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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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Specific knockdown of PLD1 in BALB/c mice using siRNA.(A) Western blot analysis shows the knockdown of murine phospholipase D1 (PLD1) in BALB/c mice PBMNCs, by the use of specific siRNA for PLD1 (4 µg/mouse). (B) Western blot analysis shows the expression of murine phospholipase D2 (PLD2) in the same set of samples as indicated. Lysates probed for PLD1 and PLD2 from PBMCs were obtained from: untreated mice which served as control (Control); mice injected with siRNA-PLD1 (siRNA-PLD1); and from mice injected with scrambled siRNA (Scr-siRNA). In addition, α-tubulin was probed for loading control.
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pone-0010506-g001: Specific knockdown of PLD1 in BALB/c mice using siRNA.(A) Western blot analysis shows the knockdown of murine phospholipase D1 (PLD1) in BALB/c mice PBMNCs, by the use of specific siRNA for PLD1 (4 µg/mouse). (B) Western blot analysis shows the expression of murine phospholipase D2 (PLD2) in the same set of samples as indicated. Lysates probed for PLD1 and PLD2 from PBMCs were obtained from: untreated mice which served as control (Control); mice injected with siRNA-PLD1 (siRNA-PLD1); and from mice injected with scrambled siRNA (Scr-siRNA). In addition, α-tubulin was probed for loading control.

Mentions: We and others have shown that intravenous administration of siRNA is known to result in a significant knockdown of the gene products in mice [32]–[34]. Figure 1a shows a significant decrease in the protein level of murine phospholipase D1 (mPLD1) in peripheral blood leukocytes of mice, following intravenous administration of siRNA (4 µg/mouse) to knockdown mPLD1 (Fig. 1A). Since both the PLD isoforms are expressed in BALB/c mice [35], we determined the isoform-specific knockdown of PLD1 by evaluating the expression of PLD2 as well, following the above-mentioned siRNA intravenous administration. The level of mPLD2 expression remained unaffected subsequent to siRNA treatment specific for PLD1 (Fig. 1B), indicating the efficiency and specificity of the isoform-specific siRNA knockdown of PLD1 in vivo.


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

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

Specific knockdown of PLD1 in BALB/c mice using siRNA.(A) Western blot analysis shows the knockdown of murine phospholipase D1 (PLD1) in BALB/c mice PBMNCs, by the use of specific siRNA for PLD1 (4 µg/mouse). (B) Western blot analysis shows the expression of murine phospholipase D2 (PLD2) in the same set of samples as indicated. Lysates probed for PLD1 and PLD2 from PBMCs were obtained from: untreated mice which served as control (Control); mice injected with siRNA-PLD1 (siRNA-PLD1); and from mice injected with scrambled siRNA (Scr-siRNA). In addition, α-tubulin was probed for loading control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2864766&req=5

pone-0010506-g001: Specific knockdown of PLD1 in BALB/c mice using siRNA.(A) Western blot analysis shows the knockdown of murine phospholipase D1 (PLD1) in BALB/c mice PBMNCs, by the use of specific siRNA for PLD1 (4 µg/mouse). (B) Western blot analysis shows the expression of murine phospholipase D2 (PLD2) in the same set of samples as indicated. Lysates probed for PLD1 and PLD2 from PBMCs were obtained from: untreated mice which served as control (Control); mice injected with siRNA-PLD1 (siRNA-PLD1); and from mice injected with scrambled siRNA (Scr-siRNA). In addition, α-tubulin was probed for loading control.
Mentions: We and others have shown that intravenous administration of siRNA is known to result in a significant knockdown of the gene products in mice [32]–[34]. Figure 1a shows a significant decrease in the protein level of murine phospholipase D1 (mPLD1) in peripheral blood leukocytes of mice, following intravenous administration of siRNA (4 µg/mouse) to knockdown mPLD1 (Fig. 1A). Since both the PLD isoforms are expressed in BALB/c mice [35], we determined the isoform-specific knockdown of PLD1 by evaluating the expression of PLD2 as well, following the above-mentioned siRNA intravenous administration. The level of mPLD2 expression remained unaffected subsequent to siRNA treatment specific for PLD1 (Fig. 1B), indicating the efficiency and specificity of the isoform-specific siRNA knockdown of PLD1 in vivo.

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