Pooled data from three separate experiments are shown. on cytoprotective HSP25/27, the E3 ubiquitin ligase Parkin and HDAC6 and promotes chlamydial antigen generation for presentation on MHC I. We propose that this novel mito-xenophagic pathway linking innate and adaptive immunity is critical for effective DC-mediated anti-bacterial resistance. Introduction Chlamydiae are Gram-negative obligate intracellular bacteria that infect mainly epithelial mucosae, causing a broad spectrum of diseases in humans and animals1. Within membrane-bound vacuoles called inclusions, they undergo a biphasic developmental cycle alternating between infectious, but metabolically inactive elementary bodies (EBs) and non-infectious metabolically active reticulate bodies (RBs)1. is the causative agent of psittacosis, a widespread contamination in psittacine birds and domestic poultry1. Zoonotic disease transmission of the microbe to humans has also been reported2, leading to life-threatening pneumonia with systemic bacterial spread, myocarditis, hepatitis, and encephalitis1. is usually regularly detected in non-avian domestic animals as well as in rodents and wildlife1. Non-avian strains can cause abortion and chronic obstructive pulmonary disease1. Chlamydiae induce cell-mediated immune responses in humans and mice3. Such immune responses are initiated by dendritic cells (DCs), which perform a sentinel function by internalizing antigens in peripheral tissues. Within secondary lymphoid organs, DCs then process and display these antigens on surface MHC molecules to stimulate CD4+ and CD8+ T cells. DCs are among the first professional antigen presenting cells (APCs) encountered by chlamydia4, and cytotoxic CD8+ T cells, primed by infected DCs, likely play an important role in the effective anti-chlamydial immune response3. However, the mechanisms by which chlamydial antigens are processed for MHC I presentation are poorly comprehended. Autophagy mediates the lysosomal degradation of cytosolic material including protein aggregates (aggrephagy) and damaged mitochondria (mitophagy). To achieve this, a membrane called phagophore engulfs cytosolic content and isolates it into a sealed double membrane-bound autophagosome. This then matures along the endocytic pathway before fusing with lysosomes5. Autophagy is also an important defence mechanism that functionally links to downstream activation of the innate and adaptive immune system5. Selective autophagosomal degradation of foreign microbes, termed xenophagy, is usually involved in the degradation of bacteria located in the cytosol and in vacuolar compartments. The molecular mechanisms underlying cargo selection and regulation of autophagy and xenophagy are only partly comprehended, but likely rely on cargo-specific receptors on autophagic membranes5. We previously established a mouse model for non-avian contamination6 and identified an autophagy-dependent immune defence pathway in DCs, in which chlamydial antigens are generated via Trichodesmine autophagosomal degradation of cytosolically released microbes following host-mediated disruption Rabbit Polyclonal to MLKL of their inclusions6. Here, we unravel how infected DCs destabilise chlamydial compartments by metabolic switch and use mito-xenophagy to degrade this material for MHC I cross-presentation. We further identify a Trichodesmine TNF-/cPLA2/AA axis involved in regulating this pathway and the components of the autophagy machinery responsible for executing this process. Results Dendritic cell-derived TNF- drives cPLA2-dependent disruption and autophagic clearance of chlamydial compartments By using C57BL/6 mice, JAWSII cells (an established BM-derived Trichodesmine mouse DC line with homogeneous and consistent cell culture properties)7 and the non-avian strain DC158 as a model system for infection, we could demonstrate that chlamydia from structurally disintegrated inclusions are targeted for autophagy and the generation of MHC I-presented peptide antigens6. Based on this, we proposed that autophagy constitutes a crucial pathway in the intracellular defence against chlamydia in infected DCs. Indeed, chlamydial contamination induces autophagy in DCs, as shown by LC3-I-to-LC3-II conversion (Fig.?1A) and autophagy-specific Cyto-ID Green labelling (Fig.?1B,C). This induction was substantially reduced Trichodesmine by knockdown of crucial autophagy factors such as Beclin-1 and Atg7 (Fig.?1D,E). Strikingly, interference with autophagy drastically increased both the number of chlamydia-positive DCs as well as their bacterial load (Fig.?1F). Moreover, autophagy-impaired DCs displayed poor stimulation of chlamydia-specific CD8+ T Trichodesmine cells (Fig.?1G). It should be noted that during the course of the respective antigen presentation experiments (48?hpi), siRNA-mediated silencing of Beclin-1 and Atg7 did not affect expression and/or infection-dependent induction of surface MHC I (H-2Kb and H-2Db), CD80, CD86, PD-L1 or PD-L2. Thus, in flow cytometry studies (Suppl. Fig.?S1A,B and C) no measureable differences were observed for surface MHC I and coregulatory molecules.