrationABSTRACT
Afferent lymphatic vessels fulfill essential immune functions by transporting leukocytes and lymph-borne antigen to draining lymph nodes (dLNs). An important cell type migrating through lymphatic vessels are dendritic cells (DCs). DCs reside in peripheral tissues like the skin, where they take up antigen and transport it via the lymphatic vascular network to dLNs for subsequent presentation to T cells. As such, DCs play a key role in the induction of adaptive immune responses during infection and vaccination, but also for the maintenance of tolerance. Although the migratory pattern of DCs has been known for long time, interactions between DCs and lymphatic vessels are only now starting to be unraveled at the cellular level. In particular, new tools for visualizing lymphatic vessels in combination with time-lapse microscopy have recently generated valuable insights into the process of DC migration to dLNs. In this review we summarize and discuss current approaches for visualizing DCs and lymphatic vessels in tissues for imaging applications. Furthermore, we review the current state of knowledge about DC migration towards, into and within lymphatic vessels, particularly focusing on the cellular interactions that take place between DCs and the lymphatic endothelium.
Subject(s)
Dendritic Cells/metabolism , Endothelium, Lymphatic/metabolism , Lymph Nodes/metabolism , Lymphatic Vessels/metabolism , Animals , Cell Communication , Cell Movement , Dendritic Cells/cytology , Endothelium, Lymphatic/cytology , Humans , Microscopy, Confocal , Time-Lapse ImagingABSTRACT
We show for the first time that the ventral diverticulum of the mosquito gut (impermeable sugar storage organ) harbors microorganisms. The gut diverticulum from newly emerged and non-fed Aedes aegypti was dissected under aseptic conditions, homogenized and plated on BHI medium. Microbial isolates were identified by sequencing of 16S rDNA for bacteria and 28S rDNA for yeast. A direct DNA extraction from Ae. aegypti gut diverticulum was also performed. The bacterial isolates were: Bacillus sp., Bacillus subtilis and Serratia sp. The latter was the predominant bacteria found in our isolations. The yeast species identified was Pichia caribbica.
Subject(s)
Animals , Aedes/microbiology , Bacillus/isolation & purification , Gastrointestinal Tract/microbiology , Pichia/isolation & purification , Serratia/isolation & purification , Bacillus/genetics , DNA, Bacterial/genetics , DNA, Ribosomal/genetics , Pichia/genetics , /genetics , /genetics , Serratia/geneticsABSTRACT
We show for the first time that the ventral diverticulum of the mosquito gut (impermeable sugar storage organ) harbors microorganisms. The gut diverticulum from newly emerged and non-fed Aedes aegypti was dissected under aseptic conditions, homogenized and plated on BHI medium. Microbial isolates were identified by sequencing of 16S rDNA for bacteria and 28S rDNA for yeast. A direct DNA extraction from Ae. aegypti gut diverticulum was also performed. The bacterial isolates were: Bacillus sp., Bacillus subtilis and Serratia sp. The latter was the predominant bacteria found in our isolations. The yeast species identified was Pichia caribbica.