Transcriptome Analysis of Spleen in Chickens Administered β-glucan Derived From Yeast Cell Wall

Background: Our previous study shown that oral administration of a product contained yeast cell wall polysaccharides enhanced immune responses elicited by Newcastle disease virus and changed microbial community of cecum in chickens. Results: The present study was design to investigate the potential molecular mechanism in relation to the immunomodulation of β-glucan in chickens. Using RNA-sequence (RNA-seq) technique, we identied 198 DEGs in spleen in chickens after oral administration of β-glucan. In addition, these DEGs were signicantly enriched in 205 GO terms and 7 KEGG pathways. Conclusions: β-glucan might regulate chicken immune system by regulating expression of genes involved in cognition, cytokines, binding, enzyme activities and multiple signaling pathway.


Background
Yeast cell wall polysaccharides, mainly composed of β-glucan and mannan-oligosaccharides (MOS), are derived from the cell wall of Saccharomyces cerevisiae [1]. They are extensively used as growth promoters, antimicrobial agents or immunomodulator in poultry for the purpose of improving production and health [2,3,4]. We have previously shown that oral administration of PW220, a product contained yeast cell wall polysaccharides, enhanced immune responses elicited by Newcastle disease virus and changed microbial community of cecum in chickens [5]. However, the underlying molecular mechanism remained obscure.
RNA sequencing (RNA-seq) is a high through-put technology that can be employed to detect quantitative gene expression and to analyze different expression pro les at the whole transcriptome level [6]. With the enhanced sensitivity and declining cost, RNA-seq has been widely used in livestock and poultry research [7,8]. Because the spleen is one of the dominating sites for priming of the primary immune responses, in the present study, transcriptome sequencing was performed to investigate the effect of β-glucan on gene expression in chicken spleen and to explore the potential signal transduction pathways.

Materials And Methods
Reagents β-glucan with purity ≥ 70% derived from yeast cell wall was provided by Angel Yeast Co. LTD (Yichang, China). PrimeScript™RT Master Mix and SYBR ® Premix Ex Taq™ II (Tli RNaseH Plus) were purchased from Takara Bio INC (Dalian, China).

Birds and Experimental Design
One-day-old White-feather silky chickens (male) were purchased from Sichuan Lihua Poultry Co., Ltd.
(Chongqing, China) and randomly allocated into two groups. Group 1 was orally administered β-glucan at a dose of 20 mg/kg of BW (the dose was chosen based on a preliminary experiments) in drinking water for 7 d before vaccination; group 2 was administered saline as a control. On the day following administration of β-glucan, each bird was received intraocular and intranasal immunization with a live vaccine of NDV at a dose of 2 × 10 3 EID, according to the manufacturer's instruction. On 2 weeks post immunization, chickens were humanly killed by cervical dislocation and the whole spleen was immediately frozen and stored at −196°C in liquid nitrogen for sequence analysis.

RNA extraction and cDNA Library Construction
The total RNA of the spleen was extracted using TRIzol reagent (Thermo sher Scienti c). RNA was detected free of contamination and degradation using 1% agarose gel, and the RNA purity was estimated using a NanoPhotometer® spectrophotometer (IMPLEN, Westlake Village, CA, USA). RNA concentration was measured using a Qubit ® RNA Assay Kit in Qubit® 2.0 Flurometer (Life Technologies, Carlsbad, CA, USA), and RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). The results showed that the RNA was intact and free of DNA contamination. The libraries were sequenced using Illumina HiSeq platform with a PE150 strategy.

Transcriptome analysis
Transcriptome sequencing, sequence assembly, and data analysis are provided by Novogene Bioinformatics Technology Co. Ltd. (Beijing, China). The transcriptome analysis process was performed as follows: (a) mRNA was puri ed from total RNA using poly-T oligo-attached magnetic beads; Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). (b) First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse. Transcriptase (RNase H-); Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. (c) Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities; After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. (d) In order to select cDNA fragments of preferentially 250~300 bp in length, the library fragments were puri ed with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 µl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were puri ed (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. TruSeq PE Cluster Kit v3-cBot-HS (Illumina) was employed to carry out the cluster of the index-coded samples. Then, the sequencing was executed on an Illumina Novaseq platform, and 150 bp paired-end reads were produced. Reference genome and gene model annotation les were downloaded from the genome website (ftp://ftp.ensembl.org/pub/release-98/fasta/gallus_gallus/and ftp://ftp.ensembl.org/pub/re-lease-98/gtf/gallus_gallus/). Using HISAT2 (v2.0.5) to build the index of the reference genome and align paired-end clean reads with the reference genome. Feature Counts v1.5.0-p3 was used to count the reads numbers mapped to each gene, and then FPKM of each gene was calculated basing the length of the gene and its reads count. DESeq2 R package (1.16.1) was used to exporting the differential expression between the H with the control group. Genes with P < 0.05 and |log2(foldchange)| > 1 were de ned as differential expression genes (DEGs).

Real-time Quantitative PCR Validation
Two DEGs that were up-regulated and four DEGs that were down-regulated in the comparison H vs. Control were selected to validate the transcriptome sequencing results using RT-qPCR. PrimeScript™RT Master Mix (Takara, Dalian, China) was used to convert RNA into cDNA on a T100™ thermal cycler (Bio-Rad, USA). Information of primers were provided in Table S3. The Chicken β-actin was served as the internal control gene. RT-qPCR with SYBR®Premix Ex Taq™ II (Tli RNaseH Plus) (Takara, Dalian, China) on selected genes was carried out on a Multiple Real-Time PCR System (AB, USA). A relative quantitative method (2− △△CT ) was employed to evaluate the quantitative variation. All samples were analyzed in triplicate.

Results And Discussion
RNA sequencing data analysis To detect differences between H and Control, RNA-seq of splenic samples was performed using the Illumina sequencing platform. Four individual samples were included for each group, and they were marked as C1, C2, C3, C4 and H1, H2, H3, H4, respectively. As shown in supplementary materials Table1

Differentially expressed genes
As shown in Fig.1A, there were 198 differentially expressed genes in total, including 47 up-regulated and 151 down-regulated genes. Detailed description of DEG will be shown in supplementary material Table   S2. The distribution of differentially expressed genes was depicted in the heatmap (Fig.1B). The clustered biological replicates of DEGs indicated good reproducibility of treatment.

Gene Ontology classi cation and KEGG enrichment analysis
According to the Gene Ontology (GO) classi cation system, the differentially expressed genes were classi ed into three main functional categories: biological process, cellular component and molecular function. Fig.2 displayed top 10 GO terms in three categories. Genes involved in the "humoral immune response", "response to chemokine", "antimicrobial humoral response", "defense response to bacterium", "antimicrobial humoral immune response mediated by antimicrobial peptide", "defense response to Gram-negative bacterium", "defense response to Gram-positive bacterium" were predominant in the biological process category. Moreover, a signi cant proportion of the genes were involved in "CCR chemokine receptor binding", "chemokine receptor binding", "lipopolysaccharide binding" in the category of molecular function. In addition, "mitochondrial respiratory chain complex I", "NADH dehydrogenase complex", "respiratory chain" were the predominant enriched terms in the cell components category.
Immunomodulatory effect of polysaccharides from yeast cell wall have been demonstrated effective to change immune response and altered gut microbiota composition in chickens [9,10]. In our previous study, a yeast cell wall product contained β-glucan and MOS was found effectively to enhance intestinal IgA response to NDV vaccination and modulated the cecum microbiota by oral route [5]. However, only a few reports analyzed the mRNA expression of spleen after oral administration of yeast polysaccharides.
RNA-seq based transcriptome analysis is a tool that allows a deep understanding of complicated physiological pathways. β-glucan may exhibit its effects on the immune function of chicken by differentially expressed genes in relation to innate and adaptive immunity in lymphocyte. Several pattern recognition receptors target β-glucan were enriched on the surface of immune cells [11]. After recognition of β-glucan, these receptors stimulate tyrosine kinase and nuclear factor κB, which consequently induced secretion of proin ammatory cytokines and activate immune reaction [12,13,14,15]. Recently, transcriptome analysis was used to study the mechanism of a vegetable oil adjuvant in mice [16]. They found DEGs in spleen were enriched in immune response related GO terms such as "humoral immune response", "antimicrobial humoral response" and "defense response to bacterium", which were consistent with our results.
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the differentially expressed genes was also performed. The results indicated that these genes were mainly classi ed into seven pathways included "Neuroactive ligand-receptor interaction", "Gap junction", "MAPK signaling pathway", "ABC transporters", "Biosynthesis of amino acids", "Drug metabolism", "Protein export". These results indicated that β-glucan regulate immune response through multiple pathways (Fig.3). Similar results were also reported by others [17 18] Interestingly, genes encoding cathelicidin including CATH3, CATH1, CATH2, and genes encoding betadefensin such as AvBD6, AvBD7, AvBD1, and AvBD4 were signi cantly down-regulated in the comparison H vs. Control. Host Defense Peptides (HDPs) are important effector molecules of the innate immune system [19] . These peptides have been found in a wide range of animals from mammals to birds. The chicken has four cathelicidins, including CATH1, CATH2, CATH3 and CATHB1, which were able to e ciently kill a variety of bacteria [20]. Avian beta-defensins (AvBDs), also known as gallinacins, are small cationic peptides having three cysteine disul de bonds between their cysteine residues and play essential roles in the innate immune system [21]. The decreased expression of cathelicidins and betadefensin might be explained by the feedback regulation, in which the population of Bacteroides and Parabacteroides were reduced by yeast cell wall polysaccharide [5]. Similar results were found in other studies. Yu reported that supplementation of yeast β-glucan in broiler chicken inhibited Salmonella infection and reduced HDPs expression by quantitative real-time PCR analysis [22].

Con rmation of differentially expressed genes by qRT-PCR
We veri ed the 6 DEGs which were up-or down-regulated using real-time quantitative PCR. The result showed that the RT-qPCR data were consistent with the RNA-seq data in general, indicating the reliability of the sequencing results (Fig.4).
In summary, this study identi ed 198 DEGs in spleen in chickens after oral administration of β-glucan. GO and KEGG pathway enrichment uncovered 205 signi cantly enriched GO terms and 7 KEGG pathways. βglucan might regulate chicken immune system by changing expression of genes involved in cognition, cytokines, binding, enzyme activities and multiple signaling pathway.   RT-qPCR con rmation of selected DEG candidates. Data are expressed as mean ± SE.