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 Ca 2+ channel TRPML1/MCOLN1 in contact with NAADP, causing Ca 2+ efflux from the lysosome, calcineurin activation, and TFEB nuclear import. This drives TFEBdependent 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..
Previously, we observed increased transcription levels of specific cytochrome P450 monooxygenase (P450) and adenosine triphosphate binding cassette (ABC) transporter genes in human body lice, Pediculus humanus humanus, following exposure to ivermectin using the non-invasive induction assay, which resulted in tolerance. To confirm the roles of these genes in induction and tolerance, the robust genetic model insect Drosophila melanogaster was chosen. Orthologous genes corresponding to the body louse P450 (Cyp9f2, Cyp6g2 and Cyp9h1) and ABC transporter (Mrp1, GC1824 as an ABCB type and CG3327 as an ABCG type) genes were selected for in vivo bioassay. Following a brief treatment with a sublethal dose of ivermectin, the mortality response was significantly slower, indicating the presence of tolerance. Concurrently, the transcription levels of Cyp9f2 and Mrp1 at 3 h and those of Cyp6g2, Cyp9h1, Mrp1, CG1824 and CG3327 at 6 h post-treatment were upregulated, indicating gene induction. In behavioural bioassay using GAL4/UAS-RNA interference transgenic fly lines, increased susceptibility to ivermectin was observed following heat shock in the Cyp9f2 , Cyp6g2 , Cyp9h1 , Mrp1 or CG3327-knockdown flies. Considering that these five genes are orthologous to those which had the largest over-expression level following ivermectin-induced tolerance in the body louse, the current results suggest that they are also associated with ivermectin detoxification in D. melanogaster and that body lice and D. melanogaster are likely to share, in part, similar mechanisms of tolerance to ivermectin.
21Macrophages are a key and heterogenous class of phagocytic cells of the innate 22immune system, which act as sentinels in peripheral tissues and are mobilized 23 during infection. Macrophage activation in the presence of bacterial cells and 24 molecules entails specific and complex programs of gene expression. How such 25 triggers elicit the gene expression programs is incompletely understood. We pre-26 viously discovered that transcription factor TFEB is a key contributor to macro-27 phage activation during bacterial phagocytosis. However, the mechanism linking 28 phagocytosis of bacterial cells to TFEB activation remained unknown. In this ar-29 ticle, we describe a previously unknown pathway that links phagocytosis with the 30 activation of TFEB and related transcription factor TFE3 in macrophages. We 31 find that phagocytosis of bacterial cells causes an NADPH oxidase (PHOX)-32 dependent oxidative burst, which activates enzyme CD38 and generates NAADP 33 in the maturing phagosome. Phago-lysosome fusion brings Ca 2+ channel 34 TRPML1/MCOLN1 in contact with NAADP, causing Ca 2+ efflux from the lyso-35 some, calcineurin activation, and TFEB nuclear import. This drives TFEB-36 dependent expression of important pro-inflammatory cytokines, such as IL-1α, IL-37 1β, and IL-6. Thus, our findings reveal that TFEB activation is a key regulatory 38 event for the activation of macrophages. These findings have important implica-39 tions for infections, cancer, obesity, and atherosclerosis. 40 tion), which is predominantly pro-inflammatory, to "alternative" activation (a.k.a. 48 M2 polarization), which is predominantly pro-resolution 2 . The activation state of 49 the macrophage has great functional consequences for health and disease. 50 51 Because transcriptional regulation contributes greatly to the macrophage phe-52 notype, understanding the transcription factors that are involved is of paramount 53 importance. Similarly, it is imperative to fully elucidate the signaling pathways 54 that regulate such transcription factors under conditions of homeostasis and dis-55 ease. Much work has characterized important signaling from the Toll-like recep-56 tors (TLRs), which recognize molecules produced by pathogens such as bacteri-57 al cell wall components and trigger the activation of transcription factors NF-κB, 58 IRF3, and AP1 3 . All three examples of transcription factors are subject to multi-59 ple regulatory layers, nuclear-cytoplasmic shuttling being a major mechanism of 60 regulation. 61 62 4We recently identified TFEB as a transcription factor that is important for cyto-63 kine and chemokine gene induction in macrophages following bacterial infection 64 4 . We found that TFEB resides in the cytosol in resting murine macrophages, but 65 is imported to the nucleus after short infection with Staphylococcus aureus or 66 Salmonella Typhimurium 4,5 . Moreover, cells defective in TFEB expression exhibit 67 defective induction of important pro-inflammatory cytokines, including TNF-α, IL-68 1β, and IL-6 4 . Subsequent stud...
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