The innate immune response requires precise spatiotemporal regulation for organisms to ensure effective pathogen clearance while avoiding detrimental overactivation. Although the core components of the Drosophila Toll pathway are well-established, the post-transcriptional regulatory networks, particularly those involving non-coding RNAs (ncRNAs), remain incompletely understood to date. Here, we elucidate a novel tripartite feedback loop comprising the long noncoding RNA (lncRNA) CR42715, the microRNA (miRNA) miR-965-3p, and the transcription factor (TF) Dif that dynamically modulates Drosophila Toll signaling. Firstly, our results demonstrate that upon Gram-positive bacterial challenge, Dif activates CR42715 expression, which acts as a competitive endogenous RNA (ceRNA) by sponging miR-965-3p to alleviate miR-965-3p-mediated repression of Dif and enhance Dif protein synthesis, thus facilitating Toll signaling immune responses. Secondly, disruption of this feedback loop via genetic manipulation of CR42715 or miR-965-3p leads to dysregulated AMP expression and compromised host survival. Thirdly, the temporal expression analysis reveals that CR42715 is rapidly induced early in infection to boost immunity, while miR-965-3p expression increases later, ensuring timely signal attenuation, which suggests that this dynamic Dif/CR42715/miR-965-3p feedback loop can ensure robust early-phase antimicrobial peptide production while preventing excessive late-phase immunity. Collectively, we unveil a novel TF-lncRNA-miRNA feedback loop that acts as a rheostat to ensure an effective immune response.

The Drosophila Imd pathways are well-known mechanisms involved in innate immunity responsible for Gram-negative (G-) bacterial infection. The intensity and durability of immunity need to be finely regulated to keep sufficient immune activation meanwhile avoid excessive immune response. In this study, we firstly demonstrated that miR-190 can downregulate the expression levels of antimicrobial peptides (AMPs) in the Imd immune pathway after Escherichia coli infection using the miR-190 overexpression flies and the miR-190KO/+ flies. Secondly, miR-190 overexpression significantly reduces while miR-190 KO increases Drosophila survival rates upon lethal Enterobacter cloacae infection. Thirdly, we further demonstrated that miR-190 negatively regulates innate immune responses by directly targeting both RA/RB and RC isoforms of Tab2. In addition, the dynamic expression pattern of AMPs (Dpt, AttA, CecA1), miR-190 and Tab2 in the wild-type flies reveals that miR-190 play an important role in Drosophila immune homeostasis restoration at the late stage of E. coli infection. Collectively, our study reveals that miR-190 can downregulate the expression of AMPs by targeting Tab2 and promote immune homeostasis restoration in Drosophila Imd pathway. Our study provides new insights into the regulatory mechanism of animal innate immune homeostasis.

The regulation and maintenance of immune homeostasis are essential for animal survival, but the molecular mechanisms are not fully understood. Here, we used the model organism Drosophila melanogaster to uncover a potential mechanism by which the nuclear factor-κB transcription factor Relish and miR-100 cooperatively regulate innate immune homeostasis. We first demonstrated in vitro and in vivo that miR-100 can negatively regulate the immune responses of the Imd pathway by inhibiting the expression of TAK1-associated binding protein 2 (Tab2) gene. Second, we found that Relish, an important transcription factor in the Drosophila Imd pathway, could not only modulate the expressions of antimicrobial peptides (AMPs) to promote immune responses, but also bind to the promoter region of miR-100 and activate its transcription to inhibit immune responses. Third, the dynamic expression of genes profiling indicated that the Relish/miR-100/Tab2 regulatory axis could contribute to innate immune homeostasis in Drosophila. Together, our findings reveal the dual role of Relish in immune regulation, that is, Relish promotes the expression of AMPs to resist pathogen infection in the early immune response, while in the late immune stages, Relish readjusts the expression of miR-100 to negatively control immune responses to avoid excessive immunity thus maintaining immunohomeostasis. Meanwhile, our study provides a new perspective for further understanding the complex regulatory mechanism of immune homeostasis in animals.

The NF-κB/Relish, as a core transcription factor of Drosophila immune deficiency (Imd) pathway, activates the transcriptions of antimicrobial peptides (AMPs) to combat gram-negative bacterial infections, but its role in regulating miRNA expression during immune response has less been reported. We here describe a negative feedback loop of Imd signaling mediated by Relish/miR-275/Dredd that controls Drosophila immune homeostasis after Escherichia coli (E. coli) infection. Our results demonstrate that Relish may directly activate the transcription of miR-275 via binding to its promoter in vitro and vivo, particularly miR-275 further inhibits the expression of Dredd through binding to its 3'UTR to negatively control Drosophila Imd immune response. Remarkably, the ectopic expression of miR-275 significantly reduces Drosophila lifespan. More importantly, our work uncovers a new mechanism by which Relish can flexibly switch its role to maintain Drosophila immune response and homeostasis during infection. Collectively, our study not only reveals the functional duality of Relish in regulating immune response of Drosophila Imd pathway, but also provides a new insight into the maintenance of animal innate immune homeostasis.

The strength and duration of the NF-κB signaling response must be tightly modulated to avoid an inadequate or excessive immune response. Relish, a core NF-κB transcription factor of the Drosophila Imd pathway, can control the expression of antimicrobial peptides, including Dpt and AttA, to defend against Gram-negative bacterial infections, but whether Relish may regulate miRNA expression to participate in the immune response remains unclear. In this study, taking advantage of Drosophila S2 cells and different overexpression/knockout/knockdown flies, we first found that Relish could directly activate the expression of miR-308 to negatively regulate the immune response and promote the survival of Drosophila during Enterobacter cloacae infection. Second, our results demonstrated that Relish-mediated expression of miR-308 could suppress target gene Tab2 to attenuate the Drosophila Imd pathway signal during the middle and late stages of the immune response. Third, we detected the dynamic expression patterns of Dpt, AttA, Relish, miR-308, and Tab2 in wild-type flies after E. coli infection, which further revealed that the feedback regulatory loop of Relish-miR-308-Tab2 plays a crucial role in the immune response and homeostasis maintenance of the Drosophila Imd pathway. Overall, our present study not only illustrates an important mechanism by which this Relish-miR-308-Tab2 regulatory axis can negatively control the Drosophila immune response and participate in homeostasis maintenance but also provides new insights into the dynamic regulation of the NF-κB/miRNA expression network of animal innate immunity.

MicroRNAs (miRNAs) are a class of ~22 nt non-coding RNA molecules in metazoans capable of down-regulating target gene expression by binding to the complementary sites in the mRNA transcripts. Many individual miRNAs are implicated in a broad range of biological pathways, but functional characterization of miRNA clusters in concert is limited. Here, we report that miR-959–962 cluster (miR-959/960/961/962) can weaken Drosophila immune response to bacterial infection evidenced by the reduced expression of antimicrobial peptide Drosomycin (Drs) and short survival within 24 h upon infection. Each of the four miRNA members is confirmed to contribute to the reduced Drs expression and survival rate of Drosophila. Mechanically, RT-qPCR and Dual-luciferase reporter assay verify that tube and dorsal (dl) mRNAs, key components of Toll pathway, can simultaneously be targeted by miR-959 and miR-960, miR-961, and miR-962, respectively. Furthermore, miR-962 can even directly target to the 3′ untranslated region (UTR) of Toll. In addition, the dynamic expression pattern analysis in wild-type flies reveals that four miRNA members play important functions in Drosophila immune homeostasis restoration at the late stage of Micrococcus luteus (M. luteus) infection. Taken together, our results identify four miRNA members from miR-959–962 cluster as novel suppressors of Toll signaling and enrich the repertoire of immune-modulating miRNA in Drosophila.