Metabolite-based inter-kingdom communication controls intestinal tissue recovery following chemotherapeutic injury.

Cytotoxic chemotherapies have devastating side effects, particularly within the gastrointestinal tract. Gastrointestinal toxicity includes the death and damage of the epithelium and an imbalance in the intestinal microbiota, otherwise known as dysbiosis. Whether dysbiosis is a direct contributor to tissue toxicity is a key area of focus. Here, from both mammalian and bacterial perspectives, we uncover an intestinal epithelial cell death-Enterobacteriaceae signaling axis that fuels dysbiosis. Specifically, our data demonstrate that chemotherapy-induced epithelial cell apoptosis and the purine-containing metabolites released from dying cells drive the inter-kingdom transcriptional re-wiring of the Enterobacteriaceae, including fundamental shifts in bacterial respiration and promotion of purine utilization-dependent expansion, which in turn delays the recovery of the intestinal tract. Inhibition of epithelial cell death or restriction of the Enterobacteriaceae to homeostatic levels reverses dysbiosis and improves intestinal recovery. These findings suggest that supportive therapies that maintain homeostatic levels of Enterobacteriaceae may be useful in resolving intestinal disease.

TL1A and IL-18 synergy promotes GM-CSF-dependent thymic granulopoiesis in mice.

Acute systemic inflammation critically alters the function of the immune system, often promoting myelopoiesis at the expense of lymphopoiesis. In the thymus, systemic inflammation results in acute thymic atrophy and, consequently, impaired T-lymphopoiesis. The mechanism by which systemic inflammation impacts the thymus beyond suppressing T-cell development is still unclear. Here, we describe how the synergism between TL1A and IL-18 suppresses T-lymphopoiesis to promote thymic myelopoiesis. The protein levels of these two cytokines were elevated in the thymus during viral-induced thymus atrophy infection with murine cytomegalovirus (MCMV) or pneumonia virus of mice (PVM). In vivo administration of TL1A and IL-18 induced acute thymic atrophy, while thymic neutrophils expanded. Fate mapping with Ms4a3-Cre mice demonstrated that thymic neutrophils emerge from thymic granulocyte-monocyte progenitors (GMPs), while Rag1-Cre fate mapping revealed a common developmental path with lymphocytes. These effects could be modeled ex vivo using neonatal thymic organ cultures (NTOCs), where TL1A and IL-18 synergistically enhanced neutrophil production and egress. NOTCH blockade by the LY411575 inhibitor increased the number of neutrophils in the culture, indicating that NOTCH restricted steady-state thymic granulopoiesis. To promote myelopoiesis, TL1A, and IL-18 synergistically increased GM-CSF levels in the NTOC, which was mainly produced by thymic ILC1s. In support, TL1A- and IL-18-induced granulopoiesis was completely prevented in NTOCs derived from Csf2rb-/- mice and by GM-CSFR antibody blockade, revealing that GM-CSF is the essential factor driving thymic granulopoiesis. Taken together, our findings reveal that TL1A and IL-18 synergism induce acute thymus atrophy while  promoting extramedullary thymic granulopoiesis in a NOTCH and GM-CSF-controlled manner.

Oxylipins and metabolites from pyroptotic cells act as promoters of tissue repair.

Pyroptosis is a lytic cell death mode that helps limit the spread of infections and is also linked to pathology in sterile inflammatory diseases and autoimmune diseases1-4. During pyroptosis, inflammasome activation and the engagement of caspase-1 lead to cell death, along with the maturation and secretion of the inflammatory cytokine interleukin-1ß (IL-1ß). The dominant effect of IL-1ß in promoting tissue inflammation has clouded the potential influence of other factors released from pyroptotic cells. Here, using a system in which macrophages are induced to undergo pyroptosis without IL-1ß or IL-1α release (denoted Pyro-1), we identify unexpected beneficial effects of the Pyro-1 secretome. First, we noted that the Pyro-1 supernatants upregulated gene signatures linked to migration, cellular proliferation and wound healing. Consistent with this gene signature, Pyro-1 supernatants boosted migration of primary fibroblasts and macrophages, and promoted faster wound closure in vitro and improved tissue repair in vivo. In mechanistic studies, lipidomics and metabolomics of the Pyro-1 supernatants identified the presence of both oxylipins and metabolites, linking them to pro-wound-healing effects. Focusing specifically on the oxylipin prostaglandin E2 (PGE2), we find that its synthesis is induced de novo during pyroptosis, downstream of caspase-1 activation and cyclooxygenase-2 activity; further, PGE2 synthesis occurs late in pyroptosis, with its release dependent on gasdermin D pores opened during pyroptosis. As for the pyroptotic metabolites, they link to immune cell infiltration into the wounds, and polarization to CD301+ macrophages. Collectively, these data advance the concept that the pyroptotic secretome possesses oxylipins and metabolites with tissue repair properties that may be harnessed therapeutically.

CXCL16-dependent scavenging of oxidized lipids by islet macrophages promotes differentiation of pathogenic CD8+ T cells in diabetic autoimmunity.

The pancreatic islet microenvironment is highly oxidative, rendering ß cells vulnerable to autoinflammatory insults. Here, we examined the role of islet resident macrophages in the autoimmune attack that initiates type 1 diabetes. Islet macrophages highly expressed CXCL16, a chemokine and scavenger receptor for oxidized low-density lipoproteins (OxLDLs), regardless of autoimmune predisposition. Deletion of Cxcl16 in nonobese diabetic (NOD) mice suppressed the development of autoimmune diabetes. Mechanistically, Cxcl16 deficiency impaired clearance of OxLDL by islet macrophages, leading to OxLDL accumulation in pancreatic islets and a substantial reduction in intra-islet transitory (Texint) CD8+ T cells displaying proliferative and effector signatures. Texint cells were vulnerable to oxidative stress and diminished by ferroptosis; PD-1 blockade rescued this population and reversed diabetes resistance in NOD.Cxcl16-/- mice. Thus, OxLDL scavenging in pancreatic islets inadvertently promotes differentiation of pathogenic CD8+ T cells, presenting a paradigm wherein tissue homeostasis processes can facilitate autoimmune pathogenesis in predisposed individuals.

Rapid unleashing of macrophage efferocytic capacity via transcriptional pause release.

During development, inflammation or tissue injury, macrophages may successively engulf and process multiple apoptotic corpses via efferocytosis to achieve tissue homeostasis1. How macrophages may rapidly adapt their transcription to achieve continuous corpse uptake is incompletely understood. Transcriptional pause/release is an evolutionarily conserved mechanism, in which RNA polymerase (Pol) II initiates transcription for 20-60 nucleotides, is paused for minutes to hours and is then released to make full-length mRNA2. Here we show that macrophages, within minutes of corpse encounter, use transcriptional pause/release to unleash a rapid transcriptional response. For human and mouse macrophages, the Pol II pause/release was required for continuous efferocytosis in vitro and in vivo. Interestingly, blocking Pol II pause/release did not impede Fc receptor-mediated phagocytosis, yeast uptake or bacterial phagocytosis. Integration of data from three genomic approaches-precision nuclear run-on sequencing, RNA sequencing, and assay for transposase-accessible chromatin using sequencing (ATAC-seq)-on efferocytic macrophages at different time points revealed that Pol II pause/release controls expression of select transcription factors and downstream target genes. Mechanistic studies on transcription factor EGR3, prominently regulated by pause/release, uncovered EGR3-related reprogramming of other macrophage genes involved in cytoskeleton and corpse processing. Using lysosomal probes and a new genetic fluorescent reporter, we identify a role for pause/release in phagosome acidification during efferocytosis. Furthermore, microglia from egr3-deficient zebrafish embryos displayed reduced phagocytosis of apoptotic neurons and fewer maturing phagosomes, supporting defective corpse processing. Collectively, these data indicate that macrophages use Pol II pause/release as a mechanism to rapidly alter their transcriptional programs for efficient processing of the ingested apoptotic corpses and for successive efferocytosis.

Autophagy critically controls skin inflammation and apoptosis-induced stem cell activation.

Macroautophagy/autophagy is a cellular recycling program regulating cell survival and controlling inflammatory responses in a context-dependent manner. Here, we demonstrate that keratinocyte-selective ablation of Atg16l1, an essential autophagy mediator, results in exacerbated inflammatory and neoplastic skin responses. In addition, mice lacking keratinocyte autophagy exhibit precocious onset of hair follicle growth, indicating altered activation kinetics of hair follicle stem cells (HFSCs). These HFSCs also exhibit expanded potencies in an autophagy-deficient context as shown by de novo hair follicle formation and improved healing of abrasion wounds. ATG16L1-deficient keratinocytes are markedly sensitized to apoptosis. Compound deletion of RIPK3-dependent necroptotic and CASP8-dependent apoptotic responses or of TNFRSF1A/TNFR1 reveals that the enhanced sensitivity of autophagy-deficient keratinocytes to TNF-dependent cell death is driving altered activation of HFSCs. Together, our data demonstrate that keratinocyte autophagy dampens skin inflammation and tumorigenesis but curtails HFSC activation by restraining apoptotic responses.Abbreviations: ATG16L1: autophagy related 16 like 1; DMBA: 2,4-dimethoxybenzaldehyde; DP: dermal papilla; EpdSCs: epidermal stem cells; Gas6: growth arrest specific 6; HF: hair follicle; HFSC: hair follicle stem cell; IFE: interfollicular epidermis; KRT5: keratin 5; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; PMK: primary mouse keratinocyte; RIPK3: receptor-interacting serine-threonine kinase 3; scRNAseq: single-cell RNA-sequencing; SG: sebaceous gland; TEWL: transepidermal water loss; TPA: 12-O-tetradecanoylphorbol-13-acetate; TNF: tumor necrosis factor; TNFRSF1A/TNFR1: tumor necrosis factor receptor superfamily, member 1a; UMAP: uniform manifold approximation and projection.

Amount of Pannexin 1 in Smooth Muscle Cells Regulates Sympathetic Nerve-Induced Vasoconstriction.

BACKGROUND: Panx1 (pannexin 1) forms high conductance channels that secrete ATP upon stimulation. The role of Panx1 in mediating constriction in response to direct sympathetic nerve stimulation is not known. Additionally, it is unknown how the expression level of Panx1 in smooth muscle cells (SMCs) influences α-adrenergic responses. We hypothesized that the amount of Panx1 in SMCs dictates the levels of sympathetic constriction and blood pressure. METHODS: To test this hypothesis, we used genetically modified mouse models enabling expression of Panx1 in vascular cells to be varied. Electrical field stimulation on isolated arteries and blood pressure were assessed. RESULTS: Genetic deletion of SMC Panx1 prevented constriction by electric field stimulation of sympathetic nerves. Conversely, overexpression of Panx1 in SMCs using a ROSA26 transgenic model increased sympathetic nerve-mediated constriction. Connexin 43 hemichannel inhibitors did not alter constriction. Next, we evaluated the effects of altered SMC Panx1 expression on blood pressure. To do this, we created mice combining a global Panx1 deletion, with ROSA26-Panx1 under the control of an inducible SMC specific Cre (Myh11). This resulted in mice that could express only human Panx1, only in SMCs. After tamoxifen, these mice had increased blood pressure that was acutely decreased by the Panx1 inhibitor spironolactone. Control mice genetically devoid of Panx1 did not respond to spironolactone. CONCLUSIONS: These data suggest Panx1 in SMCs could regulate the extent of sympathetic nerve constriction and blood pressure. The results also show the feasibility humanized Panx1-mouse models to test pharmacological candidates.

Chimeric efferocytic receptors improve apoptotic cell clearance and alleviate inflammation.

Our bodies turn over billions of cells daily via apoptosis and are in turn cleared by phagocytes via the process of "efferocytosis." Defects in efferocytosis are now linked to various inflammatory diseases. Here, we designed a strategy to boost efferocytosis, denoted "chimeric receptor for efferocytosis" (CHEF). We fused a specific signaling domain within the cytoplasmic adapter protein ELMO1 to the extracellular phosphatidylserine recognition domains of the efferocytic receptors BAI1 or TIM4, generating BELMO and TELMO, respectively. CHEF-expressing phagocytes display a striking increase in efferocytosis. In mouse models of inflammation, BELMO expression attenuates colitis, hepatotoxicity, and nephrotoxicity. In mechanistic studies, BELMO increases ER-resident enzymes and chaperones to overcome protein-folding-associated toxicity, which was further validated in a model of ER-stress-induced renal ischemia-reperfusion injury. Finally, TELMO introduction after onset of kidney injury significantly reduced fibrosis. Collectively, these data advance a concept of chimeric efferocytic receptors to boost efferocytosis and dampen inflammation.

Drugging the efferocytosis process: concepts and opportunities.

The daily removal of billions of apoptotic cells in the human body via the process of efferocytosis is essential for homeostasis. To allow for this continuous efferocytosis, rapid phenotypic changes occur in the phagocytes enabling them to engulf and digest the apoptotic cargo. In addition, efferocytosis is actively anti-inflammatory and promotes resolution. Owing to its ubiquitous nature and the sheer volume of cell turnover, efferocytosis is a point of vulnerability. Aberrations in efferocytosis are associated with numerous inflammatory pathologies, including atherosclerosis, cancer and infections. The recent exciting discoveries defining the molecular machinery involved in efferocytosis have opened many avenues for therapeutic intervention, with several agents now in clinical trials.

Cancer cells dying from ferroptosis impede dendritic cell-mediated anti-tumor immunity.

Immunogenic cell death significantly contributes to the success of anti-cancer therapies, but immunogenicity of different cell death modalities widely varies. Ferroptosis, a form of cell death that is characterized by iron accumulation and lipid peroxidation, has not yet been fully evaluated from this perspective. Here we present an inducible model of ferroptosis, distinguishing three phases in the process-'initial' associated with lipid peroxidation, 'intermediate' correlated with ATP release and 'terminal' recognized by HMGB1 release and loss of plasma membrane integrity-that serves as tool to study immune cell responses to ferroptotic cancer cells. Co-culturing ferroptotic cancer cells with dendritic cells (DC), reveals that 'initial' ferroptotic cells decrease maturation of DC, are poorly engulfed, and dampen antigen cross-presentation. DC loaded with ferroptotic, in contrast to necroptotic, cancer cells fail to protect against tumor growth. Adding ferroptotic cancer cells to immunogenic apoptotic cells dramatically reduces their prophylactic vaccination potential. Our study thus shows that ferroptosis negatively impacts antigen presenting cells and hence the adaptive immune response, which might hinder therapeutic applications of ferroptosis induction.

Pannexin 1 drives efficient epithelial repair after tissue injury.

Epithelial tissues such as lung and skin are exposed to the environment and therefore particularly vulnerable to damage during injury or infection. Rapid repair is therefore essential to restore function and organ homeostasis. Dysregulated epithelial tissue repair occurs in several human disease states, yet how individual cell types communicate and interact to coordinate tissue regeneration is incompletely understood. Here, we show that pannexin 1 (Panx1), a cell membrane channel activated by caspases in dying cells, drives efficient epithelial regeneration after tissue injury by regulating injury-induced epithelial proliferation. Lung airway epithelial injury promotes the Panx1-dependent release of factors including ATP, from dying epithelial cells, which regulates macrophage phenotype after injury. This process, in turn, induces a reparative response in tissue macrophages that includes the induction of the soluble mitogen amphiregulin, which promotes injury-induced epithelial proliferation. Analysis of regenerating lung epithelium identified Panx1-dependent induction of Nras and Bcas2, both of which positively promoted epithelial proliferation and tissue regeneration in vivo. We also established that this role of Panx1 in boosting epithelial repair after injury is conserved between mouse lung and zebrafish tailfin. These data identify a Panx1-mediated communication circuit between epithelial cells and macrophages as a key step in promoting epithelial regeneration after injury.

Endothelial pannexin-1 channels modulate macrophage and smooth muscle cell activation in abdominal aortic aneurysm formation.

Pannexin-1 (Panx1) channels have been shown to regulate leukocyte trafficking and tissue inflammation but the mechanism of Panx1 in chronic vascular diseases like abdominal aortic aneurysms (AAA) is unknown. Here we demonstrate that Panx1 on endothelial cells, but not smooth muscle cells, orchestrate a cascade of signaling events to mediate vascular inflammation and remodeling. Mechanistically, Panx1 on endothelial cells acts as a conduit for ATP release that stimulates macrophage activation via P2X7 receptors and mitochondrial DNA release to increase IL-1ß and HMGB1 secretion. Secondly, Panx1 signaling regulates smooth muscle cell-dependent intracellular Ca2+ release and vascular remodeling via P2Y2 receptors. Panx1 blockade using probenecid markedly inhibits leukocyte transmigration, aortic inflammation and remodeling to mitigate AAA formation. Panx1 expression is upregulated in human AAAs and retrospective clinical data demonstrated reduced mortality in aortic aneurysm patients treated with Panx1 inhibitors. Collectively, these data identify Panx1 signaling as a contributory mechanism of AAA formation.

Live cell tracking of macrophage efferocytosis during Drosophila embryo development in vivo.

Apoptosis of cells and their subsequent removal through efferocytosis occurs in nearly all tissues during development, homeostasis, and disease. However, it has been difficult to track cell death and subsequent corpse removal in vivo. We developed a genetically encoded fluorescent reporter, CharON (Caspase and pH Activated Reporter, Fluorescence ON), that could track emerging apoptotic cells and their efferocytic clearance by phagocytes. Using Drosophila expressing CharON, we uncovered multiple qualitative and quantitative features of coordinated clearance of apoptotic corpses during embryonic development. When confronted with high rates of emerging apoptotic corpses, the macrophages displayed heterogeneity in engulfment behaviors, leading to some efferocytic macrophages carrying high corpse burden. Overburdened macrophages were compromised in clearing wound debris. These findings reveal known and unexpected features of apoptosis and macrophage efferocytosis in vivo.

OTULIN maintains skin homeostasis by controlling keratinocyte death and stem cell identity.

OTULIN is a deubiquitinase that specifically cleaves linear ubiquitin chains. Here we demonstrate that the ablation of Otulin selectively in keratinocytes causes inflammatory skin lesions that develop into verrucous carcinomas. Genetic deletion of Tnfr1, knockin expression of kinase-inactive Ripk1 or keratinocyte-specific deletion of Fadd and Mlkl completely rescues mice with OTULIN deficiency from dermatitis and tumorigenesis, thereby identifying keratinocyte cell death as the driving force for inflammation. Single-cell RNA-sequencing comparing non-lesional and lesional skin reveals changes in epidermal stem cell identity in OTULIN-deficient keratinocytes prior to substantial immune cell infiltration. Keratinocytes lacking OTULIN display a type-1 interferon and IL-1ß response signature, and genetic or pharmacologic inhibition of these cytokines partially inhibits skin inflammation. Finally, expression of a hypomorphic mutant Otulin allele, previously shown to cause OTULIN-related autoinflammatory syndrome in humans, induces a similar inflammatory phenotype, thus supporting the importance of OTULIN for restraining skin inflammation and maintaining immune homeostasis.

A20 deficiency in myeloid cells protects mice from diet-induced obesity and insulin resistance due to increased fatty acid metabolism.

Obesity-induced inflammation is a major driving force in the development of insulin resistance, type 2 diabetes (T2D), and related metabolic disorders. During obesity, macrophages accumulate in the visceral adipose tissue, creating a low-grade inflammatory environment. Nuclear factor κB (NF-κB) signaling is a central coordinator of inflammatory responses and is tightly regulated by the anti-inflammatory protein A20. Here, we find that myeloid-specific A20-deficient mice are protected from diet-induced obesity and insulin resistance despite an inflammatory environment in their metabolic tissues. Macrophages lacking A20 show impaired mitochondrial respiratory function and metabolize more palmitate both in vitro and in vivo. We hypothesize that A20-deficient macrophages rely more on palmitate oxidation and metabolize the fat present in the diet, resulting in a lean phenotype and protection from metabolic disease. These findings reveal a role for A20 in regulating macrophage immunometabolism.

Microbes exploit death-induced nutrient release by gut epithelial cells.

Regulated cell death is an integral part of life, and has broad effects on organism development and homeostasis1. Malfunctions within the regulated cell death process, including the clearance of dying cells, can manifest in diverse pathologies throughout various tissues including the gastrointestinal tract2. A long appreciated, yet elusively defined relationship exists between cell death and gastrointestinal pathologies with an underlying microbial component3-6, 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.

ELMO1 signaling is a promoter of osteoclast function and bone loss.

Osteoporosis affects millions worldwide and is often caused by osteoclast induced bone loss. Here, we identify the cytoplasmic protein ELMO1 as an important 'signaling node' in osteoclasts. We note that ELMO1 SNPs associate with bone abnormalities in humans, and that ELMO1 deletion in mice reduces bone loss in four in vivo models: osteoprotegerin deficiency, ovariectomy, and two types of inflammatory arthritis. Our transcriptomic analyses coupled with CRISPR/Cas9 genetic deletion identify Elmo1 associated regulators of osteoclast function, including cathepsin G and myeloperoxidase. Further, we define the 'ELMO1 interactome' in osteoclasts via proteomics and reveal proteins required for bone degradation. ELMO1 also contributes to osteoclast sealing zone on bone-like surfaces and distribution of osteoclast-specific proteases. Finally, a 3D structure-based ELMO1 inhibitory peptide reduces bone resorption in wild type osteoclasts. Collectively, we identify ELMO1 as a signaling hub that regulates osteoclast function and bone loss, with relevance to osteoporosis and arthritis.

Pannexin 1 channels facilitate communication between T cells to restrict the severity of airway inflammation.

Allergic airway inflammation is driven by type-2 CD4+ T cell inflammatory responses. We uncover an immunoregulatory role for the nucleotide release channel, Panx1, in T cell crosstalk during airway disease. Inverse correlations between Panx1 and asthmatics and our mouse models revealed the necessity, specificity, and sufficiency of Panx1 in T cells to restrict inflammation. Global Panx1-/- mice experienced exacerbated airway inflammation, and T-cell-specific deletion phenocopied Panx1-/- mice. A transgenic designed to re-express Panx1 in T cells reversed disease severity in global Panx1-/- mice. Panx1 activation occurred in pro-inflammatory T effector (Teff) and inhibitory T regulatory (Treg) cells and mediated the extracellular-nucleotide-based Treg-Teff crosstalk required for suppression of Teff cell proliferation. Mechanistic studies identified a Salt-inducible kinase-dependent phosphorylation of Panx1 serine 205 important for channel activation. A genetically targeted mouse expressing non-phosphorylatable Panx1S205A phenocopied the exacerbated inflammation in Panx1-/- mice. These data identify Panx1-dependent Treg:Teff cell communication in restricting airway disease.

Efferocytosis by Paneth cells within the intestine.

Apoptotic cells are quickly and efficiently engulfed and removed via the process of efferocytosis by either professional phagocytes, such as macrophages, or non-professional phagocytes, including epithelial cells.1,2 In addition to debris removal, a key benefit of efferocytosis is that phagocytes engulfing apoptotic cells release anti-inflammatory mediators3,4 that help reduce local tissue inflammation;5 conversely, accumulation of uncleared apoptotic cells predisposes to a pro-inflammatory tissue milieu.6-8 Due to their high proliferative capacity, intestinal epithelial cells (iECs) are sensitive to inflammation, irradiation, and chemotherapy-induced DNA damage, leading to apoptosis. Mechanisms of iEC death in the context of irradiation has been studied,9,10 but phagocytosis of dying iECs is poorly understood. Here, we identify an unexpected efferocytic role for Paneth cells, which reside in intestinal crypts and are linked to innate immunity and maintenance of the stem cell niche in the crypt.11,12 Through a series of studies spanning in vitro efferocytosis, ex vivo intestinal organoids ("enteroids"), and in vivo Cre-mediated deletion of Paneth cells, we show that Paneth cells mediate apoptotic cell uptake of dying neighbors. The relevance of Paneth-cell-mediated efferocytosis was revealed ex vivo and in mice after low-dose cesium-137 (137Cs) irradiation, mimicking radiation therapies given to cancer patients often causing significant apoptosis of iECs. These data advance a new concept that Paneth cells can act as phagocytes and identify another way in which Paneth cells contribute to the overall health of the intestine. These observations also have implications for individuals undergoing chemotherapy or chronic inflammatory bowel disease.