Immune-inducible non-coding RNA molecule lincRNA-IBIN connects immunity and metabolism in Drosophila melanogaster

Abstract

Non-coding RNAs have important roles in regulating physiology, including immunity. Here, we performed transcriptome profiling of immune-responsive genes in Drosophila melanogaster during a Gram-positive bacterial infection, concentrating on long non-coding RNA (lncRNA) genes. The gene most highly induced by a Micrococcus luteus infection was CR44404, named Induced by Infection (lincRNA-IBIN). lincRNA-IBIN is induced by both Gram-positive and Gram-negative bacteria in Drosophila adults and parasitoid wasp Leptopilina boulardi in Drosophila larvae, as well as by the activation of the Toll or the Imd pathway in unchallenged flies. We show that upon infection, lincRNA-IBIN is expressed in the fat body, in hemocytes and in the gut, and its expression is regulated by NF-κB signaling and the chromatin modeling brahma complex. In the fat body, overexpression of lincRNA-IBIN affected the expression of Toll pathway -mediated genes. Notably, overexpression of lincRNA-IBIN in unchallenged flies elevated sugar levels in the hemolymph by enhancing the expression of genes important for glucose retrieval. These data show that lncRNA genes play a role in Drosophila immunity and indicate that lincRNA-IBIN acts as a link between innate immune responses and metabolism.

Fig 1. lincRNA-IBIN (CR44404) expression is strongly induced by Gram-positive and Gram-negative bacteria and its expression is regulated by the Toll and Imd pathways and a functional BAP complex.

A) In a whole transcriptome analysis, 28 genes were more than 18-fold upregulated after a M. luteus infection. The highest upregulation in infected flies was found in a long non-coding RNA gene, CR44404 (lincRNA-IBIN). p-value <0.005 in all selected genes (S1 Table). B) 16 upregulated lncRNA genes have more than a threefold expression change in response to a M. luteus infection in adult flies. p-value <0.05 in all selected lncRNA genes (S2 Table). C) lincRNA-IBIN expression is induced in Drosophila adults within two hours of an infection by M. luteus or E. cloacae. For fold-induction values, expression values in uninfected samples were set to 1. D) M. luteus-induced lincRNA-IBIN expression is dependent on the Toll pathway (the Toll pathway adaptor protein MyD88) function. E) E. cloacae-induced lincRNA-IBIN expression is dependent on the Imd pathway (Relish) function. In D and E, for fold-induction values, expression values in uninfected w, MyD88^IR samples were set to 1 F) lincRNA-IBIN expression is induced in Drosophila adults upon silencing of the Drosophila inhibitor of κB factor cactus. G) lincRNA-IBIN expression is induced in Drosophila larvae with the constitutively active form of the Toll receptor, Toll^10b. In F and G, for fold-induction values, expression values in uninfected/untreated w samples were set to 1. H) lincRNA-IBIN expression is also modestly induced by the ubiquitous overexpression of Imd with daughterless-GAL4 (da>Imd) in Drosophila larvae. For fold-induction values, the expression value of w,da> was set to 1. I-J) Both M. luteus and E. cloacae-induced lincRNA-IBIN expression is dependent on the functional chromatin remodeling BAP complex. In I and J, for fold-induction values, expression values in uninfected w, osa^IR samples were set to 1.

Fig 2. lincRNA-IBIN expression is induced in immunogenic tissues and its cellular localization is mainly nuclear.

A-B) lincRNA-IBIN is induced in the larval fat body and hemocytes after a L. boulardi infection and is dependent on the expression of the BAP complex member osa in these tissues. For fold-induction values, expression values in uninfected w, osa^IR samples were set to 1. C) qPCR for hemocyte-specific Hml (i) and fat body-specific Lsp1α (ii) was carried out to confirm the purity of the tissue fractions. lincRNA-IBIN was not found in the plasma fraction in large quantities (iii). D) RNA FISH performed in larval hemocytes shows that lincRNA-IBIN is mainly located in the nucleus; pink labelling (lincRNA-IBIN) co-localizes with blue nuclear labelling (DAPI). i) Negative control (without lincRNA-IBIN probes), ii) hemocytes from w¹¹¹⁸ larvae showing the basal expression level and localization of lincRNA-IBIN, iii) hemocytes from larvae overexpressing lincRNA-IBIN¹ (HH>lincRNA-IBIN¹) and infected with L. boulardi.

Fig 3. Overexpressing lincRNA-IBIN improves the survival of Drosophila adults from an infection and the expression of selected target genes.

A) UAS-lincRNA-IBIN (CR44404) overexpression with the C564-GAL4 driver (C564>lincRNA-IBIN¹ and C564>lincRNA-IBIN⁷) significantly increases the expression of lincRNA-IBIN in uninfected flies measured with qPCR, as does an infection with M. luteus. B) lincRNA-IBIN overexpression (C564>lincRNA-IBIN⁷) improves the survival of the flies after an infection with M. luteus + E. faecalis. C-D) lincRNA-IBIN overexpression increases the expression of two Toll pathway target genes IM1 (C) and Drosomycin (D) after a M. luteus infection. MyD88 knockdown flies were used as a negative control and cactus knockdown flies as a positive control. In A, C and D, for fold-induction values, expression values in uninfected w, lincRNA-IBIN⁷ samples were set to 1. E) E. cloacae-induced endogenous lincRNA-IBIN expression is lost upon Relish RNAi. lincRNA-IBIN overexpression is shown in the Relish RNAi background. F) Upon a E. cloacae infection, lincRNA-IBIN overexpression gives a statistically significant survival advantage to flies with a Relish RNAi background. G) Attacin C (AttC) is not produced in Relish RNAi flies, but with IBIN overexpression a very small amount of AttC is induced. H) Diptericin A (DptA) is not produced with or without lincRNA-IBIN overexpression in flies with a Relish RNAi background upon a E. cloacae infection. In E, G and H, for fold-induction values, expression values in uninfected w, Rel^IR samples were set to 1.

Fig 4. Transcriptome analysis of lincRNA-IBIN overexpressing flies reveals changes in metabolic genes that result in increased glucose levels in the hemolymph.

A) lincRNA-IBIN⁷ overexpression with the C564-GAL4 driver increases lincRNA-IBIN expression in uninfected flies by 161-fold. This induction is not visible upon a E. cloacae or M. luteus infection, since the infection induces strong lincRNA-IBIN expression. B) Cluster analysis of genes up-and downregulated in C564>lincRNA-IBIN flies compared to w; lincRNA-IBIN controls. The major cluster of induced genes consists of glycoside hydrolase enzymes, whereas enzymes involved in proteolysis form the main downregulated cluster. Clustering was done using GO Biological Process and InterPro protein classification (GO BP/InterPro). C,D) Expression differences of selected Drosophila genes involved in metabolism with and without lincRNA-IBIN overexpression and with and without an infection based on the transcriptome analysis. E) lincRNA-IBIN is induced in the Drosophila adult midgut by a E. cloacae septic injury 24h post infection. F) Workflow of the collection of hemolymph for glucose and trehalose measurements. G,H) Circulating hemolymph glucose levels are elevated upon G) infection and H) overexpression of lincRNA-IBIN in the adult midgut.

Fig 5. Schematic representation of lincRNA-IBIN functions in Drosophila larvae and adults.