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Global Gene Networks in Porcine Alveolar Macrophage Cells, 3D4/31, Stimulated with Antigenic Epitopes of Actinobacillus pleuropneumoniae ApxIA, IIA and IVA

ABSTRACT: Purpose: The goals of this study are to examine the host response with porcine alveolar macrophages (PAMs) cells, 3D4/31 by stimulation of four antigenic epitopes of App exotoxins, ApxIA, IIA and IVA. Methods: total RNA profiles of porcine alveolar macrophage cells stimulated with ApxIA Ct, ApxIIA Nt, ApxIVA C1, ApxIVA C2 and DPBS were generated by transcriptome sequencing, in duplicate, using HiSeq4000 platform.The sequence reads that passed quality filters were analyzed at the transcript isoform level with TopHat followed by Cufflinks. qRT–PCR validation was performed using SYBR Green assays Results: RNA sequencing yielded 617 million 101 bp paired-end clean reads, varying from 28 to 32 million for each sample. Among the clean reads, approximately 95.24% clean reads were uniquely mapped onto the version 11.1 of the sus scrofa. Gene expression was quantified through FPKM conversion of raw reads. The 15,269 genes (mean FPKM of the 10 samples > 0) were considered as expressed in PAMs over the 30,511 gene annotations. RNA-seq data confirmed stable expression of 1 known housekeeping genes, and 5 of these were validated with qRT–PCR. RNA-seq data had a linear relationship with qRT–PCR for more than four orders of magnitude and a goodness of fit (R2) of 0.906. Conclusions: Transcriptional responses of the porcine alveolar macrophages (PAMs) stimulated with the antigenic epitopes of ApxIA, ApxIIA, and ApxIVA were analyzed to identify the precious mechanism of Apx toxins in the host. This study will be foundational to understanding the host response by Apx toxins.

ORGANISM(S): Sus scrofa

PROVIDER: GSE116263 | GEO | 2018/06/27

REPOSITORIES: GEO

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Project description:Purpose:This study was aimed to profile the transcriptome changes during Toxoplasma infection with porcine alveolar macrophages. for this purpose, Porcine alveolar macrophages (PAMs,3D4/21) were infected by Toxoplasma Me49 strain in vitro. The transcriptome changes were captured by dual RNA-seq analyses. Methods: Transcriptomic profiles of infection samples (Me49 infected PAMs) and controls (Me49 and PAMs, respectively) were generated by deep sequencing, in triplicate, using Illumina HiseqPE150. The sequence reads that passed quality filters were analyzed at the transcript level with TopHat (v2.0.12) followed by HTSeq (v0.6.1). The Cufflinks (v2.1.1) was used to construct and identify novel transcripts. qRT–PCR validation was performed using SYBR Green assays. Results: Using an optimized data analysis workflow, More than 30 million paired-end reads were generated for the Me49 controls and 93% of the clean reads were mapped to the Me49 reference genome. Nearly 2~3 times more reads were produced in the PAMs alone or infected PAMs samples. Among these, 60% to 80% of clean reads were mapped to the Sus Scrofa genome. In the infection groups, approximately 16% of total clean reads were aligned to the Me49 reference genome. RNA-seq data indicated the remarkable transcripts changes of both sides during infection process. More than 800 and 1800 genes (fold change ≥2 and adjusted p value <0.05) were significantly regulated in PAMS and Me49, respectively. Altered expression of 15 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. The differentially expressed genes in Toxoplasma were mainly related to metabolic process and macromolecule synthesis as reflected by enhanced activity of FASII pathway. Further analyses of differentially expressed genes in PAMs uncovered unique responses of porcine macrophages to Toxoplasma infection. These analyses provide novel insights into Toxoplasma interaction with it’s hosts. Conclusions: Our study represents the first detailed dual analyses of porcine alveolar macrophages and Me49 with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here provide the overall transcripts changes both PAMs and Me49. The results showed the unexpected responses of PAMs to Toxoplasma infection. These widen our understanding of interactions between Toxoplasma and it’s hosts.

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Global Gene Networks in Porcine Alveolar Macrophage Cells, 3D4/31, Stimulated with Antigenic Epitopes of Actinobacillus pleuropneumoniae ApxIA, IIA and IVA

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