Date on Master's Thesis/Doctoral Dissertation
Microbiology and Immunology
Microbiology and Immunology, PhD
Macrophages; Inflammation; Protein kinases
AMP-activated protein kinase, AMPK, is a conserved serine/threonine kinase with a critical function in the regulation of metabolic pathways in eukaryotic cells. Recently, AMPK has been shown to play an additional role as a regulator of inflammatory activity in leukocytes. Treatment of macrophages with chemical AMPK activators, or forced expression of a constitutively active form of AMPK, results in polarization to an antiinflammatory phenotype. Additionally, we reported previously that stimulation of macrophages with antiinflammatory cytokines such as IL-10, IL-4 and TGF-ß results in rapid activation of AMPK, suggesting that AMPK contributes to the suppressive function of these cytokines. In the current study we investigated the role of AMPK in the regulation of macrophage antiatherogenic functions and demonstrated a new mechanism for AMPK’s role in mediating IL-10-induced gene expression and antiinflammatory effects. The expression of dominant negative (DN-) AMPKa1 in macrophages resulted in a spontaneous obese phenotype in mice associated with fatty liver and heart enlargement. The expression of constitutively active (CA-) AMPKa1 in macrophages resulted in decreased IL-6 production but increased expression of ABCA1 and ApoE. Compared to wild-type macrophages, AMPKa1-deficient macrophages failed to express atheroprotective genes including ApoE, LXRa, and ABCA1 in response to IL-10. Mechanistic studies revealed that IL-10-stimulated wild-type macrophages displayed rapid activation of PI3K and its downstream target Akt, an effect that was not seen in macrophages generated from AMPKa1-deficient mice. Treatment with the PI3K inhibitor LY294002 blocked IL-10’s ability to induce Akt activation but not AMPK activation, suggesting that IL-10-mediated activation of AMPK is independent of PI3K. CA-AMPKa1 macrophages displayed elevated PI3K and CREB activation in response to IL-10 compared to the empty vector transfected macrophages. IL-10 stimulation resulted in increased mTORC1 activity, an Akt downstream target, an effect that was reduced in AMPKa1-deficient mice. IL-10 induced phosphorylation of both Tyr705 and Ser727 residues of STAT3 in an AMPKa1-dependent manner, and these phosphorylation events were blocked by inhibition of CaMKKß, an upstream activator of AMPK, and by the mTORC1 inhibitor rapamycin, respectively. The impaired STAT3 phosphorylation in response to IL-10 observed in AMPKa1-deficient macrophages was accompanied by reduced SOCS3 expression and an inadequacy of IL-10 to suppress LPS-induced proinflammatory cytokine production. Overall, our data demonstrate that AMPKa1 is required for IL-10 activation of the PI3K/Akt/mTORC1 and STAT3/SOCS3 antiinflammatory pathways regulating macrophage polarization, a mechanism with broad-reaching applicability in immune homeostasis and in inflammation-associated diseases such as atherosclerosis, autoimmune diseases, and obesity.
Zhu, Yanfang, "Regulation of macrophage inflammatory signaling pathways by AMP-activated protein kinase." (2014). Electronic Theses and Dissertations. Paper 1649.