Common and specific downstream signaling targets controlled by Tlr2 and Tlr5 innate immune signaling in zebrafish
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Although the responses to many pathogen associated molecular patterns (PAMPs) in cell cultures and extracted organs are well characterized, there is little known of transcriptome responses to PAMPs in whole organisms. To characterize this in detail, we have performed RNAseq analysis of responses of zebrafish embryos to injection of PAMPs in the caudal vein at one hour after exposure. We have compared two ligands that in mammals have been shown to specifically activate the TLR2 and TLR5 receptors: Pam3CSK4 and flagellin, respectively.
We identified a group of 80 common genes that respond with high stringency selection to stimulations with both PAMPs, which included several well-known immune marker genes such as il1b and tnfa. Surprisingly, we also identified sets of 48 and 42 genes that specifically respond to either Pam3CSK4 or flagellin, respectively, after a comparative filtering approach. Remarkably, in the Pam3CSK4 specific set, there was a set of transcription factors with more than 2 fold-change, as confirmed by qPCR analyses, including cebpb, fosb, nr4a1 and egr3. We also showed that the regulation of the Pam3CSK4 and flagellin specifically responding sets is inhibited by knockdown of tlr2 or tlr5, respectively.
Our studies show that Pam3CSK4 and flagellin can stimulate the Tlr2 and Tlr5 signaling pathways leading to common and specific responses in the zebrafish embryo system.
KeywordsTLR signaling Zebrafish TLR2 TLR5 Flagellin Pam3CSK4 Innate immunity
The innate immune system is referred to as the first line in host defense against invading pathogens [1, 2]. Its highly developed ability to recognize microbial patterns and host-derived danger signals relies on so-called pattern recognition receptors (PRRs), especially on the Toll-like receptors (TLRs) [3, 4, 5]. In humans, the TLR family is composed of 10 members, which are located at the cell surface with the exception of TLR3, 7, 8 and 9, which are localized on intracellular endosomal membranes [6, 7, 8]. The TLRs are involved in the recognition of a wide variety of ligands, including pathogen associated molecular patterns (PAMPs), such as bacterial cell wall components and viral RNA, as well as damage associated molecular patterns (DAMPs) [9, 10]. This leads to subsequent intracellular signal transduction, triggering the production of inflammatory cytokines and chemokines, but it can also lead to anti-inflammatory responses as has been recently shown for TLR10 acting as a heterodimer with TLR2 [11, 12].
The recognition of PAMPs and DAMPs by different TLRs is directed by structurally conserved leucine-rich repeats (LRR) motifs of the TLRs ectodomains (ECDs) [13, 14]. Cell-surface TLRs can mediate binding to PAMPs by homodimerizing, like TLR4 and TLR5 that recognize lipopolysaccharide (LPS)  and flagellin , respectively. In contrast, other TLRs form heterodimers like TLR2 that associates with TLR1, TLR6 or TLR10, in conjunction recognizing lipopeptides and lipoproteins [12, 17, 18]. The diversity of TLR2 dimer combinations is thought to be responsible for its extensive recognition ability, ranging from the diverse components of various pathogens to the host heat-shock protein 70 . TLR2 plays an important role in resistance to the infection induced by Mycobacterium tuberculosis (Mtb) [19, 20]. For example, McBride et al. demonstrated that TLR2 knockout mice show enhanced cell infiltration and inflammation in lungs upon low dose chronic infection with Mtb, and fail to stably control the bacterial burden . A series of components of Mtb can trigger the TLR2 signaling pathway upon infection, such as triacylated lipoprotein LprG, LpqH and PhoS1 [22, 23], and glycolipid lipoarabinomannan . Pam3CysSerLys4 (Pam3CSK4) is a synthetic triacylated lipopeptide that mimics the triacylated lipoprotein of mycobacteria and classical gram-positive bacteria, which can be recognized by TLR2/TLR1 heterodimers and induce the production and release of pro- and anti-inflammatory cytokines (IL-6, IL-12, TNF-α and IL-10), chemokines (IL-8) and interferon (IFN-γ) [25, 26, 27, 28]. Most of these studies on the responses of the TLR2 signaling pathway have been performed in cell culture systems. A notable exception is the reported transcriptome response of mouse mononuclear phagocytes recruited to lungs challenged with Pam3CSK4 as measured by micro-arrays . As another example 8 day old mice were treated with Pam3CSK4 and analyzed for the expression of several inflammatory genes using qPCR .
The zebrafish embryonic model has much potential to study the ligand specificity of TLRs at the organism level . Importantly, zebrafish offers the possibility to study the innate immune system separated from the adaptive immune system in their embryonic and larval stages (up to 3-4 weeks post fertilization) [31, 32, 33]. To date, a number of TLR signaling pathway mediators have been identified and studied in zebrafish such as the adaptor proteins Myd88, Tirap (Mal), Trif and Sarm1 [34, 35, 36, 37, 38], and the downstream signaling intermediates Irak and Traf6 [39, 40].
In work previously published by our group, we demonstrated that the function and regulation of the zebrafish homologs of human TLR5, tlr5a and tlr5b, are conserved with their mammalian counterparts. Both tlr5a and tlr5b are strongly up-regulated in response to Salmonella typhimurium infection . Furthermore, in the same study it was shown that knockdown of these two genes prevented or weakened the activation of genes for several inflammatory mediators like mmp9, cxcl-C1c, il1b and il8 upon flagellin stimulation .
In this study, we aimed to study TLR2 function, in comparison with TLR5, in zebrafish using transcriptome analysis. Injection into the blood stream of the tlr2/tlr1 ligand Pam3CSK4, was followed by transcriptome profiling to characterize key genes involved in the early response to this PAMPs. In addition, by comparing the transcriptome response towards treatment with flagellin, we were able to discriminate non-specific immune responsive genes from a set of genes which are regulated by tlr2 but not by tlr5.
Wild-type zebrafish of the AB/TL strain were handled in compliance with the local animal welfare regulations and maintained according to standard protocols (zfin. org). Embryos were raised in egg water (60 g/ml Instant Ocean sea salts) at 28.5 °C. For the duration of bacterial injections, embryos were kept under anesthesia in egg water containing 0.02 % buffered 3-aminobenzoic acid ethyl ester (Tricaine). The breeding of adult fish was approved by the local animal welfare committee (DEC) of the University of Leiden. All protocols adhered to the international guidelines specified by the EU Animal Protection Directive 2010/63/EU.
Morpholino oligonucleotides (Gene Tools) were diluted to desired concentrations in 1× Danieu’s buffer (58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca (NO3)2, 5.0 mM HEPES (pH 7.6)) containing 1× phenol red (Sigma-Aldrich). For knockdown experiments, tlr2 ATG-morpholino (tlr2 mo, Additional file 1: Table S1) was injected with the optimal concentration at 0.5 mM and 1 nl volume per embryo at 0 ~ 2 cell stage. tlr5 translation was blocked via injecting 1 nl of the tlr5a and tlr5b ATG-morpholinos at a dose of 0.1 mM and 0.5 mM at 0 ~ 2 cell stage, as previously published by Stockhammer and coworkers . Control embryos were injected with the standard control morpholino (Sc mo, Additional file 1: Table S1).
For the ligands injection assay, purified Pam3CSK4 (InvivoGen) and flagellin from S. typhimurium (Flagellin FliC VacciGrade™, Invitrogen) were respectively diluted to 1 mg/ml and 100 μg/ml in sterile water. For their administration, 1 nl of the ligands was injected into the blood stream at 27 hpf, and sterile water was used as control. Injections were perormed using a FemtoJet microinjector (Eppendorf) and a micromanipulator with pulled microcapillary pipettes.
RNA isolation, cDNA synthesis and qPCR
RNA was extracted using TRIzol Reagent (Life Technologies) and purified by column according to the manufacturer’s instructions of RNeasy MinElute Cleanup Kit (Qiagen). The concentration and quality of RNA were detected by NANODROP 2000/2000c (Thermo Scientific). 1 μg cDNA synthesis reactions and qPCR were performed as described in the manufacturer’s instructions (iScript™ cDNA Synthesis Kit and iQ™ SYBR® Green Supermix, BioRad) and normalized against the expression of ppial as a housekeeping gene . PCR analysis was performed using the following protocol: 95 °C 3 min, 40 cycles real time of 95 °C 15 s and 60 °C 45 s, and final melting curve of 81 cycles from 95 °C 1 min to 55 °C 10 s. Results were analyzed using the ΔΔCt method. Primer sequences used can be found in Additional file 1: Table S1.
Deep sequencing and data analyzing
Triplicates of 10 ~ 20 embryos of AB/TL or tlr morphants from three injection conditions, Pam3CSK4, flagellin or water injection, were homogenized in 500 μl of Trizol reagent (Qiagen). Total RNA was extracted and column-purified according to the manufacturer’s instructions of the RNeasy MinElute Cleanup Kit (Qiagen). The subsequent sample preparation and Illumina RNA sequencing were as previously described . RNA samples were treated with DNaseI (Life Technologies) to remove residual genomic DNA. RNA integrity was analyzed by Lab-on-a-chip analysis (Agilent, Amstelveen, The Netherlands). A total of 2 μg of RNA was used to make RNAseq libraries using the Illumina TruSeq RNA Sample Preparation Kit v2 (Illumina, Inc., San Diego, CA, USA). The manufacturer’s instructions were followed with the exception of two modifications. In the adapter ligation step, 1 μl, instead of 2.5 μl, adaptor was used. In the library size-selection step, the library fragments were isolated with a double Ampure XP purification with a 0.7× beads to library ratio (Beckman Coulter, Woerden, The Netherlands). The resulting mRNAseq library was sequenced using an Illumina HiSeq2500 Instrument (Illumina, Inc.) according to the manufacturer’s instructions with a read length of 2 × 50 nucleotides. Image analysis and base-calling were done using the Illumina HCS version 2.0.12. The raw data has been submitted to the GEO database (accession number GSE64570). The total number of reads for each sample is summarized in Additional file 2: Table S4 and quality control was according to the sequencing company guidelines (ZF-sceens.com). The data was analyzed using the GeneTiles software (http://www.genetiles.com)  using a cut-off setting of 2 fold-change and a p-value <0.01. In brief, Genetiles used fastq files as input for the program Bowtie2  to align the reads to the zebrafish genome (obtained from Ensembl version Zv9). Subsequently, the programs SAMtools , DESeq and DEXSeq [46, 47] are used for data processing. The complete data processing pipeline for Genetiles, including the used parameters, is available for download at www.genetiles.com and can also be found in Veneman et al . Using these settings we have mapped the numbers of reads as shown in Additional file 2: Table S4. The triplicate data sets of Pam3CSK4, flagellin and control treatments were mapped to 27104, 26583, and 26409 ENSEMBL genes, respectively. The difference between the mapped reads of the individual samples compared with the mapped reads of triplicate samples was always lower than 12 % (Additional file 2: Table S4). GO analysis was performed using the software package DAVID available at http://david.abcc.ncifcrf.gov/home.jsp .
The immune response of zebrafish embryos to injection of PAMPs in the caudal vein
The function of Tlr5 and Tlr2 in the immune response towards flagellin and Pam3CSK4
The function of tlr2 in recognition of Pam3CSK4 was tested in the same manner. Our results showed that tlr2 morphants did not exhibit up-regulation of il1b expression upon Pam3CSK4 stimulation, while the control morphants did (Fig. 2b). In contrast, tlr2 morphants stimulated with flagellin, still showed a significant induction of il1b expression (Fig. 2c).
Identification of a common response gene set for Pam3CSK4 and flagellin stimulation
Since comparisons of the transcriptome response to PAMPs that activate different TLRs has not been described before in a whole organism we decided to perform RNA deep sequencing (RNAseq) of embryos treated with flagellin and Pam3CSK4 at 1 hpi.
Identification of gene sets that are specifically regulated by Pam3CSK4 and flagellin
This study unexpectedly revealed that there is a relatively large group of genes that are only induced or repressed by either Pam3CSK4 or flagellin (Fig. 3b). To further test whether this specifically induced group is completely unaffected by the other PAMP treatment we used a rigorous filtering approach as shown in Fig. 3d and e. For this approach, the DEGs from the Pam3CSK4 (264 genes) and flagellin (306 genes) stimulation groups were compared with the group of genes that were not affected by flagellin (22611 genes) and Pam3CSK4 (22391 genes), respectively, with a cut-off setting at <1.4-fold change and p > 0.05. By taking the overlap of these sets we thereby exclude genes that were inducible by the other ligand even at very low stringency. This resulted in a set of 48 genes (Fig. 3d) for which the response is specific to Pam3CSK4 and a set of 42 genes (Fig. 3e) for which the response is specific to flagellin. GO analysis indicated that these two groups contain different categories (Fig. 3d, e). Most notably, genes with high fold-change (>2) from the Pam3CSK4 specific group include many transcription factors involved in the TLR signaling pathway, such as junbb, cebpb, fosb, fosl1a, egr3 and nr4a1. In the flagellin specific group of genes this is not the case and an obvious enriched category could not be identified.
Function of the tlr2 and tlr5a genes in the transcriptome responses to Pam3CSK4 and flagellin
Discussion and Conclusion
The signaling pathways underlying recognition of PAMPs have been studied intensively and this has led to a broad understanding of key regulators of innate immunity based on studies of cell cultures and the use of knockout rodent mutants. The new possibilities for analysis of transcriptomes using RNA deep sequencing make it highly attractive to analyze the responses of an entire test animal model at the system biology level. In this manuscript we have chosen the zebrafish embryo model for such an approach and have included functional analysis of Tlr5 and Tlr2 in the response towards two well-known PAMPs, flagellin and Pam3CSK4.
The results show that there is a relatively limited overlap between the transcriptome responses towards flagellin and Pam3CSK4 (Fig. 3b). The overlap includes well known downstream immune mediators that were previously shown to be induced by flagellin  such as il1b, tnfa, irak3, mmp9, cxcl-c1c and il8. In contrast, il6 and il10, that are associated with an anti-inflammatory response, were induced much stronger by Pam3CSK4 than by flagellin. A relatively much larger group of genes showed a differential response to flagellin or Pam3CSK4, including a group of genes of which the transcription is specific for activation by one of the two treatments (Fig. 4).
GO terms analysis of the genes specifically regulated by these two PAMPs show that there is an enriched category of transcription factors in the Pam3CSK4-specific group, of which most of genes are up-regulated and only one is down-regulated. Additionally, a less enriched category of immune response genes is found in this group as well, which include a down-regulated CRP (C-reactive protein). For the flagellin-specific group of genes, there are only five genes under the GO-term regulation of transcription of which two are down-regulated. Many of the genes specifically induced by Pam3CSK4 have also been shown to be strongly regulated by infection in the zebrafish embryo system  and therefore we would like to further study this group of genes in more detail in future research.
For functional analysis of the transcriptome response towards flagellin and Pam3CSK4 we used morpholinos that were selected on basis of their blocking effect on downstream signaling using qPCR and subsequently confirmed by RNAseq analysis. Surprisingly for tlr5 we found that a morpholino against each of the two copies of this gene, tlr5a and tlr5b had an effect on downstream signaling, with the tlr5a morpholino giving a complete block of induction of il1b by injection with flagellin and a partial effect of the tlr5b morpholino. These data suggests that these two tlr5 copies function in concert, perhaps by forming heterodimers.
We thank Ulrike Nehrdich, Davy de Witt and Laura van Hulst for fish caretaking and Hulya Ozupek, Dr. Hans Jansen and Dr. Ron Dirks (ZF-screens B. V.) for assistance with RNAseq analysis. We also want to thank to Dr. Jan de Sonneville and Kees-Jan van der Kolk for making the Genetiles software available.
S. Y. was supported by a grant from the China Scholarship Council (CSC) and R. M. J by the European Marie-Curie Initial Training Network FishForPharma (contract number PITN-GA-2011-289209).
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