Microbial Pathogenesis 66 (2014) 24e28

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A novel Pseudomonas aeruginosa-derived effector cooperates with flagella to mediate the upregulation of interleukin 8 in human epithelial cells Yong-Jae Kim a, Se-Hwan Paek a, Shouguang Jin b, Beom Seok Park c, **, Un-Hwan Ha a, * a b c

Department of Biotechnology and Bioinformatics, Korea University, Sejong 339-700, Republic of Korea Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, USA Department of Biomedical Laboratory Science, Eulji University, Seongnam 461-713, Republic of Korea

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 September 2013 Received in revised form 5 December 2013 Accepted 9 December 2013 Available online 19 December 2013

Infection with Pseudomonas aeruginosa results in a massive accumulation of neutrophils in response to prolonged and sustained expression of inflammatory mediators. The major chemokine associated with this excessive neutrophil recruitment is IL-8, the accumulation of which is a hallmark of cornea and cystic fibrosis airway inflammation. To date, several P. aeruginosa-associated and extracellular factors required for the stimulation of IL-8 expression have been identified. Here, we report a novel effector molecule, nucleoside diphosphate kinase (Ndk), which increases the expression of IL-8 by translocating into host cells. The induction appears to be dependent on both the kinase activity of Ndk and an additional bacterial factor, flagellin, via an NF-kB signaling pathway. This study demonstrates the role of a novel effector, Ndk, which is capable of inducing prominent inflammatory chemokine IL-8 expression with the aid of flagellin during P. aeruginosa infection. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Epithelial cells IL-8 Pseudomonas aeruginosa

1. Introduction Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes acute and chronic infections, particularly in immunocompromised patients [1]. Infection with P. aeruginosa is associated with the development and progression of corneal damage as well as pulmonary decline in patients with cystic fibrosis, which is characterized by a massive accumulation of neutrophils [2,3]. P. aeruginosa infection potently induces proinflammatory mediators, such as interleukin-1 (IL-1), IL-6, IL-8, and IL-10 [4e8]. The accumulation of IL-8, a major chemokine associated with excessive neutrophil recruitment from the vasculature into the lumen of the airways, in turn contributes to tissue destruction, particularly at the early stage of P. aeruginosa colonization [9e11]. Since P. aeruginosa induces prolonged expression of IL-8 and this sustained IL-8 expression contributes to the recruitment of neutrophils [7], there is a growing interest in developing therapies against IL-8, with the aim of reducing the excessive inflammatory

response in patients. However, key questions remain unanswered about the P. aeruginosa factors, epithelial cell receptors, and signaling pathways that govern the initiation of this response. To date, it has been reported that IL-8 expression is induced by a number of P. aeruginosa-associated and extracellular products, including pilin, flagellin, pyocyanin, nitrite reductase, phospholipase C, autoinducers, and ExoU [12e17]. Nucleoside diphosphate kinase (Ndk; PA3807) has been reported to be cytotoxic when incubated with eukaryotic cells [18,19]. However, it has not been determined whether the Ndk has an effect on the expression of inflammatory cytokines. Here, we show that translocated Ndk into host cells is involved in the expression of IL-8 in epithelial cells. The induction appears to be dependent on the presence of flagellin, a well-known pathogen-associated molecular pattern (PAMP), as well as the kinase activity of Ndk via the NF-kB signaling pathway. The present study brings new insights into the roles of Ndk and flagellin in the induction of IL-8 expression during hostePseudomonas interaction. 2. Materials and methods

* Corresponding author. Tel.: þ82 44 860 1418; fax: þ82 44 860 1411. ** Corresponding author. Tel.: þ82 31 740 7200; fax: þ82 31 740 7354. E-mail addresses: [email protected] (B.S. Park), [email protected] (U.-H. Ha). 0882-4010/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.micpath.2013.12.001

2.1. Reagents Lipopolysaccharide (LPS), ultrapure flagellin, MG132 and caffeic acid phenethylester (CAPE) were purchased from SigmaeAldrich

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(St. Louis, MO, USA), InvivoGen (San Diego, CA, USA), A.G. Scientific (San Diego, CA, USA) and Calbiochem (Darmstadt, Germany), respectively. 2.2. Bacterial strains and culture conditions P. aeruginosa was grown in Luria broth (LB) or on LB agar plates at 37  C. Mutant strain PAKD7 is a deletion mutant, with deletion of all three known type III secreted effectors (ExoSTY), the T3SS repressor (PopN), the T2SS secretin (XcpQ), and two quorum sensing genes (LasR & LasI). PAKD8 is a mutant strain lacking Ndk, produced via homologous recombination from PAKD7, and the deletion of ndk was confirmed by PCR analysis (data not shown). PAK isogenic fliC-deficient mutant (fliC mt) was described in a previous report [20]. To obtain the pellet, bacterial cells were harvested at 10,000  g for 20 min at 4  C after overnight broth culture. The bacterial pellet was resuspended in phosphate-buffered saline (PBS) for the preparation of live bacteria, and the suspension was heated at 65  C for 10 min to obtain heat-killed (Hk) bacteria. 2.3. Cell culture All media described below were supplemented with 10% heatinactivated fetal bovine serum (HyClone, Thermo Scientific), penicillin (100 units/ml) and streptomycin (0.1 mg/ml). A549 (human alveolar epithelial) cells were cultured in RPMI-1640 (HyClone, Thermo Scientific). Mouse embryonic fibroblast (MEF) wild-type (WT) and IKKb/ cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, HyClone, Thermo Scientific) as previously described [21]. Unless specified, A549 cells were exposed to bacteria for 4 h in this study. Cells were maintained at 37  C in a humidified 5% CO2 air-jacketed incubator. 2.4. Plasmids and transfections

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using a CFX96 Real-Time PCR System (Bio-Rad, Hercules, CA, USA) using the following thermal conditions: stage 1, 50  C for 2 min and 95  C for 10 min; stage 2, 95  C for 15 s and 60  C for 1 min. Stage 2 was repeated for 40 cycles. Relative quantities of mRNA were calculated using the comparative CT method and normalized to human GAPDH (50 -CCCTCCAAAATCAAGTGG-30 and 50 -CCATCCACAGTCTTCTGG-30 ) or mouse GAPDH (50 -TGTGTCCGTCGGGATCTGA30 and 50 -CCTGCTTCACCACCTTCTTGAT-30 ) mRNA levels for each reaction. 2.6. Immunoblot analysis Cells were lysed on ice for 10 min in 20 mM TriseHCl, pH 7.4, 50 mM NaCl, 50 mM sodium pyrophosphate, 30 mM NaF, 5 mM zinc chloride, 2 mM iodoacetic acid, and 1% Triton X-100. The lysates were centrifuged at 15,000  g for 15 min at 4  C, and protein concentrations were measured using the bicinchoninic acid (BCA) method (Pierce, Rockford, IL, USA). Proteins were separated in SDSe PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked in TBS (10 mM TriseHCl, pH 7.5, 150 mM NaCl) with 5% nonfat dry milk for 1 h and incubated with primary IKKb antibody (Cell Signaling, Danvers, MA, USA) and bactin antibody (Santa Cruz Biotech, Dallas, TX, USA) for 16 h at 4  C. The immunoblots were washed and incubated with appropriate secondary antibodies and visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA). 2.7. Statistical analysis Data are presented as means  standard deviation (SD). The Student t test was used to perform statistical analysis. Values of p < 0.05 were considered statistically significant. 3. Results

(50 -ACC

P. aeruginosa ndk gene was PCR amplified with primers GGA TCC GCC ATG GCA CTG CAA CGC ACC CTG TCC ATC ATC-30 and 50 -ACC GAA TTC TCA GCG AAT GCG CTC GCA GAC TTC GGT AGC CGC30 ). The ndk gene was cloned into the eukaryotic expression vector pcDNA3.1(þ) using BamHI and EcoRI restriction sites located at the end of each primer, resulting in pDNNDK. For the construction of pDNNDKH117Q, the histidine H117 of P. aeruginosa NDK was mutated to glutamine (H117Q) through site directed mutagenesis. Both constructed plasmids were prepared using the EndoFree Plasmid Maxi Kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions. Transfection with 1.5 mg of plasmid was performed by electroporation using a pipette-type microporator (NeonÔ transfection system, Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Transfected cells were incubated for 48 h in RPMI-1640 supplemented with 10% fetal bovine serum at 37  C, and they were pretreated for 4 h prior to lysis for quantitative (real-time) reverse-transcriptase PCR (qRTPCR) analysis.

3.1. Ndk is involved in the upregulation of IL-8 expression Ndk has been reported to be cytotoxic when incubated with eukaryotic cells [18,19]. However, it has not been determined whether Ndk is capable of eliciting inflammatory responses. To examine this, the expression level of IL-8, a key chemokine causing excessive neutrophil infiltration, was quantified in A549 epithelial cells following incubation with two live P. aeruginosa strains, PAKD7 and PAKD8, at a multiplicity of infection (MOI) of 5. As shown in Fig. 1A, PAKD7 clearly induced IL-8 expression by 4 h posttreatment whereas PAKD8, which lacks Ndk, did not, indicating

2.5. qRT-PCR analysis Total RNA was isolated using TRIzolÒ reagent (Invitrogen, Grand Island, NY, USA) following the manufacturer’s instructions. SYBR Green PCR Master Mix (KAPA Biosystems, Woburn, MA, USA) was used for qRT-PCR. Synthesis of cDNA from total RNA was performed using ReverTra Ace qPCR RT kit (Toyobo, Japan). Primer sequence information was as follows: IL-8 primers, 50 -AACATGACTTCCAAGCTGGCC-30 and 50 -TTATGAATTCTCAGCCCTCTTC-30 ; MIP-2 primers, 50 -ATCCAGAGCTTGAGTGTGACGC-30 and 50 -AAGGCAAACTTTTTGACCGCC-30 . Reactions were conducted and quantified

Fig. 1. Ndk is involved in the upregulation of IL-8 expression. A, Cells were treated with either live PAKD7 or PAKD8 at an MOI of 5. B, Cells were treated with either heatkilled PAKD7 (Hk D7) or PAKD8 (Hk D8) strain at an MOI of 50 or 100. After treatment, the mRNA level of IL-8 was measured by qRT-PCR analysis. Data in AeB are expressed as mean  SD (n ¼ 3). *, p < 0.05 vs. control. MOI, multiplicity of infection.

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Fig. 2. IL-8 upregulation requires both the kinase activity of Ndk and P. aeruginosa-derived molecules. A, Cells were transfected with pDNNDK, ndk cloned into the eukaryotic expression vector pcDNA3.1(þ). B, Transfected cells were treated with the Hk D8 strain at an MOI of 20. C, Cells were transfected with either pDNNDK or pDNNDKH117Q followed by treatment with Hk D8 at an MOI of 20. The mRNA level of IL-8 was measured by qRT-PCR analysis. Data in AeC are expressed as mean  SD (n ¼ 3). *, p < 0.05 vs. group transfected with vector only (B) or group transfected with pDNNDK (C).

that Ndk is involved in inducing chemokine expression. By contrast, both heat-killed (Hk) strains at an MOI of 50 and 100 induced the expression to a similar degree (Fig. 1B), implying that Ndk from live bacteria is necessary for induction and other factors associated with bacteria could play a role in the induction at higher MOI. 3.2. IL-8 upregulation requires both the kinase activity of Ndk and P. aeruginosa-derived molecules Previously, it was reported that Ndk is secreted via T1SS [22], and this led us to examine the effects of culture supernatants. However, supernatants obtained from both PAKD7 and PAKD8 strains still induced IL-8 expression, suggesting that secreted Ndk does not play a role (data not shown). Recently, Ndk was assumed to be one of the effectors translocated into cells via T3SS in P. aeruginosa (Shouguang Jin, unpublished data). Based on this, we examined the effect of Ndk in A549 cells by transfection with a construct consisting of ndk (pDNNDK) cloned into a eukaryotic expression vector (pcDNA3.1(þ); Invitrogen). However, the transfected ndk did not induce the expression of chemokines, as shown in Fig. 2A, suggesting that Ndk might require additional factors to induce chemokine expression. To investigate this, we transfected cells with pDNNDK and then treated them with the Hk PAKD8 strain. As shown in Fig. 2B, expression was clearly increased in the presence of both pDNNDK and Hk PAKD8, implying that Ndkmediated induction does indeed require additional factors, possibly present on the bacterial membrane. Ndk is an enzyme that catalyzes the exchange of phosphate groups between different nucleoside diphosphates [23], and the kinase activity of Ndk is the

cause of cytotoxicity in macrophages, through disruption of ATP concentrations [24]. Since we found that Ndk plays a role in inducing chemokine expression, we were interested in whether the kinase activity contributes to the induction. To test this, we determined the effect of the Ndk construct (pDNNDK) on the kinase activity-deficient Ndk (pDNNDKH117Q), which had been generated by site-directed mutagenesis of a conserved histidine residue (H117) of the P. aeruginosa Ndk. As shown in Fig. 2C, cells transfected with pDNNDKH117Q exhibited a clear reduction in IL-8 expression when compared with transfection with pDNNDK, indicating that the induction effect of Ndk is dependent on its kinase activity. Taken together, the expression of IL-8 is mediated by both the kinase activity of Ndk and Pseudomonas-associated factors. 3.3. Flagellin is an additional factor involved in the upregulation of IL-8 P. aeruginosa displays a number of PAMPs on the membrane, and we were interested in identifying potent factors required for chemokine expression. One of the major factors in the outermost membrane of Gram-negative bacteria is LPS. However, the treatment of Ndk-transfected cells with LPS did not increase the expression of IL-8 (Fig. 3A). Next, we tested the effect of flagellin, another important PAMP molecule associated with the pathogenesis of P. aeruginosa, by using a heat-killed PAK isogenic fliC-deficient mutant (Hk fliC mt). As shown in Fig. 3B, IL-8 expression was reduced by the treatment of Hk fliC mt in Ndk-transfected cells, indicating that flagellin plays a role in inducing chemokine expression. To confirm the requirement for flagellin further, Ndk-

Fig. 3. Flagellin is an additional factor involved in the upregulation of IL-8. Cells were transfected with pDNNDK. A, The transfected cells were treated with LPS at the indicated concentrations. B, The transfected cells were treated with either the Hk D8 or heat-killed PAK isogenic fliC-deficient mutant (Hk fliC mt) strain at an MOI of 20. C, The transfected cells were treated with flagellin at the indicated concentrations. The mRNA level of IL-8 was measured by qRT-PCR analysis. Data in AeC are expressed as mean  SD (n ¼ 3). *, p < 0.05 vs. group treated with Hk D8 (B) or group transfected with vector (C).

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transfected cells were treated with purified flagellin. As shown in Fig. 3C, chemokine expression was increased in response to treatment with flagellin in the presence of pDNNDK. Taken together, flagellin is an additional Pseudomonas factor required for the induction of IL-8 expression. 3.4. Upregulation of IL-8 is mediated by the NF-kB pathway Nuclear factor-kappa B (NF-kB) is a key transcription factor that initiates inflammatory responses during airway infection [25]. To determine whether IL-8 expression in response to PAKD7 is mediated by the NF-kB signaling pathway, cells were pretreated with MG132, a chemical inhibitor of NF-kB, prior to the induction of IL-8 expression by P. aeruginosa. As shown in Fig. 4A, PAKD7mediated expression was significantly reduced by MG132 pretreatment. Since MG132 is known to strongly inhibit the activity of proteasome and consequently perturb the physiological responses of cell, we applied another NF-kB inhibitor, CAPE to examine its effect. As predicted, IL-8 expression was clearly inhibited by CAPE pretreatment. To further confirm this result, we used a more specific approach to inhibit NF-kB signaling. Fig. 4B shows that overexpressing IKKb DN abrogated IL-8 expression in A549 cells. Furthermore, upregulation of IL-8 was assessed in WT and IKKb/ MEFs (Fig. 4C, upper panel). The absence of IKKb protein was confirmed by immunoblot analysis (Fig. 4C, lower panel). As shown in Fig. 4C, the induction of macrophage inflammatory protein 2 (MIP-2), a murine counterpart of IL-8, was abolished in IKKb/ MEF, indicating that the expression of IL-8 in response to P. aeruginosa is mediated by the NF-kB pathway. Together, these data suggest that P. aeruginosa PAKD7 uses the NF-kB pathway to induce IL-8 transcription. 4. Discussion IL-8 is produced at basal levels under physiologic conditions, but proinflammatory stimuli such as P. aeruginosa infection in epithelial cells can induce increased IL-8 expression, leading to a marked recruitment of neutrophils to infected tissues. To date, it has been reported that IL-8 expression is induced by several P. aeruginosaassociated or -secreted factors including pilin, flagellin, pyocyanin, nitrite reductase, phospholipase C, autoinducers, and ExoU [12e17]. Here, we report Ndk as a new inducing factor that is translocated from bacterial cytoplasm into host cells through T3SS, similarly to ExoU, which is a unique effector molecule found in P. aeruginosa strain PA103 but not in strain PAK [26e28].

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P. aeruginosa is a motile pathogen with a unipolar flagellum that is a hollow cylinder made up of a globular protein, flagellin. Pulmonary infection of mice with P. aeruginosa devoid of flagella resulted in reduced airway colonization and decreased mortality compared with mice infected with flagellated P. aeruginosa [29]. This indicates that flagella play important roles in motility, adherence, and activation of host inflammation during infection. Previously, flagellin was considered to be sufficient for the induction of IL-8 expression by pathogens such as P. aeruginosa and Salmonella typhimurium [30,31]. In contrast to this, however, we did not observe clear induction of IL-8 expression upon treatment of A549 cells with ultrapure flagellin alone (Fig. 3C). This could be explained by the shorter treatment time (4 h) used in this study compared with the longer incubation time used in other studies [12,32]. However, in the presence of Ndk, a 4 h treatment with flagella was sufficient for induction of IL-8 expression (Fig. 3B), indicating cooperative effects between Ndk and flagellin at the early stage of infection. As shown in Fig. 4B and C, IL-8 expression was not completely reduced by transfection with IKKb DN or in IKKb/ cells, suggesting the possible involvement of other signaling pathways. Previously, it was reported that the potential role of bacterial flagellin as a trigger for inflammation in epithelial cells is mediated through toll-like receptor 5 (TLR5), a pattern recognition receptor specific for bacterial flagellin, via the NF-kB and p38 MAPK signaling pathways [33,34]. This suggests that p38 MAPK could be involved in the expression of IL-8 mediated by Ndk and flagellin. In agreement with this suggestion, we also observed a reduction of IL-8 expression in response to pretreatment with SB203580, a specific chemical inhibitor of the activity of p38 (data not shown). In conclusion, the massive influx of neutrophils into Pseudomonas-infected sites is stimulated by an excessive inflammatory response caused by the production and release of inflammatory mediators, the most important of which include prolonged and sustained expression of IL-8 [7,35]. Accordingly, there is a growing interest in developing novel anti-inflammatory strategies against IL-8, with the aim of reducing the excessive inflammatory response through a therapeutic intervention based on the neutralization of inflammatory mediators with specific antibodies or receptor antagonists, or inhibition of the intracellular signaling cascades that result in their production. To accomplish this, it is important to understand which bacterial products contribute to the recruitment of neutrophils during infection so that therapies can be designed to minimize this response. In this study, we found that a novel effector molecule, Ndk, is required for the expression of IL-8, thus bringing

Fig. 4. Upregulation of IL-8 is mediated by the NF-kB pathway. A, Cells were pretreated for 1 h at the indicated concentration of either MG132 or CAPE followed by treatment with PAKD7 at an MOI of 5. The mRNA level of IL-8 was measured by qRT-PCR analysis. B, Overexpressing of IKKb DN repressed the PAKD7-mediated induction of IL-8 expression. C, PAKD7 induced MIP-2 transcription in WT but not in IKKb/ MEFs (upper panel). The absence of IKKb was confirmed by immunoblot analysis (lower panel). Data in AeC are expressed as mean  SD (n ¼ 3). Immunoblot data in C are representative of three separate experiments. *, p < 0.05 vs. mock (A and B) or WT (C).

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new insight into Pseudomonas infection and the causes of inflammatory responses. Acknowledgments This work was supported by Basic Science Research Program (2010-0009047), the Converging Research Center Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (No. 2013K000249), and the BK21 plus program of the Ministry of Education, Korea. The authors have no financial conflict of interest. References [1] Chastre J, Fagon JY. Ventilator-associated pneumonia. Am J Respir Crit Care Med 2002;165:867e903. [2] Willcox MD. Pseudomonas aeruginosa infection and inflammation during contact lens wear: a review. Optom Vis Sci 2007;84:273e8. [3] Lyczak JB, Cannon CL, Pier GB. Lung infections associated with cystic fibrosis. Clin Microbiol Rev 2002;15:194e222. [4] Epelman S, Bruno TF, Neely GG, Woods DE, Mody CH. Pseudomonas aeruginosa exoenzyme S induces transcriptional expression of proinflammatory cytokines and chemokines. Infect Immun 2000;68:4811e4. [5] Sawa T, Corry DB, Gropper MA, Ohara M, Kurahashi K, Wiener-Kronish JP. IL10 improves lung injury and survival in Pseudomonas aeruginosa pneumonia. J Immunol 1997;159:2858e66. [6] Schultz MJ, Rijneveld AW, Florquin S, Edwards CK, Dinarello CA, van der Poll T. Role of interleukin-1 in the pulmonary immune response during Pseudomonas aeruginosa pneumonia. Am J Physiol Lung Cell Mol Physiol 2002;282:L285e 90. [7] Joseph T, Look D, Ferkol T. NF-kappaB activation and sustained IL-8 gene expression in primary cultures of cystic fibrosis airway epithelial cells stimulated with Pseudomonas aeruginosa. Am J Physiol Lung Cell Mol Physiol 2005;288:L471e9. [8] Kube D, Sontich U, Fletcher D, Davis PB. Proinflammatory cytokine responses to P. aeruginosa infection in human airway epithelial cell lines. Am J Physiol Lung Cell Mol Physiol 2001;280:L493e502. [9] Bonfield TL, Panuska JR, Konstan MW, Hilliard KA, Hilliard JB, Ghnaim H, et al. Inflammatory cytokines in cystic fibrosis lungs. Am J Respir Crit Care Med 1995;152:2111e8. [10] Muhlebach MS, Reed W, Noah TL. Quantitative cytokine gene expression in CF airway. Pediatr Pulmonol 2004;37:393e9. [11] Oka M, Norose K, Matsushima K, Nishigori C, Herlyn M. Overexpression of IL-8 in the cornea induces ulcer formation in the SCID mouse. Brit J Ophthalmol 2006;90:612e5. [12] DiMango E, Zar HJ, Bryan R, Prince A. Diverse Pseudomonas aeruginosa gene products stimulate respiratory epithelial cells to produce interleukin-8. J Clin Invest 1995;96:2204e10. [13] Sar B, Oishi K, Wada A, Hirayama T, Matsushima K, Nagatake T. Nitrite reductase from Pseudomonas aeruginosa released by antimicrobial agents and complement induces interleukin-8 production in bronchial epithelial cells. Antimicrob Agents Chemother 1999;43:794e801. [14] Denning GM, Wollenweber LA, Railsback MA, Cox CD, Stoll LL, Britigan BE. Pseudomonas pyocyanin increases interleukin-8 expression by human airway epithelial cells. Infect Immun 1998;66:5777e84.

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A novel Pseudomonas aeruginosa-derived effector cooperates with flagella to mediate the upregulation of interleukin 8 in human epithelial cells.

Infection with Pseudomonas aeruginosa results in a massive accumulation of neutrophils in response to prolonged and sustained expression of inflammato...
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