The results revealed that infection of induced a significant decrease in the TEER values of the cells (Figure 1A)

The results revealed that infection of induced a significant decrease in the TEER values of the cells (Figure 1A). we found that infection disrupted the barrier functions of NPTr cells. G-ALPHA-q By performing RNA sequencing (RNA-Seq), we determined 30 differentially expressed genes (DEGs), including the vascular endothelial growth factor A (VEGFA) encoding gene is the causative agent of multiple diseases in a variety of domestic and wild animals and in humans (1). According to the capsular antigens, strains are classified into five serogroups (A, B, D, E, and F) (1C3). In pigs, strains, in particular those belonging to serogroups A and D, are commonly associated with the development of respiratory disorders, such as progressive atrophic rhinitis (PAR) and pneumonia (4). also plays a crucial role in porcine respiratory disease complex (PRDC) and hemorrhagic septicemia (5). This pathogen is proposed to be a threat of high impact to the pig industry, as evidenced by its prevalence: 8.0% in diseased pigs with pneumonia or PAR in China, 10.3C15.6% in pigs with pneumonia in Korea, and 15.6% of isolated respiratory pathogens in the United States (6). However, current knowledge about the pathogenesis of swine is limited. In mammals, respiratory tracts such as the trachea and bronchi are areas where strains commonly colonize (7). The mammalian respiratory epithelium forms the first line of defense against the invasion and stimuli of respiratory pathogens (8, 9). As an important respiratory pathogen, infection might induce the dysfunction of the host epithelial barriers, thereby contributing to the bacterial invasion and the development of many K+ Channel inhibitor inflammatory disorders of the airways and lungs (9). However, the effects of infection on the epithelial barriers and the responses of mammalian respiratory epithelial cells to infection are still not well-known. Recently, several technologies such as RNA sequencing (RNA-Seq) have emerged as powerful tools to analyze gene expression and have been widely used in pathogenChost interaction studies (10). To elucidate the genes in mammalian tracheal epithelial cells in response K+ Channel inhibitor to infection, we used pig tracheal epithelial cells as a model and performed RNA-Seq to identify the differentially expressed genes (DEGs) in the cells during bacterial infection. Materials and Methods Bacterial Strains, Cells, and Growth Conditions HN05 is a serogroup D strain which was isolated from the trachea of a pig with respiratory disorders in Hunan Province, China, in 2010 2010. We have determined its whole genome sequence and deposited it to GenBank with the accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”PPVF00000000″,”term_id”:”1840207193″,”term_text”:”PPVF00000000″PPVF00000000 (1). Strain HN05 was cultured in tryptic K+ Channel inhibitor soy broth (TSB) medium (Becton, Dickinson and Company, Sparks, MD, USA) supplemented with 5% bovine serum at 37C for 8C12 h. Newborn pig tracheal epithelial (NPTr) cells (established following serial culture of primary cells derived from tracheal tissues and were kindly gifted by Prof. Hongbo Zhou at Huazhong Agricultural University, Wuhan, China) (11) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, ThermoFisher, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco, ThermoFisher, Waltham, MA, USA) under 5% CO2 atmosphere at 37C. Cell Culture and Bacterial Infection Monolayer cells (2.5 106/2 ml per well) in each well of a six-well plate (Corning, Corning, NY, USA) were washed with phosphate-buffered saline (PBS) three times and were incubated with fresh DMEM (2 ml/well). HN05 (2.5 108/100 l per well) was inoculated into three wells of the plate, while PBS (100 l/well) was added into the other three wells. The plate was incubated under 5% CO2 atmosphere at 37C for 4 h. The medium in each well of the plate was discarded and the cells were washed with PBS, followed by the addition of 1 1 ml Trizol into each well of the plate. This bacterial infection assay was performed three times at separate time points. In each independent experiment, three wells of cells were treated with either or PBS. RNAs extracted from bacterial infected cells and/or PBS-treated cells in these three wells were pooled and were regarded as one sample for further use. RNA Isolation, Construction and Sequencing of Transcriptome Library Total RNAs from the bacterial infected cells and the PBS-treated cells were isolated using the Trizol reagent protocol (Invitrogen, ThermoFisher, Waltham, MA, USA). The quantity and quality of.