Role Of C5a In The Pathogenesis Of Sepsis

Sepsis is a life-threatening medical condition caused by various microorganisms entering the human bloodstream triggering an uncontrolled inflammatory reaction, with 1.5 million septic patients per year in industrialized countries and a very high mortality rate (30-50%)。How to stop the hyper-reactive inflammatory state of sepsis timely and restore the immunobalance is still the focus of sepsis treatment.During the onset of sepsis, a lot of cytokines including TNF-α,LPS and IL-6 were released. They are also major therapeutic targets in treating sepsis, but in practice, antibody aiming at TNF-αand LPS has not resulted in expected results, and deletion of IL-6 still didn't decrease the mortality. The pathogenesis of sepsis is complex in clinic, so effective therapy of Sepsis is still to be identified. Investigating the mechanism and finding effective treatment strategy is of special importance.In the past decade, the role of complement system in sepsis was identified. Complement system is strongly activated during sepsis. As a result, a large amount of C5a is produced. C5a is one of the most potent inflammatory peptides, with a broad spectrum of functions. It has also been found to be a vasodilator. and strong chemoattractant for neutrophils, monocytes and macrophages. C5a stimulates the synthesis and release of proinflammatory cytokines such as TNF-α, IL-β, IL-6, and IL-8. It has been reported that blockade of C5a or its receptor C5aR can inhibit the development of sepsis, and greatly improves survivals. C5a has been recognized as a key player in the pathogenesis of sepsis, but a lot of work should be done to identify the mechanism by which C5a mediated inflammation in sepsis.IL-17 is a newly reported cytokine which is involved in autoimmune disease and chronic infection disease. However, the role of IL-17 in acute infection disease such as sepsis is not clear. Our recognition of IL-17 is derived from a newly detected CD4+T cells (Th17 cells). Apart from Th17 cells,γδT cells was also identified as another important IL-17-secreting cells. Until now, the mechanism of IL-17 in autoimmune disease is well identified, but in infection environment, the mechanism of IL-17 secretion and the possibility of using IL-17 as a new target for treatment are still to be identified.Dendritic cells (DC) are professional antigen-presenting cells and serve a guarding role in the immune system. DC release cytokines, which polarize T helper (Th) toward Th1 or Th2. It is believed that DC determines the results of the immune response during infection. In sepsis, DCs become rapidly mature but fail to initiate a protective Th1-type immune response. It is known that DC functions are compromised by sepsis. As C5aR is expressed on DC, it provided a perfect candidate for studying the effects of C5a.In order to recognize the pathogenesis of sepsis comprehensively, in our research, we firstly established a mouse sepsais (cecal ligation puncture) model and inspected the change of IL-6,IFN-γ,TNF-αand IL-17. In our model, we found C5a and IL-17 play an important role in sepsis. Moreover, C5a can regulate IL-17 secretion in vivo. These findings provided a new pathway for investigation mechanism of sepsis and may open up a new chapter for complement research. Some details of this research are followed.(1) Establishment and Identification of CLP modelTo investigate the pathogenesis of sepsis, we established the CLP model in mouse. After sepsis happened, we found that the animal survival condition is serious, combined with low survival rate , decreased weight, swelled spleen and pathologic change was also found in spleen and thymus, apoptosis appeared in splenocytes and thymocytes, a lot of cytokine including TNF-α,IL-6,INF-γ,IL-17 increased. Those results confirmed our sepsis model. it provided a firm foundation for our subsequent research.(2) Role of C5a in sepsis and it's relationship with cytokine IL-17In the part, we focused on the role of C5a and IL-17 in sepsis and the relationship between them. Firstly, we prepared the polyclonal anti-C5a antibody, which was identified by SDS-PAGE and antigen binding assay. After C5a blocked, the survival of sepsis is significantly increased when compared with antibody control. Anti–C5a antibody can also decreased IL-17 secretion. These results confirmed the importance of C5a and IL-17 in sepsis, and indicated a new IL-17 induction way in which C5a might be involved.(3) Role of DC cells in C5a modulated IL-17 secretionIt is known that DC functions are compromised by sepsis. More recently, it has been found that C5aR is expressed on DC. Thus, DC serves as a perfect candidate for C5a to regulate immune system. In our vitro experiment, we found C5a can mediated IL-17 production by activation DC cells. Through interaction with C5aR expressed on DC cells, C5a can significantly increase IL-17 production in virtue of IL-6 and TGF-β. We also ascertained that IL-17 is mainly produced byγδT cells. In this study we confirmed that IL-17 could be induced by C5a in vivo.. (4) Mechanism of C5a induced IL-17 production in sepsisAs we have ascertained that IL-17 could be modulated by C5a, in the following experiment, we focused on the mechanism of IL-17 induction in vivo. By microbeads selection, we firstly confirmed that in sepsis IL-17 was mainly produced byγδT cells. After sepsis happened, both the percentage ofγδT cells and IL-17 secretion capacity increased, anti-C5a antibody administration reduced the capacity of IL-17 production ofγδT cells, but it did not affect the percentage ofγδT cells. In vivo, we identified that IL-17 was secreted mainly byγδT cells.C5a can modulate IL-17 production through indirectly interaction withγδT cells.In this study, we confirmed C5a and IL-17 play important roles in sepsis. Moreover, C5a might modulate IL-17 through indirectly interacting withγδT cells. In vitro, we also identified through interaction with C5aR expressed on DC cells, C5a Can significantly increased IL-17 production in IL-6 and TGF-βdependent way. Here our investigation on the relationship between C5a and IL-17 will provided much needed insight into the immunological networks among the key players in the pathogenesis of sepsis. This line of work will help explain how C5a crosses the bridge of innate immunity to regulate the adaptive immune system, which may open up a new chapter for complement research, and will be helpful for developing more effective therapeutic strategies for sepsis and other immunological diseases.