Signaling pathways involved in LPS induced TNFalpha production in human adipocytes

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

Autologous Fat Grafting in the Breast: Critical Points and Technique Improvements

International audienceBreast augmentation or reconstruction is a major challenge in esthetic and reconstructive surgery. While autologous fat grafting (AFG) provides a natural filler and seems easy to harvest, AFG in breast surgery is still problematic especially due to the high resorption rate associated with megavolume transfer. Despite this pending issue, there is growing interest in this method, which is becoming more and more widespread, as can be seen by the recent increase in the number of clinical studies. This review aims to highlight recent knowledge in the technique of AFG to the breast and recent refined procedures to improve fat viability and long-term success of the graft.Methods Clinical publications and trials of AFG to the breast from the past 5 years were examined. Attention was focused on the different AFG steps and the clinical out- comes, in order to highlight the strengths and weaknesses of the available protocols.Results Recent studies have concentrated on new techniques to improve fat viability and graft intake. However, all of these studies use different protocols at each step of the procedure. Furthermore, results may vary depending on the technique used for fat harvesting and processing.Conclusion This review points out the recent advances in breast AFG techniques and their associated outcomes andcomplications. The bibliography has been carefully examined to reach a consensus so that recommendations could be made for each step of the technique with the aim of improving graft viability and long-term volume maintenance

Combined therapy for critical limb ischaemia: Biomimetic PLGA microcarriers potentiates the pro-angiogenic effect of adipose tissue stromal vascular fraction cells

International audienceWe propose a regenerative solution in the treatment of critical limb ischaemia (CLI). Poly‐lactic/glycolic acid microcarriers were prepared and coated with laminin to be sterilized through γ‐irradiation of 25 kGy at low temperature. Stromal vascular fraction (SVF) cells were extracted through enzymatic digestion of adipose tissue. Streptozotocin‐induced diabetic mice underwent arteriotomy and received an administration of SVF cells combined or not with biomimetic microcarriers. Functional evaluation of the ischaemic limb was then reported, and tissue reperfusion was evaluated through fluorescence molecular tomography. Microcarriers were stable and functional after γ‐irradiation until at least 12 months of storage. Mice that received an injection of SVF cells in the ischaemic limb have 22% of supplementary blood supply within this limb 7 days after surgery compared with vehicle, whereas no difference was observed at Day 14. With the combined therapy, the improvement of blood flow is significantly higher compared with vehicle, of about 31% at Day 7 and of about 11% at Day 14. Injection of SVF cells induces a significant 27% decrease of necrosis compared with vehicle. This effect is more important when SVF cells were mixed with biomimetic microcarriers: −37% compared with control. Although SVF cells injection leads to a non‐significant 22% proprioception recovery, the combined therapy induces a significant recovery of about 27% compared with vehicle. We show that the combination of SVF cells from adipose tissue with laminin‐coated poly‐lactic/glycolic acid microcarriers is efficient for critical limb ischaemia therapy in a diabetic mouse model

Presence of TLR2, TLR4, TLR6 on human adipocyte surface

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Fatty acids do not pay the toll: effect of SFA and PUFA on human adipose tissue and mature adipocytes inflammation

<p>Abstract</p> <p>Background</p> <p>On the basis that high fat diet induces inflammation in adipose tissue, we wanted to test the effect of dietary saturated and polysunsaturated fatty acids on human adipose tissue and adipocytes inflammation. Moreover we wanted to determine if TLR2 and TLR4 are involved in this pathway.</p> <p>Methods</p> <p>Human adipose tissue and adipocytes primary cultures were treated with endotoxin-free BSA conjugated with SFA (lauric acid and palmitic acid - LA and PA) and PUFA (eicosapentaeneic acid, docosahexaenoic acid and oleic acid - EPA, DHA and OA) with or without LPS. Cytokines were then assayed by ELISA (TNF-alpha, IL-6 and MCP-1). In order to determine if TLR2 and TLR4 are activated by fatty acid (FA), we used HEK-Blue cells transfected by genes from TLR2 or TLR4 pathways associated with secreted alkaline phosphatase reporter gene.</p> <p>Results</p> <p>None of the FA tested in HEK-Blue cells were able to activate TLR2 or TLR4, which is concordant with the fact that after FA treatment, adipose tissue and adipocytes cytokines levels remain the same as controls. However, all the PUFA tested: DHA, EPA and to a lesser extent OA down-regulated TNF-alpha, IL-6 and MCP-1 secretion in human adipose tissue and adipocytes cultures.</p> <p>Conclusions</p> <p>This study first confirms that FA do not activate TLR2 and TLR4. Moreover by using endotoxin-free BSA, both SFA and PUFA tested were not proinflammatory in human adipose tissue and adipocytes model. More interestingly we showed that some PUFA exert an anti-inflammatory action in human adipose tissue and adipocytes model. These results are important since they clarify the relationship between dietary fatty acids and inflammation linked to obesity.</p

Human Adipocyte and its Participation in Innate Immunity

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Presence of functional TLR2 and TLR4 on human adipocytes

International audienceIn addition to the well-known role of adipose tissue in energy metabolism, it has recently been demonstrated that this tissue can secrete a large array of molecules, including inflammatory cytokines. Furthermore, recent studies suggest that adipose cells can behave as immune cells. Therefore, the aim of this study was to determine the presence of the two most prominent ‘pattern recognition receptors’ for bacterial and fungal cell wall components, TLR2 and TLR4 on human adipose cells, as well as to assess their functionality. We demonstrated that TLR2 and TLR4 were expressed at relatively high levels (compared to a monocyte cell line) on the surface of human adipose cells. Stimulation of human adipocytes with lipopolysaccharide (LPS), or with lipoteichoic acid (LTA), two specific ligands of TLR4 and TLR2, respectively, induced a strong increase in TNFα production. The specificity of the response was demonstrated by the use of anti-TLR4 and anti-TLR2 blocking antibodies, which were able to decrease LPS- or LTA-induced TNFα secretion. Thus, it is clear that these receptors are functional in human adipocytes. This study adds weight to the argument that human fat tissue plays a potential role in innate immunity

VIP and PACAP analogs regulate therapeutic targets in high-risk neuroblastoma cells.

International audienceNeuroblastoma (NB) is a pediatric cancer. New therapies for high-risk NB aim to induce cell differentiation and to inhibit MYCN and ALK signaling in NB. The vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP) are 2 related neuropeptides sharing common receptors. The level of VIP increases with NB differentiation. Here, the effects of VIP and PACAP analogs developed for therapeutic use were studied in MYCN-amplified NB SK-N-DZ and IMR-32 cells and in Kelly cells that in addition present the F1174L ALK mutation. As previously reported by our group in IMR-32 cells, VIP induced neuritogenesis in SK-N-DZ and Kelly cells and reduced MYCN expression in Kelly but not in SK-N-DZ cells. VIP decreased AKT activity in the ALK-mutated Kelly cells. These effects were PKA-dependent. IMR-32, SK-NDZ and Kelly cells expressed the genes encoding the 3 subtypes of VIP and PACAP receptors, VPAC1, VPAC2 and PAC1. In parallel to its effect on MYCN expression, VIP inhibited invasion in IMR-32 and Kelly cells. Among the 3 PACAP analogs tested, [Hyp(2)]PACAP-27 showed higher efficiency than VIP in Kelly cells. These results indicate that VIP and PACAP analogs act on molecular and cellular processes that could reduce aggressiveness of high-risk NB

Human Adipocyte and Its Participation in Innate Immunity

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