PKC-epsilon and TLR4 synergistically regulate resistin-mediated inflammation in human macrophages
Mary C Zuniga1, Gayatri Raghuraman1, Elizabeth Hitchner1
1Department of Vascular Surgery, VA Palo Alto Health Care System, Palo Alto, CA, USA.
Background And Aims:
Resistin has been associated with atherosclerotic inflammation and cardiovascular complications. We and others have previously shown that PKC-epsilon (PKCε) is involved in resistin-induced smooth muscle cell (VSMC) dysfunction at a high pathological concentration. This study aimed to evaluate the role and potential pathways of resistin at a physiological concentration, in atherosclerosis-related inflammation.
Methods:
Plasma from patients with atherosclerosis was analyzed for resistin concentration. Patients were divided into tertiles based on resistin levels and cytokines were compared between tertiles. Macrophages were then treated with resistin in the presence or absence of PKCε inhibitor and/or TLR4 blocking-antibody, and their inflammatory state was evaluated with ELISA, RT-PCR, immunocytochemistry, and Western blot.
Results:
We observed significant associations between plasma resistin levels and TNF-α, IL-6, IL-12, MIP-1α, MIP-1β, and CD40L. Our in vitro analyses revealed that resistin activated PKCε via TLR4. This was followed by NF-kB activation and induction of a pro-inflammatory phenotype in macrophages, significantly upregulating CD40, downregulating CD206 and stimulating gene expression and secretion of the inflammatory cytokines, for which we found association in our plasma analysis. Resistin also induced persistent TRAM and CD40L upregulation up to 36 h after resistin treatment. PKCε and TLR4 inhibitors suppressed gene expression to levels similar to control, especially when used in combination.
Conclusions:
Resistin, at a physiological concentration, exacerbates the inflammatory response of macrophages. PKCε is a key upstream mediator in resistin-induced inflammation that may interact synergistically with TLR4 to promote NF-kB activation, while TRAM is an important signal. PKCε and TRAM may represent novel molecular targets for resistin-associated chronic atherosclerotic inflammation.
Insights
Physiological resistin levels worsen macrophage inflammation via PKCε and TLR4 activation, leading to NF-kB signaling. PKCε and TRAM are potential therapeutic targets for atherosclerotic inflammation.
Area of Science:
- Immunology
- Cardiovascular Biology
- Molecular Medicine
Background:
- Resistin is linked to atherosclerotic inflammation and cardiovascular issues.
- Previous studies implicated Protein Kinase C-epsilon (PKCε) in resistin-induced vascular smooth muscle cell (VSMC) dysfunction at high concentrations.
Purpose of the Study:
- To investigate resistin's role and pathways in atherosclerosis-related inflammation at physiological concentrations.
- To elucidate the molecular mechanisms linking resistin to macrophage inflammatory responses.
Main Methods:
- Analyzed plasma resistin and cytokine levels in atherosclerosis patients.
- Treated macrophages with resistin, alone or with PKCε inhibitor and/or TLR4 blocking-antibody.
- Evaluated inflammatory markers using ELISA, RT-PCR, immunocytochemistry, and Western blot.
Main Results:
- Plasma resistin correlated with inflammatory cytokines (TNF-α, IL-6, IL-12, MIP-1α, MIP-1β, CD40L).
- In vitro, resistin activated PKCε via TLR4, leading to NF-kB activation and a pro-inflammatory macrophage phenotype.
- Resistin upregulated CD40, downregulated CD206, and induced persistent TRAM and CD40L expression.
Conclusions:
- Resistin at physiological concentrations exacerbates macrophage inflammation.
- PKCε acts as a key mediator, potentially interacting with TLR4 to drive NF-kB activation.
- PKCε and TRAM are promising molecular targets for treating resistin-associated chronic atherosclerotic inflammation.
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