The Role And Mechanism Of PD-1 Induced Immunosupression In Susceptibility To Infection In The Aged Following Hip Fracture Surgery

Hip fracture is one of the most common osteoporotic fractures in the aged. The incidence increases continuously with the aging process in our country. A lot of clinical data show that pulmonary infection is one of the most common complications and the MODS caused by pulmonary infection is the most common reason for death. The immune disorders caused by trauma and surgical strike are closely related to complications such as pulmonary infection, sepsis and MODS. The post-traumatic immune disorders usually manifests the obvious inhibition of biological processes including antigen recognition, antigen presentation, T cell activation and inflammatory factors secretion, the decrease of pathogens clearance and the increase of infectious susceptibility, which has liability to serious complications such as pulmonary infection, sepsis and MODS. As the weakness of the immune compensation ability induced by aging, when the aged suffer from trauma and surgical strike, the possibility and degree of immune inhibition is greater and deeper and the infectious complications resulting in death is easier. Currently, Most of the researches related to post-traumatic immune inhibition only reported the observation of a phenomenon instead of mechanism research or effective intervention measures. The researches at home and abroad for the post-traumatic immune inhibition are extremely rare, which is inconsistent with the characteristic of the aging of population across the world. Therefore, it is undoubtedly significant to decrease the post-operation pulmonary infection and mortality for exploring the molecular mechanisms and the new intervention targets of the immune inhibition after the operation of the old patients with hip fracture.Programmed death-1(PD-1) is one of the important immunosuppressant molecules which mainly expresses on the surface of T cells and plays a negative regulation role in functions of T cells. Lots of researches indicated that PD-1 plays an important role in the processes of the immune inhibition caused by tumor, chronic viral infection and sepsis. Experiments in vivo and in vitro confirmed that PD-1 gene knockout or treatment of anti PD-1 antibody can reverse the functional failure and maintain the function of T cells to raise survival rate of experimental animals. Drugs to block PD-1 have already been used in clinical to enhance immune functions of patients with advanced malignant tumors and get good results. But, it is unclear for the function and molecular mechanisms of PD-1 in the development of post-operation immune inhibition and infectious complications. We assume that hip fracture and operation can result in immune disorders and increased PD-1 expression of T cell and consequently lead to inhibited immune responses, decreased immunity function of T cells and the ratio of bacterial clearance and increased the infectious susceptibility in the aged. Maybe anti-PD-1 antibody treatment could reverse the post-operation immune inhibition, restore the anti-infection ability and decrease pulmonary infection rate and mortality.1. Objective1.1 To establish a model of femur proximal fracture and femoral internal fixation with intramedullary nail in aged animal and to understand the variations of inflammatory immune response and the susceptibility to bacteria after operation of hip fracture.1.2 To confirm the change of PD-1 expression of T cells in peripheral blood of old patients with hip fracture in perioperative period and to analyze the correlation between the changes and infectious complications.1.3 To confirm the role of PD-1 blocker to enhance the immune function, restore T cells function and decrease the infection rate and mortality post the operation in the aged mice with hip fracture.2. Methods2.1 To establish animal models of proximal femoral fractures in rats by a blunt guillotine ramming apparatus under the guidance of C-arm so that the femoral internal fixation with intramedullary nail in clinical can be simulated by open reduction and internal fixation with Kirschner wires. Levels of inflammatory factors(TNF-α、IL-1β、IL-6、IL-10)in peripheral blood, protein concentration and inflammatory cells counts(total cells, macrophages, lymphocytes and neutrophils) in bronchoalveolar lavage fluid,the activity of casepase-3 in lung tissue, pathological examination of lung tissue and tissue injury score were test at 1, 3 and 5 days after operation. Then the P. aeruginosa were inoculated in trachea at 1, 3, 5 days after operation. Bacterial load in blood and bronchoalveolar lavage fluid were examined 24 h after inoculation and 14-day mortality is observed.2.2 From August 2013 to June 2014, the clinical materials of 119 old patients who had suffered from intertrochanteric fractures of femur in our department were collected. The PD-1 expression of peripheral T-cell of them was tested by flow cytometry at 1 day after admission, 1, 3, 5 days after operation.2.3 To replicate the model of proximal femoral fractures and femoral internal fixation with intramedullary nail in mice, and the mice were intervented by PD-1 blocker immediately after operation. The PD-1 expression and the apoptosis of T cell in spleen tissue and lung tissue were tested by flow cytometry and immunoflurescent method, the levels of inflammatory factors(IFN-γ、IL-10、IL-2) in peripheral blood were examined by Elisa. Then the P. aeruginosa were inoculated to the injured rats which were intervented by PD-1 blocker. Bacterial load were evaluated 24 h after inoculation and 14-day mortality was observed.3. Results3.1 The model of proximal femoral fractures and femoral internal fixation with intramedullary nail in rats has obvious advantages as simple operation, good controllability and a high success rate. The 14-day survival rate can reach to 90% with low variation. Hip fracture and the strike of femoral internal fixation with intramedullary nail can lead to obvious systemic inflammatory response syndrome and lung injury in aged rats during the acute phase(1-3days) after injury and surgery, which included significantly increased levels of inflammatory factors in plasma(IL-6、TNF-α、IL-1β、IL-10),protein concentration and inflammatory cells counts(macrophages, lymphocytes and neutrophils) in bronchoalveolar lavage fluid, enhanced activity of casepase-3 in lung tissue and aggravated histological changes. But the changes above were self-limited and recovered to normal levels at 7 days after operation without interference. The susceptibility to bacteria increased significantly at 1 3 days after the operation of hip fracture in the aged mice which included the significantly increased bacterial load in blood and bronchoalveolar lav age fluid after the P. aeruginosa instilled, as well as the decreased 14-day survival rate.3.2 The PD-1 expression of T cell in peripheral blood significantly increased in aged patients after femoral internal fixation with intramedullary nail and returned to pre-operation level until the 5th day after operation. In the old patients who suffered from hip fracture with infectious complication after operation, the PD-1 expression of T-cell in peripheral blood increased continuously and reached at the peak at the 3th day after operation and returned to pre-operation level at the 5th day after operation. In the patients without infectious complication, the PD-1 expression of T-cell in peripheral blood increased transiently at the 1th day after operation. The difference of PD-1 expression of T cell between the 1th day and the 3th day(PD-1D3-D1) after operation can be used to predict the incidence of infectious complication in 28 days after operation and has high sensitivity and specificity, which greater than 3.4% has good value of warming.The sensitivity and specificity of PD-1D3-D1(The difference of PD-1 expression of T cell between the 1th day and the 3th day) was high in predicting the incidence of infectious complication in 28 days after operation. It had better predicting value when > 3.4%.3.3 Hip fracture and the operation strike can significantly increase the PD-1 expression of CD3+ T cell in spleen and lung and the apoptosis of T cell in aged rats. The PD-1 expression and the apoptosis of lymphocyte were inhibited by injecting anti PD-1 antibody via caudal vein in advance. The anti-PD-1 antibody treatment can significantly enhance the ability of anti-infection, decrease the degree of immune inhibition, partially restore the secretion functions of T cell, as well as decrease the bacterial load in blood and bronchoalveolar lavage fluid in aged rats after the operation of hip fracture. And the 14-day survival rate increased from 25%-33.3% to 58.3%.4. Conclusions4.1 Hip fracture and femoral internal fixation with intramedullary nail in aged rats induced a systemic inflammatory response and lung injury associated with increased susceptibility to infection and impaired immune function during the acute phase(1-3 days) after injury and surgery.4.2 The PD-1 expression of T cell in peripheral blood significantly increased after femoral internal fixation with intramedullary nail in aged patients. The change of PD-1D3-D1(the difference of PD-1 expression on the T cell between the 1th day and the 3th day) may be a reliable predictor for infectious complications in aged patients of hip fracture.4.3 PD-1 blocker can significantly enhance the ability of anti-infection, partially restore T cells functions, and decrease the susceptibility to bacteria and mortality.