Changes Of Dendritic Cell Phenotype And Its Clinical Significance In Patients With Severe Community-acquired Pneumonia

Background: Severe community-acquired pneumonia(severe community- acquired pneumonia, SCAP) high mortality, poor prognosis, a major cause of death of patients is infection, and systemic inflammatory response after infection, and then cause sepsis(sepsis),severe sepsis(severe sepsis), septic shock(sepsis shock), triggered multiple organ dysfunction. To participate in the immune system involved, so the study of severe pneumonia immunological mechanisms has attracted great attention from researchers.Dendritic cells(dendritic cells, DC) is the body for Free power system is an important component. As antigen presenting cells, which initiate and control the adaptive immune response against respiratory pathogens. In addition, they function in a variety of the innate immune system, reducing the gap between innate and adaptive immunity. However, changes in severe community acquired pneumonia and its role in peripheral blood dendritic cell subsets and their phenotype is still not very clear.Objective: observational study of patients with severe community-acquired pneumonia in peripheral blood dendritic cell phenotype and its associated functional molecules, analyze the correlation with clinical indicators of severe pneumonia among patients. In addition, by observing the different time points of severe pneumonia model mice spleen, lung changes in dendritic cells, and to explore dendritic cells(DCs) and phenotypic subgroup of severe community-acquired pneumonia in early immune hyperthyroidism and late stages of immunosuppression and its effects.Methods: Flow cytometry SCAP patients and healthy controls frequency and peripheral blood cell surface m DC and p DC expression levels of costimulatory molecules CD80, CD86 and co-inhibitory molecule PD-L1, and to analyze the frequency and DC oxygenation in patients with SCAP index(Pa O2 / Fi O2), blood lactate(Lac) correlation. The72 male SPF Kunming mice were randomly divided into severe pneumonia group(n = 36)and healthy controls(n = 36), each group was randomly divided into six groups(4h group,12 h group, 1d group, 3d group, 5d group, 7d group), severe pneumonia group and the healthy control group were 6 mice per group. By intratracheal injection of Klebsiella pneumoniae method to produce a mouse model of severe pneumonia. In saline alternative Klebsiella pneumoniae was at the hour injection. Sacrificed at different time points in each group of mice, the removal of the lungs and spleen of mice, according to the different groups separately formalin-fixed, cryopreservation, enzymatic digestion or homogenized. Spleen pathological changes detected severe pneumonia group HE staining of mice at different time points, immunohistochemistry was observed in the spleen of mice CD205, CD86 distribution of dendritic cells, flow cytometry assay expression CDllc / CD86 positive cells mark and changes in peripheral blood T cell subsets, with terminal deoxyribonucleotide transferase-mediated biotinylated d UTP nick labeling technique(TUNEL) of severe pneumonia at different time points mice spleen dendritic cells undergo apoptosis original bit detection. Determination of TNF-α concentration in serum of mice were lung homogenates and by the ELISA method. The mice were observed by light microscopy gross pathological changes in lung tissue, and its CD86, CD205 immunohistochemistry, CDllc immunofluorescence labeled mouse lung tissue DCs. Density gradient centrifugation mouse lung tissue mononuclear cells using MACS MACS mouse lung tissue mononuclear cells DCs, DCs were identified identification and DCs cell surface phenotype by flow cytometry techniques. Lung low density mononuclear cells isolated by density gradient centrifugation,macs sorting DCS flow cytometry DCS identification and immunophenotypic analysis.Detection of IL-10 content in DCs cells by ELISA assay.Results:(1) SCAP peripheral blood dendritic cell subsets m DC and p DC relatively healthy control group significantly decreased the expression of frequency(patient group were 0.37 ±0.19 and 0.19 ± 0 12, the control group was 0.51 ± 0.18 and 0.29 ± 0.13, P <0.05).(2) severe pneumonia in patients with m DC frequency and oxygenation index(Pa O2 /Fi O2), blood lactate(Lac) was negatively correlated(r =-0.5878, P <0.0001; r =-0.4628, P= 0.003).(3) SCAP peripheral blood of patients with co-stimulatory dendritic cell surface molecules CD80, CD86 expression relative to healthy controls frequency decreased. Related cosuppression molecule PDL1 relatively healthy control group, the expression of the frequency increase.(4) normal mouse spleen DC content is less, mainly in the spleen marginal zone.Experimental 4h severe pneumonia murine splenic DC cells were significantly hyperplasia and functionally active, CD86 + / CDIlc + DC accounted spleen mononuclear cells was significantly increased compared there was a significant difference(P <0.01) and the normal group; a significant increase in CD205 cells, especially in fringe significant, patchy distribution, the normal control group, the difference was significant(P <0.01); CD86 cells was significantly increased, with the most abundant edges, diffuse, visible DC s brown staining of the membrane was branch shape to extend around, and the normal control group,the difference was significant(P <0.01); white pulp and red pulp with scattered number of TUNEL-positive cells were distributed small pieces increased significantly, showing a large,spotty or nested distribution; CD4 + T cells were significantly decreased, while the CD8 + T cells were significantly increased, CD4 + / CD8 + ratio decreased, and the normal control group, the difference was significant(P <0.05).(5) The model of severe pneumonia in mice 12 ~ 24 h group CD86 + / CDIlc + DC ratio decreased slightly, compared with normal group there are significant differences(P <0.05);CD205 cells slightly reduced compared with 4 h group, distribution is sparse, but still significant more than the normal control group(P <0.05); CD4 + T cell levels continue to the next bottoming out, and CD8 + T cell levels also decreased, CD4 + / CD8 + ratio decreased,and the normal control group, the difference was significant(P <0.01). 3 ~ 5d groups CD86+ / CDllc + DC expression ratio close to normal; CD205 breakdown close to normal control group.(6) The model of severe pneumonia in mice 3 ~ 5d apoptosis of spleen cells than 12 ~24h decreased slightly more than the normal control group; 3-5d CD4 + T cell levels increased, CD4 + / CD8 + ratio rebounded, but still lower than the normal control group.(7) The model of severe pneumonia in mice 5 ~ 7d group splenic CD86 + / CDIlc + DCIX expression was significantly decreased compared there was a significant difference(P <0.01)and normal group; cells in the CD205 increased significantly again, flaky diffuse, and normal control group and experimental group 3 ~ 5d and the difference was significant(P<0.05); CD86 cytoplasmic membrane and some dark brown, no dendritic membrane protrusion, positive cells content than the 3 ~ 5d group slightly increased, 4h increase far less obvious, CD + 86 cells with normal control group and experimental group 3 ~ 5d difference was not significant; 5 ~ 7d apoptotic cells increased again, see the white pulp and red pulp large number of TUNEL-positive withered apoptotic cells; CD4 + T cell levels decrease again, and CD8 + T cells were significantly increased, CD4 + / CD8 + ratio upside down,with the normal control group, the difference was significant(P <0.01).(8) The model of severe pneumonia mouse model 3-5d group, lung tissue fluid and serum TNF-α content than the control group significantly increased by about 3-5 times, with a very significant difference(P<0.01).(9) The mouse model of severe pneumonia group 3-5d, 5-7d group, DCs incubated supernatant significantly increased IL-10 levels than the control group, the difference was statistically significant(P<0.05).(10) A mouse model of severe pneumonia, lung tissue significantly increased the number of CDllc + DCs, 12-24 h group CDllc + cell processes increased significantly concentrated in the blood vessels in the peri-alveolar septa, 5-7d group CDllc + cells reached the highest value, but fuzzy cell structure partly apoptosis.(11) A mouse model of severe pneumonia, CD205 positive cells in 4h group and the normal control group, the difference was not statistically significant(P>0.05), and significantly increased 12-24 h group, a statistically significant difference with the control group(P <0.01), CD205 and CD11 c were significantly increased to about 1: 1, 5-7d group,CD205-positive cells was significantly decreased comparison 12-24h(P<0.05).(12) The mouse model of severe pneumonia, 12-24 h group, CD86-positive cells was significantly increased, with statistical significance(P<0.01) and normal control group and5-7d group differences. CD86-positive cells was significantly decreased compared 12-24 h in5-7d, the difference was significant(P<0.05).Conclusions:(1) Patients with severe community-acquired pneumonia in peripheral blood dendritic cell subsets was significantly reduced. Costimulatory molecules CD80, CD86 expression decreased, suggesting the presence of dendritic cells in patients with SCAP mature dysfunction, antigen-presenting capacity decreased, resulting in reduced immune response.Cosuppression molecule PDL1 expression increased, indicating that SCAP patients immunosuppression is a major cause of poor infection control, and even the occurrence of secondary infection.(2) severe pneumonia in immunocompetent mice spleen DCs undergo a process of evolution from the inhibition to hyperactivity, early after infection spleen dendritic cells in immune activation and immune suppression advanced disease is closely related to SCAP.(3) Early severe pneumonia in mice, raising a large number of interstitial lung DCs,activation, leading to hyperactive immune response, inflammation disorders. Late in mice with severe pneumonia, interstitial lung DCs reduce the activity(expression of CD86 and CD205 declining proportion), a lot of negative feedback regulation of the cytokine IL-10 secretion by inducing immune suppression stage and promote the occurrence and development.