Calcium is a universal second messenger present in all eukaryotic cells. The mobilization and storage of Ca2+ ions drives a number of signaling-related processes, stress-responses or metabolic changes, all of which are relevant for the development of immune cells and their adaption to pathogens. Here, we introduce the FRET-reporter mouse YellowCaB expressing the genetically encoded calcium indicator TN-XXL in B lymphocytes. Calcium-induced conformation change of TN-XXL results in FRET-donor quenching measurable by two-photon fluorescence lifetime imaging. For the first time, using our novel numerical analysis, we extract absolute cytoplasmic calcium concentrations in activated B cells during affinity maturation in vivo. We show that calcium in activated B cells is highly dynamic and that activation introduces a persistent calcium heterogeneity to the lineage. A characterization of absolute calcium concentrations present at any time within the cytosol is therefore of great value for the understanding of long-lived beneficial and detrimental (auto)immunity.
Source data for flow cytometric Analysis, in vitro confocal imaging, ratiometric in vivo Imaging and fluorescence lifetime in vivo Imaging are deposited at Dryad Digital Repository 10.5061/dryad.cc2fqz63d. Analyzed absolute calcium concentration for all cells measured out of 5 experiments have also been deposited there. Source code for phasor based analysis of fluorescence lifetime data has been provided with full submission upload and will be made available to the public via github after publication.
Intravital quantification of cytoplasmic B cell calcium reveals dynamic signaling across B cell differentiation stagesDryad Digital Repository, doi:10.5061/dryad.cc2fqz63d.
- Helena Radbruch
- Anja E Hauser
- Raluca A Niesner
- Anja E Hauser
- Lars Nitschke
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: The study was approved by the Berlin Landesamt für Gesundheit und Soziales under the registration # G00158/16. All surgeries and experimental procedures were conducted following the principle of minimization of suffering and 3R means were used where possible.
- Michael L Dustin, University of Oxford, United Kingdom
© 2021, Ulbricht et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Septic shock is characterized by an excessive inflammatory response depicted in a cytokine storm that results from invasive bacterial, fungi, protozoa, and viral infections. Non-canonical inflammasome activation is crucial in the development of septic shock promoting pyroptosis and proinflammatory cytokine production via caspase-11 and gasdermin D (GSDMD). Here, we show that NAD+ treatment protected mice toward bacterial and lipopolysaccharide (LPS)-induced endotoxic shock by blocking the non-canonical inflammasome specifically. NAD+ administration impeded systemic IL-1β and IL-18 production and GSDMD-mediated pyroptosis of macrophages via the IFN-β/STAT-1 signaling machinery. More importantly, NAD+ administration not only improved casp-11 KO (knockout) survival but rendered wild type (WT) mice completely resistant to septic shock via the IL-10 signaling pathway that was independent from the non-canonical inflammasome. Here, we delineated a two-sided effect of NAD+ blocking septic shock through a specific inhibition of the non-canonical inflammasome and promoting immune homeostasis via IL-10, underscoring its unique therapeutic potential.
Mechanical force is critical for the interaction between an αβ T cell receptor (TCR) and a peptide-bound major histocompatibility complex (pMHC) molecule to initiate productive T-cell activation. However, the underlying mechanism remains unclear. We use all-atom molecular dynamics simulations to examine the A6 TCR bound to HLA-A*02:01 presenting agonist or antagonist peptides under different extensions to simulate the effects of applied load on the complex, elucidating their divergent biological responses. We found that TCR α and β chains move asymmetrically, which impacts the interface with pMHC, in particular the peptide-sensing CDR3 loops. For the wild-type agonist, the complex stabilizes in a load-dependent manner while antagonists destabilize it. Simulations of the Cβ FG-loop deletion, which reduces the catch bond response, and simulations with in silico mutant peptides further support the observed behaviors. The present results highlight the combined role of interdomain motion, fluctuating forces, and interfacial contacts in determining the mechanical response and fine peptide discrimination by a TCR, thereby resolving the conundrum of nearly identical crystal structures of TCRαβ-pMHC agonist and antagonist complexes.