Pneumolysin-Dependent and -Independent Gene Expression Identified by cDNA Microarray Analysis of THP-1 Human Mononuclear Cells Stimulated by<i>Streptococcus pneumoniae</i>

Rogers, P. David; Thornton, Justin A.; Barker, Katherine S.; McDaniel, D. Olga; Sacks, Gordon S.; Swiatlo, Edwin; McDaniel, Larry S.

doi:10.1128/iai.71.4.2087-2094.2003

Cited by 71 publications

(47 citation statements)

References 57 publications

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“…Previously, we used THP-1 cells to look at monocyte gene expression in response to S. pneumoniae D39 and its PLYnegative mutant PLN (32). Although ICAM-1 was not found to be significantly up-regulated in that study, recent microarray analysis indicated a significant increase in ICAM-1 expression in THP-1 cells exposed to D39 (data not shown).…”

Section: Discussionmentioning

confidence: 91%

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THP-1 Monocytes Up-Regulate Intercellular Adhesion Molecule 1 in Response to Pneumolysin fromStreptococcus pneumoniae

Thornton¹,

McDaniel

2005

Infect Immun

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Pneumolysin (PLY) is a major virulence factor of Streptococcus pneumoniae that elicits a variety of proinflammatory responses from cells of the host immune system. Intercellular adhesion molecule 1 (ICAM-1) is a cell adhesion molecule involved in leukocyte trafficking toward inflammatory stimuli in extravascular sites. In this study, we evaluated the effect of PLY on expression of ICAM-1 in THP-1 monocytic cells exposed to S. pneumoniae. Exposure of cells to PLY-expressing S. pneumoniae strain WU2 for 6 h led to significantly higher levels of ICAM-1 message than those in cells exposed to either medium alone or ⌬PLY1, a PLY-negative isogenic mutant of WU2. Cells exposed to purified recombinant PLY also showed a dose-dependent increase in ICAM-1 mRNA compared to cells exposed to medium alone. Exposure to recombinant PLY containing a single amino acid substitution (Trp4333Phe) that decreases cytolytic activity did not increase ICAM-1 mRNA to levels seen with wild-type PLY. In addition, THP-1 cells exposed to wild-type strain WU2 or D39 had increased ICAM-1 on their surface compared to cells exposed to medium alone or their PLY-negative isogenic mutants ⌬PLY1 and ⌬PLY2, respectively. These data indicate that PLY induces transcription and production of a cell adhesion molecule involved in the inflammatory response that may play a role in pneumococcal infection.

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Section: Discussionmentioning

confidence: 91%

“…Also, monocytes exposed to PLY had increased expression of inflammatory cytokines TNF-␣ and IL-1␤ (13). Using cDNA microarray analysis of the THP-1 monocytic cell line exposed to S. pneumoniae, we previously reported PLY-dependent upregulation of many genes including those for the chemokines IL-8, MCP-3, and MIP-1␤ (32).…”

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confidence: 99%

THP-1 Monocytes Up-Regulate Intercellular Adhesion Molecule 1 in Response to Pneumolysin fromStreptococcus pneumoniae

Thornton¹,

McDaniel

2005

Infect Immun

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“…cis 7,10,13,16,8,11,14,17-EPA were purchased from Sigma Chemical Co. (St. Louis, Mo.) and made to stock concentrations of 25 mg/ml in ethanol.…”

Section: Pneumolysin (418 Ng/ml)-mediated Influx Of Camentioning

confidence: 99%

“…These agents may have potential in attenuating the proinflammatory properties of this pneumococcal toxin.Notwithstanding complement-activating properties, poreforming interactions with neutrophils and monocytes, resulting in influx of Ca 2ϩ , have been implicated in the proinflammatory activities of the pneumococcal toxin, pneumolysin (1,2,3,4,11). Rather than contributing to the eradication of the infection, however, the resultant, predominantly neutrophil-mediated inflammatory response appears to favor persistence and extrapulmonary dissemination of the pneumococcus (7, 9).…”

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Docosahexaenoic Acid and Eicosapentaenoic Acid Antagonize the Proinflammatory Interactions of Pneumolysin with Human Neutrophils

et al. 2004

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Pneumolysin (4.18 ng/ml)-mediated influx of Ca2؉ and augmentation of the chemoattractant-activated generation of reactive oxidants was antagonized by pretreatment of human neutrophils with the omega-3 polyunsaturated fatty acids docosahexaenoic acid and eicosapentaenoic acid (1.25 to 5 g/ml). These agents may have potential in attenuating the proinflammatory properties of this pneumococcal toxin.Notwithstanding complement-activating properties, poreforming interactions with neutrophils and monocytes, resulting in influx of Ca 2ϩ , have been implicated in the proinflammatory activities of the pneumococcal toxin, pneumolysin (1,2,3,4,11). Rather than contributing to the eradication of the infection, however, the resultant, predominantly neutrophil-mediated inflammatory response appears to favor persistence and extrapulmonary dissemination of the pneumococcus (7, 9).Although no pharmacological antagonists of pneumolysin have, to our knowledge, been described, we reasoned that agents, such as the omega-3 polyunsaturated fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), which increase neutrophil membrane fluidity and stability may interfere with the proinflammatory, pore-forming activities of the toxin. The present study was designed to investigate the potential of DHA and EPA to neutralize two related proinflammatory activities of pneumolysin, these being the toxinmediated influx of Ca 2ϩ into human neutrophils and Ca 2ϩ -dependent sensitization of these cells for increased production of toxic reactive oxidants (4).Endotoxin-free (Ͻ2 pg/ml) recombinant pneumolysin (specific activity, 4 ϫ 10 6 hemolytic units/mg) was expressed in Escherichia coli and purified from cell extracts as previously described (12).Neutrophils were isolated from heparinized (5 U of preservative-free heparin/ml) venous blood of healthy, adult human volunteers (4) and resuspended to 10 7 neutrophils/ml in phosphate-buffered saline (0.15 M, pH 7.4).cis 7,10,13,16,8,11,14,17-EPA were purchased from Sigma Chemical Co. (St. Louis, Mo.) and made to stock concentrations of 25 mg/ml in ethanol. Dilutions were made in ethanol, and these agents were used at final concentrations of 1.25, 2.5, and 5 g/ml in a final ethanol concentration of 0.1%. All control systems included in the experiments described below contained 0.1% ethanol, which did not interfere with the biological activity of pneumolysin.Fura-2/AM (Sigma) was used as the fluorescent Ca 2ϩ -sensitive indicator to measure alterations in neutrophil cytosolic Ca 2ϩ as previously described (4). The neutrophils were preloaded with fura-2/AM (final concentration, 2 M) for 30 min at 37°C in phosphate-buffered saline, washed, and resuspended in Hanks balanced salt solution (pH 7.4, 1.25 mM CaCl 2 ; Highveld Biological Pty. Ltd., Johannesburg, South Africa). The cells (1 ϫ 10 6 /ml) were then preincubated for 10 min at 37°C with or without DHA or EPA (1.25 to 5 g/ml) and transferred thereafter to cuvettes, maintained at 37°C in a Hitachi 650 10S fluorescence spectrophotometer with e...

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“…Thus far, only a few studies have taken this approach to identify host factors involved in pneumococcal colonization and subsequent disease. Rogers et al have analyzed the transcriptional response of a human monocytic cell line to pneumococci, focusing on pneumolysin-dependent and -independent gene expression (44). They identified 40 genes that were responsive independent of pneumolysin and 142 pneumolysin-responsive genes, including those encoding IL-8, MIP-1␤, lysozyme, and caspases 4 and 6 (44).…”

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Analysis of the In Vitro Transcriptional Response of Human Pharyngeal Epithelial Cells to AdherentStreptococcus pneumoniae: Evidence for a Distinct Response to Encapsulated Strains

2007

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Infection of the human host by Streptococcus pneumoniae begins with colonization of the nasopharynx, which is mediated by the adherence of bacteria to the respiratory epithelium. Several studies have indicated an important role for the pneumococcal capsule in this process. Here, we used microarrays to characterize the in vitro transcriptional response of human pharyngeal epithelial Detroit 562 cells to the adherence of serotype 2 encapsulated strain D39, serotype 19F encapsulated strain G54, serotype 4 encapsulated strain TIGR4, and their nonencapsulated derivatives (⌬cps). In total, 322 genes were found to be upregulated in response to adherent pneumococci. Twenty-two genes were commonly induced, including those encoding several cytokines (e.g., interleukin 1␤ [IL-1␤] and IL-6), chemokines (e.g., IL-8 and CXCL1/2), and transcriptional regulators (e.g., FOS), consistent with an innate immune response mediated by Toll-like receptor signaling. Interestingly, 85% of genes were induced specifically by one or more encapsulated strains, suggestive of a capsule-dependent response. Importantly, purified capsular polysaccharides alone had no effect. Over a third of these loci encoded products predicted to be involved in transcriptional regulation and signal transduction, in particular mitogenactivated protein kinase signaling pathways. Real-time PCR of a subset of 10 genes confirmed the microarray data and showed a time-dependent upregulation of, especially, innate immunity genes. The downregulation of epithelial genes was most pronounced upon adherence of D39⌬cps, as 68% of the 161 genes identified were repressed only by this nonencapsulated strain. In conclusion, we identified a subset of host genes specifically induced by encapsulated strains during in vitro adherence and have demonstrated the complexity of interactions occurring during the initial stages of pneumococcal infection.

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Pneumolysin-Dependent and -Independent Gene Expression Identified by cDNA Microarray Analysis of THP-1 Human Mononuclear Cells Stimulated byStreptococcus pneumoniae

Cited by 71 publications

References 57 publications

THP-1 Monocytes Up-Regulate Intercellular Adhesion Molecule 1 in Response to Pneumolysin fromStreptococcus pneumoniae

THP-1 Monocytes Up-Regulate Intercellular Adhesion Molecule 1 in Response to Pneumolysin fromStreptococcus pneumoniae

Docosahexaenoic Acid and Eicosapentaenoic Acid Antagonize the Proinflammatory Interactions of Pneumolysin with Human Neutrophils

Analysis of the In Vitro Transcriptional Response of Human Pharyngeal Epithelial Cells to AdherentStreptococcus pneumoniae: Evidence for a Distinct Response to Encapsulated Strains

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