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Orientia tsutsugamushi induced endothelial cell activation via the NOD1-IL-32 pathway
Microbial Pathogenesis 49 (2010) 95e104
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Orientia tsutsugamushi induced endothelial cell activation via the NOD1-IL-32 pathway Kyung-Ah Cho a, Yoon Hee Jun b, Jee Won Suh a, Jae-Seung Kang c, Hee Jung Choi b, So-Youn Woo a, * a
Department of Microbiology, School of Medicine, Ewha Womans University, 911-1 Mok-Dong, Yangcheon-Gu, Seoul 158-710, Republic of Korea Division of Infectious Disease, Department of Internal Medicine, School of Medicine, Ewha Womans University Mokdong Hospital, 911-1 Mok-Dong, Yangcheon-Gu, Seoul 158-710, Republic of Korea c Department of Microbiology, Inha Research Institute for Medical Science, Inha University College of Medicine, Jungsuk B/D, 3rd street, Shinheung-Dong, Choong-Gu, Incheon 400-712, Republic of Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 March 2010 Received in revised form 27 April 2010 Accepted 4 May 2010 Available online 12 May 2010
Orientia tsutsugamushi (OT), the causative agent of scrub typhus, is an obligate intracellular bacterium. In order to verify the inflammatory responses involved in the pathogenesis of scrub typhus, we assessed the cytokine profile of the human endothelial cell line, ECV304, after OT infection. We noted that CCL5, CCL17, IL-1a, IL-6, IL-8, IL-10, IL-15, TNF-a and TNF-b were strongly induced in response to OT. Additionally, IL-32, the candidate modulator for the induction of IL-6 and IL-8, was increased significantly with OT infection and these increases coincided with NOD1 pathway activation. Thus, we hypothesized that NOD1 pathway and IL-32 might act on cytokine release in endothelial cells as a modulator of the inflammation caused by OT infection. NOD1 siRNA resulted in a reduction in IL-32 levels, and also reduced IL-1b, IL-6, IL-8, and ICAM-1 expression in OT-infected ECV304 cells. These changes in IL-1b, IL-6, IL-8, and ICAM-1 induced by NOD1 knockdown were reversed as the result of IL-32 treatment. This indicated that OT infection activated the NOD1 pathway followed by IL-32 secretion, thus resulting in the production and expression of IL-1b, IL-6, IL-8, and ICAM-1. Therefore, IL-32 might perform a role upstream of the inflammatory reaction in endothelial cells of OT infection. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: NOD1 IL-32 Orientia tsutsugamushi Endothelial cells
1. Introduction Orientia tsutsugamushi (OT) is the causative agent of scrub typhus, also referred to as tsutsugamushi disease, a serious public health problem in Asia. OT is maintained in nature by transovarian transmission in trombiculid mites, primarily of the genus Leptotrombidium. After hatching, infected larval mites (chiggers, the only stage that feeds on an animal host) inoculate organisms into the skin during feeding. Occasionally, infected mites or rat fleas bite humans and cause scrub typhus, characterized focal or disseminated vasculitis and perivasculitis, which may involve the lungs, heart, liver, spleen, and central nervous systems [1]. The illness varies in severity from mild and selflimited to fatal. After an incubation period spanning 6e21 days, the onset of disease is characterized by fever, rash, eschar,
Abbreviations: OT, Orientia tsutsugamushi; TARC, Thymus and activation regulated chemokine; NOD, Nucleotide-binding and oligomerization domain; NLR, NOD-like receptor. * Corresponding author. Tel.: þ82 2 2650 5740; fax: þ82 2 2653 8891. E-mail address: [email protected] (S.-Y. Woo). 0882-4010/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2010.05.001
headache, myalgia, cough, and gastrointestinal symptoms. Severe cases typically include prominent encephalitis and interstitial pneumonia as key features of vascular injury [2,3]. During OT disease progression, OT was located in endothelial cells in all the involved organs [4], and also infected a variety of cells, including macrophages, polymorphonuclear leukocytes (PMNs), and lymphocytes. This subsequently results in local inflammation followed by dissemination to the whole body [4,5]. The pathogenesis of inflammation in scrub typhus involves immune and inflammatory mediators including cytokines, prostaglandins, leukotrienes, and kinins from infected and immune cells. Inflammatory cytokines, such as TNF-a, IL-1b, and IL-6 were increased markedly in patients with scrub typhus, and these cytokines were reduced after doxycycline treatment [6]. These cytokines are attributable to the high fever occurring in most scrub typhus patients. Additionally, OT-infected macrophages express CCL2 (MCP-1), CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), and CCL8 (MIP-2) [3]. Such host responses against microorganisms involve the activation of specialized pattern recognition receptors in the cells and lead to the production of inflammatory mediators.
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Nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) comprises a large family of intracellular pattern recognition receptors (PRRs), which are characterized by the presence of conserved NOD. On the basis of the N-terminal domains, NLRs can be classified into three subfamilies referred to as the caspase recruitment domain (CARD)-containing NODs, pyrin (PYD)-containing NALPs, or baculovirus inhibitor repeat (BIR)containing NAIPs [7]. Owing to their location, NLRs can be activated by intracellular pathogens to initiate innate immune responses. For example, the role of NOD proteins in the response to intracellular pathogens such as Chlamydophila pneumoniae and Listeria monocytogenes [8,9] has been previously reported. Whereas NOD1 has been shown to recognize peptidoglycan containing meso-diaminopimelic acid, which is found principally in gram-negative bacteria [10], NOD2 detects muramyl dipeptide (MurNAc-LAla-DisoGln), which is conserved in basically all types of peptidoglycan [11]. Although such differences exist in sensing pathogens, NOD1 and NOD2 share the ability to activate downstream signaling, such as NF-kB and mitogen-activated protein kinases (MAPKs) [7,12] and eventually lead to the propagation of inflammation via the secretion of proinflammatory cytokines. There is increasing evidence to suggest that IL-32, one of the proinflammatory cytokines, induces IL-1a, IL-1b, IL-6, and TNFa via the NF-kB and p38 MAPK pathways [13,14]. IL-32 is constitutively expressed in endothelial cells and functions as a regulator of endothelial inflammation by affecting the expression of intercellular adhesion molecules 1 (ICAM-1) on endothelial cells with IL-1b [14]. Thus, we noted the possible contribution of NLR, particularly NOD1, and IL-32 to tsutsugamushi disease, due to their own ability to modulate immune responses in the endothelial cells. In an effort to gain insight into the pathogenesis of scrub typhus in relation to vascular injury, we focused on delineating the inflammatory responses in OT-infected endothelial cells. Therefore, we evaluated the cytokine profiles from OT-infected ECV304 endothelial cells, and the molecular mechanism of NOD1-IL-32 interaction, which may possibly be involved in the activation of endothelial cells after OT infection using NOD1 siRNA. 2. Results 2.1. Expression profile of cytokines and chemokines in OT-infected ECV304 cells In order to identify the possible inflammatory mediators from OT-infected endothelial cells, we compared the simultaneous expression profiles of cytokines and chemokines from ECV304 cells (as control) and OT-infected ECV304 cells using a cytokine antibody array (Fig. 1A and B). CXCL1-3 (Gro), CCL5 (RANTES), CCL17 (TARC), IL-1a, IL-6, IL-8, IL-10, IL-15, TNF-a and TNF-b were induced more profoundly in OT-infected ECV304 cells in those of the ECV304 extract-treated group, as controls (Fig. 1C). As it was reported that IL-32 was expressed constitutively in endothelial cells and IL-32 performs a function in inflammation by inducing proinflammatory cytokines such as IL-1b, IL-6, and IL-8, we compared the levels of IL-32 expression between ECV304 cells and OT-infected ECV304 cells via immunoblotting and ELISA. The average levels of IL-32 production in ECV304 cells were 7.2 2.1 pg/ ml. We determined that IL-32 was generated and secreted more in OT-infected cells (ranges 14.2 1.8 pg/ml) than in the control group (Fig. 2 and Supplementary Figs. 2 and 3). Thus, we noted that OT infection of ECV304 cells induced the expression of proinflammatory cytokines, including IL-1a, IL-6, IL-8, IL-10, and IL-32 in endothelial cells.
2.2. OT-infected ECV304 cells produce more NOD1 and active forms of caspase-1 and IL-1b Next, we considered that the increase in IL-32 expression might influence the endothelial cell functions after OT infection, and we evaluated the possible upstream signaling pathway by which IL-32 is induced in ECV304 cells. First, we compared NOD1 expression after OT infection in ECV304 cells. In the presence of OT in ECV304 cells, we determined that OT-infected ECV304 cells generated more NOD1. As the activation of NOD1 results in the production of pro-IL-1b, we compared the expression levels of pro-IL-1b, as well as the active forms. We determined that the active form of IL-1b was increased by OT infection. We then compared the expression levels of caspase-1. As is shown in Fig. 3, the pro and cleaved forms of caspase-1 were increased in cases of OT infection. It was reported that NOD1 enhances pro-IL1b processing by interacting pro-caspase-1 [15]. Therefore we determined that NOD1 was expressed more abundantly in OT-infected cells accompanying increases in the levels of active forms of caspase-1 (Fig. 3). Therefore, we considered that OT infection resulted in NOD1 activation and affected IL-1b production. As shown in Fig. 3, OT infection lead to increase of NOD1, caspase-1, as well as IL-1b. For the cytokines and cell adhesion molecules from OT-infected endothelial cells, possibly induced by IL-32, we determined the levels of IL-6, IL-8, and ICAM-1, and compared the expression levels. Because we had observed increases in the levels of IL-6 and IL-8 after OT infection by cytokine array, we determined that the average production levels of IL-6 and IL-8 by ELISA were 1860 280 pg/ml and 960 190 pg/ml, respectively (Fig. 4A and B). The upregulated expression of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells was noted in OT-infected cells (Fig. 4C). 2.3. OT-infected ECV304 cells induced IL-32 via NOD1 pathway Although IL-32 was identified as a regulator of endothelial inflammation [14], the sequential relationship between IL-32 and NOD1 in cases of OT infection of endothelial cells was unclear. Therefore, we focused on the role of NOD1 in OT-infected endothelial cells and compared the changes in endothelial activation with or without OT infection. In order to determine whether or not NOD1 is involved in the induction of IL-32 secretion in ECV304 cells, we effected a reduction in NOD1 expression via siRNA transfection. As is shown in Fig. 5A, NOD1 siRNA reduced the expression of endogenous NOD1, as confirmed by RT-PCR (by 78%) and immunoblotting (by 53%). The reduction of NOD1 affected the expression and secretion of IL-32 from OT-infected ECV304 cells (Fig. 5B). NOD1 knockdown also reduced the secretion of IL-1b (Fig. 5C). Therefore, according to these results in OT-infected ECV304 cells, we concluded that NOD1-mediated the induction of IL-32 and IL-1b. 2.4. NOD1 knockdown decreased the production and expression of IL-6, IL-8 and ICAM-1 in ECV304 cells In order to evaluate the effects of NOD1 on the production of IL32-inducing cytokines and endothelial cell activation, we compared the expression of IL-6, IL-8, and ICAM-1 after NOD1 knockdown. When endogenous NOD1 levels were reduced by siRNA transfection, we noted a reduction of IL-6, IL-8, and ICAM-1 in OT-infected ECV304 cells (Fig. 6). Thus, we concluded that OT infection increased the level of NOD1 in endothelial cells, followed by the induction of IL-32, IL-6, IL-8, and ICAM-1.
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Fig. 1. Cytokine expression profile of ECV304 cells infected with OT. (A) RayBioÒ Human Cytokine Antibody Array 3 Map shows cytokines and chemokines corresponding to each of the spots. (B) ECV304 cell infected with OT lysates was subjected to cytokine antibody array (right panel). ECV304 cells cultured with non-infected ECV304 extract were used as controls (left panel). Each cytokine is represented by duplicate spots in the shown locations and the cytokine array image represents the results of one of two independent experiments that demonstrated similar expression patterns. The array membranes were exposed for 1 min after the addition of ECL solution and acquired the images. (C) The results of the cytokine antibody array kit were converted to pixel densities. Each spot of the scanned images were digitized and calculated for the pixel densities. The average net optical intensity SEM for each pair of cytokine spots is shown. *P < 0.05.
2.5. Effect of IL-32 on ECV304 cells in OT infection In order to evaluate the direct effect of IL-32 on IL-1b, IL-6, IL-8, and ICAM-1 production in OT-infected ECV304 cells, we incubated
the OT-infected ECV304 cells with or without IL-32 after transfection of NOD1 siRNA. We verified that IL-32 treatment induced increases in IL-1b, IL-6, IL-8, and ICAM-1 production even after NOD1 knockdown (Fig. 7). Therefore it is suggested that the
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Fig. 2. IL-32 is increased by OT infection in ECV304 cells. (A) IL-32 production in OTinfected ECV304 cells was assessed by immunoblotting, and one representative experiment of three is shown. (B) IL-32 secretion into culture media was measured via ELISA. The data are expressed as the means SEM (n ¼ 9). *P < 0.05.
upregulation of IL-32 occurs downstream of the NOD1 pathway, and that IL-32 directly affected the expression of inflammatory modulators of endothelial cells, including IL-1b, IL-6, IL-8, and ICAM-1. 3. Discussion In this study, we have demonstrated that the IL-32 secretion of ECV304 endothelial cells after OT infection occurred downstream of the NOD1 pathway, and that such increased IL-32 levels affected the secretion of proinflammatory cytokines, such as IL-1b, IL-6, and IL-8, as well as ICAM-1 expression in endothelial cells. Inflammation is initiated by OT-infected macrophages and endothelial cells in the dermis. Although mononuclear cells including lymphocytes, macrophages, and neutrophils were observed in the eschars and rashes associated with scrub typhus, inactive tissue macrophages could support growth, and activated macrophages and lymphocytes are essential for protection. Because early host inflammatory responses appear to be crucial for determining the outcome of the progression of the diseases of OTinfected hosts [16], it is important to analyze the regulatory mechanism pertaining to the propagation of the inflammation process via cytokines and cellular influx to the OT infection site. A variety of cytokines and chemokines have been produced in experimental mouse infection [17]. According to the results of human studies, the level of TNF-a in the serum is increased significantly during acute phase as well as convalescent phase [18], and we also noted an increase in the concentration of IFN-g and IL-10 in scrub typhus patients [6]. Activated monocytes and macrophages are the principal sources of chemokines [19], and it was reported that CCL3, CCL4 (macrophage inflammatory protein 1a/b, MIP-1a/b), CCL5 (RANTES) and CCL2 (macrophage chemoattractant protein 1, MCP-1) were induced via NF-kB activation [3];
endothelial cells infected with OT also generate CCL2 and CXCL8 (IL-8) independent of NF-kB activation [2]. In this study, we noted increases in the levels of CXCL1 to 3 (Gro), CCL5, CCL17, IL-1a, IL-6, IL-8, IL-10, IL-15, TNF-a and TNF-b in an OT-infected endothelial cell line (Fig. 1). An increase in IL-10 secretion, as well as that of other proinflammatory cytokines, has been noted. IL-10 is a potent immunosuppressive cytokine that inhibits the production of many cytokines, including IL-2, IFN-g, IL-4, IL-3, IL-1, GM-CSF, and TNF-a. It may be that OT activates the proinflammatory and modulatory pathways, via IL-10, in the early stages of infection. Endothelial cells are key participants in the inflammation process. The activation of endothelial cells is stimulated by proinflammatory cytokines including TNF-a and IL-1, which are released from infected sites and result in the upregulation of cell adhesion molecules, such as P-selectin, E-selectin, ICAM-1, and VCAM-1, to promote cellular influx via transendothelial migration as well as the production of cytokines such as IL-6 and IL-8 to initiate and propagate local inflammatory responses. In patients with scrub typhus, the serum levels of soluble L- and E-selectin were correlated with the symptoms of the disease. These results are suggestive of the activation of mononuclear as well as endothelial cells during OT infection [20]. We demonstrated that the expression of ICAM-1 was increased markedly in our OT-infected cells (Fig. 4C), and this increase in ICAM-1 expression was induced by IL-32 secreted from OT-infected endothelial cells (Fig. 7D). However, the endothelial cells themselves are the targets of OT infection, and this should be considered a direct effect on OT-infected endothelial cells during the immune reaction. Innate immunity to microbial pathogens relies on the specific host-detection of pathogen-derived molecules. In the case of intracellular pathogens, such as rickettsia and chlamydia, sensing via intracellular pattern recognition molecules is mandatory. The two major pattern recognition molecule families are the Toll-like receptor family and the NOD-like receptor (NLR) family. The NLR family is composed of 22 intracellular molecular pattern recognition molecules sharing a central NACHT domain (stands for NAIP, CIITA, HET-E and TP1) and a carboxy-terminal leucine-rich repeat (LRR) region [21]. One subfamily includes NOD1 and NOD2, both of which harbor an N-terminal caspase recruitment domain (CARD) that activates NF-kB signaling. Another subfamily includes the pyrin domain-containing (NLRP) proteins. NLRP proteins are essential for the regulation of caspase-1 activation due to inflammasome formation via the N-terminal pyrin domain [22]. NOD1 and NOD2 sense peptidoglycan polymers from the cell wall components and NLRP3 (NALP3, or cryopyrin) detects a variety of pathogen-associated molecular patterns (muramyl dipeptide, poly (I-C), double-stranded RNA, bacterial RNA, or pore-forming toxins) and danger-associated molecular patterns (extracellular ATP, uric acid, asbestos, silica, or aluminum hydroxide). The activation of NOD1 and NOD2 triggers the recruitment of the adapter protein receptor-interacting serineethreonine kinase 2 (RIPK2 or RIP2), followed by the activation of NF-kB and mitogen-activated protein kinases (MAPKs). NALP3 activation converges on the formation and activation of inflammasomes, consisting of NALP3, ASC, and caspase-1. The NALP3 inflammasome then cleaves pro-IL-1b, proIL-18, and pro-IL-33 to mature IL-1b, IL-18 and IL-33, respectively. NOD1 is expressed in a variety of tissues, but NOD2 is expressed principally in leukocytes, dendritic cells, and epithelial cells [23]. For endothelial cell activation, it has been reported that Chlamydophila pneumonia activates NOD1 and leads to the production of IL-8 [8]. In this study, we also demonstrated increased levels of NOD1 in OT-infected endothelial cells (Fig. 3). The expression level of the mature form of IL-1b was increased in OT-infected cells with an increase in the active form of caspase-1. Therefore, we considered that NOD1 sensed the OT component in endothelial cells, and
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Fig. 3. OT induces NOD1 pathway activation. ECV304 cells were infected with OT and maintained for 6 days. The protein expression of NOD1 (A) was analyzed via immunoblotting (top) and the band densities were quantified by dividing the pixel densities of actin bands (bottom). The protein expression of caspase-1 (B) and IL-1b (C) was analyzed via immunoblotting (top) for the precursor forms as well as the active forms. Band densities of active forms of each protein were quantified by dividing the pixel densities of the actin band (bottom). The relative values of pixel densities are shown as the means SEM of three independent experiments.
NOD1 activated the downstream pathway of NF-kB, thereby resulting in the production of the pro-form of IL-1b. This pro-IL-1b can be matured by activated caspase-1 via the NOD1 pathway. However, which ingredient of OT binds to and stimulates NOD1 remains to be clearly determined. O. tsutsugamushi (OT), an etiologic agent of scrub typhus, is a member of the Rickettsiaceae family, and is related phylogenetically to the genus Rickettsia with 16S rRNA sequence homology of 90.2e90.6%. However, the composition of the cell wall differs substantially; i.e, the cell wall of Rickettsia harbors a large quantity of lipopolysaccharides (LPS) and outermembrane proteins, but Orientia lacks both the peptidoglycan layer and LPS, containing only the major strain-variable 56-kDa protein as well as the antigenically variable 110-, 47-, and 25-kDa proteins [24]. Cellular infection involves bacterial interaction with the cell-surface heparin sulfate of syndecan-4 [25], but the bacterial receptor for attachment to the target cells remains unknown. Because of our findings that OT induced NOD1-mediated endothelial activation, we speculated that peptidoglycan-like structures existed in Orientia, or that there is some yet-to-be-discovered effect of antigenic proteins in OT. In addition, NOD1 senses bacterial growth though the recognition of peptidoglycan fragments released by growing bacteria in which
undertake peptidoglycan remodeling [26]. In that case, we might use OT deleted for enzymes involved in peptidoglycan remodeling, such as AmpG [27] to reveal the relation between peptidoglycan remodeling and IL-32 secretion from OT-infected endothelial cells. In our experiments, NOD1 silencing by siRNA resulted in a reduction in IL-32, IL-6, IL-8, and ICAM-1 in ECV304 cells (Figs. 5 and 6), and this reduction in cytokine markers of endothelial cell activation was reversed by supplementation with IL-32. This shows that NOD1 is necessary for the activation of endothelial cells to initiate local inflammation. Additionally, the administration of IL-32 to endothelial cells is sufficient to increase cytokine production in endothelial cells, but the production levels were not as high as in the OT-infected cells (Fig. 7). By way of contrast with a previous report showing that IL-32 synergized with NOD1 and NOD2 ligands for IL-1b and IL-6 production through a caspase-1 dependent pathway [28], we determined herein that the production of IL-32 was dependent on NOD1, and also that it affected IL-1b secretion. Therefore, our data indicate that the signaling pathway might begin with the activation of NOD1 after OT infection, and that NOD1 leads to IL-32 production, resulting in increases in the levels of pro-IL-1b, IL-6, and IL-8, as well as ICAM-1 expression.
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Fig. 4. The expression of IL-6, IL-8, and ICAM-1 are upregulated in OT-exposed ECV304 cells. (A and B) IL-6 and IL-8 levels were measured via ELISA and increased significantly in response to OT. Values are provided as means SEM (n ¼ 9). (C) ECV304 cells expressing ICAM-1 were analyzed via flow cytometry (left) and the mean fluorescence intensities of ICAM-1 (right) are shown as the means SEM (n ¼ 3). *P < 0.05. Filled histogram indicates staining with isotype antibody (mouse IgG1) for the background.
In endothelial cells, it was noted that IL-1b induced IL-32 production, followed by an increase in the levels of ICAM-1, IL-1a, IL-6 and IL-8 expression [14]. We noted that IL-32-induced IL-1b expression in OT-infected endothelial cells, and this IL-1b reciprocally affected IL-32 secretion. Therefore, IL-32 and IL-1b might form a positive feedback loop on endothelial cells during the process of inflammation. Additionally, the production of IL-32 by mycobacteria is dependent on active IL-18, and is induced by active caspase-1. Furthermore, IL-18 is a key stimulant for the induction of IFN-g, and IFN-g induces IL-32 secretion [29]. In short, it is possible that caspase-1 activation via NOD1 can be attributed to IL-32 production and IL-32 might be a common downstream cytokine activated by the NOD pathway during intracellular infection. Although the cytokine and chemokine profile results demonstrated that OT infection results in the immune reaction via soluble mediators from infected tissues or cells, OT exploits immune evasion mechanisms for survival. One of the regulatory mechanisms employed by OT involves the suppression of the production of proinflammatory cytokines, including IL-6 and TNF-a, via the induction of IL-10 secretion from infected macrophages [30,31]. According to our results, OT-infected ECV304 cells expressed IL-10 at higher levels (Fig. 1C). Recently, it was also reported that the proinflammatory cytokine IL-32 promotes the production of the anti-inflammatory cytokine IL-10 [32]. Thus, in this experiment we focused principally on the proinflammatory effects of IL-32 on endothelial cells; however, the overall effect of IL-32 in disease progression will require further study. IL-32 was identified as an IL-18-induced cytokine and has previously been recognized as a natural killer cell transcript 4 (NK4) [33]. IL-32 has major representative transcripts of four isoforms (a, b, g, and d). All isoforms are biologically active, but
IL-32g was the most active [34]. IL-32 induces a variety of cytokines e human TNF-a, IL-8 in THP-1 monocytic cells, and mouse TNF-a and MIP-2 in RAW264.7 macrophage cells [35]. Therefore, IL-32 functions as a proinflammatory cytokine by stimulating TNFa, IL-1b, and IL-8 production, and by activating the NF-kB and p38 MAPK pathways [35]. In synovial fluids from rheumatoid arthritis patients, the concentration of IL-32 is increased markedly as compared to that seen in osteoarthritis patients. Moreover, the production of IL-32 in synovial fibroblasts is induced by TNF-a stimulation via the Syk and PKCd pathways [36]. Several positive and negative regulatory mechanisms of inflammation are mediated by IL-32 other than those described above. For example, infection with influenza virus in patients results in elevated concentrations of IL-32 and prostaglandin E2. The knockdown of IL-32 by siRNA inhibited the release of iNOS and NO by the dsRNA of the influenza virus in A549 cells (human lung epithelial cells). This suggests that IL-32 functions upstream of dsRNA-triggered iNOS production [37]. IL-32 has been reported to be capable of inducing osteoclast differentiation independent of the RANK/ RANKL pathway. Although IL-32 increases the secretion of proinflammatory cytokines, i.e, TNF-a, IL-6, LIGHT, CCL3 (MIP-1a), VEGF, IFN-g, and IL-4, which are known to positively influence osteoclastogenesis, IL-32 alone proved unable to induce the activation of newly-formed multinucleated cells into bone-absorbing osteoclasts [38]. In summary, we determined that intracellular OT infection led to the activation of NOD1, and was involved in endothelial cell activation via IL-32, possibly propagating inflammation via the induction of IL-1b, IL-6, IL-8, and ICAM-1 in endothelial cells. This suggested that the NOD1-IL-32 pathway may function as a modulator of endothelial immune response in OT infection. These results may contribute to our understanding of the inflammatory process of the endothelium in scrub typhus.
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Fig. 5. Silencing NOD1 in OT-infected ECV304 cells impairs the expression of IL-32 and IL-1b. (A) Endogenous NOD1 was reduced by specific siRNA. The levels of mRNA (left) and protein (right) of NOD1 were quantified after 3 days of culture. (B) NOD1 knockdown resulted in a reduction in IL-32 levels in OT-infected samples. The expression levels were detected and compared via immunoblotting (left) and ELISA (right). (C) ECV304 cells with NOD1 knockdown evidence downregulated IL-1b expression in response to OT. Data in (B) and (C) represent means SEM (n ¼ 9). *P < 0.05.
4. Materials and Methods 4.1. Cell culture and infection of O. tsutsugamushi The Boryong serotype of OT (strain MF) was cultured in ECV304 cells, as previously described [3]. When the infected cells evidenced the maximal cytopathogenic effects, the cells were collected and disrupted via vortexing with glass beads of 1.0 mm diameters (G1152, Sigma, St. Louis, MO), followed by 5 min of centrifugation at 1500 rpm. The supernatant was collected and used to infect the ECV304 cells. The titer of OT inoculums was determined microscopically and infected cell counting units (ICU) were calculated as follows: ICU ¼ total number of cells used for inoculation percentage of infected cells fold of dilution/100. Then a total of 2 106 ICU of OT was used to infect ECV304 cells
cultured in 6-well plates. The growth of OT was determined by immunofluorescent staining of OT-infected ECV304 cells with fluorescein isothiocyanate (FITC)-conjugated FS15 monoclonal antibody [30] (Supplementary Fig. 1). 4.2. Cytokine antibody array For the profile of secreted or produced cytokines of OT-infected ECV304 cells, a RayBioÒ Human Cytokine Antibody Array 3 (RayBiotech, Inc., Norcross, GA) was used, in accordance with the manufacturer’s instructions. In brief, the membranes were incubated with blocking buffer followed by incubation with cell culture supernatant or cell lysates. Next, we added diluted biotin-conjugated anti-cytokines and horseradish peroxidase (HRP)-conjugated streptavidin for probing. We proceeded with the detection reaction
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Fig. 6. NOD1 knockdown affects the expression levels of IL-6, IL-8 and ICAM-1 in ECV304 cells after OT infection. Secretion levels of IL-6 (A) and IL-8 (B) were reduced by NOD1 knockdown in OT-infected ECV304 cells. C. The levels of ICAM-1 were measured by flow cytometry (left) and mean fluorescence intensities of ICAM-1 (right). The data shown are representative of three independent experiments. Data are expressed as the means SEM. *P < 0.05. Filled histogram indicates staining with isotype antibody (mouse IgG1) for the background.
and exposed the array to LAS-3000 (Fujifilm, Japan). We then analyzed the pixel densities of the dotted spots using Un-Scan-ItÔ (Silk Scientific, Inc., Orem, UT) software. 4.3. Small interfering RNA (siRNA) silencing of NOD1 expression ECV304 cells at 60e70% confluence were transfected with 80 pmol of siRNA oligonucleotides (Santa Cruz Biotechnology, Santa Cruz, CA) using siRNA transfection reagent (Santa Cruz Biotechnology) to reduce endogenous NOD1 expression. Cells transfected with either a siRNA oligonucleotide against NOD1 or a non-targeted control siRNA oligonucleotide were maintained at 37 C under standard tissue culture conditions. Seven hours after transfection, the cells were incubated with or without OT. ECV304 cells were maintained for 3 days and the cell lysates and supernatants were harvested for Western blotting and ELISA, respectively. In order to evaluate IL-32-induced cytokine responses, rhIL-32g (#4690-IL/CF, R&D Systems, Minneapolis, MN) was added at 100 ng/ml to each experimental group.
4.5. Immunoblot Cells were lysed in a lysis buffer (1% Triton X-100, 150 mM NaCl, 20 mM Tris, pH 7.5) with protease inhibitors and 10 mg of proteins were separated via electrophoresis on 12% SDS-polyacrylamide gels. The resolved proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore Co., Billerica, MA). The membranes were blocked with 5% skim milk and then incubated with primary antibodies, anti-caspase-1 (Rabbit polyclonal, #2225, Cell Signaling Technology, Danvers, MA), anti-IL-1b (Rabbit polyclonal, #2022, Cell Signaling Technology), anti-NOD1 (Rabbit polyclonal, #3545, Cell Signaling Technology), and anti-IL-32 (clone KU32-56, BioLegend, San Diego, CA) antibodies. Anti-IL-32 antibody (clone KU32-56) [13] was also generously provided by Do-Young Yoon at Konkuk University (Seoul, Korea). After washing, the membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit IgG secondary antibody and the signals were visualized via enhanced chemiluminescence using a Thermo ECL kit (Thermo Fisher Scientific Inc., Waltham, MA) in accordance with the manufacturer’s instructions.
4.4. Reverse transcription-PCR
4.6. ELISA
Total RNA from ECV304 cells, transfected with NOD1-specific siRNA or non-specific control siRNA, was isolated with a RiboPureÔ Kit (Ambion, Austin, TX) and reverse-transcribed using AMV reverse-transcriptase (Promega, Madison, WI). The generated cDNA was amplified using NOD1-specific primer pairs (sense: 50 -GGG GTG ACC AGC AGT CCT AT-30 , anti-sense: 50 -GAA GGG AGG ATA GCA GGA CG-30 , 175 bp). In order to confirm equal amounts of RNA in each experiment, all samples were checked for and compared with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA expression. The primer sequence for the GAPDH was as follows: sense 50 - GTC TTC TCC ACC ATG GAG AAG GCT -30 and anti-sense 50 CAT GCC AGT GAG CTT CCC GTT CA- 30 (395 bp).
Levels of IL-1b, IL-6, and IL-8 in ECV304 supernatant infected with or without OT were quantified with OptEIA kit (BD PharMingen, San Diego, CA) and IL-32 levels with human IL-32a ELISA MAXÔ kit from BioLegend. NOD1-silenced ECV304 cells were also infected with OT and the cytokine concentrations were measured. 4.7. Flow cytometry ECV304 cells were analyzed for the cell-surface molecule, ICAM-1, after each designed experiment. The cells were washed in PBS and stained with phycoerythrin (PE)-conjugated antiintercellular adhesion molecule-1 (ICAM-1, clone HA58, mouse
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Fig. 7. IL-32 reverses the effects of NOD1 knockdown on the expression of IL-1b, IL-6, IL-8, and ICAM-1. (A) Decreased IL-1b secretion as the result of NOD1 silencing is reversed by IL-32 treatment. IL-32 treatment was shown to have an independent ability to induce IL-1b as compared to those levels in unstimulated cells. (B) IL-6 secretion is reduced in NOD1reduced cells, and is recovered after IL-32 treatment. IL-32 treatment was also shown to have an independent ability to induce IL-6 secretion as compared to those measured in unstimulated cells. (C) IL-32 treatment reverses the downregulated IL-8 secretion in NOD1-silenced cells and also was shown to have an independent ability to induce IL-8 as compared to what was noted in unstimulated cells. (D) ICAM-1 expression on ECV304 cells is increased in response to IL-32 in NOD1-silenced cells, but evidences no statistical difference as compared to what was noted in unstimulated cells. The data shown are representative of three independent experiments. The data are expressed as the means SEM. *P < 0.05.
IgG1, BD PharMingen) for 15 min at room temperature. The level of non-specific staining was determined using PE-conjugated mouse IgG1 as an isotype control. Samples were acquired on a FACSCalibur system (BD Biosciences, San Jose, CA) and were analyzed using CellQuest software (BD Biosciences). 4.8. Statistics The values are expressed as the means SEM. Non-parametric ManneWhitney tests were applied to analyze the results for significant differences (at P < 0.05), using GraphPad Prism software (GraphPad Software Inc., San Diego, CA). Acknowledgments We thank professor Do-Young Yoon at Konkuk University (Seoul, Korea) for providing us with the anti-IL-32 antibody.
Appendix. Supporting information Supporting information associated with this article can be found, in the online version, at doi:10.1016/j.micpath.2010.05.001.
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[6] Chung DR, Lee YS, Lee SS. Kinetics of inflammatory cytokines in patients with scrub typhus receiving doxycycline treatment. J Infect 2008;56:44e50. [7] Kanneganti TD, Lamkanfi M, Nunez G. Intracellular NOD-like receptors in host defense and disease. Immunity 2007;27:549e59. [8] Opitz B, Forster S, Hocke AC, Maass M, Schmeck B, Hippenstiel S, et al. Nod1mediated endothelial cell activation by Chlamydophila pneumoniae. Circ Res 2005;96:319e26. [9] Opitz B, Puschel A, Beermann W, Hocke AC, Forster S, Schmeck B, et al. Listeria monocytogenes activated p38 MAPK and induced IL-8 secretion in a nucleotide-binding oligomerization domain 1-dependent manner in endothelial cells. J Immunol 2006;176:484e90. [10] Greiffenberg L, Goebel W, Kim KS, Daniels J, Kuhn M. Interaction of Listeria monocytogenes with human brain microvascular endothelial cells: an electron microscopic study. Infect Immun 2000;68:3275e9; Slaghuis J, Goetz M, Engelbrecht F, Goebel W. Inefficient replication of Listeria innocua in the cytosol of mammalian cells. J Infect Dis 2004;189:393e401. [11] Rose F, Zeller SA, Chakraborty T, Domann E, Machleidt T, Kronke M, et al. Human endothelial cell activation and mediator release in response to Listeria monocytogenes virulence factors. Infect Immun 2001;69:897e905. [12] Opitz B, Puschel A, Schmeck B, Hocke AC, Rosseau S, Hammerschmidt S, et al. Nucleotide-binding oligomerization domain proteins are innate immune receptors for internalized Streptococcus pneumoniae. J Biol Chem 2004;279:36426e32. [13] Arisoy M, Aysev D, Ekim M, Ozel D, Kose SK, Ozsoy ED, et al. Detection of virulence factors of Escherichia coli from children by multiplex polymerase chain reaction. Int J Clin Pract 2006;60:170e3. [14] Nold-Petry CA, Nold MF, Zepp JA, Kim SH, Voelkel NF, Dinarello CA. IL-32dependent effects of IL-1beta on endothelial cell functions. Proc Natl Acad Sci U S A 2009;106:3883e8. [15] Yoo NJ, Park WS, Kim SY, Reed JC, Son SG, Lee JY, et al. Nod1, a CARD protein, enhances pro-interleukin-1beta processing through the interaction with procaspase-1. Biochem Biophys Res Commun 2002;299:652e8. [16] Nacy CA, Groves MG. Macrophages in resistance to rickettsial infections: early host defense mechanisms in experimental scrub typhus. Infect Immun 1981;31:1239e50. [17] Koh YS, Yun JH, Seong SY, Choi MS, Kim IS. Chemokine and cytokine production during Orientia tsutsugamushi infection in mice. Microb Pathog 2004;36:51e7. [18] Iwasaki H, Takada N, Nakamura T, Ueda T. Increased levels of macrophage colony-stimulating factor, gamma interferon, and tumor necrosis factor alpha in sera of patients with Orientia tsutsugamushi infection. J Clin Microbiol 1997;35:3320e2. [19] Baggiolini M, Dewald B, Moser B. Interleukin-8 and related chemotactic cytokineseCXC and CC chemokines. Adv Immunol 1994;55:97e179. [20] Paris DH, Jenjaroen K, Blacksell SD, Phetsouvanh R, Wuthiekanun V, Newton PN, et al. Differential patterns of endothelial and leucocyte activation in ‘typhuslike’ illnesses in Laos and Thailand. Clin Exp Immunol 2008;153:63e7. [21] Fritz JH, Ferrero RL, Philpott DJ, Girardin SE. Nod-like proteins in immunity, inflammation and disease. Nat Immunol 2006;7:1250e7; Werts C, Girardin SE, Philpott DJ. TIR, CARD and PYRIN: three domains for an antimicrobial triad. Cell Death Diff 2006;13:798e815. [22] Martinon F, Burns K, Tschopp J. The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Mol Cells 2002;10:417e26.
[23] Chamaillard M, Girardin SE, Viala J, Philpott DJ. Nods, Nalps and Naip: intracellular regulators of bacterial-induced inflammation. Cell Microbiol 2003;5:581e92; Inohara N, Nunez G. NODs: intracellular proteins involved in inflammation and apoptosis. Nat Rev Immunol 2003;3:371e82. [24] Hanson B. Identification and partial characterization of Rickettsia tsutsugamushi major protein immunogens. Infect Immun 1985;50:603e9; Oaks EV, Rice RM, Kelly DJ, Stover CK. Antigenic and genetic relatedness of eight Rickettsia tsutsugamushi antigens. Infect Immun 1989;57:3116e22; Tamura A, Ohashi N, Urakami H, Takahashi K, Oyanagi M. Analysis of polypeptide composition and antigenic components of Rickettsia tsutsugamushi by polyacrylamide gel electrophoresis and immunoblotting. Infect Immun 1985;48:671e5. [25] Ihn KS, Han SH, Kim HR, Huh MS, Seong SY, Kang JS, et al. Cellular invasion of Orientia tsutsugamushi requires initial interaction with cell surface heparan sulfate. Microb Pathog 2000;28:227e33; Kim HR, Choi MS, Kim IS. Role of Syndecan-4 in the cellular invasion of Orientia tsutsugamushi. Microb Pathog 2004;36:219e25. [26] Franchi L, Warner N, Viani K, Nunez G. Function of Nod-like receptors in microbial recognition and host defense. Immunol Rev 2009;227:106e28. [27] Cheng Q, Park JT. Substrate specificity of the AmpG permease required for recycling of cell wall anhydro-muropeptides. J Bacteriol 2002;184:6434e6. [28] Netea MG, Azam T, Ferwerda G, Girardin SE, Walsh M, Park JS, et al. IL-32 synergizes with nucleotide oligomerization domain (NOD) 1 and NOD2 ligands for IL-1beta and IL-6 production through a caspase 1-dependent mechanism. Proc Natl Acad Sci U S A 2005;102:16309e14. [29] Netea MG, Azam T, Lewis EC, Joosten LA, Wang M, Langenberg D, et al. Mycobacterium tuberculosis induces interleukin-32 production through a caspase- 1/IL-18/interferon-gamma-dependent mechanism. PLoS Med 2006; 3:e277. [30] Kim MJ, Kim MK, Kang JS. Orientia tsutsugamushi inhibits tumor necrosis factor alpha production by inducing interleukin 10 secretion in murine macrophages. Microb Pathog 2006;40:1e7. [31] Kim MK, Kang JS. Orientia tsutsugamushi suppresses the production of inflammatory cytokines induced by its own heat-stable component in murine macrophages. Microb Pathog 2001;31:145e50. [32] Kang JW, Choi SC, Cho MC, Kim HJ, Kim JH, Lim JS, et al. A proinflammatory cytokine interleukin-32beta promotes the production of an anti-inflammatory cytokine interleukin-10. Immunology 2009;128:e532e40. [33] Dahl CA, Schall RP, He HL, Cairns JS. Identification of a novel gene expressed in activated natural killer cells and T cells. J Immunol 1992;148:597e603. [34] Choi JD, Bae SY, Hong JW, Azam T, Dinarello CA, Her E, et al. Identification of the most active interleukin-32 isoform. Immunology 2009;126:535e42. [35] Kim SH, Han SY, Azam T, Yoon DY, Dinarello CA. Interleukin-32: a cytokine and inducer of TNFalpha. Immunity 2005;22:131e42. [36] Mun SH, Kim JW, Nah SS, Ko NY, Lee JH, Kim JD, et al. Tumor necrosis factor alpha-induced interleukin-32 is positively regulated via the Syk/protein kinase Cdelta/JNK pathway in rheumatoid synovial fibroblasts. Arthritis Rheum 2009;60:678e85. [37] Li W, Yang F, Liu Y, Gong R, Liu L, Feng Y, et al. Negative feedback regulation of IL-32 production by iNOS activation in response to dsRNA or influenza virus infection. Eur J Immunol 2009;39:1019e24. [38] Mabilleau G, Sabokbar A. Interleukin-32 promotes osteoclast differentiation but not osteoclast activation. PLoS One 2009;4:e4173.
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Orientia tsutsugamushi induced endothelial cell activation via the NOD1-IL-32 pathway Kyung-Ah Cho a, Yoon Hee Jun b, Jee Won Suh a, Jae-Seung Kang c, Hee Jung Choi b, So-Youn Woo a, * a
Department of Microbiology, School of Medicine, Ewha Womans University, 911-1 Mok-Dong, Yangcheon-Gu, Seoul 158-710, Republic of Korea Division of Infectious Disease, Department of Internal Medicine, School of Medicine, Ewha Womans University Mokdong Hospital, 911-1 Mok-Dong, Yangcheon-Gu, Seoul 158-710, Republic of Korea c Department of Microbiology, Inha Research Institute for Medical Science, Inha University College of Medicine, Jungsuk B/D, 3rd street, Shinheung-Dong, Choong-Gu, Incheon 400-712, Republic of Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 March 2010 Received in revised form 27 April 2010 Accepted 4 May 2010 Available online 12 May 2010
Orientia tsutsugamushi (OT), the causative agent of scrub typhus, is an obligate intracellular bacterium. In order to verify the inflammatory responses involved in the pathogenesis of scrub typhus, we assessed the cytokine profile of the human endothelial cell line, ECV304, after OT infection. We noted that CCL5, CCL17, IL-1a, IL-6, IL-8, IL-10, IL-15, TNF-a and TNF-b were strongly induced in response to OT. Additionally, IL-32, the candidate modulator for the induction of IL-6 and IL-8, was increased significantly with OT infection and these increases coincided with NOD1 pathway activation. Thus, we hypothesized that NOD1 pathway and IL-32 might act on cytokine release in endothelial cells as a modulator of the inflammation caused by OT infection. NOD1 siRNA resulted in a reduction in IL-32 levels, and also reduced IL-1b, IL-6, IL-8, and ICAM-1 expression in OT-infected ECV304 cells. These changes in IL-1b, IL-6, IL-8, and ICAM-1 induced by NOD1 knockdown were reversed as the result of IL-32 treatment. This indicated that OT infection activated the NOD1 pathway followed by IL-32 secretion, thus resulting in the production and expression of IL-1b, IL-6, IL-8, and ICAM-1. Therefore, IL-32 might perform a role upstream of the inflammatory reaction in endothelial cells of OT infection. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: NOD1 IL-32 Orientia tsutsugamushi Endothelial cells
1. Introduction Orientia tsutsugamushi (OT) is the causative agent of scrub typhus, also referred to as tsutsugamushi disease, a serious public health problem in Asia. OT is maintained in nature by transovarian transmission in trombiculid mites, primarily of the genus Leptotrombidium. After hatching, infected larval mites (chiggers, the only stage that feeds on an animal host) inoculate organisms into the skin during feeding. Occasionally, infected mites or rat fleas bite humans and cause scrub typhus, characterized focal or disseminated vasculitis and perivasculitis, which may involve the lungs, heart, liver, spleen, and central nervous systems [1]. The illness varies in severity from mild and selflimited to fatal. After an incubation period spanning 6e21 days, the onset of disease is characterized by fever, rash, eschar,
Abbreviations: OT, Orientia tsutsugamushi; TARC, Thymus and activation regulated chemokine; NOD, Nucleotide-binding and oligomerization domain; NLR, NOD-like receptor. * Corresponding author. Tel.: þ82 2 2650 5740; fax: þ82 2 2653 8891. E-mail address: [email protected] (S.-Y. Woo). 0882-4010/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2010.05.001
headache, myalgia, cough, and gastrointestinal symptoms. Severe cases typically include prominent encephalitis and interstitial pneumonia as key features of vascular injury [2,3]. During OT disease progression, OT was located in endothelial cells in all the involved organs [4], and also infected a variety of cells, including macrophages, polymorphonuclear leukocytes (PMNs), and lymphocytes. This subsequently results in local inflammation followed by dissemination to the whole body [4,5]. The pathogenesis of inflammation in scrub typhus involves immune and inflammatory mediators including cytokines, prostaglandins, leukotrienes, and kinins from infected and immune cells. Inflammatory cytokines, such as TNF-a, IL-1b, and IL-6 were increased markedly in patients with scrub typhus, and these cytokines were reduced after doxycycline treatment [6]. These cytokines are attributable to the high fever occurring in most scrub typhus patients. Additionally, OT-infected macrophages express CCL2 (MCP-1), CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), and CCL8 (MIP-2) [3]. Such host responses against microorganisms involve the activation of specialized pattern recognition receptors in the cells and lead to the production of inflammatory mediators.
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Nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) comprises a large family of intracellular pattern recognition receptors (PRRs), which are characterized by the presence of conserved NOD. On the basis of the N-terminal domains, NLRs can be classified into three subfamilies referred to as the caspase recruitment domain (CARD)-containing NODs, pyrin (PYD)-containing NALPs, or baculovirus inhibitor repeat (BIR)containing NAIPs [7]. Owing to their location, NLRs can be activated by intracellular pathogens to initiate innate immune responses. For example, the role of NOD proteins in the response to intracellular pathogens such as Chlamydophila pneumoniae and Listeria monocytogenes [8,9] has been previously reported. Whereas NOD1 has been shown to recognize peptidoglycan containing meso-diaminopimelic acid, which is found principally in gram-negative bacteria [10], NOD2 detects muramyl dipeptide (MurNAc-LAla-DisoGln), which is conserved in basically all types of peptidoglycan [11]. Although such differences exist in sensing pathogens, NOD1 and NOD2 share the ability to activate downstream signaling, such as NF-kB and mitogen-activated protein kinases (MAPKs) [7,12] and eventually lead to the propagation of inflammation via the secretion of proinflammatory cytokines. There is increasing evidence to suggest that IL-32, one of the proinflammatory cytokines, induces IL-1a, IL-1b, IL-6, and TNFa via the NF-kB and p38 MAPK pathways [13,14]. IL-32 is constitutively expressed in endothelial cells and functions as a regulator of endothelial inflammation by affecting the expression of intercellular adhesion molecules 1 (ICAM-1) on endothelial cells with IL-1b [14]. Thus, we noted the possible contribution of NLR, particularly NOD1, and IL-32 to tsutsugamushi disease, due to their own ability to modulate immune responses in the endothelial cells. In an effort to gain insight into the pathogenesis of scrub typhus in relation to vascular injury, we focused on delineating the inflammatory responses in OT-infected endothelial cells. Therefore, we evaluated the cytokine profiles from OT-infected ECV304 endothelial cells, and the molecular mechanism of NOD1-IL-32 interaction, which may possibly be involved in the activation of endothelial cells after OT infection using NOD1 siRNA. 2. Results 2.1. Expression profile of cytokines and chemokines in OT-infected ECV304 cells In order to identify the possible inflammatory mediators from OT-infected endothelial cells, we compared the simultaneous expression profiles of cytokines and chemokines from ECV304 cells (as control) and OT-infected ECV304 cells using a cytokine antibody array (Fig. 1A and B). CXCL1-3 (Gro), CCL5 (RANTES), CCL17 (TARC), IL-1a, IL-6, IL-8, IL-10, IL-15, TNF-a and TNF-b were induced more profoundly in OT-infected ECV304 cells in those of the ECV304 extract-treated group, as controls (Fig. 1C). As it was reported that IL-32 was expressed constitutively in endothelial cells and IL-32 performs a function in inflammation by inducing proinflammatory cytokines such as IL-1b, IL-6, and IL-8, we compared the levels of IL-32 expression between ECV304 cells and OT-infected ECV304 cells via immunoblotting and ELISA. The average levels of IL-32 production in ECV304 cells were 7.2 2.1 pg/ ml. We determined that IL-32 was generated and secreted more in OT-infected cells (ranges 14.2 1.8 pg/ml) than in the control group (Fig. 2 and Supplementary Figs. 2 and 3). Thus, we noted that OT infection of ECV304 cells induced the expression of proinflammatory cytokines, including IL-1a, IL-6, IL-8, IL-10, and IL-32 in endothelial cells.
2.2. OT-infected ECV304 cells produce more NOD1 and active forms of caspase-1 and IL-1b Next, we considered that the increase in IL-32 expression might influence the endothelial cell functions after OT infection, and we evaluated the possible upstream signaling pathway by which IL-32 is induced in ECV304 cells. First, we compared NOD1 expression after OT infection in ECV304 cells. In the presence of OT in ECV304 cells, we determined that OT-infected ECV304 cells generated more NOD1. As the activation of NOD1 results in the production of pro-IL-1b, we compared the expression levels of pro-IL-1b, as well as the active forms. We determined that the active form of IL-1b was increased by OT infection. We then compared the expression levels of caspase-1. As is shown in Fig. 3, the pro and cleaved forms of caspase-1 were increased in cases of OT infection. It was reported that NOD1 enhances pro-IL1b processing by interacting pro-caspase-1 [15]. Therefore we determined that NOD1 was expressed more abundantly in OT-infected cells accompanying increases in the levels of active forms of caspase-1 (Fig. 3). Therefore, we considered that OT infection resulted in NOD1 activation and affected IL-1b production. As shown in Fig. 3, OT infection lead to increase of NOD1, caspase-1, as well as IL-1b. For the cytokines and cell adhesion molecules from OT-infected endothelial cells, possibly induced by IL-32, we determined the levels of IL-6, IL-8, and ICAM-1, and compared the expression levels. Because we had observed increases in the levels of IL-6 and IL-8 after OT infection by cytokine array, we determined that the average production levels of IL-6 and IL-8 by ELISA were 1860 280 pg/ml and 960 190 pg/ml, respectively (Fig. 4A and B). The upregulated expression of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells was noted in OT-infected cells (Fig. 4C). 2.3. OT-infected ECV304 cells induced IL-32 via NOD1 pathway Although IL-32 was identified as a regulator of endothelial inflammation [14], the sequential relationship between IL-32 and NOD1 in cases of OT infection of endothelial cells was unclear. Therefore, we focused on the role of NOD1 in OT-infected endothelial cells and compared the changes in endothelial activation with or without OT infection. In order to determine whether or not NOD1 is involved in the induction of IL-32 secretion in ECV304 cells, we effected a reduction in NOD1 expression via siRNA transfection. As is shown in Fig. 5A, NOD1 siRNA reduced the expression of endogenous NOD1, as confirmed by RT-PCR (by 78%) and immunoblotting (by 53%). The reduction of NOD1 affected the expression and secretion of IL-32 from OT-infected ECV304 cells (Fig. 5B). NOD1 knockdown also reduced the secretion of IL-1b (Fig. 5C). Therefore, according to these results in OT-infected ECV304 cells, we concluded that NOD1-mediated the induction of IL-32 and IL-1b. 2.4. NOD1 knockdown decreased the production and expression of IL-6, IL-8 and ICAM-1 in ECV304 cells In order to evaluate the effects of NOD1 on the production of IL32-inducing cytokines and endothelial cell activation, we compared the expression of IL-6, IL-8, and ICAM-1 after NOD1 knockdown. When endogenous NOD1 levels were reduced by siRNA transfection, we noted a reduction of IL-6, IL-8, and ICAM-1 in OT-infected ECV304 cells (Fig. 6). Thus, we concluded that OT infection increased the level of NOD1 in endothelial cells, followed by the induction of IL-32, IL-6, IL-8, and ICAM-1.
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Fig. 1. Cytokine expression profile of ECV304 cells infected with OT. (A) RayBioÒ Human Cytokine Antibody Array 3 Map shows cytokines and chemokines corresponding to each of the spots. (B) ECV304 cell infected with OT lysates was subjected to cytokine antibody array (right panel). ECV304 cells cultured with non-infected ECV304 extract were used as controls (left panel). Each cytokine is represented by duplicate spots in the shown locations and the cytokine array image represents the results of one of two independent experiments that demonstrated similar expression patterns. The array membranes were exposed for 1 min after the addition of ECL solution and acquired the images. (C) The results of the cytokine antibody array kit were converted to pixel densities. Each spot of the scanned images were digitized and calculated for the pixel densities. The average net optical intensity SEM for each pair of cytokine spots is shown. *P < 0.05.
2.5. Effect of IL-32 on ECV304 cells in OT infection In order to evaluate the direct effect of IL-32 on IL-1b, IL-6, IL-8, and ICAM-1 production in OT-infected ECV304 cells, we incubated
the OT-infected ECV304 cells with or without IL-32 after transfection of NOD1 siRNA. We verified that IL-32 treatment induced increases in IL-1b, IL-6, IL-8, and ICAM-1 production even after NOD1 knockdown (Fig. 7). Therefore it is suggested that the
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Fig. 2. IL-32 is increased by OT infection in ECV304 cells. (A) IL-32 production in OTinfected ECV304 cells was assessed by immunoblotting, and one representative experiment of three is shown. (B) IL-32 secretion into culture media was measured via ELISA. The data are expressed as the means SEM (n ¼ 9). *P < 0.05.
upregulation of IL-32 occurs downstream of the NOD1 pathway, and that IL-32 directly affected the expression of inflammatory modulators of endothelial cells, including IL-1b, IL-6, IL-8, and ICAM-1. 3. Discussion In this study, we have demonstrated that the IL-32 secretion of ECV304 endothelial cells after OT infection occurred downstream of the NOD1 pathway, and that such increased IL-32 levels affected the secretion of proinflammatory cytokines, such as IL-1b, IL-6, and IL-8, as well as ICAM-1 expression in endothelial cells. Inflammation is initiated by OT-infected macrophages and endothelial cells in the dermis. Although mononuclear cells including lymphocytes, macrophages, and neutrophils were observed in the eschars and rashes associated with scrub typhus, inactive tissue macrophages could support growth, and activated macrophages and lymphocytes are essential for protection. Because early host inflammatory responses appear to be crucial for determining the outcome of the progression of the diseases of OTinfected hosts [16], it is important to analyze the regulatory mechanism pertaining to the propagation of the inflammation process via cytokines and cellular influx to the OT infection site. A variety of cytokines and chemokines have been produced in experimental mouse infection [17]. According to the results of human studies, the level of TNF-a in the serum is increased significantly during acute phase as well as convalescent phase [18], and we also noted an increase in the concentration of IFN-g and IL-10 in scrub typhus patients [6]. Activated monocytes and macrophages are the principal sources of chemokines [19], and it was reported that CCL3, CCL4 (macrophage inflammatory protein 1a/b, MIP-1a/b), CCL5 (RANTES) and CCL2 (macrophage chemoattractant protein 1, MCP-1) were induced via NF-kB activation [3];
endothelial cells infected with OT also generate CCL2 and CXCL8 (IL-8) independent of NF-kB activation [2]. In this study, we noted increases in the levels of CXCL1 to 3 (Gro), CCL5, CCL17, IL-1a, IL-6, IL-8, IL-10, IL-15, TNF-a and TNF-b in an OT-infected endothelial cell line (Fig. 1). An increase in IL-10 secretion, as well as that of other proinflammatory cytokines, has been noted. IL-10 is a potent immunosuppressive cytokine that inhibits the production of many cytokines, including IL-2, IFN-g, IL-4, IL-3, IL-1, GM-CSF, and TNF-a. It may be that OT activates the proinflammatory and modulatory pathways, via IL-10, in the early stages of infection. Endothelial cells are key participants in the inflammation process. The activation of endothelial cells is stimulated by proinflammatory cytokines including TNF-a and IL-1, which are released from infected sites and result in the upregulation of cell adhesion molecules, such as P-selectin, E-selectin, ICAM-1, and VCAM-1, to promote cellular influx via transendothelial migration as well as the production of cytokines such as IL-6 and IL-8 to initiate and propagate local inflammatory responses. In patients with scrub typhus, the serum levels of soluble L- and E-selectin were correlated with the symptoms of the disease. These results are suggestive of the activation of mononuclear as well as endothelial cells during OT infection [20]. We demonstrated that the expression of ICAM-1 was increased markedly in our OT-infected cells (Fig. 4C), and this increase in ICAM-1 expression was induced by IL-32 secreted from OT-infected endothelial cells (Fig. 7D). However, the endothelial cells themselves are the targets of OT infection, and this should be considered a direct effect on OT-infected endothelial cells during the immune reaction. Innate immunity to microbial pathogens relies on the specific host-detection of pathogen-derived molecules. In the case of intracellular pathogens, such as rickettsia and chlamydia, sensing via intracellular pattern recognition molecules is mandatory. The two major pattern recognition molecule families are the Toll-like receptor family and the NOD-like receptor (NLR) family. The NLR family is composed of 22 intracellular molecular pattern recognition molecules sharing a central NACHT domain (stands for NAIP, CIITA, HET-E and TP1) and a carboxy-terminal leucine-rich repeat (LRR) region [21]. One subfamily includes NOD1 and NOD2, both of which harbor an N-terminal caspase recruitment domain (CARD) that activates NF-kB signaling. Another subfamily includes the pyrin domain-containing (NLRP) proteins. NLRP proteins are essential for the regulation of caspase-1 activation due to inflammasome formation via the N-terminal pyrin domain [22]. NOD1 and NOD2 sense peptidoglycan polymers from the cell wall components and NLRP3 (NALP3, or cryopyrin) detects a variety of pathogen-associated molecular patterns (muramyl dipeptide, poly (I-C), double-stranded RNA, bacterial RNA, or pore-forming toxins) and danger-associated molecular patterns (extracellular ATP, uric acid, asbestos, silica, or aluminum hydroxide). The activation of NOD1 and NOD2 triggers the recruitment of the adapter protein receptor-interacting serineethreonine kinase 2 (RIPK2 or RIP2), followed by the activation of NF-kB and mitogen-activated protein kinases (MAPKs). NALP3 activation converges on the formation and activation of inflammasomes, consisting of NALP3, ASC, and caspase-1. The NALP3 inflammasome then cleaves pro-IL-1b, proIL-18, and pro-IL-33 to mature IL-1b, IL-18 and IL-33, respectively. NOD1 is expressed in a variety of tissues, but NOD2 is expressed principally in leukocytes, dendritic cells, and epithelial cells [23]. For endothelial cell activation, it has been reported that Chlamydophila pneumonia activates NOD1 and leads to the production of IL-8 [8]. In this study, we also demonstrated increased levels of NOD1 in OT-infected endothelial cells (Fig. 3). The expression level of the mature form of IL-1b was increased in OT-infected cells with an increase in the active form of caspase-1. Therefore, we considered that NOD1 sensed the OT component in endothelial cells, and
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Fig. 3. OT induces NOD1 pathway activation. ECV304 cells were infected with OT and maintained for 6 days. The protein expression of NOD1 (A) was analyzed via immunoblotting (top) and the band densities were quantified by dividing the pixel densities of actin bands (bottom). The protein expression of caspase-1 (B) and IL-1b (C) was analyzed via immunoblotting (top) for the precursor forms as well as the active forms. Band densities of active forms of each protein were quantified by dividing the pixel densities of the actin band (bottom). The relative values of pixel densities are shown as the means SEM of three independent experiments.
NOD1 activated the downstream pathway of NF-kB, thereby resulting in the production of the pro-form of IL-1b. This pro-IL-1b can be matured by activated caspase-1 via the NOD1 pathway. However, which ingredient of OT binds to and stimulates NOD1 remains to be clearly determined. O. tsutsugamushi (OT), an etiologic agent of scrub typhus, is a member of the Rickettsiaceae family, and is related phylogenetically to the genus Rickettsia with 16S rRNA sequence homology of 90.2e90.6%. However, the composition of the cell wall differs substantially; i.e, the cell wall of Rickettsia harbors a large quantity of lipopolysaccharides (LPS) and outermembrane proteins, but Orientia lacks both the peptidoglycan layer and LPS, containing only the major strain-variable 56-kDa protein as well as the antigenically variable 110-, 47-, and 25-kDa proteins [24]. Cellular infection involves bacterial interaction with the cell-surface heparin sulfate of syndecan-4 [25], but the bacterial receptor for attachment to the target cells remains unknown. Because of our findings that OT induced NOD1-mediated endothelial activation, we speculated that peptidoglycan-like structures existed in Orientia, or that there is some yet-to-be-discovered effect of antigenic proteins in OT. In addition, NOD1 senses bacterial growth though the recognition of peptidoglycan fragments released by growing bacteria in which
undertake peptidoglycan remodeling [26]. In that case, we might use OT deleted for enzymes involved in peptidoglycan remodeling, such as AmpG [27] to reveal the relation between peptidoglycan remodeling and IL-32 secretion from OT-infected endothelial cells. In our experiments, NOD1 silencing by siRNA resulted in a reduction in IL-32, IL-6, IL-8, and ICAM-1 in ECV304 cells (Figs. 5 and 6), and this reduction in cytokine markers of endothelial cell activation was reversed by supplementation with IL-32. This shows that NOD1 is necessary for the activation of endothelial cells to initiate local inflammation. Additionally, the administration of IL-32 to endothelial cells is sufficient to increase cytokine production in endothelial cells, but the production levels were not as high as in the OT-infected cells (Fig. 7). By way of contrast with a previous report showing that IL-32 synergized with NOD1 and NOD2 ligands for IL-1b and IL-6 production through a caspase-1 dependent pathway [28], we determined herein that the production of IL-32 was dependent on NOD1, and also that it affected IL-1b secretion. Therefore, our data indicate that the signaling pathway might begin with the activation of NOD1 after OT infection, and that NOD1 leads to IL-32 production, resulting in increases in the levels of pro-IL-1b, IL-6, and IL-8, as well as ICAM-1 expression.
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Fig. 4. The expression of IL-6, IL-8, and ICAM-1 are upregulated in OT-exposed ECV304 cells. (A and B) IL-6 and IL-8 levels were measured via ELISA and increased significantly in response to OT. Values are provided as means SEM (n ¼ 9). (C) ECV304 cells expressing ICAM-1 were analyzed via flow cytometry (left) and the mean fluorescence intensities of ICAM-1 (right) are shown as the means SEM (n ¼ 3). *P < 0.05. Filled histogram indicates staining with isotype antibody (mouse IgG1) for the background.
In endothelial cells, it was noted that IL-1b induced IL-32 production, followed by an increase in the levels of ICAM-1, IL-1a, IL-6 and IL-8 expression [14]. We noted that IL-32-induced IL-1b expression in OT-infected endothelial cells, and this IL-1b reciprocally affected IL-32 secretion. Therefore, IL-32 and IL-1b might form a positive feedback loop on endothelial cells during the process of inflammation. Additionally, the production of IL-32 by mycobacteria is dependent on active IL-18, and is induced by active caspase-1. Furthermore, IL-18 is a key stimulant for the induction of IFN-g, and IFN-g induces IL-32 secretion [29]. In short, it is possible that caspase-1 activation via NOD1 can be attributed to IL-32 production and IL-32 might be a common downstream cytokine activated by the NOD pathway during intracellular infection. Although the cytokine and chemokine profile results demonstrated that OT infection results in the immune reaction via soluble mediators from infected tissues or cells, OT exploits immune evasion mechanisms for survival. One of the regulatory mechanisms employed by OT involves the suppression of the production of proinflammatory cytokines, including IL-6 and TNF-a, via the induction of IL-10 secretion from infected macrophages [30,31]. According to our results, OT-infected ECV304 cells expressed IL-10 at higher levels (Fig. 1C). Recently, it was also reported that the proinflammatory cytokine IL-32 promotes the production of the anti-inflammatory cytokine IL-10 [32]. Thus, in this experiment we focused principally on the proinflammatory effects of IL-32 on endothelial cells; however, the overall effect of IL-32 in disease progression will require further study. IL-32 was identified as an IL-18-induced cytokine and has previously been recognized as a natural killer cell transcript 4 (NK4) [33]. IL-32 has major representative transcripts of four isoforms (a, b, g, and d). All isoforms are biologically active, but
IL-32g was the most active [34]. IL-32 induces a variety of cytokines e human TNF-a, IL-8 in THP-1 monocytic cells, and mouse TNF-a and MIP-2 in RAW264.7 macrophage cells [35]. Therefore, IL-32 functions as a proinflammatory cytokine by stimulating TNFa, IL-1b, and IL-8 production, and by activating the NF-kB and p38 MAPK pathways [35]. In synovial fluids from rheumatoid arthritis patients, the concentration of IL-32 is increased markedly as compared to that seen in osteoarthritis patients. Moreover, the production of IL-32 in synovial fibroblasts is induced by TNF-a stimulation via the Syk and PKCd pathways [36]. Several positive and negative regulatory mechanisms of inflammation are mediated by IL-32 other than those described above. For example, infection with influenza virus in patients results in elevated concentrations of IL-32 and prostaglandin E2. The knockdown of IL-32 by siRNA inhibited the release of iNOS and NO by the dsRNA of the influenza virus in A549 cells (human lung epithelial cells). This suggests that IL-32 functions upstream of dsRNA-triggered iNOS production [37]. IL-32 has been reported to be capable of inducing osteoclast differentiation independent of the RANK/ RANKL pathway. Although IL-32 increases the secretion of proinflammatory cytokines, i.e, TNF-a, IL-6, LIGHT, CCL3 (MIP-1a), VEGF, IFN-g, and IL-4, which are known to positively influence osteoclastogenesis, IL-32 alone proved unable to induce the activation of newly-formed multinucleated cells into bone-absorbing osteoclasts [38]. In summary, we determined that intracellular OT infection led to the activation of NOD1, and was involved in endothelial cell activation via IL-32, possibly propagating inflammation via the induction of IL-1b, IL-6, IL-8, and ICAM-1 in endothelial cells. This suggested that the NOD1-IL-32 pathway may function as a modulator of endothelial immune response in OT infection. These results may contribute to our understanding of the inflammatory process of the endothelium in scrub typhus.
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Fig. 5. Silencing NOD1 in OT-infected ECV304 cells impairs the expression of IL-32 and IL-1b. (A) Endogenous NOD1 was reduced by specific siRNA. The levels of mRNA (left) and protein (right) of NOD1 were quantified after 3 days of culture. (B) NOD1 knockdown resulted in a reduction in IL-32 levels in OT-infected samples. The expression levels were detected and compared via immunoblotting (left) and ELISA (right). (C) ECV304 cells with NOD1 knockdown evidence downregulated IL-1b expression in response to OT. Data in (B) and (C) represent means SEM (n ¼ 9). *P < 0.05.
4. Materials and Methods 4.1. Cell culture and infection of O. tsutsugamushi The Boryong serotype of OT (strain MF) was cultured in ECV304 cells, as previously described [3]. When the infected cells evidenced the maximal cytopathogenic effects, the cells were collected and disrupted via vortexing with glass beads of 1.0 mm diameters (G1152, Sigma, St. Louis, MO), followed by 5 min of centrifugation at 1500 rpm. The supernatant was collected and used to infect the ECV304 cells. The titer of OT inoculums was determined microscopically and infected cell counting units (ICU) were calculated as follows: ICU ¼ total number of cells used for inoculation percentage of infected cells fold of dilution/100. Then a total of 2 106 ICU of OT was used to infect ECV304 cells
cultured in 6-well plates. The growth of OT was determined by immunofluorescent staining of OT-infected ECV304 cells with fluorescein isothiocyanate (FITC)-conjugated FS15 monoclonal antibody [30] (Supplementary Fig. 1). 4.2. Cytokine antibody array For the profile of secreted or produced cytokines of OT-infected ECV304 cells, a RayBioÒ Human Cytokine Antibody Array 3 (RayBiotech, Inc., Norcross, GA) was used, in accordance with the manufacturer’s instructions. In brief, the membranes were incubated with blocking buffer followed by incubation with cell culture supernatant or cell lysates. Next, we added diluted biotin-conjugated anti-cytokines and horseradish peroxidase (HRP)-conjugated streptavidin for probing. We proceeded with the detection reaction
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Fig. 6. NOD1 knockdown affects the expression levels of IL-6, IL-8 and ICAM-1 in ECV304 cells after OT infection. Secretion levels of IL-6 (A) and IL-8 (B) were reduced by NOD1 knockdown in OT-infected ECV304 cells. C. The levels of ICAM-1 were measured by flow cytometry (left) and mean fluorescence intensities of ICAM-1 (right). The data shown are representative of three independent experiments. Data are expressed as the means SEM. *P < 0.05. Filled histogram indicates staining with isotype antibody (mouse IgG1) for the background.
and exposed the array to LAS-3000 (Fujifilm, Japan). We then analyzed the pixel densities of the dotted spots using Un-Scan-ItÔ (Silk Scientific, Inc., Orem, UT) software. 4.3. Small interfering RNA (siRNA) silencing of NOD1 expression ECV304 cells at 60e70% confluence were transfected with 80 pmol of siRNA oligonucleotides (Santa Cruz Biotechnology, Santa Cruz, CA) using siRNA transfection reagent (Santa Cruz Biotechnology) to reduce endogenous NOD1 expression. Cells transfected with either a siRNA oligonucleotide against NOD1 or a non-targeted control siRNA oligonucleotide were maintained at 37 C under standard tissue culture conditions. Seven hours after transfection, the cells were incubated with or without OT. ECV304 cells were maintained for 3 days and the cell lysates and supernatants were harvested for Western blotting and ELISA, respectively. In order to evaluate IL-32-induced cytokine responses, rhIL-32g (#4690-IL/CF, R&D Systems, Minneapolis, MN) was added at 100 ng/ml to each experimental group.
4.5. Immunoblot Cells were lysed in a lysis buffer (1% Triton X-100, 150 mM NaCl, 20 mM Tris, pH 7.5) with protease inhibitors and 10 mg of proteins were separated via electrophoresis on 12% SDS-polyacrylamide gels. The resolved proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore Co., Billerica, MA). The membranes were blocked with 5% skim milk and then incubated with primary antibodies, anti-caspase-1 (Rabbit polyclonal, #2225, Cell Signaling Technology, Danvers, MA), anti-IL-1b (Rabbit polyclonal, #2022, Cell Signaling Technology), anti-NOD1 (Rabbit polyclonal, #3545, Cell Signaling Technology), and anti-IL-32 (clone KU32-56, BioLegend, San Diego, CA) antibodies. Anti-IL-32 antibody (clone KU32-56) [13] was also generously provided by Do-Young Yoon at Konkuk University (Seoul, Korea). After washing, the membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit IgG secondary antibody and the signals were visualized via enhanced chemiluminescence using a Thermo ECL kit (Thermo Fisher Scientific Inc., Waltham, MA) in accordance with the manufacturer’s instructions.
4.4. Reverse transcription-PCR
4.6. ELISA
Total RNA from ECV304 cells, transfected with NOD1-specific siRNA or non-specific control siRNA, was isolated with a RiboPureÔ Kit (Ambion, Austin, TX) and reverse-transcribed using AMV reverse-transcriptase (Promega, Madison, WI). The generated cDNA was amplified using NOD1-specific primer pairs (sense: 50 -GGG GTG ACC AGC AGT CCT AT-30 , anti-sense: 50 -GAA GGG AGG ATA GCA GGA CG-30 , 175 bp). In order to confirm equal amounts of RNA in each experiment, all samples were checked for and compared with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA expression. The primer sequence for the GAPDH was as follows: sense 50 - GTC TTC TCC ACC ATG GAG AAG GCT -30 and anti-sense 50 CAT GCC AGT GAG CTT CCC GTT CA- 30 (395 bp).
Levels of IL-1b, IL-6, and IL-8 in ECV304 supernatant infected with or without OT were quantified with OptEIA kit (BD PharMingen, San Diego, CA) and IL-32 levels with human IL-32a ELISA MAXÔ kit from BioLegend. NOD1-silenced ECV304 cells were also infected with OT and the cytokine concentrations were measured. 4.7. Flow cytometry ECV304 cells were analyzed for the cell-surface molecule, ICAM-1, after each designed experiment. The cells were washed in PBS and stained with phycoerythrin (PE)-conjugated antiintercellular adhesion molecule-1 (ICAM-1, clone HA58, mouse
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Fig. 7. IL-32 reverses the effects of NOD1 knockdown on the expression of IL-1b, IL-6, IL-8, and ICAM-1. (A) Decreased IL-1b secretion as the result of NOD1 silencing is reversed by IL-32 treatment. IL-32 treatment was shown to have an independent ability to induce IL-1b as compared to those levels in unstimulated cells. (B) IL-6 secretion is reduced in NOD1reduced cells, and is recovered after IL-32 treatment. IL-32 treatment was also shown to have an independent ability to induce IL-6 secretion as compared to those measured in unstimulated cells. (C) IL-32 treatment reverses the downregulated IL-8 secretion in NOD1-silenced cells and also was shown to have an independent ability to induce IL-8 as compared to what was noted in unstimulated cells. (D) ICAM-1 expression on ECV304 cells is increased in response to IL-32 in NOD1-silenced cells, but evidences no statistical difference as compared to what was noted in unstimulated cells. The data shown are representative of three independent experiments. The data are expressed as the means SEM. *P < 0.05.
IgG1, BD PharMingen) for 15 min at room temperature. The level of non-specific staining was determined using PE-conjugated mouse IgG1 as an isotype control. Samples were acquired on a FACSCalibur system (BD Biosciences, San Jose, CA) and were analyzed using CellQuest software (BD Biosciences). 4.8. Statistics The values are expressed as the means SEM. Non-parametric ManneWhitney tests were applied to analyze the results for significant differences (at P < 0.05), using GraphPad Prism software (GraphPad Software Inc., San Diego, CA). Acknowledgments We thank professor Do-Young Yoon at Konkuk University (Seoul, Korea) for providing us with the anti-IL-32 antibody.
Appendix. Supporting information Supporting information associated with this article can be found, in the online version, at doi:10.1016/j.micpath.2010.05.001.
References [1] Jeong YJ, Kim S, Wook YD, Lee JW, Kim KI, Lee SH. Scrub typhus: clinical, pathologic, and imaging findings. Radiographics 2007;27:161e72. [2] Cho NH, Seong SY, Choi MS, Kim IS. Expression of chemokine genes in human dermal microvascular endothelial cell lines infected with Orientia tsutsugamushi. Infect Immun 2001;69:1265e72. [3] Cho NH, Seong SY, Huh MS, Han TH, Koh YS, Choi MS, et al. Expression of chemokine genes in murine macrophages infected with Orientia tsutsugamushi. Infect Immun 2000;68:594e602. [4] Moron CG, Popov VL, Feng HM, Wear D, Walker DH. Identification of the target cells of Orientia tsutsugamushi in human cases of scrub typhus. Mod Pathol 2001;14:752e9. [5] Rikihisa Y, Ito S. Intracellular localization of Rickettsia tsutsugamushi in polymorphonuclear leukocytes. J Exp Med 1979;150:703e8.
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