FcγRⅢB Accelerates The Progression Of Sepsis By Inducing Neutrophil Apoptosis

IntroductionSepsis is characterized by a whole-body inflammatory state (Systemic Inflammatory Response Syndrome) induced by infection. As a serious medical condition, sepsis is the most important cause of acute kidney injury which increases patient morbidity and predicts higher mortality. Many clinical trials in sepsis failed in recent decades, which showed that single-drug therapy was not effective in sepsis patients. On the other hand, individual difference might be one key reason for the failure of these clinical trials. Unknown pathological mechanisms, susceptibility gene and effective therapies might be the hotspots in research field for sepsis.Sepsis is a disesase of the microcirculation. Disturbance of the delicate balance between oxygen delivery and oxygen consumption to the tissues could be associated with sepsis. Multiple orgen failure with a high probability of death would develop if this microcirculatory state of hypoperfusion is not reversed in a timely manner. Neutrophils account for 50% to 70% of all circulating leukocytes in humans and they are the first line of host defense against a wild range of infectious pathogens. Neutrophil apoptosis is closely related to the progression of sepsis. Proinflammatory response predominates in early phase of sepsis, whereas anti-inflammatory response becomes predominant in late phase of sepsis. Neutrophil apoptosis is essential for resolution of inflammation during early phase because they are the key source of proinflammatory cytokines. However, neutrophils would lose their ability of extravasation and recruitment to sites of infection if their apoptosis occurred in late phase. Moreover, most of immune cells would apoptosis in late phase, including macrophages. Apoptotic bodies from neutrophils would not be cleaned by apoptotic macrophages. Thus these apoptotic bodies would be another source of inflammation, which would lead to vascular occlusion and further impede tissue adequate oxygenation.FcγRⅢB is mainly expressed on neutrophils and its deficiency is related to infection resistance. Furthermore, FcγRⅢB is associated with neutrophil apoptosis regulation. We want to find how FcγRⅢB affects neutrophils’ viability in late phase of sepsis and whether it would influence the progression of sepsis.Fcgr3B gene, encodes FcγRⅢB, has the feature of copy number variation. Copy number variation is related to disease susceptibility and individual differences. Low Fcgr3B copy number is associated with systemic lupus erythematosus. We want to identify the role of FcγRⅢB played in sepsis by analyzing Fcgr3B copy number variation in severe sepsis patients.FcγRⅢB is GPI-anchored protein and thus lacks any obvious means of signal transduction upon cross-linking. FcγRⅢB would transduct signal if it was cross-linked with FcγRⅢB or Mac-1. Recent researches indicated that FcγRⅢB alone would internalize soluble immune complexes through a mechanism used by GPI-anchored receptors and fluid-phase endocytosis, whereas FcγRⅢB cross-linked with FcγRⅢB would internalize insoluble immune complexes. Moreover, FcγRⅢB alone would impede neutrophil spontaneous apoptosis in normal environment when it binds its autoantibodies. FcγRⅢB cross-linked with Mac-1 would mediate neutrophil tethered to intravascularly deposited IC and promote neutrophil rolling and adhesion to the vessel wall. We want to find how FcγRⅢB affects neutrophil apoptosis and phagocytosis in late phase of sepsis and whether it would influence the progression of sepsis. Meanwhile we want to verify this relationship between FcγRⅢB and neutrophil apoptosis in vitro and find some key regulating factors.Part I Fcgr3B copy number variation is associated with severe sepsisObjective We want to identify the role of FcγRⅢB played in sepsis by analyzing Fcgr3B copy number variation in severe sepsis patients and healthy volunteers.Methods 468 severe sepsis patients and 450 healthy volunteers were enrolled. We assayed Fcgr3B copy number in all individuals by real-time PCR and compared the difference of Fcgr3B copy number variation between severe sepsis patients and healthy volunteers.Results The frequencies of Fcgr3B gene 0,1,2,3,4,5, and>6 copies in severe sepsis patients were 0.2%,18.6%,51.3%,26.5%,1.9%,0.4%, and 1.1% respectively. The frequencies of Fcgr3B gene 0,1,2,3,4,5, and>6 copies in healthy volunteers were 2.4%,39.3%,47.3%,6.9%,2.7%,0.9%, and 0.4% respectively. We divided all sepsis patients and healthy volunteers into three groups as low copies group (<2 copies),2 copies group and high copies group (>2 copies) by Fcgr3B copy number. The frequencies of Fcgr3B gene<2 copies,2 copies, and>2 copies in severe sepsis patients were 18.8%,51.3%, and 29.9% respectively. The frequencies of Fcgr3B gene<2 copies, 2 copies, and>2 copies in healthy volunteers were 41.8%,47.3%, and 10.9% respectively. The frequency of Fcgr3B high copies in severe sepsis patients was significantly higher than healthy volunteers (χ2(df)=84.01(2), P<0.0001). By logistic regression analysis, possession of a copy number of Fcgr3B more than two copies was associated with an increased risk of developing severe sepsis (OR=2.536, 95%CI=1.745-3.686, P<0.0001). Possession of a copy number of Fcgr3B less than two copies was associated with a decreased risk of developing severe sepsis (OR=0.415, 95%CI=0.304-0.568, P<0.0001).Conclusions Fcgr3B high copy number (>2 copies) was associated with an increased risk of developing severe sepsis in this Chinese population.Part II Role of neutrophil FcγRⅢB played in mice sepsis modelObjective We want to find how FcγRⅢB affects neutrophil apoptosis and phagocytosis in late phase of sepsis and whether it would influence the progression of sepsis.Methods Fcgr3B+Fcerlg+/+, Fcgr3B+Fcerlg-/-and Fcgr3B+Fcgr2A+Fcerlg-/-C57BL/6 mice belonged to experimental groups which expressed FcγRⅢB. Fcerlg+/+ and Fcer1g-/-C57BL/6 mice belonged to control groups which did not express FcγRⅢB. All five kinds of mice were randomly divided into cecal ligation and puncture (CLP)-induced sepsis model group and sham group. Survival, serum lactate and TNF-a production, neutrophil apoptosis, and neutrophil phagocytosis ability were evaluated.Results Our study showed that decreased survival of Fcgr3B+ mice after CLP compared with Fcgr3B- mice. Serum lactate levels, TNF-a levels and neutrophil apoptosis level were increased in Fcgr3B+ mice after CLP compared with Fcgr3B-mice. Neutrophil phagocytosis ability of Fcgr3B+ sepsis mice was worse than Fcgr3B-sepsis mice.Conclusions FcγRⅢB has a deleterious role in CLP-induced polymicrobial sepsis by inducing neutrophil apoptosis, affecting neutrophil phagocytosis ablity, worsening innate immunity and promoting the progression of sepsis.Part Ⅲ Relationship between FcγRⅢB and neutrophil apoptosis in vitroObjective Second part of our study indicated that FcγRⅢB has a deleterious role in CLP-induced polymicrobial sepsis by inducing neutrophil apoptosis. We designed this part of study to verify this relationship between FcγRⅢBB and neutrophil apoptosis in vitro.Methods Bone marrow neutrophils from Fcgr3B+Fcerlg+/+, Fcgr3B+Fcerlg-/-, Fcgr3B+Fcgr2A+Fcerlg-/-, Fcerlg+/+(WT) and Fcerlg-/- mice were divided into four groups in average:blank control, TNF-α, TNF-α plus second antibody, and TNF-α plus FcγRⅢB cross-linking. Neutrophil apoptosis, caspase 3 expression, caspase 8 expression and SHIP expression were evaluated.Results Our study showed that FcγRⅢB cross-linking plus TNF-a increased neutrophil apoptosis level, cleaved caspase 3 expression level, cleaved caspase 8 expression level, SHIP expression level and phosphor-SHIP expression level in Fcgr3B+mice. However, FcγRⅢB cross-linking plus TNF-α showed no effect on neutrophils from Fcgr3B-mice.Conclusions FcγRⅢB plus TNF-α induced neutrophil apoptosis in vitro by activating caspase 3, caspase 8 and SHIP.