The expression of fibronectin is significantly suppressed in macrophages to exert a protective effect against Staphylococcus aureus infection
© The Author(s). 2017
Received: 6 May 2016
Accepted: 7 April 2017
Published: 13 April 2017
Fibronectin (Fn) plays a major role in the attachment of Staphylococcus aureus to host cells by bridging staphylococcal fibronectin-binding proteins (FnBPs) and cell-surface integrins. A previous study demonstrated that the phagocytosis of S. aureus by macrophages is enhanced in the presence of exogenous Fn. We recently found that FnBPs overexpression also enhances phagocytic activity. The effect of S. aureus infection on the expression of macrophage Fn was investigated.
The level of Fn secreted by monocytes (THP-1), macrophages, human lung adenocarcinoma (A549) cells, and hepatocellular carcinoma (HepG2) cells in response to S. aureus infection was determined by Western blotting and it was significantly suppressed only in macrophages. The activation of signaling pathways associated with Fn regulation in macrophages and HepG2 cells was also investigated by Western blotting. Erk was activated in both macrophages and HepG2 cells, whereas Src-JNK-c-Jun signaling was only activated in macrophages. A significant decrease in macrophage viability was observed in response to S. aureus infection in the presence of exogenous Fn.
The Src-JNK-c-Jun signaling pathway was activated in macrophages in response to S. aureus infection and resulted in the suppression of Fn expression. This suppression may play a protective role in macrophages against S. aureus infection. This study provides the first demonstration that Fn is suppressed in macrophages by S. aureus infection.
Staphylococcus aureus is one of the leading causes of healthcare-associated and community-acquired infections . This pathogen adheres to and invades host tissues mediated by the interaction between its surface adhesins and the host extracellular matrix (ECM). For example, bacterial attachment can be achieved through fibronectin (Fn) bridge between staphylococcal fibronectin-binding proteins (FnBPs) and the host’s cell-surface integrins . We recently demonstrated that the expression of FnBP is upregulated by antibiotic stress, leading to an increase in cytotoxicity through an increase in bacterial attachment .
There are two principal types of Fn, the plasmatic and cell-surface-bound forms, which are generated from the same gene (FN1) transcript by alternative splicing . Only the plasmatic form can bind to and be modified by FnBPs of which the process is essential for integrin binding . This glycoprotein is an important component of the ECM and is functionally implicated in the regulation of several cellular processes, including cell adhesion, migration, as well as tissue repair and the recruitment of inflammatory cells . Fn is majorly produced by hepatocytes, and part of its production is mediated by fibroblasts, monocytes, macrophages, and endothelial cells . It has been reported that the expression of Fn is upregulated by NF-κB and downregulated by the c-Jun/c-Jun homodimer [8, 9]. The regulation of Fn expression is also associated with fibronectin-binding integrins through the modulation of Rho-GTP loading . Several host cell signaling pathways such as focal adhesion kinase (FAK) and steroid receptor coactivator (Src), which are the intracellular tyrosine protein kinase, are activated by the formation of a Fn bridge that links FnBPs and integrins . Once FAK is activated, downstream signaling pathways, such as those associated with MAPK-Erk kinases and Src-c-Jun NH2-terminal kinase (JNK)-c-Jun, which are key regulators controlling the expression of genes related to inflammation and cell adhesion, are also activated [12–14]. In addition, the expression level of plasma Fn is decreased and can be used as a biomarker for the evaluation of sepsis [15, 16]. It has been shown that the treatment of macrophages with lipopolysaccharide (LPS), the endotoxin of Gram-negative bacteria, leads to a decrease in Fn expression [16, 17]. However, the underlying mechanism remains unclear.
We recently found that the expression of FnBPs in S. aureus was increased after the application of the sub-lethal doses of cell wall active antibiotics . This increased FnBP expression enhanced phagocytosis of S. aureus by macrophages, similarly to the effect obtained in other studies with the addition of exogenous Fn . We proposed that the expression of Fn by macrophages would be increased in response to S. aureus infection to catch more bacterial cells, and this hypothesis was investigated in the present study. Unexpectedly, we found that the expression of Fn was significantly suppressed and the mechanism underlying was studied.
The allelic replacement of the fnbAB genes by a spectinomycin cassette (spc) was performed by introducing the pMAfnb plasmid into S. aureus strain ATCC12598 to generate the fnbAB-knockout strain SJC1221, according to a method described elsewhere . The spc restricted by BamHI and BglII was flanked by the upstream and downstream arms of the PCR product. The upstream arm originating from the 5′ end of fnbA was amplified by the primer pairs fnbA-F (5′-ATGACCATGGCTACTTGTCTTTGATCTCCGCTATTGT-3′) and fnbA-R (5′-AATTAGATCTCTTGTGTACAAGGGTTTCTGATGACTTG-3′) with BglII and NcoI restriction sites. The downstream arm was restricted by the primer pairs fnbB-F (5′-AGGATCCCTTCATAGTGTCATTG-3′) and fnbB-R (5′-GAGTCGACTGGTACAATCGAAG-3′) with BamHI and SalI restriction sites from the 3′ end of the fnbB. The constructed upstream arm-spc-downstream arm DNA fragment was then cloned into pMAD to yield pMAfnb.
The human lung adenocarcinoma (A549) and hepatocellular carcinoma (HepG2) cell lines were maintained in DMEM, whereas the human acute monocytic leukemia (THP-1) cell line was maintained in RPMI 1640 medium. THP-1 cells (5 × 106/ml) were differentiated using 10 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, USA) for 2 days, and after the PMA-containing media was removed, the cells were incubated in fresh RPMI 1640 for an additional day . All cell lines were cultured in serum-free media 1 day prior to bacterial infection. All the cell lines originally purchased from Bioresource Collection and Research Center (BCRC, Taiwan) were kindly given by Professor Hsin-Chih Lai in this department.
Infection of cells
The bacterial infection of the cells was performed by inoculating with different S. aureus strains at a multiplicity of infection (MOI) of 25. Under specified conditions, Fn (200 or 400 μg/ml) was added to the medium to determine the macrophage viability after infection. Cells treated with LPS (100 μg/ml) were used as an experimental control. To determine the Fn-associated signaling in response to bacterial infection, the cells were pre-treated with several signaling inhibitors such as SP600125 (10 μM, JNK inhibitor), SB203580 (25 μM, p38 inhibitor), PP2 (25 μM, Src inhibitor), and PD98059 (25 μM, Erk kinase inhibitor) for 15 min prior to bacterial infection. Concentrations of those inhibitors used in this experiment were according to studies described elsewhere [21–24]. The infection was stopped at indicated time points, and the infected cells were washed twice with PBS. After the addition of gentamycin (50 μg/ml) and lysostaphin (10 μg/ml), the cells were incubated for an additional hour to kill and remove the extracellular bacteria. All the chemicals and antibiotics were purchased from Sigma-Aldrich.
The level of secreted Fn and the activation of intracellularly Fn-associated signaling pathways were detected by Western blotting analysis using samples from cell culture supernatants and whole-cell lysates, respectively. Cells were pelleted by centrifugation, and supernatants containing the secreted Fn were removed and filtrated. The pelleted cells were lysed in lysis buffer as described elsewhere, and the concentration of protein in supernatant or cell lysate was determined . Equal amounts of protein (3 μg/lane) were applied to SDS-PAGE gels. The membranes were probed with primary antibody (rabbit monoclonal) against fibronectin (Sigma-Aldrich), Src, Src-P, c-Jun, c-Jun-P (Ser73), p44/42 MAPK (Erk1/2), p44/42-P (Thr202/Tyr204) MAPK (Erk1/2), p38, p38-P (Thr180/Tyr182) or GAPDH (Cell Signaling Technology, Danvers, USA). After washing with TBST, the membranes were probed with secondary horse anti-rabbit HRP-conjugated antibody (Cell Signaling) for 1 h and developed using ECL reagents (Thermo Scientific, Waltham, USA). The blots were photographed and quantified by densitometry using ImageJ software. The results were presented as fold-change expression levels relative to that of the untreated cells and all of the samples were tested in three independent experiments.
S. aureus infected or LPS treated macrophages were collected and pelleted 30 and 60 min post-infection and frozen on dry ice immediately. Total RNA was extracted from cell pellets using the TRIzol (Invitrogen, Waltham, USA) method followed by RQ1 RNase-free DNase (Promega, Madison, USA) treatment to eliminate any remaining DNA. mRNA levels of FN1 were determined by RT-PCR with the iScript Reverse Transcription Supermix (Bio-Rad, Hercules, USA). The expression levels of FN1 were normalized against the GAPDH expression level and quantified by densitometry using ImageJ software. The results were presented as fold-change expression levels relative to that of the untreated cells and all of the samples were tested in three independent experiments.
Determination of cell viability
The viability of macrophages after S. aureus infection in the presence of exogenous Fn was determined using the MTT assay with MTT (3- (4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; 0.5 mg/ml; Sigma-Aldrich), as described previously . Four hours post-infection, 100 μl of MTT solution was added to each well, and the plates were incubated at 37 °C for 4 h. The purple formazan crystals were dissolved by adding 100 μl of DMSO, and the absorbance at A570 was spectrophotometrically measured with a reference wavelength of A690. The results are expressed as the percent absorbance of each experimental well as a function of the absorbance of the well containing untreated cells. Three wells were measured for each experimental condition, and each experimental condition was tested in three independent experiments. The attached viable cells were observed by crystal violet staining following to 1 h gentamycin and lysostaphin treatment to eliminate the extracellular bacteria. The cells were fixed by 1% formaldehyde followed by staining with PBS containing 0.5% crystal violet solution (Sigma-Aldrich) for 20 min at room temperature. The cells were gently washed with deionized water and air-dried. The attached damage cells were also visualized by propidium iodide (PI) staining with a commercial apoptosis detection kit (BioVision, Milpitas, USA) by microscopy according to the manufacturer’s instructions.
A one-way ANOVA with Games-Howell post-hoc test was used to analyze the experimental data and to compare means. Error bars in the bar graphs of the relative densitometric quantification or cell viability represent as the standard deviation from three experimental repetitions. P-values of less than 0.05 were considered statistically significant.
Expression of fn by macrophages was suppressed by S. aureus infection
Reduction in fn expression in macrophages was mediated by c-Jun phosphorylation through the activation of Src signaling
Viability of macrophages after S. aureus infection was decreased in the presence of exogenous fn
In this present study, the expression level of secreted Fn was found to be significantly suppressed in macrophages by S. aureus infection. This suppression has also been observed in macrophages treated with either exotoxins or heat-killed S. aureus, indicating that the Fn suppression mediated by S. aureus is multifactorial. A mitigation of Fn suppression was detected in the co-cultured of FnBP-deficient S. aureus strain with macrophages, implying that the FnBP-mediated bacteria-host interaction may contribute to the Fn suppression observed in macrophages. Fn suppression in macrophages was found to be mediated only through the activation of the Src-JNK-c-Jun signaling pathway. The physiological role underlying Fn suppression may reduce the cytotoxicity caused by S. aureus infection. To the best of our understanding, this study provides the first demonstration of Fn suppression in macrophages upon bacterial infection, at least by S. aureus.
S. aureus infection-mediated Fn suppression was only observed in macrophages among the distinct types of cells employed in this study (Fig. 1). The activation of Erk, p38 and Src-JNK-c-Jun signaling pathways in macrophages and hepatocytes (HepG2) was then investigated. The activation of Erk and p38 was detected in both cells, whereas Src-JNK-c-Jun signaling was only activated in macrophages upon S. aureus infection (Fig. 3). The expression of Fn was not altered in the presence of Erk and p38 inhibitors, suggesting that both signaling are not involved in the suppression of Fn. However, the expression of Fn was recovered to a basal level by pre-treatment with a JNK inhibitor, indicating that the observed Fn suppression is mediated through the Src-JNK signaling (Fig. 4). Though Src inhibitor (PP2) showed cytotoxicity against macrophages but expression level of Fn was still higher than PP2-free counterpart indicating that the abrogation of the Fn suppression by PP2 was not due to the cell death. It has been demonstrated that α-hemolysin triggered the activation of FAK and Src signaling through the mediation of integrins . Therefore, the Fn suppression as also observed when macrophages were incubated with bacterial culture medium (Fig. 1 and Additional file 4: Figure S4). S. aureus infection plays distinct roles in Fn expression in macrophages and others cells, and this difference is likely due to differences in CD14 expression. CD14 is expressed mainly by macrophages and dendritic cells and acts as a co-receptor together with TLRs for the recognition of PAMPs, such as LPS [33, 37, 38]. LPS, which was used as an experimental control in our study, has been shown to downregulate Fn expression in macrophages, but the underlying mechanism remains unclear and no follow-up study has been reported . LPS also triggers Src signaling and the accumulation of c-Jun, leading to the overexpression of cytokines [39, 40]. As described above, the association of TLRs, CD14, and the FAK complex is required for Src activation [31, 33]. Nevertheless, LPS was used as an experimental technique control and the difference of the suppressive mechanism between S. aureus infection and LPS treatment should be further elucidated.
Through the time course study, the strongest suppression of FN1 transcription and secreted Fn expression was observed at 30 min and 2 h-post infection, respectively (Figs. 1 and 2). We may not rule out that the decreased Fn expression was partially due to the effects on cell viability. Only approximately 10% of the macrophages had been killed following to S. aureus infection (4 h post-infection; Fig. 5a) but a five-fold reduction of the Fn secretion was already observed 2 h post-infection (Fig. 1), as well as the downregulation of the FN1 transcription was observed (Fig. 2). Suppressed Fn expression upon S. aureus infection was only observed in macrophages, not in other cell types, indicating that cell viability had less effect on the Fn expression (Fig. 1). In addition, equal amount of the total exoproteins were loaded per sample for Western blotting (Additional file 1: Figure S1) and the relatively low levels of Fn expression was not caused from the decreased sample loading.
The interesting finding in this study is that the suppression of FN expression in macrophages may play a protective role against S. aureus infection (Fig. 5). The suppression of Fn may reduce the attachment of bacteria to macrophages through the FnBP-Fn-integrin connection. Therefore, the addition of exogenous Fn had no significant effect on the macrophage viability upon infection with FnBP deficient strain SJC1221. Previous studies have demonstrated that the production of TNF-α was increased from macrophages infected by group B streptococci in the presence of exogenous Fn . A large release of TNF-α has been shown to be associated with harmful pathophysiological effects and increased mortality from septic shock . In addition, the abnormally diminished concentration of plasma Fn has been used as a biomarker in evaluating sepsis . The reduction in the plasma Fn concentration observed in patients with sepsis may arise from intravascular coagulation and cryofibrinogenemia, which is likely due to increased plasma Fn catabolism secondary to the intravascular formation of fibrin . However, the mechanisms underlying this reduction remain unclear. Therefore, a reduction in the Fn concentration may be beneficial to the host, at least to macrophages, after S. aureus infection. Macrophages are majorly found in tissues through the differentiation of monocytes when they leave the blood. Thus, such protective mechanisms may be relevant to localized infection in the tissues. However, Fn is also present in the tissues and the effect of tissue Fn on the macrophages against S. aureus infection remains to be elucidated further. An animal model which reflects tissue infection for evaluating the local Fn expression level by immunofluorescent assay can be conducted. In addition, fibroblasts are widely dispersed in connective tissues and the effect of S. aureus infection on the expression of Fn by fibroblasts can be studied in the future.
Our study provides the first demonstration that Fn is suppressed in macrophages by S. aureus infection. This S. aureus infection induced suppression of Fn expression from macrophages was mediated by Src-JNK-c-Jun signaling pathway. This suppression may play a protective role in macrophages against S. aureus infection.
Human lung adenocarcinoma cells
Focal adhesion kinase
Hepatocellular carcinoma cells
Multiplicity of infection
- Spc :
steroid receptor coactivator
human acute monocytic leukemia
This study was kindly supported by the Ministry of Science and Technology, Taiwan (grant No. MOST103-2320-B-182-022), and Research Program of Chang Gung Memorial Hospital (grant No. CMRPD1C0851 ~ 2), Taoyuan, Taiwan.
Availability of data and material
Data supporting this findings can be found in the main paper or additional supporting files.
HYC and JCS made substantial contributions to conception and design. HYC acquisition of data, and analysis and interpretation of data. MHL and CCC conceived of the study, and participated in its design and coordination and helped to draft the manuscript. HYC and JCS gave final approval of the version to be submitted and any revised version. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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