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Pannexin 1 (Panx1) is a membrane channel implicated in numerous physiological and pathophysiological processes via its ability to support release of ATP and other cellular metabolites for local intercellular signaling. However, to date, there has been no direct demonstration of large molecule permeation via the Panx1 channel itself, and thus the permselectivity of Panx1 for different molecules remains unknown. To address this, we expressed, purified, and reconstituted Panx1 into proteoliposomes and demonstrated that channel activation by caspase cleavage yields a dye-permeable pore that favors flux of anionic, large-molecule permeants (up to ~1 kDa). Large cationic molecules can also permeate the channel, albeit at a much lower rate. We further show that Panx1 channels provide a molecular pathway for flux of ATP and other anionic (glutamate) and cationic signaling metabolites (spermidine). These results verify large molecule permeation directly through caspase-activated Panx1 channels that can support their many physiological roles.

Regulated cell death is an integral part of life, and has broad effects on organism development and homeostasis.sup.1. Malfunctions within the regulated cell death process, including the clearance of dying cells, can manifest in diverse pathologies throughout various tissues including the gastrointestinal tract.sup.2. A long appreciated, yet elusively defined relationship exists between cell death and gastrointestinal pathologies with an underlying microbial component.sup.3-6, but the direct effect of dying mammalian cells on bacterial growth is unclear. Here we advance a concept that several Enterobacteriaceae, including patient-derived clinical isolates, have an efficient growth strategy to exploit soluble factors that are released from dying gut epithelial cells. Mammalian nutrients released after caspase-3/7-dependent apoptosis boosts the growth of multiple Enterobacteriaceae and is observed using primary mouse colonic tissue, mouse and human cell lines, several apoptotic triggers, and in conventional as well as germ-free mice in vivo. The mammalian cell death nutrients induce a core transcriptional response in pathogenic Salmonella, and we identify the pyruvate formate-lyase-encoding pflB gene as a key driver of bacterial colonization in three contexts: a foodborne infection model, a TNF- and A20-dependent cell death model, and a chemotherapy-induced mucositis model. These findings introduce a new layer to the complex host-pathogen interaction, in which death-induced nutrient release acts as a source of fuel for intestinal bacteria, with implications for gut inflammation and cytotoxic chemotherapy treatment. Intestinal microorganisms exploit nutrients released by apoptotic gut epithelial cells for growth., Author(s): Christopher J. Anderson [sup.1] [sup.2] , Christopher B. Medina [sup.3] , Brady J. Barron [sup.3] , Laura Karvelyte [sup.1] , Tania Løve Aaes [sup.1] [sup.2] , Irina Lambertz [sup.1] [...]

Caspase-dependent apoptosis accounts for approximately 90% of homeostatic cell turnover in the body.sup.1, and regulates inflammation, cell proliferation, and tissue regeneration.sup.2-4. How apoptotic cells mediate such diverse effects is not fully understood. Here we profiled the apoptotic metabolite secretome and determined its effects on the tissue neighbourhood. We show that apoptotic lymphocytes and macrophages release specific metabolites, while retaining their membrane integrity. A subset of these metabolites is also shared across different primary cells and cell lines after the induction of apoptosis by different stimuli. Mechanistically, the apoptotic metabolite secretome is not simply due to passive emptying of cellular contents and instead is a regulated process. Caspase-mediated opening of pannexin 1 channels at the plasma membrane facilitated the release of a select subset of metabolites. In addition, certain metabolic pathways continued to remain active during apoptosis, with the release of only select metabolites from a given pathway. Functionally, the apoptotic metabolite secretome induced specific gene programs in healthy neighbouring cells, including suppression of inflammation, cell proliferation, and wound healing. Furthermore, a cocktail of apoptotic metabolites reduced disease severity in mouse models of inflammatory arthritis and lung-graft rejection. These data advance the concept that apoptotic cells are not inert cells waiting for removal, but instead release metabolites as 'good-bye' signals to actively modulate outcomes in tissues. Apoptotic cells communicate with neighbouring cells by the regulated release of specific metabolites, and a cocktail of select apoptotic metabolites reduces disease severity in mouse models of inflammatory arthritis and lung transplant rejection., Author(s): Christopher B. Medina [sup.1] [sup.2] , Parul Mehrotra [sup.3] , Sanja Arandjelovic [sup.1] [sup.2] , Justin S. A. Perry [sup.1] [sup.2] , Yizhan Guo [sup.4] , Sho Morioka [sup.1] [...]

Development and routine tissue homeostasis require a high turnover of apoptotic cells. These cells are removed by professional and non-professional phagocytes via efferocytosis.sup.1. How a phagocyte maintains its homeostasis while coordinating corpse uptake, processing ingested materials and secreting anti-inflammatory mediators is incompletely understood.sup.1,2. Here, using RNA sequencing to characterize the transcriptional program of phagocytes actively engulfing apoptotic cells, we identify a genetic signature involving 33 members of the solute carrier (SLC) family of membrane transport proteins, in which expression is specifically modulated during efferocytosis, but not during antibody-mediated phagocytosis. We assessed the functional relevance of these SLCs in efferocytic phagocytes and observed a robust induction of an aerobic glycolysis program, initiated by SLC2A1-mediated glucose uptake, with concurrent suppression of the oxidative phosphorylation program. The different steps of phagocytosis.sup.2--that is, 'smell' ('find-me' signals or sensing factors released by apoptotic cells), 'taste' (phagocyte-apoptotic cell contact) and 'ingestion' (corpse internalization)--activated distinct and overlapping sets of genes, including several SLC genes, to promote glycolysis. SLC16A1 was upregulated after corpse uptake, increasing the release of lactate, a natural by-product of aerobic glycolysis.sup.3. Whereas glycolysis within phagocytes contributed to actin polymerization and the continued uptake of corpses, lactate released via SLC16A1 promoted the establishment of an anti-inflammatory tissue environment. Collectively, these data reveal a SLC program that is activated during efferocytosis, identify a previously unknown reliance on aerobic glycolysis during apoptotic cell uptake and show that glycolytic by-products of efferocytosis can influence surrounding cells.Distinct transcriptional programs are activated during different stages of apoptotic cell engulfment, including a unique program of genes coding for solute carrier proteins and enzymes in the glycolytic pathway., Author(s): Sho Morioka [sup.1] [sup.2] , Justin S. A. Perry [sup.1] [sup.2] , Michael H. Raymond [sup.1] [sup.3] , Christopher B. Medina [sup.1] [sup.2] , Yunlu Zhu [sup.4] , Liyang [...]

PD-1+TCF-1+ stem-like CD8 T cells act as critical resource cells for maintaining T cell immunity in chronic viral infections and cancer. In addition, they provide the proliferative burst of effector CD8 T cells after programmed death protein 1 (PD-1)–directed immunotherapy. However, it is not known whether checkpoint blockade diminishes the number of these stem-like progenitor cells as effector cell differentiation increases. To investigate this, we used the mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection. Treatment of chronically infected mice with either αPD-1 or αPD-L1 antibody not only increased effector cell differentiation from the virus-specific stem-like CD8 T cells but also increased their proliferation so their numbers were maintained. The increased self-renewal of LCMV-specific stem-like CD8 T cells was mTOR dependent. We used microscopy to understand the division of these progenitor cells and found that after PD-1 blockade, an individual dividing cell could give rise to a differentiated TCF-1− daughter cell alongside a self-renewing TCF-1+ sister cell. This asymmetric division helped to preserve the number of stem-like cells. Moreover, we found that the PD-1+TCF-1+ stem-like CD8 T cells retained their transcriptional program and their in vivo functionality in terms of responding to viral infection and to repeat PD-1 blockade. Together, our results demonstrate that PD-1 blockade does not deplete the stem-like population despite increasing effector differentiation. These findings have implications for PD-1–directed immunotherapy in humans. Editor's summary: Stem-like CD8 T cells develop during periods of persistent antigen exposure. During chronic infection and cancer, therapies can stimulate their differentiation into antigen-specific cells with effector function; yet factors controlling the integrity of the stem-like T cell pool were unknown. Studying chronic viral infection in mice, Humblin et al. found that the co-stimulatory molecule CD28 regulated metabolism of stem-like CD8 T cells expressing the inhibitory receptor PD-1 to promote self-renewal and differentiation. In companion work, Gill et al. found that PD-1 blockade, while promoting effector cell differentiation, likewise stimulated the self-renewal of these stem-like CD8 T cells, maintaining the size and functionality of the progenitor pool. Thus, immune checkpoint therapies may rejuvenate immune responses without diminishing the durability of the stem-like CD8 T cells that respond to treatment. —Sarah H. Ross [ABSTRACT FROM AUTHOR]