Splenic microRNA Expression Profiles and Integration Analyses Involved in Host Responses to Salmonella enteritidis Infection in Chickens

Abstract

To understand the role of miRNAs in regulating genes involved in the host response to Salmonella enteritidis (SE) infection, next generation sequencing was applied to explore the altered splenic expression of microRNAs (miRNAs) and deregulated genes in specific-pathogen-free chickens. Birds were either infected or not (controls, C) and those challenged with SE were evaluated 24 h later and separated into two groups on the basis of the severity of clinical symptoms and blood load of SE: resistant (R, SE challenged-slight clinical symptoms and <105 cfu / 10 μL), and susceptible (S, SE challenged-severe clinical symptoms and >107 cfu/10 μL). Thirty-two differentially expressed (DE) miRNAs were identified in spleen, including 16 miRNAs between S and C, 13 between R and C, and 13 between S and R. Through integration analysis of DE miRNAs and mRNA, a total of 273 miRNA-target genes were identified. Functional annotation analysis showed that Apoptosis and NOD-like receptor signaling pathway and adaptive immune response were significantly enriched (P < 0.05). Interestingly, apoptosis pathway was significantly enriched in S vs. C, while NOD-like receptor pathway was enriched in R vs. C (P < 0.05). Two miRNAs, gga-miR-101-3p and gga-miR-155, in the hub positions of the miRNA-mRNA regulatory network, were identified as candidates potentially associated with SE infection. These 2 miRNAs directly repressed luciferase reporter gene activity via binding to 3′-untranslated regions of immune-related genes IRF4 and LRRC59; over-expressed gga-miR-155 and interference gga-miR-101-3p in chicken HD11 macrophage cells significantly altered expression of their target genes and decreased the production of pro-inflammatory cytokines. These findings facilitate better understanding of the mechanisms of host resistance and susceptibility to SE infection in chickens.

Messenger RNA Sequencing and Pathway Analysis Provide Novel Insights Into the Susceptibility to Salmonella enteritidis Infection in Chickens

“…Specific-pathogen-free chicks (White Leghorn) were obtained from the Beijing Laboratory Animal Research Center and were treated as described earlier ( Gou et al, 2012 ; Li et al, 2017a ). Groups of 30 SE-challenged chicks were initially screened at 0.5, 1, 2, 4, 6, and 8-days post infection at 3 days of age; 24-h post infection was found to be optimal for showing differences (clinical symptoms and bacterial burden) between the 3 groups to best expose potential differences in mRNA expression.…”

“…Sample Preparation Kit (Illumina, San Diego, CA, United States), and paired-end sequencing was performed on an Illumina Hiseq2000 by LC-BIO (LC Sciences, Houston, TX, United States) and 100 bp paired-end reads were generated. Quality control of reads was determined by FastQC software (v0.10.1), details of which were described earlier ( Li et al, 2017a ). In brief, clear data were obtained from the raw reads, eliminating contamination with sequencing adapters or poly-N and low quality reads ( Q values < 20), along with potential residual ribosome RNA.…”

“…The spleen plays a major role in detecting cell damage during bacterial infection and in the pathogenic mechanisms of bacterial clearance ( Altamura et al, 2001 ). Increasing evidence suggests that the spleen plays a greater role in immune function in avian than in mammalian species, and is responsible for an immediate immune reaction after recognizing pathogens by filtering antigens from the blood ( Smith and Hunt, 2004 ; Li et al, 2017a ).…”

“…Although there have been several previous studies focusing on the splenic transcriptome following infection with Salmonella enterica ( Zhou and Lamont, 2007 ; Matulova et al, 2012 ), avian pathogenic Escherichia coli (APEC) ( Sandford et al, 2011 ; Nie et al, 2012 ) and virus ( Wang et al, 2006 ; Haq et al, 2010 ; Smith et al, 2011 ), little is known about immune-related genes and pathways between resistant and susceptible birds during the course of SE pathogenesis. This paper identifies genes and pathways that are differently expressed in susceptible versus resistant chickens, after challenge with SE, to aid understanding of host immune resistance to SE infection; an earlier report ( Li et al, 2017a ) presented differences that were apparent at the miRNA level.…”

What Makes the Harderian Gland Transcriptome Different From Other Chicken Immune Tissues? A Gene Expression Comparative Analysis

“…In human cell lines, mir-200b was shown to inhibit the TLR4 pathway ( Wendlandt et al, 2012 ), mir-221 increased the expression of NF -κβ and STAT3 ( Liu et al, 2014 ), and mir-193b overexpression resulted in increased autophagy ( Nyhan et al, 2016 ). In chickens, mir-1764 decreased the expression levels of the inflammatory cytokine STAT1 ( Jeong et al, 2013 ), and mir-30d may regulate IRF4 ( Li et al, 2017 ). Avian influenza impacted the expression levels of several miRNAs in the chicken trachea and lung including the following miRNAs significant in the current study: let-7a-1 , let-7f , let-7j , mir-1b , mir-30d , mir-34c , mir-101-2 , mir-144 , mir-200b , and mir-451 ( Wang et al, 2009 ).…”