- Email: [email protected]
Porcine CD14 gene silencing partially inhibited the bacterial immune response mediated by TLR4 signaling pathway
Gene 628 (2017) 267–274
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Research paper
Porcine CD14 gene silencing partially inhibited the bacterial immune response mediated by TLR4 signaling pathway
MARK
Chaohui Daia,1, Haifei Wanga,1, Guoqiang Zhub,c, Shenglong Wua,c, Wenbin Baoa,c,⁎ a Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu 225009, China b College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009, China c Joint International Research Laboratory of Agriculture & Agri-Product Safety, Yangzhou University, Jiangsu, Yangzhou 225009, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Pig CD14 Gene silencing TLR4 signaling pathway E. coli Immune response
Cluster of differentiation antigen 14 (CD14) is the membrane receptor protein in Toll-like Receptor 4 (TLR4) signaling pathway, which plays an important regulation role in not only innate immune response but also adaptive immune response. In this study, the pig kidney epithelial cell (PK15) line with CD14 gene silencing mediated by lentivirus was established and cells of CD14-RNAi and NC group were exposed to three kinds of Escherichia coli (E. coli F18ab, E. coli F18ac and E. coli K88ac) and LPS. Then qPCR and western blot were used to detect expression levels of TLR4 signaling pathway-related genes. Finally, ELISA was used to detect the level of proinflammatory cytokines in the cell culture supernatant. The results showed that the expression level of TLR4 signaling pathway-related genes in the entire signal pathway had obvious increases when cells were exposed to the stimulation induced by E. coli and LPS. In addition, the expression levels of CD14-RNAi group were overall significantly lower than NC group (P < 0.05 or P < 0.01), which was the same with the release levels of proinflammatory cytokines. This study revealed that pig CD14 gene silencing partially inhibited immune response to E. coli F18 invasion mediated by TLR4 signaling pathway.
1. Introduction Toll-like receptors (TLRs) were found in drosophila's Toll proteins, which can not only participate in regulating the formation of dorsal ventral polarity in embryonic drosophilas but also directly mediate drosophila's natural (inherent) immune response to microbial infection (Medzhitov, 2001). Toll-like receptors gene family belongs to the type I transmembrane protein receptors in mammals, which widely distributes in the organization such as gastrointestinal tract and respiratory tract; TLRs play an important role in not only adaptive immune but also innate immune (Beutler, 2005). TLR4, as the main receptor for recognizing the bacterial lipopolysaccharide (LPS) and signaling pathway mediated by TLR4, belongs to signal transduction pattern recognition receptor (PRR) in TLRs family and plays an important role in a variety of inflammatory reactions (Poltorak et al., 1998).
Cluster of differentiation antigen 14 (CD14), an essential protein in TLR4 signaling pathway, is a kind of specific surface marker of monocytes, neutrophils and macrophages cells; it belongs to one of members of the cell surface glycoprotein family and has been proved to be a high affinity receptor of LPS which is endotoxin of gram-negative bacterium (Wright et al., 1990). The main biological activities of CD14 are as a receptor of LPS to recognize and combine with LPS or LPS/LBP complexes and mediate LPS inflammatory cells reaction. When the concentration of LPS is low (≤ 100 ng/mL), its effect on the activation of cells is completely mediated by its receptor CD14; and when concentration is high, its activation is in part mediated by CD14 (Perera et al., 1997). Its action mechanism is approximately when gram-negative bacteria invades the body, a variety of bacterial products such as LPS and lipoteichoic acid (LTA) mediated by CD14 signaling pathway activates immunocompetent cells like mononuclear macrophages and endothelial cells, which leads to release of cytokines and other
Abbreviations: CD14, cluster of differentiation antigen 14; mCD14, membrane CD14; sCD14, soluble CD14; TLR4, Toll-like receptor 4; MyD88, myeloid differentiation protein; IFN-α, interferon-alpha; TNF-α, tumor necrosis factor-alpha; IL-1β, interleukin-1β; MD2, myeloid differentiation protein 2; PRR, pattern recognition receptor; E. coli, Escherichia coli; LPS, lipopolysaccharide; LBP, LPS-binding protein; LTA, lipoteichoic acid; qPCR, quantitative real-time PCR; ATCC, American Type Culture Collection; DMEM, Dulbecco's modified eagle medium; FBS, fetal bovine serum; SYBR, Synergy Brands, Inc; GPI, glycosylphosphatidyl phthalide lipids; MHC, major histocompatibility complex ⁎ Corresponding author at: College of Animal Science and Technology, Yangzhou University, 48 East Wenhui Ave, Yangzhou, Jiangsu 225009, China. E-mail address: [email protected] (W. Bao). 1 These authors contributed equally to this study. http://dx.doi.org/10.1016/j.gene.2017.07.055 Received 14 January 2017; Received in revised form 26 April 2017; Accepted 19 July 2017 Available online 20 July 2017 0378-1119/ © 2017 Elsevier B.V. All rights reserved.
Gene 628 (2017) 267–274
C. Dai et al.
inflammatory mediators and finally presents the response to the bacterial infection (Chen, 2000). Plenty of clinical studies have indicated that the expression level of CD14 gene has a close relation with human sepsis, traumatic disease, disease of immune system, diseases of the blood system, digestive system diseases and so on (Carrillo et al., 2001; Frey et al., 1992; Gluck et al., 2001; Nockher and Scherberich, 1997; Sakr et al., 2008). Fung and Mollnes (2012) has found that the combined use of complement inhibitors and the inhibitor of CD14 signaling pathway was a kind of effective method for the prevention and treatment of inflammation caused by endotoxin LPS. Diarrhea and edema disease in weaned piglets are the important infectious diseases that can lead to the deaths of weaned piglets, which causes enormous economic loss to the pig industry. The enteropathogenic Escherichia coli (EPEC) is the main pathogenic factor of these two kinds of diseases, which results in intestinal diseases mainly through the release of endotoxin LPS. CD14 is the major receptor for recognition of LPS, so functional analysis of CD14 gene could be conducive to investigate the molecular mechanism of diarrhea and edema disease in weaned piglets caused by EPEC. Pig CD14 gene is located on chromosome 2 q28, including two exons and one intron and encoding 373 amino acid sequence, whose structure has high homology with humans, mice, rabbits, horses and cattle. Liu et al. (2008) analyzed the polymorphism of pig CD14 gene and found that there were three potential polymorphic loci (− 61, 587 and 1246) in porcine CD14 gene, and they analyzed the relationship between three kinds of genotypes at − 61 site and parts of the immune traits like IgG and DTH. Sun et al. (2015) found that under the condition of stimulation induced by LPS, the transcription of CD14 gene in pig small intestinal epithelial cells (IPEC-J2) significantly raised with extending of time, which showed that LPS was released in the IPEC-J2 cells with E. coli infection and CD14 gene played an essential role in mediating the inflammatory reactions and immune response infected by E. coli with releasing LPS. In our previous study, transcriptome analysis of duodenum of E. coli F-18 resistant and sensitive full sibs in Meishan weaning piglets was conducted and we found that TLR4 signaling pathway and CD14 gene were associated with resistance to E. coli in weaning piglets (Wu et al., 2016). In order to investigate the immune regulation roles of TLR4 signaling pathway and CD14 gene in the process of E. coli infection, a PK15 cell line with stable CD14 gene silencing was established in this study. Then three kinds of E. coli (F18ab, F18ac and K88ac) and LPS with three kinds of concentration (50 ng/ mL, 100 ng/mL and 1000 ng/mL) were used to stimulate cells to detect the expression changes of key genes in the TLR4 signaling pathway and the release levels of proinflammatory factor in downstream, which aims to explore the influence of CD14 gene silencing on the immune response mediated by TLR4 signaling pathway so as to provide a basis for further function and mechanism research of CD14 gene.
purchased from Jackson (PA, USA). LPS was purchased from SigmaAldrich (MO, USA). Porcine IL-1β and TNF-α ELISA kits were purchased from AssayPro (MO, USA). 2.2. Primer design and sequence synthesis The qPCR primers of TLR4 signaling pathway related genes were designed based on the sequences of these genes from the pig available in the GenBank database using Primer Express 2.0 software (Table 1). All of primers were synthesized by Sangon Biotech (Shanghai, China) Co., Ltd. 2.3. Establishment of PK15 cell line with CD14 gene silencing PK15 cells were incubated with DMEM culture medium containing 10% FBS in an incubator under the conditions of 37 °C and 5% CO2. When the coverage of cells reached ~80%, transfections were performed with four lentivirus vector constructed in our previous research (Wu et al., 2016) by pGLV3-CD14-1, pGLV3-CD14-2, pGLV3-CD14-3 and pGLV3-CD14-4, a negative control group pGLV3- NC and a blank control group without lentivirus infection, each treatment had three repeats. Infected cells were cultured at 37 °C in a 5% CO2 incubator overnight and then cells expressing green fluorescent protein were observed after culture for 24 h. 10 μg/mL puromycin was used to screen the positive cells every 24 h and cell total RNA and protein were extracted for analyzing the transcription and translation level to detected the interference of CD14 gene. Then several positive monoclonal cells were picked to establish the PK15 cell line with CD14 gene silencing. 2.4. E. coli stimulation in PK15 cells of CD14-RNAi group and NC group PK15 cells of CD14-RNAi group and NC group were inoculated to 12-well plates at a density of 5.0 × 105 cells per well, and cells were incubated for 24 h in an incubator under the conditions of 37 °C and 5% CO2. E. coli F18ab, E. coli F18ac, and E. coli K88ac fimbriae standard strains were respectively inoculated to LB culture medium and incubated for 12 h at a rotating speed of 200 rev/min on a shaking table. After centrifugation for 5 min at a rotating speed of 4000 rev/min, the supernatant of three kinds of E. coli were filtered through 0.22 μm filters to collect the bacteria supernatant and PBS buffer solution was used to resuspend the bacteria precipitation and then wash and centrifuge for three times. Cells with CD14-RNAi group and NC group were exposed to the bacteria supernatant of E. coli F18ab, E. coli F18ac, and E. coli K88ac for 4, 8 and 12 h, three parallel replicates were used for each group. 2.5. LPS induce in PK15 cells of CD14-RNAi group and NC group
2. Materials and methods
When the cells of CD14-RNAi group and NC group were 80% confluent, they were exposed to 50 ng/mL, 100 ng/mL and 1000 ng/mL LPS (Sigma-Aldrich, MO, USA) for 2, 4 and 6 h, three parallel replicates were used for each group. At the same time, CD14-RNAi cells and NC cells without stimulation treatment were set as control group respectively. Then cell total RNA and protein were extracted for transcript and translation levels of TLR4 signaling related genes. And cells culture supernatants were collected for ELISA analysis.
2.1. Reagents and materials The pig kidney cell line (PK15) was obtained from the American Type Culture Collection (ATCC, USA). E. coli F18ab, E. coli F18ac, and E. coli K88ac fimbriae standard strains were provided by the veterinary laboratory at the Institute of Microbiology, University of Pennsylvania. Fetal bovine serum (FBS) and Dulbecco's modified eagle medium (DMEM) were purchased from Gibco (NY, USA). Trizol was purchased from Invitrogen (CA, USA). HiScript® II Q Select RT SuperMix for qPCR and AceQ® qPCR SYBR® Green Master Mix were purchased from Vazyme Biotech Co., Ltd. (Nanjing, China). Total protein extraction kit and BCA protein detection kit were purchased from Nanjing Keygen Technology Development Co., Ltd. (Nanjing, China). Primary antibodies-CD14 (1:400), MyD88 (1:800), IFN-α (1:600), IL-1β (1:600), TLR4 (1:1000), TNF-α (1:1000) and β-actin (1:4000)-were purchased from Abcam (Cambridge, UK). Second antibody (IgG-HRP, 1:3000) was
2.6. Quantitative real-time PCR (qPCR) Total RNA was extracted using the Trizol Reagent (Takara, China), according to the manufacturer's instructions. RNA was reversed to cDNA and was used as a template to detect and analyze the transcript levels of CD14 and TLR4 signaling related genes. The 50-μL reaction mixture for cDNA synthesis contained the following: 10 μL 5× HiScript® II Q RT SuperMix, 2000 ng total RNA, and RNase-free H2O to make up the final volume of 50 μL. The reaction was carried out at 268
Gene 628 (2017) 267–274
C. Dai et al.
Fig. 1. Establishment of PK15 cell line with CD14 gene silencing. (A) The expression of green fluorescent protein (GFP) in PK15 cells infected by lentivirus solution for 24 h under a fluorescence microscope (100×); the lower panels are the corresponding cells in the absence of fluorescence exposure, which shows the overall density of cells. (B) The mRNA levels of CD14 in four kinds of lentivirus (pGLV3-CD14-1, pGLV3-CD14-2, pGLV3-CD14-3, pGLV3-CD14-4 and pGLV3-NC) and Blank cells without any treatments were determined by qPCR detection. **, P < 0.01. (C) The protein expression levels of CD14 in four kinds of lentivirus and Blank cells without any treatments were determined by western blot. The integrated option density (IOD) of each kind of protein was calculated by ImageJ and gray value was defined as IOD (target protein)/IOD (β-actin), and the average of gray values of Blank group was defined as 1 to homogenize different groups to analyze the relative protein expression level of each kind of protein; *, P < 0.05. (D) The expression of green fluorescent protein (GFP) in PK15 cells with CD14 silencing and NC group cultured from monoclonal cells respectively under a fluorescence microscope (100×); the right-hand panels are the corresponding cells in the absence of fluorescence exposure, which shows the overall density of cells. Each treatment has tree repetitions.
2.8. Cytokines detection of IL-1β and TNF-α in the TLR4 signaling pathway
37 °C for 15 min and then at 85 °C for 5 s. QPCR (PrimeScriptTM RT Master Mix, Vazyme) was carried out as follows: 10 μL SYBR Premix ExTaq™ II (2 ×), 0.4 μL PCR Forward Primer (10 μM), 0.4 μL PCR Reverse Primer (10 μM), 0.4 μL ROX Reference Dye II (50 ×), 2 μL cDNA, and ddH2O to make the final volume 20 μL, each sample had three repeats. The house keeping genes ACTB and GAPDH were used as references control to normalize the expression level, and 2− ΔΔCt method was used to calculate the relative transcription level. We performed qPCR using the ABI 7500 system (Applied Biosystems, USA). The reaction conditions were 95 °C for 15 s, followed by 40 cycles of 95 °C for 5 s and 62 °C for 34 s. The dissociation curve was analyzed after amplification. The reaction conditions were 95 °C for 15 s, 62 °C for 60 s, 95 °C for 15 s, and 60 °C for 15 s.
In PK15 cells between RNAi group and control group, we measured the level of two cytokines in the TLR4 signaling pathway including interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) using the ELISA kits (AssayPro, USA). The assay procedure and standard curve generation were carried out according to the instructions provided with the kit. 2.9. Statistical analysis The 2− ΔΔCt method (Livak and Schmittgen, 2001) was used to detect the transcript levels of genes, and the internal reference genes GAPDH and ACTB were used to normalize gene expression levels. SPSS 17.0 including one-way ANOVA method was provided for difference analysis of transcript and protein levels of genes and release levels of cytokines between CD14-RNAi group and NC group. Each independent experiment or treatment was performed with three repeats and the data was counted as x ± SD.
2.7. Western blot analysis BCA kit (Nanjing Keygen Technology Development Co., LTD) was used to normalize protein levels. 10% SDS PAGE was used to treat all protein samples in each group, 10 μL protein was added to loading hole and ran the electrophoresis for 70 min under the condition of 160 V. Blotting: proteins were transferred to PVDF membranes and immunoblotted with antibodies to CD14 (1:400), MyD88 (1:800), IFN-α (1:600), IL-1β (1:600), TLR4 (1:1000), TNF-α (1:1000) and β-actin (1:4000). The secondary antibody was horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG (Abcam, UK, 1:3000).
3. Results 3.1. Establishment of PK15 cell line with CD14 gene silencing PK15 cells infected by four kinds of interference vectors and a negative control lentivirus for 24 h could express highly green fluorescent 269
Gene 628 (2017) 267–274
C. Dai et al.
Fig. 2. Effect of CD14 gene silencing on the TLR4 signaling pathway without any stimulation. Expression differences of TLR4 signaling pathway related genes in PK15 cells with CD14 gene silencing in normal state were detected by qPCR (A) and western blot (B). The integrated option density (IOD) of each kind of protein was calculated by ImageJ and gray value was defined as IOD (target protein)/IOD (β-actin), and the average of gray values of NC group of each protein was defined as 1 to homogenize different groups to analyze the relative protein expression level; **, P < 0.01; *, P < 0.05. Each treatment has tree repetitions.
3.2. Influence of CD14 gene silencing on TLR4 signaling pathway under normal physiological condition in PK15 cells
protein (Fig. 1A), which illustrated that lentivirus had integrated into PK15 cells efficiently. Cells total RNA and total protein were collected after screened by puromycin for 24 h (namely transfection for 48 h) to analyze the interference efficiency of different interference vectors. Results showed that four kinds of interference vectors could significantly reduce transcription level of CD14 gene in PK15 cells. The interference efficiency of pGLV3-CD14-1, pGLV3-CD14-2, pGLV3CD14-3 and pGLV3-CD14-4 were 35.90%, 44.17%, 69.91% and 35.90%, respectively (Fig. 1B). Protein expression levels of pGLV3CD14-3 and pGLV3-CD14-4 were also significantly reduced (Fig. 1C). PK15 cells infected by pGLV3-CD14-3 with the highest interference efficiency were diluted to cultivate positive monoclonal cells. And amplification culture of monoclonal cells could express green fluorescent protein stably after continuous puromycin screening (Fig. 1D), which suggested that PK15 cell line with stable CD14 gene silencing has been established successfully and could be used for further research.
When the convergence rate of PK15 cells with CD14-RNAi group and NC group reached to 80–90%, cells total RNA and protein were extracted to analyze mRNA and protein expression level. Taking the average expression level of each gene negative control group as reference 1, the relative expression level of each gene after CD14 gene silencing was calculated according to 2− ΔΔCt (Livak and Schmittgen, 2001). QPCR results show that the mRNA expression levels of TLR4 pathway-related genes (MyD88, IFN-α, IL-1, TLR4 and TNF-α) were not significant changes (P > 0.05) after CD14 gene silencing (Fig. 2A). Western blot results showed that there was no significant change (P > 0.05) in the protein expression level (Fig. 2B).
270
Gene 628 (2017) 267–274
C. Dai et al.
Fig. 3. Effect of CD14 gene silencing on the TLR4 signaling pathway with stimulation by E. coli strain and supernatant. The abscissa below and the left ordinate represent four kinds of cells with different treatments and their relative mRNA expression level; the data showed extremely significant difference with different capital letters (P < 0.01) and significant difference with different little letters (P < 0.05). The upper abscissa and the right ordinate represent four different stimulation time points of bacterial culture supernatants and their relative mRNA expression level; ** showed extremely significant difference between CD14-RNAi group and NC group at the same time point (P < 0.01) and * showed significant difference (P < 0.05). (A), (B) and (C) represented the expression of TLR4 signaling pathway related genes between CD14-RNAi group and NC group with stimulation by strain and bacterial culture supernatants from E. coli F18ab, E. coli F18ac and E. coli K88ac, respectively. Each treatment has tree repetitions.
fold) and decreased later (within 2-fold). What's interesting was that the expression levels of CD14-RNAi group were always lower than NC group (P < 0.05 or P < 0.01).
3.3. Influence of CD14 gene silencing on TLR4 signaling pathway in PK15 cells stimulated by E. coli PK15 cells of CD14-RNAi group, NC group and Blank group were exposed to E. coli strain of F18ab, F18ac and K88ac with the concentration of 109 CFU/mL for 4 h and stimulated by bacterial supernatants for 4 h, 8 h and 12 h. Then qPCR was used to analyze mRNA expression levels of TLR4 signaling pathway-related genes and variation trend with time. As the results showed in Fig. 3, the mRNA expression levels of key genes in TLR4 signaling pathway after three kinds of E. coli stimulation presented a consistent pattern: the mRNA expression level of TLR4 signaling pathway-related genes in cells of blank group and NC group significantly increased compared with cells without stimulation, while expression levels in cells of CD14-RNAi group were close to cells without stimulation (P > 0.05) even downregulated (P < 0.05 or P < 0.01) except MyD88 gene. With the increase of stimulation time induced by bacterial supernatants, the expression level of TNF-α showed a trend of rising (> 3-fold), while which of MyD88, IFN-α, IL-1 and TLR4 increased first (overall > 2-
3.4. Influence of CD14 gene silencing on TLR4 signaling pathway in PK15 cells induced by LPS PK15 cells of CD14-RNAi group and NC group were exposed to LPS solution with the concentration of 50, 100 and 1000 ng/mL for 2 h, 4 h and 6 h. Then qPCR was used to analyze mRNA expression variation between CD14-RNAi group and NC group of TLR4 signaling pathwayrelated genes and variation trend with time increasing. Results showed that the expression levels of all genes in TLR4 signaling pathway in both CD14-RNAi group and NC group showed a trend of rising with the increase of time (overall > 2-fold), and the expression levels of CD14RNAi group were always lower than NC group (P < 0.05 or P < 0.01). Fig. 4 showed the details of expression change of all genes.
271
Gene 628 (2017) 267–274
C. Dai et al.
Fig. 4. Effect of CD14 gene silencing on the TLR4 signaling pathway with stimulation by LPS. Expression differences of TLR4 signaling pathway related genes in PK15 cells between CD14 RNAi group and NC group induced by LPS with different concentration at different time points were detected by qPCR. * showed significant difference between CD14-RNAi group and NC group at the same time point (P < 0.05). ** showed extremely significant difference between CD14-RNAi group and NC group at the same time point (P < 0.01). (A), (B) and (C) represented the expression of TLR4 signaling pathway-related genes in PK15 cells between CD14-RNAi group and NC group induced by LPS with concentration of 50 ng/mL, 100 ng/mL and 1000 ng/mL, respectively. Each treatment has tree repetitions.
related genes increased first and decreased later and reached the peak at 4 h or 8 h then down-regulated at 12 h, which illustrated that time point leading to constant immune response of body was between 4 and 8 h. When PK15 cells were induced by LPS with three different concentration at three time points, the expression levels of signaling pathway-related genes up-regulated with the increase of time while the degree of up-regulation of CD14-RNAi group was greatly lower than NC group, which was consistent with the results of bacterial stimulation. These results suggested that CD14, as the membrane receptor protein in TLR4 signaling pathway, participated in immune regulation through synergistic regulation with key genes involved in immune response pathway in the pathogenic stimulation such as LPS and played an important regulating role in the whole signaling pathway. The reduction of mRNA transcription level of CD14 gene led to lower translation level of receptor proteins, causing lower transcription level of key molecules with synergy in membranal signaling pathway. MyD88 as an initial adapter protein for TLRs/IL-1 signaling pathways, could mediate most members of TLRs family downstream signaling transduction including TLR4 (O'Neill and Bowie, 2007). In this study, the mRNA level of MyD88 was not significantly lower after CD14 gene silencing, which may relate to its regulation function in other signaling pathways. In the body's normal physiological state, the mRNA expression level of MyD88 can remain in a relatively stable state under the coordination of multiple signal pathways. ELISA results showed that the release level of IL-1β up-regulated continuously with the increase of stimulation time induced by bacterial supernatant and LPS. The release level of TNF-α up-regulated continuously with the increase of stimulation time induced by LPS but increased first and decreased later induced by bacterial supernatants, which reached the highest level at 8 h. It is obvious that the levels of TNF-α and IL-1β in cell culture supernatant of CD14-RNAi group were always lower than NC group, which was consistent with previous results. Interleukin-1β (IL-1β) and tumor necrosis factor (TNF) are two kinds of important cytokines in immune response (Marchand-Adam et al., 2005; Szydlowski et al., 2011), so the above results suggested that CD14 do play an important regulating role not only in TLR4 signaling pathway but also in the immune process of E. coli F18 invasion. The
3.5. Changes of proinflammatory cytokine levels in cell culture supernatant with CD14 gene silencing Cell culture supernatant with different treatments were collected to analyze proinflammatory cytokines levels in downstream of TLR4 signaling pathway (TNF-α and IL-1β) in PK15 cells between CD14-RNAi group and NC group by ELISA kit. ELISA scale was used to make the standard curves of TNF-α and IL-1β and then regression equations were obtained (Fig. S1). The results of proinflammatory cytokines levels showed that the level of IL-1β presented a trend of continuous rising (overall > 3-fold) with the increase of stimulation time induced by bacterial supernatant and LPS (Fig. 5A). The release level of TNF-α presented a trend of continuous rising (P < 0.05 or P < 0.01) with the increase of stimulation time induced by LPS but increased first and decreased later (P < 0.05) induced by bacterial supernatants (Fig. 5B). As a whole, the levels of TNF-α and IL-1β in cell culture supernatant of CD14-RNAi group were always lower than NC group (P < 0.05 or P < 0.01). 4. Discussion This study established PK15 cell line with stable CD14 gene silencing, which played an important role in intestinal immune response and pathogen defense caused by pathogenic microorganisms. With CD14 gene silencing, the expression levels of TLR4 signaling pathway-related genes did not change significantly and the entire signaling pathway was in a steady state. It meant that CD14 RNAi mediated by lentivirus interfered the expression of CD14 gene specifically but it had no negative influence on the physiological state of cell itself, which revealed that RNAi technology mediated by lentivirus had been applied to the stable gene silencing maturely (Abbas-Terki et al., 2002). We used three kinds of pathogenic E. coli F18ab, F18ac and K88ac to stimulate PK15 cells of CD14-RNAi group and NC group and found that the expression levels of all genes in entire TLR4 signaling pathway up-regulated significantly, while the degree of up-regulation of CD14-RNAi group was greatly lower than NC group. With the increase of stimulation time induced by bacterial supernatant, the expression levels of signaling pathway272
Gene 628 (2017) 267–274
C. Dai et al.
Fig. 5. Effect of CD14 gene silencing on the release of immune proinflammatory factors. Expression differences of proinflammatory factors of IL-1β (A) and TNF-α (B) in TLR4 signaling pathway in PK15 cells between CD14 RNAi group and NC group under the treatment of stimulation by E. coli supernatant as well as LPS with the increase of time were detected by ELISA. LPS-50, LPS-100 and LPS-1000 represented LPS with concentration of 50 ng/mL, 100 ng/mL and 1000 ng/mL, respectively. * showed significant difference between CD14-RNAi group and NC group at the same time point (P < 0.05). Each treatment has tree repetitions.
was not suitable for Chinese local pig breeds (Bao et al., 2008). Based on previous results of transcriptome sequencing and the research results in this study, it is necessary to view CD14 gene as a new candidate gene of resistance to E. coli. We will research specifically the regulation role of CD14 gene in the process of E. coli invasion in Chinese local pig breeds, which aims to investigate the feasibility of making CD14 gene as a genetic marker of resistance to colibacillosis in weaning piglets especially Chinese local pig breeds. The final purpose of our study is to make an important breakthrough in bottleneck of molecular breeding of resistance to colibacillosis in Chinese local pig breeds at present.
down-regulation of these two kinds of cytokines inhibited the immune response mediated by TLR4 signaling pathway, which partially led to immunosuppression at last. What's interesting was that most expression level changes ranged from 1.5 to 2.5-fold, which might be considered physiologically relevant. With the prolongation of bacterial stimulation, the cell body will start the appropriate defense mechanism (such as secreting some anti-inflammatory factors) to prevent the infinitely elevation of expression of proinflammatory factors and genes. In our previous study, glycosphingolipid biosynthesis-globo series and Toll-like signaling pathway were found to have important regulation role in resistance to E. coli F18 in Sutai piglets (Bao et al., 2012). Then we found TLR4 signaling pathway and CD14 gene could be associated with resistance of Chinese native weaning piglets to E. coli by transcriptome analysis of duodenum in E. coli F18-resistant and E. coli F18-sensitive individuals of Meishan piglets (Chinese local pig breed) (Wu et al., 2016). This study further verified that CD14 gene and TLR4 signaling pathway played an important regulation role in resistance to E. coli invasion. Previous research achieved the disease-resistant breeding in foreign pig breeds based on the polymorphism on M307 loci of α-1, 2-Fucosyltransferasegene (FUT1) in glycosphingolipid biosynthesis-globo series (Meijerink et al., 1997) while this genetic marker
5. Conclusions This study established successfully a PK15 cell line with CD14 gene silencing and illustrated that CD14 gene silencing would not have effect on TLR4 signaling pathway and the entire signaling pathway was in a steady state when cells were under normal physiological condition. However, pig CD14 gene silencing partially inhibited immune response to E. coli F18 invasion mediated by TLR4 signaling pathway. This study also further revealed that CD14 gene played an important regulation role not only in TLR 4 signaling pathway but also in the immune 273
Gene 628 (2017) 267–274
C. Dai et al.
Table 1 The primer information of genes for qPCR. Gene
Sequence of the primer
Length of products (bp)
Accession no.
CD14
F: 5′-CCTCAGACTCCGTAATGTG-3′ R: 5′-CCGGGATTGTCAGATAGG-3′ F: 5′-GTGCCGTCGGATGGTAGT-3′ R: 5′-CAGTGATGAACCGCAGGAT-3′ F: 5′-CCTGGACCACAGAAGGGA-3′ R: 5′-TCTCATGCACCAGAGCCA-3′ F: 5′-TGATTGTGGCAAAGGAGGA-3′ R: 5′-TTGGGTCATCATCACAGACG-3′ F: 5′-CAGATAAGCGAGGCCGTCATT-3′ R: 5′-TTGCAGCCCACAAAAAGCA-3′ F: 5′-CGACTCAGTGCCGAGATCAA-3′ R: 5′-CCTGCCCAGATTCAGCAAAG-3′ F: 5′-ACATCATCCCTGCTTCTACTGG-3′ R: 5′-CTCGGACGCCTGCTTCAC-3′ F: 5′-TGGCGCCCAGCACGATGAAG-3′ R: 5′-GATGGAGGGGCCGGACTCGT-3′
180
AB267810
173
EU056736
92
X57191
63
NM_001005149
113
AB232527
58
X54001
188
AF017079
149
XM_003124280.3
MyD88 IFN-α IL-1β TLR4 TNF-α GAPDH ACTB
Res. 6, 351–358. Liu, H.Z., Li, X.Y., Liu, B., Yu, M., Ma, Y.H., Chu, M.X., Li, K., 2008. Tissue distribution, SHP detection and association study with immune traits of porcine LBP and CD14 genes. Asian Australas. J. Anim. Sci. 21, 1080–1087. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using realtime quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25, 402–408. Marchand-Adam, S., Plantier, L., Bernuau, D., Legrand, A., Cohen, M., Marchal, J., Soler, P., Leseche, G., Mal, H., Aubier, M., Dehoux, M., Crestani, B., 2005. Keratinocyte growth factor expression by fibroblasts in pulmonary fibrosis: poor response to interleukin-1beta. Am. J. Respir. Cell Mol. Biol. 32, 470–477. Medzhitov, R., 2001. Toll-like receptors and innate immunity. Nat. Rev. Immunol. 1, 135–145. Meijerink, E., Fries, R., Vogeli, P., Masabanda, J., Wigger, G., Stricker, C., Neuenschwander, S., Bertschinger, H.U., Stranzinger, G., 1997. Two alpha(1,2) fucosyltransferase genes on porcine chromosome 6q11 are closely linked to the blood group inhibitor (S) and Escherichia coli F18 receptor (ECF18R) loci. Mamm. Genome 8, 736–741. Nockher, W.A., Scherberich, J.E., 1997. Expression and release of the monocyte lipopolysaccharide receptor antigen CD14 are suppressed by glucocorticoids in vivo and in vitro. J. Immunol. 158, 1345–1352. O'Neill, L.A., Bowie, A.G., 2007. The family of five: TIR-domain-containing adaptors in Toll-like receptor signalling. Nat. Rev. Immunol. 7, 353–364. Perera, P.Y., Vogel, S.N., Detore, G.R., Haziot, A., Goyert, S.M., 1997. CD14-dependent and CD14-independent signaling pathways in murine macrophages from normal and CD14 knockout mice stimulated with lipopolysaccharide or taxol. J. Immunol. 158, 4422–4429. Poltorak, A., He, X., Smirnova, I., Liu, M.Y., Van Huffel, C., Du, X., Birdwell, D., Alejos, E., Silva, M., Galanos, C., Freudenberg, M., Ricciardi-Castagnoli, P., Layton, B., Beutler, B., 1998. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282, 2085–2088. Sakr, Y., Burgett, U., Nacul, F.E., Reinhart, K., Brunkhorst, F., 2008. Lipopolysaccharide binding protein in a surgical intensive care unit: a marker of sepsis? Crit. Care Med. 36, 2014–2022. Sun, L., Xia, R.W., Yin, X.M., Yu, L.H., Zhu, G.Q., Wu, S.L., Bao, W.B., 2015. Analysis of differential expression of TLR4 and TLR4 signaling pathway genes under lipopolysaccharide-induced pig intestinal epithelial cells. Acta Veterinaria et. Zootechnica Sinica 46, 1095–1101. Szydlowski, M., Buszka, A., Mackowski, M., Lechniak, D., Switonski, M., 2011. Polymorphism of genes encoding cytokines IL6 and TNF is associated with pig fatness. Livest. Sci. 136, 150–156. Wright, S.D., Ramos, R.A., Tobias, P.S., Ulevitch, R.J., Mathison, J.C., 1990. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 249, 1431–1433. Wu, Z.C., Liu, Y., Dong, W.H., Zhu, G.Q., Wu, S.L., Bao, W.B., 2016. CD14 in the TLRs signaling pathway is associated with the resistance to E. coli F18 in Chinese domestic weaned piglets. Sci Rep 6.
process of E. coli F18 invasion, which was expected to be as a molecular marker of piglet diarrhea caused by E. coli. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2017.07.055. Declaration of interest The authors declare that they have no conflict. Acknowledgement This work was supported by the National Natural Science Foundation of China (31572360, 31372285), the Science and Technology Supporting Project of Jiangsu Province (BE2016315, BE2014357), and the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions. References Abbas-Terki, T., Blanco-Bose, W., Deglon, N., Pralong, W., Aebischer, P., 2002. Lentiviralmediated RNA interference. Hum. Gene Ther. 13, 2197–2201. Bao, W.B., Wu, S.L., Musa, H.H., Zhu, G.Q., Chen, G.H., 2008. Genetic variation at the alpha-1-fucosyltransferase (FUT1) gene in Asian wild boar and Chinese and Western commercial pig breeds. J. Anim. Breed. Genet. 125, 427–430. Bao, W.B., Ye, L., Pan, Z.Y., Zhu, J., Du, Z.D., Zhu, G.Q., Huang, X.G., Wu, S.L., 2012. Microarray analysis of differential gene expression in sensitive and resistant pig to Escherichia coli F18. Anim. Genet. 43, 525–534. Beutler, B., 2005. The toll-like receptors: analysis by forward genetic methods. Immunogenetics 57, 385–392. Carrillo, E.H., Gordon, L., Goode, E., Davis, E., Polk, H.C., 2001. Early elevation of soluble CD14 may help identify trauma patients at high risk for infection. J. Trauma 50, 810–816. Chen, L.H., 2000. Molecular CD14's receptor function and its signal transmission. Foreign Medical Sciences 23, 137–140. Frey, E.A., Miller, D.S., Jahr, T.G., Sundan, A., Bazil, V., Espevik, T., Finlay, B.B., Wright, S.D., 1992. Soluble CD14 participates in the response of cells to lipopolysaccharide. J. Exp. Med. 176, 1665–1671. Fung, S.C., Mollnes, T.E., 2012. Methods and compositions for the prevention and treatment of sepsis: U.S. Patent 8,329,169[P]. 2012-12-11. Gluck, T., Silver, J., Epstein, M., Cao, P., Farber, B., Goyert, S.M., 2001. Parameters influencing membrane CD14 expression and soluble CD14 levels in sepsis. Eur. J. Med.
274
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Research paper
Porcine CD14 gene silencing partially inhibited the bacterial immune response mediated by TLR4 signaling pathway
MARK
Chaohui Daia,1, Haifei Wanga,1, Guoqiang Zhub,c, Shenglong Wua,c, Wenbin Baoa,c,⁎ a Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu 225009, China b College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009, China c Joint International Research Laboratory of Agriculture & Agri-Product Safety, Yangzhou University, Jiangsu, Yangzhou 225009, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Pig CD14 Gene silencing TLR4 signaling pathway E. coli Immune response
Cluster of differentiation antigen 14 (CD14) is the membrane receptor protein in Toll-like Receptor 4 (TLR4) signaling pathway, which plays an important regulation role in not only innate immune response but also adaptive immune response. In this study, the pig kidney epithelial cell (PK15) line with CD14 gene silencing mediated by lentivirus was established and cells of CD14-RNAi and NC group were exposed to three kinds of Escherichia coli (E. coli F18ab, E. coli F18ac and E. coli K88ac) and LPS. Then qPCR and western blot were used to detect expression levels of TLR4 signaling pathway-related genes. Finally, ELISA was used to detect the level of proinflammatory cytokines in the cell culture supernatant. The results showed that the expression level of TLR4 signaling pathway-related genes in the entire signal pathway had obvious increases when cells were exposed to the stimulation induced by E. coli and LPS. In addition, the expression levels of CD14-RNAi group were overall significantly lower than NC group (P < 0.05 or P < 0.01), which was the same with the release levels of proinflammatory cytokines. This study revealed that pig CD14 gene silencing partially inhibited immune response to E. coli F18 invasion mediated by TLR4 signaling pathway.
1. Introduction Toll-like receptors (TLRs) were found in drosophila's Toll proteins, which can not only participate in regulating the formation of dorsal ventral polarity in embryonic drosophilas but also directly mediate drosophila's natural (inherent) immune response to microbial infection (Medzhitov, 2001). Toll-like receptors gene family belongs to the type I transmembrane protein receptors in mammals, which widely distributes in the organization such as gastrointestinal tract and respiratory tract; TLRs play an important role in not only adaptive immune but also innate immune (Beutler, 2005). TLR4, as the main receptor for recognizing the bacterial lipopolysaccharide (LPS) and signaling pathway mediated by TLR4, belongs to signal transduction pattern recognition receptor (PRR) in TLRs family and plays an important role in a variety of inflammatory reactions (Poltorak et al., 1998).
Cluster of differentiation antigen 14 (CD14), an essential protein in TLR4 signaling pathway, is a kind of specific surface marker of monocytes, neutrophils and macrophages cells; it belongs to one of members of the cell surface glycoprotein family and has been proved to be a high affinity receptor of LPS which is endotoxin of gram-negative bacterium (Wright et al., 1990). The main biological activities of CD14 are as a receptor of LPS to recognize and combine with LPS or LPS/LBP complexes and mediate LPS inflammatory cells reaction. When the concentration of LPS is low (≤ 100 ng/mL), its effect on the activation of cells is completely mediated by its receptor CD14; and when concentration is high, its activation is in part mediated by CD14 (Perera et al., 1997). Its action mechanism is approximately when gram-negative bacteria invades the body, a variety of bacterial products such as LPS and lipoteichoic acid (LTA) mediated by CD14 signaling pathway activates immunocompetent cells like mononuclear macrophages and endothelial cells, which leads to release of cytokines and other
Abbreviations: CD14, cluster of differentiation antigen 14; mCD14, membrane CD14; sCD14, soluble CD14; TLR4, Toll-like receptor 4; MyD88, myeloid differentiation protein; IFN-α, interferon-alpha; TNF-α, tumor necrosis factor-alpha; IL-1β, interleukin-1β; MD2, myeloid differentiation protein 2; PRR, pattern recognition receptor; E. coli, Escherichia coli; LPS, lipopolysaccharide; LBP, LPS-binding protein; LTA, lipoteichoic acid; qPCR, quantitative real-time PCR; ATCC, American Type Culture Collection; DMEM, Dulbecco's modified eagle medium; FBS, fetal bovine serum; SYBR, Synergy Brands, Inc; GPI, glycosylphosphatidyl phthalide lipids; MHC, major histocompatibility complex ⁎ Corresponding author at: College of Animal Science and Technology, Yangzhou University, 48 East Wenhui Ave, Yangzhou, Jiangsu 225009, China. E-mail address: [email protected] (W. Bao). 1 These authors contributed equally to this study. http://dx.doi.org/10.1016/j.gene.2017.07.055 Received 14 January 2017; Received in revised form 26 April 2017; Accepted 19 July 2017 Available online 20 July 2017 0378-1119/ © 2017 Elsevier B.V. All rights reserved.
Gene 628 (2017) 267–274
C. Dai et al.
inflammatory mediators and finally presents the response to the bacterial infection (Chen, 2000). Plenty of clinical studies have indicated that the expression level of CD14 gene has a close relation with human sepsis, traumatic disease, disease of immune system, diseases of the blood system, digestive system diseases and so on (Carrillo et al., 2001; Frey et al., 1992; Gluck et al., 2001; Nockher and Scherberich, 1997; Sakr et al., 2008). Fung and Mollnes (2012) has found that the combined use of complement inhibitors and the inhibitor of CD14 signaling pathway was a kind of effective method for the prevention and treatment of inflammation caused by endotoxin LPS. Diarrhea and edema disease in weaned piglets are the important infectious diseases that can lead to the deaths of weaned piglets, which causes enormous economic loss to the pig industry. The enteropathogenic Escherichia coli (EPEC) is the main pathogenic factor of these two kinds of diseases, which results in intestinal diseases mainly through the release of endotoxin LPS. CD14 is the major receptor for recognition of LPS, so functional analysis of CD14 gene could be conducive to investigate the molecular mechanism of diarrhea and edema disease in weaned piglets caused by EPEC. Pig CD14 gene is located on chromosome 2 q28, including two exons and one intron and encoding 373 amino acid sequence, whose structure has high homology with humans, mice, rabbits, horses and cattle. Liu et al. (2008) analyzed the polymorphism of pig CD14 gene and found that there were three potential polymorphic loci (− 61, 587 and 1246) in porcine CD14 gene, and they analyzed the relationship between three kinds of genotypes at − 61 site and parts of the immune traits like IgG and DTH. Sun et al. (2015) found that under the condition of stimulation induced by LPS, the transcription of CD14 gene in pig small intestinal epithelial cells (IPEC-J2) significantly raised with extending of time, which showed that LPS was released in the IPEC-J2 cells with E. coli infection and CD14 gene played an essential role in mediating the inflammatory reactions and immune response infected by E. coli with releasing LPS. In our previous study, transcriptome analysis of duodenum of E. coli F-18 resistant and sensitive full sibs in Meishan weaning piglets was conducted and we found that TLR4 signaling pathway and CD14 gene were associated with resistance to E. coli in weaning piglets (Wu et al., 2016). In order to investigate the immune regulation roles of TLR4 signaling pathway and CD14 gene in the process of E. coli infection, a PK15 cell line with stable CD14 gene silencing was established in this study. Then three kinds of E. coli (F18ab, F18ac and K88ac) and LPS with three kinds of concentration (50 ng/ mL, 100 ng/mL and 1000 ng/mL) were used to stimulate cells to detect the expression changes of key genes in the TLR4 signaling pathway and the release levels of proinflammatory factor in downstream, which aims to explore the influence of CD14 gene silencing on the immune response mediated by TLR4 signaling pathway so as to provide a basis for further function and mechanism research of CD14 gene.
purchased from Jackson (PA, USA). LPS was purchased from SigmaAldrich (MO, USA). Porcine IL-1β and TNF-α ELISA kits were purchased from AssayPro (MO, USA). 2.2. Primer design and sequence synthesis The qPCR primers of TLR4 signaling pathway related genes were designed based on the sequences of these genes from the pig available in the GenBank database using Primer Express 2.0 software (Table 1). All of primers were synthesized by Sangon Biotech (Shanghai, China) Co., Ltd. 2.3. Establishment of PK15 cell line with CD14 gene silencing PK15 cells were incubated with DMEM culture medium containing 10% FBS in an incubator under the conditions of 37 °C and 5% CO2. When the coverage of cells reached ~80%, transfections were performed with four lentivirus vector constructed in our previous research (Wu et al., 2016) by pGLV3-CD14-1, pGLV3-CD14-2, pGLV3-CD14-3 and pGLV3-CD14-4, a negative control group pGLV3- NC and a blank control group without lentivirus infection, each treatment had three repeats. Infected cells were cultured at 37 °C in a 5% CO2 incubator overnight and then cells expressing green fluorescent protein were observed after culture for 24 h. 10 μg/mL puromycin was used to screen the positive cells every 24 h and cell total RNA and protein were extracted for analyzing the transcription and translation level to detected the interference of CD14 gene. Then several positive monoclonal cells were picked to establish the PK15 cell line with CD14 gene silencing. 2.4. E. coli stimulation in PK15 cells of CD14-RNAi group and NC group PK15 cells of CD14-RNAi group and NC group were inoculated to 12-well plates at a density of 5.0 × 105 cells per well, and cells were incubated for 24 h in an incubator under the conditions of 37 °C and 5% CO2. E. coli F18ab, E. coli F18ac, and E. coli K88ac fimbriae standard strains were respectively inoculated to LB culture medium and incubated for 12 h at a rotating speed of 200 rev/min on a shaking table. After centrifugation for 5 min at a rotating speed of 4000 rev/min, the supernatant of three kinds of E. coli were filtered through 0.22 μm filters to collect the bacteria supernatant and PBS buffer solution was used to resuspend the bacteria precipitation and then wash and centrifuge for three times. Cells with CD14-RNAi group and NC group were exposed to the bacteria supernatant of E. coli F18ab, E. coli F18ac, and E. coli K88ac for 4, 8 and 12 h, three parallel replicates were used for each group. 2.5. LPS induce in PK15 cells of CD14-RNAi group and NC group
2. Materials and methods
When the cells of CD14-RNAi group and NC group were 80% confluent, they were exposed to 50 ng/mL, 100 ng/mL and 1000 ng/mL LPS (Sigma-Aldrich, MO, USA) for 2, 4 and 6 h, three parallel replicates were used for each group. At the same time, CD14-RNAi cells and NC cells without stimulation treatment were set as control group respectively. Then cell total RNA and protein were extracted for transcript and translation levels of TLR4 signaling related genes. And cells culture supernatants were collected for ELISA analysis.
2.1. Reagents and materials The pig kidney cell line (PK15) was obtained from the American Type Culture Collection (ATCC, USA). E. coli F18ab, E. coli F18ac, and E. coli K88ac fimbriae standard strains were provided by the veterinary laboratory at the Institute of Microbiology, University of Pennsylvania. Fetal bovine serum (FBS) and Dulbecco's modified eagle medium (DMEM) were purchased from Gibco (NY, USA). Trizol was purchased from Invitrogen (CA, USA). HiScript® II Q Select RT SuperMix for qPCR and AceQ® qPCR SYBR® Green Master Mix were purchased from Vazyme Biotech Co., Ltd. (Nanjing, China). Total protein extraction kit and BCA protein detection kit were purchased from Nanjing Keygen Technology Development Co., Ltd. (Nanjing, China). Primary antibodies-CD14 (1:400), MyD88 (1:800), IFN-α (1:600), IL-1β (1:600), TLR4 (1:1000), TNF-α (1:1000) and β-actin (1:4000)-were purchased from Abcam (Cambridge, UK). Second antibody (IgG-HRP, 1:3000) was
2.6. Quantitative real-time PCR (qPCR) Total RNA was extracted using the Trizol Reagent (Takara, China), according to the manufacturer's instructions. RNA was reversed to cDNA and was used as a template to detect and analyze the transcript levels of CD14 and TLR4 signaling related genes. The 50-μL reaction mixture for cDNA synthesis contained the following: 10 μL 5× HiScript® II Q RT SuperMix, 2000 ng total RNA, and RNase-free H2O to make up the final volume of 50 μL. The reaction was carried out at 268
Gene 628 (2017) 267–274
C. Dai et al.
Fig. 1. Establishment of PK15 cell line with CD14 gene silencing. (A) The expression of green fluorescent protein (GFP) in PK15 cells infected by lentivirus solution for 24 h under a fluorescence microscope (100×); the lower panels are the corresponding cells in the absence of fluorescence exposure, which shows the overall density of cells. (B) The mRNA levels of CD14 in four kinds of lentivirus (pGLV3-CD14-1, pGLV3-CD14-2, pGLV3-CD14-3, pGLV3-CD14-4 and pGLV3-NC) and Blank cells without any treatments were determined by qPCR detection. **, P < 0.01. (C) The protein expression levels of CD14 in four kinds of lentivirus and Blank cells without any treatments were determined by western blot. The integrated option density (IOD) of each kind of protein was calculated by ImageJ and gray value was defined as IOD (target protein)/IOD (β-actin), and the average of gray values of Blank group was defined as 1 to homogenize different groups to analyze the relative protein expression level of each kind of protein; *, P < 0.05. (D) The expression of green fluorescent protein (GFP) in PK15 cells with CD14 silencing and NC group cultured from monoclonal cells respectively under a fluorescence microscope (100×); the right-hand panels are the corresponding cells in the absence of fluorescence exposure, which shows the overall density of cells. Each treatment has tree repetitions.
2.8. Cytokines detection of IL-1β and TNF-α in the TLR4 signaling pathway
37 °C for 15 min and then at 85 °C for 5 s. QPCR (PrimeScriptTM RT Master Mix, Vazyme) was carried out as follows: 10 μL SYBR Premix ExTaq™ II (2 ×), 0.4 μL PCR Forward Primer (10 μM), 0.4 μL PCR Reverse Primer (10 μM), 0.4 μL ROX Reference Dye II (50 ×), 2 μL cDNA, and ddH2O to make the final volume 20 μL, each sample had three repeats. The house keeping genes ACTB and GAPDH were used as references control to normalize the expression level, and 2− ΔΔCt method was used to calculate the relative transcription level. We performed qPCR using the ABI 7500 system (Applied Biosystems, USA). The reaction conditions were 95 °C for 15 s, followed by 40 cycles of 95 °C for 5 s and 62 °C for 34 s. The dissociation curve was analyzed after amplification. The reaction conditions were 95 °C for 15 s, 62 °C for 60 s, 95 °C for 15 s, and 60 °C for 15 s.
In PK15 cells between RNAi group and control group, we measured the level of two cytokines in the TLR4 signaling pathway including interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) using the ELISA kits (AssayPro, USA). The assay procedure and standard curve generation were carried out according to the instructions provided with the kit. 2.9. Statistical analysis The 2− ΔΔCt method (Livak and Schmittgen, 2001) was used to detect the transcript levels of genes, and the internal reference genes GAPDH and ACTB were used to normalize gene expression levels. SPSS 17.0 including one-way ANOVA method was provided for difference analysis of transcript and protein levels of genes and release levels of cytokines between CD14-RNAi group and NC group. Each independent experiment or treatment was performed with three repeats and the data was counted as x ± SD.
2.7. Western blot analysis BCA kit (Nanjing Keygen Technology Development Co., LTD) was used to normalize protein levels. 10% SDS PAGE was used to treat all protein samples in each group, 10 μL protein was added to loading hole and ran the electrophoresis for 70 min under the condition of 160 V. Blotting: proteins were transferred to PVDF membranes and immunoblotted with antibodies to CD14 (1:400), MyD88 (1:800), IFN-α (1:600), IL-1β (1:600), TLR4 (1:1000), TNF-α (1:1000) and β-actin (1:4000). The secondary antibody was horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG (Abcam, UK, 1:3000).
3. Results 3.1. Establishment of PK15 cell line with CD14 gene silencing PK15 cells infected by four kinds of interference vectors and a negative control lentivirus for 24 h could express highly green fluorescent 269
Gene 628 (2017) 267–274
C. Dai et al.
Fig. 2. Effect of CD14 gene silencing on the TLR4 signaling pathway without any stimulation. Expression differences of TLR4 signaling pathway related genes in PK15 cells with CD14 gene silencing in normal state were detected by qPCR (A) and western blot (B). The integrated option density (IOD) of each kind of protein was calculated by ImageJ and gray value was defined as IOD (target protein)/IOD (β-actin), and the average of gray values of NC group of each protein was defined as 1 to homogenize different groups to analyze the relative protein expression level; **, P < 0.01; *, P < 0.05. Each treatment has tree repetitions.
3.2. Influence of CD14 gene silencing on TLR4 signaling pathway under normal physiological condition in PK15 cells
protein (Fig. 1A), which illustrated that lentivirus had integrated into PK15 cells efficiently. Cells total RNA and total protein were collected after screened by puromycin for 24 h (namely transfection for 48 h) to analyze the interference efficiency of different interference vectors. Results showed that four kinds of interference vectors could significantly reduce transcription level of CD14 gene in PK15 cells. The interference efficiency of pGLV3-CD14-1, pGLV3-CD14-2, pGLV3CD14-3 and pGLV3-CD14-4 were 35.90%, 44.17%, 69.91% and 35.90%, respectively (Fig. 1B). Protein expression levels of pGLV3CD14-3 and pGLV3-CD14-4 were also significantly reduced (Fig. 1C). PK15 cells infected by pGLV3-CD14-3 with the highest interference efficiency were diluted to cultivate positive monoclonal cells. And amplification culture of monoclonal cells could express green fluorescent protein stably after continuous puromycin screening (Fig. 1D), which suggested that PK15 cell line with stable CD14 gene silencing has been established successfully and could be used for further research.
When the convergence rate of PK15 cells with CD14-RNAi group and NC group reached to 80–90%, cells total RNA and protein were extracted to analyze mRNA and protein expression level. Taking the average expression level of each gene negative control group as reference 1, the relative expression level of each gene after CD14 gene silencing was calculated according to 2− ΔΔCt (Livak and Schmittgen, 2001). QPCR results show that the mRNA expression levels of TLR4 pathway-related genes (MyD88, IFN-α, IL-1, TLR4 and TNF-α) were not significant changes (P > 0.05) after CD14 gene silencing (Fig. 2A). Western blot results showed that there was no significant change (P > 0.05) in the protein expression level (Fig. 2B).
270
Gene 628 (2017) 267–274
C. Dai et al.
Fig. 3. Effect of CD14 gene silencing on the TLR4 signaling pathway with stimulation by E. coli strain and supernatant. The abscissa below and the left ordinate represent four kinds of cells with different treatments and their relative mRNA expression level; the data showed extremely significant difference with different capital letters (P < 0.01) and significant difference with different little letters (P < 0.05). The upper abscissa and the right ordinate represent four different stimulation time points of bacterial culture supernatants and their relative mRNA expression level; ** showed extremely significant difference between CD14-RNAi group and NC group at the same time point (P < 0.01) and * showed significant difference (P < 0.05). (A), (B) and (C) represented the expression of TLR4 signaling pathway related genes between CD14-RNAi group and NC group with stimulation by strain and bacterial culture supernatants from E. coli F18ab, E. coli F18ac and E. coli K88ac, respectively. Each treatment has tree repetitions.
fold) and decreased later (within 2-fold). What's interesting was that the expression levels of CD14-RNAi group were always lower than NC group (P < 0.05 or P < 0.01).
3.3. Influence of CD14 gene silencing on TLR4 signaling pathway in PK15 cells stimulated by E. coli PK15 cells of CD14-RNAi group, NC group and Blank group were exposed to E. coli strain of F18ab, F18ac and K88ac with the concentration of 109 CFU/mL for 4 h and stimulated by bacterial supernatants for 4 h, 8 h and 12 h. Then qPCR was used to analyze mRNA expression levels of TLR4 signaling pathway-related genes and variation trend with time. As the results showed in Fig. 3, the mRNA expression levels of key genes in TLR4 signaling pathway after three kinds of E. coli stimulation presented a consistent pattern: the mRNA expression level of TLR4 signaling pathway-related genes in cells of blank group and NC group significantly increased compared with cells without stimulation, while expression levels in cells of CD14-RNAi group were close to cells without stimulation (P > 0.05) even downregulated (P < 0.05 or P < 0.01) except MyD88 gene. With the increase of stimulation time induced by bacterial supernatants, the expression level of TNF-α showed a trend of rising (> 3-fold), while which of MyD88, IFN-α, IL-1 and TLR4 increased first (overall > 2-
3.4. Influence of CD14 gene silencing on TLR4 signaling pathway in PK15 cells induced by LPS PK15 cells of CD14-RNAi group and NC group were exposed to LPS solution with the concentration of 50, 100 and 1000 ng/mL for 2 h, 4 h and 6 h. Then qPCR was used to analyze mRNA expression variation between CD14-RNAi group and NC group of TLR4 signaling pathwayrelated genes and variation trend with time increasing. Results showed that the expression levels of all genes in TLR4 signaling pathway in both CD14-RNAi group and NC group showed a trend of rising with the increase of time (overall > 2-fold), and the expression levels of CD14RNAi group were always lower than NC group (P < 0.05 or P < 0.01). Fig. 4 showed the details of expression change of all genes.
271
Gene 628 (2017) 267–274
C. Dai et al.
Fig. 4. Effect of CD14 gene silencing on the TLR4 signaling pathway with stimulation by LPS. Expression differences of TLR4 signaling pathway related genes in PK15 cells between CD14 RNAi group and NC group induced by LPS with different concentration at different time points were detected by qPCR. * showed significant difference between CD14-RNAi group and NC group at the same time point (P < 0.05). ** showed extremely significant difference between CD14-RNAi group and NC group at the same time point (P < 0.01). (A), (B) and (C) represented the expression of TLR4 signaling pathway-related genes in PK15 cells between CD14-RNAi group and NC group induced by LPS with concentration of 50 ng/mL, 100 ng/mL and 1000 ng/mL, respectively. Each treatment has tree repetitions.
related genes increased first and decreased later and reached the peak at 4 h or 8 h then down-regulated at 12 h, which illustrated that time point leading to constant immune response of body was between 4 and 8 h. When PK15 cells were induced by LPS with three different concentration at three time points, the expression levels of signaling pathway-related genes up-regulated with the increase of time while the degree of up-regulation of CD14-RNAi group was greatly lower than NC group, which was consistent with the results of bacterial stimulation. These results suggested that CD14, as the membrane receptor protein in TLR4 signaling pathway, participated in immune regulation through synergistic regulation with key genes involved in immune response pathway in the pathogenic stimulation such as LPS and played an important regulating role in the whole signaling pathway. The reduction of mRNA transcription level of CD14 gene led to lower translation level of receptor proteins, causing lower transcription level of key molecules with synergy in membranal signaling pathway. MyD88 as an initial adapter protein for TLRs/IL-1 signaling pathways, could mediate most members of TLRs family downstream signaling transduction including TLR4 (O'Neill and Bowie, 2007). In this study, the mRNA level of MyD88 was not significantly lower after CD14 gene silencing, which may relate to its regulation function in other signaling pathways. In the body's normal physiological state, the mRNA expression level of MyD88 can remain in a relatively stable state under the coordination of multiple signal pathways. ELISA results showed that the release level of IL-1β up-regulated continuously with the increase of stimulation time induced by bacterial supernatant and LPS. The release level of TNF-α up-regulated continuously with the increase of stimulation time induced by LPS but increased first and decreased later induced by bacterial supernatants, which reached the highest level at 8 h. It is obvious that the levels of TNF-α and IL-1β in cell culture supernatant of CD14-RNAi group were always lower than NC group, which was consistent with previous results. Interleukin-1β (IL-1β) and tumor necrosis factor (TNF) are two kinds of important cytokines in immune response (Marchand-Adam et al., 2005; Szydlowski et al., 2011), so the above results suggested that CD14 do play an important regulating role not only in TLR4 signaling pathway but also in the immune process of E. coli F18 invasion. The
3.5. Changes of proinflammatory cytokine levels in cell culture supernatant with CD14 gene silencing Cell culture supernatant with different treatments were collected to analyze proinflammatory cytokines levels in downstream of TLR4 signaling pathway (TNF-α and IL-1β) in PK15 cells between CD14-RNAi group and NC group by ELISA kit. ELISA scale was used to make the standard curves of TNF-α and IL-1β and then regression equations were obtained (Fig. S1). The results of proinflammatory cytokines levels showed that the level of IL-1β presented a trend of continuous rising (overall > 3-fold) with the increase of stimulation time induced by bacterial supernatant and LPS (Fig. 5A). The release level of TNF-α presented a trend of continuous rising (P < 0.05 or P < 0.01) with the increase of stimulation time induced by LPS but increased first and decreased later (P < 0.05) induced by bacterial supernatants (Fig. 5B). As a whole, the levels of TNF-α and IL-1β in cell culture supernatant of CD14-RNAi group were always lower than NC group (P < 0.05 or P < 0.01). 4. Discussion This study established PK15 cell line with stable CD14 gene silencing, which played an important role in intestinal immune response and pathogen defense caused by pathogenic microorganisms. With CD14 gene silencing, the expression levels of TLR4 signaling pathway-related genes did not change significantly and the entire signaling pathway was in a steady state. It meant that CD14 RNAi mediated by lentivirus interfered the expression of CD14 gene specifically but it had no negative influence on the physiological state of cell itself, which revealed that RNAi technology mediated by lentivirus had been applied to the stable gene silencing maturely (Abbas-Terki et al., 2002). We used three kinds of pathogenic E. coli F18ab, F18ac and K88ac to stimulate PK15 cells of CD14-RNAi group and NC group and found that the expression levels of all genes in entire TLR4 signaling pathway up-regulated significantly, while the degree of up-regulation of CD14-RNAi group was greatly lower than NC group. With the increase of stimulation time induced by bacterial supernatant, the expression levels of signaling pathway272
Gene 628 (2017) 267–274
C. Dai et al.
Fig. 5. Effect of CD14 gene silencing on the release of immune proinflammatory factors. Expression differences of proinflammatory factors of IL-1β (A) and TNF-α (B) in TLR4 signaling pathway in PK15 cells between CD14 RNAi group and NC group under the treatment of stimulation by E. coli supernatant as well as LPS with the increase of time were detected by ELISA. LPS-50, LPS-100 and LPS-1000 represented LPS with concentration of 50 ng/mL, 100 ng/mL and 1000 ng/mL, respectively. * showed significant difference between CD14-RNAi group and NC group at the same time point (P < 0.05). Each treatment has tree repetitions.
was not suitable for Chinese local pig breeds (Bao et al., 2008). Based on previous results of transcriptome sequencing and the research results in this study, it is necessary to view CD14 gene as a new candidate gene of resistance to E. coli. We will research specifically the regulation role of CD14 gene in the process of E. coli invasion in Chinese local pig breeds, which aims to investigate the feasibility of making CD14 gene as a genetic marker of resistance to colibacillosis in weaning piglets especially Chinese local pig breeds. The final purpose of our study is to make an important breakthrough in bottleneck of molecular breeding of resistance to colibacillosis in Chinese local pig breeds at present.
down-regulation of these two kinds of cytokines inhibited the immune response mediated by TLR4 signaling pathway, which partially led to immunosuppression at last. What's interesting was that most expression level changes ranged from 1.5 to 2.5-fold, which might be considered physiologically relevant. With the prolongation of bacterial stimulation, the cell body will start the appropriate defense mechanism (such as secreting some anti-inflammatory factors) to prevent the infinitely elevation of expression of proinflammatory factors and genes. In our previous study, glycosphingolipid biosynthesis-globo series and Toll-like signaling pathway were found to have important regulation role in resistance to E. coli F18 in Sutai piglets (Bao et al., 2012). Then we found TLR4 signaling pathway and CD14 gene could be associated with resistance of Chinese native weaning piglets to E. coli by transcriptome analysis of duodenum in E. coli F18-resistant and E. coli F18-sensitive individuals of Meishan piglets (Chinese local pig breed) (Wu et al., 2016). This study further verified that CD14 gene and TLR4 signaling pathway played an important regulation role in resistance to E. coli invasion. Previous research achieved the disease-resistant breeding in foreign pig breeds based on the polymorphism on M307 loci of α-1, 2-Fucosyltransferasegene (FUT1) in glycosphingolipid biosynthesis-globo series (Meijerink et al., 1997) while this genetic marker
5. Conclusions This study established successfully a PK15 cell line with CD14 gene silencing and illustrated that CD14 gene silencing would not have effect on TLR4 signaling pathway and the entire signaling pathway was in a steady state when cells were under normal physiological condition. However, pig CD14 gene silencing partially inhibited immune response to E. coli F18 invasion mediated by TLR4 signaling pathway. This study also further revealed that CD14 gene played an important regulation role not only in TLR 4 signaling pathway but also in the immune 273
Gene 628 (2017) 267–274
C. Dai et al.
Table 1 The primer information of genes for qPCR. Gene
Sequence of the primer
Length of products (bp)
Accession no.
CD14
F: 5′-CCTCAGACTCCGTAATGTG-3′ R: 5′-CCGGGATTGTCAGATAGG-3′ F: 5′-GTGCCGTCGGATGGTAGT-3′ R: 5′-CAGTGATGAACCGCAGGAT-3′ F: 5′-CCTGGACCACAGAAGGGA-3′ R: 5′-TCTCATGCACCAGAGCCA-3′ F: 5′-TGATTGTGGCAAAGGAGGA-3′ R: 5′-TTGGGTCATCATCACAGACG-3′ F: 5′-CAGATAAGCGAGGCCGTCATT-3′ R: 5′-TTGCAGCCCACAAAAAGCA-3′ F: 5′-CGACTCAGTGCCGAGATCAA-3′ R: 5′-CCTGCCCAGATTCAGCAAAG-3′ F: 5′-ACATCATCCCTGCTTCTACTGG-3′ R: 5′-CTCGGACGCCTGCTTCAC-3′ F: 5′-TGGCGCCCAGCACGATGAAG-3′ R: 5′-GATGGAGGGGCCGGACTCGT-3′
180
AB267810
173
EU056736
92
X57191
63
NM_001005149
113
AB232527
58
X54001
188
AF017079
149
XM_003124280.3
MyD88 IFN-α IL-1β TLR4 TNF-α GAPDH ACTB
Res. 6, 351–358. Liu, H.Z., Li, X.Y., Liu, B., Yu, M., Ma, Y.H., Chu, M.X., Li, K., 2008. Tissue distribution, SHP detection and association study with immune traits of porcine LBP and CD14 genes. Asian Australas. J. Anim. Sci. 21, 1080–1087. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using realtime quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25, 402–408. Marchand-Adam, S., Plantier, L., Bernuau, D., Legrand, A., Cohen, M., Marchal, J., Soler, P., Leseche, G., Mal, H., Aubier, M., Dehoux, M., Crestani, B., 2005. Keratinocyte growth factor expression by fibroblasts in pulmonary fibrosis: poor response to interleukin-1beta. Am. J. Respir. Cell Mol. Biol. 32, 470–477. Medzhitov, R., 2001. Toll-like receptors and innate immunity. Nat. Rev. Immunol. 1, 135–145. Meijerink, E., Fries, R., Vogeli, P., Masabanda, J., Wigger, G., Stricker, C., Neuenschwander, S., Bertschinger, H.U., Stranzinger, G., 1997. Two alpha(1,2) fucosyltransferase genes on porcine chromosome 6q11 are closely linked to the blood group inhibitor (S) and Escherichia coli F18 receptor (ECF18R) loci. Mamm. Genome 8, 736–741. Nockher, W.A., Scherberich, J.E., 1997. Expression and release of the monocyte lipopolysaccharide receptor antigen CD14 are suppressed by glucocorticoids in vivo and in vitro. J. Immunol. 158, 1345–1352. O'Neill, L.A., Bowie, A.G., 2007. The family of five: TIR-domain-containing adaptors in Toll-like receptor signalling. Nat. Rev. Immunol. 7, 353–364. Perera, P.Y., Vogel, S.N., Detore, G.R., Haziot, A., Goyert, S.M., 1997. CD14-dependent and CD14-independent signaling pathways in murine macrophages from normal and CD14 knockout mice stimulated with lipopolysaccharide or taxol. J. Immunol. 158, 4422–4429. Poltorak, A., He, X., Smirnova, I., Liu, M.Y., Van Huffel, C., Du, X., Birdwell, D., Alejos, E., Silva, M., Galanos, C., Freudenberg, M., Ricciardi-Castagnoli, P., Layton, B., Beutler, B., 1998. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282, 2085–2088. Sakr, Y., Burgett, U., Nacul, F.E., Reinhart, K., Brunkhorst, F., 2008. Lipopolysaccharide binding protein in a surgical intensive care unit: a marker of sepsis? Crit. Care Med. 36, 2014–2022. Sun, L., Xia, R.W., Yin, X.M., Yu, L.H., Zhu, G.Q., Wu, S.L., Bao, W.B., 2015. Analysis of differential expression of TLR4 and TLR4 signaling pathway genes under lipopolysaccharide-induced pig intestinal epithelial cells. Acta Veterinaria et. Zootechnica Sinica 46, 1095–1101. Szydlowski, M., Buszka, A., Mackowski, M., Lechniak, D., Switonski, M., 2011. Polymorphism of genes encoding cytokines IL6 and TNF is associated with pig fatness. Livest. Sci. 136, 150–156. Wright, S.D., Ramos, R.A., Tobias, P.S., Ulevitch, R.J., Mathison, J.C., 1990. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 249, 1431–1433. Wu, Z.C., Liu, Y., Dong, W.H., Zhu, G.Q., Wu, S.L., Bao, W.B., 2016. CD14 in the TLRs signaling pathway is associated with the resistance to E. coli F18 in Chinese domestic weaned piglets. Sci Rep 6.
process of E. coli F18 invasion, which was expected to be as a molecular marker of piglet diarrhea caused by E. coli. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2017.07.055. Declaration of interest The authors declare that they have no conflict. Acknowledgement This work was supported by the National Natural Science Foundation of China (31572360, 31372285), the Science and Technology Supporting Project of Jiangsu Province (BE2016315, BE2014357), and the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions. References Abbas-Terki, T., Blanco-Bose, W., Deglon, N., Pralong, W., Aebischer, P., 2002. Lentiviralmediated RNA interference. Hum. Gene Ther. 13, 2197–2201. Bao, W.B., Wu, S.L., Musa, H.H., Zhu, G.Q., Chen, G.H., 2008. Genetic variation at the alpha-1-fucosyltransferase (FUT1) gene in Asian wild boar and Chinese and Western commercial pig breeds. J. Anim. Breed. Genet. 125, 427–430. Bao, W.B., Ye, L., Pan, Z.Y., Zhu, J., Du, Z.D., Zhu, G.Q., Huang, X.G., Wu, S.L., 2012. Microarray analysis of differential gene expression in sensitive and resistant pig to Escherichia coli F18. Anim. Genet. 43, 525–534. Beutler, B., 2005. The toll-like receptors: analysis by forward genetic methods. Immunogenetics 57, 385–392. Carrillo, E.H., Gordon, L., Goode, E., Davis, E., Polk, H.C., 2001. Early elevation of soluble CD14 may help identify trauma patients at high risk for infection. J. Trauma 50, 810–816. Chen, L.H., 2000. Molecular CD14's receptor function and its signal transmission. Foreign Medical Sciences 23, 137–140. Frey, E.A., Miller, D.S., Jahr, T.G., Sundan, A., Bazil, V., Espevik, T., Finlay, B.B., Wright, S.D., 1992. Soluble CD14 participates in the response of cells to lipopolysaccharide. J. Exp. Med. 176, 1665–1671. Fung, S.C., Mollnes, T.E., 2012. Methods and compositions for the prevention and treatment of sepsis: U.S. Patent 8,329,169[P]. 2012-12-11. Gluck, T., Silver, J., Epstein, M., Cao, P., Farber, B., Goyert, S.M., 2001. Parameters influencing membrane CD14 expression and soluble CD14 levels in sepsis. Eur. J. Med.
274