下呼吸道流感嗜血杆菌定植通过Toll样受体4影响哮喘小鼠免疫失衡

摘要/Abstract

摘要：

目的：观察下呼吸道流感嗜血杆菌定植对哮喘小鼠气道炎症和免疫失衡的影响,并研究其信号通路。方法：32只C57BL/6野生型（wild type,WT）和32只Toll样受体4（,TLR4）基因敲除（TLR4^-/-）小鼠为实验对象,其中C57BL/6 WT小鼠,随机分为对照（NC）组和哮喘（AC）组、注菌（NS）组、哮喘注菌（AS）组4组,每组8只。32只TLR4^-/-小鼠处理及分组同上。对AC组、AS组,分别用卵清蛋白（ovalbumin,OVA）致敏和激发,制备慢性哮喘小鼠模型;对NS、AS组,方经气道注入流感嗜血杆菌琼脂菌珠,制备气道定植菌模型。采用酶联免疫吸附法检测小鼠血清中IL-17、IL-10水平,用流式细胞仪检测小鼠脾脏单个核细胞中Th17、Treg及TLR4⁺细胞数,并进行三者相关性分析。结果：C57BL/6 WT小鼠中,与AC组、NS组和NC组相比,AS组IL-17水平、IL-17/IL-10比值、Th17、Th17/Treg比值、TLR4⁺细胞水平升高,IL-10、Treg降低,提示下呼吸道流感嗜血杆菌定植加重了哮喘小鼠的气道炎症和免疫失衡。与C57BL/6 WT的AS小鼠相比,TLR4^-/-小鼠的AS组IL-17、IL-17/IL-10比值、Th17、Th17/Treg比值、TLR4⁺细胞降低,IL-10水平、Treg数升高,说明下呼吸道流感嗜血杆菌定植对气道的影响可能是通过TLR4通路来调控。脾脏单个核细胞中,表达TLR4的CD4⁺T淋巴细胞比例与Th17比例呈正相关（r=0.912,P<0.05）,而与Treg细胞比例呈负相关（r=0.689,P<0.05）。下呼吸道流感嗜血杆菌气道定植后,可能是通过TLR4影响Th17和Treg的平衡来影响免疫平衡。结论：下呼吸道流感嗜血杆菌定植可能通过TLR4的信号转导加重哮喘小鼠的免疫失衡,进而参与了哮喘病情的进展。

关键词:流感嗜血杆菌,哮喘,TLR4,Th17/Treg,IL-17/IL-10

Abstract:

Objective:To observe the effect ofHaemophilusinfluenzae colonizing in lower respiratory tract on airway inflammation and immune imbalance in asthmatic mice, and to study the signaling pathway.Methods:Thirty-two C57BL/6 wild type (WT) and 32 TLR4 gene knockout (TLR4^-/-) mice were used. The C57BL/6 mice were randomly divided into control group (NC), asthma group (AC), inoculation group (NS) and asthma inoculation group (AS), and there were 8 mice in each group. Thirty-two TLR4^-/-mice were grouped same as above. Mice in AC and AS group were sensitized and stimulated by ovalbumin (OVA) to make chronice asthma model. TheHemophilusinfluenzae AGAR bead was infused into the airway to prepare the airway colonization model. The levels of IL-17 and IL-10 in serum were measured by ELISA. Th17, Treg and TLR4+ cells in spleen nonocytes were isolated and detected by flow cytometry, and the association analysis among them was performed.Results:In C57BL/6 wild type mice, compared with AC, NS and NC group, IL-17, IL-17/IL-10, Th17 and TLR4⁺cell increased, and IL-10 and Treg decreased significantly in AS group, which indicated Haemophilus influenza colonization worsen airway inflammation and led to immune imbalance. Compared with AS group of C57BL/6, IL-17, IL-17/IL-10, Th17, Th17/Treg, TLR4+ cells were decreased, and IL-10 and Treg increased in AS Group of TLR4^-/mice, which implicatedHaemophilus influenzacolonization in lower airway might play effect through TLR4^-/-signaling pathway. Correlation analysis showed that ratio of CD4⁺T lymphocyte with TLR4 expression was positively correlated with Th17 ratio(r=0.912, 0.723,P<0.05, respectively), while was negatively correlated with ratio of Treg cells (r=0.689,P<0.05), indicating thatHaemophilusinfluenzae colonzing in the lower respiratory tract, may affect the balance of Th17 and Treg to influence the immune balance through TLR4.Conclusions:Haemophilusinfluenzae colonization in lower respiratory tract may aggravate the immune imbalance of asthmatic mice through signal transduction of TLR4, which led to the progression of asthma.

Key words:Haemophilus,Asthma,TLR4,Th17/Treg,IL-17/IL-10

中图分类号:

R541.7

徐程, 徐欣欣, 田烨, 范嘉盈, 宋珍, 杨玲. 下呼吸道流感嗜血杆菌定植通过Toll样受体4影响哮喘小鼠免疫失衡[J]. 诊断学理论与实践, 2022, 21(04): 470-475.

XU Cheng, XU Xinxin, TIAN Ye, FAN Jiaying, SONG Zhen, YANG Ling. Effect ofHaemophilusinfluenzae colonizing in lower respiratory tract on immune imbalance through TLR4 signaling pathway in asthmatic mice[J]. Journal of Diagnostics Concepts & Practice, 2022, 21(04): 470-475.

图/表5

表1

血清 IL-17、IL-10、IL-10/IL-17变化（n=8,$\bar{x}±s$）

指标	WT	TLR4^-/-	t值	P值
IL-17（pg/mL）
NC组	5.58±1.10	5.89±1.41	-2.123	0.087
NS组	4.09±2.08	5.96±1.82	-6.623	0.061
AC组	23.96±4.86^a	16.49±5.14^a	-2.030	0.098
AS组	58.55±15.84^a,b	24.19±10.07^a,b,c	3.438	0.018
F值	16.305	13.276
P值	0.000	0.000
IL-10（pg/mL）
NC组	199.55±83.00	590.66±37.29^c	-3.150	0.025
NS组	165.00±48.00	401.69±122.63^c	-3.625	0.015
AC组	80.07±33.42^a	100.61±6.61^a	-0.265	0.801
AS组	50.51±21.10^a,b	72.70±24.04^a	1.042	0.345
F值	2.486	5.515
P值	0.064	0.064
IL-10/IL-17比值
NC组	77.20±19.23	84.493±11.42	-1.273	0.259
NS组	48.16±19.56	68.36±7.39^c	-1.905	0.015
AC组	6.09±1.75^a	7.99±1.88^a	4.255	0.108
AS组	0.37±0.98^a,b	5.73±1.99^a,c	-3.620	0.015
F值	34.596	204.898
P值	0.000	0.000

表1

图1

表2

脾脏单个核细胞中Th17、Treg细胞占CD4+T细胞的比例（n=8,$\bar{x}±s$）

指标	WT	TLR4^-/-	t值	P值
CD4⁺IL-17⁺T（%）
NC组	0.03±0.07	0.08±0.07	-2.252	0.074
NS组	0.247±0.06	0.12±0.05	2.522	0.053
AC组	0.328±0.18^a	0.21±0.14^a	2.286	0.071
AS组	2.3±0.02^a,b	0.25±0.09^a,c	8.847	0.000
F值	15.285	14.653
P值	<0.001	<0.001
CD4⁺CD25⁺Foxp3⁺T（%）
NC组	13.33±1.34	12.42±0.95	1.083	0.328
NS组	10.35±2.6	10.49±2.31	-0.115	0.913
AC组	9.56±0.92^a	10.51±1.00^a	-1.791	0.133
AS组	7.03±0.98^a,b	8.26±0.66^a	-2.183	0.081
F值	67.829	3.870
P值	<0.001	0.025
Treg/Th17比值
NC组	88.45±6.63	102.62±23.05	-1.815	0.129
NS组	70.38±23.95	98.59±28.70	-2.485	0.055
AC组	33.94±8.94^a	48.27±12.59^a	-2.529	0.053
AS组	3.34±0.66^a,b	28.8±2.6^a,b,c	-26.131	0.000
F值	49.792	21.071
P值	<0.001	<0.001

表2

图2

图3

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