- Email: [email protected]
Identification and functional characterization of IRAK-4 in grass carp (Ctenopharyngodon idellus)
Accepted Manuscript Identification and functional characterization of IRAK-4 in grass carp (Ctenopharyngodon idellus) Chuxin Wu, Xiaowen Xu, Xiaoping Zhi, Zeyin Jiang, Yinping Li, Xiaofen Xie, Xingxing Chen, Chengyu Hu PII:
S1050-4648(19)30041-5
DOI:
https://doi.org/10.1016/j.fsi.2019.01.031
Reference:
YFSIM 5876
To appear in:
Fish and Shellfish Immunology
Received Date: 21 October 2018 Revised Date:
11 January 2019
Accepted Date: 23 January 2019
Please cite this article as: Wu C, Xu X, Zhi X, Jiang Z, Li Y, Xie X, Chen X, Hu C, Identification and functional characterization of IRAK-4 in grass carp (Ctenopharyngodon idellus), Fish and Shellfish Immunology (2019), doi: https://doi.org/10.1016/j.fsi.2019.01.031. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Identification and functional characterization of IRAK-4 in grass
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carp (Ctenopharyngodon idellus)
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Chuxin Wu a, Xiaowen Xu b, Xiaoping Zhi a, Zeyin Jiang b, Yinping Li b, Xiaofen
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Xie b, Xingxing Chen b, Chengyu Hu b*
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a
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b
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Nanchang 330031, China
Yuzhang Normal University, Nanchang 330103, China
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Chengyu Hu, Ph.D.
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* Corresponding author:
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Department of Bioscience, College of Life Science, Nanchang University,
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Department of Bioscience, College of Life Science, Nanchang University, Nanchang
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330031, China.
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Fax: +86-791-8396-9530, E-mail: [email protected]
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ACCEPTED MANUSCRIPT Abstract
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IL-1R-associated kinase 4 (IRAK4), a central TIR signaling mediator in innate
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immunity, can initiate a cascade of signaling events and lead to induction of
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inflammatory target gene expression eventually. In the present study, we cloned and
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characterized an IRAK4 orthologue from grass carp (Ctenopharyngodon idella). The
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full length cDNA of CiIRAK4 was 2057 bp with an ORF of 1422 bp encoding a
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polypeptide of 472 amino acids. Multiple alignments showed that IRAK4s were
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highly conserved among different species. Phylogenetic tree analysis revealed that
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CiIRAK4 shared high homologous with zebra fish IRAK4. Expression analysis
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indicated that CiIRAK4 was widely expressed in all tested tissues. It was
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significantly up-regulated after treatment with poly I:C, especially obvious in liver
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and spleen. Also, CiIRAK4 could be induced by poly I:C and LPS in CIK cells.
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Fluorescence microscopy assays showed that CiIRAK4 localized in the cytoplasm.
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RNAi-mediated knockdown and overexpression assays indicated that CiIRAK4
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might have little effect on NF-kappa B p65 translocation from cytoplasm to nucleus，
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indicating that CiIRAK4 was dispensable for activation of NF-kappa B p65. In
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addition, IRAK4 promoted IRF5 nuclear translocation, which has nothing to do with
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the interaction between IRAK4 and IRF5. It suggested that fish IRAK4 kinase
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regulated IRF5 activity through indirect ways.
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Keywords: IRAK4; NF-kappa B p65; IRF5; function; grass carp
ACCEPTED MANUSCRIPT 1. Introduction
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The innate immune system is the first line of host defense against pathogens,
42
particularly important for lower vertebrates to defense against viral or microbial
43
invasion [1, 2]. The recognition of invading pathogens is a pivotal step to trigger
44
intracellular signaling cascades in host cells, and pattern-recognition receptors
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(PRRs) play a critical role in this process [3]. Different PRRs react with specific
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pathogen-associated molecular partterns (PAMPs), activate specific signaling
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pathways, and lead to distinct antipathogen responses [4]. Toll-like receptors (TLRs),
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the first identified and the most well characterized PRRs, can trigger innate immune
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responses and prime antigen-specific adaptive immunity [5, 6]. To date, 10 and 12
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functional TLRs have been identified in human and mouse respectively, and they are
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evolutionarily conserved from worm to mammals [7]. Containing intracellular
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Toll–interleukin 1 (IL-1) receptor (TIR) domains, TLRs recruit a specific set of
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adaptor molecules, such as MyD88 and TRIF, and initiate distinct signaling
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pathways [8]. In the MyD88-dependent pathway, IL-1 receptor-associated kinases
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(IRAKs) play a critical role in linking TLR signaling to the nuclear factor κB
56
(NF-kappa B) pathway [8, 9].
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In mammals, IRAKs is comprised of four family members: IRAK1, IRAK2, IRAK3
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(IRAKM), and IRAK4 [10-13]. Although all of them contain an N-terminal death
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domain (DD), a proST domain, and a conserved central kinase domain, only IRAK1
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and IRAK4 have real kinase activity, while IRAK2 and IRAK3 are inactive
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pseudokinases [14, 15]. In the MyD88-dependent pathway, after PAMPs binding
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with their TLRs, the adaptor protein MyD88 is recruited to the receptors. Then,
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MyD88 recruits the first protein kinase IRAK4 via interactions between their
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N-terminal
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trans-autophosphorylation
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phosphorylates the downstream kinases IRAK1 and IRAK2, which results in
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formation of the TRAF6-TAK1-IKK signalosome ultimately leads to the activation
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of the NF-kappa B and Jun N-terminal kinase pathway [17-19].
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So far, some IRAK4 homologous cDNA were cloned from zebrafish (Danio rerio)
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[20], roughskin sculpin (Trachidermus fasciatus) [21], half-smooth tongue sole
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(Cynoglossus semilaevis) [22], rainbow trout (Oncorhynchus mykiss) [23], grouper
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(Epinephelus coioides) [24], rock bream (Oplegnathus fasciatus) [25], and large
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yellow croaker (Larimichthys crocea) [26]. In zebrafish, DrIRAK4 contains the
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conserved domains that shared with insect and mammalian gene. Also, mRNA
75
expression of DrIRAK4 is significantly upregulated after exposure of zebrafish to
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Edwardsiella tarda [20]. It is suggested that fish posses some conserved
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TLR-signaling pathways similar to mammals. However, previous studies found that
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the functions of some fish IRAK4 were quite intriguing. Rainbow trout IRAK4
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significantly quenched TLR2-mediated signaling in HEK-293T cells [23]. When
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co-expressed with MyD88, grouper and large yellow croaker IRAK4 impaired
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MyD88 induced NF-kappa B activation [24, 26]. These results demonstrated that the
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functions of fish IRAK4 might be diverse.
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In the present study, we cloned and identified the full-length cDNA sequence of
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grass carp (Ctenopharyngodon idellus) IRAK4 (CiIRAK4). The expression patterns
85
analysis showed that the mRNA level of CiIRAK4 was dramatically up-regulated
domains, [13,
and
IRAK4
16].
The
undergoes activated
dimerization
IRAK4
and
subsequently
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distributed entirely in the cytoplasm in CIK cells. Furthermore, nuclear-cytosol
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extraction assays found overexpressing CiIRAK4 had very little effect on the nuclear
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translocation of NF-kappa B p65 in CIK cells. However, CiIRAK4 could promote
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IRF5 translocation to the nucleus, which has nothing to do with the interaction
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between IRAK4 and IRF5.
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2. Materials and Methods
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2.1. Reagents, Antibodies, and kits
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Medium 199 and DMEM were purchased from Corning, and DAPI was
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commercially acquired from Sangon Biotech. Polyclonal antibody for Histone H3
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was purchased from Affinity Biosciences, and mouse monoclonal antibody against
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GFP was purchased from Abmart, respectively. Goat anti-rabbit IgG-HRP and goat
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anti-mouse IgG-HRP were purchased from ZSGB-BIO. Anti-Flag and anti-GFP
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agarose conjugate were purchased from Sigma. Polyclonal antibodies for grass carp
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p65 (Suppl. Fig. 1) and GAPDH [27] were prepared and preserved in our laboratory.
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Small interfering RNA (siRNA) for IRAK4 and negative control RNA oligo were
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purchased from Shanghai GenePharma. Poly I:C and LPS were purchased from
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Sigma. HiperFect transfection reagent was purchased from QIAGEN. FuGENE®6
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Transfection Reagent was purchased from Promega. RNA simple Total RNA Kit was
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purchased from Tiangen. Super Script III reverse polymerase was purchased from
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Invitrogen. Script RT reagent kit with gDNA Eraser Perfect Real Time was
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purchased from TaKaRa. Nuclear and Cytoplasmic Protein Extraction Kit was
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purchased from Beyotime.
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2.2. Fish, cells and vectors
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Grass carps (about 20 g body weight) were obtained from Nanchang Shenlong
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Fisheries Development (Jiangxi, China) and acclimatized to the laboratory
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conditions for two weeks in a quarantine area. The CIK cell lines were kindly gifted
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by Professor Pin Nie, Institute of Hydrobiology, Chinese Academy of Sciences. CIK
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cells were cultured at 28 °C in M199 containing 10 % FCS, 100 µg/ml penicillin,
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and 100 µg/ml streptomycin. pEASY®-T1 and pcDNA3.1 (+) were purchased from
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TransGen and Invitrogen, respectively. p3×FLAG-MYC-CMV and pEGFPC1 were
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purchased from Sigma and Promega, respectively.
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2.3. Cloning and sequence analysis of grass carp IRAK4 cDNA
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Grass carp IRAK4 full length cDNA sequence (CiIRAK4) was obtained by
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RACE-PCR. Briefly, total RNA was extracted from CIK cells using RNA simple
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Total RNA Kit. SMART cDNA was prepared using Super Script III reverse
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polymerase according to manufacturer’s protocol. Based on the partial sequence of
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IRAK4 from Grass Carp Genome Database (GCGD) (CI_GC_18320), four
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degenerate
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3′-IRAK4-Inner (Table 1) were designed. The 5′-end was amplified by using the
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primer pair 5′-IRAK4-Out/UPM and 5′-IRAK4-Inner/NUP. The 3′-end was
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amplified by the primers 3′-IRAK4-Out/UPM, 3′-IRAK4-Inner/NUP. The PCR
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cycling conditions were: 95 °C/3 min, 35 cycles of 95 °C/30 s, 56 °C/30 s, 72 °C/1.5
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primers
5′-IRAK4-Out,
5′-IRAK4-Inner,
3′-IRAK4-Out
and
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transformed to E. coli strains DH5α for sequencing by Sangon Biotech.
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Polypeptide sequence prediction was determined by online-software ORF Finder
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(http://www.ncbi.nlm.nih.Gov/gorf/gorf.html). Known IRAK4s from different
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species were searched with NCBI BLAST (http://www.ncbi.nlm.nih.gov/ blast). The
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domain
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(http://smart.emblheidelberg.de/smart/saveuserpreferences.pl). Multiple alignments
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were generated using Clustal X 2.0 and the results were subsequently edited by using
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GeneDoc program. The phylogenetic tree was constructed by the Neighbor-Joining
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algorithm implemented in MEGA software version 6.0 (http://www.megasoftware.
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net).
of
CiIRAK4
were
predicted
from
SMART program
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2.4. Plasmids construction
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The open reading frame (ORF) of CiIRAK4 was inserted into pcDNA3.1 (+) vector
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to construct the expression plasmid pcDNA3.1/CiIRAK4 using for over-expression
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experiment.
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p3×FLAG-MYC-CMV
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co-immunoprecipitation assay, respectively. In addition, the ORF of CiIRF5 was
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cloned into pEGFP-C1. DNA sequencing confirmed all constructs. The primers used
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for plasmid construction are shown in Table 1.
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and
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2.5. Q-PCR analysis of CiIRAK4 mRNA expression in grass carp tissues and cells
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To determine the expression profile of CiIRAK4, grass carp were injected 10 mg/g
ACCEPTED MANUSCRIPT bodyweight Poly I:C. The control group fish were injected with PBS. After
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challenged with Poly I:C for 0 h, 6 h, 12 h, 24 h, 48 h and 72 h, total RNA were
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extracted from liver, spleen, kidney, brain, intestine and eye tissues by using RNA
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simple Total RNA Kit, respectively. CIK cells were seeded in 6-well plates to obtain
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80% confluence, then stimulated with poly I:C or LPS. Total RNA were extracted at
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different time (0 h, 6 h, 12 h, 24 h, 48 h and 72 h) post-stimulation of poly I:C or
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LPS. cDNA was reverse transcribed using the Prime Script RT reagent kit with
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gDNA Eraser Perfect Real Time. Quantitative real-time PCR (Q-PCR) was
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performed to detect the expression of CiIRAK4 with β-actin as an internal reference
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gene. It was performed as described in our previous study [28]. The primers for
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CiIRAK4 amplification were listed in Table 1.
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2.6. Knockdown and overexpression of CiIRAK4
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In RNAi-mediated gene knockdown assay, the specific siRNA sequence against
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CiIRAK4 and negative control RNA oligo were designed and synthesized from
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Shanghai GenePharma (Table 1). The RNAi-mediated knockdown was performed
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according to the respective protocol guidelines as described in our previous study
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[27].
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For overexpression of CiIRAK4, CIK cells were seeded in 6-well plates overnight. 6
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µl FuGENE®6 Transfection Reagent were diluted in 100 µl M199 (free of FCS) and
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incubated for 5 min. At the same time, 2 µg plasmids of pcDNA3.1/CiIRAK4 or
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pcDNA3.1 (+) were diluted in 100 µl M199 (free of FCS). Added the diluted
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FuGENE®6 Transfection Reagent to the diluted plasmids and mixed by vortexing.
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The mixture was incubated for 20 min at room temperature and dropped onto the
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cells.
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2.7. Extraction of cytoplasmic and nuclear proteins
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CIK cells were seeded in T25 culture flasks to reach ~80% confluence. Cells were
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harvested at 60 min post LPS treatment or 24 h post transfection and the cytoplasmic
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and nuclear proteins were extracted from CIK cells using the Nuclear and
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Cytoplasmic Protein Extraction Kit (Beyotime) according to the manufacturer’s
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instructions as described in our previous study [29]. Nuclear-cytoplasm translocation
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of Cip65 was detected with the primary antibody against p65 by Western blot as
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described in Materials and methods 2.9.
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2.8. Subcellular localization analysis
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CIK cells were plated on microscopic petri dishes to obtain 70-80% confluence, and
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transfected with pEGFP-C1/CiIRAK4. Meanwhile, CIK or HEK-293T cells were
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co-transfected with pEGFP-C1/CiIRF5 and pcDNA3.1/CiIRAK4. After transfection
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for 36 h, the cells were washed three times with PBS and fixed with 4% (v/v)
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paraformaldehyde for 15 min at room temperature. Then, the cells were dyed with
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DAPI (0.1 µg/ml) and examined under a confocal microscope (Leica).
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Meanwhile, the cytoplasmic and nuclear proteins were extracted from CIK cells as
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described in Materials and methods 2.7. Nuclear-cytoplasm translocation of CiIRF5
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was detected with the primary antibody against mouse monoclonal antibody against
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GFP by western blot.
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2.9. Western blotting
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For Western blot analysis, CIK cells were washed twice with PBS and lysed.
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Proteins were detected by western blotting with different antibody as described in
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our previous study [27]. The membrane was exposed to a chemiluminescence
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Imaging System (CLINX, China).
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2.10. Co-immunoprecipitation assay
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In Co-IP experiments, HEK-293T cells were co-transfected with pEGFP-C1/CiIRF5
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and p3×FLAG/CiIRAK4. At 36 h post-transfection, the cells were lysed and the
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cellular debris was removed by centrifugation at 12,000 g for 10 min at 4 °C. The
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supernatant was incubated with anti-Flag or anti-GFP agarose conjugate overnight at
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4 °C. Then, the beads were detected by immunoblotting with the indicated antibody.
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It was performed using the same strategy as described in our previous study [28].
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3. Results
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3.1. Sequence and phylogenetic analysis of CiIRAK4
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In this study, the full-length cDNA of IRAK4 in grass carp (CiIRAK4) had been
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cloned. CiIRAK4 was 2057 bp in length with 170 bp of 5′UTR and 465 bp of 3′UTR.
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The open reading frame was 1422 bp that could code a 472 amino acids peptide
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(Suppl. Fig. 2). The putative CiIRAK4 protein possessed an N-terminal death
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domain (DD) and a protein kinase domain (P kinase).
ACCEPTED MANUSCRIPT To better understand the sequence similarity of CiIRAK4 and that in other species,
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amino acid sequence multiple alignments were done. As shown in Fig. 1, IRAK4
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proteins showed highly conserved among different species, especially their death
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domain and protein kinase domain. For instance, CiIRAK4 has a similarity of 66%
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with IRAK4 of Danio rerio, 66% with Ictalurus punctatus, 54% with Gallus gallus,
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55% with Rattus norvegicus, 62% with Salmo salar, 55% with Homo sapiens, which
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revealed that CiIRAK4 shared homology with other fishes except with Danio rerio
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(65% similarity), and that homology was also found among mammals (Fig. 1).
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Meanwhile, a phylogenetic tree was constructed with 18 known IRAK4 members
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from mammals, amphibian, and fish. The results showed that CiIRAK4 shared high
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homologous (> 90% bootstrap value) with other fish IRAK4, especially with zebra
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fish. All of the fish IRAK4 sequences clustered together as well as diverged from
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their counterparts in other species (Fig. 2).
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3.2. Expression analysis of CiIRAK4 in tissues and cells of grass carp
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Q-PCR analysis was used to evaluate the expression profile of CiIRAK4 mRNA in
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different tissues. As shown in Fig. 3A, CiIRAK4 was expressed ubiquitously in all
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detected tissues (eye, liver, spleen, kidney, brain and intestine), with its higher
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expression in the liver. After injection with poly I:C, the expression levels of
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CiIRAK4 peaked at 6 h post-treatment in most tested tissues, especially obvious in
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liver (74.46-fold) and spleen (10.72-fold) (Fig. 3A). Then, it declined rapidly and
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reached normal or lower levels at 24 h post-treatment. It reduced continually and
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came to the lowest level at 72 h post-treatment.
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ACCEPTED MANUSCRIPT In addition, the expression patterns of CiIRAK4 in CIK cells were examined by
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Q-PCR. When CIK cells were challenged with poly I:C, the level of CiIRAK4
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mRNA was increased rapidly (4.6-fold) at 6 h post-treatment, and reached the peak
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(21.56-fold) at 48 h post-treatment (Fig. 3B). By contrast, CiIRAK4 was
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up-regulated and reached the peak (46.73-fold) at 12 h after CIK cells were treated
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with LPS (Fig. 3B).
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3.3. Subcellular localization of CiIRAK4
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To investigate the subcellular localization of CiIRAK4, pEGFP-C1/CiIRAK4 was
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transfected into CIK cells. The results revealed that CiIRAK4 was distributed
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entirely in the cytoplasm, whereas GFP empty protein was located in both the
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cytoplasm and the nucleus (Fig. 4A). Also, it was confirmed by Western blotting
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analysis (Fig. 4B). When CIK cells were transfected with pEGFP-C1/CiIRAK4,
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GFP-IRAK4 fusion protein was detected by GFP antibody at the correct molecular
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weight.
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3.4. Effect of CiIRAK4 on NF-kappa B p65
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To explore the effect of CiIRAK4 on NF-kappa B p65, knockdown and
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overexpression of CiIRAK4 assays were preformed. The level of CiIRAK4 mRNA
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was diminished in the cells transfected with siRNA against CiIRAK4 gene,
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confirming the siRNA specifically knocked down IRAK4 in CIK cells. When the
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cells transfected with pcDNA3.1/CiIRAK4, IRAK4 mRNA level was found to be
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up-regulated for 3.66-fold compared to the control (Suppl. Fig. 3).
ACCEPTED MANUSCRIPT After CIK cells were transfected with pcDNA3.1/CiIRAK4 or siRNA against
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CiIRAK4 gene, the cytoplasmic and nuclear proteins were extracted. As shown in
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Fig. 5, compared with the mock, CIK cells treated with LPS for 60 min could be
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observed a part of NF-kappa B p65 translocation into the nucleus. When CiIRAK4
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was overexpressed in CIK cells, it displayed that only a small number of NF-kappa B
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p65 were translocated into the nucleus. Meanwhile, as CiIRAK4 knockdown in CIK
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cells, the proportion of NF-kappa B p65 nuclear translocation was similar to that of
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negative control RNA oligo and vector (pcDNA3.1) transfection alone. These data
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suggested that CiIRAK4 might have little effect on NF-kappa B p65 nuclear
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translocation.
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3.5. IRAK4 promotes IRF5 nuclear translocation
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Recently, IRAK4 was verified to have a minor effect on the activation and nuclear
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translocation of NF-kappa B p65 [30]. Also, IRAK4 kinase activity was required for
284
IRF5 nuclear translocation but redundant for NF-kappa B [31]. Then, we next
285
investigated whether CiIRAK4 could promote IRF5 translocation to the nucleus.
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pcDNA3.1/CiIRAK4 and pEGFP-C1/CiIRF5 were cotransfected into CIK cells. As
287
shown in Fig. 6A, some green granules were obviously filled in the nuclear regions,
288
suggesting that CiIRF5 was translocated to the nucleus when cells cotransfected with
289
CiIRAK4. Also, this result could be found in HEK-293T cells (Suppl. Fig. 4).
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Consistent with the observation above, as shown in Fig. 6B, GFP-IRF5 could be
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found
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pEGFP-C1/CiIRF5 alone. Correspondingly, when CIK cells were co-expressed
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the
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were
transfected
with
ACCEPTED MANUSCRIPT CiIRF5 with CiIRAK4, some of GFP-IRF5 were translocated from the cytoplasm to
294
the nucleus (Fig. 6B).
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Subsequently, co-immunoprecipitation was used to detect if IRF5 nuclear
296
translocation controlled by IRAK4 related to the interaction between IRAK4 and
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IRF5. p3×FLAG/CiIRAK4 and pEGFP-C1/CiIRF5 were co-transfected into
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HEK-293T cells. As shown in Fig. 7, IRAK4 could not directly interact with IRF5.
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These results suggested that IRAK4 promoted IRF5 nuclear translocation via
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activating some other signal factors.
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4. Discussion
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IRAK4 plays a pivotal role in the signaling pathways of TLRs/IL-1Rs. It can initiate
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a cascade of signaling events and leads to induction of inflammatory target gene
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expression eventually [32]. To date, some homologs of IRAK4 have been found in
307
some different species of fish except in mammals. In this study, a novel teleost
308
IRAK4 cDNA was identified from grass carp. Similar to other IRAK4 subfamily
309
member, CiIRAK4 protein contained an N-terminal death domain (DD) and a
310
C-terminal kinase domain. Customarily, MyD88 activates IRAK4 by binding to
311
certain sites in the IRAK4 death domain and transducts signals to the downstream
312
factors of the TLRs pathway [33]. In addition, the alignment and phylogenetic
313
analysis also demonstrated that IRAK4 was highly conserved in vertebrates, and
314
grass carp IRAK4 shared the highest homology with zebra fish IRAK4.
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In consistent with the previous reports, CiIRAK4 was constitutively expressed in
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ACCEPTED MANUSCRIPT different tested tissues, with the higher expression level in the immune-related
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tissues, such as liver, spleen and kidney. It indicated that CiIRAK4 was essential for
318
immune defense. Meanwhile, the expression of CiIRAK4 was significantly
319
up-regulated at 6 hours after poly I:C stimulation, especially in the liver and spleen.
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Similarly, The expression of large yellow croaker IRAK4 was significantly induced
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by poly I:C treatment in the spleen and liver tissues [26, 34]. Additionally, some
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studies revealed that the expression of fish IRAK4 is different for various PAMPs.
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Rock bream IRAK4 was up-regulated after infection of rock bream iridovirus (RBIV)
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[25], but large yellow croaker and trout IRAK4 showed no significant change after
325
infection of V. parahemolyticus and Aeromonas salmonicida, respectively [23, 26].
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Zebrafish IRAK4 could be observably induced by Edwardsiella tarda, whereas it was
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down-regulated after infection with snakehead rhabdovirus (SHRV) [20]. Similarly,
328
as revealed in the present study, CiIRAK4 mRNA was induced by poly I:C and LPS
329
in CIK cells. The level of CiIRAK4 mRNA was elevated first at 6 h after poly I:C
330
treatment and declined to the normal levels, then it reached the peak at 48 h.
331
However, CiIRAK4 mRNA was up-regulated significantly and peaked at 12 h after
332
LPS infection (Fig. 3B). It could be postulated that recognition of pathogenic pattern
333
receptors by different signaling pathways might be responsible for the differential
334
expression of IRAK4 in fish.
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In MyD88-dependent pathway, upon binding of ligands, TLRs recruit the adaptor
336
molecule MyD88 through the TIR domain. Then, MyD88 recruits IRAK4 and
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initiates an intracellular signaling cascade [17]. It's no doubt that IRAK4 is located in
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the cytoplasm in cells. Similar to other fish species [23, 24, 26], when GFP-tagged
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ACCEPTED MANUSCRIPT CiIRAK4 was expressed in CIK cells, it could be obviously detected that IRAK4
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distributed entirely in the cytoplasm, which was confirmed by Western blot result by
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using a GFP-specific antibody (Fig. 4).
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It is undoubted that IRAK4 plays the critical roles in TLRs signaling. Paradoxically,
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the function of IRAK4 is quite diverse. In mammals, Yang et al. found that human
344
IFN-alpha/beta and -lambda could be induced by TLR-3 and TLR-4 agonists in
345
IRAK-4-deficient cells. In contrast, TLR-7, -8, and -9-mediated induction of
346
IFN-alpha/beta and -lambda was strictly IRAK-4 dependent [35]. Recent studies
347
have demonstrated that IRAK4 played a dual role in Myddosome formation and
348
TLR signaling. On the one hand, IRAK4 had a critical scaffold function in
349
Myddosome formation, and the kinase activity of IRAK4 was dispensable for
350
Myddosome assembly and activation of the NF-kappa B and MAPK pathways. On
351
the other, IRAK4 was essential for MyD88-dependent production of inflammatory
352
cytokines [30]. In teleost fishes, IRAK4 seems to perform different functions. In
353
zebra fish, overexpression of IRAK-4 in ZFL cells resulted in NF-kappa
354
B-dependent luciferase expression, indicating that zfIRAK-4 could activate the
355
NF-kappa B signal transduction pathway [20]. In contrast, previous studies found
356
that grouper and large yellow croaker IRAK4 could impair MyD88-mediated
357
NF-kappa B activation in HEK-293T cells [24, 26]. In the present study, we
358
identified that CiIRAK4 was unnecessary for nuclear translocation of NF-kappa B
359
p65 (Fig. 5), which might imply that CiIRAK4 has little effects on the activation of
360
NF-kappa B p65.
361
Recent studies from primary human monocytes revealed that IRAK4 kinase
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ACCEPTED MANUSCRIPT activated TAK1 and IKK, and the activated IKK then phosphorylated IRF5 and
363
induced nuclear translocation of IRF5. During this process, IRAK4 kinase activity
364
was required for IRF5 nuclear translocation but redundant for NF-kappa B [31].
365
Here, when CIK cells were cotransfected with CiIRF5 and CiIRAK4, it could be
366
noticed that IRAK4 promoted nuclear translocation of some IRF5 (Fig. 6), which has
367
nothing to do with the interaction between IRAK4 and IRF5 (Fig. 7). It suggested
368
that fish IRAK4 kinase regulated IRF5 activity through indirect ways. Therefore, it
369
could be postulated that CiIRAK4 was dispensable for activation of the NF-kappa B
370
pathways, nevertheless, it was critical for TLR7/8 cytokine responses via IRF5 in grass
371
carp. Further work will be needed to test this hypothesis.
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Acknowledgements
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This work was supported by research grants from the National Natural Science
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Foundation of China (31560594) and the Science & Technology Foundations of
377
Education Department of Jiangxi (GJJ161303).
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myddosome formation and Toll-like receptor signaling, J. Biol. Chem. 293
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Figure Legends
486
Fig. 1. Multiple alignment of CiIRAK4 with the amino acid sequences of known
488
typical organisms IRAK4s by Clustal X 2.0 program.
489
Identical (shaded in black) and similar (shaded in gray and light gray) residues
490
identified by the ClustalW program are indicated. Both death domain (DD) and
491
protein kinase domain (Pkinase) are indicated with arrows.
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Fig. 2. Phylogenetic analysis of IRAK4s.
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A neighbour-joining tree was constructed by MEGA 6.0. All sequences used for
495
analysis were derived from GenBank and their accession numbers were shown in
496
parentheses. The bootstrap confidence values shown at the nodes of the tree were
497
based on 1000 bootstrap replications.
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Fig. 3. Expression of CiIRAK4 in response to poly I:C or LPS induction.
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(A) Expression analysis of CiIRAK4 mRNA in grass carp tissues (eye, liver, spleen,
501
kidney, brain, and intestine) at various time following infected with poly I:C. Eye
502
(treatment for 0 h) was used as the calibrator (B) Expression analysis of CiIRAK4
503
mRNA in CIK cells at various time following induced with poly I:C or LPS. The
504
data shown were derived from a representative experiment reported as the mean (n
505
=3)±S.D. * and ** represented significant (p<0.05) and highly significant (p<0.01),
506
respectively.
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ACCEPTED MANUSCRIPT Fig. 4. Subcellular localization of CiIRAK4.
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(A) CIK cells were seeded on microscopy dishes and transiently transfected with 2
509
µg of pEGFP-C1/CiIRAK4 plasmids. After 36 h of transfection, the cells were fixed
510
and examined with confocal microscopy. The CIK cells transfected with pEGFP-C1
511
were used as control. Green staining represents the CiIRAK4 protein signal, and blue
512
staining indicates the nucleus region. The results are representative of three
513
independent experiments. Scale bars represent 25 µM. (B) The expression of GFP
514
fusion proteins were confirmed by Western blotting analysis using mouse
515
monoclonal antibody against GFP.
516
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Fig. 5. CiIRAK4 is dispensable for the nuclear translocation of NF-kappa B p65.
518
CIK cells were seeded in T25 culture flasks overnight. LPS treatment,
519
RNAi-mediated gene knockdown and overexpression of CiIRAK4 assays were
520
preformed, respectively. Cells were harvested at 60 min post LPS treatment or 24 h
521
post transfection. The cytoplasmic and nuclear proteins were extracted from these
522
cells for Western blot. GAPDH and Histone were used as internal references for
523
cytoplasmic and nuclear proteins, respectively. The intensity of bands on a Western
524
blot was measured by Image J (https://imagej.nih.gov/ij/). Results were shown by
525
columns. Values were mean ± SD of three experiments. C, cytoplasmic proteins; N,
526
nuclear proteins; mock, untreated cells; siRNA, RNAi-mediated gene knockdown of
527
CiIRAK4; NC, negative control RNA oligo.
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ACCEPTED MANUSCRIPT Fig. 6. IRAK4 promotes IRF5 nuclear translocation.
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(A) CIK cells were seeded on microscopy dishes and co-transfected with
531
pEGFP-C1/CiIRF5 and pcDNA3.1/CiIRAK4. After transfection for 36 h, the cells
532
were fixed and examined with confocal microscopy. The transfection of
533
pEGFP-C1/CiIRF5 alone into CIK cells acted as a control. Green granules in
534
nucleus represent the nuclear translocation of CiIRF5 protein (indicated with arrow).
535
The results are representative of three independent experiments. Scale bars represent
536
25 µM. (B) At the same time, the CIK cells were harvested and subjected to
537
separation of subcellular components. The indicated proteins in the cytoplasmic (C)
538
and nuclear (N) fraction were then tested by for Western blot. GAPDH and Histone
539
were used as internal references for cytoplasmic and nuclear proteins, respectively.
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Fig. 7. IRAK4 can not directly interact with IRF5.
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HEK-293T cells seeded in 10 cm2 dishes were co-transfected with 5 µg
543
p3×FLAG/CiIRAK4 and pEGFP-C1/CiIRF5 plasmid. At 48 h post-transfection, the
544
whole-cell lysates were immunoprecipitated (IP) with either anti-Flag affinity gel (A)
545
or anti-GFP-agarose beads (B). The immunoprecipitates and cell lysates were then
546
analyzed by immunoblotting (IB) with the anti-Flag and anti-GFP antibody,
547
respectively.
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ACCEPTED MANUSCRIPT Table 1 Primers used for all the experiments. Name
Sequence (5’ to 3’)
Application
5′-IRAK4-Out
TGCTGAACGGGTTTAGTGAC
3′-IRAK4-Out
RI PT
5′-IRAK4-Inner GATGAGCCCGCTTTAGTGTGG GAGATGCTGTTCGACTGGG
Race-PCR
3′-IRAK4-Inner ATTCTGATCCGACACCAGCTG
CTAATACGACTCACTATAGGGCAAAGCAGTGGTATCAACGCAGAGT
Short
CTAATACGACTCACTATA
NUP
AAGCAGTGGTATCAACGCAGAG
IRAK4-ORF-F
ATGTGTGACGTCACGCGGG
IRAK4-ORF-R
TCATCCAGAGCTCTGGGAATG
IRAK4-RT-F
CTCCACACTGAGAGCTTTATC
IRAK4-RT-R
ATGTGCAGCTGTGTGTATCT
β-actin-F
CACTGTGCCCATCTACGAG
β-actin-R
CCATCTCCTGCTCGAAGTC
3.1-IRAK4-F
GCGGTACCATGTGTGACGTCACGCGGG
3.1-IRAK4-R
CGGAATTCTCATCCAGAGCTCTGGGAATG
GFP-IRAK4-F
CGGAATTCTATGTGTGACGTCACGCGGG
GFP-IRAK4-R
GCGGTACCTCATCCAGAGCTCTGGGAATG
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Flag-IRAK4-F
CGGAATTCTATGTGTGACGTCACGCGGG
Flag-IRAK4-R
GCGGTACCTCATCCAGAGCTCTGGGAATG
GFP-IRF5-F
CGGAATTCTATGAGCGGTCAACCACGGAG
GFP-IRF5-F
GCGGTACCTTAGTGGATGTTTTGAGGCC
IRAK4 siRNA
Q-PCR
Eukaryotic vector construction
GGAGGUGGAGAGGAUGUAUTT AUACAUCCUCUCCACCUCCTT
negative control
ORF cloning
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Long
UUCUCCGAACGUGUCACGUTT ACGUGACACGUUCGGAGAATT
Knockdown
ACCEPTED MANUSCRIPT
Fig. 1
Death domain (DD)
RI PT
C. idellus D. rerio E. coioides O. mykiss S. salar C. semilaevis L. crocea I. punctatus X. tropicalis B. taurus M. musculus H. sapiens
M AN U TE D
AC C
C. idellus D. rerio E. coioides O. mykiss S. salar C. semilaevis L. crocea I. punctatus X. tropicalis B. taurus M. musculus H. sapiens
EP
C. idellus D. rerio E. coioides O. mykiss S. salar C. semilaevis L. crocea I. punctatus X. tropicalis B. taurus M. musculus H. sapiens
SC
C. idellus D. rerio E. coioides O. mykiss S. salar C. semilaevis L. crocea I. punctatus X. tropicalis B. taurus M. musculus H. sapiens
C. idellus D. rerio E. coioides O. mykiss S. salar C. semilaevis L. crocea I. punctatus X. tropicalis B. taurus M. musculus H. sapiens
Protein kinase domain (Pkinase)
ACCEPTED MANUSCRIPT
Protein kinase domain (Pkinase)
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C. idellus D. rerio E. coioides O. mykiss S. salar C. semilaevis L. crocea I. punctatus X. tropicalis B. taurus M. musculus H. sapiens
SC
C. idellus D. rerio E. coioides O. mykiss S. salar C. semilaevis L. crocea I. punctatus X. tropicalis B. taurus M. musculus H. sapiens
RI PT
C. idellus D. rerio E. coioides O. mykiss S. salar C. semilaevis L. crocea I. punctatus X. tropicalis B. taurus M. musculus H. sapiens
ACCEPTED MANUSCRIPT
AC C
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Fig. 2
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A
AC C
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B
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DAPI
GFP
SC TE D
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GFP-IRAK4 B
Merged
RI PT
pEGFP-C1
A
AC C
EP
GFP-IRAK4
pEGFP-C1
ACCEPTED MANUSCRIPT Fig. 5 Mock C
LPS N
C
pcDNA3.1/IRAK4 N
C
pcDNA3.1
N
C
p65
Histone
siRNA C
N
NC C
N
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p65
SC
RI PT
GAPDH
GAPDH
N C
siR N A
pc D N A 3. 1
LP pc S D N A 3. 1/ IR A K 4
C on tr l
AC C
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Histone
N
ACCEPTED MANUSCRIPT Fig. 6
A
DAPI
SC
GFP-IRF5
TE D
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IRAK4+GFP-IRF5
IRF5
B
AC C
GAPDH Histone
IRAK4+IRF5
N
EP
C GFP-IRF5
Merged
RI PT
GFP
C
N
pcDNA3.1+IRF5 C
N
ACCEPTED MANUSCRIPT Fig. 7
A
＋ －
－ ＋
＋ ＋
RI PT
IP: GFP
GFP-IRF5 Flag-IRAK4
IB: Flag
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IB: GFP (IRF5) Input
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Input
－ ＋
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IP: GFP
＋ －
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B
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IB: Flag (IRAK4)
＋ ＋
Flag-IRAK4 GFP-IRF5
IB: Flag
IB: GFP (IRF5)
IB: Flag (IRAK4)
ACCEPTED MANUSCRIPT Highlights
An IRAK4 orthologue from grass carp (Ctenopharyngodon idella) was cloned.
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CiIRAK4 shared high homologous with zebra fish IRAK4. CiIRAK4 was significantly up-regulated after treatment with poly I:C and LPS.
CiIRAK4 was located in the cytoplasm.
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CiIRAK4 was dispensable for activation of NF-kappa B p65. However,
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IRAK4 promoted IRF5 nuclear translocation, which has nothing to do with the
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interaction between IRAK4 and IRF5.
S1050-4648(19)30041-5
DOI:
https://doi.org/10.1016/j.fsi.2019.01.031
Reference:
YFSIM 5876
To appear in:
Fish and Shellfish Immunology
Received Date: 21 October 2018 Revised Date:
11 January 2019
Accepted Date: 23 January 2019
Please cite this article as: Wu C, Xu X, Zhi X, Jiang Z, Li Y, Xie X, Chen X, Hu C, Identification and functional characterization of IRAK-4 in grass carp (Ctenopharyngodon idellus), Fish and Shellfish Immunology (2019), doi: https://doi.org/10.1016/j.fsi.2019.01.031. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Identification and functional characterization of IRAK-4 in grass
2
carp (Ctenopharyngodon idellus)
3
Chuxin Wu a, Xiaowen Xu b, Xiaoping Zhi a, Zeyin Jiang b, Yinping Li b, Xiaofen
4
Xie b, Xingxing Chen b, Chengyu Hu b*
5 6
a
7
b
8
Nanchang 330031, China
Yuzhang Normal University, Nanchang 330103, China
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Chengyu Hu, Ph.D.
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* Corresponding author:
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Department of Bioscience, College of Life Science, Nanchang University,
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12
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Department of Bioscience, College of Life Science, Nanchang University, Nanchang
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330031, China.
18
Fax: +86-791-8396-9530, E-mail: [email protected]
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ACCEPTED MANUSCRIPT Abstract
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IL-1R-associated kinase 4 (IRAK4), a central TIR signaling mediator in innate
21
immunity, can initiate a cascade of signaling events and lead to induction of
22
inflammatory target gene expression eventually. In the present study, we cloned and
23
characterized an IRAK4 orthologue from grass carp (Ctenopharyngodon idella). The
24
full length cDNA of CiIRAK4 was 2057 bp with an ORF of 1422 bp encoding a
25
polypeptide of 472 amino acids. Multiple alignments showed that IRAK4s were
26
highly conserved among different species. Phylogenetic tree analysis revealed that
27
CiIRAK4 shared high homologous with zebra fish IRAK4. Expression analysis
28
indicated that CiIRAK4 was widely expressed in all tested tissues. It was
29
significantly up-regulated after treatment with poly I:C, especially obvious in liver
30
and spleen. Also, CiIRAK4 could be induced by poly I:C and LPS in CIK cells.
31
Fluorescence microscopy assays showed that CiIRAK4 localized in the cytoplasm.
32
RNAi-mediated knockdown and overexpression assays indicated that CiIRAK4
33
might have little effect on NF-kappa B p65 translocation from cytoplasm to nucleus，
34
indicating that CiIRAK4 was dispensable for activation of NF-kappa B p65. In
35
addition, IRAK4 promoted IRF5 nuclear translocation, which has nothing to do with
36
the interaction between IRAK4 and IRF5. It suggested that fish IRAK4 kinase
37
regulated IRF5 activity through indirect ways.
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Keywords: IRAK4; NF-kappa B p65; IRF5; function; grass carp
ACCEPTED MANUSCRIPT 1. Introduction
41
The innate immune system is the first line of host defense against pathogens,
42
particularly important for lower vertebrates to defense against viral or microbial
43
invasion [1, 2]. The recognition of invading pathogens is a pivotal step to trigger
44
intracellular signaling cascades in host cells, and pattern-recognition receptors
45
(PRRs) play a critical role in this process [3]. Different PRRs react with specific
46
pathogen-associated molecular partterns (PAMPs), activate specific signaling
47
pathways, and lead to distinct antipathogen responses [4]. Toll-like receptors (TLRs),
48
the first identified and the most well characterized PRRs, can trigger innate immune
49
responses and prime antigen-specific adaptive immunity [5, 6]. To date, 10 and 12
50
functional TLRs have been identified in human and mouse respectively, and they are
51
evolutionarily conserved from worm to mammals [7]. Containing intracellular
52
Toll–interleukin 1 (IL-1) receptor (TIR) domains, TLRs recruit a specific set of
53
adaptor molecules, such as MyD88 and TRIF, and initiate distinct signaling
54
pathways [8]. In the MyD88-dependent pathway, IL-1 receptor-associated kinases
55
(IRAKs) play a critical role in linking TLR signaling to the nuclear factor κB
56
(NF-kappa B) pathway [8, 9].
57
In mammals, IRAKs is comprised of four family members: IRAK1, IRAK2, IRAK3
58
(IRAKM), and IRAK4 [10-13]. Although all of them contain an N-terminal death
59
domain (DD), a proST domain, and a conserved central kinase domain, only IRAK1
60
and IRAK4 have real kinase activity, while IRAK2 and IRAK3 are inactive
61
pseudokinases [14, 15]. In the MyD88-dependent pathway, after PAMPs binding
62
with their TLRs, the adaptor protein MyD88 is recruited to the receptors. Then,
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MyD88 recruits the first protein kinase IRAK4 via interactions between their
64
N-terminal
65
trans-autophosphorylation
66
phosphorylates the downstream kinases IRAK1 and IRAK2, which results in
67
formation of the TRAF6-TAK1-IKK signalosome ultimately leads to the activation
68
of the NF-kappa B and Jun N-terminal kinase pathway [17-19].
69
So far, some IRAK4 homologous cDNA were cloned from zebrafish (Danio rerio)
70
[20], roughskin sculpin (Trachidermus fasciatus) [21], half-smooth tongue sole
71
(Cynoglossus semilaevis) [22], rainbow trout (Oncorhynchus mykiss) [23], grouper
72
(Epinephelus coioides) [24], rock bream (Oplegnathus fasciatus) [25], and large
73
yellow croaker (Larimichthys crocea) [26]. In zebrafish, DrIRAK4 contains the
74
conserved domains that shared with insect and mammalian gene. Also, mRNA
75
expression of DrIRAK4 is significantly upregulated after exposure of zebrafish to
76
Edwardsiella tarda [20]. It is suggested that fish posses some conserved
77
TLR-signaling pathways similar to mammals. However, previous studies found that
78
the functions of some fish IRAK4 were quite intriguing. Rainbow trout IRAK4
79
significantly quenched TLR2-mediated signaling in HEK-293T cells [23]. When
80
co-expressed with MyD88, grouper and large yellow croaker IRAK4 impaired
81
MyD88 induced NF-kappa B activation [24, 26]. These results demonstrated that the
82
functions of fish IRAK4 might be diverse.
83
In the present study, we cloned and identified the full-length cDNA sequence of
84
grass carp (Ctenopharyngodon idellus) IRAK4 (CiIRAK4). The expression patterns
85
analysis showed that the mRNA level of CiIRAK4 was dramatically up-regulated
domains, [13,
and
IRAK4
16].
The
undergoes activated
dimerization
IRAK4
and
subsequently
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87
distributed entirely in the cytoplasm in CIK cells. Furthermore, nuclear-cytosol
88
extraction assays found overexpressing CiIRAK4 had very little effect on the nuclear
89
translocation of NF-kappa B p65 in CIK cells. However, CiIRAK4 could promote
90
IRF5 translocation to the nucleus, which has nothing to do with the interaction
91
between IRAK4 and IRF5.
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2. Materials and Methods
94
2.1. Reagents, Antibodies, and kits
95
Medium 199 and DMEM were purchased from Corning, and DAPI was
96
commercially acquired from Sangon Biotech. Polyclonal antibody for Histone H3
97
was purchased from Affinity Biosciences, and mouse monoclonal antibody against
98
GFP was purchased from Abmart, respectively. Goat anti-rabbit IgG-HRP and goat
99
anti-mouse IgG-HRP were purchased from ZSGB-BIO. Anti-Flag and anti-GFP
100
agarose conjugate were purchased from Sigma. Polyclonal antibodies for grass carp
101
p65 (Suppl. Fig. 1) and GAPDH [27] were prepared and preserved in our laboratory.
102
Small interfering RNA (siRNA) for IRAK4 and negative control RNA oligo were
103
purchased from Shanghai GenePharma. Poly I:C and LPS were purchased from
104
Sigma. HiperFect transfection reagent was purchased from QIAGEN. FuGENE®6
105
Transfection Reagent was purchased from Promega. RNA simple Total RNA Kit was
106
purchased from Tiangen. Super Script III reverse polymerase was purchased from
107
Invitrogen. Script RT reagent kit with gDNA Eraser Perfect Real Time was
108
purchased from TaKaRa. Nuclear and Cytoplasmic Protein Extraction Kit was
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purchased from Beyotime.
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2.2. Fish, cells and vectors
112
Grass carps (about 20 g body weight) were obtained from Nanchang Shenlong
113
Fisheries Development (Jiangxi, China) and acclimatized to the laboratory
114
conditions for two weeks in a quarantine area. The CIK cell lines were kindly gifted
115
by Professor Pin Nie, Institute of Hydrobiology, Chinese Academy of Sciences. CIK
116
cells were cultured at 28 °C in M199 containing 10 % FCS, 100 µg/ml penicillin,
117
and 100 µg/ml streptomycin. pEASY®-T1 and pcDNA3.1 (+) were purchased from
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TransGen and Invitrogen, respectively. p3×FLAG-MYC-CMV and pEGFPC1 were
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purchased from Sigma and Promega, respectively.
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2.3. Cloning and sequence analysis of grass carp IRAK4 cDNA
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Grass carp IRAK4 full length cDNA sequence (CiIRAK4) was obtained by
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RACE-PCR. Briefly, total RNA was extracted from CIK cells using RNA simple
124
Total RNA Kit. SMART cDNA was prepared using Super Script III reverse
125
polymerase according to manufacturer’s protocol. Based on the partial sequence of
126
IRAK4 from Grass Carp Genome Database (GCGD) (CI_GC_18320), four
127
degenerate
128
3′-IRAK4-Inner (Table 1) were designed. The 5′-end was amplified by using the
129
primer pair 5′-IRAK4-Out/UPM and 5′-IRAK4-Inner/NUP. The 3′-end was
130
amplified by the primers 3′-IRAK4-Out/UPM, 3′-IRAK4-Inner/NUP. The PCR
131
cycling conditions were: 95 °C/3 min, 35 cycles of 95 °C/30 s, 56 °C/30 s, 72 °C/1.5
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primers
5′-IRAK4-Out,
5′-IRAK4-Inner,
3′-IRAK4-Out
and
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133
transformed to E. coli strains DH5α for sequencing by Sangon Biotech.
134
Polypeptide sequence prediction was determined by online-software ORF Finder
135
(http://www.ncbi.nlm.nih.Gov/gorf/gorf.html). Known IRAK4s from different
136
species were searched with NCBI BLAST (http://www.ncbi.nlm.nih.gov/ blast). The
137
domain
138
(http://smart.emblheidelberg.de/smart/saveuserpreferences.pl). Multiple alignments
139
were generated using Clustal X 2.0 and the results were subsequently edited by using
140
GeneDoc program. The phylogenetic tree was constructed by the Neighbor-Joining
141
algorithm implemented in MEGA software version 6.0 (http://www.megasoftware.
142
net).
of
CiIRAK4
were
predicted
from
SMART program
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2.4. Plasmids construction
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The open reading frame (ORF) of CiIRAK4 was inserted into pcDNA3.1 (+) vector
146
to construct the expression plasmid pcDNA3.1/CiIRAK4 using for over-expression
147
experiment.
148
p3×FLAG-MYC-CMV
149
co-immunoprecipitation assay, respectively. In addition, the ORF of CiIRF5 was
150
cloned into pEGFP-C1. DNA sequencing confirmed all constructs. The primers used
151
for plasmid construction are shown in Table 1.
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localization
pEGFP-C1
and
assay
and
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2.5. Q-PCR analysis of CiIRAK4 mRNA expression in grass carp tissues and cells
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To determine the expression profile of CiIRAK4, grass carp were injected 10 mg/g
ACCEPTED MANUSCRIPT bodyweight Poly I:C. The control group fish were injected with PBS. After
156
challenged with Poly I:C for 0 h, 6 h, 12 h, 24 h, 48 h and 72 h, total RNA were
157
extracted from liver, spleen, kidney, brain, intestine and eye tissues by using RNA
158
simple Total RNA Kit, respectively. CIK cells were seeded in 6-well plates to obtain
159
80% confluence, then stimulated with poly I:C or LPS. Total RNA were extracted at
160
different time (0 h, 6 h, 12 h, 24 h, 48 h and 72 h) post-stimulation of poly I:C or
161
LPS. cDNA was reverse transcribed using the Prime Script RT reagent kit with
162
gDNA Eraser Perfect Real Time. Quantitative real-time PCR (Q-PCR) was
163
performed to detect the expression of CiIRAK4 with β-actin as an internal reference
164
gene. It was performed as described in our previous study [28]. The primers for
165
CiIRAK4 amplification were listed in Table 1.
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2.6. Knockdown and overexpression of CiIRAK4
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In RNAi-mediated gene knockdown assay, the specific siRNA sequence against
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CiIRAK4 and negative control RNA oligo were designed and synthesized from
170
Shanghai GenePharma (Table 1). The RNAi-mediated knockdown was performed
171
according to the respective protocol guidelines as described in our previous study
172
[27].
173
For overexpression of CiIRAK4, CIK cells were seeded in 6-well plates overnight. 6
174
µl FuGENE®6 Transfection Reagent were diluted in 100 µl M199 (free of FCS) and
175
incubated for 5 min. At the same time, 2 µg plasmids of pcDNA3.1/CiIRAK4 or
176
pcDNA3.1 (+) were diluted in 100 µl M199 (free of FCS). Added the diluted
177
FuGENE®6 Transfection Reagent to the diluted plasmids and mixed by vortexing.
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The mixture was incubated for 20 min at room temperature and dropped onto the
179
cells.
180
2.7. Extraction of cytoplasmic and nuclear proteins
182
CIK cells were seeded in T25 culture flasks to reach ~80% confluence. Cells were
183
harvested at 60 min post LPS treatment or 24 h post transfection and the cytoplasmic
184
and nuclear proteins were extracted from CIK cells using the Nuclear and
185
Cytoplasmic Protein Extraction Kit (Beyotime) according to the manufacturer’s
186
instructions as described in our previous study [29]. Nuclear-cytoplasm translocation
187
of Cip65 was detected with the primary antibody against p65 by Western blot as
188
described in Materials and methods 2.9.
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CIK cells were plated on microscopic petri dishes to obtain 70-80% confluence, and
192
transfected with pEGFP-C1/CiIRAK4. Meanwhile, CIK or HEK-293T cells were
193
co-transfected with pEGFP-C1/CiIRF5 and pcDNA3.1/CiIRAK4. After transfection
194
for 36 h, the cells were washed three times with PBS and fixed with 4% (v/v)
195
paraformaldehyde for 15 min at room temperature. Then, the cells were dyed with
196
DAPI (0.1 µg/ml) and examined under a confocal microscope (Leica).
197
Meanwhile, the cytoplasmic and nuclear proteins were extracted from CIK cells as
198
described in Materials and methods 2.7. Nuclear-cytoplasm translocation of CiIRF5
199
was detected with the primary antibody against mouse monoclonal antibody against
200
GFP by western blot.
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2.9. Western blotting
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For Western blot analysis, CIK cells were washed twice with PBS and lysed.
204
Proteins were detected by western blotting with different antibody as described in
205
our previous study [27]. The membrane was exposed to a chemiluminescence
206
Imaging System (CLINX, China).
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2.10. Co-immunoprecipitation assay
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In Co-IP experiments, HEK-293T cells were co-transfected with pEGFP-C1/CiIRF5
210
and p3×FLAG/CiIRAK4. At 36 h post-transfection, the cells were lysed and the
211
cellular debris was removed by centrifugation at 12,000 g for 10 min at 4 °C. The
212
supernatant was incubated with anti-Flag or anti-GFP agarose conjugate overnight at
213
4 °C. Then, the beads were detected by immunoblotting with the indicated antibody.
214
It was performed using the same strategy as described in our previous study [28].
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3. Results
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3.1. Sequence and phylogenetic analysis of CiIRAK4
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In this study, the full-length cDNA of IRAK4 in grass carp (CiIRAK4) had been
220
cloned. CiIRAK4 was 2057 bp in length with 170 bp of 5′UTR and 465 bp of 3′UTR.
221
The open reading frame was 1422 bp that could code a 472 amino acids peptide
222
(Suppl. Fig. 2). The putative CiIRAK4 protein possessed an N-terminal death
223
domain (DD) and a protein kinase domain (P kinase).
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225
amino acid sequence multiple alignments were done. As shown in Fig. 1, IRAK4
226
proteins showed highly conserved among different species, especially their death
227
domain and protein kinase domain. For instance, CiIRAK4 has a similarity of 66%
228
with IRAK4 of Danio rerio, 66% with Ictalurus punctatus, 54% with Gallus gallus,
229
55% with Rattus norvegicus, 62% with Salmo salar, 55% with Homo sapiens, which
230
revealed that CiIRAK4 shared homology with other fishes except with Danio rerio
231
(65% similarity), and that homology was also found among mammals (Fig. 1).
232
Meanwhile, a phylogenetic tree was constructed with 18 known IRAK4 members
233
from mammals, amphibian, and fish. The results showed that CiIRAK4 shared high
234
homologous (> 90% bootstrap value) with other fish IRAK4, especially with zebra
235
fish. All of the fish IRAK4 sequences clustered together as well as diverged from
236
their counterparts in other species (Fig. 2).
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3.2. Expression analysis of CiIRAK4 in tissues and cells of grass carp
239
Q-PCR analysis was used to evaluate the expression profile of CiIRAK4 mRNA in
240
different tissues. As shown in Fig. 3A, CiIRAK4 was expressed ubiquitously in all
241
detected tissues (eye, liver, spleen, kidney, brain and intestine), with its higher
242
expression in the liver. After injection with poly I:C, the expression levels of
243
CiIRAK4 peaked at 6 h post-treatment in most tested tissues, especially obvious in
244
liver (74.46-fold) and spleen (10.72-fold) (Fig. 3A). Then, it declined rapidly and
245
reached normal or lower levels at 24 h post-treatment. It reduced continually and
246
came to the lowest level at 72 h post-treatment.
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248
Q-PCR. When CIK cells were challenged with poly I:C, the level of CiIRAK4
249
mRNA was increased rapidly (4.6-fold) at 6 h post-treatment, and reached the peak
250
(21.56-fold) at 48 h post-treatment (Fig. 3B). By contrast, CiIRAK4 was
251
up-regulated and reached the peak (46.73-fold) at 12 h after CIK cells were treated
252
with LPS (Fig. 3B).
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3.3. Subcellular localization of CiIRAK4
255
To investigate the subcellular localization of CiIRAK4, pEGFP-C1/CiIRAK4 was
256
transfected into CIK cells. The results revealed that CiIRAK4 was distributed
257
entirely in the cytoplasm, whereas GFP empty protein was located in both the
258
cytoplasm and the nucleus (Fig. 4A). Also, it was confirmed by Western blotting
259
analysis (Fig. 4B). When CIK cells were transfected with pEGFP-C1/CiIRAK4,
260
GFP-IRAK4 fusion protein was detected by GFP antibody at the correct molecular
261
weight.
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3.4. Effect of CiIRAK4 on NF-kappa B p65
264
To explore the effect of CiIRAK4 on NF-kappa B p65, knockdown and
265
overexpression of CiIRAK4 assays were preformed. The level of CiIRAK4 mRNA
266
was diminished in the cells transfected with siRNA against CiIRAK4 gene,
267
confirming the siRNA specifically knocked down IRAK4 in CIK cells. When the
268
cells transfected with pcDNA3.1/CiIRAK4, IRAK4 mRNA level was found to be
269
up-regulated for 3.66-fold compared to the control (Suppl. Fig. 3).
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271
CiIRAK4 gene, the cytoplasmic and nuclear proteins were extracted. As shown in
272
Fig. 5, compared with the mock, CIK cells treated with LPS for 60 min could be
273
observed a part of NF-kappa B p65 translocation into the nucleus. When CiIRAK4
274
was overexpressed in CIK cells, it displayed that only a small number of NF-kappa B
275
p65 were translocated into the nucleus. Meanwhile, as CiIRAK4 knockdown in CIK
276
cells, the proportion of NF-kappa B p65 nuclear translocation was similar to that of
277
negative control RNA oligo and vector (pcDNA3.1) transfection alone. These data
278
suggested that CiIRAK4 might have little effect on NF-kappa B p65 nuclear
279
translocation.
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3.5. IRAK4 promotes IRF5 nuclear translocation
282
Recently, IRAK4 was verified to have a minor effect on the activation and nuclear
283
translocation of NF-kappa B p65 [30]. Also, IRAK4 kinase activity was required for
284
IRF5 nuclear translocation but redundant for NF-kappa B [31]. Then, we next
285
investigated whether CiIRAK4 could promote IRF5 translocation to the nucleus.
286
pcDNA3.1/CiIRAK4 and pEGFP-C1/CiIRF5 were cotransfected into CIK cells. As
287
shown in Fig. 6A, some green granules were obviously filled in the nuclear regions,
288
suggesting that CiIRF5 was translocated to the nucleus when cells cotransfected with
289
CiIRAK4. Also, this result could be found in HEK-293T cells (Suppl. Fig. 4).
290
Consistent with the observation above, as shown in Fig. 6B, GFP-IRF5 could be
291
found
292
pEGFP-C1/CiIRF5 alone. Correspondingly, when CIK cells were co-expressed
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only in
the
cytoplasm
when
CIK
cells
were
transfected
with
ACCEPTED MANUSCRIPT CiIRF5 with CiIRAK4, some of GFP-IRF5 were translocated from the cytoplasm to
294
the nucleus (Fig. 6B).
295
Subsequently, co-immunoprecipitation was used to detect if IRF5 nuclear
296
translocation controlled by IRAK4 related to the interaction between IRAK4 and
297
IRF5. p3×FLAG/CiIRAK4 and pEGFP-C1/CiIRF5 were co-transfected into
298
HEK-293T cells. As shown in Fig. 7, IRAK4 could not directly interact with IRF5.
299
These results suggested that IRAK4 promoted IRF5 nuclear translocation via
300
activating some other signal factors.
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4. Discussion
303
IRAK4 plays a pivotal role in the signaling pathways of TLRs/IL-1Rs. It can initiate
305
a cascade of signaling events and leads to induction of inflammatory target gene
306
expression eventually [32]. To date, some homologs of IRAK4 have been found in
307
some different species of fish except in mammals. In this study, a novel teleost
308
IRAK4 cDNA was identified from grass carp. Similar to other IRAK4 subfamily
309
member, CiIRAK4 protein contained an N-terminal death domain (DD) and a
310
C-terminal kinase domain. Customarily, MyD88 activates IRAK4 by binding to
311
certain sites in the IRAK4 death domain and transducts signals to the downstream
312
factors of the TLRs pathway [33]. In addition, the alignment and phylogenetic
313
analysis also demonstrated that IRAK4 was highly conserved in vertebrates, and
314
grass carp IRAK4 shared the highest homology with zebra fish IRAK4.
315
In consistent with the previous reports, CiIRAK4 was constitutively expressed in
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317
tissues, such as liver, spleen and kidney. It indicated that CiIRAK4 was essential for
318
immune defense. Meanwhile, the expression of CiIRAK4 was significantly
319
up-regulated at 6 hours after poly I:C stimulation, especially in the liver and spleen.
320
Similarly, The expression of large yellow croaker IRAK4 was significantly induced
321
by poly I:C treatment in the spleen and liver tissues [26, 34]. Additionally, some
322
studies revealed that the expression of fish IRAK4 is different for various PAMPs.
323
Rock bream IRAK4 was up-regulated after infection of rock bream iridovirus (RBIV)
324
[25], but large yellow croaker and trout IRAK4 showed no significant change after
325
infection of V. parahemolyticus and Aeromonas salmonicida, respectively [23, 26].
326
Zebrafish IRAK4 could be observably induced by Edwardsiella tarda, whereas it was
327
down-regulated after infection with snakehead rhabdovirus (SHRV) [20]. Similarly,
328
as revealed in the present study, CiIRAK4 mRNA was induced by poly I:C and LPS
329
in CIK cells. The level of CiIRAK4 mRNA was elevated first at 6 h after poly I:C
330
treatment and declined to the normal levels, then it reached the peak at 48 h.
331
However, CiIRAK4 mRNA was up-regulated significantly and peaked at 12 h after
332
LPS infection (Fig. 3B). It could be postulated that recognition of pathogenic pattern
333
receptors by different signaling pathways might be responsible for the differential
334
expression of IRAK4 in fish.
335
In MyD88-dependent pathway, upon binding of ligands, TLRs recruit the adaptor
336
molecule MyD88 through the TIR domain. Then, MyD88 recruits IRAK4 and
337
initiates an intracellular signaling cascade [17]. It's no doubt that IRAK4 is located in
338
the cytoplasm in cells. Similar to other fish species [23, 24, 26], when GFP-tagged
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ACCEPTED MANUSCRIPT CiIRAK4 was expressed in CIK cells, it could be obviously detected that IRAK4
340
distributed entirely in the cytoplasm, which was confirmed by Western blot result by
341
using a GFP-specific antibody (Fig. 4).
342
It is undoubted that IRAK4 plays the critical roles in TLRs signaling. Paradoxically,
343
the function of IRAK4 is quite diverse. In mammals, Yang et al. found that human
344
IFN-alpha/beta and -lambda could be induced by TLR-3 and TLR-4 agonists in
345
IRAK-4-deficient cells. In contrast, TLR-7, -8, and -9-mediated induction of
346
IFN-alpha/beta and -lambda was strictly IRAK-4 dependent [35]. Recent studies
347
have demonstrated that IRAK4 played a dual role in Myddosome formation and
348
TLR signaling. On the one hand, IRAK4 had a critical scaffold function in
349
Myddosome formation, and the kinase activity of IRAK4 was dispensable for
350
Myddosome assembly and activation of the NF-kappa B and MAPK pathways. On
351
the other, IRAK4 was essential for MyD88-dependent production of inflammatory
352
cytokines [30]. In teleost fishes, IRAK4 seems to perform different functions. In
353
zebra fish, overexpression of IRAK-4 in ZFL cells resulted in NF-kappa
354
B-dependent luciferase expression, indicating that zfIRAK-4 could activate the
355
NF-kappa B signal transduction pathway [20]. In contrast, previous studies found
356
that grouper and large yellow croaker IRAK4 could impair MyD88-mediated
357
NF-kappa B activation in HEK-293T cells [24, 26]. In the present study, we
358
identified that CiIRAK4 was unnecessary for nuclear translocation of NF-kappa B
359
p65 (Fig. 5), which might imply that CiIRAK4 has little effects on the activation of
360
NF-kappa B p65.
361
Recent studies from primary human monocytes revealed that IRAK4 kinase
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ACCEPTED MANUSCRIPT activated TAK1 and IKK, and the activated IKK then phosphorylated IRF5 and
363
induced nuclear translocation of IRF5. During this process, IRAK4 kinase activity
364
was required for IRF5 nuclear translocation but redundant for NF-kappa B [31].
365
Here, when CIK cells were cotransfected with CiIRF5 and CiIRAK4, it could be
366
noticed that IRAK4 promoted nuclear translocation of some IRF5 (Fig. 6), which has
367
nothing to do with the interaction between IRAK4 and IRF5 (Fig. 7). It suggested
368
that fish IRAK4 kinase regulated IRF5 activity through indirect ways. Therefore, it
369
could be postulated that CiIRAK4 was dispensable for activation of the NF-kappa B
370
pathways, nevertheless, it was critical for TLR7/8 cytokine responses via IRF5 in grass
371
carp. Further work will be needed to test this hypothesis.
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Acknowledgements
374
This work was supported by research grants from the National Natural Science
376
Foundation of China (31560594) and the Science & Technology Foundations of
377
Education Department of Jiangxi (GJJ161303).
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References
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dimerization and trans-autophosphorylation are induced by Myddosome
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assembly, Mol. Cell. 55 (2014) 891-903.
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[17] S. Vollmer, S. Strickson, T. Zhang, N. Gray, K.L. Lee, V.R. Rao, et al., The
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mechanism of activation of IRAK1 and IRAK4 by interleukin-1 and Toll-like
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receptor agonists, Biochem. J. 474 (2017) 2027-2038. [18] S.R. Hubbard, IRAK4 activation: a cautious embrace, Mol. Cell. 55 (2014) 805-806. [19] J.L. Ryan Ferrao, Elisa Bergamin, Hao Wu, Structural insights into the assembly
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of large oligomeric signalosomes in the toll-like receptor-interleukin-1 receptor
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superfamily. Sci. Signal. 5 (2012) re3.
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Identification and characterization of IL-1 receptor-associated kinase-4
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Immunol. 32 (2012) 609-615.
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(IRAK4)-encoding gene in salmonid fish: the functional copy is rearranged in
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Oncorhynchus mykiss and that factor can impair TLR signaling in mammalian
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cells, Fish. Shellfish. Immunol. 36 (2014) 206-214.
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IRAK-4 in grouper (Epinephelus coioides) that impairs MyD88-dependent
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NF-kappaB activation, Dev. Comp. Immunol. 45 (2014) 190-197. [25] N. Umasuthan, S.D. Bathige, I. Whang, B.S. Lim, C.Y. Choi, J. Lee, Insights
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into molecular profiles and genomic evolution of an IRAK4 homolog from rock
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transcriptional expression, Fish. Shellfish. Immunol. 43 (2015) 436-448.
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pathogen-induced
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(Oplegnathus
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functional characterization of IRAK4 in large yellow croaker (Larimichthys
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CCL20 and Bcl-xl, Fish. Shellfish. Immunol. 80 (2018) 214-222.
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performs as an activator for IFN expression, Dev. Comp. Immunol. 86 (2018)
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[30] D. De Nardo, K.R. Balka, Y. Cardona Gloria, V.R. Rao, E. Latz, S.L. Masters,
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Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a dual role in
ACCEPTED MANUSCRIPT 466
myddosome formation and Toll-like receptor signaling, J. Biol. Chem. 293
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(2018) 15195-15207 [31] L. Cushing, A. Winkler, S.A. Jelinsky, K. Lee, W. Korver, R. Hawtin, et al.,
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475
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pathways involved in the antiviral response, PLoS. One. 9 (2014) e97471.
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Figure Legends
486
Fig. 1. Multiple alignment of CiIRAK4 with the amino acid sequences of known
488
typical organisms IRAK4s by Clustal X 2.0 program.
489
Identical (shaded in black) and similar (shaded in gray and light gray) residues
490
identified by the ClustalW program are indicated. Both death domain (DD) and
491
protein kinase domain (Pkinase) are indicated with arrows.
SC
RI PT
487
M AN U
492
Fig. 2. Phylogenetic analysis of IRAK4s.
494
A neighbour-joining tree was constructed by MEGA 6.0. All sequences used for
495
analysis were derived from GenBank and their accession numbers were shown in
496
parentheses. The bootstrap confidence values shown at the nodes of the tree were
497
based on 1000 bootstrap replications.
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Fig. 3. Expression of CiIRAK4 in response to poly I:C or LPS induction.
500
(A) Expression analysis of CiIRAK4 mRNA in grass carp tissues (eye, liver, spleen,
501
kidney, brain, and intestine) at various time following infected with poly I:C. Eye
502
(treatment for 0 h) was used as the calibrator (B) Expression analysis of CiIRAK4
503
mRNA in CIK cells at various time following induced with poly I:C or LPS. The
504
data shown were derived from a representative experiment reported as the mean (n
505
=3)±S.D. * and ** represented significant (p<0.05) and highly significant (p<0.01),
506
respectively.
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ACCEPTED MANUSCRIPT Fig. 4. Subcellular localization of CiIRAK4.
508
(A) CIK cells were seeded on microscopy dishes and transiently transfected with 2
509
µg of pEGFP-C1/CiIRAK4 plasmids. After 36 h of transfection, the cells were fixed
510
and examined with confocal microscopy. The CIK cells transfected with pEGFP-C1
511
were used as control. Green staining represents the CiIRAK4 protein signal, and blue
512
staining indicates the nucleus region. The results are representative of three
513
independent experiments. Scale bars represent 25 µM. (B) The expression of GFP
514
fusion proteins were confirmed by Western blotting analysis using mouse
515
monoclonal antibody against GFP.
516
M AN U
SC
RI PT
507
Fig. 5. CiIRAK4 is dispensable for the nuclear translocation of NF-kappa B p65.
518
CIK cells were seeded in T25 culture flasks overnight. LPS treatment,
519
RNAi-mediated gene knockdown and overexpression of CiIRAK4 assays were
520
preformed, respectively. Cells were harvested at 60 min post LPS treatment or 24 h
521
post transfection. The cytoplasmic and nuclear proteins were extracted from these
522
cells for Western blot. GAPDH and Histone were used as internal references for
523
cytoplasmic and nuclear proteins, respectively. The intensity of bands on a Western
524
blot was measured by Image J (https://imagej.nih.gov/ij/). Results were shown by
525
columns. Values were mean ± SD of three experiments. C, cytoplasmic proteins; N,
526
nuclear proteins; mock, untreated cells; siRNA, RNAi-mediated gene knockdown of
527
CiIRAK4; NC, negative control RNA oligo.
528
AC C
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517
ACCEPTED MANUSCRIPT Fig. 6. IRAK4 promotes IRF5 nuclear translocation.
530
(A) CIK cells were seeded on microscopy dishes and co-transfected with
531
pEGFP-C1/CiIRF5 and pcDNA3.1/CiIRAK4. After transfection for 36 h, the cells
532
were fixed and examined with confocal microscopy. The transfection of
533
pEGFP-C1/CiIRF5 alone into CIK cells acted as a control. Green granules in
534
nucleus represent the nuclear translocation of CiIRF5 protein (indicated with arrow).
535
The results are representative of three independent experiments. Scale bars represent
536
25 µM. (B) At the same time, the CIK cells were harvested and subjected to
537
separation of subcellular components. The indicated proteins in the cytoplasmic (C)
538
and nuclear (N) fraction were then tested by for Western blot. GAPDH and Histone
539
were used as internal references for cytoplasmic and nuclear proteins, respectively.
M AN U
SC
RI PT
529
TE D
540
Fig. 7. IRAK4 can not directly interact with IRF5.
542
HEK-293T cells seeded in 10 cm2 dishes were co-transfected with 5 µg
543
p3×FLAG/CiIRAK4 and pEGFP-C1/CiIRF5 plasmid. At 48 h post-transfection, the
544
whole-cell lysates were immunoprecipitated (IP) with either anti-Flag affinity gel (A)
545
or anti-GFP-agarose beads (B). The immunoprecipitates and cell lysates were then
546
analyzed by immunoblotting (IB) with the anti-Flag and anti-GFP antibody,
547
respectively.
AC C
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541
ACCEPTED MANUSCRIPT Table 1 Primers used for all the experiments. Name
Sequence (5’ to 3’)
Application
5′-IRAK4-Out
TGCTGAACGGGTTTAGTGAC
3′-IRAK4-Out
RI PT
5′-IRAK4-Inner GATGAGCCCGCTTTAGTGTGG GAGATGCTGTTCGACTGGG
Race-PCR
3′-IRAK4-Inner ATTCTGATCCGACACCAGCTG
CTAATACGACTCACTATAGGGCAAAGCAGTGGTATCAACGCAGAGT
Short
CTAATACGACTCACTATA
NUP
AAGCAGTGGTATCAACGCAGAG
IRAK4-ORF-F
ATGTGTGACGTCACGCGGG
IRAK4-ORF-R
TCATCCAGAGCTCTGGGAATG
IRAK4-RT-F
CTCCACACTGAGAGCTTTATC
IRAK4-RT-R
ATGTGCAGCTGTGTGTATCT
β-actin-F
CACTGTGCCCATCTACGAG
β-actin-R
CCATCTCCTGCTCGAAGTC
3.1-IRAK4-F
GCGGTACCATGTGTGACGTCACGCGGG
3.1-IRAK4-R
CGGAATTCTCATCCAGAGCTCTGGGAATG
GFP-IRAK4-F
CGGAATTCTATGTGTGACGTCACGCGGG
GFP-IRAK4-R
GCGGTACCTCATCCAGAGCTCTGGGAATG
EP
AC C
Flag-IRAK4-F
CGGAATTCTATGTGTGACGTCACGCGGG
Flag-IRAK4-R
GCGGTACCTCATCCAGAGCTCTGGGAATG
GFP-IRF5-F
CGGAATTCTATGAGCGGTCAACCACGGAG
GFP-IRF5-F
GCGGTACCTTAGTGGATGTTTTGAGGCC
IRAK4 siRNA
Q-PCR
Eukaryotic vector construction
GGAGGUGGAGAGGAUGUAUTT AUACAUCCUCUCCACCUCCTT
negative control
ORF cloning
TE D
M AN U
SC
Long
UUCUCCGAACGUGUCACGUTT ACGUGACACGUUCGGAGAATT
Knockdown
ACCEPTED MANUSCRIPT
Fig. 1
Death domain (DD)
RI PT
C. idellus D. rerio E. coioides O. mykiss S. salar C. semilaevis L. crocea I. punctatus X. tropicalis B. taurus M. musculus H. sapiens
M AN U TE D
AC C
C. idellus D. rerio E. coioides O. mykiss S. salar C. semilaevis L. crocea I. punctatus X. tropicalis B. taurus M. musculus H. sapiens
EP
C. idellus D. rerio E. coioides O. mykiss S. salar C. semilaevis L. crocea I. punctatus X. tropicalis B. taurus M. musculus H. sapiens
SC
C. idellus D. rerio E. coioides O. mykiss S. salar C. semilaevis L. crocea I. punctatus X. tropicalis B. taurus M. musculus H. sapiens
C. idellus D. rerio E. coioides O. mykiss S. salar C. semilaevis L. crocea I. punctatus X. tropicalis B. taurus M. musculus H. sapiens
Protein kinase domain (Pkinase)
ACCEPTED MANUSCRIPT
Protein kinase domain (Pkinase)
M AN U TE D
AC C
EP
C. idellus D. rerio E. coioides O. mykiss S. salar C. semilaevis L. crocea I. punctatus X. tropicalis B. taurus M. musculus H. sapiens
SC
C. idellus D. rerio E. coioides O. mykiss S. salar C. semilaevis L. crocea I. punctatus X. tropicalis B. taurus M. musculus H. sapiens
RI PT
C. idellus D. rerio E. coioides O. mykiss S. salar C. semilaevis L. crocea I. punctatus X. tropicalis B. taurus M. musculus H. sapiens
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
Fig. 2
ACCEPTED MANUSCRIPT Fig. 3
M AN U
SC
RI PT
A
AC C
EP
TE D
B
ACCEPTED MANUSCRIPT Fig. 4
DAPI
GFP
SC TE D
M AN U
GFP-IRAK4 B
Merged
RI PT
pEGFP-C1
A
AC C
EP
GFP-IRAK4
pEGFP-C1
ACCEPTED MANUSCRIPT Fig. 5 Mock C
LPS N
C
pcDNA3.1/IRAK4 N
C
pcDNA3.1
N
C
p65
Histone
siRNA C
N
NC C
N
M AN U
p65
SC
RI PT
GAPDH
GAPDH
N C
siR N A
pc D N A 3. 1
LP pc S D N A 3. 1/ IR A K 4
C on tr l
AC C
EP
TE D
Histone
N
ACCEPTED MANUSCRIPT Fig. 6
A
DAPI
SC
GFP-IRF5
TE D
M AN U
IRAK4+GFP-IRF5
IRF5
B
AC C
GAPDH Histone
IRAK4+IRF5
N
EP
C GFP-IRF5
Merged
RI PT
GFP
C
N
pcDNA3.1+IRF5 C
N
ACCEPTED MANUSCRIPT Fig. 7
A
＋ －
－ ＋
＋ ＋
RI PT
IP: GFP
GFP-IRF5 Flag-IRAK4
IB: Flag
SC
IB: GFP (IRF5) Input
AC C
Input
－ ＋
TE D
IP: GFP
＋ －
EP
B
M AN U
IB: Flag (IRAK4)
＋ ＋
Flag-IRAK4 GFP-IRF5
IB: Flag
IB: GFP (IRF5)
IB: Flag (IRAK4)
ACCEPTED MANUSCRIPT Highlights
An IRAK4 orthologue from grass carp (Ctenopharyngodon idella) was cloned.
RI PT
CiIRAK4 shared high homologous with zebra fish IRAK4. CiIRAK4 was significantly up-regulated after treatment with poly I:C and LPS.
CiIRAK4 was located in the cytoplasm.
SC
CiIRAK4 was dispensable for activation of NF-kappa B p65. However,
M AN U
IRAK4 promoted IRF5 nuclear translocation, which has nothing to do with the
AC C
EP
TE D
interaction between IRAK4 and IRF5.