f AIP56 (apoptosis-inducing protein of 56 kDa) is a metalloprotease AB toxin secreted by Photobacterium damselae subsp. piscicida that acts by cleaving NF-B. During infection, AIP56 spreads systemically and depletes phagocytes by postapoptotic secondary necrosis, impairing the host phagocytic defense and contributing to the genesis of infection-associated necrotic lesions. Here we show that mouse bone marrow-derived macrophages (mBMDM) intoxicated by AIP56 undergo NF-B p65 depletion and apoptosis. Similarly to what was reported for sea bass phagocytes, intoxication of mBMDM involves interaction of AIP56 C-terminal region with cell surface components, suggesting the existence of a conserved receptor. Biochemical approaches and confocal microscopy revealed that AIP56 undergoes clathrin-dependent endocytosis, reaches early endosomes, and follows the recycling pathway. Translocation of AIP56 into the cytosol requires endosome acidification, and an acidic pulse triggers translocation of cell surface-bound AIP56 into the cytosol. Accordingly, at acidic pH, AIP56 becomes more hydrophobic, interacting with artificial lipid bilayer membranes. Altogether, these data indicate that AIP56 is a short-trip toxin that reaches the cytosol using an acidic-pH-dependent mechanism, probably from early endosomes. Usually, for short-trip AB toxins, a minor pool reaches the cytosol by translocating from endosomes, whereas the rest is routed to lysosomes for degradation. Here we demonstrate that part of endocytosed AIP56 is recycled back and released extracellularly through a mechanism requiring phosphoinositide 3-kinase (PI3K) activity but independent of endosome acidification. So far, we have been unable to detect biological activity of recycled AIP56, thereby bringing into question its biological relevance as well as the importance of the recycling pathway.A IP56 (apoptosis-inducing protein of 56 kDa) is a plasmidencoded toxin of Photobacterium damselae subsp. piscicida (1), a Gram-negative bacterium that infects economically important fish species (2, 3) and is considered one of the most relevant pathogens in mariculture (2-5). In acute infections, a rapid septicemia develops, causing very high mortalities (2, 4, 6). Histopathological analysis of the diseased animals revealed cytotoxic alterations (4, 7-10) that were found to result from pathogeninduced apoptotic death of macrophages and neutrophils (11) and later associated with the activity of AIP56 (1). Indeed, it has been shown that in infected fish, the toxin is systemically distributed and depletes macrophages and neutrophils by postapoptotic secondary necrosis (12), leading to the impairment of the phagocytic defense of the host and consequently favoring P. damselae subsp. piscicida survival and dissemination. Furthermore, the occurrence of a secondary necrotic process in which the phagocytes undergoing apoptosis lyse and release their cytotoxic contents contributes to the genesis of the infection-associated necrotic lesions (12, 13). These observations, together with the fac...

Cortical (cTEC) and medullary (mTEC) thymic epithelial cells establish key microenvironments for T-cell differentiation and arise from thymic epithelial cell progenitors (TEP). However, the nature of TEPs and the mechanism controlling their stemness in the postnatal thymus remain poorly defined. Using TEC clonogenic assays as a surrogate to survey TEP activity, we found that a fraction of cTECs generates specialized clonal-derived colonies, which contain cells with sustained colony-forming capacity (ClonoTECs). These ClonoTECs are EpCAM+MHCII-Foxn1lo cells that lack traits of mature cTECs or mTECs but co-express stem-cell markers, including CD24 and Sca-1. Supportive of their progenitor identity, ClonoTECs reintegrate within native thymic microenvironments and generate cTECs or mTECs in vivo. Strikingly, the frequency of cTECs with the potential to generate ClonoTECs wanes between the postnatal and young adult immunocompetent thymus, but it is sustained in alymphoid Rag2-/-Il2rg-/-counterparts. Conversely, transplantation of wild-type bone marrow hematopoietic progenitors into Rag2-/-Il2rg-/-mice and consequent restoration of thymocyte-mediated TEC differentiation diminishes the frequency of colony-forming units within cTECs. Our findings provide evidence that the cortical epithelium contains a reservoir of epithelial progenitors whose abundance is dynamically controlled by continual interactions with developing thymocytes across lifespan.

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Thymic epithelial cells (TECs) are the main regulators of T lymphocyte development and selection, requiring a three-dimensional (3D) environment to properly perform these biological functions. The aim of this work was to develop a 3D culture substrate that allows the survival and proliferation of TECs. Thus, electrospun fibrous meshes (eFMs) were functionalized with fibronectin, one of the major extracellular matrix (ECM) proteins of the thymus. For that, highly porous eFMs were activated using oxygen plasma treatment followed by amine insertion, which allows the immobilization of fibronectin through EDC/NHS chemistry. The medullary TECs presented increased proliferation, viability, and protein synthesis when cultured on fibronectin-functionalized eFMs (FN-eFMs). These cells showed a spread morphology, with increased migration toward the inner layers of FN-eFMs and the production of thymic ECM proteins, such as collagen type IV and laminin. These results suggest that FN-eFMs are an effective substrate for supporting thymic cell cultures.

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