Toll-mediated airway homeostasis is essential for fly survival upon injection of RasV12-GFP oncogenic cells.

Toll signaling is well known for its pivotal role in the host response against the invasion of external pathogens. Here, we investigate the potential involvement of Toll signaling in the intersection between the host and oncogenic cells. We show that loss of myeloid differentiation factor 88 (Myd88) leads to drastic fly death after the injection of RasV12-GFP oncogenic cells. Transcriptomic analyses show that challenging flies with oncogenic cells or bacteria leads to distinct inductions of Myd88-dependent genes. We note that downregulation of Myd88 in the tracheal system accounts for fly mortality, and ectopic tracheal complementation of Myd88 rescues the survival defect in Myd88 loss-of-function mutants following RasV12-GFP injection. Further, molecular and genetic evidence indicate that Toll signaling modulates fly resistance to RasV12-GFP cells through mediating airway function in a rolled-dependent manner. Collectively, our data indicate a critical role of Toll signaling in tracheal homeostasis and host survival after the injection of oncogenic cells.

Drosophila eIF3f1 mediates host immune defense by targeting dTak1.

Eukaryotic translation initiation factors have long been recognized for their critical roles in governing the translation of coding RNAs into peptides/proteins. However, whether they harbor functional activities at the post-translational level remains poorly understood. Here, we demonstrate that eIF3f1 (eukaryotic translation initiation factor 3 subunit f1), which encodes an archetypal deubiquitinase, is essential for the antimicrobial innate immune defense of Drosophila melanogaster. Our in vitro and in vivo evidence indicate that the immunological function of eIF3f1 is dependent on the N-terminal JAMM (JAB1/MPN/Mov34 metalloenzymes) domain. Mechanistically, eIF3f1 physically associates with dTak1 (Drosophila TGF-beta activating kinase 1), a key regulator of the IMD (immune deficiency) signaling pathway, and mediates the turnover of dTak1 by specifically restricting its K48-linked ubiquitination. Collectively, these results provide compelling insight into a noncanonical molecular function of a translation initiation factor that controls the post-translational modification of a target protein.

Ubiquitin C-Terminal Hydrolase L5 Plays an Essential Role in the Fly Innate Immune Defense against Bacterial Infection.

BACKGROUND: Drosophila ubiquitin carboxy-terminal hydrolase L5 (Uch-L5) functions as a critical component of the 26S proteasome to mediate degradation of polyubiquitinated proteins. It was recently shown to modulate tissue/organ development by targeting the Smoothened protein in the hedgehog pathway. However, whether it plays a role in controlling organismal immune response remains largely unknown. METHODS: Reverse transcription plus quantitative polymerase chain reaction (RT-qPCR), dual-luciferase, and Western blot assays were used to explore the potential function of Uch-L5 in the innate immune regulation in cultured Drosophila S2 cells. Further genetic manipulations and bacterial infections were conducted to confirm the findings in vivo. RESULTS: Silencing of Uch-L5 antagonizes the immune deficiency (IMD) but not the Toll innate immune signaling both in vitro and in vivo. Moreover, Uch-L5 positively contributes to the Drosophila innate immune response via its N-terminal Uch domain, which is the catalytical triad executing its deubiquitinase activity. CONCLUSIONS: Our studies shed light on a novel function of the deubiquitinase Uch-L5 in governing the anti-microbial defense in Drosophila.

Dietary Supplementation of Aspirin Promotes Drosophila Defense against Viral Infection.

Aspirin, also known as acetylsalicylic acid, is widely consumed as a pain reliever and an anti-inflammatory as well as anti-platelet agent. Recently, our studies using the animal model of Drosophila demonstrated that the dietary supplementation of aspirin renovates age-onset intestinal dysfunction and delays organismal aging. Nevertheless, it remains probable that aspirin plays functional roles in other biological activities, for instance antiviral defense reactions. Intriguingly, we observed that the replications of several types of viruses were drastically antagonized in Drosophila macrophage-like S2 cells with the addition of aspirin. Further in vivo experimental approaches illustrate that adult flies consuming aspirin harbor higher resistances to viral infections with respect to flies without aspirin treatment. Mechanistically, aspirin positively contributes to the Drosophila antiviral defense largely through mediating the STING (stimulator of interferon genes) but not the IMD (immune deficiency) signaling pathway. Collectively, our studies uncover a novel biological function of aspirin in modulating Drosophila antiviral immunity and provide theoretical bases for exploring new antiviral treatments in clinical trials.

Single Cell Analysis of the Fate of Injected Oncogenic RasV12 Cells in Adult Wild Type Drosophila.

We have injected dish-cultured oncogenic RasV12 cells into adult male flies and analyzed by single cell transcriptomics their destiny within the host after 11 days. We identified in the preinjection samples and in the 11-day postinjection samples in all 16 clusters of cells, of which 5 disappeared during the experiment in the host. The other cell clusters expanded and expressed genes involved in the regulation of cell cycle, metabolism, and development. In addition, three clusters expressed genes related to inflammation and defense. Predominant among these were genes coding for phagocytosis and/or characteristic for plasmatocytes (the fly equivalent of macrophages). A pilot experiment indicated that the injection into flies of oncogenic cells, in which two of most strongly expressed genes had been previously silenced by RNA interference, into flies resulted in a dramatic reduction of their proliferation in the host flies as compared to controls. As we have shown earlier, the proliferation of the injected oncogenic cells in the adult flies is a hallmark of the disease and induces a wave of transcriptions in the experimental flies. We hypothesize that this results from a bitter dialogue between the injected cells and the host, while the experiments presented here should contribute to deciphering this dialogue.

E3 ligase Cul2 mediates Drosophila early germ cell differentiation through targeting Bam.

Drosophila ovary has been one of the most mature and excellent systems for studying the in vivo regulatory mechanisms of stem cell fate determination. It has been well-known that the bone morphogenetic protein (BMP) signaling released by the niche cells promotes the maintenance of germline stem cells (GSCs) through inhibiting the transcription of the bag-of-marbles (bam) gene, which encodes a key factor for GSC differentiation. However, whether Bam is regulated at the post-translational level remains largely unknown. Here we show that the E3 ligase Cullin-2 (Cul2) is involved in modulating Bam ubiquitination, which occurs probably at multiple lysine residues of Bam's C-terminal region. Genetic evidence further supports the notion that Cul2-mediated Bam ubiquitination and turnover are essential for GSC maintenance and proper germline development. Collectively, our data not only uncovers a novel regulatory mechanism by which Bam is controlled at the post-translational level, but also provides new insights into how Cullin family protein determines the differentiation fate of early germ cells.

A Toll-dependent Bre1/Rad6-cact feedback loop in controlling host innate immune response.

The Toll signaling pathway was initially identified for its involvement in the control of early embryogenesis. It was later shown to be also part of a major innate immune pathway controlling the expression of anti-microbial peptides in many eukaryotes including humans; cactus, the essential negative regulator of this pathway in flies, was found to be induced in parallel to the Toll-dependent activation process during immune defenses. We were interested in the mechanisms of this dual effect and provide here evidence that upon pathogenic stimuli, dorsal, one of the transcription factors of the fly Toll pathway, can induce the expression of the E3 ligase Bre1. We further show that Bre1 complexes with the E2 Rad6 to mono-ubiquitinate histone H2B and to promote the transcription of cactus to achieve homeostasis of the Toll immune response. Our studies characterize a Toll signal-dependent regulatory machinery in governing the Toll pathway in Drosophila.

Corrigendum: A feedback regulatory loop involving dTrbd/dTak1 in controlling IMD signaling in Drosophila Melanogaster.

[This corrects the article DOI: 10.3389/fimmu.2022.932268.].

A Feedback Regulatory Loop Involving dTrbd/dTak1 in Controlling IMD Signaling in Drosophila Melanogaster.

Negative regulators of the inflammatory responses are essential for the maintenance of immune homeostasis and organismal fitness. In Drosophila, the deubiquitinase (Dub) dTrbd selectively restricts the K63-linked ubiquitination modification of dTak1, a pivotal kinase of the IMD signaling pathway, to regulate the IMD innate immune response. However, which domain and how it functions to enable dTrbd's activity remain unexplored. Here, we provide compelling evidence showing that the NZF domain of dTrbd is essential for its association with dTak1. Meanwhile, the Linker region of dTrbd is involved in modulating its condensation, a functional state representing the Dub enzymatical activity of dTrbd. Of interest, the activated IMD signals following bacterial stimuli enhance the dTrbd/dTak1 interaction, as well as the condensate assembly and Dub enzymatical activity of dTrbd. Collectively, our studies shed light on the dual mechanisms by which the IMD signaling-mediated feedback loop of dTrbd/dTak1 precisely regulates the innate immune response in Drosophila.

Endoplasmic reticulum-associated protein degradation contributes to Toll innate immune defense in Drosophila melanogaster.

In Drosophila, the endoplasmic reticulum-associated protein degradation (ERAD) is engaged in regulating pleiotropic biological processes, with regard to retinal degeneration, intestinal homeostasis, and organismal development. The extent to which it functions in controlling the fly innate immune defense, however, remains largely unknown. Here, we show that blockade of the ERAD in fat bodies antagonizes the Toll but not the IMD innate immune defense in Drosophila. Genetic approaches further suggest a functional role of Me31B in the ERAD-mediated fly innate immunity. Moreover, we provide evidence that silence of Xbp1 other than PERK or Atf6 partially rescues the immune defects by the dysregulated ERAD in fat bodies. Collectively, our study uncovers an essential function of the ERAD in mediating the Toll innate immune reaction in Drosophila.

Aspirin Positively Contributes to Drosophila Intestinal Homeostasis and Delays Aging through Targeting Imd.

The intestine, a high-turnover tissue, plays a critical role in regulating aging and health in both vertebrates and invertebrates. Maintaining the epithelial barrier function of the intestine by preserving innate immune homeostasis significantly delays aging and prevents mortality. In an effort to explore effective chemicals and materials that can improve intestinal integrity, we performed a nonbiased screen utilizing Drosophila as an animal model. We showed that long-term uptake of aspirin markedly prevented age-onset gut leakage, the over-proliferation of intestinal stem cells, and the dysbiosis of commensal microbiota in fruit flies. Mechanistically, aspirin efficiently downregulated chronic activation of intestinal immune deficiency signaling during aging. Furthermore, our in vivo and in vitro biochemical analyses indicated that aspirin is a negative modulator in control of the K63-linked ubiquitination of Imd. Our findings uncover a novel regulatory mechanism by which aspirin positively modulates intestinal homeostasis, thus delaying aging, in Drosophila.

Theaflavin-regulated Imd condensates control Drosophila intestinal homeostasis and aging.

Black tea is the most widely consumed tea drink in the world and has consistently been reported to possess anti-aging benefits. However, whether theaflavins, one type of the characteristic phytochemicals in black tea extracts, are involved in regulating aging and lifespan in consumers remains largely unknown. In this study, we show that theaflavins play a beneficial role in preventing age-onset intestinal leakage and dysbiosis, thus delaying aging in Drosophila. Mechanistically, theaflavins regulate the condensate assembly of Imd to negatively govern the overactivation of Imd signals in fruit fly intestines. In addition, theaflavins prevent DSS-induced colitis in mice, suggesting theaflavins play a role in modulating intestinal integrity. Overall, our study reveals a molecular mechanism by which theaflavins regulate gut homeostasis likely through controlling Imd coalescence.

LC Domain-Mediated Coalescence Is Essential for Otu Enzymatic Activity to Extend Drosophila Lifespan.

In eukaryotic cells, RNA-binding proteins (RBPs) interact with RNAs to form ribonucleoprotein complexes (RNA granules) that have long been thought to regulate RNA fate or activity. Emerging evidence suggests that some RBPs not only bind RNA but also possess enzymatic activity related to ubiquitin regulation, raising important questions of whether these RBP-formed RNA granules regulate ubiquitin signaling and related biological functions. Here, we show that Drosophila Otu binds RNAs and coalesces to membrane-less biomolecular condensates via its intrinsically disordered low-complexity domain, and coalescence represents a functional state for Otu exerting deubiquitinase activity. Notably, coalescence-mediated enzymatic activity of Otu is positively regulated by its bound RNAs and co-partner Bam. Further genetic analysis reveals that the Otu/Bam deubiquitinase complex and dTraf6 constitute a feedback loop to maintain intestinal immune homeostasis during aging, thereby controlling longevity. Thus, regulated biomolecular condensates may represent a mechanism that controls dynamic enzymatic activities and related biological processes.

Early and late stage profiles for a chemotaxis model with density-dependent jump probability.

In this paper, we derive a chemotaxis model with degenerate diffusion and density-dependent chemotactic sensitivity, and we provide a more realistic description of cell migration process for its early and late stages. Different from the existing studies focusing on the case of non-degenerate diffusion, this model with degenerate diffusion causes us some essential difficulty on the boundedness estimates and the propagation behavior of its compact support. In the presence of logistic damping, for the early stage before tumour cells spread to the whole domain, we first estimate the expanding speed of tumour region as O(t^ß) for 0 < ß < 1/2 . Then, for the late stage of cell migration, we further prove that the asymptotic profile of the original system is just its corresponding steady state. The global convergence of the original weak solution to the steady state with exponential rate O(e^(-ct)) for some c < 0 is also obtained.

Membrane targeting of inhibitory Smads through palmitoylation controls TGF-ß/BMP signaling.

TGF-ß/BMP (bone morphogenetic protein) signaling pathways play conserved roles in controlling embryonic development, tissue homeostasis, and stem cell regulation. Inhibitory Smads (I-Smads) have been shown to negatively regulate TGF-ß/BMP signaling by primarily targeting the type I receptors for ubiquitination and turnover. However, little is known about how I-Smads access the membrane to execute their functions. Here we show that Dad, the Drosophila I-Smad, associates with the cellular membrane via palmitoylation, thereby targeting the BMP type I receptor for ubiquitination. By performing systematic biochemistry assays, we characterized the specific cysteine (Cys556) essential for Dad palmitoylation and membrane association. Moreover, we demonstrate that dHIP14, a Drosophila palmitoyl acyl-transferase, catalyzes Dad palmitoylation, thereby inhibiting efficient BMP signaling. Thus, our findings uncover a modification of the inhibitory Smads that controls TGF-ß/BMP signaling activity.

Bam-dependent deubiquitinase complex can disrupt germ-line stem cell maintenance by targeting cyclin A.

Drosophila germ-line stem cells (GSCs) provide an excellent model to study the regulatory mechanisms of stem cells in vivo. Bag of marbles (bam) has been demonstrated to be necessary and sufficient to promote GSC and cystoblast differentiation. Despite extensive investigation of its regulation and genetic functions, the biochemical nature of the Bam protein has been unknown. Here, we report that Bam is an ubiquitin-associated protein and controls the turnover of cyclin A (CycA). Mechanistically, we found that Bam associated with Otu to form a deubiquitinase complex that stabilized CycA by deubiquitination, thus providing a mechanism to explain how ectopic expression of Bam in GSCs promotes differentiation. Collectively, our findings not only identify a biochemical function of Bam, which contributes to GSC fate determination, but also emphasizes the critical role of proper expression of cyclin proteins mediated by both ubiquitination and deubiquitination pathways in balancing stem cell self-renewal and differentiation.

Ci antagonizes Hippo signaling in the somatic cells of the ovary to drive germline stem cell differentiation.

Many stem cell populations are tightly regulated by their local microenvironment (niche), which comprises distinct types of stromal cells. However, little is known about mechanisms by which niche subgroups coordinately determine the stem cell fate. Here we identify that Yki, the key Hippo pathway component, is essential for escort cell (EC) function in promoting germline differentiation in Drosophila ovary. We found that Hedgehog (Hh) signals emanating primarily from cap cells support the function of ECs, where Cubitus interruptus (Ci), the Hh signaling effector, acts to inhibit Hippo kinase cascade activity. Mechanistically, we found that Ci competitively interacts with Hpo and impairs the Hpo-Wts signaling complex formation, thereby promoting Yki nuclear localization. The actions of Ci ensure effective Yki signaling to antagonize Sd/Tgi/Vg-mediated default repression in ECs. This study uncovers a mechanism explaining how subgroups of niche cells coordinate to determine the stem cell fate via Hh-Hippo signaling crosstalk, and enhances our understanding of mechanistic regulations of the oncogenic Yki/YAP signaling.

Cell-surface localization of Pellino antagonizes Toll-mediated innate immune signalling by controlling MyD88 turnover in Drosophila.

Innate immunity mediated by Toll signalling has been extensively studied, but how Toll signalling is precisely controlled in balancing innate immune responses remains poorly understood. It was reported that the plasma membrane localization of Drosophila MyD88 is necessary for the recruitment of cytosolic adaptor Tube to the cell surface, thus contributing to Toll signalling transduction. Here we demonstrate that Drosophila Pellino functions as a negative regulator in Toll-mediated signalling. We show that Pellino accumulates at the plasma membrane upon the activation of Toll signalling in a MyD88-dependent manner. Moreover, we find that Pellino is associated with MyD88 via its CTE domain, which is necessary and sufficient to promote Pellino accumulation at the plasma membrane where it targets MyD88 for ubiquitination and degradation. Collectively, our study uncovers a mechanism by which a feedback regulatory loop involving MyD88 and Pellino controls Toll-mediated signalling, thereby maintaining homeostasis of host innate immunity.