The Role Of KLF2 And S1PR1 In Migration Of Neutrophils In Asthma Patients

Objective: By observing the percentage of peripheral blood neutrophils of normal subjects and patients with asthma, investigating the expression of KLF2 and S1PR1 in neutrophils isolated from peripheral blood, and calculating its migration rate,we aimed to explore the effect of KLF2 and S1PR1 on neutrophils’ migration ability, thus providing new ideas for the prevention and treatment of bronchial asthma.Methods: During April 2014- March 2016, 29 asthma patients with acute exacerbation were collected from Mawangdui hospital of hunan province and Third Hospital of Changsha.Then according to their severity,patients were separated into three groups, mild asthma group(L group),moderate asthma group(M group), severe asthma group(S group).Healthy volunteers, whose gender and age match with asthma patients,were selected as healthy control group(C group). Peripheral blood were collected for extracting neutrophils within 6 hours, followed by Diff Quick staining.The location of KLF2 protein in neutrophils were identified by immunofluorescence.The relative m RNA expression level of KLF2 and S1PR1 in neutrophils were tested by Realtime-PCR,while the relative protein expression level of KLF2 and S1PR1 in neutrophils were tested by Western-blot. Migaration rate of neutrophils were tested by transwell chambers.Results:1. Peripheral blood leukocyte differential counts of each groups. The ratio of neutrophils in healthy control group, mild, moderate,severe asthma group tend to increased(61.35(51.90 ~ 65.70), 62.54(54.75~66.12), 70.20(65.34 ~75.50), 89.84(73.35~ 94.57)). there is a statistical difference between control group, mild asthma group and severe asthma group; there is also a statistical difference between control group, mild asthma group and moderate asthma group(P <0.05).2. Neutrophils isolation and identification We separated peripheral blood neutrophils by gradient centrifugation,then resuspended in PBS. Cells were stained by Diff Quick Dyeing liquid,then observed through oil mirror : the nucleus of neutrophil were kidney-type, rod-shaped or lobulated. Results showed that the neutrophils’ purity is more than 95%, while its survival rate identified by trypan blue staining is greater than 90%.3. Location of KLF2 protein in neutrophils isolated from peripheral blood The results of immunofluorescence showed: KLF2 was expressed in neutrophils in peripheral blood, mainly located in the nucleus,meanwhile there is also little expression in cytoplasm.4.The expression of m RNA of KLF2 and S1PR1 in neutrophils of each group For control group, mild,moderate and severe asthma group, the relative expression of m RNA of KLF2 in neutrophils gradully decreased(1.00(0.96~1.05)、0.80(0.77~0.85)、0.49(0.45~0.52)、0.33(0.32~0.35)),but the relative expression of m RNA of S1PR1 in neutrophils gradully increased(0.99(0.76~1.21)、2.45(2.11~2.81)、3.68(3.41~5.50)、7.52(6.89~7.98)). For hese two factors,there is a statistical difference between moderate, severe asthma group and control group(p<0.01); there is also a statistical difference between severe asthma group and mild asthma group(P<0.01).5.The expression of protein of KLF2 and S1PR1 in neutrophils of each group Results of Western blot showed that: For control group, mild,moderate and severe asthma group, the relative expression of m RNA of KLF2 in neutrophils gradully decreased(0.515(0.390~0.605),0.450(0.385~0.545),0.370(0.310~0.430),0.260(0.245~0.300)),while the relative expression of m RNA of S1PR1 increased( 0.250(0.233~0.345),0.350(0.290~0.435),0.400(0.360~0.410),0.540(0.490~0.615)). For the relative expression of m RNA of KLF2,moderate and severe asthma group were lower than control group(p<0.05), and severe asthma group were lower than mild asthma group(P<0.01). But for the relative expressions of protein of S1PR1, moderate and severe asthma group were higher than control group(p<0.05); severe asthma group were higher than mild asthma group(P<0.05).6. Migration of neutrophils isolated from peripheral blood in each groups According to our previous Preliminary experiments results,we chose 0.1 u M S1 P to finish migration experiments.For control group,mild,moderate and severe asthma group, the migration rate of neutrophils were increased(4.28(3.04~5.06),10.35(9.40~11.18),16.05(14.70~18.15),20.70(19.05~22.80)), and mild, moderate and severe asthma group were higher than control group(p<0.05); severe asthma group were higher than mild asthma group(P<0.05). 7. Pearson correlation analysis on the m RNA and protein exression of KLF2 and S1PR1 The resluts showed there is a negative correlation on the m RNA exression between KLF2 and S1PR1, according to Pearson correlation analysis(r =-0.891, P <0.01). And there is a negative correlation on the protein exression between KLF2 and S1PR1, according to Pearson correlation analysis(r =-0.671, P <0.01).8. Relationship between the protein exression level of KLF2, S1PR1 and the migration of neutrophils We found a negatively correlation between the protein expression level of KLF2 and the migration of neutrophils(r =-0.664, P < 0.01),while there is a positively correlation between the protein expression level of S1PR1 and the migration of neutrophils(r = 0.810, P <0.01).Conclusions:1. The expression of KLF2 in neutrophils is decreased with the aggravation of asthma severity, but the expression of KLF2 in neutrophils is increased, meanwhile the migration rate of neutrophils gradually increased.2. Downregulation of KLF2 in neutrophils of asthma patients may upregulates the expression of S1PR1 to promote the migration of neutrophils.