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Haemophilus parasuis induces activation of NF-κB and MAP kinase signaling pathways mediated by toll-like receptors
Molecular Immunology 65 (2015) 360–366
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Haemophilus parasuis induces activation of NF-B and MAP kinase signaling pathways mediated by toll-like receptors Yushan Chen a , Ting Liu a , Paul Langford b , Kexin Hua a , Shanshan Zhou a , Yajun Zhai a , Hongde Xiao c , Rui Luo a , Dingren Bi a , Hui Jin a,∗ , Rui Zhou a,∗ a
State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China Section of Paediatrics, Imperial College London, St Mary’s Campus, London W2 1PG, UK c Hubei Center for Animal Disease Control and Prevention, Wuhan 430070, China b
a r t i c l e
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Article history: Received 4 December 2014 Received in revised form 5 February 2015 Accepted 11 February 2015 Keywords: Haemophilus parasuis Inflammatory response NF-B pathway MAPK pathway TLRs
a b s t r a c t Glässer’s disease in pigs caused by Haemophilus parasuis is characterized by a severe membrane inflammation. In our previous study, we have identified activation of the transcription factor NF-B after H. parasuis infection of porcine epithelial cells. In this study, we found that H. parasuis infection also contributed to the activation of p38/JNK MAPK pathway predominantly linked to inflammation, but not the ERK MAPK pathway associated with growth, differentiation and development. Inhibition of NF-B, p38 and JNK but not ERK activity significantly reduced IL-8 and CCL4 expression by H. parasuis. We also found TLR1, TLR2, TLR4 and TLR6 were required for NF-B, p38 and JNK MAPK activation. Furthermore, MyD88 and TRIF signaling cascades were essential for H. parasuis-induced NF-B activation. These results provided new insights into the molecular pathways underlying the inflammatory response induced by H. parasuis. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction The consequences of infections caused by Haemophilus parasuis in pigs are great economic losses in the swine industry worldwide, due to expensive antibiotic treatment and mortality of animals in acute form of the disease (Nedbalcova et al., 2006; Olvera et al., 2007). This Gram-negative organism firstly establishes infection in the upper respiratory tract but, under certain circumstances, which occurred sporadically and stress associated, some strains can constitutes an initial step to cause pneumonia (Aragon, 2013; Baumann and Bilkei, 2002; Costa-Hurtado and Aragon, 2013; Moller and Kilian, 1990). In disease outbreaks, virulent strains spread to internal organs such as brain, heart, spleen, lung, kidney, intestine and lymph node (Vahle et al., 1997; Wang et al., 2006; Yin et al., 2004), causing systemic invasion characterized by polyserositis syndrome including polyserositis, pleuritis, peritonitis, meningitis, pericarditis and arthritis, which is associated with substantial morbidity and mortality of piglets (Brockmeier, 2004; Oliveira and Pijoan, 2004). Multiplication of H. parasuis inside the host induces a strong inflammatory reaction mediated by inflammatory cytokines such
∗ Corresponding authors. Tel.: +86 27 87280147; fax: +86 27 87280208. E-mail address: [email protected] (H. Jin). http://dx.doi.org/10.1016/j.molimm.2015.02.016 0161-5890/© 2015 Elsevier Ltd. All rights reserved.
as IL-8 and macrophage inflammatory protein-1 (MIP-1/CCL4) by recruitment of immune cells to the site of infection to initiate innate immune responses (Bouchet et al., 2008, 2009; Chen et al., 2012; Costa-Hurtado et al., 2013). IL-8 is associated with serous membrane inflammation and meningitis after H. parasuis infection (Bouchet et al., 2008, 2009). CCL4, a chemoattractant for monocytes and other immune cells, effectively induces the adhesion of circulating leukocytes for their extravasation bound with proteoglycan on endothelia, and is associated with exudative inflammation (Bystry et al., 2001; Ren et al., 2010; Tanaka et al., 1993). IL-8 and CCL4 were subject to transcription factor NF-B transcriptional regulation (Ali and Mann, 2004; Vallabhapurapu and Karin, 2009). In a recent study, we have demonstrated that H. parasuis infection activated the NF-B pathway and induced the up-regulation of IL-8 andCCL4 (Chen et al., 2012). However, whether there were common pathways involved in activation was not investigated. Toll-like receptors (TLRs) are the first line of defense against invading pathogens, being the important pattern recognition receptors for detecting and responding to microbial ligands upstream of the NF-B pathway (Doyle and O’Neill, 2006; Kawai and Akira, 2007). It has been reported that TLR2 recognizes bacterial lipopeptides in combination with TLR1 and TLR6, and TLR4 recognizes LPS from most Gram-negative species (Kawai and Akira,
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2007). Helicobacter pylori infection induced NF-B activation via TLR2 and TLR5 but not TLR4 (Smith et al., 2003), suggesting that diverse bacteria activate the NF-B pathway in different ways. After the identification of NF-B activation following H. parasuis infection (Chen et al., 2012), here we determine the TLRs by which H. parasuis mediate NF-B activation and inflammatory cytokine upregulation by the NF-B and mitogen-activated protein kinases (MAPKs) pathways. Early activation of TLRs by pathogen-associated molecular patterns (PAMPs) and their downstream signaling pathways, such as MAPKs and NF-B, have been found to be critical for host innate and adaptive immune responses, including expression of inflammatory cytokines (Doyle and O’Neill, 2006; Kawai and Akira, 2007; Kyriakis and Avruch, 2012). MAPK signaling cascades are involved in many cellular processes such as inflammation, proliferation, differentiation, stress responses and apoptosis (Kyriakis and Avruch, 2012; Platanias, 2003). There are three major groups of MAPK: the p38 MAP kinases (p38), the c-Jun amino-terminal kinase (JNK), and the extracellular signal-regulated protein kinase 1/2 (ERK), all of which are involved in the regulation of inflammation (Cargnello and Roux, 2011). The recent study suggested that MAPK signaling pathway involved in activating immune and inflammatory response upon H. parasuis infection (Wang et al., 2012). Understanding the cellular signaling pathways leading to inflammation in response to H. parasuis infection may provide new therapeutic strategies for treatment.
2. Materials and methods 2.1. Bacterial strain and culture conditions H. parasuis SH0165 strain was cultured in tryptic soy broth (TSB; Difco Laboratories, Detroit, MI, USA) supplemented with 1 mg/ml nicotinamide adenine dinucleotide (NAD) and 5% bovine serum. After incubation at 37 ◦ C overnight with circular agitation (200 rpm), bacteria were harvested by centrifugation at 5000 × g for 15 min, washed three times with PBS and resuspended in fresh DMEM medium.
2.2. Cell culture Porcine kidney (PK-15) cells were cultured and maintained in Dulbecco’s Modified Eagle Media (DMEM, Gibco), supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U/mL penicillin, 10 g/mL streptomycin sulfate and incubated at 37 ◦ C in a humidified 5% CO2 incubator. The antibiotics have been removed before transfection and cells were washed three times with PBS before inoculating with H. parasuis.
2.3. RNA exaction and real-time quantitative analysis Total cellular RNA was extracted from PK-15 cells by using TRIzol reagent (Invitrogen). RNA was reverse-transcribed into cDNA using reverse transcriptase (Roche), and cDNA amplification was performed by the SYBR green PCR assay (Roche). Individual transcripts in each sample were assayed three times and normalized to the porcine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA level. Nucleotide sequences of the primers used for Q-PCR are presented in Table S2. In experiments designed to block NF-B and MAPK signaling, PK-15 cell were pre-treated for 1 h with inhibitors prior to H. parasuis (MOI of 10) infection, and RNA samples were collected after 24 h infection.
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2.4. Reagents and siRNA Mitogen-activated protein kinase (MAPK) inhibitors SB203580, SP600125, U0126 and NF-B inhibitor BAY-117082 were purchased from Sigma–Aldrich (St. Louis, MO, USA). Cells were pretreated with 0.2, 1, 5 or 10 M inhibitor for 1 h before infection with H. parasuis at a MOI of 10. Negative control siRNA, TLR1, TLR2, TLR4, TLR5, TLR6, MyD88, TRIF, TRAF6, IRAK1, IRAK4, TAK1 and TAB1 siRNA oligonucleotide were purchased from GenePharma (China) and the sequences are listed in Table S1. siRNA (40 pmol) was transfected into PK-15 cells using Lipofectamine 2000 (Invitrogen) in siRNA knockdown experiments. 2.5. Western blotting PK-15 cells were cultured in 60 mm-dishes and were infected or mock-infected with H. parasuis. At the indicated time, cells were harvested by adding lysis buffer with proteinase inhibitor cocktail and phosphatase inhibitor cocktail (Sigma–Aldrich). Extracted proteins were separated by SDS-PAGE and electrophoretically transferred to polyvinyldifluoride membranes (Millipore). The blots were blocked with 5% nonfat dry milk and then incubated with primary antibody overnight at 4 ◦ C. After three washes, the blots were subsequently incubated with the secondary antibody horseradish peroxidase (HRP) conjugate solution for 1 h at room temperature. Finally, the signal was detected using an ECL chemiluminescence detection kit (Bio-rad). Polyclonal antibodies against NF-B p65, phospho-NF-B p65 (S536), p38, phosphop38 (Thr180/Tyr182), JNK, phospho-JNK (Thr183/Tyr185), ERK, phospho-ERK (Thr202/Tyr204) were obtained from Cell Signaling Technology (USA). Polyclonal antibodies against TLR1, TLR2, TLR4 and TLR6 were purchased from Santa Cruz Biotechnology and monoclonal antibodies -actin was purchased from Beyotime (China). Polyclonal antibodies against MyD88, TRIF, IRAK4, IRAK1, TRAF6, TAK1 and TAB1 were obtained from Cell Signaling Technology (USA). 2.6. Transfection and luciferase reporter assay Transient transfection was performed by using Lipofectamine 2000 (Invitrogen). PK-15 cells were seeded in 24-well plates and incubated until the cells were grown to 60–70% confluency. Cells were transfected with 40 pmol/well negative control siRNA or other siRNA. Twenty-four hours later, PK-15 cells were infected or mockinfected with H. parasuis at a MOI of 10. At the indicated time points, cell lysates were analyzed by Western blotting or QPCR. In selected experiments, cells were co-transfected with 100 ng/well of NF-B luciferase reporter plasmid (pNF-B-Luc) and 100 ng/well of the Renilla luciferase construct pRL-TK (Promega) together with 40 pmol/well negative control or each siRNA. Twenty-four hours later, cells were infected or mock-infected with H. parasuis at a MOI of 10. Cells were harvested at the indicated time point and luciferase activity was measured using a dual-luciferase Assay System (Promega) according to the manufacturer’s directions. Data represent relative firefly luciferase activity normalized to Renilla luciferase activity. 2.7. Statistical analysis Data are expressed as mean ± SEM. Statistical analysis was performed using one-way analysis of variance (ANOVA) without interaction terms followed by Dunnett’s or Duncan’s test for multiple comparisons. A P-value less than 0.05 was considered significant and a P-value less than 0.01 was considered highly significant.
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3. Results 3.1. H. parasuis infection activated p38 and JNK MAPK pathway We originally identified that H. parasuis infection activated the NF-B pathway through IB␣ degradation and the phosphorylation of p65 in PK-15 cells (Chen et al., 2012). Thus, we sought to determine whether H. parasuis infection could activate the MAPK pathway, including three subfamilies of MAP kinases, p38, JNK1/2 and ERK1/2. H. parasuis infection induced the phosphorylation of p38 and JNK1/2 in a time-dependent manner. On the contrary, no ERK1/2 phosphorylation could be detected under the same experimental conditions (Fig. 1), whereas ERK1/2 activation is inducible in PK-15 cell by TNF␣ (data not shown). 3.2. NF-B and p38/JNK MAPK pathways were required for induction of IL-8 and CCL4 by H. parasuis in PK-15 cells We have recently demonstrated that H. parasuis infection induced expression of the inflammatory cytokines IL-8 and CCL4 in PK-15 cells (Chen et al., 2012). To determine whether there is a role for NF-B in induction of IL-8 and CCL4 by H. parasuis, we used the NF-B specific inhibitor BAY11-7082. As shown in Fig. 2A, cells treated with BAY11-7082 exhibited a reduced ability to up-regulate IL-8 and CCL4 transcription after H. parasuis infection. Previous studies have shown that the MAPK signaling pathways play an important role in the inflammatory response (Kyriakis and Avruch, 2012). We next sought to examine the role of the MAPK signaling cascades in the transcriptional regulation of IL-8 and CCL4 after H. parasuis infection. PK-15 cells were treated with SB202190 (a specific inhibitor of p38), SP600125 (a specific inhibitor of JNK), or U0126 (a specific inhibitor of ERK1/2), followed by H. parasuis infection. As shown in Fig. 2B and C, treatment with SB202190 and
Fig. 1. H. parasuis infection activates p38 and JNK in PK-15 cells. PK-15 cells were inoculated with DMEM (control, CON) or H. parasuis (HPS) at a MOI of 10 and whole cell extracts were prepared at indicated time points and subjected to Western blot analysis with antibodies specific for p38, phosphorylated p38, JNK, phosphorylated JNK, ERK, phosphorylated ERK. Anti--actin was included as a positive control for sample loading.
SP600125 significantly attenuated IL-8 and CCL4 transcription in a dose-dependent manner, but treatment with U0126 did not affect IL-8 and CCL4 transcription in H. parasuis-infected PK-15 cells. 3.3. H. parasuis infection activated NF-B via TLR signaling pathway TLR1, 2, 4, 5 and 6 have been shown to play a critical role in the recognition of various bacterial components with host cells (Kawai and Akira, 2007; Kopp and Medzhitov, 2003). To determine
Fig. 2. NF-B and p38/JNK MAPK pathways regulate H. parasuis-induced IL-8 and CCL4 transcription in PK-15 cells. (A) PK-15 cells were treated with NF-B inhibitor, BAY11-7082 (0.2, 1, 5 and 10 M) or DMSO (control), followed by un-infected or infection with H. parasuis (HPS, MOI of 10) for 12 h. PK-15 cells were harvested and real-time RT-PCR was performed as described in methods. *P < 0.05 or **P < 0.01 compared with DMSO plus H. parasuis (HPS) infection. (B) and (C) were performed similarly to that described in (A) expect for the inhibitors: p38 inhibitor, SB202190 (0.2, 1, 5 and 10 M), JNK inhibitor, SP600125 (0.2, 1, 5 and 10 M), or ERK inhibitor, U0126 (0.2, 1, 5 and 10 M) were used. **P < 0.01 as compared with DMSO plus H. parasuis (HPS) infection.
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Fig. 3. Involvement of TLR signaling cascades in NF-B activation by H. parasuis. (A, C and E) PK-15 cells were transfected with pNF-B-Luc and pRL-TK together with siRNAs of negative control, TLR1/2/4/5/6, MyD88, TRIF, TRAF6, IRAK1, IRAK4, TAK1 or TAB1 with/without H. parasuis infection at a MOI of 10. PK-15 cells were harvested at 24 h post-infection and luciferase activity measured. **P < 0.01 as compared with negative control siRNA plus H. parasuis infection. (B, D and F) PK-15 cells were transfected with negative control siRNA, TLR1, 2, 4, 5, 6, MyD88, TRIF, TRAF6, IRAK1, IRAK4, TAK1 or TAB1 siRNA and un-infected or infected with H. parasuis at a MOI of 10. Whole cell extracts were prepared at 24 h post-infection and subjected to Western blot analysis with antibodies specific for NF-B p65 or phospho-NF-B p65. Anti--actin was included as a control for sample loading.
whether TLRs are involved in mediating H. parasuis-induced NF-B activation, NF-B luciferase reporter assay was performed. PK-15 cells were transfected with pNF-B-Luc and pRL-TK together with TLR1, 2, 4, 5 or 6 siRNAs. TLR5 was used as a negative control because there is no bacterial flagellin in H. parasuis. We first confirmed the efficiency of TLR1, 2, 4, 5 and 6 siRNA in reducing endogenous TLR expression in PK-15 cells transfected with TLR siRNA or negative control siRNA. The expression of TLR1, 2, 4, 5 and 6 was markedly reduced by TLR siRNA (Fig. S1). As expected, TLR1, 2, 4 and 6 but not TLR5 knockdown significantly inhibited the induction of NFB after H. parasuis infection (Fig. 3A). Because phosphorylation of p65 has been shown to be important in NF-B-dependent transcriptional activity (Chen and Greene, 2004), we also assessed the phosphorylation of p65 in PK-15 cells transfected with TLR siRNA, followed by H. parasuis infection. TLR1, 2, 4 and 6, but not TLR5, siRNA reduced the phosphorylation of p65 in H. parasuis-infected PK-15 cells (Fig. 3B). These results indicated that TLR1, 2, 4 and 6 are involved in H. parasuis-induced NF-B activation. Among the known downstream signaling molecules in TLR signaling pathway, the adaptor molecule MyD88 or TRIF appears are among the first molecules to be recruited to the TLR receptor complex (Kawai and Akira, 2007). Thus, we next sought to determine the role of MyD88 and TRIF in H. parasuis-induced NF-B
activation. As shown in Fig. 3C and D, H. parasuis-induced NF-B activation was decreased after MyD88 or TRIF siRNA transfection. We further investigated whether TRAF6, IRAK1, IRAK4, TAK1 and TAB1, which are key adaptor molecules for NF-B activation in the TLR signaling pathway, are required for induction of NF-B. As shown in Fig. 3E and F, siRNA knockdown of TRAF6, IRAK1, IRAK4, TAK1 or TAB1 inhibited the activation of NF-B by H. parasuis. These data suggested that H. parasuis infection activated NF-B via TLR signaling pathway. 3.4. H. parasuis infection activated p38 and JNK MAPK pathway via TLR1, TLR2, TLR4 and TLR6 As shown in Fig. 4, transfection with TLR1, 2, 4 and 6, but not TLR5, siRNA inhibited the phosphorylation of p38 and JNK, but not ERK. The results suggest that H. parasuis induces activation of the p38 and JNK MAPK pathways by TLR1, 2, 4 and 6. 3.5. H. parasuis infection triggered the production of IL8 and CCL4 by TLR1, TLR2, TLR4 and TLR6 We have identified that H. parasuis infection activated NFB via TLR signaling pathways and induced expression of the
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to H. parasuis infection. As shown in Fig. 5, cells transfected with TLR1, TLR2, TLR4 or TLR6 siRNA, but not TLR5 siRNA had a reduced up-regulation of IL8 and CCL4 transcription. These results substantiate our findings that H. parasuis infection induces NF-B activation and downstream cytokine secretion in PK-15 cells via TLR1, TLR2, TLR4 and TLR6.
4. Discussion
Fig. 4. Involvement of TLR1, TLR2, TLR4 and TLR6 in p38 and JNK activation by H. parasuis. PK-15 cells were transfected with negative control (DMEM, CON) or TLR1, 2, 4, 5 or 6 siRNA and un-infected or infected with H. parasuis (HPS) at a MOI of 10. Whole cell extracts were prepared at 24 h post-infection and subjected to Western blot analysis with antibodies specific for p38, phosphorylated p38, JNK, phosphorylated JNK, ERK and phosphorylated ERK. Anti--actin was included as a control for sample loading. p-p38, p-JNK1/2 and p-ERK1/2 protein levels were quantitated by densitometric analysis using ImageJ software. Densitometric values for p-p38, p-JNK and p-ERK were normalized to p38, JNK and ERK respectively. Finally, all the values were normalized to siNegative/CON, which was set to a value of 1.0.
proinflammatory cytokines IL-8 and CCL4. To determine which TLR(s) were involved in signal transduction from the epithelial cell surface to mediate cytoplasmic IL-8 and CCL4 expression, we transfected PK-15 cells with TLR1, TLR2, TLR4, TLR5 or TLR6 siRNA prior
Some studies have demonstrated that H. parasuis induces host inflammatory responses (Bouchet et al., 2008, 2009; Chen et al., 2012). However, the signaling mechanisms underlying induction of the inflammatory responses have remained largely unknown. In this study, we demonstrate that H. parasuis infection induced IL-8 and CCL4 transcription via the NF-B and p38/JNK MAPK pathway in PK-15 cells. We also identified that TLR1, 2, 4 and 6 were involved in H. parasuis-induced NF-B activation and downstream transcription of the cytokines IL-8 and CCL4. Moreover, H. parasuis induced NF-B via MyD88 and TRIF signaling cascades (Fig. 6). The recent studies demonstrated H. parasuis can adhere and invade PK-15 cells (Frandoloso et al., 2012; Zhang et al., 2012, 2013), a well-established epithelial cell line of porcine kidney origin, which were applied in this study and proved to be an appropriate cell line for studying on the NF-B pathway activated by H. parasuis (Chen et al., 2012; Liu et al., 2014). In our previous study we showed that H. parasuis activated NF-B (Chen et al., 2012). Addiditionally, the results from this study showed that the bacterium induced activation of the p38 MAPK and JNK MAPK pathways (Figs. 1 and 2). In our study, no ERK pathway activation was found after infection with H. parasuis (Figs. 1, 2 and 4). One possibility is that JNK and p38 kinases are more responsive to stress stimuli ranging from osmotic shock to cytokine stimulation, while ERK1/2 is preferentially activated in response to growth factors and phorbolesters (Roux and Blenis, 2004). It has been previously reported that MEK1/2 were ERK-specific MAP2Ks, MKK3/4/6 were JNK-specific MAP2Ks, and MKK4/7 were p38-specific MAP2Ks while MKK4 can phosphorylate and activate both p38 and JNK (Kyriakis and Avruch, 2012). We speculate that H. parasuis infection activates MKK3/4/6/7 instead of MEK1/2 but further work is required to determine this. Our data also suggest that H. parasuis-induced IL-8 and CCL4 expression in PK15 cells is dependent on both the NF-B and MAPK-p38/JNK pathways (Fig. 2). This is consistent with other data in the literature. For example, it was reported that p38 and JNK had a role in promoting cytokine release via posttranscriptional regulation of mRNAs encoding proinflammatory proteins
Fig. 5. H. parasuis infection activated IL-8 and CCL4 transcription via TLR1, TLR2, TLR4 and TLR6. (A) PK-15 cells were transfected with negative control (DMEM, CON) or TLR1, 2, 4, 5 or 6 siRNA and un-infected or infected with H. parasuis at a MOI of 10 for 12 h. PK-15 cells were harvested and real-time RT-PCR for IL8 was performed as described in methods. (B) The experiments were performed similarly to those described in (A) expect that CCL4 transcription was measured. **P < 0.01 as compared with negative control siRNA plus H. parasuis infection.
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Fig. 6. Schematic representation of the signaling pathways involved in H. parasuis (HPS) induced IL-8 and CCL4 expression. As indicated, H. parasuis up-regulates IL-8 and CCL4 transcription via TLR-MyD88/TRIF-NF-B and TLR-p38/JNK MAPK signaling pathways. The dotted lines indicate indirect connections.
(Kyriakis and Avruch, 2012). Additionally, Islam et al. (2006) and Kang et al. (2007) demonstrated that p38, JNK and NF-B activation was required for induction of IL-8 and proinflammatory gene expression in the monocyte and intestinal epithelial cells after infection with Yersinia enterocolitica (Grassl et al., 2003). Gonsalves and Kalra (2010) showed that endothelin-1-mediated p38 and JNK1 MAPK signaling led to the up-regulation of CCL4 in monocytic cells. Our data indicates that there is also an important role for NFB and MAPK-p38/JNK pathways in the induction of IL-8 and CCL4 in porcine cells after H. parasuis infection. The results of luciferase reporter assays and Western-blots show that TLR1, 2, 4 and 6, but not TLR5, are involved in H. parasuisinduced NF-B activation (Fig. 3A and B). Also Fig. 4 shows that TLR1, 2, 4 and 6, but not TLR5, are involved in p38 and JNK MAPK activation. We speculate that H. parasuis outer membrane proteins diacylated lipopeptides activate TLR2 and TLR6, triacylated lipopeptides activate TLR1 and TLR2, and H. parasuis LPS activate TLR4. H. parasuis outer membrane protein P2 (OmpP2), outer membrane protein P5 (OmpP5) and outer membrane autotransporter adhesin AidA which are associated with adherence and invasion may involve in activating TLR1, 2 and 6. Using RNA interference experiments, we found that H. parasuisinduced NF-B activation depends on the adaptor proteins MyD88, TRIF, IRAK4, IRAK1, TRAF6, TAK1 and TAB1, which are key adaptor proteins in the TLR-NF-B signaling cascade (Kawai and Akira, 2007). In particular, TRAF6 participates in both MyD88- and TRIFdependent pathways, and is an important central regulator of the TLR-NF-B pathway and subsequent recruitment of a protein kinase complex involving TAK1 and TAB1 to transmit signaling downstream (Adhikari et al., 2007). H. parasuis induced both the MyD88- and TRIF-dependent pathways (Fig. 3C and D). All
TLRs (expect TLR3) can activate the MyD88-dependent pathway, while TLR4 can recruit four adaptor proteins and activate both the MyD88- and the TRIF-dependent pathways (Kawai and Akira, 2011). These two pathways have different kinetics. First, TLR4 initially recruits TIRAP and MyD88 to transmit signals accumulating in NF-B and MAPK activation and the induction of inflammatory cytokines. Subsequently TLR4 is endocytosed and delivered to intracellular vesicles to form a complex with TRAM and TRIF, leading to the activation of NF-B and IRF3 and the induction of type I IFN and inflammatory cytokine productions (Kawai and Akira, 2011). In this study, TLR1, 2, 4 and 6 were involved in activating TLR/MyD88- and TRIF-NF-B signaling pathway after H. parasuis infection. In summary, we have demonstrated that H. parasuis infection activated TLR mediating NF-B and p38/JNK MAPK signaling pathways to induce downstream inflammatory cytokines IL-8 and CCL4 transcription. This study brings new insights into the molecular pathogenesis of H. parasuis-induced inflammation and has identified host pathways with potential for novel therapeutic intervention for the alleviation of Glasser’s disease.
Acknowledgments This work was supported by grants from National Natural Science Foundation of China (NSFC, grant no. 31201931), National Basic Research Program (973 Program, grant no. 2012CB 18802), Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP, grant no. 20110146120005), Fundamental Research Funds for the Central Universities (FRFCU, grant no. 2010QC003).
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Islam, Z., Gray, J.S., Pestka, J.J., 2006. p38 Mitogen-activated protein kinase mediates IL-8 induction by the ribotoxin deoxynivalenol in human monocytes. Toxicol. Appl. Pharmacol. 213, 235–244. Kang, S.S., Woo, S.S., Im, J., Yang, J.S., Yun, C.H., Ju, H.R., Son, C.G., Moon, E.Y., Han, S.H., 2007. Human placenta promotes IL-8 expression through activation of JNK/SAPK and transcription factors NF-kappaB and AP-1 in PMA-differentiated THP-1 cells. Int. Immunopharmacol. 7, 1488–1495. Kawai, T., Akira, S., 2007. Signaling to NF-kappaB by toll-like receptors. Trends Mol. Med. 13, 460–469. Kawai, T., Akira, S., 2011. Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity 34, 637–650. Kopp, E., Medzhitov, R., 2003. Recognition of microbial infection by toll-like receptors. Curr. Opin. Immunol. 15, 396–401. Kyriakis, J.M., Avruch, J., 2012. Mammalian MAPK signal transduction pathways activated by stress and inflammation: a 10-year update. Physiol. Rev. 92, 689–737. Liu, C., Wang, Y., Zhang, H., Cheng, S., Charreyre, C., Audonnet, J.C., Chen, P., He, Q., 2014. Porcine coronin 1A contributes to nuclear factor-kappa B (NF-kappaB) inactivation during Haemophilus parasuis infection. PLOS ONE 9, e103904. Moller, K., Kilian, M., 1990. V factor-dependent members of the family Pasteurellaceae in the porcine upper respiratory tract. J. Clin. Microbiol. 28, 2711–2716. Nedbalcova, K., Satran, P., Jaglic, Z., Ondriasova, R., Kucerova, Z., 2006. Haemophilus parasuis and Glässer’s disease in pigs: a review. Vet. Med.-Czech. 51, 168–179. Oliveira, S., Pijoan, C., 2004. Haemophilus parasuis: new trends on diagnosis, epidemiology and control. Vet. Microbiol. 99, 1–12. Olvera, A., Segales, J., Aragon, V., 2007. Update on the diagnosis of Haemophilus parasuis infection in pigs and novel genotyping methods. Vet. J. 174, 522–529. Platanias, L.C., 2003. Map kinase signaling pathways and hematologic malignancies. Blood 101, 4667–4679. Ren, M., Guo, Q., Guo, L., Lenz, M., Qian, F., Koenen, R.R., Xu, H., Schilling, A.B., Weber, C., Ye, R.D., Dinner, A.R., Tang, W.J., 2010. Polymerization of MIP-1 chemokine (CCL3 and CCL4) and clearance of MIP-1 by insulin-degrading enzyme. EMBO J. 29, 3952–3966. Roux, P.P., Blenis, J., 2004. ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiol. Mol. Biol. Rev. 68, 320–344. Smith Jr., M.F., Mitchell, A., Li, G., Ding, S., Fitzmaurice, A.M., Ryan, K., Crowe, S., Goldberg, J.B., 2003. Toll-like receptor (TLR) 2 and TLR5, but not TLR4, are required for Helicobacter pylori-induced NF-kappa B activation and chemokine expression by epithelial cells. J. Biol. Chem. 278, 32552–32560. Tanaka, Y., Adams, D.H., Hubscher, S., Hirano, H., Siebenlist, U., Shaw, S., 1993. T-cell adhesion induced by proteoglycan-immobilized cytokine MIP-1 beta. Nature 361, 79–82. Vahle, J.L., Haynes, J.S., Andrews, J.J., 1997. Interaction of Haemophilus parasuis with nasal and tracheal mucosa following intranasal inoculation of cesarean derived colostrum deprived (CDCD) swine. Can. J. Vet. Res. 61, 200–206. Vallabhapurapu, S., Karin, M., 2009. Regulation and function of NF-kappaB transcription factors in the immune system. Annu. Rev. Immunol. 27, 693–733. Wang, Y., Liao, J., Tang, R., 2006. Diagnosis and prevention of Haemophilus parasuis. China J. Anim. Husb. Vet. Med. 33, 106–107. Wang, Y., Liu, C., Fang, Y., Liu, X., Li, W., Liu, S., Liu, Y., Liu, Y., Charreyre, C., Audonnet, J.C., Chen, P., He, Q., 2012. Transcription analysis on response of porcine alveolar macrophages to Haemophilus parasuis. BMC Genomics 13, 68. Yin, X., Jiang, P., Deng, Y., Tang, J., 2004. Isolation and identification of Haemophilus parasuis. Anim. Husb. Vet. Med. 36, 6–8. Zhang, B., He, Y., Xu, C., Xu, L., Feng, S., Liao, M., Ren, T., 2012. Cytolethal distending toxin (CDT) of the Haemophilus parasuis SC096 strain contributes to serum resistance and adherence to and invasion of PK-15 and PUVEC cells. Vet. Microbiol. 157, 237–242. Zhang, B., Xu, C., Zhang, L., Zhou, S., Feng, S., He, Y., Liao, M., 2013. Enhanced adherence to and invasion of PUVEC and PK-15 cells due to the overexpression of RfaD, ThyA and Mip in the DeltaompP2 mutant of Haemophilus parasuis SC096 strain. Vet. Microbiol. 162, 713–723.
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Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
Haemophilus parasuis induces activation of NF-B and MAP kinase signaling pathways mediated by toll-like receptors Yushan Chen a , Ting Liu a , Paul Langford b , Kexin Hua a , Shanshan Zhou a , Yajun Zhai a , Hongde Xiao c , Rui Luo a , Dingren Bi a , Hui Jin a,∗ , Rui Zhou a,∗ a
State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China Section of Paediatrics, Imperial College London, St Mary’s Campus, London W2 1PG, UK c Hubei Center for Animal Disease Control and Prevention, Wuhan 430070, China b
a r t i c l e
i n f o
Article history: Received 4 December 2014 Received in revised form 5 February 2015 Accepted 11 February 2015 Keywords: Haemophilus parasuis Inflammatory response NF-B pathway MAPK pathway TLRs
a b s t r a c t Glässer’s disease in pigs caused by Haemophilus parasuis is characterized by a severe membrane inflammation. In our previous study, we have identified activation of the transcription factor NF-B after H. parasuis infection of porcine epithelial cells. In this study, we found that H. parasuis infection also contributed to the activation of p38/JNK MAPK pathway predominantly linked to inflammation, but not the ERK MAPK pathway associated with growth, differentiation and development. Inhibition of NF-B, p38 and JNK but not ERK activity significantly reduced IL-8 and CCL4 expression by H. parasuis. We also found TLR1, TLR2, TLR4 and TLR6 were required for NF-B, p38 and JNK MAPK activation. Furthermore, MyD88 and TRIF signaling cascades were essential for H. parasuis-induced NF-B activation. These results provided new insights into the molecular pathways underlying the inflammatory response induced by H. parasuis. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction The consequences of infections caused by Haemophilus parasuis in pigs are great economic losses in the swine industry worldwide, due to expensive antibiotic treatment and mortality of animals in acute form of the disease (Nedbalcova et al., 2006; Olvera et al., 2007). This Gram-negative organism firstly establishes infection in the upper respiratory tract but, under certain circumstances, which occurred sporadically and stress associated, some strains can constitutes an initial step to cause pneumonia (Aragon, 2013; Baumann and Bilkei, 2002; Costa-Hurtado and Aragon, 2013; Moller and Kilian, 1990). In disease outbreaks, virulent strains spread to internal organs such as brain, heart, spleen, lung, kidney, intestine and lymph node (Vahle et al., 1997; Wang et al., 2006; Yin et al., 2004), causing systemic invasion characterized by polyserositis syndrome including polyserositis, pleuritis, peritonitis, meningitis, pericarditis and arthritis, which is associated with substantial morbidity and mortality of piglets (Brockmeier, 2004; Oliveira and Pijoan, 2004). Multiplication of H. parasuis inside the host induces a strong inflammatory reaction mediated by inflammatory cytokines such
∗ Corresponding authors. Tel.: +86 27 87280147; fax: +86 27 87280208. E-mail address: [email protected] (H. Jin). http://dx.doi.org/10.1016/j.molimm.2015.02.016 0161-5890/© 2015 Elsevier Ltd. All rights reserved.
as IL-8 and macrophage inflammatory protein-1 (MIP-1/CCL4) by recruitment of immune cells to the site of infection to initiate innate immune responses (Bouchet et al., 2008, 2009; Chen et al., 2012; Costa-Hurtado et al., 2013). IL-8 is associated with serous membrane inflammation and meningitis after H. parasuis infection (Bouchet et al., 2008, 2009). CCL4, a chemoattractant for monocytes and other immune cells, effectively induces the adhesion of circulating leukocytes for their extravasation bound with proteoglycan on endothelia, and is associated with exudative inflammation (Bystry et al., 2001; Ren et al., 2010; Tanaka et al., 1993). IL-8 and CCL4 were subject to transcription factor NF-B transcriptional regulation (Ali and Mann, 2004; Vallabhapurapu and Karin, 2009). In a recent study, we have demonstrated that H. parasuis infection activated the NF-B pathway and induced the up-regulation of IL-8 andCCL4 (Chen et al., 2012). However, whether there were common pathways involved in activation was not investigated. Toll-like receptors (TLRs) are the first line of defense against invading pathogens, being the important pattern recognition receptors for detecting and responding to microbial ligands upstream of the NF-B pathway (Doyle and O’Neill, 2006; Kawai and Akira, 2007). It has been reported that TLR2 recognizes bacterial lipopeptides in combination with TLR1 and TLR6, and TLR4 recognizes LPS from most Gram-negative species (Kawai and Akira,
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2007). Helicobacter pylori infection induced NF-B activation via TLR2 and TLR5 but not TLR4 (Smith et al., 2003), suggesting that diverse bacteria activate the NF-B pathway in different ways. After the identification of NF-B activation following H. parasuis infection (Chen et al., 2012), here we determine the TLRs by which H. parasuis mediate NF-B activation and inflammatory cytokine upregulation by the NF-B and mitogen-activated protein kinases (MAPKs) pathways. Early activation of TLRs by pathogen-associated molecular patterns (PAMPs) and their downstream signaling pathways, such as MAPKs and NF-B, have been found to be critical for host innate and adaptive immune responses, including expression of inflammatory cytokines (Doyle and O’Neill, 2006; Kawai and Akira, 2007; Kyriakis and Avruch, 2012). MAPK signaling cascades are involved in many cellular processes such as inflammation, proliferation, differentiation, stress responses and apoptosis (Kyriakis and Avruch, 2012; Platanias, 2003). There are three major groups of MAPK: the p38 MAP kinases (p38), the c-Jun amino-terminal kinase (JNK), and the extracellular signal-regulated protein kinase 1/2 (ERK), all of which are involved in the regulation of inflammation (Cargnello and Roux, 2011). The recent study suggested that MAPK signaling pathway involved in activating immune and inflammatory response upon H. parasuis infection (Wang et al., 2012). Understanding the cellular signaling pathways leading to inflammation in response to H. parasuis infection may provide new therapeutic strategies for treatment.
2. Materials and methods 2.1. Bacterial strain and culture conditions H. parasuis SH0165 strain was cultured in tryptic soy broth (TSB; Difco Laboratories, Detroit, MI, USA) supplemented with 1 mg/ml nicotinamide adenine dinucleotide (NAD) and 5% bovine serum. After incubation at 37 ◦ C overnight with circular agitation (200 rpm), bacteria were harvested by centrifugation at 5000 × g for 15 min, washed three times with PBS and resuspended in fresh DMEM medium.
2.2. Cell culture Porcine kidney (PK-15) cells were cultured and maintained in Dulbecco’s Modified Eagle Media (DMEM, Gibco), supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U/mL penicillin, 10 g/mL streptomycin sulfate and incubated at 37 ◦ C in a humidified 5% CO2 incubator. The antibiotics have been removed before transfection and cells were washed three times with PBS before inoculating with H. parasuis.
2.3. RNA exaction and real-time quantitative analysis Total cellular RNA was extracted from PK-15 cells by using TRIzol reagent (Invitrogen). RNA was reverse-transcribed into cDNA using reverse transcriptase (Roche), and cDNA amplification was performed by the SYBR green PCR assay (Roche). Individual transcripts in each sample were assayed three times and normalized to the porcine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA level. Nucleotide sequences of the primers used for Q-PCR are presented in Table S2. In experiments designed to block NF-B and MAPK signaling, PK-15 cell were pre-treated for 1 h with inhibitors prior to H. parasuis (MOI of 10) infection, and RNA samples were collected after 24 h infection.
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2.4. Reagents and siRNA Mitogen-activated protein kinase (MAPK) inhibitors SB203580, SP600125, U0126 and NF-B inhibitor BAY-117082 were purchased from Sigma–Aldrich (St. Louis, MO, USA). Cells were pretreated with 0.2, 1, 5 or 10 M inhibitor for 1 h before infection with H. parasuis at a MOI of 10. Negative control siRNA, TLR1, TLR2, TLR4, TLR5, TLR6, MyD88, TRIF, TRAF6, IRAK1, IRAK4, TAK1 and TAB1 siRNA oligonucleotide were purchased from GenePharma (China) and the sequences are listed in Table S1. siRNA (40 pmol) was transfected into PK-15 cells using Lipofectamine 2000 (Invitrogen) in siRNA knockdown experiments. 2.5. Western blotting PK-15 cells were cultured in 60 mm-dishes and were infected or mock-infected with H. parasuis. At the indicated time, cells were harvested by adding lysis buffer with proteinase inhibitor cocktail and phosphatase inhibitor cocktail (Sigma–Aldrich). Extracted proteins were separated by SDS-PAGE and electrophoretically transferred to polyvinyldifluoride membranes (Millipore). The blots were blocked with 5% nonfat dry milk and then incubated with primary antibody overnight at 4 ◦ C. After three washes, the blots were subsequently incubated with the secondary antibody horseradish peroxidase (HRP) conjugate solution for 1 h at room temperature. Finally, the signal was detected using an ECL chemiluminescence detection kit (Bio-rad). Polyclonal antibodies against NF-B p65, phospho-NF-B p65 (S536), p38, phosphop38 (Thr180/Tyr182), JNK, phospho-JNK (Thr183/Tyr185), ERK, phospho-ERK (Thr202/Tyr204) were obtained from Cell Signaling Technology (USA). Polyclonal antibodies against TLR1, TLR2, TLR4 and TLR6 were purchased from Santa Cruz Biotechnology and monoclonal antibodies -actin was purchased from Beyotime (China). Polyclonal antibodies against MyD88, TRIF, IRAK4, IRAK1, TRAF6, TAK1 and TAB1 were obtained from Cell Signaling Technology (USA). 2.6. Transfection and luciferase reporter assay Transient transfection was performed by using Lipofectamine 2000 (Invitrogen). PK-15 cells were seeded in 24-well plates and incubated until the cells were grown to 60–70% confluency. Cells were transfected with 40 pmol/well negative control siRNA or other siRNA. Twenty-four hours later, PK-15 cells were infected or mockinfected with H. parasuis at a MOI of 10. At the indicated time points, cell lysates were analyzed by Western blotting or QPCR. In selected experiments, cells were co-transfected with 100 ng/well of NF-B luciferase reporter plasmid (pNF-B-Luc) and 100 ng/well of the Renilla luciferase construct pRL-TK (Promega) together with 40 pmol/well negative control or each siRNA. Twenty-four hours later, cells were infected or mock-infected with H. parasuis at a MOI of 10. Cells were harvested at the indicated time point and luciferase activity was measured using a dual-luciferase Assay System (Promega) according to the manufacturer’s directions. Data represent relative firefly luciferase activity normalized to Renilla luciferase activity. 2.7. Statistical analysis Data are expressed as mean ± SEM. Statistical analysis was performed using one-way analysis of variance (ANOVA) without interaction terms followed by Dunnett’s or Duncan’s test for multiple comparisons. A P-value less than 0.05 was considered significant and a P-value less than 0.01 was considered highly significant.
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3. Results 3.1. H. parasuis infection activated p38 and JNK MAPK pathway We originally identified that H. parasuis infection activated the NF-B pathway through IB␣ degradation and the phosphorylation of p65 in PK-15 cells (Chen et al., 2012). Thus, we sought to determine whether H. parasuis infection could activate the MAPK pathway, including three subfamilies of MAP kinases, p38, JNK1/2 and ERK1/2. H. parasuis infection induced the phosphorylation of p38 and JNK1/2 in a time-dependent manner. On the contrary, no ERK1/2 phosphorylation could be detected under the same experimental conditions (Fig. 1), whereas ERK1/2 activation is inducible in PK-15 cell by TNF␣ (data not shown). 3.2. NF-B and p38/JNK MAPK pathways were required for induction of IL-8 and CCL4 by H. parasuis in PK-15 cells We have recently demonstrated that H. parasuis infection induced expression of the inflammatory cytokines IL-8 and CCL4 in PK-15 cells (Chen et al., 2012). To determine whether there is a role for NF-B in induction of IL-8 and CCL4 by H. parasuis, we used the NF-B specific inhibitor BAY11-7082. As shown in Fig. 2A, cells treated with BAY11-7082 exhibited a reduced ability to up-regulate IL-8 and CCL4 transcription after H. parasuis infection. Previous studies have shown that the MAPK signaling pathways play an important role in the inflammatory response (Kyriakis and Avruch, 2012). We next sought to examine the role of the MAPK signaling cascades in the transcriptional regulation of IL-8 and CCL4 after H. parasuis infection. PK-15 cells were treated with SB202190 (a specific inhibitor of p38), SP600125 (a specific inhibitor of JNK), or U0126 (a specific inhibitor of ERK1/2), followed by H. parasuis infection. As shown in Fig. 2B and C, treatment with SB202190 and
Fig. 1. H. parasuis infection activates p38 and JNK in PK-15 cells. PK-15 cells were inoculated with DMEM (control, CON) or H. parasuis (HPS) at a MOI of 10 and whole cell extracts were prepared at indicated time points and subjected to Western blot analysis with antibodies specific for p38, phosphorylated p38, JNK, phosphorylated JNK, ERK, phosphorylated ERK. Anti--actin was included as a positive control for sample loading.
SP600125 significantly attenuated IL-8 and CCL4 transcription in a dose-dependent manner, but treatment with U0126 did not affect IL-8 and CCL4 transcription in H. parasuis-infected PK-15 cells. 3.3. H. parasuis infection activated NF-B via TLR signaling pathway TLR1, 2, 4, 5 and 6 have been shown to play a critical role in the recognition of various bacterial components with host cells (Kawai and Akira, 2007; Kopp and Medzhitov, 2003). To determine
Fig. 2. NF-B and p38/JNK MAPK pathways regulate H. parasuis-induced IL-8 and CCL4 transcription in PK-15 cells. (A) PK-15 cells were treated with NF-B inhibitor, BAY11-7082 (0.2, 1, 5 and 10 M) or DMSO (control), followed by un-infected or infection with H. parasuis (HPS, MOI of 10) for 12 h. PK-15 cells were harvested and real-time RT-PCR was performed as described in methods. *P < 0.05 or **P < 0.01 compared with DMSO plus H. parasuis (HPS) infection. (B) and (C) were performed similarly to that described in (A) expect for the inhibitors: p38 inhibitor, SB202190 (0.2, 1, 5 and 10 M), JNK inhibitor, SP600125 (0.2, 1, 5 and 10 M), or ERK inhibitor, U0126 (0.2, 1, 5 and 10 M) were used. **P < 0.01 as compared with DMSO plus H. parasuis (HPS) infection.
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Fig. 3. Involvement of TLR signaling cascades in NF-B activation by H. parasuis. (A, C and E) PK-15 cells were transfected with pNF-B-Luc and pRL-TK together with siRNAs of negative control, TLR1/2/4/5/6, MyD88, TRIF, TRAF6, IRAK1, IRAK4, TAK1 or TAB1 with/without H. parasuis infection at a MOI of 10. PK-15 cells were harvested at 24 h post-infection and luciferase activity measured. **P < 0.01 as compared with negative control siRNA plus H. parasuis infection. (B, D and F) PK-15 cells were transfected with negative control siRNA, TLR1, 2, 4, 5, 6, MyD88, TRIF, TRAF6, IRAK1, IRAK4, TAK1 or TAB1 siRNA and un-infected or infected with H. parasuis at a MOI of 10. Whole cell extracts were prepared at 24 h post-infection and subjected to Western blot analysis with antibodies specific for NF-B p65 or phospho-NF-B p65. Anti--actin was included as a control for sample loading.
whether TLRs are involved in mediating H. parasuis-induced NF-B activation, NF-B luciferase reporter assay was performed. PK-15 cells were transfected with pNF-B-Luc and pRL-TK together with TLR1, 2, 4, 5 or 6 siRNAs. TLR5 was used as a negative control because there is no bacterial flagellin in H. parasuis. We first confirmed the efficiency of TLR1, 2, 4, 5 and 6 siRNA in reducing endogenous TLR expression in PK-15 cells transfected with TLR siRNA or negative control siRNA. The expression of TLR1, 2, 4, 5 and 6 was markedly reduced by TLR siRNA (Fig. S1). As expected, TLR1, 2, 4 and 6 but not TLR5 knockdown significantly inhibited the induction of NFB after H. parasuis infection (Fig. 3A). Because phosphorylation of p65 has been shown to be important in NF-B-dependent transcriptional activity (Chen and Greene, 2004), we also assessed the phosphorylation of p65 in PK-15 cells transfected with TLR siRNA, followed by H. parasuis infection. TLR1, 2, 4 and 6, but not TLR5, siRNA reduced the phosphorylation of p65 in H. parasuis-infected PK-15 cells (Fig. 3B). These results indicated that TLR1, 2, 4 and 6 are involved in H. parasuis-induced NF-B activation. Among the known downstream signaling molecules in TLR signaling pathway, the adaptor molecule MyD88 or TRIF appears are among the first molecules to be recruited to the TLR receptor complex (Kawai and Akira, 2007). Thus, we next sought to determine the role of MyD88 and TRIF in H. parasuis-induced NF-B
activation. As shown in Fig. 3C and D, H. parasuis-induced NF-B activation was decreased after MyD88 or TRIF siRNA transfection. We further investigated whether TRAF6, IRAK1, IRAK4, TAK1 and TAB1, which are key adaptor molecules for NF-B activation in the TLR signaling pathway, are required for induction of NF-B. As shown in Fig. 3E and F, siRNA knockdown of TRAF6, IRAK1, IRAK4, TAK1 or TAB1 inhibited the activation of NF-B by H. parasuis. These data suggested that H. parasuis infection activated NF-B via TLR signaling pathway. 3.4. H. parasuis infection activated p38 and JNK MAPK pathway via TLR1, TLR2, TLR4 and TLR6 As shown in Fig. 4, transfection with TLR1, 2, 4 and 6, but not TLR5, siRNA inhibited the phosphorylation of p38 and JNK, but not ERK. The results suggest that H. parasuis induces activation of the p38 and JNK MAPK pathways by TLR1, 2, 4 and 6. 3.5. H. parasuis infection triggered the production of IL8 and CCL4 by TLR1, TLR2, TLR4 and TLR6 We have identified that H. parasuis infection activated NFB via TLR signaling pathways and induced expression of the
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to H. parasuis infection. As shown in Fig. 5, cells transfected with TLR1, TLR2, TLR4 or TLR6 siRNA, but not TLR5 siRNA had a reduced up-regulation of IL8 and CCL4 transcription. These results substantiate our findings that H. parasuis infection induces NF-B activation and downstream cytokine secretion in PK-15 cells via TLR1, TLR2, TLR4 and TLR6.
4. Discussion
Fig. 4. Involvement of TLR1, TLR2, TLR4 and TLR6 in p38 and JNK activation by H. parasuis. PK-15 cells were transfected with negative control (DMEM, CON) or TLR1, 2, 4, 5 or 6 siRNA and un-infected or infected with H. parasuis (HPS) at a MOI of 10. Whole cell extracts were prepared at 24 h post-infection and subjected to Western blot analysis with antibodies specific for p38, phosphorylated p38, JNK, phosphorylated JNK, ERK and phosphorylated ERK. Anti--actin was included as a control for sample loading. p-p38, p-JNK1/2 and p-ERK1/2 protein levels were quantitated by densitometric analysis using ImageJ software. Densitometric values for p-p38, p-JNK and p-ERK were normalized to p38, JNK and ERK respectively. Finally, all the values were normalized to siNegative/CON, which was set to a value of 1.0.
proinflammatory cytokines IL-8 and CCL4. To determine which TLR(s) were involved in signal transduction from the epithelial cell surface to mediate cytoplasmic IL-8 and CCL4 expression, we transfected PK-15 cells with TLR1, TLR2, TLR4, TLR5 or TLR6 siRNA prior
Some studies have demonstrated that H. parasuis induces host inflammatory responses (Bouchet et al., 2008, 2009; Chen et al., 2012). However, the signaling mechanisms underlying induction of the inflammatory responses have remained largely unknown. In this study, we demonstrate that H. parasuis infection induced IL-8 and CCL4 transcription via the NF-B and p38/JNK MAPK pathway in PK-15 cells. We also identified that TLR1, 2, 4 and 6 were involved in H. parasuis-induced NF-B activation and downstream transcription of the cytokines IL-8 and CCL4. Moreover, H. parasuis induced NF-B via MyD88 and TRIF signaling cascades (Fig. 6). The recent studies demonstrated H. parasuis can adhere and invade PK-15 cells (Frandoloso et al., 2012; Zhang et al., 2012, 2013), a well-established epithelial cell line of porcine kidney origin, which were applied in this study and proved to be an appropriate cell line for studying on the NF-B pathway activated by H. parasuis (Chen et al., 2012; Liu et al., 2014). In our previous study we showed that H. parasuis activated NF-B (Chen et al., 2012). Addiditionally, the results from this study showed that the bacterium induced activation of the p38 MAPK and JNK MAPK pathways (Figs. 1 and 2). In our study, no ERK pathway activation was found after infection with H. parasuis (Figs. 1, 2 and 4). One possibility is that JNK and p38 kinases are more responsive to stress stimuli ranging from osmotic shock to cytokine stimulation, while ERK1/2 is preferentially activated in response to growth factors and phorbolesters (Roux and Blenis, 2004). It has been previously reported that MEK1/2 were ERK-specific MAP2Ks, MKK3/4/6 were JNK-specific MAP2Ks, and MKK4/7 were p38-specific MAP2Ks while MKK4 can phosphorylate and activate both p38 and JNK (Kyriakis and Avruch, 2012). We speculate that H. parasuis infection activates MKK3/4/6/7 instead of MEK1/2 but further work is required to determine this. Our data also suggest that H. parasuis-induced IL-8 and CCL4 expression in PK15 cells is dependent on both the NF-B and MAPK-p38/JNK pathways (Fig. 2). This is consistent with other data in the literature. For example, it was reported that p38 and JNK had a role in promoting cytokine release via posttranscriptional regulation of mRNAs encoding proinflammatory proteins
Fig. 5. H. parasuis infection activated IL-8 and CCL4 transcription via TLR1, TLR2, TLR4 and TLR6. (A) PK-15 cells were transfected with negative control (DMEM, CON) or TLR1, 2, 4, 5 or 6 siRNA and un-infected or infected with H. parasuis at a MOI of 10 for 12 h. PK-15 cells were harvested and real-time RT-PCR for IL8 was performed as described in methods. (B) The experiments were performed similarly to those described in (A) expect that CCL4 transcription was measured. **P < 0.01 as compared with negative control siRNA plus H. parasuis infection.
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Fig. 6. Schematic representation of the signaling pathways involved in H. parasuis (HPS) induced IL-8 and CCL4 expression. As indicated, H. parasuis up-regulates IL-8 and CCL4 transcription via TLR-MyD88/TRIF-NF-B and TLR-p38/JNK MAPK signaling pathways. The dotted lines indicate indirect connections.
(Kyriakis and Avruch, 2012). Additionally, Islam et al. (2006) and Kang et al. (2007) demonstrated that p38, JNK and NF-B activation was required for induction of IL-8 and proinflammatory gene expression in the monocyte and intestinal epithelial cells after infection with Yersinia enterocolitica (Grassl et al., 2003). Gonsalves and Kalra (2010) showed that endothelin-1-mediated p38 and JNK1 MAPK signaling led to the up-regulation of CCL4 in monocytic cells. Our data indicates that there is also an important role for NFB and MAPK-p38/JNK pathways in the induction of IL-8 and CCL4 in porcine cells after H. parasuis infection. The results of luciferase reporter assays and Western-blots show that TLR1, 2, 4 and 6, but not TLR5, are involved in H. parasuisinduced NF-B activation (Fig. 3A and B). Also Fig. 4 shows that TLR1, 2, 4 and 6, but not TLR5, are involved in p38 and JNK MAPK activation. We speculate that H. parasuis outer membrane proteins diacylated lipopeptides activate TLR2 and TLR6, triacylated lipopeptides activate TLR1 and TLR2, and H. parasuis LPS activate TLR4. H. parasuis outer membrane protein P2 (OmpP2), outer membrane protein P5 (OmpP5) and outer membrane autotransporter adhesin AidA which are associated with adherence and invasion may involve in activating TLR1, 2 and 6. Using RNA interference experiments, we found that H. parasuisinduced NF-B activation depends on the adaptor proteins MyD88, TRIF, IRAK4, IRAK1, TRAF6, TAK1 and TAB1, which are key adaptor proteins in the TLR-NF-B signaling cascade (Kawai and Akira, 2007). In particular, TRAF6 participates in both MyD88- and TRIFdependent pathways, and is an important central regulator of the TLR-NF-B pathway and subsequent recruitment of a protein kinase complex involving TAK1 and TAB1 to transmit signaling downstream (Adhikari et al., 2007). H. parasuis induced both the MyD88- and TRIF-dependent pathways (Fig. 3C and D). All
TLRs (expect TLR3) can activate the MyD88-dependent pathway, while TLR4 can recruit four adaptor proteins and activate both the MyD88- and the TRIF-dependent pathways (Kawai and Akira, 2011). These two pathways have different kinetics. First, TLR4 initially recruits TIRAP and MyD88 to transmit signals accumulating in NF-B and MAPK activation and the induction of inflammatory cytokines. Subsequently TLR4 is endocytosed and delivered to intracellular vesicles to form a complex with TRAM and TRIF, leading to the activation of NF-B and IRF3 and the induction of type I IFN and inflammatory cytokine productions (Kawai and Akira, 2011). In this study, TLR1, 2, 4 and 6 were involved in activating TLR/MyD88- and TRIF-NF-B signaling pathway after H. parasuis infection. In summary, we have demonstrated that H. parasuis infection activated TLR mediating NF-B and p38/JNK MAPK signaling pathways to induce downstream inflammatory cytokines IL-8 and CCL4 transcription. This study brings new insights into the molecular pathogenesis of H. parasuis-induced inflammation and has identified host pathways with potential for novel therapeutic intervention for the alleviation of Glasser’s disease.
Acknowledgments This work was supported by grants from National Natural Science Foundation of China (NSFC, grant no. 31201931), National Basic Research Program (973 Program, grant no. 2012CB 18802), Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP, grant no. 20110146120005), Fundamental Research Funds for the Central Universities (FRFCU, grant no. 2010QC003).
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