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Impact of anti-inflammatory agents on the gene expression profile of stimulated human neutrophils: unraveling endogenous resolution pathways.

St-Onge M, Dumas A, Michaud A, Laflamme C, Dussault AA, Pouliot M - PLoS ONE (2009)

Bottom Line: Genes encoding transcription factors, enzymes and regulatory proteins, as well as secreted cytokines/chemokines showed differential expression.We have identified a set of genes that may be part of important resolution pathways that interfere with cell activation.Identification of these pathways will improve understanding of the capacity of tissues to terminate inflammatory responses and contribute to the development of therapeutic strategies based on endogenous resolution.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy-Physiology, Faculty of Medicine, Centre de Recherche en Rhumatologie et Immunologie du CHUQ, Laval University, Quebec City, Quebec, Canada.

ABSTRACT
Adenosine, prostaglandin E(2), or increased intracellular cyclic AMP concentration each elicit potent anti-inflammatory events in human neutrophils by inhibiting functions such as phagocytosis, superoxide production, adhesion and cytokine release. However, the endogenous molecular pathways mediating these actions are poorly understood. In the present study, we examined their impact on the gene expression profile of stimulated neutrophils. Purified blood neutrophils from healthy donors were stimulated with a cocktail of inflammatory agonists in the presence of at least one of the following anti-inflammatory agents: adenosine A(2A) receptor agonist CGS 21680, prostaglandin E(2), cyclic-AMP-elevating compounds forskolin and RO 20-1724. Total RNA was analyzed using gene chips and real-time PCR. Genes encoding transcription factors, enzymes and regulatory proteins, as well as secreted cytokines/chemokines showed differential expression. We identified 15 genes for which the anti-inflammatory agents altered mRNA levels. The agents affected the expression profile in remarkably similar fashion, suggesting a central mechanism limiting cell activation. We have identified a set of genes that may be part of important resolution pathways that interfere with cell activation. Identification of these pathways will improve understanding of the capacity of tissues to terminate inflammatory responses and contribute to the development of therapeutic strategies based on endogenous resolution.

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Impact of a combination of anti-inflammatory agents on gene expression in stimulated human neutrophils.Cells were pretreated with CGS 21680 (1 µM), PGE2 (10 µM), RO-20-1724 (10 µM) and forskolin (50 µM) simultaneously, then stimulated as described in Materials and Methods for 30 min at 37°C, or for 2 h where indicated (λ). Top panels show genes that are up-regulated by the anti-inflammatory treatments and bottom ones show genes that are down-regulated. The dotted line indicates the level of expression observed in un-stimulated cells ( = 1). Values are ratios of mRNA levels (treated cells/un-stimulated cells) as determined by real-time PCR, averaged±SEM for three independent experiments performed under identical conditions with a different single donor of cells. *Significantly higher than in un-stimulated samples. **Significantly higher than in un-stimulated samples and in samples stimulated in the absence of anti-inflammatory agent. “#” Significantly higher than in un-stimulated samples but significantly lower than in samples stimulated in the absence of anti-inflammatory agent.
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pone-0004902-g003: Impact of a combination of anti-inflammatory agents on gene expression in stimulated human neutrophils.Cells were pretreated with CGS 21680 (1 µM), PGE2 (10 µM), RO-20-1724 (10 µM) and forskolin (50 µM) simultaneously, then stimulated as described in Materials and Methods for 30 min at 37°C, or for 2 h where indicated (λ). Top panels show genes that are up-regulated by the anti-inflammatory treatments and bottom ones show genes that are down-regulated. The dotted line indicates the level of expression observed in un-stimulated cells ( = 1). Values are ratios of mRNA levels (treated cells/un-stimulated cells) as determined by real-time PCR, averaged±SEM for three independent experiments performed under identical conditions with a different single donor of cells. *Significantly higher than in un-stimulated samples. **Significantly higher than in un-stimulated samples and in samples stimulated in the absence of anti-inflammatory agent. “#” Significantly higher than in un-stimulated samples but significantly lower than in samples stimulated in the absence of anti-inflammatory agent.

Mentions: Gene chips and real-time PCR showed similar effects of PGE2 or pharmacological elevation of intracellular cAMP on most of the genes affected by A2AR engagement, suggesting that even when distinct receptors are engaged, signaling pathways eventually merge and cAMP-dependent processes take part in a central anti-inflammatory response. In order to address this point specifically, we next stimulated neutrophils in the simultaneous presence of all three types of anti-inflammatory agent. Messenger RNA levels of the 15 genes identified earlier were determined by real-time PCR. This experiment produced essentially the same result as obtained with each anti-inflammatory strategy alone (Figure 3), further advocating for an important role of these genes in limiting cell activation. Indeed, no additive or synergistic effect was obtained for the majority of the genes. The exceptions were NR4A3 and DUSP5, for which the simultaneous presence of the anti-inflammatory agents proved more potent than any individual agent. Overall, these results support the concept of a relative redundancy between the distinct anti-inflammatory agents and more specifically their participation in a central and largely cAMP-dependent cellular immunomodulatory response.


Impact of anti-inflammatory agents on the gene expression profile of stimulated human neutrophils: unraveling endogenous resolution pathways.

St-Onge M, Dumas A, Michaud A, Laflamme C, Dussault AA, Pouliot M - PLoS ONE (2009)

Impact of a combination of anti-inflammatory agents on gene expression in stimulated human neutrophils.Cells were pretreated with CGS 21680 (1 µM), PGE2 (10 µM), RO-20-1724 (10 µM) and forskolin (50 µM) simultaneously, then stimulated as described in Materials and Methods for 30 min at 37°C, or for 2 h where indicated (λ). Top panels show genes that are up-regulated by the anti-inflammatory treatments and bottom ones show genes that are down-regulated. The dotted line indicates the level of expression observed in un-stimulated cells ( = 1). Values are ratios of mRNA levels (treated cells/un-stimulated cells) as determined by real-time PCR, averaged±SEM for three independent experiments performed under identical conditions with a different single donor of cells. *Significantly higher than in un-stimulated samples. **Significantly higher than in un-stimulated samples and in samples stimulated in the absence of anti-inflammatory agent. “#” Significantly higher than in un-stimulated samples but significantly lower than in samples stimulated in the absence of anti-inflammatory agent.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654409&req=5

pone-0004902-g003: Impact of a combination of anti-inflammatory agents on gene expression in stimulated human neutrophils.Cells were pretreated with CGS 21680 (1 µM), PGE2 (10 µM), RO-20-1724 (10 µM) and forskolin (50 µM) simultaneously, then stimulated as described in Materials and Methods for 30 min at 37°C, or for 2 h where indicated (λ). Top panels show genes that are up-regulated by the anti-inflammatory treatments and bottom ones show genes that are down-regulated. The dotted line indicates the level of expression observed in un-stimulated cells ( = 1). Values are ratios of mRNA levels (treated cells/un-stimulated cells) as determined by real-time PCR, averaged±SEM for three independent experiments performed under identical conditions with a different single donor of cells. *Significantly higher than in un-stimulated samples. **Significantly higher than in un-stimulated samples and in samples stimulated in the absence of anti-inflammatory agent. “#” Significantly higher than in un-stimulated samples but significantly lower than in samples stimulated in the absence of anti-inflammatory agent.
Mentions: Gene chips and real-time PCR showed similar effects of PGE2 or pharmacological elevation of intracellular cAMP on most of the genes affected by A2AR engagement, suggesting that even when distinct receptors are engaged, signaling pathways eventually merge and cAMP-dependent processes take part in a central anti-inflammatory response. In order to address this point specifically, we next stimulated neutrophils in the simultaneous presence of all three types of anti-inflammatory agent. Messenger RNA levels of the 15 genes identified earlier were determined by real-time PCR. This experiment produced essentially the same result as obtained with each anti-inflammatory strategy alone (Figure 3), further advocating for an important role of these genes in limiting cell activation. Indeed, no additive or synergistic effect was obtained for the majority of the genes. The exceptions were NR4A3 and DUSP5, for which the simultaneous presence of the anti-inflammatory agents proved more potent than any individual agent. Overall, these results support the concept of a relative redundancy between the distinct anti-inflammatory agents and more specifically their participation in a central and largely cAMP-dependent cellular immunomodulatory response.

Bottom Line: Genes encoding transcription factors, enzymes and regulatory proteins, as well as secreted cytokines/chemokines showed differential expression.We have identified a set of genes that may be part of important resolution pathways that interfere with cell activation.Identification of these pathways will improve understanding of the capacity of tissues to terminate inflammatory responses and contribute to the development of therapeutic strategies based on endogenous resolution.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy-Physiology, Faculty of Medicine, Centre de Recherche en Rhumatologie et Immunologie du CHUQ, Laval University, Quebec City, Quebec, Canada.

ABSTRACT
Adenosine, prostaglandin E(2), or increased intracellular cyclic AMP concentration each elicit potent anti-inflammatory events in human neutrophils by inhibiting functions such as phagocytosis, superoxide production, adhesion and cytokine release. However, the endogenous molecular pathways mediating these actions are poorly understood. In the present study, we examined their impact on the gene expression profile of stimulated neutrophils. Purified blood neutrophils from healthy donors were stimulated with a cocktail of inflammatory agonists in the presence of at least one of the following anti-inflammatory agents: adenosine A(2A) receptor agonist CGS 21680, prostaglandin E(2), cyclic-AMP-elevating compounds forskolin and RO 20-1724. Total RNA was analyzed using gene chips and real-time PCR. Genes encoding transcription factors, enzymes and regulatory proteins, as well as secreted cytokines/chemokines showed differential expression. We identified 15 genes for which the anti-inflammatory agents altered mRNA levels. The agents affected the expression profile in remarkably similar fashion, suggesting a central mechanism limiting cell activation. We have identified a set of genes that may be part of important resolution pathways that interfere with cell activation. Identification of these pathways will improve understanding of the capacity of tissues to terminate inflammatory responses and contribute to the development of therapeutic strategies based on endogenous resolution.

Show MeSH
Related in: MedlinePlus