| Background and objectivesSepsis, a common complication occurred after trauma, shock and surgery, is a highly progressive severe disease. Sepsis and its associated multiple organ dysfunction are the main cause of death in the intensive care unit. Currently, there is no specific therapeutic method in the treatment of sepsis. Macrophage, the main component in the innate immune system, plays a key role in the initiation of inflammatory response and clearance of invading microbes. Recent studies demonstrated that macrophage dysfunction contributed to the progression of sepsis. In the early phase, macrophage was activated by various foreign materials and subsequently produced large mount of inflammatory mediators, which attracted the migration of neutrophils and activated the systematic inflammatory response. Moreover, macrophage releases certain cytokines, processes and presents specific antigens, which help in the activation of lymphocytes in acquired immunity. It is believed that macrophage underwent dysfunction following activation in sepsis, which is characterized by lessened cytokines production and impaired phagocytic ability. There is increasing evidence which indicates that PD-1:B7H1 pathway contributes to sepsis induced immunosuppression. Both PD-1 and B7H1 deficient mice have higher survival rate during sepsis which is associated with ameliorated organ injury, reduced cytokines productions and enhanced bacterial clearance. Notably, the dysfunction of macrophage in sepsis was mitigated in PD-1-/- and B7H1-/- mice, characterizing by increased migration of macrophage in infection site and enhanced cytokines production. Macrophage phagocytic capacity, which was impaired following CLP, was also protected in PD-1-/- mice. In septic patients, our previous studies also found that blockade of PD-1:B7H1 pathway improved the monocytes functions with higher cytokines production in septic patients. In summary, PD-1:B7H1 may play an important role in macrophage dysfunction during sepsis. PD-1 and B7H1 seems to have different effects on the modulation of macrophage functions. How the expressions of PD-1 and B7H1 on macrophage were regulated and the effects on macrophage functions and phenotype remained to be determined.In the present study, we used both bone marrow derived macrophage(BMDM) and peritoneal macrophage(PM) cultured from wild type, PD-1-/- and B7H1-/- mice. We designed this experiment in aim to determine cytokines production, antigens presenting molecules expression, macrophage polarization and phagocytic ability in PD-1 and B7H1 deficient mice. To further explore the role of PD-1:B7H1 pathway in sepsis, we also looked at the macrophage activation and phenotype in mice subjected to polymicrobial sepsis. Materials and methods1. In BMDM cultivation, bone marrow was harvested from age-matched male C57BL/6, PD-1-/- and B7H1-/- mice(n=8 in each group) and cultured with M-CSF for 7days. For PM, C57BL/6, PD-1-/- and B7H1-/- mice(n=8 in each group) were intra-peritoneally injected with 1ml 3% thioglucollate, and peritoneal lavage was harvested 4 days post injection. Macrophage was stained with F4/80 and detected by flow cytometry.2. Both BMDM and PM were treated with various concentrations of LPS for 24 hours. Supernatants were collected, and expressions of TNF-α, IL-6, IL-10 and IL-12p70 were determined by ELISA. C57BL/6, PD-1-/- and B7H1-/- mice(n=6 in each group) were intra-peritoneally injected with 5mg/kg LPS. 8 hours later, blood and peritoneal lavage were harvested and examined by ELISA.3. BMDM and PM from each group were treated with LPS or IFN-γ for 24 hours, membranous expression of CD80, CD86, CD40 and MHC-II were determined by flow cytometry. Phosphorylation of STAT-1 and STAT-3 in IFN-γ stimulated BMDM were measured with western-blotting.4. Age-matched male C57BL/6, PD-1-/- and B7H1-/- mice were randomly distributed to control and CLP group(n=6 in each group). Expression of CD80, CD86, CD40 and MHCII on peritoneal macrophage and splenic macrophage were determined with flow cytometry. Serum IFN-γ and IL-12p70 expressions in naive mice were examined by ELISA. Phosphorylation of STAT-4 in peritoneal macrophage was determined by western-blotting.5. BMDM were cultured and stimulated with IFN-γ or IL-4 for 24 h to induce polarization. Total RNA and protein were collected and processed. Expression of i-NOS, arginase, CD206 and YM-1 were determined using real-time PCR and western blotting.6. BMDM were cultured and stimulated with or without LPS, then p Hrodo RED E. coli Bio Particles Conjugate were added. Samples were determined by flow cytometry. Results1. Both BMDM and PM cultured from WT, PD-1-/- and B7H1-/- mice grew well with the purity at 80-95%.2. With low concentration of LPS(1ng/ml) stimulation, BMDM and PM from PD-1 or B7H1 deficient mice produced higher levels of cytokines compared with wide type group. Upon high concentration of LPS(100ng/ml) stimulation, there was no difference among groups. In vivo, there were higher levels of TNF-α,IL-6,IL-10 and IL-12p70 in serum samples from PD-1 or B7H1 deficient mice. In parallel, there were higher levels of IL-6 and IL-12p70 in peritoneal lavage. TNF-α and IL-10 expression in peritoneal lavage were too low to be detected.3. CD80 and MHC-II expression on BMDM from B7H1-/- group were lower than that in wild type group, and CD80 remains lower upon LPS or IFN-γ stimulation. MHC-II expression was much higher in PM in B7H1-/- group compared with wild type group. There were much higher expressions of CD80, CD86 and MHC-II on PM in PD-1-/- group. There was no difference regarding the expression of phos-STAT-1 or phos-STAT-3 with or without IFN-γ stimulation among wild type and PD-1-/- or B7H1-/- group.4. There was much higher MHC-II expression on PM in naive PD-1-/- and B7H1-/- mice compared to wild type mice. Expressions of CD80 on PM and SM were higher than that in wild type mice. Compared to wild type mice, there were higher levels of IL-12p70 in the serum from PD-1-/- and B7H1-/- mice, and there was no difference regarding IFN-γ. Accordingly, the expression of pho-STAT-4 was higher in PM from PD-1-/- and B7H1-/-. Compared to naive mice, sepsis induced a great loss of CD86, CD40 and MHC-II expression on PM, while it led to increased expression of CD80 on SM. 24 h after CLP, there were comparable expression levels of antigen presenting molecules among wild type, PD-1-/- and B7H1-/- mice.5. Compared to wild type group, IFN-γ induced comparable expression levels of i-NOS m RNA and protein. IL-4 induced significant up-regulation of arginase, CD206 and YM-1, and there was no significant difference among groups except for the higher CD206 protein expression in B7H1-/- group.6. Pretreatment with LPS did not change the phagocytosis capacity in macrophage. There was no significant difference regarding phagocytosis ability among wild type, PD-1-/- and B7H1-/- groups. ConclusionsPD-1:B7H1 pathways contributed to the down-regulation in LPS induced cytokines productions. PD-1 or B7H1 gene deficiency had different effects on the expression of antigen presenting molecules, which depended on macrophage compartments and stimulation methods. The higher levels of IL-12p70 in PD-1-/- and B7H1-/- mice seems to be involved in the modulation of macrophage phenotype through STAT-4 pathway. PD-1:B7H1 pathway seems to have no effect on macrophage polarization and phagocytosis. and there was no significant difference among groups except for the higher CD206 protein expression in B7H1-/- group. |
PDF Full Text Request