Gαᵢ₁ and Gαᵢ₃ regulate macrophage polarization by forming a complex containing CD14 and Gab1

Significance In this study, we demonstrate that guanine nucleotide-binding protein G(i) subunit alpha-1 and alpha-3 (Gα ᵢ₁/₃) regulate the downstream signaling pathways of Toll-like receptor 4 (TLR4). We show that Gα ᵢ₁/₃ form complexes containing the pattern recognition receptor (PRR) CD14 and growth factor receptor binding 2 (Grb2)-associated binding protein (Gab1), which are required for activation of PI3K-Akt signaling and NF-κB activation. Besides, Gα ᵢ₁/₃ act at both the plasma membrane and the endosome levels and are involved in TLR4 endocytosis. Furthermore, Gα ᵢ₁/₃ participated in the induction of M1 polarization of macrophages, and their decreased expression contributed to LPS tolerance. Thus, Gα ᵢ₁/₃ are important in controlling inflammation. Heterotrimeric G proteins have been implicated in Toll-like receptor 4 (TLR4) signaling in macrophages and endothelial cells. However, whether guanine nucleotide-binding protein G(i) subunit alpha-1 and alpha-3 (Gα ᵢ₁/₃) are required for LPS responses remains unclear, and if so, the underlying mechanisms need to be studied. In this study, we demonstrated that, in response to LPS, Gα ᵢ₁/₃ form complexes containing the pattern recognition receptor (PRR) CD14 and growth factor receptor binding 2 (Grb2)-associated binding protein (Gab1), which are required for activation of PI3K-Akt signaling. Gα ᵢ₁/₃ deficiency decreased LPS-induced TLR4 endocytosis, which was associated with decreased phosphorylation of IFN regulatory factor 3 (IRF3). Gα ᵢ₁/₃ knockdown in bone marrow-derived macrophage cells (Gα ᵢ₁/₃ KD BMDMs) exhibited an M2-like phenotype with significantly suppressed production of TNF-α, IL-6, IL-12, and NO in response to LPS. The altered polarization coincided with decreased Akt activation. Further, Gα ᵢ₁/₃ deficiency caused LPS tolerance in mice. In vitro studies revealed that, in LPS-tolerant macrophages, Gα ᵢ₁/₃ were down-regulated partially by the proteasome pathway. Collectively, the present findings demonstrated that Gα ᵢ₁/₃ can interact with CD14/Gab1, which modulates macrophage polarization in vitro and in vivo.