Phosphoproteomic analysis of Fc gamma receptor Iib deficient macrophages in enhancing the severity of endotoxin tolerance

Abstract

Sepsis is a severe response to systemic infection and one of the leading causes of death in patients world-wide. Bacterial endotoxin (lipopolysaccharide; LPS), is a potent inducer of inflammation that has been associated with the pathophysiology of sepsis. The repeated LPS exposure can induce a suppressive effect as refer to endotoxin tolerance. Fc gamma receptor IIb (CD32b), the only inhibitory receptor among FcGR family, associates with systemic lupus erythematosus (FcGRIIb defunctioning polymorphisms) which are commonly found in Asian populations. Because of an inhibitory nature of the signaling, endotoxin tolerance in lupus with FcGRIIb deficient (FcGRIIb-/-) is interesting and might be translated into the infection in lupus patients. Therefore, we aim to explore the endotoxin tolerance in FcGRIIb-/- mice and the underlying mechanisms. Indeed, several characteristics of endotoxin tolerance using two sequential LPS stimulations in FcGRIIb-/- bone marrow-derived macrophage (BMM) including cytokine production, phagocytosis and killing activity were lower than the wild-type (WT) cell. For in vivo, cecal ligation and puncture (CLP) induce sepsis after LPS preconditioning was more severe in FcGRIIb -/- mice, as measured by mortality rate, bacteria count in blood, serum cytokines, creatinine (kidney injury) and alanine transaminase (liver damage). Then the phosphoproteomic analysis, using a dimethyl labeling method, in endotoxin tolerance of FcGRIIb-/- macrophage revealed the prominently decreased protein kinase C-β type II (PKCBII) in comparison with WT cell. In addition, the validation analysis including western blot analysis of macrophage in vitro and the immunoblot of spleen from FcGRIIb-/- mice with sequential LPS administration demonstrated the lower PKCBII in LPS tolerance FcGRIIb-/- groups. The impact of FcGRIIb and endotoxin tolerance association with sepsis might lead to the new strategies for the prevention and/or treatment of bacterial infections in patients with lupus in the future