Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection.

Programmed cell death contributes to host defense against pathogens. To investigate the relative importance of pyroptosis, necroptosis, and apoptosis during Salmonella infection, we infected mice and macrophages deficient for diverse combinations of caspases-1, -11, -12, and -8 and receptor interacting serine/threonine kinase 3 (RIPK3). Loss of pyroptosis, caspase-8-driven apoptosis, or necroptosis had minor impact on Salmonella control. However, combined deficiency of these cell death pathways caused loss of bacterial control in mice and their macrophages, demonstrating that host defense can employ varying components of several cell death pathways to limit intracellular infections. This flexible use of distinct cell death pathways involved extensive cross-talk between initiators and effectors of pyroptosis and apoptosis, where initiator caspases-1 and -8 also functioned as executioners when all known effectors of cell death were absent. These findings uncover a highly coordinated and flexible cell death system with in-built fail-safe processes that protect the host from intracellular infections.Wellcome Trus

Effective priming of herpes simplex virusspecific CD8+ T cells in vivo does not require infected dendritic cells

Resolution of virus infections depends on the priming of virus-specific CD8+ T cells by dendritic cells (DC). While this process requires major histocompatibility complex (MHC) class I-restricted antigen presentation by DC, the relative contribution to CD8+ T cell priming by infected DC is less clear. We have addressed this question in the context of a peripheral infection with herpes simplex virus 1 (HSV). Assessing the endogenous, polyclonal HSV-specific CD8+ T cell response, we found that effective in vivo T cell priming depended on the presence of DC subsets specialized in cross-presentation, while Langerhans cells and plasmacytoid DC were dispensable. Utilizing a novel mouse model that allows for the in vivo elimination of infected DC, we also demonstrated in vivo that this requirement for cross-presenting DC was not related to their infection but instead reflected their capacity to cross-present HSV-derived antigen. Taking the results together, this study shows that infected DC are not required for effective CD8+ T cell priming during a peripheral virus infection. IMPORTANCE The ability of some DC to present viral antigen to CD8+ T cells without being infected is thought to enable the host to induce killer T cells even when viruses evade or kill infected DC. However, direct experimental in vivo proof for this notion has remained elusive. The work described in this study characterizes the role that different DC play in the induction of virus-specific killer T cell responses and, critically, introduces a novel mouse model that allows for the selective elimination of infected DC in vivo. Our finding that HSV-specific CD8+ T cells can be fully primed in the absence of DC infection shows that cross-presentation by DC is indeed sufficient for effective CD8+ T cell priming during a peripheral virus infection.Our research is supported by the National Health and Medical Research Council of Australia. P. Whitney is supported by an Overseas Biomedical Fellowship (NHMRC) and a MDHS Faculty Fellowship (University of Melbourne). T. Gebhardt is supported by a fellowship from the Sylvia and Charles Viertel Charitable Foundation. D. Tscharke is supported by a Senior Research Fellowship (NHMRC)

The role of T cell help in shaping Dendritic cell function

Completed under a Cotutelle arrangement between the University of Melbourne and University of Bonn© 2019 Elise Marie Elisabeth GressierCD8+ T cell priming depends on antigen presentation by dendritic cells (DCs) and their capacity to communicate contextual cues associated with antigen acquisition. DCs often also require additional signals from helper CD4+ T cells, which upon mediation via CD40-CD40L further modulate the communication of contextual cue to the responding CD8+T cells. The present study was designed to explore the kinetics and molecular mechanisms underpinning this helper-dependent modulation of DC function. To address this, we employed an in vitro system of bone marrow (BM)-derived equivalents of CD8+ DCs (eCD8+ DCs) and we assessed the role of different CD40 signalling components in driving their IFN-aA-induced cytokine and chemokine responses by using flow cytometry, mass spectrometry-based proteomics, real time PCR and RNA sequencing. This brought to light remarkable and distinct patterns of gene regulation through which CD4+ T cells triggered CD40 and thereby amplified the capacity of IFN-aA to induce or downregulate a broad range of genes. We also observed an unexpected pattern of gene regulation: some genes required both T cell help and IFN-aA stimulations but could not be induced by ‘help’ or IFN-a alone. By varying the exposure time, we further discovered that eCD8+ DCs required 1-2 hours of IFN-aA to become responsive to CD40 triggering. Once this pre-activated state was achieved, CD40 stimulation rapidly amplified responses with remarkably fast kinetics. Combining proteomics and RNA sequencing data presented in this thesis suggests a complex interplay between the IFN-aA signalling pathway involving IRFs transcription factors and the NF-kB signalling pathway. These findings not only reveal new insights into how T cell help adjusts the responsiveness of DC to innate stimuli, but also reveal that this can occur with remarkable speed, which aligns with in vivo imaging studies describing very brief interactions between eCD8+ DCs and CD4+ T cells during CD8+ T cell priming

Molecular dissection of plasmacytoid dendritic cell activation in vivo during a viral infection

International audiencePlasmacytoid dendritic cells (pDC) are the major source of type I interferons (IFN-I) during viral infections, in response to triggering of endosomal Toll-like receptors (TLRs) 7 or 9 by viral single-stranded RNA or unmethylated CpG DNA, respectively. Synthetic ligands have been used to disentangle the underlying signaling pathways. The adaptor protein AP3 is necessary to transport molecular complexes of TLRs, synthetic CpG DNA, and MyD88 into endosomal compartments allowing interferon regulatory factor 7 (IRF7) recruitment whose phosphorylation then initiates IFN-I production. High basal expression of IRF7 by pDC and its further enhancement by positive IFN-I feedback signaling appear to be necessary for robust cytokine production. In contrast, we show here that in vivo during mouse cytomegalovirus (MCMV) infection pDC produce high amounts of IFN-I downstream of the TLR9-to-MyD88-to-IRF7 signaling pathway without requiring IFN-I positive feedback, high IRF7 expression, or AP3-driven endosomal routing of TLRs. Hence, the current model of the molecular requirements for professional IFN-I production by pDC, established by using synthetic TLR ligands, does not strictly apply to a physiological viral infection