Transcriptome Profiling and Functional Analyses of the Zebrafish Embryonic Innate Immune Response to Salmonella Infection

Abstract

Due to the clear separation of innate immunity from adaptive responses, the externally developing zebrafish embryo represents a useful in vivo model for identification of innate host determinants of the response to bacterial infection. Here we performed a time-course transcriptome profiling study and gene ontology analysis of the embryonic innate immune response to infection with two model Salmonella strains that elicit either a lethal infection or an attenuated response. The transcriptional response to infection with both the lethal strain and the avirulent LPS O-Ag mutant strain showed clear conservation with host responses detected in other vertebrate models and human cells, including induction of genes encoding cell surface receptors, signaling intermediates, transcription factors, and inflammatory mediators. Furthermore, our study led to the identification of a large set of novel immune response genes and infection markers, the future functional characterization of which will support vertebrate genome annotation. From the time series and bacterial strain comparisons, matrix metalloproteinase genes, including mmp9, were among the most consistent infection-responsive genes. Purified Salmonella flagellin also strongly induced mmp9 expression. Using knockdown analysis, we showed that this gene was downstream of the zebrafish homologs of the flagellin receptor TLR5 and the adaptor MyD88. Additionally, flagellin-mediated induction of other inflammation markers, including il1b, il8, and cxcl-C1c, was reduced upon Tlr5 knockdown as well as expression of irak3, a putative negative TLR pathway regulator. Finally, we showed that induction of il1b, mmp9, and irak3 requires Myd88-dependent signaling, while ifn1 and il8 were induced Myd88 independently during Salmonella infection.

Regulation of immunity and disease resistance by commensal microbes and chromatin modifications during zebrafish development

“…Intriguingly, several studies with zebrafish larvae have suggested that commensal microbiota colonize the larvae as soon as they hatch, but all assign great importance to the role played by gut microbiota in modifying innate immunity, nutrient metabolism, impaired intestinal differentiation, and function when microbes invade the internal organs (19, 22, 36-38, 40, 53). Also, in contrast to our work, it previously was proposed that host defense mechanisms, such as proinflammatory cytokines, are inducible mostly upon recognition of virulence factors of pathogenic bacteria (9,52,(54)(55)(56). However, it should be noted that spontaneous infection was not recorded in the present study; thus the "inflammatory state" observed in CONR fish was transient, rapidly returning to basal levels, marked here by the levels of IL-1β expression similar to those observed in the GF fish 24 h later (i.e., at 96 hpf).…”

Covariation between behaviour and physiology indicators of coping style in zebrafish (Danio rerio)

“…The relative expression levels of GRa and GRb were evaluated using qPCR according to Stockhammer et al (2009). Briefly, five proactive and five reactive fish were killed immediately after separation according to coping style, in an overdose of clove oil (10% in ethanol, O30% in water).…”

The Involvement of Cholesterol in Sepsis and Tolerance to Lipopolysaccharide Highlighted by the Transcriptome Analysis of Zebrafish (Danio rerio)

“…In the past few years, this species has been gaining importance as a model for many human diseases because it has important advantages: its fertilized embryos are transparent, fluorescent tools are available to study host-pathogen interactions, it is suitable for largescale genetic analyses, and there is a clear temporal separation between the innate and adaptive immune systems. [5][6][7][8][9][10] Moreover, we can investigate processes in vivo by using the complete organism and by studying the global response. Therefore, to determine the associated transcriptome profile and to identify the candidate genes associated with sepsis and tolerance, we studied the effects of a LPS treatment or the combination of this lethal treatment preceded by the administration of a sublethal concentration of LPS, which results in a tolerized state.…”