Background & Aims

Although glycogen synthase kinase 3 β (Gsk3β) has been found to play key roles in ischemia and reperfusion injuries (IRI) of different organs, underlying mechanisms remain ambiguous, partially due to that its chemical inhibitors do not differentiate Gsk3β from its α isoform and have no target cell selectivity in vivo.

Methods

As global Gsk3β gene KO is embryonically lethal, we created myeloid specific Gsk3β KO mice using LyzM-Cre/Loxp-Gsk3β system to studied its roles in regulating macrophages response against IR in a murine liver partial warm ischemia model (90m ischemia in cephalad lobes). Activation and differentiation of both Kuppfer cells (KCs) and bone marrow derived macrophages (BMMs) were studied in vitro.

Results

Myeloid specific Gsk3β KO was confirmed by Western blot analysis of liver parenchymal, non-parenchymal cells, as well as bone marrow derived-macrophages (BMMs). These KO mice were protected from liver IRI (sALT and liver histological analysis) with diminished pro-inflammatory immune responses (measured by qRT-PCR of TNF-a, IL-6, IL-10, CXCL10 gene expressions and MPO assays), as compared with WT controls. Gsk3β deficient BMMs expressed higher levels of M2 (Arginase and MRC1), lower levels of M1 (iNOS) markers at the resting state, and responded to TLR stimulations with comparable levels of TNF-a, IL-6, lower levels of IL-12/23, but much higher levels of IL-10, productions, as compared with WT cells. The IL-10 high phenotype and elevated M2 marker expressions in Gsk3β deficient BMMs were sustained even under M1 polarization condition (IFN-γ). The inhibitory phosphorylation of AMPKa at Thr479 was diminished in LPS-stimulated Gsk3β deficient BMMs, with simultaneous increase of its activating phosphorylation at Thr172. More prominently, Gsk3β deficiency resulted in a significantly higher level of induction of SHP, the newly identified innate immune negative regulator, upon TLR stimulation. Inhibition of AMPK (Compound C) attenuated the IL-10 high phenotype and SHP induction in Gsk3β deficient BMMs.

Conclusions

Gsk3β promotes liver pro-inflammatory immune activation by limiting the induction of SHP in activated macrophages via the AMPK signaling pathway.

CITATION INFORMATION: Zhou H, Zhu J, Yue S, Busuttil R, Kupiec-Weglinski J, Wang X, Zhai Y. Gsk3β Regulates Macrophage Pro-Inflammatory Immune Activation via AMPK-Mediated Induction of Small Heterodimer Partner (SHP). Am J Transplant. 2016;16 (suppl 3).

« Back to 2016 American Transplant Congress