Genomic analysis of between-cow variation in dermal fibroblast response to lipopolysaccharide
Genomic analysis of between-cow variation in dermal fibroblast response to lipopolysaccharide
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DOI：
10.3168/jds.2011-5251
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发表时间：
2012-07-01
影响因子：
3.5
通讯作者：
Kerr, D. E.
中科院分区：
文献类型：
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作者：
Kandasamy, S.;Kerr, D. E.
先天性免疫应答在防御乳腺炎病原体中起着重要作用。鉴定奶牛先天免疫信号传导中存在的变异和变异的潜在分子原因可能有助于设计新的乳腺炎控制策略。真皮成纤维细胞已被用作模型细胞类型，以探索奶牛之间的变化，在细胞产生IL-8的能力，响应脂多糖（LPS）治疗，这种反应似乎与动物的能力，在体内的挑战与LPS或大肠杆菌乳腺炎。在这项研究中，奶牛的原代真皮成纤维细胞培养和基于微阵列的基因组分析被用来研究对LPS的可变反应的原因。从我们收集的88头奶牛的成纤维细胞培养物中选择2头奶牛的成纤维细胞培养物，其中一头具有低反应表型（LR阵列），另一头具有高反应表型（HR阵列）。与HRarray成纤维细胞培养物相比，LRarray成纤维细胞培养物响应于24小时LPS处理产生的IL-8和IL-6蛋白约少5倍。从2个培养物的3个重复在8小时LPS处理之前和之后获得的RNA的基因组分析揭示了组合的LPS诱导的321个转录物的差异表达，表明成纤维细胞的稳健的响应能力。在基础条件下，微阵列分析显示，与HR阵列成纤维细胞相比，LR阵列成纤维细胞中toll样受体4（TLR 4）的表达低2倍，这与TLR 4-MyD 88依赖性和TLR 4-TRIF依赖性途径（IL-8、IL-6、SAA 3、CCL 20、MX 1、IRF 1和ISG 20）调节的基因的表达显著降低有关。通过定量PCR分析（QPCR）证实并扩展了TLR 4的培养物间差异表达，其揭示了LRarray成纤维细胞培养物中TLR 4的表达低33倍。LPS处理后，TLR 4表达的差异增加到几乎50倍，并且与IL-8和IL-6表达降低8倍以上相关。在TLR 4基因的近端1，300-bp启动子区域中没有发现DNA序列变异，并且微阵列分析没有揭示基础条件下或暴露于LPS后TLR 4表达减少的分子解释。LR阵列成纤维细胞培养物对LPS的减弱的先天免疫应答可能是由降低的TLR 4受体表达引起的。此外，原代真皮成纤维细胞可用于检查关键免疫应答途径中奶牛之间差异的根本原因。
The innate immune response plays a major role in defense against mastitis-causing pathogens. Identification of existing variation in innate immune signaling among cows and the underlying molecular causes for the variation may help in design of new mastitis control strategies. The dermal fibroblast has been used as a model cell type to explore between-cow variation in the ability of cells to produce IL-8 in response to lipopolysaccharide (LPS) treatment, and this response appears related to an animal's ability to respond to in vivo challenge with LPS or Escherichia coli mastitis. In this study, primary dermal fibroblast cultures of cows and microarray-based genomic analysis were used to investigate the cause(s) for the variable response to LPS. Fibroblast cultures from 2 cows, one with a low response phenotype (LRarray) and another with a high response phenotype (HRarray), were selected from our collection of fibroblast cultures established from 88 cows. The LRarray fibroblast culture produced approximately 5-fold less IL-8 and IL-6 protein in response to 24-h LPS treatment than the HRarray fibroblast culture. Genomic analysis of RNA obtained from 3 replicates of the 2 cultures before and after 8-h LPS treatment revealed a combined LPS-induced differential expression of 321 transcripts, indicating the robust response capability of the fibroblast cell. Under basal conditions, the microarray analysis revealed 2-fold less expression of toll-like receptor 4 (TLR4) in the LRarray, ay fibroblasts compared with the HRarray fibroblasts, and this was associated with a marked reduction in expression of genes regulated by the TLR4-MyD88-dependent and TLR4-TRIF-dependent pathways (IL-8, IL-6, SAA3, CCL20, MX1, IRF1, and ISG20). The between-culture differential expression of TLR4 was confirmed and extended by quantitative PCR analysis (QPCR) that revealed a 33-fold lower expression of TLR4 in the LRarray fibroblast culture. After LPS treatment, the difference in TLR4 expression increased to almost 50-fold and was associated with more than 8-fold lower expression of IL-8 and IL-6. No DNA sequence variations were identified in the proximal 1,300-bp promoter region of the TLR4 gene, and microarray analysis did not reveal a molecular explanation for the reduced TLR4 expression under either basal conditions or following exposure to LPS. The attenuated innate immune response of the LRarray fibroblast culture to LPS may be caused by reduced TLR4 receptor expression. Also, the primary dermal fibroblast cells can be used to examine underlying causes for between-cow variations in key immune response pathways.