Dataset Information

Spatiotemporal and functional behavior of airway dendritic cells visualized by two-photon microscopy.

ABSTRACT: Airway mucosal dendritic cells (DCs), located beneath the epithelium of the conducting airways, are believed to be specialized for immunosurveillance via sampling of antigens from the airway luminal surface. However, the dynamics of airway DC activity has not yet been visualized. We used two-photon microscopy to illuminate the endogenous mucosal DC network in the airways of mice. To characterize DC behavior, we used lung section preparations and an intravital microscopic approach. DCs displayed a heterogeneous movement pattern according to their localization within the airway mucosa: sessile intraepithelial DCs with a dendritiform shape exhibited active probing movements and occasionally formed transepithelial extensions into the airway lumen. In contrast, DCs within the deeper layers of the mucosal tissue migrated fast in an amoeboid manner, without probing movements, and slowed down after aeroallergen challenge. Strikingly, neither of these two mucosal DC populations ingested fluorescently labeled antigens after antigen administration to the airways in the steady state, in contrast to alveolar macrophage/DC populations in the lung periphery. Our results provide a first description of the dynamic behavior of airway mucosal DCs, with their exact role in antigen sampling remaining unclear.

SUBMITTER: Veres TZ

PROVIDER: S-EPMC3157232 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Spatiotemporal and functional behavior of airway dendritic cells visualized by two-photon microscopy.

Veres Tibor Z TZ Voedisch Sabrina S Spies Emma E Tschernig Thomas T Braun Armin A

The American journal of pathology 20110625 2

Airway mucosal dendritic cells (DCs), located beneath the epithelium of the conducting airways, are believed to be specialized for immunosurveillance via sampling of antigens from the airway luminal surface. However, the dynamics of airway DC activity has not yet been visualized. We used two-photon microscopy to illuminate the endogenous mucosal DC network in the airways of mice. To characterize DC behavior, we used lung section preparations and an intravital microscopic approach. DCs displayed ...[more]

PMID: 21708113

Similar Datasets

Project description:The lacrimal gland (LG) is the main source of the tear film aqueous layer and its dysfunction results in dry eye disease (DED), a chronic immune-mediated disorder of the ocular surface. The desiccating stress (DS) murine model that mimics human DED, results in LG dysfunction, immune cell infiltration, and consequently insufficient tear production. To date, the immune cell kinetics in DED are poorly understood. The purpose of this study was to develop a murine model of intravital multi-photon microscopy (IV-MPM) for the LG, and to investigate the migratory kinetics and 3D morphological properties of conventional dendritic cells (cDCs), the professional antigen presenting cells of the ocular surface, in DED. Mice were placed in a controlled environmental chamber with low humidity and increased airflow rate for 2 and 4 weeks to induce DED, while control naïve transgenic mice were housed under standard conditions. DED mice had significantly decreased tear secretion and increased fluorescein staining (p < 0.01) compared to naïve controls. Histological analysis of the LG exhibited infiltrating mononuclear and polymorphonuclear cells (p < 0.05), as well as increased LG swelling (p < 0.001) in DED mice compared to controls. Immunofluorescence staining revealed increased density of cDCs in DED mice (p < 0.001). IV-MPM of the LG demonstrated increased density of cDCs in the LGs of DED mice, compared with controls (p < 0.001). cDCs were more spherical in DED at both time points compared to controls (p < 0.001); however, differences in surface area were found at 2 weeks in DED compared with naïve controls (p < 0.001). Similarly, 3D cell volume was significantly lower at 2 weeks in DED vs. the naïve controls (p < 0.001). 3D instantaneous velocity and mean track speed were significantly higher in DED compared to naïve mice (p < 0.001). Finally, the meandering index, an index for directionality, was significant increased at 4 weeks after DED compared with controls and 2 weeks of DED (p < 0.001). Our IV-MPM study sheds light into the 3D morphological alterations and cDC kinetics in the LG during DED. While in naïve LGs, cDCs exhibit a more dendritic morphology and are less motile, they became more spherical with enhanced motility during DED. This study shows that IV-MPM represents a robust tool to study immune cell trafficking and kinetics in the LG, which might elucidate cellular alterations in immunological diseases, such as DED.

Project description:The future of nanomedicines in oncology requires leveraging more than just the passive drug accumulation in tumors through the enhanced permeability and retention effect. Promising results combining mild hyperthermia (HT) with lyso-thermosensitive liposomal doxorubicin (LTSL-DOX) has led to improved drug delivery and potent antitumor effects in pre-clinical studies. The ultimate patient benefit from these treatments can only be realized when robust methods of HT can be achieved clinically. One of the most promising methods of non-invasive HT is the use of focused ultrasound (FUS) with MRI thermometry for anatomical targeting and feedback. MRI-guided focused ultrasound (MRgFUS) is limited by respiratory motion and large blood vessel cooling. In order to translate exciting pre-clinical results to the clinic, novel heating approaches capable of overcoming the limitations on clinical MRgFUS+HT must be tested and evaluated on their ability to locally release drug from LTSL-DOX. Methods: In this work, a new system is described to integrate focused ultrasound (FUS) into a two-photon microscopy (2PM) setting to image the release of drug from LTSL-DOX in real-time during FUS+HT in vivo. A candidate scheme for overcoming the limitations of respiratory motion and large blood vessel cooling during MRgFUS+HT involves applying FUS+HT to 42°C in short ~30s bursts. The spatiotemporal drug release pattern from LTSL-DOX as a result is quantified using 2PM and compared against continuous (3.5min and 20min at 42°C) FUS+HT schemes and unheated controls. Results: It was observed for the first time in vivo that these short duration temperature elevations could produce substantial drug release from LTSL-DOX. Ten 30s bursts of FUS+HT was able to achieve almost half of the interstitial drug concentration as 20min of continuous FUS+HT. There was no significant difference between the intravascular area under the concentration-time curve for ten 30s bursts of FUS+HT and 3.5min of continuous FUS+HT. Conclusion: We have successfully combined 2PM with FUS+HT for imaging the release of DOX from LTSL-DOX in vivo in real-time, which will permit the investigation of FUS+HT heating schemes to improve drug delivery from LTSL-DOX. We have evaluated the ability to release DOX in short 30s FUS+HT bursts to 42°C as a method to overcome limitations on clinical MRgFUS+HT and have found that such exposures are capable of releasing measurable amounts of drug. Such an exposure has the potential to overcome limitations that hamper conventional MRgFUS+HT treatments in targets that are associated with substantial tissue motion.

Project description:BackgroundT helper (Th) 17 cells are a highly plastic subset of T cells, which in the context of neuroinflammation, are able to acquire pathogenic features originally attributed to Th1 cells (resulting in so called ex-Th17 cells). Thus, a strict separation between the two T cell subsets in the context of experimental autoimmune encephalomyelitis (EAE) is difficult. High variability in culture and EAE induction protocols contributed to previous conflicting results concerning the differential contribution of Th1 and Th17 cells in EAE. Here, we systematically evaluate the role of different T cell differentiation and transfer protocols for EAE disease development and investigate the functional dynamics of encephalitogenic T cells directly within the inflamed central nervous system (CNS) tissue.MethodsWe compiled the currently used EAE induction protocols reported in literature and investigated the influence of the different Th1 and Th17 differentiation protocols as well as EAE induction protocols on the EAE disease course. Moreover, we assessed the cytokine profile and functional dynamics of both encephalitogenic Th1 and Th17 cells in the inflamed CNS using flow cytometry and intravital two-photon laser scanning microscopy. Lastly, we used astrocyte culture and adoptive transfer EAE to evaluate the impact of Th1 and Th17 cells on astrocyte adhesion molecule expression in vitro and in vivo.ResultsWe show that EAE courses are highly dependent on in vitro differentiation and transfer protocols. Moreover, using genetically encoded reporter mice (B6.IL17A-EGFP.acRFP x 2d2/2d2.RFP), we show that the motility of interferon (IFN)γ-producing ex-Th17 cells more closely resembles Th1 cells than Th17 cells in transfer EAE. Mechanistically, IFNγ-producing Th1 cells selectively induce the expression of cellular adhesion molecules I-CAM1 while Th1 as well as ex-Th17 induce V-CAM1 on astrocytes.ConclusionsThe behavior of ex-Th17 cells in EAE lesions in vivo resembles Th1 rather than Th17 cells, underlining that their change in cytokine production is associated with functional phenotype alterations of these cells.

Project description:Pluripotent stem cell-derived organoid technologies have opened avenues to preclinical basic science research, drug discovery, and transplantation therapy in organ systems. Stem cell-derived organoids follow a time course similar to species-specific organ gestation in vivo. However, heterogeneous tissue yields, and subjective tissue selection reduce the repeatability of organoid-based scientific experiments and clinical studies. To improve the quality control of organoids, we introduced a live imaging technique based on two-photon microscopy to non-invasively monitor and characterize retinal organoids' (RtOgs') long-term development. Fluorescence lifetime imaging microscopy (FLIM) was used to monitor the metabolic trajectory, and hyperspectral imaging was applied to characterize structural and molecular changes. We further validated the live imaging experimental results with endpoint biological tests, including quantitative polymerase chain reaction (qPCR), single-cell RNA sequencing, and immunohistochemistry. With FLIM results, we analyzed the free/bound nicotinamide adenine dinucleotide (f/b NADH) ratio of the imaged regions and found that there was a metabolic shift from glycolysis to oxidative phosphorylation. This shift occurred between the second and third months of differentiation. The total metabolic activity shifted slightly back toward glycolysis between the third and fourth months and stayed relatively stable between the fourth and sixth months. Consistency in organoid development among cell lines and production lots was examined. Molecular analysis showed that retinal progenitor genes were expressed in all groups between days 51 and 159. Photoreceptor gene expression emerged around the second month of differentiation, which corresponded to the shift in the f/b NADH ratio. RtOgs between 3 and 6 months of differentiation exhibited photoreceptor gene expression levels that were between the native human fetal and adult retina gene expression levels. The occurrence of cone opsin expression (OPN1 SW and OPN1 LW) indicated the maturation of photoreceptors in the fourth month of differentiation, which was consistent with the stabilized level of f/b NADH ratio starting from 4 months. Endpoint single-cell RNA and immunohistology data showed that the cellular compositions and lamination of RtOgs at different developmental stages followed those in vivo.

Dataset Information

Spatiotemporal and functional behavior of airway dendritic cells visualized by two-photon microscopy.

Publications

Spatiotemporal and functional behavior of airway dendritic cells visualized by two-photon microscopy.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets