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Innate lymphoid cells (ILCs) are well-characterized immune cells that play key roles in host defense and tissue homeostasis. Yet, how the three-dimensional (3D) genome organization underlies the development and functions of ILCs is unknown. Herein, we carried out an integrative analysis of the 3D genome structure, chromatin accessibility and gene expression in mature ILCs. Our results revealed that the local 3D configuration of the genome is rewired specifically at loci associated with ILC biology to promote their development and functional differentiation. Importantly, we demonstrated that the ontogenesis of ILC2s and the progression of allergic airway inflammation are determined by a unique local 3D configuration of the region containing the ILC-lineage-defining factor Id2, which is characterized by multiple interactions between the Id2 promoter and distal regulatory elements bound by the transcription factors GATA-3 and RORα, unveiling the mechanism whereby the Id2 expression is specifically controlled in group 2 ILCs.

Multi-enhancer hubs are spatial clusters of enhancers which have been recently characterized across numerous developmental programs. Yet, the functional relevance of these three-dimensional (3D) structures is poorly understood. Here we show that the multiplicity of enhancers interacting with the transcription factorEts1is essential to control the precise expression level of this gene in response to cellular cues, and the failure to do so can lead to allergic diseases. Focusing on T cells as a model, we identified a highly connected multi-enhancer hub at theEts1locus, comprising a noncoding regulatory element that is a hotspot for sequence variation associated with allergic diseases. We deleted this hotspot and found that the multi-enhancer connectivity is dispensable for T cell development but required for CD4+T helper (Th1) differentiation in response to changes in the cytokine milieu. Mice lacking this hotspot are thus protected from Th1-mediated colitis but demonstrate an overt allergic response to house dust mites, a T cell-mediated response which is dampened by Th1 cells. Mechanistically, the multi-enhancer hub controls the expression level ofEts1that is dispensable for the active enhancer landscape but required for the Th1-specific genome topology through recruitment of CTCF. Together, we establish a paradigm for the functional and mechanistic relevance of multi-enhancer hubs controlling cellular competence to respond specifically to an inductive cue.

Cell death plays a critical role in inflammatory responses. During pyroptosis, inflammatory caspases cleave Gasdermin D (GSDMD) to release an N-terminal fragment that generates plasma membrane pores that mediate cell lysis and IL-1 cytokine release. Terminal cell lysis and IL-1β release following caspase activation can be uncoupled in certain cell types or in response to particular stimuli, a state termed hyperactivation. However, the factors and mechanisms that regulate terminal cell lysis downstream of GSDMD cleavage remain poorly understood. In the course of studies to define regulation of pyroptosis during Yersinia infection, we identified a line of Card19-deficient mice (Card19lxcn) whose macrophages were protected from cell lysis and showed reduced apoptosis and pyroptosis, yet had wild-type levels of caspase activation, IL-1 secretion, and GSDMD cleavage. Unexpectedly, CARD19, a mitochondrial CARD-containing protein, was not directly responsible for this, as an independently-generated CRISPR/Cas9 Card19 knockout mouse line (Card19Null) showed no defect in macrophage cell lysis. Notably, Card19 is located on chromosome 13, immediately adjacent to Ninj1, which was recently found to regulate cell lysis downstream of GSDMD activation. RNA-seq and western blotting revealed that Card19lxcn BMDMs have significantly reduced NINJ1 expression, and reconstitution of Ninj1 in Card19lxcn immortalized BMDMs restored their ability to undergo cell lysis in response to caspase-dependent cell death stimuli. Card19lxcn mice exhibited increased susceptibility to Yersinia infection, whereas independently-generated Card19Null mice did not, demonstrating that cell lysis itself plays a key role in protection against bacterial infection, and that the increased infection susceptibility of Card19lxcn mice is attributable to loss of NINJ1. Our findings identify genetic targeting of Card19 being responsible for off-target effects on the adjacent gene Ninj1, disrupting the ability of macrophages to undergo plasma membrane rupture downstream of gasdermin cleavage and impacting host survival and bacterial control during Yersinia infection., Author summary Programmed cell death is critical for regulating tissue homeostasis and host defense against infection. Pyroptosis is an inflammatory form of programmed cell death that couples cell lysis with release of inflammatory cytokines. Cell lysis is triggered by activation of particular intracellular pore forming proteins, but how regulation of cell lysis occurs is not well understood. Genetic targeting of Card19 on chromosome 13 resulted in decreased expression of the adjacent gene, Ninj1, which was recently found to regulate terminal lysis events in response to cell death-inducing stimuli. Consistently, macrophages from Card19-deficient mice were resistant to multiple forms of cell death in response to a variety of inflammatory stimuli, including canonical and non-canonical inflammasome activation, as well as triggers of cell-extrinsic apoptosis. Notably, Card19-deficient mice were more susceptible to Yersinia infection, indicating that cell lysis contributes to control of bacterial infections. Our data provide new insight into the impact of terminal cell lysis on control of bacterial infection and highlight the role of additional factors that regulate lytic cell death downstream of gasdermin cleavage.

The transcriptional programs that regulate CD8 T-cell differentiation and function in the context of viral infections or tumor immune surveillance have been extensively studied; yet how long noncoding RNAs (lncRNAs) and the loci that transcribe them contribute to the regulation of CD8 T cells during viral infections remains largely unexplored. Here, we report that transcription of the lncRNA Morrbid is specifically induced by T-cell receptor (TCR) and type I IFN stimulation during the early stages of acute and chronic lymphocytic choriomeningitis virus (LCMV) infection. In response to type I IFN, the Morrbid RNA and its locus control CD8 T cell expansion, survival, and effector function by regulating the expression of the proapoptotic factor, Bcl2l11 , and by modulating the strength of the PI3K–AKT signaling pathway. Thus, our results demonstrate that inflammatory cue-responsive lncRNA loci represent fundamental mechanisms by which CD8 T cells are regulated in response to pathogens and potentially cancer.

Cell death plays a critical role in inflammatory responses. During pyroptosis, inflammatory caspases cleave Gasdermin D (GSDMD) to release an N-terminal fragment that generates plasma membrane pores that mediate cell lysis and IL-1 cytokine release. Terminal cell lysis and IL-1β release following caspase activation can be uncoupled in certain cell types or in response to particular stimuli, a state termed hyperactivation. However, the factors and mechanisms that regulate terminal cell lysis downstream of GSDMD cleavage remain poorly understood. In the course of studies to define regulation of pyroptosis during Yersinia infection, we identified a line of Card19-deficient mice (Card19lxcn) whose macrophages were protected from cell lysis and showed reduced apoptosis and pyroptosis, yet had wild-type levels of caspase activation, IL-1 secretion, and GSDMD cleavage. Unexpectedly, CARD19, a mitochondrial CARD-containing protein, was not directly responsible for this, as two independently-generated CRISPR/Cas9 Card19 knockout mice showed no defect in macrophage cell lysis, and expression of CARD19 in Card19lxcn macrophages did not restore cell lysis. Card19 is located on chromosome 13, adjacent to Ninj1, which was recently reported to regulate cell lysis downstream of GSDMD activation. Intriguingly, RNA-seq and western blotting revealed that Card19lxcn BMDMs are hypomorphic for NINJ1 expression, and reconstitution of Ninj1 in Card19lxcn immortalized BMDMs restored cell lysis. Card19lxcn mice exhibited significantly increased susceptibility to Yersinia infection, demonstrating that cell lysis itself plays a key role in protection against bacterial infection. Our findings identify genetic targeting of Card19 being responsible for off-target effects on the adjacent Ninj1 gene, thereby disrupting the ability of macrophages to undergo plasma membrane rupture downstream of gasdermin cleavage and impacting host survival and bacterial control during Yersinia infection.Author SummaryProgrammed cell death is critical for regulating tissue homeostasis and host defense against infection. Pyroptosis is an inflammatory form of programmed cell death that couples cell lysis with release of inflammatory cytokines. Cell lysis is triggered by activation of particular intracellular pore forming proteins, but how regulation of cell lysis occurs is not well understood. Genetic targeting of Card19 on chromosome 13 resulted in decreased expression of the adjacent gene, Ninj1 which was recently found to regulate terminal lysis events in response to cell death-inducing stimuli. We found that macrophages from Card19-deficient mice were resistant to multiple forms of cell death in response to a variety of inflammatory stimuli, including canonical and non-canonical inflammasome activation, as well as triggers of cell-extrinsic apoptosis. Notably, Card19-deficient mice were more susceptible to Yersinia infection, indicating that cell lysis contributes to control of bacterial infections. Our data provide new insight into the impact of terminal cell lysis on control of bacterial infection and highlight the role of additional factors that regulate lytic cell death downstream of gasdermin cleavage.

SummaryEfficient cross-presentation of antigens by dendritic cells (DCs) is critical for initiation of anti-tumour immune responses. Yet, several steps of antigen intracellular traffic during cross-presentation are incompletely understood: in particular, the molecular mechanisms and the relative importance of antigen import from endocytic compartments into the cytosol. Here, we asked whether antigen import into the cytosol is rate-limiting for cross-presentation and anti-tumour immunity. By screening 700 FDA-approved drugs, we identified 37 import enhancers. We focused on prazosin and tamoxifen, and generated proteomic organellar maps of drug-treated DCs, covering the subcellular localisations of over 2000 proteins. By combining organellar mapping, quantitative proteomics, microscopy, and bioinformatics, we conclude that import enhancers undergo lysosomal trapping leading to membrane permeation and antigen release into the cytosol. Enhancing antigen import facilitates cross-presentation of both soluble and cell-associated antigens. Systemic administration of prazosin also led to reduced growth of MC38 tumours and to a synergistic effect with checkpoint immunotherapy in a melanoma model. Thus, inefficient antigen import into the cytosol limits antigen cross-presentation, restraining the potency of anti-tumour immune responses and efficacy of checkpoint blockers.HighlightsQuinazolamine derivatives are potent enhancers of antigen import into the cytosolAntigen import enhancement occurs as a consequence of lysosomal trappingAntigen import enhancers facilitate cross-presentation and synergise with PD-L1Proteomic organellar maps of cDC1s as a tool to detect biological effects of drugs

Summary Cross-presentation of antigens by dendritic cells (DCs) is critical for initiation of anti-tumor immune responses. Yet, key steps involved in trafficking of antigens taken up by DCs remain incompletely understood. Here, we screen 700 US Food and Drug Administration (FDA)-approved drugs and identify 37 enhancers of antigen import from endolysosomes into the cytosol. To reveal their mechanism of action, we generate proteomic organellar maps of control and drug-treated DCs (focusing on two compounds, prazosin and tamoxifen). By combining organellar mapping, quantitative proteomics, and microscopy, we conclude that import enhancers undergo lysosomal trapping leading to membrane permeation and antigen release. Enhancing antigen import facilitates cross-presentation of soluble and cell-associated antigens. Systemic administration of prazosin leads to reduced growth of MC38 tumors and to a synergistic effect with checkpoint immunotherapy in a melanoma model. Thus, inefficient antigen import into the cytosol limits antigen cross-presentation, restraining the potency of anti-tumor immune responses and efficacy of checkpoint blockers., Graphical Abstract, Highlights • Quinazolinamine derivatives are potent enhancers of antigen import into the cytosol • Antigen import enhancement occurs as a consequence of lysosomal trapping • Antigen import enhancers facilitate cross-presentation and synergize with anti-PD-L1 • Proteomic organellar maps can be used as a tool to detect biological effects of drugs, Kozik et al. apply a screening strategy to identify drugs that facilitate endosome-to-cytosol import of antigens in dendritic cells during cross-presentation. They characterize selected compounds using proteomic organellar mapping, live-cell imaging, in vitro cross-presentation assays, and tumor models and demonstrate that enhancing import facilitates cross-presentation and synergizes with checkpoint-based immunotherapy.

The transcriptional programs that regulate CD8 T-cell differentiation and function in the context of viral infections or tumor immune surveillance have been extensively studied; yet how long noncoding RNAs (lncRNAs) and the loci that transcribe them contribute to the regulation of CD8 T cells during viral infections remains largely unexplored. Here, we report that transcription of the lncRNA Morrbid is specifically induced by T-cell receptor (TCR) and type I IFN stimulation during the early stages of acute and chronic lymphocytic choriomeningitis virus (LCMV) infection. In response to type I IFN, the Morrbid RNA and its locus control CD8 T cell expansion, survival, and effector function by regulating the expression of the proapoptotic factor, Bcl2l11, and by modulating the strength of the PI3K–AKT signaling pathway. Thus, our results demonstrate that inflammatory cue-responsive lncRNA loci represent fundamental mechanisms by which CD8 T cells are regulated in response to pathogens and potentially cancer.

Significance CD4 T cells are major regulators of immune responses against both self and pathogens. Understanding pathways that govern CD4 T cell differentiation and regulation are thus key for the discovery of new immunoregulatory drug targets. Here, we have identified an epigenetic pathway that regulates the expression of a set of proteins that determine T cell responsiveness. By silencing enhancers distal to a set of genes known to be involved in regulatory T cell function, the epigenetic modifiers TRIM28 and HP1β/γ regulate T cell receptor signaling. This leads to defective metabolic reprograming and inefficient effector differentiation of naive T cells. This mechanism provides an exciting opportunity to regulate T cell responsivity in both autoimmunity and T cell-based immunodeficiencies., Naive CD4+ T lymphocytes differentiate into different effector types, including helper and regulatory cells (Th and Treg, respectively). Heritable gene expression programs that define these effector types are established during differentiation, but little is known about the epigenetic mechanisms that install and maintain these programs. Here, we use mice defective for different components of heterochromatin-dependent gene silencing to investigate the epigenetic control of CD4+ T cell plasticity. We show that, upon T cell receptor (TCR) engagement, naive and regulatory T cells defective for TRIM28 (an epigenetic adaptor for histone binding modules) or for heterochromatin protein 1 β and γ isoforms (HP1β/γ, 2 histone-binding factors involved in gene silencing) fail to effectively signal through the PI3K–AKT–mTOR axis and switch to glycolysis. While differentiation of naive TRIM28−/− T cells into cytokine-producing effector T cells is impaired, resulting in reduced induction of autoimmune colitis, TRIM28−/− regulatory T cells also fail to expand in vivo and to suppress autoimmunity effectively. Using a combination of transcriptome and chromatin immunoprecipitation-sequencing (ChIP-seq) analyses for H3K9me3, H3K9Ac, and RNA polymerase II, we show that reduced effector differentiation correlates with impaired transcriptional silencing at distal regulatory regions of a defined set of Treg-associated genes, including, for example, NRP1 or Snai3. We conclude that TRIM28 and HP1β/γ control metabolic reprograming through epigenetic silencing of a defined set of Treg-characteristic genes, thus allowing effective T cell expansion and differentiation into helper and regulatory phenotypes.

The gut microbiota is a key environmental determinant of mammalian metabolism. Regulation of white adipose tissue (WAT) by the gut microbiota is a process critical to maintaining metabolic fitness, and gut dysbiosis can contribute to the development of obesity and insulin resistance (IR). However, how the gut microbiota regulates WAT function remains largely unknown. Here, we show that tryptophan-derived metabolites produced by the gut microbiota controlled the expression of the miR-181 family in white adipocytes in mice to regulate energy expenditure and insulin sensitivity. Moreover, we show that dysregulation of the gut microbiota-miR-181 axis was required for the development of obesity, IR, and WAT inflammation in mice. Our results indicate that regulation of miR-181 in WAT by gut microbiota-derived metabolites is a central mechanism by which host metabolism is tuned in response to dietary and environmental changes. As we also found that MIR-181 expression in WAT and the plasma abundance of tryptophan-derived metabolites were dysregulated in a cohort of obese human children, the MIR-181 family may represent a potential therapeutic target to modulate WAT function in the context of obesity.

Carpier et al. show that LAT trafficking to the immune synapse depends on endosome-to-Golgi/TGN retrograde transport and is controlled by Rab6 and Syntaxin-16. Moreover, they show that this retrograde pathway controls the TCR-induced activation of T lymphocytes., The adapter molecule linker for activation of T cells (LAT) orchestrates the formation of signalosomes upon T cell receptor (TCR) stimulation. LAT is present in different intracellular pools and is dynamically recruited to the immune synapse upon stimulation. However, the intracellular traffic of LAT and its function in T lymphocyte activation are ill defined. We show herein that LAT, once internalized, transits through the Golgi–trans-Golgi network (TGN), where it is repolarized to the immune synapse. This retrograde transport of LAT depends on the small GTPase Rab6 and the target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (t-SNARE) Syntaxin-16, two regulators of the endosome-to-Golgi/TGN retrograde transport. We also show in vitro in Syntaxin-16– or Rab6-silenced human cells and in vivo in CD4+ T lymphocytes of the Rab6 knockout mouse that this retrograde traffic controls TCR stimulation. These results establish that the retrograde traffic of LAT from the plasma membrane to the Golgi-TGN controls the polarized delivery of LAT at the immune synapse and T lymphocyte activation., Graphical Abstract

© 2015 European Association for the Study of the Liver. Background & Aims Liver fibrosis, an important health concern associated to chronic liver injury that provides a permissive environment for cancer development, is characterized by accumulation of extracellular matrix components mainly derived from activated hepatic stellate cells (HSCs). Axl, a receptor tyrosine kinase and its ligand Gas6, are involved in cell differentiation, immune response and carcinogenesis. Methods HSCs were obtained from WT and Axl-/- mice, treated with recombinant Gas6 protein (rGas6), Axl siRNAs or the Axl inhibitor BGB324, and analyzed by western blot and real-time PCR. Experimental fibrosis was studied in CCl4-treated WT and Axl-/- mice, and in combination with Axl inhibitor. Gas6 and Axl serum levels were measured in alcoholic liver disease (ALD) and hepatitis C virus (HCV) patients. Results In primary mouse HSCs, Gas6 and Axl levels paralleled HSC activation. rGas6 phosphorylated Axl and AKT prior to HSC phenotypic changes, while Axl siRNA silencing reduced HSC activation. Moreover, BGB324 blocked Axl/AKT phosphorylation and diminished HSC activation. In addition, Axl-/- mice displayed decreased HSC activation in vitro and liver fibrogenesis after chronic damage by CCl4 administration. Similarly, BGB324 reduced collagen deposition and CCl4-induced liver fibrosis in mice. Importantly, Gas6 and Axl serum levels increased in ALD and HCV patients, inversely correlating with liver functionality. Conclusions The Gas6/Axl axis is required for full HSC activation. Gas6 and Axl serum levels increase in parallel to chronic liver disease progression. Axl targeting may be a therapeutic strategy for liver fibrosis management., This study was funded by grants from the Instituto de Salud Carlos III (FIS PI12/00110, PI09/00056 to A.M., FIS PI13/00374 to M.M., PI12/01265 to J.C., PI14/00320 to P.S-B. and PI11/0325 to J.F.C.), Ministerio de Economía y Competitividad (BFU2010-22185 to P.G.F., SAF2012/34831 to J.F.C. and SAF2011-23031 to C.G.R.) and co-funded by FEDER (Fondo Europeo de Desarrollo Regional, Unión Europea. “Una manera de hacer Europa”); center grant P50-AA-11999 from Research Center for Liver and Pancreatic Diseases, (US NIAAA to J.F.C.); National Institutes of Health (R01 AI089824 to C.V.R); Fundació la Marató de TV3 to J.F.C.; Fundación Mutua Madrileña (AP103502012 to C.G.R.) and by CIBERehd from the Instituto de Salud Carlos III

The receptor tyrosine kinases Axl and Mer, belonging to the Tyro3, Axl and Mer (TAM) receptor family, are expressed in a number of tumor cells and have well-characterized oncogenic roles. The therapeutic targeting of these kinases is considered an anticancer strategy, and various inhibitors are currently under development. At the same time, Axl and Mer are expressed in dendritic cells and macrophages and have an essential function in limiting inflammation. Inflammation is an enabling characteristic of multiple cancer hallmarks. These contrasting oncogenic and anti-inflammatory functions of Axl and Mer posit a potential paradox in terms of anticancer therapy. Here we demonstrate that azoxymethane (AOM) and dextran sulfate sodium (DSS)-induced inflammation-associated cancer is exacerbated in mice lacking Axl and Mer. Ablation of Axl and Mer signaling is associated with increased production of proinflammatory cytokines and failure to clear apoptotic neutrophils in the intestinal lamina propria, thereby favoring a tumor-promoting environment. Interestingly, loss of these genes in the hematopoietic compartment is not associated with increased colitis. Axl and Mer are expressed in radioresistant intestinal macrophages, and the loss of these genes is associated with an increased inflammatory signature in this compartment. Our results raise the possibility of potential adverse effects of systemic anticancer therapies with Axl and Mer inhibitors, and underscore the importance of understanding their tissue and cell type-specific functions in cancer.

The authors regret that in the original version of their paper, they mistakenly used the phrase OVA-expressing cells instead of OVA-secreting cells in parts of the text and cited reference Boissonnas et al. (2007. http://dx.doi.org/10.1084/jem.20061890) instead of Zeelenberg et al. (2008. http://dx.doi.org/10.1158/0008-5472.CAN-07-3163) and Sedlik et al. (2014. http://dx.doi.org/10.3402/jev.v3.24646). The Results and discussion paragraph containing the corrected references and full bibliographic information appear below. Fil: Alloatti, Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Rookhuizen, Derek C.. Institute Curie. U-932 Immunity And Cancer; Francia Fil: Joannas, Leonel. Institute Curie. U-932 Immunity And Cancer; Francia Fil: Carpier, Jean-Marie. Institute Curie. U-932 Immunity And Cancer; Francia Fil: Iborra, Salvador. Institute Curie. U-932 Immunity And Cancer; Francia Fil: Magalhaes, Joao G.. Institute Curie. U-932 Immunity And Cancer; Francia Fil: Yatim, Nader. Institut Pasteur, Paris; Francia Fil: Kozik, Patrycja. Institute Curie. U-932 Immunity And Cancer; Francia Fil: Sancho, David. Institute Curie. U-932 Immunity And Cancer; Francia Fil: Albert, Matthew L.. Institut Pasteur, Paris; Francia Fil: Amigorena, Sebastian. Institute Curie. U-932 Immunity And Cancer; Francia

It has been shown that the expression of the morphine (MOR) withdrawal syndrome precipitated by naloxone (NAL) is more intense in male mice than in females, but the reasons for this phenomenon remain uncertain. The purpose of the present study was to evaluate whether this sexual dimorphism might be due to differences in MOR and/or NAL plasma levels after a chronic treatment with MOR. Prepubertal Swiss male and female mice were rendered dependent by intraperitoneal (i.p.) injection of MOR (2 mg/kg), twice daily for 9 days. On day 10 dependent mice received NAL (6 mg/kg, i.p.) 60 min after MOR injection. Blood samples were taken at different times in order to determine MOR and NAL plasma levels by gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC), respectively. Pharmacokinetic analysis showed no differences between male and female mice either for MOR or for NAL. In conclusion, although males and females respond differentially to NAL-precipitated withdrawal, this dimorphic behavior would not be influenced by a pharmacokinetic factor. Copyright © 2007 John Wiley & Sons, Ltd.

The initiation of cytotoxic immune responses by dendritic cells (DCs) requires the presentation of antigenic peptides derived from phagocytosed microbes and infected or dead cells to CD8(+) T cells, a process called cross-presentation. Antigen cross-presentation by non-activated DCs, however, is not sufficient for the effective induction of immune responses. Additionally, DCs need to be activated through innate receptors, like Toll-like receptors (TLRs). During DC maturation, cross-presentation efficiency is first upregulated and then turned off. Here we show that during this transient phase of enhanced cross-presentation, phago-lysosome fusion was blocked by the topological re-organization of lysosomes into perinuclear clusters. LPS-induced lysosomal clustering, inhibition of phago-lysosome fusion and enhanced cross-presentation, all required expression of the GTPase Rab34. We conclude that TLR4 engagement induces a Rab34-dependent re-organization of lysosomal distribution that delays antigen degradation to transiently enhance cross-presentation, thereby optimizing the priming of CD8(+) T cell responses against pathogens. Fil: Alloatti, Andrés. Inserm; Francia. Institute Curie; Francia Fil: Kotsias, Fiorella. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Institute Curie; Francia. Inserm; Francia. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento Virologia; Argentina Fil: Pauwels, Anne Marie. University of Ghent; Bélgica Fil: Carpier, Jean Marie. Institute Curie; Francia. Inserm; Francia Fil: Jouve, Mabel. Institute Curie; Francia Fil: Timmerman, Evy. University of Ghent; Bélgica Fil: Pace, Luigia. Institute Curie; Francia. Inserm; Francia Fil: Vargas, Pablo. Institute Curie; Francia. Inserm; Francia. Centre National de la Recherche Scientifique; Francia Fil: Maurin, Mathieu. Institute Curie; Francia. Inserm; Francia Fil: Gehrmann, Ulf. Institute Curie; Francia. Inserm; Francia Fil: Joannas, Leonel. Institute Curie; Francia. Inserm; Francia Fil: Vivar, Omar I.. Institute Curie; Francia. Inserm; Francia Fil: Lennon Duménil, Ana Maria. Institute Curie; Francia. Inserm; Francia Fil: Savina, Ariel. Institute Curie; Francia. Inserm; Francia. Roche SAS; Francia Fil: Gevaert, Kris. University of Ghent; Bélgica Fil: Beyaert, Rudi. University of Ghent; Bélgica Fil: Hoffmann, Eik. Inserm; Francia. Institute Curie; Francia. University of Ghent; Bélgica Fil: Amigorena, Sebastian. Inserm; Francia. Institute Curie; Francia

Dendritic cell (DC) activation is essential for the induction of immune defense against pathogens, yet needs to be tightly controlled to avoid chronic inflammation and exaggerated immune responses. Here, we identify a mechanism of immune homeostasis by which adaptive immunity, once triggered, tempers DC activation and prevents overreactive immune responses. T cells, once activated, produced Protein S (Pros1) that signaled through TAM receptor tyrosine kinases in DCs to limit the magnitude of DC activation. Genetic ablation of Pros1 in mouse T cells led to increased expression of costimulatory molecules and cytokines in DCs and enhanced immune responses to T cell-dependent antigens, as well as increased colitis. Additionally, PROS1 was expressed in activated human T cells, and its ability to regulate DC activation was conserved. Our results identify a heretofore unrecognized, homeostatic negative feedback mechanism at the interface of adaptive and innate immunity that maintains the physiological magnitude of the immune response. Fil: Carrera Silva, Eugenio Antonio. University of Yale. School of Medicine; Estados Unidos de América; Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental; Argentina; Fil: Chan, Pamela Y.. University of Yale. School of Medicine; Estados Unidos de América; Fil: Joannas, Leonel. University of Yale. School of Medicine; Estados Unidos de América; Fil: Errasti, Andrea Emilse. Universidad de Buenos Aires. Facultad de Medicina. Cátedra de Farmacología; Argentina; University of Yale. School of Medicine; Estados Unidos de América; Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina; Fil: Gagliani, Nicola. University of Yale. School of Medicine; Estados Unidos de América; Fil: Bosurgi, Lidia. University of Yale. School of Medicine; Estados Unidos de América; Fil: Jabbour, Maurice. University Of Arizona; Estados Unidos de América; Fil: Perry, Anthony. Banner MD Anderson Cancer Center; Estados Unidos de América; Fil: Smith Chakmakova, Faye. Saint Joseph’s Hospital and Medical Center; Estados Unidos de América; Fil: Mucida, Daniel. La Jolla Institute for Allergy and Immunology; Estados Unidos de América; Fil: Cheroutre, Hilde. La Jolla Institute for Allergy and Immunology; Estados Unidos de América; Fil: Burstyn Cohen, Tal. Salk Institute for Biological Studies; Estados Unidos de América; Fil: Leighton, Jonathan A.. Mayo Clinic Arizona; Estados Unidos de América; Fil: Lemke, Greg. Salk Institute for Biological Studies; Estados Unidos de América; Fil: Ghosh, Sourav. University Of Arizona; Estados Unidos de América; Fil: Rothlin, Carla V.. University of Yale. School of Medicine; Estados Unidos de América

Background and Aims: AXL, a receptor tyrosin kinase, and its ligand GAS6 are related to processes leading to cell differentiation and carcinogenesis. The role of GAS6/AXL signaling in hepatic stellate cell (HSC) transformation during liver disease has been insufficiently addressed. Methods: HSCs were isolated from wild-type and AXL knockout (KO) mice. HSCs and LX2 human activated HSCs were exposed to recombinant GAS6, AXL inhibitor (BGB324, BerGenBio) or siRNA silencing. p-AXL, p-AKT and HSC transformation were evaluated by western blotting and qPCR. CCl4-induced fibrosis was analyzed in wild-type and AXL KO mice. Gas6 and soluble AXL (sAXL) levels were measured by ELISA in serum from alcoholic patients with normal liver, compensated cirrhosis and decompensated cirrhosis, and in control individuals (n = 10, each group). Results: Recombinant GAS6 stimulated AXL and AKT activation, inducing phenotypic changes in primary HSCs. HSCs from AXL KO mice displayed reduced capacity to be activated in vitro, and AXL inhibition (BGB324, Bergenbio) reduced HSC activation and proliferation. Similar results were obtained in LX2 cells and after RNA silencing of AXL. Chronic CCl4 administration increased serum levels of sAXL and GAS6, while AXL deficient mice exhibited reduced liver fibrosis. More importantly, AXL inhibition (BGB324) efficiently diminished CCl4-induced liver fibrosis in wild-type mice. In parallel, GAS6 and sAXL levels in serum were increased in alcoholic liver disease (ALD) patients, inversely correlating with liver functionality. Conclusions: AXL inhibitors have clinical interest since AXL targeting is effective to treat experimental liver fibrosis. In addition, GAS6 and sAXL levels could be proposed as early prognostic markers in liver disease.

Injection of effector proteins to block host innate immune signalling is a common strategy used by many pathogenic organisms to establish an infection. Pathogenic Yersinia species for example inject the acetyltransferase YopJ into target cells to inhibit NF-κB and MAPK signalling. To counteract this, detection of YopJ activity in myeloid cells promotes the assembly of a RIPK1-caspase-8 death-inducing platform that confers antibacterial defence. While recent studies revealed that caspase-8 cleaves the pore-forming protein, gasdermin D (GSDMD) to trigger pyroptosis in macrophages, whether RIPK1 activates additional substrates downstream of caspase-8 to promote host defence is unclear. Here, we report that the related gasdermin family member gasdermin E (GSDME) is activated upon detection of YopJ activity in a RIPK1 kinase-dependent manner. Specifically, GSDME promotes neutrophil pyroptosis and IL-1β release, which is critical for anti-Yersinia defence. During in vivo infection, IL-1β neutralisation increases bacterial burden in wild type but not Gsdme-deficient mice. Thus, our study establishes GSDME as an important mediator that counteracts pathogen blockade of innate immune signalling.