Genotype and Gene Expression Associations with Immune Function in Drosophila

It is now well established that natural populations of Drosophila melanogaster harbor substantial genetic variation associated with physiological measures of immune function. In no case, however, have intermediate measures of immune function, such as transcriptional activity of immune-related genes, been tested as mediators of phenotypic variation in immunity. In this study, we measured bacterial load sustained after infection of D. melanogaster with Serratia marcescens, Providencia rettgeri, Enterococcus faecalis, and Lactococcus lactis in a panel of 94 third-chromosome substitution lines. We also measured transcriptional levels of 329 immune-related genes eight hours after infection with E. faecalis and S. marcescens in lines from the phenotypic tails of the test panel. We genotyped the substitution lines at 137 polymorphic markers distributed across 25 genes in order to test for statistical associations among genotype, bacterial load, and transcriptional dynamics. We find that genetic polymorphisms in the pathogen recognition genes (and particularly in PGRP-LC, GNBP1, and GNBP2) are most significantly associated with variation in bacterial load. We also find that overall transcriptional induction of effector proteins is a significant predictor of bacterial load after infection with E. faecalis, and that a marker upstream of the recognition gene PGRP-SD is statistically associated with variation in both bacterial load and transcriptional induction of effector proteins. These results show that polymorphism in genes near the top of the immune system signaling cascade can have a disproportionate effect on organismal phenotype due to the amplification of minor effects through the cascade

A shared role for RBF1 and dCAP-D3 in the regulation of transcription with consequences for innate immunity

Previously, we discovered a conserved interaction between RB proteins and the Condensin II protein CAP-D3 that is important for ensuring uniform chromatin condensation during mitotic prophase. The Drosophila melanogaster homologs RBF1 and dCAP-D3 co-localize on non-dividing polytene chromatin, suggesting the existence of a shared, non-mitotic role for these two proteins. Here, we show that the absence of RBF1 and dCAP-D3 alters the expression of many of the same genes in larvae and adult flies. Strikingly, most of the genes affected by the loss of RBF1 and dCAP-D3 are not classic cell cycle genes but are developmentally regulated genes with tissue-specific functions and these genes tend to be located in gene clusters. Our data reveal that RBF1 and dCAP-D3 are needed in fat body cells to activate transcription of clusters of antimicrobial peptide (AMP) genes. AMPs are important for innate immunity, and loss of either dCAP-D3 or RBF1 regulation results in a decrease in the ability to clear bacteria. Interestingly, in the adult fat body, RBF1 and dCAP-D3 bind to regions flanking an AMP gene cluster both prior to and following bacterial infection. These results describe a novel, non-mitotic role for the RBF1 and dCAP-D3 proteins in activation of the Drosophila immune system and suggest dCAP-D3 has an important role at specific subsets of RBF1-dependent genes

Toll-8/Tollo Negatively Regulates Antimicrobial Response in the Drosophila Respiratory Epithelium

Barrier epithelia that are persistently exposed to microbes have evolved potent immune tools to eliminate such pathogens. If mechanisms that control Drosophila systemic responses are well-characterized, the epithelial immune responses remain poorly understood. Here, we performed a genetic dissection of the cascades activated during the immune response of the Drosophila airway epithelium i.e. trachea. We present evidence that bacteria induced-antimicrobial peptide (AMP) production in the trachea is controlled by two signalling cascades. AMP gene transcription is activated by the inducible IMD pathway that acts non-cell autonomously in trachea. This IMD-dependent AMP activation is antagonized by a constitutively active signalling module involving the receptor Toll-8/Tollo, the ligand Spätzle2/DNT1 and Ect-4, the Drosophila ortholog of the human Sterile alpha and HEAT/ARMadillo motif (SARM). Our data show that, in addition to Toll-1 whose function is essential during the systemic immune response, Drosophila relies on another Toll family member to control the immune response in the respiratory epithelium

Lack of an Antibacterial Response Defect in Drosophila Toll-9 Mutant

Toll and Toll-like receptors represent families of receptors involved in mediating innate immunity response in insects and mammals. Although Drosophila proteome contains multiple Toll paralogs, Toll-1 is, so far, the only receptor to which an immune role has been attributed. In contrast, every single mammalian TLR is a key membrane receptor upstream of the vertebrate immune signaling cascades. The prevailing view is that TLR-mediated immunity is ancient. Structural analysis reveals that Drosophila Toll-9 is the most closely related to vertebrate TLRs and utilizes similar signaling components as Toll-1. This suggests that Toll-9 could be an ancestor of TLR-like receptors and could have immune function. Consistently, it has been reported that over-expression of Toll-9 in immune tissues is sufficient to induce the expression of some antimicrobial peptides in flies. These results have led to the idea that Toll-9 could be a constitutively active receptor that maintain significant levels of antimicrobial molecules and therefore provide constant basal protection against micro-organisms. To test theses hypotheses, we generated and analyzed phenotypes associated with a complete loss-of-function allele of Toll-9. Our results suggest that Toll-9 is neither required to maintain a basal anti-microbial response nor to mount an efficient immune response to bacterial infection

The Functions of Grainy Head-Like Proteins in Animals and Fungi and the Evolution of Apical Extracellular Barriers

The Grainy head (GRH) family of transcription factors are crucial for the development and repair of epidermal barriers in all animals in which they have been studied. This is a high-level functional conservation, as the known structural and enzymatic genes regulated by GRH proteins differ between species depending on the type of epidermal barrier being formed. Interestingly, members of the CP2 superfamily of transcription factors, which encompasses the GRH and LSF families in animals, are also found in fungi – organisms that lack epidermal tissues. To shed light on CP2 protein function in fungi, we characterized a Neurospora crassa mutant lacking the CP2 member we refer to as grainy head-like (grhl). We show that Neurospora GRHL has a DNA-binding specificity similar to that of animal GRH proteins and dissimilar to that of animal LSF proteins. Neurospora grhl mutants are defective in conidial-spore dispersal due to an inability to remodel the cell wall, and we show that grhl mutants and the long-known conidial separation-2 (csp-2) mutants are allelic. We then characterized the transcriptomes of both Neurospora grhl mutants and Drosophila grh mutant embryos to look for similarities in the affected genes. Neurospora grhl appears to play a role in the development and remodeling of the cell wall, as well as in the activation of genes involved in defense and virulence. Drosophila GRH is required to activate the expression of many genes involved in cuticular/epidermal-barrier formation. We also present evidence that GRH plays a role in adult antimicrobial defense. These results, along with previous studies of animal GRH proteins, suggest the fascinating possibility that the apical extracellular barriers of some animals and fungi might share an evolutionary connection, and that the formation of physical barriers in the last common ancestor was under the control of a transcriptional code that included GRH-like proteins

Scientific Validation of Three-Dimensional Stereophotogrammetry Compared to the IGAIS Clinical Scale for Assessing Wrinkles and Scars after Laser Treatment

Measuring outcomes from treatments to the skin is either reliant upon patient’s subjective feedback or scale-based peer assessments. Three-Dimensional stereophotogrammetry intend to accurately quantify skin microtopography before and after treatments. The objective of this study is comparing the accuracy of stereophotogrammetry with a scale-based peer evaluation in assessing topographical changes to skin surface following laser treatment. A 3D stereophotogrammetry system photographed skin surface of 48 patients with facial wrinkles or scars before and three months after laser resurfacing, followed immediately by topical application of vitamin C. The software measured changes in skin roughness, wrinkle depth and scar volume. Images were presented to three observers, each independently scoring cutaneous improvement according to Investigator Global Aesthetic Improvement Scale (IGAIS). As for the results, a trend reflecting skin/scar improvement was reported by 3D SPM measurements and raters. The percentage of topographical change given by the raters matched 3D SPM findings. Agreement was highest when observers analysed 3D images. However, observers overestimated skin improvement in a nontreatment control whilst 3D SPM was precise in detecting absence of intervention. This study confirmed a direct correlation between the IGAIS clinical scale and 3D SPM and confirmed the efficacy and accuracy of the latter when assessing cutaneous microtopography alterations as a response to laser treatment