Conjugated bile acids attenuate allergen-induced airway inflammation and hyperresposiveness by inhibiting UPR transducers

© 2019 American Society for Clinical Investigation. Conjugated bile acids (CBAs), such as tauroursodeoxycholic acid (TUDCA), are known to resolve the inflammatory and unfolded protein response (UPR) in inflammatory diseases, such as asthma. Whether CBAs exert their beneficial effects on allergic airway responses via 1 arm or several arms of the UPR, or alternatively through the signaling pathways for conserved bile acid receptor, remains largely unknown. We used a house dust mite-induced (HDM-induced) murine model of asthma to evaluate and compare the effects of 5 CBAs and 1 unconjugated bile acid in attenuating allergen-induced UPR and airway responses. Expression of UPRassociated transcripts was assessed in airway brushings from human patients with asthma and healthy subjects. Here we show that CBAs, such as alanyl β-muricholic acid (AβM) and TUDCA, significantly decreased inflammatory, immune, and cytokine responses; mucus metaplasia; and airway hyperresponsiveness, as compared with other CBAs in a model of allergic airway disease. CBAs predominantly bind to activating transcription factor 6α (ATF6α) compared with the other canonical transducers of the UPR, subsequently decreasing allergen-induced UPR activation and resolving allergic airway disease, without significant activation of the bile acid receptors. TUDCA and AβM also attenuated other HDM-induced ER stress markers in the lungs of allergic mice. Quantitative mRNA analysis of airway epithelial brushings from human subjects demonstrated that several ATF6α-related transcripts were significantly upregulated in patients with asthma compared with healthy subjects. Collectively, these results demonstrate that CBA-based therapy potently inhibits the allergen-induced UPR and allergic airway disease in mice via preferential binding of the canonical transducer of the UPR, ATF6α. These results potentially suggest a novel avenue to treat allergic asthma using select CBAs

A Near-Infrared Cell Tracker Reagent for Multiscopic In Vivo Imaging and Quantification of Leukocyte Immune Responses

The complexity of the tumor microenvironment necessitates that cell behavior is studied in a broad, multi-scale context. Although tomographic and microscopy-based far and near infrared fluorescence (NIRF, >650 nm) imaging methods offer high resolution, sensitivity, and depth penetration, there has been a lack of optimized NIRF agents to label and track cells in their native environments at different scales. In this study we labeled mammalian leukocytes with VivoTag 680 (VT680), an amine reactive N-hydroxysuccinimide (NHS) ester of a (benz) indolium-derived far red fluorescent probe. We show that VT680 diffuses into leukocytes within minutes, covalently binds to cellular components, remains internalized for days in vitro and in vivo, and does not transfer fluorescence to adjacent cells. It is biocompatible, keeps cells fully functional, and fluoresces at high intensities. In a tumor model of cytotoxic T lymphocyte (CTL) immunotherapy, we track and quantify VT680-labeled cells longitudinally at the whole-body level with fluorescence-mediated molecular tomography (FMT), within tissues at single cell resolutions by multiphoton and confocal intravital microscopy, and ex vivo by flow cytometry. Thus, this approach is suitable to monitor cells at multiple resolutions in real time in their native environments by NIR-based fluorescence imaging

The impact of negative selection on thymocyte migration in the medulla

Developing thymocytes are screened for self-reactivity before they exit the thymus, but how thymocytes scan the medulla for self antigens is unclear. Using two-photon microscopy, we observed that medullary thymocytes migrated rapidly and made frequent, transient contacts with dendritic cells. In the presence of a negative selecting ligand, thymocytes slowed, became confined to areas of approximately 30 mum in diameter and had increased contact with dendritic cells surrounding confinement zones. One third of polyclonal medullary thymocytes also showed confined, slower migration and may correspond to autoreactive thymocytes. Our data suggest that many autoreactive thymocytes do not undergo immediate arrest and death after encountering a negative selecting ligand but instead adopt an altered migration program while remaining in the medullary microenvironment

Barcoding T Cell Calcium Response Diversity with Methods for Automated and Accurate Analysis of Cell Signals (MAAACS)

International audienceWe introduce a series of experimental procedures enabling sensitive calcium monitoring in T cell populations by confocal video-microscopy. Tracking and post-acquisition analysis was performed using Methods for Automated and Accurate Analysis of Cell Signals (MAAACS), a fully customized program that associates a high throughput tracking algorithm, an intuitive reconnection routine and a statistical platform to provide, at a glance, the calcium barcode of a population of individual T-cells. Combined with a sensitive calcium probe, this method allowed us to unravel the heterogeneity in shape and intensity of the calcium response in T cell populations and especially in naive T cells, which display intracellular calcium oscillations upon stimulation by antigen presenting cells

Regulation of thymocyte positive selection and motility by GIT2

Thymocytes are highly motile cells that migrate under the influence of chemokines in distinct thymic compartments as they mature. The motility of thymocytes is tightly regulated; however, the molecular mechanisms that control thymocyte motility are not well understood. Here we report that G protein–coupled receptor kinase-interactor 2 (GIT2) was required for efficient positive selection. Notably, Git2−/− double-positive thymocytes showed greater activation of the small GTPase Rac, actin polymerization and migration toward the chemokines CXCL12 (SDF-1) and CCL25 in vitro. By two-photon laser-scanning microscopy, we found that the scanning activity of Git2−/− thymocytes was compromised in the thymic cortex, which suggests GIT2 has a key role in regulating the chemokine-mediated motility of double-positive thymocytes.National Institutes of Health (U.S.) (R01AI064227)Leukemia & Lymphoma Society of Americ

Identification of genetic alterations in pancreatic cancer by the combined use of tissue microdissection and array-based comparative genomic hybridisation

Pancreatic ductal adenocarcinoma (PDAC) is characterised pathologically by a marked desmoplastic stromal reaction that significantly reduces the sensitivity and specificity of cytogenetic analysis. To identify genetic alterations that reflect the characteristics of the tumour in vivo, we screened a total of 23 microdissected PDAC tissue samples using array-based comparative genomic hybridisation (array CGH) with 1 Mb resolution. Highly stringent statistical analysis enabled us to define the regions of nonrandom genomic changes. We detected a total of 41 contiguous regions (>3.0 Mb) of copy number changes, such as a genetic gain at 7p22.2–p15.1 (26.0 Mb) and losses at 17p13.3–p11.2 (13.6 Mb), 18q21.2–q22.1 (12.0 Mb), 18q22.3–q23 (7.1 Mb) and 18q12.3–q21.2 (6.9 Mb). To validate our array CGH results, fluorescence in situ hybridisation was performed using four probes from those regions, showing that these genetic alterations were observed in 37–68% of a separate sample set of 19 PDAC cases. In particular, deletion of the SEC11L3 gene (18q21.32) was detected at a very high frequency (13 out of 19 cases; 68%) and in situ RNA hybridisation for this gene demonstrated a significant correlation between deletion and expression levels. It was further confirmed by reverse transcription–PCR that SEC11L3 mRNA was downregulated in 16 out of 16 PDAC tissues (100%). In conclusion, the combination of tissue microdissection and array CGH provided a valid data set that represents in vivo genetic changes in PDAC. Our results raise the possibility that the SEC11L3 gene may play a role as a tumour suppressor in this disease

Interaction and Modulation of Two Antagonistic Cell Wall Enzymes of Mycobacteria

Bacterial cell growth and division require coordinated cell wall hydrolysis and synthesis, allowing for the removal and expansion of cell wall material. Without proper coordination, unchecked hydrolysis can result in cell lysis. How these opposing activities are simultaneously regulated is poorly understood. In Mycobacterium tuberculosis, the resuscitation-promoting factor B (RpfB), a lytic transglycosylase, interacts and synergizes with Rpf-interacting protein A (RipA), an endopeptidase, to hydrolyze peptidoglycan. However, it remains unclear what governs this synergy and how it is coordinated with cell wall synthesis. Here we identify the bifunctional peptidoglycan-synthesizing enzyme, penicillin binding protein 1 (PBP1), as a RipA-interacting protein. PBP1, like RipA, localizes both at the poles and septa of dividing cells. Depletion of the ponA1 gene, encoding PBP1 in M. smegmatis, results in a severe growth defect and abnormally shaped cells, indicating that PBP1 is necessary for viability and cell wall stability. Finally, PBP1 inhibits the synergistic hydrolysis of peptidoglycan by the RipA-RpfB complex in vitro. These data reveal a post-translational mechanism for regulating cell wall hydrolysis and synthesis through protein–protein interactions between enzymes with antagonistic functions

The Immunological Synapse: a Dynamic Platform for Local Signaling

The immunological synapse (IS) as a concept has evolved from a static view of the junction between T cells and their antigen-presenting cell partners. The entire process of IS formation and extinction is now known to entail a dynamic reorganization of membrane domains and proteins within and adjacent to those domains. Discussion The entire process is also intricately tied to the motility machinery—both as that machinery directs “scanning” prior to T-cell receptor engagement and as it is appropriated during the ongoing developments at the IS. While the synapse often remains dynamic in order to encourage surveillance of new antigen-presenting surfaces, cytoskeletal forces also regulate the development of signals, likely including the assembly of ion channels. In both neuronal and immunological synapses, localized Ca 2+ signals and accumulation or depletion of ions in microdomains accompany the concentration of signaling molecules in the synapse. Such spatiotemporal signaling in the synapse greatly accelerates kinetics and provides essential checkpoints to validate effective cell–cell communication