Regulation Of High-density Lipoprotein And Its Main Component Apolipoprotein-AI On Group 2 Innate Lymphoid Cells In The Pathogenesis Of Allergic Rhinitis And Its Related Mechanisms

PART I: Regulation of high-density lipoprotein on group 2 innate lymphoid cells in allergic rhinitis.Objective:More and more studies have shown that dyslipidemia is closely related to allergic diseases.High density lipoprotein(HDL)usually exerts its anti-inflammatory and antioxidant effects by inhibiting the chemotaxis and activation of immune cells.The purpose of this study was to investigate the regulatory effect of HDL on type Ⅱ innate lymphoid cells(ILC2)in allergic rhinitis(AR).Methods:The levels of blood lipids and their correlation with symptom scores were analyzed in 20 patients with AR and 20 normal controls.The frequency of ILC2 and its functional phenotype IL-5+ILC2 and IL-13+ILC2 in peripheral blood of the two groups were detected by flow cytometry.Purified ILC2 was stimulated by recombinant human HDL,and the production of cytokines was detected by enzyme-linked immunosorbent assay(ELISA).The reverse transcriptase polymerase chain reaction(RT-PCR)was used to measure the m RNA levels of GATA binding protein 3(GATA3)and retinoic acid related orphan receptor α(ROR α)expressed by ILC2.The proliferation of ILC2 was measured by H-thymidine(3H-Td R)incorporation.Results:The level of HDL in AR group was obviously decreased compared with the control group(31.18±6.52 vs 68.75±16.29,P < 0.05).The frequency of ILC2 and its functional phenotype(IL-5+ILC2 and IL-13+ILC2)in peripheral blood of the AR patients were apparently reduced compared with the control group(ILC2:0.121±0.021 vs 0.026±0.01,P<0.001;IL-5+ILC2: 0.1±0.017 vs 0.026±0.008,P<0.001;IL-13+ILC2: 0.109±0.018 vs 0.029±0.007,P<0.001).In addition,the frequency of ILC2 was positively correlated with the symptom score(r=0.58,P <0.05).The level of serum HDL in patients with AR was negatively correlated with the increase of ILC2,IL-5+ILC2 and IL-13+ILC2(ILC2: r=0.59,P=0.01;IL-5+ILC2:r=0.61,P=0.01;IL-13+ILC2: r=0.52,P=0.001).HDL significantly decreased the levels of ILC2 and type Ⅱ cytokines by inhibiting the expression of GATA3 and Ro Rα(P<0.05).Conclusion:Our data provide preliminary evidence that HDL may play a negative role in ILC2 inflammation in AR,suggesting that HDL may be a promising therapeutic target for AR.PART Ⅱ: Regulation of apolipoprotein-AI on group 2 innate lymphoid cells in allergic rhinitis.Objective:Apolipoprotein(Apo)-AI is not only the main structural and functional apolipoprotein of high density lipoprotein,but also has a variety of anti-inflammatory properties.The purpose of this study was to investigate the regulatory effect of Apo-AI on type Ⅱ innate lymphoid cells(ILC2)in allergic rhinitis(AR).Methods:The protein concentrations of HDL and Apo-AI in peripheral blood of children with AR and normal children were detected by ELISA.We used reverse transcriptase polymerase chain reaction(RT-PCR)to measure the expression of ATP binding cassette transporter A1(ABCA1)in ILC2 cells of AR patients.ILC2 was isolated from mononuclear lymphocytes(PBMC)of patients with AR and cultured with Apo-AI and anti-Apo-AI.The phagocytosis of Apo-AI by ILC2 was observed by confocal laser microscopy.The expression of ABCA1 in ILC2 cells was detected by flow cytometry and RT-PCR.The expression of JAK,MAPK and ERK in ILC2 regulated by Apo-AI was detected by Western Blot assay.The AR mouse model was established and stimulated with Apo-AI.The typical symptoms of AR(sneezing and scratching times)were recorded.The nasal mucosa of mice were stained with hematoxylin-eosin(HE staining).The frequency of ILC2 in nasal mucosa and peripheral blood was detected by flow cytometry.Results:1.When compared with the controls,the concentrations of Apo-AI in serum of AR patients were obviously decreased(147.0±2.723 vs 156.2±4.689,P<0.001).The concentration of Apo-AI in peripheral blood of AR was not only negatively correlated with the frequency of ILC2 cells and their functional phenotype(IL-5+ILC2 and IL-13+ILC2)(ILC2: r=0.49,P=0.01;IL-5+ILC2: r=0.6,P<0.01;IL-13+ILC2: r=0.58,P<0.01),but also had a negative correlation with TNSS score(r=0.64,P<0.01).2.Laser confocal microscopy showed that ILC2 cells could ingest Apo-AI.Flow cytometry and RT-PCR showed that the expression of ABCA1 in ILC2 cells in AR group was lower than that in control group(flow cytometry: 0.03±0.01 vs 0.18±0.07,P<0.05;RT-PCR:77.27±11.98 vs 115.7±18.83,P=0.001).The results of Western Blot assay showed that the phosphorylation of JAK,MAPK and ERK proteins increased significantly after ILC2 was stimulated by Apo-AI.RT-PCR showed that Apo-AI up-regulated the expression of ABCA1 on the surface of ILC2 in a concentration-dependent manner.3.In the mouse model,the sneezing times and nose scratching times of AR mice were significantly higher than those of normal mice(nose scratching: 7.200±2.490 vs2.400±1.342,P<0.05;sneezing: 1.800±1.095 vs 0.400±0.547,P<0.05),but allergic symptoms could be inhibited by Apo-AI(nose scratching: 4.500±1.225 vs7.200±2.490,P<0.05;sneezing: 0667±0.817 vs 1.800±1.095,P<0.05).In addition,the HE staining of nasal mucosa of ordinary AR mice showed disordered and interrupted arrangement of epithelial cilia and obvious eosinophil infiltration in the lamina propria,while the nasal mucosa of normal control group and Apo-AI group showed clear and complete tissue structure,neat and uniform arrangement of epithelial cilia and little or no eosinophil infiltration in lamina propria.The results of flow cytometry showed that the frequency of ILC2 in nasal mucosa and peripheral blood in ordinary AR group was significantly lower than that in Apo-AI group and control group(ILC2in nasal mucosa: 26.7±9.8 vs 11.5±5.2 vs 16.8±6.3;ILC2 in peripheral blood:25.6±8.7 vs 12.8±4.6 vs 13.4±4.9,P<0.05).Conclusion:Apo-AI may play a negative role in ILC2 inflammation of AR,suggesting that Apo-A1 may be a promising therapeutic target for AR.