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Transcriptome Profiling and Functional Analyses of the Zebrafish Embryonic Innate Immune Response to Salmonella Infection¹

Oliver W. Stockhammer^*, Anna Zakrzewska^*, Zoltán Hegedûs, Herman P. Spaink^* and Annemarie H. Meijer^2,*

^* Institute of Biology, Leiden University, Leiden, The Netherlands; and ZenonBio, Szeged, Hungary, and Bioinformatics Laboratory, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary

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.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by the European Commission Sixth Framework Programs ZF-MODELS (LSHG-CT-2003-503496) and ZF-TOOLS (LSHG-CT-2006-037220).

² Address correspondence and reprint requests to Dr. Annemarie H. Meijer, Institute of Biology, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands. E-mail address: a.h.meijer{at}biology.leidenuniv.nl

³ Abbreviations used in this paper: wt, wild type; BP, biological process; CC, cellular component; GO, gene ontology; hpf, hours postfertilization; hpi, hours postinfection; MF, molecular function; qRT-PCR, quantitative RT-PCR.

⁴ The online version of this article contains supplemental material.