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Endo-lysosomes transport along microtubules and clustering in the perinuclear area are two necessary steps for microbes to activate specialized phagocyte functions. We report that RUN and FYVE domain-containing protein 3 exists as two alternative isoforms distinguishable by the presence of a C-terminal FYVE domain and by their affinity for PtdIns(3)P on endosomal membranes. The FYVE domain-bearing isoform (iRUFY3) is preferentially expressed in immune cells and up-regulated upon activation by microbes and cytokines. iRUFY3 is necessary for ARL8b+/LAMP1+ endo-lysosomes positioning in the pericentriolar organelles cloud of LPS-activated macrophages. We show that iRUFY3 controls macrophages migration, MHC II presentation and responses to Interferon-γ while being required for Intracellular Salmonella replication. Specific inactivation of Rufy3 in phagocytes leads to aggravated pathologies in mouse upon LPS injection or bacterial pneumonia. This study highlights iRUFY3 function, as a novel modulator of immunity, controlling endo-lysosomes dynamics, and ultimately regulating the function of activated phagocytes.

International audience; Lung-resident memory B cells (MBCs) provide localized protection against reinfection in respiratory airways. Currently, the biology of these cells remains largely unexplored. Here, we combined influenza and SARS-CoV-2 infection with fluorescent-reporter mice to identify MBCs regardless of antigen specificity. We found that two main transcriptionally distinct subsets of MBCs colonized the lung peribronchial niche after infection. These subsets arose from different progenitors and were both class switched, somatically mutated, and intrinsically biased in their differentiation fate toward plasma cells. Combined analysis of antigen specificity and B cell receptor repertoire segregated these subsets into “bona fide” virus-specific MBCs and “bystander” MBCs with no apparent specificity for eliciting viruses generated through an alternative permissive process. Thus, diverse transcriptional programs in MBCs are not linked to specific effector fates but rather to divergent strategies of the immune system to simultaneously provide rapid protection from reinfection while diversifying the initial B cell repertoire.

When intracellular, pathogenic Salmonella reside in a membrane compartment composed of interconnected vacuoles and tubules, the formation of which depends on the translocation of bacterial effectors into the host cell. Cytoskeletons and their molecular motors are prime targets for these effectors. In this study, we show that the microtubule molecular motor KIF1Bβ (a splice variant of KIF1B), a member of the kinesin-3 family, is a key element for the establishment of the Salmonella replication niche as its absence is detrimental to the stability of bacterial vacuoles and the formation of associated tubules. Kinesin-3 interacts with the Salmonella effector SifA but also with SKIP (also known as PLEKHM2), a host protein complexed to SifA. The interaction with SifA is essential for the recruitment of kinesin-3 on Salmonella vacuoles whereas that with SKIP is incidental. In the non-infectious context, however, the interaction with SKIP is essential for the recruitment and activity of kinesin-3 only on a fraction of the lysosomes. Finally, our results show that, in infected cells, the presence of SifA establishes a kinesin-1 and kinesin-3 recruitment pathway that is analogous to and functions independently of that mediated by the Arl8a and Arl8b GTPases. This article has an associated First Person interview with the first author of the paper.

SummaryLung-resident memory B cells (MBCs) provide localized protection against reinfection in the respiratory airways. Currently, the biology of these cells remains largely unexplored. Here, we combined influenza and SARS-CoV-2 infection with fluorescent-reporter mice to identify MBCs regardless of antigen specificity. scRNA-seq analysis and confocal imaging revealed that two main transcriptionally distinct subsets of MBCs colonize the lung peribronchial niche after infection. These subsets arise from different progenitors and are both class-switched, somatically mutated and intrinsically biased in their differentiation fate towards plasma cells. Combined analysis of antigen-specificity and B cell receptor repertoire unveiled a highly permissive selection process that segregates these subsets into “bona fide” virus-specific MBCs and “bystander” MBCs with no apparent specificity for eliciting viruses. Thus, diverse transcriptional programs in MBCs are not linked to specific effector fates but rather to divergent strategies of the immune system to simultaneously provide rapid protection from reinfection while diversifying the initial B cell repertoire.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Study of cell populations in tissues using immunofluorescence is a powerful method for both basic and medical research. Image acquisitions performed by confocal microscopy notably allow excellent lateral resolution and more than 10 parameter measurement when using spectral or multiplex imaging. Analysis of such complex images can be very challenging and easily lead to bias and misinterpretation. Here, we have developed the Shiny Analytical Plot of Histological Image Results (SAPHIR), an R shiny application for histo-cytometry using scatterplot representation of data extracted by segmentation. It offers many features, such as filtering of spurious data points, selection of cell subsets on scatterplot, visualization of scatterplot selections back into the image, statistics of selected data and data annotation. Our application allows to quickly characterize labeled cells, from their phenotype to their number and location in the tissue, as well as their interaction with other cells. SAPHIR is available from: https://github.com/elodiegermani/SAPHIR

As a first line of defence against the environment, the epidermis protect animals from infection and physical damage. In C. elegans, wounding the epidermal epithelium triggers both an immune reaction and a repair response. Exactly how these are controlled, and the degree to which they are inter-connected remains unclear. To address these questions, we established a simple system for simultaneously inflicting precise laser wounds and imaging at high spatial and temporal resolution. We show that in C. elegans, wounding provokes a rapid sealing of the plasma membrane, involving reorganisation of phosphatidylinositol 4,5- bisphosphate domains. This is followed by a radial recruitment at the wound site of EBP-2/EB1, a protein that binds the plus ends of microtubules. EB1 recruitment is accompanied by a reorganisation of microtubules, required for the subsequent recruitment of actin and wound closure. It is also required for the directed trafficking towards the site of injury of the key signalling protein SNF-12. In the absence of SNF-12 recruitment, there is an abrogation of the immune response. Our results suggest that microtubule dynamics coordinate the cytoskeletal changes required for wound repair and the concomitant activation of the innate immune response.

Combining stimulated emission depletion and fluorescence correlation spectroscopy (STED-FCS) provides a powerful and sensitive tool for studying the molecular dynamics in live cells with high spatio-temporal resolution. STED-FCS gives access to molecular diffusion characteristic at the nanoscale occurring within short period of times. However due to the incomplete suppression of fluorescence in the STED process, the STED-FCS point spread function (PSF) deviates from a Gaussian shape and challenges the analysis of the auto-correlation curves obtained by FCS. Here, we model the effect of the incomplete fluorescence suppression in STED-FCS experiments and propose a new fitting model improving the accuracy of the diffusion times and average molecule numbers measurements. The implementation of a STED module with pulsed laser source on a commercial confocal/FCS microscope allowed us to apply the STED-background corrected model to fit the STED-FCS measurements. The experimental results are in good accordance with the theoretical analysis both for the number of molecules and the diffusion time which decrease accordingly with the STED power. (C) 2018 Elsevier Inc. All rights reserved.

International audience; In innate immune responses, induction of type-I interferons (IFNs) prevents virus spreading while viral replication is delayed by protein synthesis inhibition. We asked how cells perform these apparently contradictory activities. Using single fibroblast monitoring by flow cytometry and mathematical modeling, we demonstrate that type-I IFN production is linked to cell's ability to enter dsRNA-activated PKR-dependent translational arrest and then overcome this inhibition by decreasing eIF2a phosphorylation through phosphatase 1c cofactor GADD34 (Ppp1r15a) expression. GADD34 expression, shown here to be dependent on the IRF3 transcription factor, is responsible for a biochemical cycle permitting pulse of IFN synthesis to occur in cells undergoing protein synthesis inhibition. Translation arrest is further demonstrated to be key for anti-viral response by acting synergistically with MAVS activation to amplify TBK1 signaling and IFN-b mRNA transcription , while GADD34-dependent protein synthesis recovery contributes to the heterogeneous expression of IFN observed in dsRNA-activated cells.

International audience; Cancer-germline genes in both humans and mice have been shown to encode antigens susceptible to targeting by cytotoxic CD8 T effector cells (CTL). We analysed the ability of CTL to kill different tumour cell lines expressing the same cancer-germline gene P1A (Trap1a). We previously demonstrated that CTL expressing a T-cell receptor specific for the P1A(35-43) peptide associated with H-2L(d), although able to induce regression of P1A-expressing P815 mastocytoma cells, were much less effective against P1A-expressing melanoma cells. Here, we analysed parameters of the in vitro interaction between P1A-specific CTL and mastocytoma or melanoma cells expressing similar levels of the P1A gene and of surface H-2L(d). The mastocytoma cells were more sensitive to cytolysis than the melanoma cells in vitro. Analysis by video-microscopy of early events required for target cell killing showed that similar patterns of increase in cytoplasmic Ca2+ concentration ([Ca2+]i) were induced by both types of P1A-expressing tumour cells. However, the use of CTL expressing a fluorescent granzyme B (GZMB-Tom) showed a delay in the migration of cytotoxic granules to the tumour interaction site, as well as a partially deficient GZMB-Tom exocytosis in response to the melanoma cells. Among surface molecules possibly affecting tumour-CTL interactions, the mastocytoma cells were found to express intercellular adhesion molecule-1, the ligand for LFA-1, which was not detected on the melanoma cells.

Cancer‐germline genes in both humans and mice have been shown to encode antigens susceptible to targeting by cytotoxic CD8 T effector cells (CTL). We analysed the ability of CTL to kill different tumour cell lines expressing the same cancer‐germline gene P1A (Trap1a). We previously demonstrated that CTL expressing a T‐cell receptor specific for the P1A35–43 peptide associated with H‐2Ld, although able to induce regression of P1A‐expressing P815 mastocytoma cells, were much less effective against P1A‐expressing melanoma cells. Here, we analysed parameters of the in vitro interaction between P1A‐specific CTL and mastocytoma or melanoma cells expressing similar levels of the P1A gene and of surface H‐2Ld. The mastocytoma cells were more sensitive to cytolysis than the melanoma cells in vitro. Analysis by video‐microscopy of early events required for target cell killing showed that similar patterns of increase in cytoplasmic Ca2+ concentration ([Ca2+]i) were induced by both types of P1A‐expressing tumour cells. However, the use of CTL expressing a fluorescent granzyme B (GZMB‐Tom) showed a delay in the migration of cytotoxic granules to the tumour interaction site, as well as a partially deficient GZMB‐Tom exocytosis in response to the melanoma cells. Among surface molecules possibly affecting tumour–CTL interactions, the mastocytoma cells were found to express intercellular adhesion molecule‐1, the ligand for LFA‐1, which was not detected on the melanoma cells.

Interleukin-4 boosts the capacity of dendritic cells to present endogenous antigens on MHC II and to resist bacterial infection through a mechanism shown to be partially dependent on RUFY4 expression., Autophagy is a key degradative pathway coordinated by external cues, including starvation, oxidative stress, or pathogen detection. Rare are the molecules known to contribute mechanistically to the regulation of autophagy and expressed specifically in particular environmental contexts or in distinct cell types. Here, we unravel the role of RUN and FYVE domain–containing protein 4 (RUFY4) as a positive molecular regulator of macroautophagy in primary dendritic cells (DCs). We show that exposure to interleukin-4 (IL-4) during DC differentiation enhances autophagy flux through mTORC1 regulation and RUFY4 induction, which in turn actively promote LC3 degradation, Syntaxin 17–positive autophagosome formation, and lysosome tethering. Enhanced autophagy boosts endogenous antigen presentation by MHC II and allows host control of Brucella abortus replication in IL-4–treated DCs and in RUFY4-expressing cells. RUFY4 is therefore the first molecule characterized to date that promotes autophagy and influences endosome dynamics in a subset of immune cells.

International audience; BACKGROUND & AIMS: Peyer's patches (PPs) of the small intestine are antigen sampling and inductive sites that help establish mucosal immunity. Luminal antigens are transported from the mucosal surface of PPs to the subepithelial dome (SED), through the specialized epithelial M cells of the follicle-associated epithelium. Among the SED resident dendritic cells (DCs), which are situated ideally for taking up these antigens, some express high levels of lysozyme (LysoDC) and have strong phagocytic activity. We investigated the mechanisms by which LysoDCs capture luminal antigens in vivo. METHODS: We performed 2-photon microscopy on explants of PPs from mice in which the enhanced green fluorescent protein gene was inserted into the lysozyme M locus (lys-EGFP mice), allowing fluorescence detection of LysoDC. RESULTS: LysoDC extended dendrites through M-cell-specific transcellular pores to the gut lumen. The M-cell adhesion molecules junctional adhesion molecule-A and epithelial cell adhesion molecule were recruited to sites of transcellular migration. Transcellular dendrites scanned the M-cell apical surface and the gut luminal content; they were able to take pathogenic bacteria and inert particles in the lumen before retracting back to the SED. CONCLUSIONS: We describe an antigen sampling mechanism that occurs in PPs and involves cooperation between M cells of the follicle-associated epithelium and DCs of the subepithelial dome. This process might be developed to target vaccines to the mu-cosa.

After entry into thymus, T cell progenitors migrate in the cortex and the medulla while completing their education. Recent reports have documented the dynamic and tortuous behavior of thymocytes. However, other than chemokines and/or segregated thymic substrates, the factors contributing to the dynamic patterns of thymocyte movement are poorly characterized. By combining confocal and dynamic two-photon microscopy, we demonstrate that thymocytes continuously migrate on thymic stromal cell networks. In addition to constituting “roads” for thymocytes, we observed that these networks also provide a scaffold on which dendritic cells attach themselves. These results highlight the central role of stromal microanatomy in orchestrating the multiple cellular interactions necessary for T cell migration/development within the thymus.

The superfamily of fibroblast growth factors (FGF), which counts 22 members in humans, exerts many functions during animal development and adult life. LET-756 is one of the two FGFs of the nematode C. elegans. Re-introduction of LET-756 in a null mutant strain restores viability, allowing the study of structural requirements for LET-756 trafficking and function. LET-756 protein has several regions and motifs, including a non-classical internal motif required for secretion. We show here that a main difference in the wild-type LET-756 molecule and a truncated molecule that mimics a partial loss-of-function mutant lies on subnuclear expression. Using Cos-1 cells and rescue activity we show that: (i) nuclear localization is due to various redundant NLS, one of them acting as a nucleolar localization signal; (ii) nuclear LET-756 is addressed to the speckles by a stretch of glutamine residues; (iii) nuclear LET-756 is trafficking between speckles and nucleoli; (iv) in the nucleolus, LET-756 is associated with proteins of the rRNA splicing compartment; (v) changing LET-756 secretion signal prevents its nuclear localization. We propose that LET-756 exerts its functions through a balance between secreted and nuclear forms due to two opposite addressing signals, (i) synergy of several NLS and (ii) attenuated secretion signal.