Verloren negatively regulates the expression of IMD pathway dependent antimicrobial peptides in Drosophila

Drosophila immune deficiency (IMD) pathway is similar to the human tumor necrosis factor receptor (TNFR) signaling pathway and is preferentially activated by Gram-negative bacterial infection. Recent studies highlighted the importance of IMD pathway regulation as it is tightly controlled by numbers of negative regulators at multiple levels. Here, we report a new negative regulator of the IMD pathway, Verloren (Velo). Silencing of Velo led to constitutive expression of the IMD pathway dependent antimicrobial peptides (AMPs), and Escherichia coli stimulation further enhanced the AMP expression. Epistatic analysis indicated that Velo knock-down mediated AMP upregulation is dependent on the canonical members of the IMD pathway. The immune fluorescent study using overexpression constructs revealed that Velo resides both in the nucleus and cytoplasm, but the majority (~ 75%) is localized in the nucleus. We also observed from in vivo analysis that Velo knock-down flies exhibit significant upregulation of the AMP expression and reduced bacterial load. Survival experiments showed that Velo knock-down flies have a short lifespan and are susceptible to the infection of pathogenic Gram-negative bacteria, P. aeruginosa. Taken together, these data suggest that Velo is an additional new negative regulator of the IMD pathway, possibly acting in both the nucleus and cytoplasm

Differential modulation of the cellular and humoral immune responses in Drosophila is mediated by the endosomal ARF1-Asrij axis

How multicellular organisms maintain immune homeostasis across various organs and cell types is an outstanding question in immune biology and cell signaling. In Drosophila, blood cells (hemocytes) respond to local and systemic cues to mount an immune response. While endosomal regulation of Drosophila hematopoiesis is reported, the role of endosomal proteins in cellular and humoral immunity is not well-studied. Here we demonstrate a functional role for endosomal proteins in immune homeostasis. We show that the ubiquitous trafficking protein ADP Ribosylation Factor 1 (ARF1) and the hemocyte-specific endosomal regulator Asrij differentially regulate humoral immunity. Asrij and ARF1 play an important role in regulating the cellular immune response by controlling the crystal cell melanization and phenoloxidase activity. ARF1 and Asrij mutants show reduced survival and lifespan upon infection, indicating perturbed immune homeostasis. The ARF1-Asrij axis suppresses the Toll pathway anti-microbial peptides (AMPs) by regulating ubiquitination of the inhibitor Cactus. The Imd pathway is inversely regulated- while ARF1 suppresses AMPs, Asrij is essential for AMP production. Several immune mutants have reduced Asrij expression, suggesting that Asrij co-ordinates with these pathways to regulate the immune response. Our study highlights the role of endosomal proteins in modulating the immune response by maintaining the balance of AMP production. Similar mechanisms can now be tested in mammalian hematopoiesis and immunity

Conserved Regulation of the JAK/STAT Pathway by the Endosomal Protein Asrij Maintains Stem Cell Potency

Asrij/OCIAD1 is an endosomal protein expressed in stem cells and cardiovascular lineages and aberrantly expressed in several cancers. We show that dose-dependent modulation of cytokine-dependent JAK/STAT signaling by Asrij regulates mouse embryonic stem cell pluripotency as well as Drosophila hematopoietic stem cell maintenance. Furthermore, mouse asrij can substitute for Drosophila asrij, indicating that they are true homologs. We identify a conserved region of Asrij that is necessary and sufficient for vesicular localization and function. We also show that Asrij and STAT3 colocalize in endosomes and interact biochemically. We propose that Asrij provides an endosomal scaffold for STAT3 interaction and activation, and may similarly control other circuits that maintain stemness. Thus, Asrij provides a key point of control for spatial and kinetic regulation of stem cell signals

A time course transcriptomic analysis of host and injected oncogenic cells reveals new aspects of Drosophila immune defenses

International audienceOncogenic RasV12 cells [A. Simcox et al., PLoS Genet. 4, e1000142 (2008)] injected into adult males proliferated massively after a lag period of several days, and led to the demise of the flies after 2 to 3 wk. The injection induced an early massive transcriptomic response that, unexpectedly, included more than 100 genes encoding chemoreceptors of various families. The kinetics of induction and the identities of the induced genes differed markedly from the responses generated by injections of microbes. Subsequently, hundreds of genes were up-regulated, attesting to intense catabolic activities in the flies, active tracheogenesis, and cuticulogenesis, as well as stress and inflammation-type responses. At 11 d after the injections, GFP-positive oncogenic cells isolated from the host flies exhibited a markedly different transcriptomic profile from that of the host and distinct from that at the time of their injection, including in particular up-regulated expression of genes typical for cells engaged in the classical antimicrobial response of Drosophila

ARF1–GTP regulates Asrij to provide endocytic control of Drosophila

Drosophila melanogaster larval hematopoiesis is a well-established model to study mechanisms that regulate hematopoietic niche maintenance and control of blood cell precursor (prohemocyte) differentiation. Molecules that perturb niche function affect the balance between prohemocytes and differentiated hemocytes. The conserved hemocyte-specific endosomal protein Asrij is essential for niche function and prohemocyte maintenance. Elucidating how subcellular trafficking molecules can regulate signaling presents an important challenge. Here we show that Asrij function is mediated by the Ras family GTPase Arf79F, the Drosophila homolog of ADP ribosylation factor 1 (ARF1), essential for clathrin coat assembly, Golgi architecture, and vesicular trafficking. ARF1 is expressed in the larval lymph gland and in circulating hemocytes and interacts with Asrij. ARF1-depleted lymph glands show loss of niche cells and prohemocyte maintenance with increased differentiation. Inhibiting ARF1 activation by knocking down its guanine nucleotide exchange factor (Gartenzwerg) or overexpressing its GTPAse-activating protein showed that ARF1–GTP is essential for regulating niche size and maintaining stemness. Activated ARF1 regulates Asrij levels in blood cells thereby mediating Asrij function. Asrij controls crystal cell differentiation by affecting Notch trafficking. ARF1 perturbation also leads to aberrant Notch trafficking and the Notch intracellular domain is stalled in sorting endosomes. Thus, ARF1 can regulate Drosophila blood cell homeostasis by regulating Asrij endocytic function. ARF1 also regulates signals arising from the niche and differentiated cells by integrating the insulin-mediated and PDGF-VEGF receptor signaling pathways. We propose that the conserved ARF1–Asrij endocytic axis modulates signals that govern hematopoietic development. Thus, Asrij affords tissue-specific control of global mechanisms involved in molecular traffic

ARF1–GTP regulates Asrij to provide endocytic control of Drosophila blood cell homeostasis

Ras small GTPases including ARFs act as molecular switches to modulate signaling pathways involved in hematopoiesis. Decreased guanine nucleotide exchange factor activity or increased GTPase-activating protein activation are associated with many leukemias, myelodysplastic syndromes, and myeloproliferative disorders. Drosophila is a good model to address questions related to aberrant hematopoiesis. Using Drosophila genetics and gene expression analysis we show tissue-specific function of the ubiquitously expressed endocytic protein, Drosophila ARF1 by interaction of ARF1–GTP with a blood-cell–expressed endocytic protein Asrij. The ARF1–Asrij axis brings about endosomal regulation of multiple signaling pathways in hematopoiesis