A Novel PHOX/CD38/MCOLN1/TFEB Axis Important For Macrophage Activation During Bacterial Phagocytosis [preprint]

Macrophages are a key and heterogenous class of phagocytic cells of the innate immune system, which act as sentinels in peripheral tissues and are mobilized during infection. Macrophage activation in the presence of bacterial cells and molecules entails specific and complex programs of gene expression. How such triggers elicit the gene expression programs is incompletely understood. We previously discovered that transcription factor TFEB is a key contributor to macrophage activation during bacterial phagocytosis. However, the mechanism linking phagocytosis of bacterial cells to TFEB activation remained unknown. In this article, we describe a previously unknown pathway that links phagocytosis with the activation of TFEB and related transcription factor TFE3 in macrophages. We find that phagocytosis of bacterial cells causes an NADPH oxidase (PHOX)-dependent oxidative burst, which activates enzyme CD38 and generates NAADP in the maturing phagosome. Phago-lysosome fusion brings Ca2+ channel TRPML1/MCOLN1 in contact with NAADP, causing Ca2+ efflux from the lysosome, calcineurin activation, and TFEB nuclear import. This drives TFEB-dependent expression of important pro-inflammatory cytokines, such as IL-1α, IL-1β, and IL-6. Thus, our findings reveal that TFEB activation is a key regulatory event for the activation of macrophages. These findings have important implications for infections, cancer, obesity, and atherosclerosis

Transcription factor TFEB cell-autonomously modulates susceptibility to intestinal epithelial cell injury in vivo

Understanding the transcription factors that modulate epithelial resistance to injury is necessary for understanding intestinal homeostasis and injury repair processes. Recently, transcription factor EB (TFEB) was implicated in expression of autophagy and host defense genes in nematodes and mammalian cells. However, the in vivo roles of TFEB in the mammalian intestinal epithelium were not known. Here, we used mice with a conditional deletion of Tfeb in the intestinal epithelium (Tfeb ΔIEC) to examine its importance in defense against injury. Unperturbed Tfeb ΔIEC mice exhibited grossly normal intestinal epithelia, except for a defect in Paneth cell granules. Tfeb ΔIEC mice exhibited lower levels of lipoprotein ApoA1 expression, which is downregulated in Crohn’s disease patients and causally linked to colitis susceptibility. Upon environmental epithelial injury using dextran sodium sulfate (DSS), Tfeb ΔIEC mice exhibited exaggerated colitis. Thus, our study reveals that TFEB is critical for resistance to intestinal epithelial cell injury, potentially mediated by APOA1

An Evolutionarily Conserved PLC-PKD-TFEB Pathway for Host Defense

The mechanisms that tightly control the transcription of host defense genes have not been fully elucidated. We previously identified TFEB as a transcription factor important for host defense, but the mechanisms that regulate TFEB during infection remained unknown. Here, we used C. elegans to discover a pathway that activates TFEB during infection. Gene dkf-1, which encodes a homolog of protein kinase D (PKD), was required for TFEB activation in nematodes infected with Staphylococcus aureus. Conversely, pharmacological activation of PKD was sufficient to activate TFEB. Furthermore, phospholipase C (PLC) gene plc-1 was also required for TFEB activation, downstream of Gαq homolog egl-30 and upstream of dkf-1. Using reverse and chemical genetics, we discovered a similar PLC-PKD-TFEB axis in Salmonella-infected mouse macrophages. In addition, PKCα was required in macrophages. These observations reveal a previously unknown host defense signaling pathway, which has been conserved across one billion years of evolution

Intestinal Epithelial Wnt Signaling Mediates Acetylcholine-Triggered Host Defense against Infection

Regulated antimicrobial peptide expression in the intestinal epithelium is key to defense against infection and to microbiota homeostasis. Understanding the mechanisms that regulate such expression is necessary for understanding immune homeostasis and inflammatory disease and for developing safe and effective therapies. We used Caenorhabditis elegans in a preclinical approach to discover mechanisms of antimicrobial gene expression control in the intestinal epithelium. We found an unexpected role for the cholinergic nervous system. Infection-induced acetylcholine release from neurons stimulated muscarinic signaling in the epithelium, driving downstream induction of Wnt expression in the same tissue. Wnt induction activated the epithelial canonical Wnt pathway, resulting in the expression of C-type lectin and lysozyme genes that enhanced host defense. Furthermore, the muscarinic and Wnt pathways are linked by conserved transcription factors. These results reveal a tight connection between the nervous system and the intestinal epithelium, with important implications for host defense, immune homeostasis, and cancer

Targeting the gut to prevent sepsis from a cutaneous burn

Severe burn injury induces gut barrier dysfunction and subsequently a profound systemic inflammatory response. In the present study, we examined the role of the small intestinal brush border enzyme, intestinal alkaline phosphatase (IAP), in preserving gut barrier function and preventing systemic inflammation after burn wound infection in mice. Mice were subjected to a 30% total body surface area dorsal burn with or without intradermal injection of Pseudomonas aeruginosa. Mice were gavaged with 2000 units of IAP or vehicle at 3 and 12 hours after the insult. We found that both endogenously produced and exogenously supplemented IAP significantly reduced gut barrier damage, decreased bacterial translocation to the systemic organs, attenuated systemic inflammation, and improved survival in this burn wound infection model. IAP attenuated liver inflammation and reduced the proinflammatory characteristics of portal serum. Furthermore, we found that intestinal luminal contents of burn wound–infected mice negatively impacted the intestinal epithelial integrity compared with luminal contents of control mice and that IAP supplementation preserved monolayer integrity. These results indicate that oral IAP therapy may represent an approach to preserving gut barrier function, blocking proinflammatory triggers from entering the portal system, preventing gut-induced systemic inflammation, and improving survival after severe burn injuries