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Phospholipase D1 mediates TNFalpha-induced inflammation in a murine model of TNFalpha-induced peritonitis.

Sethu S, Pushparaj PN, Melendez AJ - PLoS ONE (2010)

Bottom Line: Administration of the siRNA against PLD1 significantly reduced PLD1 levels in vivo.By contrast, in mice where PLD1 had been silenced, the TNFalpha-triggered increase in vascular permeability and phagocyte influx was substantially reduced.These novel data demonstrate a critical role for PLD1 in TNFalpha-induced inflammation in vivo and warrant further investigation.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

ABSTRACT

Background: Tumor Necrosis Factor alpha (TNFalpha) is a pleiotropic cytokine extensively studied for its role in the pathogenesis of a variety of disease conditions, including in inflammatory diseases. We have recently shown that, in vitro, that TNFalpha utilizes PLD1 to mediate the activation of NFkappaB and ERK1/2 in human monocytes. The aim of this study was to investigate the role(s) played by phospholipase D1 (PLD1) in TNFalpha-mediated inflammatory responses in vivo.

Methodology/findings: Studies were performed in vivo using a mouse model of TNFalpha-induced peritonitis. The role of PLD1 was investigated by functional genomics, utilizing a specific siRNA to silence the expression of PLD1. Administration of the siRNA against PLD1 significantly reduced PLD1 levels in vivo. TNFalpha triggers a rapid pyrogenic response, but the in vivo silencing of PLD1 protects mice from the TNFalpha-induced rise in temperature. Similarly TNFalpha caused an increase in the serum levels of IL-6, MIP-1alpha and MIP-1beta: this increase in cytokine/chemokine levels was inhibited in mice where PLD1 had been silenced. We then induced acute peritonitis with TNFalpha. Intraperitoneal injection of TNFalpha triggered a rapid increase in vascular permeability, and the influx of neutrophils and monocytes into the peritoneal cavity. By contrast, in mice where PLD1 had been silenced, the TNFalpha-triggered increase in vascular permeability and phagocyte influx was substantially reduced. Furthermore, we also show that the TNFalpha-mediated upregulation of the cell adhesion molecules VCAM and ICAM1, in the vascular endothelium, were dependent on PLD1.

Conclusions: These novel data demonstrate a critical role for PLD1 in TNFalpha-induced inflammation in vivo and warrant further investigation. Indeed, our results suggest PLD1 as a novel target for treating inflammatory diseases, where TNFalpha play key roles: these include diseases ranging from sepsis to respiratory and autoimmune diseases; all diseases with considerable unmet medical need.

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TNFα-induced vascular permeability and VCAM-1 and ICAM-1 expression in peritoneal tissues is dependent on PLD1.(A) Peritoneal lavage was collected, and the OD was measured, from mice: untreated following the i.p. injection of PBS (Basal); following the i.p. injection of TNFα (TNF); following the i.p. injection of TNFα in mice pretreated with the siRNA-PLD1 (TNF +siRNA-PLD1); following the i.p. injection of TNFα in mice pretreated with the scrambled siRNA (TNF +Scr-siRNA). Data showed as means ± SD of triplicate measurements from three different experiments. Student's t test p values (*p<0.05, **p<0.01). Six mice were used per treatment group per experiment. (B) VCAM-1 and ICAM-1 expression pattern using immunohistochemistry in peritoneal tissues after 2 h of TNFα administration. The panels indicate the peritoneal tissues from control mice (Basal); from mice injected with TNFα (TNF); from mice pretreated with the siRNA-PLD1 prior to TNFα administration (TNF+siRNA-PLD1); and from mice pretreated with the scrambled siRNA prior to TNFα administration (TNF+Scr-siRNA). Results shown are representative of three different experiments and of multiple sections and fields.
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pone-0010506-g003: TNFα-induced vascular permeability and VCAM-1 and ICAM-1 expression in peritoneal tissues is dependent on PLD1.(A) Peritoneal lavage was collected, and the OD was measured, from mice: untreated following the i.p. injection of PBS (Basal); following the i.p. injection of TNFα (TNF); following the i.p. injection of TNFα in mice pretreated with the siRNA-PLD1 (TNF +siRNA-PLD1); following the i.p. injection of TNFα in mice pretreated with the scrambled siRNA (TNF +Scr-siRNA). Data showed as means ± SD of triplicate measurements from three different experiments. Student's t test p values (*p<0.05, **p<0.01). Six mice were used per treatment group per experiment. (B) VCAM-1 and ICAM-1 expression pattern using immunohistochemistry in peritoneal tissues after 2 h of TNFα administration. The panels indicate the peritoneal tissues from control mice (Basal); from mice injected with TNFα (TNF); from mice pretreated with the siRNA-PLD1 prior to TNFα administration (TNF+siRNA-PLD1); and from mice pretreated with the scrambled siRNA prior to TNFα administration (TNF+Scr-siRNA). Results shown are representative of three different experiments and of multiple sections and fields.

Mentions: One of the characteristic events in an inflammatory response is the increase in vascular permeability, and vascular cell adhesion molecule expression. Alterations in vascular permeability were determined by i.v. injection of Evans blue dye, which binds to serum proteins and thus can be used to quantify alterations in vascular permeability. Intra-peritoneal injection of TNFα into the peritoneal cavity caused a steady influx of Evans blue into the peritoneal cavity, with a continued increase from 2 to 12 h (Fig. 3A).


Phospholipase D1 mediates TNFalpha-induced inflammation in a murine model of TNFalpha-induced peritonitis.

Sethu S, Pushparaj PN, Melendez AJ - PLoS ONE (2010)

TNFα-induced vascular permeability and VCAM-1 and ICAM-1 expression in peritoneal tissues is dependent on PLD1.(A) Peritoneal lavage was collected, and the OD was measured, from mice: untreated following the i.p. injection of PBS (Basal); following the i.p. injection of TNFα (TNF); following the i.p. injection of TNFα in mice pretreated with the siRNA-PLD1 (TNF +siRNA-PLD1); following the i.p. injection of TNFα in mice pretreated with the scrambled siRNA (TNF +Scr-siRNA). Data showed as means ± SD of triplicate measurements from three different experiments. Student's t test p values (*p<0.05, **p<0.01). Six mice were used per treatment group per experiment. (B) VCAM-1 and ICAM-1 expression pattern using immunohistochemistry in peritoneal tissues after 2 h of TNFα administration. The panels indicate the peritoneal tissues from control mice (Basal); from mice injected with TNFα (TNF); from mice pretreated with the siRNA-PLD1 prior to TNFα administration (TNF+siRNA-PLD1); and from mice pretreated with the scrambled siRNA prior to TNFα administration (TNF+Scr-siRNA). Results shown are representative of three different experiments and of multiple sections and fields.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2864766&req=5

pone-0010506-g003: TNFα-induced vascular permeability and VCAM-1 and ICAM-1 expression in peritoneal tissues is dependent on PLD1.(A) Peritoneal lavage was collected, and the OD was measured, from mice: untreated following the i.p. injection of PBS (Basal); following the i.p. injection of TNFα (TNF); following the i.p. injection of TNFα in mice pretreated with the siRNA-PLD1 (TNF +siRNA-PLD1); following the i.p. injection of TNFα in mice pretreated with the scrambled siRNA (TNF +Scr-siRNA). Data showed as means ± SD of triplicate measurements from three different experiments. Student's t test p values (*p<0.05, **p<0.01). Six mice were used per treatment group per experiment. (B) VCAM-1 and ICAM-1 expression pattern using immunohistochemistry in peritoneal tissues after 2 h of TNFα administration. The panels indicate the peritoneal tissues from control mice (Basal); from mice injected with TNFα (TNF); from mice pretreated with the siRNA-PLD1 prior to TNFα administration (TNF+siRNA-PLD1); and from mice pretreated with the scrambled siRNA prior to TNFα administration (TNF+Scr-siRNA). Results shown are representative of three different experiments and of multiple sections and fields.
Mentions: One of the characteristic events in an inflammatory response is the increase in vascular permeability, and vascular cell adhesion molecule expression. Alterations in vascular permeability were determined by i.v. injection of Evans blue dye, which binds to serum proteins and thus can be used to quantify alterations in vascular permeability. Intra-peritoneal injection of TNFα into the peritoneal cavity caused a steady influx of Evans blue into the peritoneal cavity, with a continued increase from 2 to 12 h (Fig. 3A).

Bottom Line: Administration of the siRNA against PLD1 significantly reduced PLD1 levels in vivo.By contrast, in mice where PLD1 had been silenced, the TNFalpha-triggered increase in vascular permeability and phagocyte influx was substantially reduced.These novel data demonstrate a critical role for PLD1 in TNFalpha-induced inflammation in vivo and warrant further investigation.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

ABSTRACT

Background: Tumor Necrosis Factor alpha (TNFalpha) is a pleiotropic cytokine extensively studied for its role in the pathogenesis of a variety of disease conditions, including in inflammatory diseases. We have recently shown that, in vitro, that TNFalpha utilizes PLD1 to mediate the activation of NFkappaB and ERK1/2 in human monocytes. The aim of this study was to investigate the role(s) played by phospholipase D1 (PLD1) in TNFalpha-mediated inflammatory responses in vivo.

Methodology/findings: Studies were performed in vivo using a mouse model of TNFalpha-induced peritonitis. The role of PLD1 was investigated by functional genomics, utilizing a specific siRNA to silence the expression of PLD1. Administration of the siRNA against PLD1 significantly reduced PLD1 levels in vivo. TNFalpha triggers a rapid pyrogenic response, but the in vivo silencing of PLD1 protects mice from the TNFalpha-induced rise in temperature. Similarly TNFalpha caused an increase in the serum levels of IL-6, MIP-1alpha and MIP-1beta: this increase in cytokine/chemokine levels was inhibited in mice where PLD1 had been silenced. We then induced acute peritonitis with TNFalpha. Intraperitoneal injection of TNFalpha triggered a rapid increase in vascular permeability, and the influx of neutrophils and monocytes into the peritoneal cavity. By contrast, in mice where PLD1 had been silenced, the TNFalpha-triggered increase in vascular permeability and phagocyte influx was substantially reduced. Furthermore, we also show that the TNFalpha-mediated upregulation of the cell adhesion molecules VCAM and ICAM1, in the vascular endothelium, were dependent on PLD1.

Conclusions: These novel data demonstrate a critical role for PLD1 in TNFalpha-induced inflammation in vivo and warrant further investigation. Indeed, our results suggest PLD1 as a novel target for treating inflammatory diseases, where TNFalpha play key roles: these include diseases ranging from sepsis to respiratory and autoimmune diseases; all diseases with considerable unmet medical need.

Show MeSH
Related in: MedlinePlus