Quintana Ayshea M, Landolt Gabriele A, Annis Kristina M, Hussey Gisela Soboll
Department of Clinical Sciences, Colorado State University, Fort Collins, CO 80523-1678, USA.
Vet Immunol Immunopathol. 2011 Apr 15;140(3-4):226-36. doi: 10.1016/j.vetimm.2010.12.008. Epub 2011 Jan 11.
Our understanding of innate immunity within the equine respiratory tract is limited despite growing evidence for its key role in both the immediate defense and the shaping of downstream adaptive immune responses to respiratory disease. As the first interface to undergo pathogen invasion, the respiratory epithelium is a key player in these early events and our goal was to examine the innate immune characteristics of equine respiratory epithelia and compare them to an in vitro equine respiratory epithelial cell model cultured at the air-fluid interface (AFI). Respiratory epithelial tissues, isolated epithelial cells, and four-week old cultured differentiated airway epithelial cells collected from five locations of the equine respiratory tract were examined for the expression of toll-like receptors (TLRs) and host defense peptides (HDPs) using conventional polymerase chain reaction (PCR). Cultured, differentiated, respiratory epithelial cells and freshly isolated respiratory epithelial cells were also examined for the expression of TLR3, TLR9 and major histocompatibility complex (MHC) class I and class II using fluorescence-activated cell sorting (FACS) analysis. In addition, cytokine and chemokine profiles from respiratory epithelial tissues, freshly isolated respiratory epithelial cells, and cultured, differentiated, epithelial cells from the upper respiratory tract were examined using real-time PCR. We found that respiratory epithelial tissues and isolated epithelial cells expressed TLRs 1-4 and 6-10 as well as HDPs, MxA, 2'5' OAS, β-defensin-1, and lactoferrin. In contrast, epithelial cells cultured at the AFI expressed TLRs 1-4 and 6 and 7 as well as MxA, 2'5' OAS, β-defensin-1, but had lost expression of TLRs 8-10 and lactoferrin. In addition, MHC-I and MHC-II surface expression decreased in epithelial cells cultured at the AFI compared to isolated epithelial cells whereas TLR3 and TLR9 were expressed at similar levels. Lastly, we found that equine respiratory epithelial cells express an array of pro-inflammatory, antiviral and regulatory cytokines and that after four weeks of in vitro growth conditions, equine respiratory epithelial cells cultured at the AFI retained expression of GM-CSF, IL-10, IL-8, TGF-β, TNF-α, and IL-6. In summary, we describe the development of an in vitro equine respiratory epithelial cell culture model that is morphologically similar to the equine airway epithelium and retains several key immunological properties. In the future this model will be a used to study equine respiratory viral pathogenesis and cell-to-cell interactions.
尽管越来越多的证据表明固有免疫在马呼吸道的即时防御以及对呼吸道疾病下游适应性免疫反应的形成中起着关键作用，但我们对马呼吸道内固有免疫的了解仍然有限。作为病原体入侵的首个界面，呼吸道上皮在这些早期事件中起着关键作用，我们的目标是研究马呼吸道上皮的固有免疫特征，并将其与在气液界面（AFI）培养的体外马呼吸道上皮细胞模型进行比较。使用常规聚合酶链反应（PCR）检测从马呼吸道五个部位收集的呼吸道上皮组织、分离的上皮细胞以及四周龄培养的分化气道上皮细胞中Toll样受体（TLRs）和宿主防御肽（HDPs）的表达。还使用荧光激活细胞分选（FACS）分析检测培养的、分化的呼吸道上皮细胞和新鲜分离的呼吸道上皮细胞中TLR3、TLR9以及主要组织相容性复合体（MHC）I类和II类的表达。此外，使用实时PCR检测呼吸道上皮组织、新鲜分离的呼吸道上皮细胞以及上呼吸道培养的、分化的上皮细胞的细胞因子和趋化因子谱。我们发现呼吸道上皮组织和分离的上皮细胞表达TLRs 1-4、6-10以及HDPs、Mx A、2'5' OAS、β-防御素-1和乳铁蛋白。相比之下，在AFI培养的上皮细胞表达TLRs 1-4、6和7以及Mx A、2'5' OAS、β-防御素-1，但失去了TLRs 8-10和乳铁蛋白的表达。此外，与分离的上皮细胞相比，在AFI培养的上皮细胞中MHC-I和MHC-II的表面表达降低，而TLR3和TLR9以相似水平表达。最后，我们发现马呼吸道上皮细胞表达一系列促炎、抗病毒和调节性细胞因子，并且在体外生长条件四周后，在AFI培养的马呼吸道上皮细胞保留了GM-CSF、IL-10、IL-8、TGF-β、TNF-α和IL-6的表达。总之，我们描述了一种体外马呼吸道上皮细胞培养模型的建立，该模型在形态上与马气道上皮相似，并保留了几个关键的免疫学特性。未来该模型将用于研究马呼吸道病毒发病机制和细胞间相互作用。
