Heat shock protein 10 of Chlamydophila pneumoniae induces proinflammatory cytokines through Toll-like receptor (TLR) 2 and TLR4 in human monocytes THP-1
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Inflammatory response is the first line of infection. Previous studies have suggested that Chlamydophila pneumoniae heat shock protein (CHSP) 60 is present in human atheromata, and it plays an important role on the chronic infection elicited by C. pneumoniae. Here, we demonstrated in vitro the impact of heat shock protein 10 (HSP10) of C. pneumoniae on THP-1 cells and the role of Toll-like receptors (TLRs) in the procedures of inflammatory response. The production of proinflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-6, and IL-1beta were induced by recombinant HSP10 dose-dependently, and the proinflammatory activity of HSP10 was greatly reduced by heating and deproteinization treatment. The expression of TLR4 and TLR2 on the cultured cells were determined by reverse transcriptase-polymerase chain reaction and immunofluorescence. Peritoneal macrophages isolated from wild-type (C3H/HeN) and TLR4-deficient mice (C3H/HeJ) were respectively stimulated with endotoxin-free proteins. Cytokine responses after stimulation were significantly different, depending on the presence of TLR4. The effect on cytokine expression was blocked by anti-TLR2 or anti-TLR4 MAb partially or dramatically. Thus, HSP10 of C. pneumoniae which could elicit inflammatory reactions in human monocytes may contribute to the inflammatory processes in Chlamydophila infection, and the effects were mediated by TLR4 and, to a lesser extent, TLR2.
KeywordsChlamydophila pneumoniae Heat shock protein 10 Proinflammatory cytokines Toll-like receptors THP-1
Chlamydophila pneumoniae is an obligate intracellular pathogen which spread worldwide. The seroprevalence in the adult population is about 50∼70%, and nearly everybody has been infected at least once during his or her lifetime (Miyashita et al. 2008; Jha et al. 2009). The pathogen causes acute respiratory infections such as pneumonia and bronchitis and has been implicated in chronic processes including asthma and cardiovascular disease (Grayston et al. 1993; Hahn et al. 1998; Cook 1999; Cosentini et al. 2008; Blasi et al. 2009). Although the link between C. pneumoniae and atherosclerosis is well-accepted, little has been known about the mechanisms of C. pneumoniae-induced infections.
Inflammation seems to play a key role on the acute infections and chronic inflammatory diseases, and the cytokine response is one of its remarkable features. Several studies have confirmed C. pneumoniae triggers an inflammatory response in human and animal cells (Blessing et al. 2000; Yamamoto et al. 2005; Didion 2008). The pathogen could invade and replicate in the host cells and induce activation of chronic inflammation with cytokines release such as IL-1beta, IL-6, and TNF-alpha, but the specific components of C. pneumoniae involved in these initiations is not well identified.
Heat shock proteins (HSPs) are highly conserved molecules. Because of molecular mimicry of human homologous HSP, some microbial HSPs seem to induce humoral or cellular responses and involve in pathogenesis such as inflammation and autoimmunity. It was proven that HSP60 of C. pneumoniae can be detected in human atheroma and may take part in the development of lesions and directly activate monocytes/macrophages to secrete cytokines from cells (Kol et al. 1998, LaVerda et al. 2000). But very little is known about the role of HSP10 which is genetically and physiologically linked to HSP60.
The present study aimed to determine whether HSP10 were implicated in the inflammatory responses against infection and whether the TLRs (TLR2 and TLR4) were required for C. pneumoniae HSP10 (CHSP10)-induced cytokine expression by monocyte cells. For this purpose, we incubated cultured human monocytic cells with recombinant CHSP10 with or without anti-TLR antibodies and measured the effects of CHSP10 on IL-1beta, IL-6, and TNF-alpha production.
Materials and Methods
The human monocytic cell line THP-1 (American Type Culture Collection) and human lung adenocarcinoma epithelial cell line A549 (China Center for Type Culture Collection, Wuhan University) were grown at 37°C with 5% CO2 in RPMI 1640 medium (HyClone, Logan, UT). Human kidney cell line HEK293T (China Center for Type Culture Collection, Wuhan University) was cultured in Dulbecco’s modified Eagle’s medium (HyClone); all the cells were cultured in mediums contained 10% heat-inactivated fatal bovine serum (FBS; GIBCO; Langley, OK), 2 mM l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin.
Neutralizing anti-human TLR4 MAb (HTA125), neutralizing anti-human TLR2 MAb (TL2.1), and isotype control MAb (Mouse IgG2a, κ Isotype Ctrl) as a blocking Ab were purchased from Biolegend (Clonetimes Biotech Co., Ltd, Changsa, China). CyTM2-conjugated goat anti-mouse IgG and CyTM3-conjugated goat anti-mouse IgG were obtained from Jackson ImmunoResearch Laboratories Inc. (Westgrove, PA). For reverse transcriptase reaction, the RevertAidTM First-Strand cDNA Synthesis Kit (Fermentas; Shenzhen, China) was applied. PCR amplification was performed with Taq DNA polymerase (Dongsheng Biotech; Zhejiang, China). Penicillin, ampicillin, polymyxin B (PMB), and Escherichia coli lipopolysaccharide (LPS; 0111:B4) were purchased from Sigma-Aldrich (Shanghai, China). All other chemicals were obtained from commercial sources and were of analytical or reagent grade.
Peritoneal macrophages from C3H/HeN and C3H/HeJ.
A 4 ∼ 6-wk-ld C3H/HeN (Vital River Laboratory Animal Technology Co. Ltd.) and C3H/HeJ female mice (Model Animal Research Center of Nanjing University) were fed with standard laboratory chow and water ad libitum. Macrophages were induced by injecting 1 mL of 5% sterile starch broth intraperitoneally, and peritoneal macrophages were collected 4 or 5 d later by peritoneal lavage with chilled serum-free culture medium. The macrophages were then incubated in a 5% CO2 incubator at 37°C for 2 ∼ 4 h in RPMI-1640 culture medium supplemented with 100 U/mL penicillin G and 100 μg/mL streptomycin. After three times vigorous washing, nonadherent cells were removed, and macrophages were incubated in RPMI 1640 with 10% FBS in 96-well plate (HyClone) at 1 × 104 cells per well for cytokine assays.
Recombinant CHSP10 preparation.
CHSP10 recombinant plasmid was constructed with pQE30 vector (QIAGEN; Shanghai, China) and transformed into E. coli M15, induced, and purified as described elsewhere (Jiang et al. 2008). Briefly, cells were cultured in Luria-Bertani broth containing 100 μg/mL ampicillin and 100 μg/mL kanamycin, harvested by centrifugation, and disrupted by sonication on ice; soluble His-tagged protein CHSP10 was purified by nickel-chelate affinity resin (QIAGEN) according to the manufacturer’s protocol. Elution fractions were then analyzed by sodium dodecyl polyacrilamide gel electrophoresis using 15% running gel. The concentration of the purified protein was determined by BCA Protein Assay Kit (Pierce). To exclude possible LPS contamination, proteins were collected and mixed with 50 μg/mL polymyxin B (Sigma) or treated with Detoxi-GelTM Endotoxin Removing Gel (Pierce; IL). The endotoxin concentration of recombinant HSP10 was less than 60 pg/mL, as determined by the Limulus amebocyte lysate assay (Chinese Horseshoe Crab Reagent Manufactory, Ltd., Xiamen, China). The protein was stored at −135°C until it was used.
For stimulation of THP-1 or A549 cells with HSP10, approximately 3 × 105 cells per well were seeded into 24-well plate. After incubation for indicated conditions, cells were then stimulated with HSP10 using different concentrations or in different times. Cell-free supernatants were harvested after centrifugation and was stored at −70°C until use.
Heat, deproteinized treatments of CHSP10.
Heat experiment was performed by heating CHSP10 at 56°C for 30 min or 100°C for 20 min, respectively. Deproteinized treatments were performed by mixing purified proteins with 50% nickel-chelate affinity resin and incubating at room temperature for 1 h to bind the His-tagged CHSP10, and then the mixture was loaded onto a column. Flow-through was collected to incubate with resin again and purified as described above. As controls, CHSP10 samples without heating or deproteinization were incubated with THP-1 cells. Cell-free supernatants were harvested after 24 h incubation and kept at −70°C until cytokine measurements.
RT-PCR and immunofluorescent staining.
TLR4 and TLR2 expression was analyzed in THP-1 monocytes by RT-PCR and immunofluorescent staining. Total RNA was extracted from THP-1 or HEK293T cells using the TRIzol reagent (Invitrogen; Beijing, China); cDNA was synthesized according to the manufacturer’s instructions. The primer sequences used for amplifications were as follows (Faure et al. 2000; Cheng et al. 2008): TLR4 forward, 5′-TGGATACGTTTCCTTATAAG -3′; TLR4 reverse, 5′-GAAATGGAGGCACCCCTT -3′ C; TLR2 forward, 5′-GCCAAAGTCTTGATTGATTGG -3′; TLR2 reverse, 5′-TTGAAGTTCT CCAGCTCCTG-3′; beta-actin forward, 5′-CTACAATGAGCTGCGTGTGG-3′; beta-actin reverse, 5′-AAGGAAGGCTGGAAGAGTGC-3′. PCR amplification was performed with Taq polymerase for one cycle at 94°C for 5 min, 32 cycles at 94°C for 45 s, 50°C (TLR4), 54°C (TLR2), or 52°C (beta-actin) for 45 s, and 72°C for 1 min, followed by one cycle at 72°C for 10 min. Amplification products were separated by electrophoresis on a 1.5% (w/v) agarose gel and visualized by ethidium bromide fluorescence. Surface expression of TLR4 and TLR2 on THP-1 cells was determined by using anti-human TLR4 and TLR2 MAb. THP-1 cells were cultured in the 24-well plate. After 12 ∼ 24 h, the medium was removed by centrifugation, and the cells were harvested. Cells were fixed in 4% paraformaldehyde solution for 15 min at room temperature, blocked with phosphate-buffered saline containing 1% bovine serum albumin for 30 min, and incubated with anti-TLR4 and anti-TLR2 antibodies in blocking buffer at 4°C overnight. Primary antibodies were detected with CyTM3- or CyTM2-conjugated goat anti-mouse IgG in blocking buffer at room temperature for 30 min. In between the two steps, the cells were washed three times with centrifugation. At last, the coverslips were mounted on glass slides with Vectashield (Vector Laboratories, Inc., Burlingame, CA 94010) and viewed under a fluorescence microscope (Nikon Eclipse TE 2000-S).
Cytokine response to peritoneal macrophages from C3H/HeN and C3H/HeJ mice.
Resident macrophages from either C3H/HeJ or C3H/HeN mice were cultured in 96-well plates with RPMI 1640 medium, in a volume of 100 μL. The cells were stimulated with 20 μg/mL CHSP10 for 24 h at 37°C, respectively; the secreted cytokines IL-1beta,IL-6, and TNF-alpha in the culture supernatant were tested by using mouse ELISA kit (Neobioscience; Shenzhen, China) according to the manufacturer’s instructions.
THP-1 cells were cultivated in 24-well tissue culture plates (HyClone), and blocking experiments were conducted by pretreating cells with 10 μg/mL neutralizing anti-human TLR4 MAb or/and neutralizing anti-human TLR2 MAb or isotype control MAb for 1 h at room temperature and then stimulated with CHSP10 for 24 h. The supernatants were collected and stored at −70°C until cytokine assays were performed.
Assays for IL-6, IL-1beta, and TNF-alpha.
Secretion of IL-1beta, TNF-alpha, and IL-6 was determined with the Duoset ELISA Development Systems (R&D Systems) according to the manufacturer’s instructions. The assays employ the quantitative sandwich ELISA technique, using monoclonal antibodies specific for the tested cytokines. In brief, 200 μL (IL-1beta and TNF-alpha)/100 μL (IL-6) standard, sample, or control were added to each well and incubated for 2 h at room temperature (RT). After aspirating and four washes, 200 μL conjugate/well were added and incubated for 1 ∼ 2 h at RT. Four washes later, 200 μL substrate solution was added to each well and incubated for 20 min (RT). Then, 50 μL stop solution was added, and optical density was determined using a microplate reader at 450 nm. The sensitivity of assay was 0.7 (IL-6), 1 (IL-1beta), and 1.6 pg/mL (TNF-alpha). All experiments were repeated three times.
Data and statistical analysis.
The results of all experiments were expressed as the mean ± SE of triplicate experiments. Data were compared using one-way analysis of variance combined with the Student’s t test, and P values less than 0.05 were considered statistically significant. All analyses were performed by SPSS version 13.0.
Effect of CHSP10 on proinflammatory cytokine expression in human monocytes THP-1 cells.
Stimulation of proinflammatory cytokines by heated or deproteinized CHSP10.
Stimulation experiments by CHSP10 in TLR4-deficient and wild-type mice macrophages.
The mRNA and protein expression of TLRs (TLR2 and TLR4) in THP-1 cells.
The effects of anti-TLR4 and anti-TLR2 MAb in the blocking experiments.
Chronic C. pneumoniae infection might trigger the inflammatory reactions and induce the development of atherosclerosis (Romano et al. 2006). Clinical studies reported the presence of C. pneumoniae and the antibodies against it in human serum (Davidson et al. 1998; Kuo et al. 1993; Kuo and Campbell 2000). Proinflammatory cytokines such as TNF-alpha, IL-1beta, and IL-6 can be secreted after C. pneumoniae infection (Tiran et al. 2002; Yang et al. 2003; Rodriguez et al. 2010). Since inflammatory reactions may be involved in the development and progression of atherosclerosis, the components of C. pneumoniae which activate the host cells and initiate proinflammatory cytokines needs to be defined.
Chlamydial HSPs may induce chronic disease by antigenic stimulation and host cell activation (LaVerda et al. 1999; Jha et al. 2010). CHSP60 is the most important antigen reported to be involved in the pathogenesis of chlamydial infections. It had been detected in human atheroma, and antibody against it was particularly elevated in coronary heart disease patients. Mounting research displayed that CHSP60 is an inducer of inflammatory responses to endothelial cells and macrophages (Kol et al. 1998; Kol et al. 1999; Bulut et al. 2002). HSP10 (GroES), a cochaperonin to HSP60 (GroEL) shared the same GroE operons and co-expressed the GroE complex with HSP60. It is located in cytosol, cell membrane, intercellular space, and periphery with mounting evidence demonstration (Fossati et al. 2003; Jia et al. 2011) and may interact with various surrounding environment. The association between the presence of anti-HSP10 antibodies and adult onset asthma or Chlamydia trachomatis genital tract infection (Betsou et al. 1999 LaVerda et al. 2000; Betsou et al. 2003) indicated that CHSP10 had been exposed to the host components and triggered the immune system to produce the specific antibodies. Reports on the other organisms also proved HSP10 may play a role in the inflammation-related immunopathogenic process (Ragno et al. 1996; Henderson and Pockley 2010).
Cytokines, produced by a variety of cells, exert diversified functions in immune-related diseases. The main advantage is that they can induce innate immune mechanisms, and exert cytodestructive effects to eliminate the infectious agents. While the main disadvantage is that they affect the development of inflammatory reactions, which may contribute to destruction and pathologic processes directly or indirectly. In this study, we found that HSP10 from C. pneumoniae, like other organism-derived HSP10, could stimulate THP-1 cells to secrete proinflammatory cytokines TNF-alpha, IL-1beta, and IL-6 in a dose-dependent manner. The results suggested CHSP10 could also induce the monocytes to secrete the cytokines but did not play a major role, compared with other components such as HSP60 and HSP70 (data not shown). Yet, cytokines act in networks or cascades by diverse cell types; only several selected cytokines from THP-1 cell line is a limitation in our study in that it is difficult to reflect the biological functions of cytokines in C. pneumoniae infection in vivo comprehensively and systematically.
TLRs are innate immune receptors which have been reported to mediate the recognition and binding of bacterial antigens such as HSPs (Huang et al. 2009). It is responsible for initiating innate immunity and secretion of different sets of chemokines and cytokines by host cells. TLR2 and TLR4 are the most possible candidates for mediating cell activation and cytokine releasing by LPS and HSP60 of C. pneumoniae, but some controversy remains about the mechanism of how TLR2 and TLR4 act in cells activation (Prebeck et al. 2001; Sasu et al. 2001; Bulut et al. 2002; Netea et al. 2004). Here, we have investigated whether CHSP10 is a ligand for TLR2 or/and TLR4 and whether these TLRs were involved in CHSP10-responsive human monocytes THP-1. The confirmation of TLR2 and TLR4’s existence is an important key to initiate further research about their role in cell activation. Next, we found that macrophages derived from C3H/HeJ mice that express mutant TLR4 were nearly nonresponsive to CHSP10, confirmed a role of TLR4 in CHSP10-induced cell activation. In support of the data obtained above, we showed that blockade of TLR4 significantly reduced the production of IL-1beta, IL-6, and TNF-alpha induced by CHSP10, and anti-TLR2 antibody just inhibited the proinflammatory cytokines secretion slightly. In terms of the responses, CHSP10 has been shown to stimulate production of proinflammatory cytokines through TLR4- and TLR2-mediated signals. Here, TLR4 was found to be more important than TLR2 in CHSP10-induced monocytes THP-1 activation. However, these observations cannot rule out the possibility that TLRs other than TLR2 and 4 are involved in CHSP10-induced responses to THP-1.
To our knowledge, endotoxin is able to induce the production of TNF-alpha by macrophages and monocytes via Toll-like receptor 4-mediated signal transduction pathways (Moreno et al. 2004; Youn et al. 2008). LPS contamination of the recombinant proteins is a possible factor for the effects on cells activation. Therefore, we conducted the removal of LPS from CHSP10 by Endotoxin Removing Gel or preincubation with polymyxin B. LPS is heat-resistant and is not easily destroyed by thermal treatment (Gao et al. 2006), while the protein can be broken by heat, which leads to changes of its function. A549, which was often used in vitro studies of C. pneumoniae infection (Törmäkangas et al. 2010), failed to produce TNF-alpha in our research. It is not consistent with what has been shown earlier for induction of TNF-alpha, IL-8, and IFN-gamma in A549 cells during C. pneumoniae infection (Yang et al. 2003). As cellular responses to stimuli are very complicated, and various cell lines exhibit different responses, it could be due to the different cell types used. Thus, we concluded that A549 cells may not be applicable to protein samples; in other words, it is insensitive to protein stimuli such as CHSP10. This implied that CHSP10 may not affect the epithelial cells in cytokine productions.
In summary, we demonstrated that CHSP10 can induce expression of proinflammatory cytokines TNF-alpha, IL-1beta, and IL-6 in monocytes THP-1, and this induction predominantly depended on TLR4 and was only to a minor extent, TLR2-dependent. These observations provided a better understanding of cytokine release following C. pneumoniae infection. The role of CHSP10 in the inflammatory aspects of C. pneumoniae-induced infection will need to be further clarified.
We thank the National Natural Science Foundation of China (no. 30901352), Innovative Research Team in University of Hunan province (number:  51), Hunan Provincial Innovation Foundation For Postgraduate, and Hunan Provincial training and innovation base for post-graduate, for their financial support of this study.
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