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Signaling pathways involved in LPS induced TNFalpha production in human adipocytes.

Hoareau L, Bencharif K, Rondeau P, Murumalla R, Ravanan P, Tallet F, Delarue P, Cesari M, Roche R, Festy F - J Inflamm (Lond) (2010)

Bottom Line: Cells were treated with a pathogen-associated molecular pattern: LPS, with and without several kinase inhibitors.Moreover, we demonstrate that the PI3Kinase pathway is clearly involved in the first step of the LPS-pathway.Lastly, we show that adipocytes are able to secrete a large amount of TNFalpha compared to macrophages.

View Article: PubMed Central - HTML - PubMed

Affiliation: LBGM-GEICO, Laboratoire de Biochimie et de Génétique Moléculaire - Groupe d'Etude sur l'Inflammation Chronique et l'Obésité, Université de l'île de la Réunion, 15 avenue René Cassin 97715 Saint Denis Messag Cedex, France.

ABSTRACT

Background: The development of obesity has been linked to an inflammatory process, and the role of adipose tissue in the secretion of pro-inflammatory molecules such as IL-6 or TNFalpha has now been largely confirmed. Although TNFalpha secretion by adipose cells is probably induced, most notably by TLR ligands, the activation and secretion pathways of this cytokine are not yet entirely understood. Moreover, given that macrophagic infiltration is a characteristic of obesity, it is difficult to clearly establish the level of involvement of the different cellular types present within the adipose tissue during inflammation.

Methods: Primary cultures of human adipocytes and human peripheral blood mononuclear cells were used. Cells were treated with a pathogen-associated molecular pattern: LPS, with and without several kinase inhibitors. Western blot for p38 MAP Kinase was performed on cell lysates. TNFalpha mRNA was detected in cells by RT-PCR and TNFalpha protein was detected in supernatants by ELISA assays.

Results: WE SHOW FOR THE FIRST TIME THAT THE PRODUCTION OF TNFALPHA IN MATURE HUMAN ADIPOCYTES IS MAINLY DEPENDENT UPON TWO PATHWAYS: NFkappaB and p38 MAP Kinase. Moreover, we demonstrate that the PI3Kinase pathway is clearly involved in the first step of the LPS-pathway. Lastly, we show that adipocytes are able to secrete a large amount of TNFalpha compared to macrophages.

Conclusion: This study clearly demonstrates that the LPS induced activation pathway is an integral part of the inflammatory process linked to obesity, and that adipocytes are responsible for most of the secreted TNFalpha in inflamed adipose tissue, through TLR4 activation.

No MeSH data available.


Related in: MedlinePlus

LPS induced TNFalpha protein secretion requires co-factors and is dependant upon the CD14/TLR4 pathway. Panel A: FBS effect on LPS induced TNFalpha secretion. The concentrations of TNFalpha (pg/mL) in the medium of mature adipocyte cultures treated or not with increasing concentrations of FBS and 1 μg/mL of LPS, were determined at 6 hours. The graph represents the mean ± SE of the results from two patients. ***P < 0.001%, *P < 0.05%, versus control cells. Panel B: Effect of TLR4 blocking antibody on TNFalpha secretion. The concentrations of TNFalpha (pg/mL) in the medium of mature adipocyte cultures treated with 1 μg/mL of LPS, with or without 1 h of pre-incubation with a blocking anti-TLR4 antibody (20 μg/mL, Clinisciences, Montrouge, France, clone HTA125) were determined at 6 hours. The graph represents the mean ± SE of the results from two patients. ***P < 0.001%, versus cells treated with LPS alone. Panel C: Effect of CD14 blocking antibody on TNFα secretion. The concentrations of TNFα (pg/mL) in the medium of mature adipocyte cultures treated with 1 μg/mL of LPS, with or without 1 h of pre-incubation with a blocking anti-CD14 antibody (5 μg/mL, Hycult biotechnology, clone 18D11) were determined at 6 hours. The graph represents the mean ± SE of the results from one patient (n = 6 for each condition), representative of two experiments on two different patients. ***P < 0.001%, versus cells treated with LPS alone.
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Figure 5: LPS induced TNFalpha protein secretion requires co-factors and is dependant upon the CD14/TLR4 pathway. Panel A: FBS effect on LPS induced TNFalpha secretion. The concentrations of TNFalpha (pg/mL) in the medium of mature adipocyte cultures treated or not with increasing concentrations of FBS and 1 μg/mL of LPS, were determined at 6 hours. The graph represents the mean ± SE of the results from two patients. ***P < 0.001%, *P < 0.05%, versus control cells. Panel B: Effect of TLR4 blocking antibody on TNFalpha secretion. The concentrations of TNFalpha (pg/mL) in the medium of mature adipocyte cultures treated with 1 μg/mL of LPS, with or without 1 h of pre-incubation with a blocking anti-TLR4 antibody (20 μg/mL, Clinisciences, Montrouge, France, clone HTA125) were determined at 6 hours. The graph represents the mean ± SE of the results from two patients. ***P < 0.001%, versus cells treated with LPS alone. Panel C: Effect of CD14 blocking antibody on TNFα secretion. The concentrations of TNFα (pg/mL) in the medium of mature adipocyte cultures treated with 1 μg/mL of LPS, with or without 1 h of pre-incubation with a blocking anti-CD14 antibody (5 μg/mL, Hycult biotechnology, clone 18D11) were determined at 6 hours. The graph represents the mean ± SE of the results from one patient (n = 6 for each condition), representative of two experiments on two different patients. ***P < 0.001%, versus cells treated with LPS alone.

Mentions: The activation of TNFalpha secretion by LPS is receptor specific and dependent upon the bacterial endotoxin binding to the TLR4 receptor [8]. Indeed, the presence of an anti-TLR4 antibody decreases by more than 5-fold the activator effect of LPS (Figure 5B). Nevertheless, LPS requires one or several partner components to be present in the FBS in order to activate TLR4. The absence of FBS in the culture medium strongly limits the LPS activation of TNFalpha secretion (Figure 5A). Moreover, it is highly probable that another TLR4-partner, CD14 [16], is present in the serum, as CD14 is not present on the surface of mature human adipocytes [17]. The use of anti-CD14 antibody confirms that the presence of CD14 is essential to TLR4 signalling. This is demonstrated by the 6-fold reduction in the LPS-effect brought about as a result of antibody blocking (Figure 5C).


Signaling pathways involved in LPS induced TNFalpha production in human adipocytes.

Hoareau L, Bencharif K, Rondeau P, Murumalla R, Ravanan P, Tallet F, Delarue P, Cesari M, Roche R, Festy F - J Inflamm (Lond) (2010)

LPS induced TNFalpha protein secretion requires co-factors and is dependant upon the CD14/TLR4 pathway. Panel A: FBS effect on LPS induced TNFalpha secretion. The concentrations of TNFalpha (pg/mL) in the medium of mature adipocyte cultures treated or not with increasing concentrations of FBS and 1 μg/mL of LPS, were determined at 6 hours. The graph represents the mean ± SE of the results from two patients. ***P < 0.001%, *P < 0.05%, versus control cells. Panel B: Effect of TLR4 blocking antibody on TNFalpha secretion. The concentrations of TNFalpha (pg/mL) in the medium of mature adipocyte cultures treated with 1 μg/mL of LPS, with or without 1 h of pre-incubation with a blocking anti-TLR4 antibody (20 μg/mL, Clinisciences, Montrouge, France, clone HTA125) were determined at 6 hours. The graph represents the mean ± SE of the results from two patients. ***P < 0.001%, versus cells treated with LPS alone. Panel C: Effect of CD14 blocking antibody on TNFα secretion. The concentrations of TNFα (pg/mL) in the medium of mature adipocyte cultures treated with 1 μg/mL of LPS, with or without 1 h of pre-incubation with a blocking anti-CD14 antibody (5 μg/mL, Hycult biotechnology, clone 18D11) were determined at 6 hours. The graph represents the mean ± SE of the results from one patient (n = 6 for each condition), representative of two experiments on two different patients. ***P < 0.001%, versus cells treated with LPS alone.
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Figure 5: LPS induced TNFalpha protein secretion requires co-factors and is dependant upon the CD14/TLR4 pathway. Panel A: FBS effect on LPS induced TNFalpha secretion. The concentrations of TNFalpha (pg/mL) in the medium of mature adipocyte cultures treated or not with increasing concentrations of FBS and 1 μg/mL of LPS, were determined at 6 hours. The graph represents the mean ± SE of the results from two patients. ***P < 0.001%, *P < 0.05%, versus control cells. Panel B: Effect of TLR4 blocking antibody on TNFalpha secretion. The concentrations of TNFalpha (pg/mL) in the medium of mature adipocyte cultures treated with 1 μg/mL of LPS, with or without 1 h of pre-incubation with a blocking anti-TLR4 antibody (20 μg/mL, Clinisciences, Montrouge, France, clone HTA125) were determined at 6 hours. The graph represents the mean ± SE of the results from two patients. ***P < 0.001%, versus cells treated with LPS alone. Panel C: Effect of CD14 blocking antibody on TNFα secretion. The concentrations of TNFα (pg/mL) in the medium of mature adipocyte cultures treated with 1 μg/mL of LPS, with or without 1 h of pre-incubation with a blocking anti-CD14 antibody (5 μg/mL, Hycult biotechnology, clone 18D11) were determined at 6 hours. The graph represents the mean ± SE of the results from one patient (n = 6 for each condition), representative of two experiments on two different patients. ***P < 0.001%, versus cells treated with LPS alone.
Mentions: The activation of TNFalpha secretion by LPS is receptor specific and dependent upon the bacterial endotoxin binding to the TLR4 receptor [8]. Indeed, the presence of an anti-TLR4 antibody decreases by more than 5-fold the activator effect of LPS (Figure 5B). Nevertheless, LPS requires one or several partner components to be present in the FBS in order to activate TLR4. The absence of FBS in the culture medium strongly limits the LPS activation of TNFalpha secretion (Figure 5A). Moreover, it is highly probable that another TLR4-partner, CD14 [16], is present in the serum, as CD14 is not present on the surface of mature human adipocytes [17]. The use of anti-CD14 antibody confirms that the presence of CD14 is essential to TLR4 signalling. This is demonstrated by the 6-fold reduction in the LPS-effect brought about as a result of antibody blocking (Figure 5C).

Bottom Line: Cells were treated with a pathogen-associated molecular pattern: LPS, with and without several kinase inhibitors.Moreover, we demonstrate that the PI3Kinase pathway is clearly involved in the first step of the LPS-pathway.Lastly, we show that adipocytes are able to secrete a large amount of TNFalpha compared to macrophages.

View Article: PubMed Central - HTML - PubMed

Affiliation: LBGM-GEICO, Laboratoire de Biochimie et de Génétique Moléculaire - Groupe d'Etude sur l'Inflammation Chronique et l'Obésité, Université de l'île de la Réunion, 15 avenue René Cassin 97715 Saint Denis Messag Cedex, France.

ABSTRACT

Background: The development of obesity has been linked to an inflammatory process, and the role of adipose tissue in the secretion of pro-inflammatory molecules such as IL-6 or TNFalpha has now been largely confirmed. Although TNFalpha secretion by adipose cells is probably induced, most notably by TLR ligands, the activation and secretion pathways of this cytokine are not yet entirely understood. Moreover, given that macrophagic infiltration is a characteristic of obesity, it is difficult to clearly establish the level of involvement of the different cellular types present within the adipose tissue during inflammation.

Methods: Primary cultures of human adipocytes and human peripheral blood mononuclear cells were used. Cells were treated with a pathogen-associated molecular pattern: LPS, with and without several kinase inhibitors. Western blot for p38 MAP Kinase was performed on cell lysates. TNFalpha mRNA was detected in cells by RT-PCR and TNFalpha protein was detected in supernatants by ELISA assays.

Results: WE SHOW FOR THE FIRST TIME THAT THE PRODUCTION OF TNFALPHA IN MATURE HUMAN ADIPOCYTES IS MAINLY DEPENDENT UPON TWO PATHWAYS: NFkappaB and p38 MAP Kinase. Moreover, we demonstrate that the PI3Kinase pathway is clearly involved in the first step of the LPS-pathway. Lastly, we show that adipocytes are able to secrete a large amount of TNFalpha compared to macrophages.

Conclusion: This study clearly demonstrates that the LPS induced activation pathway is an integral part of the inflammatory process linked to obesity, and that adipocytes are responsible for most of the secreted TNFalpha in inflamed adipose tissue, through TLR4 activation.

No MeSH data available.


Related in: MedlinePlus