| Objective Sepsis is a clinical problem in urgent need to be solved, with immunosuppression as a vital cause of death. C larification of the mechanisms of sepsis- induced immunosuppression will help to find new strategies to improve clinical prognosis. It has been reported that TLR4 is closely related to immune response, the cross-talk between Toll- like and chemokine receptors played an important role in immune function and inflammatory reaction. O ur previous results suggested that TLR4 facilitated the endocytosis of chemokine receptor CX3CR1 which was closely related to sepsis-induced immunosuppresion. In the following project, in vivo, ex vivo and in vitro sepsis models, “two-hit” animal model, immune tolerance cell model will be used to verify the mentioned hypotheses by immunofluorescence, q PCR, etc. The object is to clarify the mechanisms of TLR4 and TLR4-dependent C X3CR1 endocytosis in sepsis induced immunosuppression. The successful completion of the current study might find new mechanisms of sepsis and provide a potential target for clinical strategy.Methods Our study was divided into three parts: The first part was to explore the period of sepsis-induced immunosuppression in mice with sepsis after cecal ligation and puncture(C LP) and the condition after “two-hit” model.(1) Forty C57BL/6 mice was randomly divided into four groups, Group A serum and lung were obtained in sham, Group B serum and lung were obtained at the first day after C LP, Group C serum and lung were obtained at the four day after C LP, and Group D serum and lung were obtained at the seventh day after C LP. Elisa was taken to test the concentration of IL-1β, IL-6 and TNF-αin the serum above; q PCE was take n to test the m RNA levels of IL-1β, IL-6 and TNF-α with the lung tissue; HE stain was taken to test the lung tissue of every group; Western Blot was taken to test P-NFκB p65 protein of the lung tissue of every group;(2) Forty C57BL/6 mice was randomly divided into four groups: mice were operated with sham, mice at the first day after CLP were given pneumococcal pneumoniae through nasal cavity, mice at the fourth day after CLP were given pneumococcal pneumoniae through nasal cavity, mice at the seventh day after CLP were given pneumococcal pneumoniae through nasal cavity.Mortality was recorded after surgery. The second part was to explore the effect of TLR4 on immunosuppression and the way of playing a role:(1)Ten of Wild type(WT) mice(C57BL/6 mice) and ten o f TLR4 gene knockout mice(TLR4-/-), only after CLP with 25% ligation 4d, were taken with streptococcus pneumoniae "two-hit" model, observing the survival rate after 7 days of the "two-hit".(2) Forty of C57BL/6 mice with 25% ligation CLP model TLR4-/- mice were randomly divided into four groups, we used immunofluorescence to observe the endocytosis of CX3CR1 of abdominal macrophage at 0,1,4,7d after CLP. The third part was to explore the probable mechanism of endocytosis of C X3CR1 promoting immunosuppression. We inhibited the expression of CX3CR1 or β-arrestin with si RNA, or inhibited the function of clathrin with chlorpromazine, and then built a cell model of endotoxin tolerance with RAW264.7. After the second stimulation of LPS, supernates of cel s were collected and concentration of IL-1β, IL-6 and TNF-α was measured with ELISA.Results 1. The results of ELISA showed that the concentration of IL-1β, IL-6 and TNF-α decreased significantly 4 days after C LP(P<0.05). The results of q PC R showed that the expression of IL-1β, IL-6 and TNF-α of lung tissues of mice 4 days after CLP was significantly decreased(P<0.05). The results of HE staining showed that the greatest damage of lung tissues with the most infiltration of inflammatory cells was appeared on the first day after C LP. Compare with other time points, expression of P-NFκB p65 of lung tissues on the first day after CLP was the highest The survival rate of mice given suspension of pneumococcal pneumoniae through nasal cavity at the fourth day after C LP was 40%, which is lowest in the three groups. The survival rate of mice given 25 ul suspension of pneumococcal pneumoniae through nasal cavity at the first and seventh day after CLP was 60% and 80% respectively. 2. Compared with WT mice, after given suspension of pneumococcal pneumoniae through nasal cavity at the fourth day after CLP, the survival rate of TLR4-/- mice was increased significantly. The results of immunofluorescence showed obvious endocytosis of CX3CR1 in the peritoneal macrophage obtained at the fourth day after CLP.3. Compared with RAW264.7 without treatment with si RNA or chlorpromazine, inhibition of CX3CR1, β-arrestin or clathrin could increase the expression of IL-1β, IL-6 and TNF-α under the cell model of endotoxin tolerance(P<0.05).Conclusions 1. Sepsis induces immunosuppression in mice at the fourth day after cecal ligation and puncture(C LP), and the survival rate of mice with the second hit during immunosuppression was decreased. 2. TLR4 knock-out could increase the survival rate of mice with the second hit during immunosuppression; the probal reason of the phenomenon above was TLR4 promoting endocytosis of CX3CR1. 3. TLR4 promotes endocytosis of CX3CR1 probaly through the pathway of clathrin, and endocytosis of β-arrestin also participate in this process. |
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