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FimH adhesin of Escherichia coli K1 type 1 fimbriae activates BV-2 microglia
BBRC Biochemical and Biophysical Research Communications 334 (2005) 917–923 www.elsevier.com/locate/ybbrc
FimH adhesin of Escherichia coli K1 type 1 fimbriae activates BV-2 microglia Jongseok Lee a,1, Sooan Shin a,1, Ching-Hao Teng a, Suk Jin Hong b, Kwang Sik Kim a,* a
Division of Pediatric Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA b Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA Received 22 June 2005 Available online 12 July 2005
Abstract The generation of intense inflammation in the subarachnoid space in response to meningitis-causing bacteria contributes to brain dysfunction and neuronal injury in bacterial meningitis. Microglia, the major immune effector cells in the central nervous system (CNS), become activated by bacterial components to produce proinflammatory immune mediators. In this study, we showed that FimH adhesin, a tip component of type 1 fimbriae of meningitis-causing Escherichia coli K1, activated the murine microglial cell line, BV-2, which resulted in the production of nitric oxide and the release of tumor necrosis factor-a. Mitogen-activated protein kinases, ERK and p-38, and nuclear factor-jB were involved in FimH adhesin-mediated microglial activation. These findings suggest that FimH adhesin contributes to the CNS inflammatory response by virtue of activating microglia in E. coli meningitis. 2005 Elsevier Inc. All rights reserved. Keywords: Monocytes/macrophages; Bacterial; Nitric oxide; Inflammation; Signal transduction; E. Coli; TNF-a; Mitogen-activated protein kinases; Nuclear factor-jB
Bacterial meningitis is a serious disease associated with high mortality and morbidity. Despite improved diagnostic techniques, the introduction of new antibiotics, adjunctive therapies, and progress in intensive care, this disease is recognized as one of the top ten infectious causes of death worldwide [1,2]. Incomplete understanding of the pathogenesis of this disease is a major contributing factor to this neurological sequelae and high mortality. Inflammation in the subarachnoid space in response to bacteria and bacterial substances contributes to the brain dysfunction and neuronal injury in bacterial meningitis, and immune effector cells which are resident in and/or infiltrated into the CNS are responsible for eliciting inflammatory responses [3]. Microglia are resident macrophage-like cells within the CNS, constituting approximately 10–15% of the to*
1
Corresponding author. Fax: +1 410 614 1491. E-mail address: [email protected] (K.S. Kim). These authors contributed equally to this work.
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.06.180
tal cell population in the parenchyma [4]. Activated microglia are known to participate in innate immune responses, including phagocytosis and regulation of T-cell responses through antigen presentation [5]. As such, it has been suggested that microglia have the role in the first line of defense against pathogens in the CNS prior to leukocyte infiltration [6]. Although some of microglial responses against pathogens may be beneficial, proinflammatory cytokines and cytotoxic factors such as tumor necrosis factor-a (TNF-a) and nitric oxide (NO), secreted by activated microglial cells, can potentiate neuronal injury associated with CNS infection including bacterial meningitis [7–12]. Activation of microglial cell has also been shown to be involved in the pathogenesis of neuronal injury coincident with neurodegenerative diseases and HIV infection [13–18]. FimH adhesin, the tip protein of E. coli type 1 fimbriae, contains a lectin-like domain recognizing a-D-mannoside, which is shown to mediate E. coli association
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with and invasion of bladder epithelial cell and mast cell/marcrophage [19]. FimH adhesin-mediated invasion of bladder epithelial cell depends upon the activation of phosphoinositide-3-kinase (PI-3-K) and the Rho-family GTPase member Cdc42 [20,21]. Internalization of E. coli by mast cell and macrophage is initiated as a consequence of interactions of FimH adhesin with CD48, a glycosylphosphatidylinositiol (GPI)-anchored receptor [22,23]. Recently, we have shown that the FimH adhesin is involved in E. coli K1 binding to and invasion of human brain microvascular endothelial cell, which was used as in vitro BBB model [24,25]. E. coli K1 is the leading cause of gram-negative bacteria in neonatal meningitis [3]. In addition to the role as an adhesion/ invasion factor, FimH adhesin is a possible effector of immune response. For example, FimH-mediated interaction of E. coli with epithelial cell stimulates the release of proinflammatory cytokines, synergistically with other bacterial products such as lipopolysaccharide (LPS) [26– 28]. FimH also activates mast cell to produce TNF-a through interaction with CD48 [29,30], resulting in neutrophil recruitment to the bacterial infection site. [31]. Gram-positive bacterial cell walls, LPS, and CpG oligodeoxynucleotides are known bacterial products for the microglial activation [7,8,32]. However, these components can be released from bacterial cell after lysis. In the case of LPS, the lipid A, the toxic portion of LPS, is embedded in the outer membrane of intact gram-negative bacteria. When the lipid–A portion is exposed by bacteria lysis, it exerts its biological effects of endotoxin. Such bacterial lysis occurs as a result of the attack by the complement, uptake and killing by phagocytes, or killing by certain types of antibiotics [33]. In the present study, we demonstrated for the first time that the FimH adhesin, which is on the surface of intact meningitis-causing E. coli K1, is one of the bacterial macromolecules that activate microglial cell, implicating the role of FimH adhesin in the pathogenesis of E. coli meningitis. Materials and methods Cell culture. The murine microglial cell line, BV2, which exhibits phenotypic and functional properties of reactive microglial cell [34], was generously provided by McKinney (Mayo Clinic, Jacksonville, FL). The cells were grown in DulbeccoÕs modified EagleÕs medium (Invitrogen, Calsbad, CA) supplemented with 10% fetal bovine serum (Biosource, Camarillo, CA), and penicillin–streptomycin (Invitrogen). Purification of FimC and FimCH proteins. To prepare functionally active FimH adhesin, we used the method of co-purification with FimC, a periplasmic chaperon of type 1 pilus subunit proteins [35,36]. The plasmid pFimH-FimC [36], which allows co-expression of E. coli K12 FimH and FimC under the control of trc promoter, was modified to express E. coli K1 FimH. Primers 5 0 -CCTCTAGAGGAGGGATG ATTGTAATGAAACGAGT-3 0 (forward) and 5 0 -ATTCCCTGCAG TCACCTGCCCT-3 0 (reverse) were used to amplify E. coli K1 fimH region by the polymerase chain reaction (PCR). The XbaI and PstI sites (underlined) were used to clone the amplified fragment into the XbaI–PstI digested pFimH-FimC plasmid, yielding pCHT4. To
eliminate the contamination of E. coli K-12 FimH during the purification procedure, NEC026 (BL21 fim) [37] strain was used for the expression host. Expression and purification of the FimH and FimC complex (hereafter called as FimCH) and FimC protein were carried out as described previously [38]. Purified proteins were dialyzed against PBS to apply to BV2 culture. The levels of endotoxin in purified proteins determined by using Limulus amebocyte lysate (LAL, Associates of Cape Cod, E. Falmouth, MA) test were <0.25 EU/mg proteins. NO quantification. After microglia (3 · 104 cells in 100 ll/well) were treated with FimCH in 96-well plates, nitrite in culture supernatants was measured to assess NO production by microglia. Fifty microliters of the culture supernatants were mixed with 50 ll of Griess reagent (Sigma, St. Louis, MO) and incubated at 25 C for 10 min. The OD was measured at 550 nm. NaNO2 was used as the standard to calculate nitrite concentration. To examine the involvement of MAPKs and NFjB in NO production, PD98059 (inhibitor of Erk1/2 MAP kinase, Calbiochem, San Diego, CA), SB203580 (inhibitor of p38 MAP kinase, Calbiochem), or N-acetyl cystein (inhibitor of NF-jB, Sigma) was co-treated with FimCH. Reverse transcription-polymerase chain reaction (RT-PCR). Microglia plated in six-well (5 · 105 cells) were treated with FimCH for 18 h. Total RNA was extracted from cells by using Trizol (Sigma) and further purified with RNeasy kit (Qiagen, Valencia, CA). Reverse transcription was carried out using the Superscript First strand synthesis kit (Invitrogen) with a random hexamer. PCR cycles consisted of denaturation at 94 C for 30 s, annealing at 57 C for 30 s, and extension at 72 C for 1 min. The PCR primers for iNOS, TNF-a, and GAPDH genes were as follows: iNOS, 5 0 -TCACTGGGACAGCA CAGAAT-3 0 (forward) and 5 0 -TGTGTCTGCAGATGTGCTGA-3 0 (reverse); TNF-a, 5 0 -ATGAGCACAGAAAGCATGATC-3 0 (forward) and 5 0 -TACAGGCTTGTCACTCGAATT-3 0 (reverse); GAPDH, 5 0 -GTGATGGGTGTGAACCACGAG-3 0 (forward) and 5 0 -CA GTGAGCTTCCCGTTCAGCT-3 0 (reverse). The optimal cycle number for each gene under nonsaturating conditions was determined empirically. PCR product was separated by electrophoresis on 2% ethidium bromide-containing agarose gel. DNA bands of 513 bp (iNOS), 276 bp (TNF-a), and 295 bp (GAPDH) were detected. TNF-a ELISA. Microglia grown in 96-well were treated with FimCH for 18 h. TNF-a released in culture supernatants was measured by using Endogen mouse TNF-a ELISA kit (Pierce, Rockford, IL) following the manufacturerÕs instructions. Preparation of cellular extract and Western blot analysis. Microglia grown in six-well plate were treated with FimCH and lysed with the RIPA lysis buffer (Upstate, Lake Placid, NY), containing protease inhibitors cocktail (Sigma). Twenty micrograms of protein for each sample were separated by 12% or 14% SDS–PAGE and transferred to nitrocellulose membrane (Trans-Blot Transfer Medium, Bio-Rad, Hercules, CA). After blocking for 1 h in TBS-T (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) containing 5% BSA (Sigma), membranes were probed with antibodies diluted in TBS-T/5% BSA. Anti-iNOS (rabbit, Transduction Laboratories, San Diego, CA), anti-phospho-ERK (mouse), anti-phospho-p38 (rabbit), anti-phosphoJNK (rabbit), anti-total-ERK (rabbit), anti-total-p38 (rabbit), antitotal-JNK (rabbit, Cell Signaling, Beverly, MA) antibodies were used at a dilution of 1:1000 and 1:2000, respectively. Mouse anti-GAPDH antibody (HyTest, Turku, Finland) was used at a dilution of 1:3000. Membranes were incubated with primary antibodies overnight at 4 C and washed three times with TBS-T. HRP-conjugated secondary antibodies (Cell Signaling) were diluted to 1:5000 in TBS-T and incubated with membranes for 1 h. Following washing three times with TBS-T, bands were visualized by using ECL Western blotting detection reagents (Amersham Biosciences, Piscataway, NJ). Electrophoretic mobility shift assay. Nuclear extract was prepared according to the previous method [39]. The NF-jB-specific oligonucleotide 5 0 -AGCTCAGAGGGGGACTTTCCGAGAG-3 0 from the murine Igj-light chain gene enhancer was synthesized with 3 0 digoxi-
J. Lee et al. / Biochemical and Biophysical Research Communications 334 (2005) 917–923 genin (DIG), and its complimentary oligonucleotide without labeling were synthesized from Integrated DNA Technologies (Coralville, IA). Nuclear extracts (5 lg) were incubated with 20 fmol DIG-labeled DNA probe in a binding buffer (final volume, 20 ll) containing 20 mM Tris–HCl, pH 7.9, 50 mM KCl, 5 mM MgCl2, 0.5 mM EDTA, 1 mM DTT, 5% glycerol, 0.25 lg BSA, and 2 lg poly(dI–dC) (Amersham Biosciences) for 30 min at room temperature. DNA binding mixture was loaded onto Novex 6% DNA retardation gel (Invitrogen), and the DNA–protein complexes were resolved by electrophoresis. The gel was blotted into a Hybond-N+ membrane, and the membrane was crosslinked with UV-light (120 mJ in Stratalinker, Stratagene, La Jolla, CA). DIG-labeled DNA bands were visualized with AP-conjugated anti-DIG antibody and CSPD chemiluminescent reagent (Roche Applied Science, Indianapolis, IN) according to the manufacturerÕs instructions.
Results and discussion FimH adhesin induces production of nitric oxide in microglia Purified FimCH and FimC proteins are shown in Fig. 1. Sequence-based molecular weights of mature FimH (E. coli K1) and FimC (E. coli K12) proteins are 29,342 and 22,730 Da, respectively. FimH was confirmed by SDS–PAGE and Western blot analysis with anti-FimH antibody (data not shown). It is known that the NO production is a prominent marker of microglia activation [40,41]. To determine whether FimH adhesin is an effector on the microglial activation, nitrite formed from nitric oxide (NO) was measured in microglial culture supernatants in response to the treatment with purified proteins. The concentrations of nitrite from microglial culture treated FimCH for 24 h increased in a dose-dependent manner (Fig. 2A). Four hundred nanomoles of FimCH induced a similar amount of
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Fig. 1. SDS–PAGE of the purified FimCH and FimC. Proteins were purified as described in Materials and methods and subjected to 14% SDS–PAGE. A gel was stained with Coomassie blue. FimCH complex was separated to each FimH (29 kDa) and FimC (22 kDa) protein in SDS–PAGE. Lane 1, molecular mass standard; lane 2, FimCH; and lane 3, FimC.
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nitrite from microglial culture compared to LPS control (100 ng/ml). In contrast, like untreated control, FimC treatment did not increase the NO production, indicating that the FimH adhesin is the effector molecule of FimCH complex in the microglial activation. NO production from microglia treated with 200 nM of FimCH was continuously increased during the 48 h incubation period, which was similar to the result of LPS treatment (Fig. 2B). Inducible nitric oxide synthase (iNOS) is known to be responsible for the production of NO in LPS treated microglia [42]. To examine whether FimH adhesin induces the expression of iNOS, the relative quantity of mRNA encoding iNOS was determined by RTPCR. The message level of iNOS was induced within 6 h after the treatment of 200 nM FimCH (data not shown) and it was maintained upto 18 h (Fig. 2C). The synthesis of iNOS protein, detected by probing with anti-iNOS antibody in Western blot analysis, was consistent with RT-PCR result (Fig. 2D). As control, LPS induced the expression of iNOS gene and the synthesis of iNOS protein in microglial cells (Figs. 2C and D). These results show that FimH adhesin induced the expression of iNOS, which consequently increased the NO production in microglia. FimH adhesin induced the expression of TNF-a Microglia are the principle source of proinflammatory cytokines in infectious and autoimmune diseases of the CNS [43,44]. TNF-a is one of the key cytokines in activated microglia cell, as its level is elevated in the CSF of meningitis [45] and other neurodegenerative diseases including multiple sclerosis [46]. To investigate the effect of FimH adhesin on the expression of TNF-a in microglia, the level of mRNA encoding TNF-a was determined by RT-PCR. After 18 h incubation, TNF-a expression was not detected in cells treated with FimC or without treatment. In contrast, FimCH induced the expression of TNF-a similar to LPS (Fig. 3A). The amounts of secreted TNF-a protein were measured by using ELISA from the microglial culture medium. FimCH treatment increased TNF-a secretion more than 9-fold compared to FimC (Fig. 3B). This indicates that FimH adhesin induces TNF-a gene expression and TNF-a protein secretion of microglia. These findings are consistent with those of murine mast cell, where FimH adhesin is shown to induce TNF-a production [29]. FimH adhesin activates mitogen-activated protein kinases and NF-jB MAPKs are the common intracellular signaling molecules involved in the microglial activation responsible for NO production [40,41]. We examined whether FimH
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Fig. 2. FimH adhesin induces the production of nitric oxide in microglia. Microglia were treated with the indicated amounts of 200 nM of FimCH and FimC (negative control), or 100 ng/ml of LPS (positive control) for 24 h (A) or treated with FimCH (200 nM) or LPS (100 ng/ml) for indicated time periods (B). The amount of nitrite converted from NO in the culture supernatant was determined as described in Materials and methods. Values are means ± SEM of three experiments. NT means no treatment. (C) After 18 h treatment of FimC (200 nM), FimCH (200 nM), and LPS (100 ng/ ml), total RNA was isolated from microglia and the mRNA encoding for iNOS and GAPDH (internal control) was analyzed by RT-PCR. (D) Total proteins were extracted from microglia treated as the same condition with RT-PCR experiment, and Western blot experiment was performed for analysis of iNOS and GAPDH (internal control) proteins probed by anti-iNOS and anti-GAPDH antibodies, respectively. Lane 1, no treatment; lane 2, FimC; lane 3, FimCH; and lane 4, LPS.
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Fig. 3. FimH adhesin induces the production of TNF-a in microglia. Cells were treated with or without 200 nM FimC and FimCH, or 100 ng/ml LPS. (A) After 18 h incubation, total RNA was isolated and RT-PCR was performed to analyze the amount of mRNA encoding for TNF-a and GAPDH (internal control). Lane 1, no treatment; lane 2, FimC; lane 3, FimCH; and lane 4, LPS. (B) As the same condition with RT-PCR experiment, the amount of TNF-a in the cell culture supernatant was determined by ELISA. Values (means ± SEM of three experiments) are expressed in pg/ml. NT means no treatment.
adhesin activates MAPKs in microglia by examining phosphorylation of MAPKs. Treatment of FimCH (200 nM) induced phosphorylation of p44/42 MAP kinase (ERK) and p38 MAP kinase (p38) within 15 and 30 min, respectively (Fig. 4A). Jun-N-terminal kinase (JNK) activation in response to FimCH, however, was not observed in 30 min incubation. LPS activated all three MAPKs, indicating that the FimH adhesin may lead to different signaling pathway from LPS. To con-
firm whether activities of ERK and p38 were involved in microglial activation, PD98059 (ERK inhibitor) and SB203580 (p38 inhibitor) were co-treated with FimCH to measure NO production. Each inhibitor exhibited partial reduction of nitrite formation in a dose-dependent manner (Fig. 4B and C). These results indicate that the activities of ERK and p38 are in part involved in the NO production by microglia in response to FimH adhesin.
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Fig. 4. FimH adhesin activates of MAPKs in microglia. (A) Total proteins were extracted from microglia, which were treated with or without 200 nM FimC and FimCH, or 100 ng/ml LPS, after 15 min (ERK) and 30 min (p38 and JNK), and Western blot experiment was performed for analysis of activated ERK (P-ERK), p38 (P-p38), and JNK (P-JNK), which were detected by using antibodies specified for the phosphorylated form of each MAPK. To show that equivalent amount of protein was loaded in each lane, unphosphorylated MAPKs were detected by using antibodies for ERK, p38, and JNK. Lane 1, no treatment; lane 2, FimC; lane 3, FimCH; and lane 4, LPS. (B and C) Microglia were treated with or without 200 nM FimCH, or 100 ng/ml LPS for 24 h in the presence or absence of the indicated concentration of PD98059 (ERK inhibitor) or SB203580 (p38 inhibitor). The amount of nitrite converted from NO in the culture supernatant was determined as described in Materials and methods. Values are means ± SEM of three experiments. NT means no treatment.
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Fig. 5. FimH adhesin activates NF-jB in microglia. (A) Nuclear extracts were prepared from microglia treated with or without 200 nM FimC and FimCH, or 100 ng/ml LPS for 45 min. EMSA was performed as described in Materials and methods. The NF-jB-specific oligonucleotide–protein complexes and unshifted probes are marked by filled and opened arrowheads, respectively. Lane 1, no treatment; lane 2, FimC; lane 3, FimCH; and lane 4, LPS. (B) Microglia were treated without or with 200 nM FimCH, or 100 ng/ml LPS for 24 h in the presence or absence of the indicated concentration of N-acetyl cystein (NAC, NF-jB inhibitor). The amount of nitrite converted from NO in the culture supernatant was determined as described in Materials and methods. Values are means ± SEM of three experiments. NT means no treatment.
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It is known that the expressions of iNOS and TNF-a are dependent on the activity of NF-jB, a transcription factor responding to various extracellular stimulations in immune cells, including activated microglia [40,41]. The electrophoresis mobility shift assay (EMSA) showed that NF-jB was activated within 45 min with FimCH treatment, as well as LPS control (Fig. 5A). A specific inhibitor of NF-jB, N-acetyl cystein (NAC), decreased NO production in the FimCH (200 lM)-treated microglia in a dose-dependent manner (Fig. 5B). These results indicate that FimH adhesin can induce a signal transduction to NF-jB, whose activity is involved in the production of NO in microglia. In summary, FimH adhesin activated murine micorglial cell line BV-2 to produce NO and TNF-a, and the activities of ERK, p38, and NF-jB were involved in NO production in FimH adhesin-activated microglia. These results illustrate that FimH adhesin of type 1 fimbriae on the surface of E. coli can contribute to the CNS inflammatory response in part via microglial activation in E. coli meningitis.
Acknowledgments This work was supported by National Institutes of Health Grants NS-26310 and AI47225, and in part by the Post-doctoral Fellowship Program of Korea Science and Engineering Foundation (KOSEF) for J.L. and we thank Dr. Michael McKinney (Mayo Clinic, Jacksonville, FL) for providing the BV-2 cell line and Dr. David Gally (University of Edinbugh, UK) for providing NEC026 strain.
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FimH adhesin of Escherichia coli K1 type 1 fimbriae activates BV-2 microglia Jongseok Lee a,1, Sooan Shin a,1, Ching-Hao Teng a, Suk Jin Hong b, Kwang Sik Kim a,* a
Division of Pediatric Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA b Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA Received 22 June 2005 Available online 12 July 2005
Abstract The generation of intense inflammation in the subarachnoid space in response to meningitis-causing bacteria contributes to brain dysfunction and neuronal injury in bacterial meningitis. Microglia, the major immune effector cells in the central nervous system (CNS), become activated by bacterial components to produce proinflammatory immune mediators. In this study, we showed that FimH adhesin, a tip component of type 1 fimbriae of meningitis-causing Escherichia coli K1, activated the murine microglial cell line, BV-2, which resulted in the production of nitric oxide and the release of tumor necrosis factor-a. Mitogen-activated protein kinases, ERK and p-38, and nuclear factor-jB were involved in FimH adhesin-mediated microglial activation. These findings suggest that FimH adhesin contributes to the CNS inflammatory response by virtue of activating microglia in E. coli meningitis. 2005 Elsevier Inc. All rights reserved. Keywords: Monocytes/macrophages; Bacterial; Nitric oxide; Inflammation; Signal transduction; E. Coli; TNF-a; Mitogen-activated protein kinases; Nuclear factor-jB
Bacterial meningitis is a serious disease associated with high mortality and morbidity. Despite improved diagnostic techniques, the introduction of new antibiotics, adjunctive therapies, and progress in intensive care, this disease is recognized as one of the top ten infectious causes of death worldwide [1,2]. Incomplete understanding of the pathogenesis of this disease is a major contributing factor to this neurological sequelae and high mortality. Inflammation in the subarachnoid space in response to bacteria and bacterial substances contributes to the brain dysfunction and neuronal injury in bacterial meningitis, and immune effector cells which are resident in and/or infiltrated into the CNS are responsible for eliciting inflammatory responses [3]. Microglia are resident macrophage-like cells within the CNS, constituting approximately 10–15% of the to*
1
Corresponding author. Fax: +1 410 614 1491. E-mail address: [email protected] (K.S. Kim). These authors contributed equally to this work.
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.06.180
tal cell population in the parenchyma [4]. Activated microglia are known to participate in innate immune responses, including phagocytosis and regulation of T-cell responses through antigen presentation [5]. As such, it has been suggested that microglia have the role in the first line of defense against pathogens in the CNS prior to leukocyte infiltration [6]. Although some of microglial responses against pathogens may be beneficial, proinflammatory cytokines and cytotoxic factors such as tumor necrosis factor-a (TNF-a) and nitric oxide (NO), secreted by activated microglial cells, can potentiate neuronal injury associated with CNS infection including bacterial meningitis [7–12]. Activation of microglial cell has also been shown to be involved in the pathogenesis of neuronal injury coincident with neurodegenerative diseases and HIV infection [13–18]. FimH adhesin, the tip protein of E. coli type 1 fimbriae, contains a lectin-like domain recognizing a-D-mannoside, which is shown to mediate E. coli association
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with and invasion of bladder epithelial cell and mast cell/marcrophage [19]. FimH adhesin-mediated invasion of bladder epithelial cell depends upon the activation of phosphoinositide-3-kinase (PI-3-K) and the Rho-family GTPase member Cdc42 [20,21]. Internalization of E. coli by mast cell and macrophage is initiated as a consequence of interactions of FimH adhesin with CD48, a glycosylphosphatidylinositiol (GPI)-anchored receptor [22,23]. Recently, we have shown that the FimH adhesin is involved in E. coli K1 binding to and invasion of human brain microvascular endothelial cell, which was used as in vitro BBB model [24,25]. E. coli K1 is the leading cause of gram-negative bacteria in neonatal meningitis [3]. In addition to the role as an adhesion/ invasion factor, FimH adhesin is a possible effector of immune response. For example, FimH-mediated interaction of E. coli with epithelial cell stimulates the release of proinflammatory cytokines, synergistically with other bacterial products such as lipopolysaccharide (LPS) [26– 28]. FimH also activates mast cell to produce TNF-a through interaction with CD48 [29,30], resulting in neutrophil recruitment to the bacterial infection site. [31]. Gram-positive bacterial cell walls, LPS, and CpG oligodeoxynucleotides are known bacterial products for the microglial activation [7,8,32]. However, these components can be released from bacterial cell after lysis. In the case of LPS, the lipid A, the toxic portion of LPS, is embedded in the outer membrane of intact gram-negative bacteria. When the lipid–A portion is exposed by bacteria lysis, it exerts its biological effects of endotoxin. Such bacterial lysis occurs as a result of the attack by the complement, uptake and killing by phagocytes, or killing by certain types of antibiotics [33]. In the present study, we demonstrated for the first time that the FimH adhesin, which is on the surface of intact meningitis-causing E. coli K1, is one of the bacterial macromolecules that activate microglial cell, implicating the role of FimH adhesin in the pathogenesis of E. coli meningitis. Materials and methods Cell culture. The murine microglial cell line, BV2, which exhibits phenotypic and functional properties of reactive microglial cell [34], was generously provided by McKinney (Mayo Clinic, Jacksonville, FL). The cells were grown in DulbeccoÕs modified EagleÕs medium (Invitrogen, Calsbad, CA) supplemented with 10% fetal bovine serum (Biosource, Camarillo, CA), and penicillin–streptomycin (Invitrogen). Purification of FimC and FimCH proteins. To prepare functionally active FimH adhesin, we used the method of co-purification with FimC, a periplasmic chaperon of type 1 pilus subunit proteins [35,36]. The plasmid pFimH-FimC [36], which allows co-expression of E. coli K12 FimH and FimC under the control of trc promoter, was modified to express E. coli K1 FimH. Primers 5 0 -CCTCTAGAGGAGGGATG ATTGTAATGAAACGAGT-3 0 (forward) and 5 0 -ATTCCCTGCAG TCACCTGCCCT-3 0 (reverse) were used to amplify E. coli K1 fimH region by the polymerase chain reaction (PCR). The XbaI and PstI sites (underlined) were used to clone the amplified fragment into the XbaI–PstI digested pFimH-FimC plasmid, yielding pCHT4. To
eliminate the contamination of E. coli K-12 FimH during the purification procedure, NEC026 (BL21 fim) [37] strain was used for the expression host. Expression and purification of the FimH and FimC complex (hereafter called as FimCH) and FimC protein were carried out as described previously [38]. Purified proteins were dialyzed against PBS to apply to BV2 culture. The levels of endotoxin in purified proteins determined by using Limulus amebocyte lysate (LAL, Associates of Cape Cod, E. Falmouth, MA) test were <0.25 EU/mg proteins. NO quantification. After microglia (3 · 104 cells in 100 ll/well) were treated with FimCH in 96-well plates, nitrite in culture supernatants was measured to assess NO production by microglia. Fifty microliters of the culture supernatants were mixed with 50 ll of Griess reagent (Sigma, St. Louis, MO) and incubated at 25 C for 10 min. The OD was measured at 550 nm. NaNO2 was used as the standard to calculate nitrite concentration. To examine the involvement of MAPKs and NFjB in NO production, PD98059 (inhibitor of Erk1/2 MAP kinase, Calbiochem, San Diego, CA), SB203580 (inhibitor of p38 MAP kinase, Calbiochem), or N-acetyl cystein (inhibitor of NF-jB, Sigma) was co-treated with FimCH. Reverse transcription-polymerase chain reaction (RT-PCR). Microglia plated in six-well (5 · 105 cells) were treated with FimCH for 18 h. Total RNA was extracted from cells by using Trizol (Sigma) and further purified with RNeasy kit (Qiagen, Valencia, CA). Reverse transcription was carried out using the Superscript First strand synthesis kit (Invitrogen) with a random hexamer. PCR cycles consisted of denaturation at 94 C for 30 s, annealing at 57 C for 30 s, and extension at 72 C for 1 min. The PCR primers for iNOS, TNF-a, and GAPDH genes were as follows: iNOS, 5 0 -TCACTGGGACAGCA CAGAAT-3 0 (forward) and 5 0 -TGTGTCTGCAGATGTGCTGA-3 0 (reverse); TNF-a, 5 0 -ATGAGCACAGAAAGCATGATC-3 0 (forward) and 5 0 -TACAGGCTTGTCACTCGAATT-3 0 (reverse); GAPDH, 5 0 -GTGATGGGTGTGAACCACGAG-3 0 (forward) and 5 0 -CA GTGAGCTTCCCGTTCAGCT-3 0 (reverse). The optimal cycle number for each gene under nonsaturating conditions was determined empirically. PCR product was separated by electrophoresis on 2% ethidium bromide-containing agarose gel. DNA bands of 513 bp (iNOS), 276 bp (TNF-a), and 295 bp (GAPDH) were detected. TNF-a ELISA. Microglia grown in 96-well were treated with FimCH for 18 h. TNF-a released in culture supernatants was measured by using Endogen mouse TNF-a ELISA kit (Pierce, Rockford, IL) following the manufacturerÕs instructions. Preparation of cellular extract and Western blot analysis. Microglia grown in six-well plate were treated with FimCH and lysed with the RIPA lysis buffer (Upstate, Lake Placid, NY), containing protease inhibitors cocktail (Sigma). Twenty micrograms of protein for each sample were separated by 12% or 14% SDS–PAGE and transferred to nitrocellulose membrane (Trans-Blot Transfer Medium, Bio-Rad, Hercules, CA). After blocking for 1 h in TBS-T (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) containing 5% BSA (Sigma), membranes were probed with antibodies diluted in TBS-T/5% BSA. Anti-iNOS (rabbit, Transduction Laboratories, San Diego, CA), anti-phospho-ERK (mouse), anti-phospho-p38 (rabbit), anti-phosphoJNK (rabbit), anti-total-ERK (rabbit), anti-total-p38 (rabbit), antitotal-JNK (rabbit, Cell Signaling, Beverly, MA) antibodies were used at a dilution of 1:1000 and 1:2000, respectively. Mouse anti-GAPDH antibody (HyTest, Turku, Finland) was used at a dilution of 1:3000. Membranes were incubated with primary antibodies overnight at 4 C and washed three times with TBS-T. HRP-conjugated secondary antibodies (Cell Signaling) were diluted to 1:5000 in TBS-T and incubated with membranes for 1 h. Following washing three times with TBS-T, bands were visualized by using ECL Western blotting detection reagents (Amersham Biosciences, Piscataway, NJ). Electrophoretic mobility shift assay. Nuclear extract was prepared according to the previous method [39]. The NF-jB-specific oligonucleotide 5 0 -AGCTCAGAGGGGGACTTTCCGAGAG-3 0 from the murine Igj-light chain gene enhancer was synthesized with 3 0 digoxi-
J. Lee et al. / Biochemical and Biophysical Research Communications 334 (2005) 917–923 genin (DIG), and its complimentary oligonucleotide without labeling were synthesized from Integrated DNA Technologies (Coralville, IA). Nuclear extracts (5 lg) were incubated with 20 fmol DIG-labeled DNA probe in a binding buffer (final volume, 20 ll) containing 20 mM Tris–HCl, pH 7.9, 50 mM KCl, 5 mM MgCl2, 0.5 mM EDTA, 1 mM DTT, 5% glycerol, 0.25 lg BSA, and 2 lg poly(dI–dC) (Amersham Biosciences) for 30 min at room temperature. DNA binding mixture was loaded onto Novex 6% DNA retardation gel (Invitrogen), and the DNA–protein complexes were resolved by electrophoresis. The gel was blotted into a Hybond-N+ membrane, and the membrane was crosslinked with UV-light (120 mJ in Stratalinker, Stratagene, La Jolla, CA). DIG-labeled DNA bands were visualized with AP-conjugated anti-DIG antibody and CSPD chemiluminescent reagent (Roche Applied Science, Indianapolis, IN) according to the manufacturerÕs instructions.
Results and discussion FimH adhesin induces production of nitric oxide in microglia Purified FimCH and FimC proteins are shown in Fig. 1. Sequence-based molecular weights of mature FimH (E. coli K1) and FimC (E. coli K12) proteins are 29,342 and 22,730 Da, respectively. FimH was confirmed by SDS–PAGE and Western blot analysis with anti-FimH antibody (data not shown). It is known that the NO production is a prominent marker of microglia activation [40,41]. To determine whether FimH adhesin is an effector on the microglial activation, nitrite formed from nitric oxide (NO) was measured in microglial culture supernatants in response to the treatment with purified proteins. The concentrations of nitrite from microglial culture treated FimCH for 24 h increased in a dose-dependent manner (Fig. 2A). Four hundred nanomoles of FimCH induced a similar amount of
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Fig. 1. SDS–PAGE of the purified FimCH and FimC. Proteins were purified as described in Materials and methods and subjected to 14% SDS–PAGE. A gel was stained with Coomassie blue. FimCH complex was separated to each FimH (29 kDa) and FimC (22 kDa) protein in SDS–PAGE. Lane 1, molecular mass standard; lane 2, FimCH; and lane 3, FimC.
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nitrite from microglial culture compared to LPS control (100 ng/ml). In contrast, like untreated control, FimC treatment did not increase the NO production, indicating that the FimH adhesin is the effector molecule of FimCH complex in the microglial activation. NO production from microglia treated with 200 nM of FimCH was continuously increased during the 48 h incubation period, which was similar to the result of LPS treatment (Fig. 2B). Inducible nitric oxide synthase (iNOS) is known to be responsible for the production of NO in LPS treated microglia [42]. To examine whether FimH adhesin induces the expression of iNOS, the relative quantity of mRNA encoding iNOS was determined by RTPCR. The message level of iNOS was induced within 6 h after the treatment of 200 nM FimCH (data not shown) and it was maintained upto 18 h (Fig. 2C). The synthesis of iNOS protein, detected by probing with anti-iNOS antibody in Western blot analysis, was consistent with RT-PCR result (Fig. 2D). As control, LPS induced the expression of iNOS gene and the synthesis of iNOS protein in microglial cells (Figs. 2C and D). These results show that FimH adhesin induced the expression of iNOS, which consequently increased the NO production in microglia. FimH adhesin induced the expression of TNF-a Microglia are the principle source of proinflammatory cytokines in infectious and autoimmune diseases of the CNS [43,44]. TNF-a is one of the key cytokines in activated microglia cell, as its level is elevated in the CSF of meningitis [45] and other neurodegenerative diseases including multiple sclerosis [46]. To investigate the effect of FimH adhesin on the expression of TNF-a in microglia, the level of mRNA encoding TNF-a was determined by RT-PCR. After 18 h incubation, TNF-a expression was not detected in cells treated with FimC or without treatment. In contrast, FimCH induced the expression of TNF-a similar to LPS (Fig. 3A). The amounts of secreted TNF-a protein were measured by using ELISA from the microglial culture medium. FimCH treatment increased TNF-a secretion more than 9-fold compared to FimC (Fig. 3B). This indicates that FimH adhesin induces TNF-a gene expression and TNF-a protein secretion of microglia. These findings are consistent with those of murine mast cell, where FimH adhesin is shown to induce TNF-a production [29]. FimH adhesin activates mitogen-activated protein kinases and NF-jB MAPKs are the common intracellular signaling molecules involved in the microglial activation responsible for NO production [40,41]. We examined whether FimH
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Fig. 2. FimH adhesin induces the production of nitric oxide in microglia. Microglia were treated with the indicated amounts of 200 nM of FimCH and FimC (negative control), or 100 ng/ml of LPS (positive control) for 24 h (A) or treated with FimCH (200 nM) or LPS (100 ng/ml) for indicated time periods (B). The amount of nitrite converted from NO in the culture supernatant was determined as described in Materials and methods. Values are means ± SEM of three experiments. NT means no treatment. (C) After 18 h treatment of FimC (200 nM), FimCH (200 nM), and LPS (100 ng/ ml), total RNA was isolated from microglia and the mRNA encoding for iNOS and GAPDH (internal control) was analyzed by RT-PCR. (D) Total proteins were extracted from microglia treated as the same condition with RT-PCR experiment, and Western blot experiment was performed for analysis of iNOS and GAPDH (internal control) proteins probed by anti-iNOS and anti-GAPDH antibodies, respectively. Lane 1, no treatment; lane 2, FimC; lane 3, FimCH; and lane 4, LPS.
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Fig. 3. FimH adhesin induces the production of TNF-a in microglia. Cells were treated with or without 200 nM FimC and FimCH, or 100 ng/ml LPS. (A) After 18 h incubation, total RNA was isolated and RT-PCR was performed to analyze the amount of mRNA encoding for TNF-a and GAPDH (internal control). Lane 1, no treatment; lane 2, FimC; lane 3, FimCH; and lane 4, LPS. (B) As the same condition with RT-PCR experiment, the amount of TNF-a in the cell culture supernatant was determined by ELISA. Values (means ± SEM of three experiments) are expressed in pg/ml. NT means no treatment.
adhesin activates MAPKs in microglia by examining phosphorylation of MAPKs. Treatment of FimCH (200 nM) induced phosphorylation of p44/42 MAP kinase (ERK) and p38 MAP kinase (p38) within 15 and 30 min, respectively (Fig. 4A). Jun-N-terminal kinase (JNK) activation in response to FimCH, however, was not observed in 30 min incubation. LPS activated all three MAPKs, indicating that the FimH adhesin may lead to different signaling pathway from LPS. To con-
firm whether activities of ERK and p38 were involved in microglial activation, PD98059 (ERK inhibitor) and SB203580 (p38 inhibitor) were co-treated with FimCH to measure NO production. Each inhibitor exhibited partial reduction of nitrite formation in a dose-dependent manner (Fig. 4B and C). These results indicate that the activities of ERK and p38 are in part involved in the NO production by microglia in response to FimH adhesin.
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Fig. 4. FimH adhesin activates of MAPKs in microglia. (A) Total proteins were extracted from microglia, which were treated with or without 200 nM FimC and FimCH, or 100 ng/ml LPS, after 15 min (ERK) and 30 min (p38 and JNK), and Western blot experiment was performed for analysis of activated ERK (P-ERK), p38 (P-p38), and JNK (P-JNK), which were detected by using antibodies specified for the phosphorylated form of each MAPK. To show that equivalent amount of protein was loaded in each lane, unphosphorylated MAPKs were detected by using antibodies for ERK, p38, and JNK. Lane 1, no treatment; lane 2, FimC; lane 3, FimCH; and lane 4, LPS. (B and C) Microglia were treated with or without 200 nM FimCH, or 100 ng/ml LPS for 24 h in the presence or absence of the indicated concentration of PD98059 (ERK inhibitor) or SB203580 (p38 inhibitor). The amount of nitrite converted from NO in the culture supernatant was determined as described in Materials and methods. Values are means ± SEM of three experiments. NT means no treatment.
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Fig. 5. FimH adhesin activates NF-jB in microglia. (A) Nuclear extracts were prepared from microglia treated with or without 200 nM FimC and FimCH, or 100 ng/ml LPS for 45 min. EMSA was performed as described in Materials and methods. The NF-jB-specific oligonucleotide–protein complexes and unshifted probes are marked by filled and opened arrowheads, respectively. Lane 1, no treatment; lane 2, FimC; lane 3, FimCH; and lane 4, LPS. (B) Microglia were treated without or with 200 nM FimCH, or 100 ng/ml LPS for 24 h in the presence or absence of the indicated concentration of N-acetyl cystein (NAC, NF-jB inhibitor). The amount of nitrite converted from NO in the culture supernatant was determined as described in Materials and methods. Values are means ± SEM of three experiments. NT means no treatment.
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It is known that the expressions of iNOS and TNF-a are dependent on the activity of NF-jB, a transcription factor responding to various extracellular stimulations in immune cells, including activated microglia [40,41]. The electrophoresis mobility shift assay (EMSA) showed that NF-jB was activated within 45 min with FimCH treatment, as well as LPS control (Fig. 5A). A specific inhibitor of NF-jB, N-acetyl cystein (NAC), decreased NO production in the FimCH (200 lM)-treated microglia in a dose-dependent manner (Fig. 5B). These results indicate that FimH adhesin can induce a signal transduction to NF-jB, whose activity is involved in the production of NO in microglia. In summary, FimH adhesin activated murine micorglial cell line BV-2 to produce NO and TNF-a, and the activities of ERK, p38, and NF-jB were involved in NO production in FimH adhesin-activated microglia. These results illustrate that FimH adhesin of type 1 fimbriae on the surface of E. coli can contribute to the CNS inflammatory response in part via microglial activation in E. coli meningitis.
Acknowledgments This work was supported by National Institutes of Health Grants NS-26310 and AI47225, and in part by the Post-doctoral Fellowship Program of Korea Science and Engineering Foundation (KOSEF) for J.L. and we thank Dr. Michael McKinney (Mayo Clinic, Jacksonville, FL) for providing the BV-2 cell line and Dr. David Gally (University of Edinbugh, UK) for providing NEC026 strain.
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