Abstract

   One hundred and twenty one-day-old breeder cocks, included 15 cages of
   8 birds each, were fed to learn the aging's effect on chicken's thymus
   immunity. At 2 (2-W) and 40 (40-W) weeks of age, one chicken each cage
   was randomly chosen and slaughtered to get the thymus sample. The
   results showed that thymus weight and morphology of 40-W group were far
   different from that of 2-W group, and exhibited a property of
   degeneration. Considering this phenotype variance, we analyzed the
   thymus' transcriptome to investigate the molecular mechanism that had
   been implicated in this phenotype diversity with age. Pearson
   correlation coefficients and principal component analysis indicated
   that two major populations corresponding to 40-W and 2-W group were
   identified, and 1949 differentially expressed genes (DEGs, 1722
   up-regulated and 127 down-regulated) were obtained. Results of GO and
   KEGG pathway enrichment found that 4 significantly enriched KEGG
   pathways (Cytokine-cytokine receptor interaction, Intestinal immune
   network for IgA production, Toll-like receptor signaling pathway,
   AGE-RAGE signaling pathway in diabetic complications) related to
   immunoregulation were screened between 40-W and 2-W group. These
   results confirmed that thymus immunity of chickens had a strong
   age-related correlation. DEGs related to these 4 enriched KEGG pathways
   were suppressed in the thymus of 2-W group, this indicated that thymus
   immunity of 2-weeks-age chick was down-regulated. CD40 is involved in 3
   of the 4 significantly enriched pathways, and it is critical for thymus
   immune-regulation. CD40 promoter methylation level of 2-W group was
   higher than that of 40-W group, it is consistent with the
   transcriptional differences of the gene. Our study concluded that
   thymus immunity of chicken was varied with age. Compared to the 40-W
   group, thymus immunity of 2-W group was down-regulated, and in a status
   of hypo-activation on the whole, and these effects might be related to
   CD40 suppression induced by promoter hyper-methylation of the gene.

   Keywords: chicken, thymus immunity, age, CD40, DNA methylation

Introduction

   As central immunological organs, thymus is an essential organ for the
   generation of the adaptive immune system ([37]1), as well as an organ
   where T cells grow, differentiation and to be mature finally ([38]2).
   After differentiation from bone marrow, T lymphocytes will migrate into
   thymus with the assistance of chemokines, and finally become mature T
   lymphocytes ([39]3–[40]5). Meanwhile, Thymus itself can also generate a
   large pool of immature thymocyte progenitors, and further differentiate
   into CD8αα^+ intraepithelial lymphocytes, invariant natural killer T
   cells, natural Foxp3^+ regulatory T cells, and natural T helper cells
   ([41]6). These thymic immunocytes will collaborate together to be
   responsive to foreign antigens and tolerant to self, and further
   modulate the health of adaptive immune system ([42]6–[43]8). After
   long-term co-evolution, thymus could successfully deal with a great
   variety of pathogens and passed through antigen-rich environments.
   Foreign antigens, mostly coming from intestinal microbiota, can be
   incepted by thymic antigen presenting cells, and involved in antigen
   processing and presentation. And then, antigen processing and
   presentation will be implicated in differentiation and repertoire
   selection of T cell in thymus ([44]8, [45]9). This means that pathogen
   associated molecule patterns (PAMP) from microbiota are critical for
   induction and development of thymus.

   Across the lifespan, thymus has distinct developmental and
   morphological characteristic at different ages. As to mammals, previous
   research found that thymus grew and developed rapidly after birth. And
   then, the slowdown and deterioration of the thymus would begin shortly
   after teenage years ([46]10). As to humans, who will experience a
   relatively long lifespan, the immune function of lymphocyte from thymus
   shows high diversity in different life stages ([47]11, [48]12). For
   bird, as well, the functional status of thymus are changing in
   different degrees with age growth. Chick at 2-weeks-age is young, and
   its thymus is still in the developing stage. When it comes to
   40-weeks-age, thymus of adult chicken maybe in the status of
   degeneration as adult mammals. To a large extent, the functional status
   of thymus are determined by the expressed genes, so we analyze the
   genes expression at the level of transcription to learn the thymus
   immunity differences with age (2 and 40 weeks age).

   DNA methylation, as an important kind of epigenetic modification, could
   modulate genes expression at the transcriptional level ([49]13,
   [50]14). Previous researches found that DNA methylation had a
   charicteristic of high in stability, and status of DNA methylation
   could be maintained for a long time. DNA (de)methylation was a very
   time-intensive process (more than 1 week), and so, DNA methylation
   could stabilize the status of genes transcription (activated or
   suppressed) ([51]15–[52]17). Genes transcription and functional status
   of thymus could also be last out a long period. We hypothesized that
   genes transcriptional changes of thymus with age were related to their
   differential DNA methylation. So, we analyzed the genes transcriptome,
   as well as promoter methylation of core regulator in thymus to learn
   the thymus immunity at different developmental stages.

Materials and methods

Animals

   The animal protocol for our study was approved strictly by the Animal
   Care and Use Committee of the College of Animal Science and Technology
   of the Northwest A&F University (Shaanxi, China). 120 one-day-old Avein
   chickens were purchased from Poultry Breeding co., LTD of Beijing
   (Beijing, China), and fed in 15 cages, 8 birds each cage. All the
   chickens had a corn-soybean meal diet (Table [53]S1), and drank water
   freely. At 2 (2-W) and 40 (40-W) weeks of age, one chicken each cage
   was randomly chosen and slaughtered. The second thymus on the right was
   collected to analyze the morphology and weight of thymus. Then we
   quickly collected the thymus on the left, and frozen immediately in
   liquid nitrogen, and stored at −80°C for RNA-seq, qRT-PCR and BSP
   analysis.

RNA-seq library preparation and quality control analysis

   Total RNA was extracted from thymus by using TRIzol reagent (Takara)
   following manufacturer's instructions, and then treated with DNase I to
   remove the genomic DNA. The purity and quantity of total RNA were
   analyzed using Nanodrop equipment. Messenger RNA (mRNA) was isolated
   using magnetic beads with Oligo (dT) and fragmented into short
   fragments with the fragmentation buffer. Then cDNA was synthesized
   using the mRNA fragments as templates, after that, the cDNA was
   purified and resolved with EB buffer for end reparation and single
   nucleotide A (adenine) addition. After agarose gel electrophoresis, the
   suitable fragments were selected for the PCR amplification as
   templates. Quality of the sample library was checked on a 2100
   Bioanalyser (Agilent, Waldbroon, Germany), and the related indexes
   include clean reads number, base percentage composition along reads and
   so on all had been calculated and assessed. At last, the sample library
   was sequenced using Illumina HiSeq^TM 2000.

Alignment of clean reads to the reference sequences

   Then, we aligned the pair-end clean raw reads to gallus reference
   genome
   (ftp.ncbi.nih.gov/genomes/Gallus_gallus/Assembledchromosomes/seq) and
   reference genes
   (/ifs1/pub/database/ftp.ncbi.nih.gov/genomes/Gallus_gallus/RNA/rna.fa.g
   z) by SOAP-aligner/SOAP2. No more than 5 mismatches each read are
   allowed in the alignment. Then, we calculated the distributions of
   reads on reference genes and the gene coverage distribution to assess
   the quality of alignment.

Screening and significant test for differentially-expressed genes (DEGs)

   The relative transcriptional level of unigene was calculated by using
   reads per kb per million reads (RPKM) method. We can screen DEGs
   between 40-W group and 2-W group on the basis of genes' RPKM (Table
   [54]S2). In this study, a rigorous formula was applied to categorize
   different expression levels of genes. Divided each gene's RPKM of 40-W
   group by that of 2-W group, and we get fold change of each unigene. For
   further analysis, DEGs were selected where (i) the absolute values of
   Log2 (fold change) >1, (ii) the p-value (Independent sample T-test) was
   < 0.05, and (iii) the average RPKM was >0.5.

Hierarchical cluster analysis

   Hierarchical cluster analysis was performed to analyze the genes
   expression pattern of the DEGs on the basis of the Z-score values, and
   was performed using the Euclidean distance on the cluster 3.0 software.
   The Z-score of each thymus sample was calculated, respectively using
   the formula: Z = (x–μ)/σ, x is the RPKM of the transcript, μ and σ
   represent the mean and standard deviation of the transcript,
   respectively.

GO and KEGG pathway enrichment analysis of DEGs

   Gene Ontology (GO) is an international standard gene functional
   classification system to describe the properties of genes and their
   products, which includes three aspects: molecular function, cellular
   component and biological process. The Kyoto Encyclopedia of Genes and
   Genomes (KEGG) pathway enrichment analysis for the DEGs ([55]18) was
   conducted to identify significantly enriched metabolic pathways or
   signal transduction pathways in DEGs comparing with the whole genome
   background. GO and KEGG enrichment analysis for the screened DEGs was
   carried out using online KOBAS 3.0
   ([56]http://kobas.cbi.pku.edu.cn/anno_iden.php) software.

Validation the transcription of DEGs using qRT-PCR

   The total RNA extracted from thymus organs of both 2-W and 40-W groups
   was used for fluorescent quantitative PCR analysis (qRT-PCR). The cDNA
   was synthesized using the PrimeScript RT reagent Kit (TaKaRa). The
   qRT-PCR was performed with SYBR® Premix Ex Taq TM II (TaKaRa) following
   the protocol provided by manufacturer on an IQ5 quantitative PCR
   instrument (Bio-Rad Laboratories, Hercules, CA), and programmed as
   follows: 95°C for 5 min; 40 cycles of 95°C for 10 s, 60°C for 30 s,
   72°C for 30 s; and 72°C for 5 min. The 20 μL RT-qPCR mixture included:
   10 μL of SYBR Premix Ex Taq II (2 ×), 1 μL of forward primer (10 μM), 1
   μL of reverse primer (10 μM), 1 μL of template DNA, and 7 μL of
   ultrapure water. The reference gene, β-actin was used as an internal
   expression control. All samples were run in triplicate, and 2^−ΔΔCt
   method was used to calculate the transcriptional level of the selected
   DEGs. The primer sequences (Table [57]S3) for the 8 DEGs (TLR1, TLR4,
   TLR5, CD40, AP-1, IL8, TACI, and PIGR) were designed using online
   NCBI/Primer-BLAST software
   ([58]https://www.ncbi.nlm.nih.gov/tools/primer-blast/).

Bisulfite sequencing PCR (BSP)

   Genomic DNA was extracted from the thymus using QIAamp DNA Mini Kit
   (51306; Qiagen) following the manufacturer's protocol, and the
   extracted DNA was quantified using the NanoDrop ND-1000
   spectrophotometer (Thermo Scientific). Bisulfate treatment of the DNA
   samples (~200 ng/sample) was performed using the EZ DNA
   Methylation-Gold™ Kit (A-D5005; Zymo research, USA). The modified DNA
   samples were diluted in 10 μL of ddH[2]O and served as templates for
   PCR amplification. And the BSP primers (Figure 6A) for promoter CpG
   island (−848 ~ −648, 17 CpGs) of CD40 were designed using online
   MethPrimer software ([59]19). The 20 uL BSP-PCR mixture contained the
   following: 10 μL of 2 × Premix Taq^TM (R004A; Takara), 1 μL of template
   DNA, 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM),
   and 7 μL of ultrapure water. And the BSP-PCR was performed in a DNA
   Engine Thermal Cycler (Bio-Rad, USA) using the following program: 3 min
   at 95°C followed by 35 cycles of denaturation for 30 s at 95°C,
   annealing for 30 s at 60°C and extension for 30 s at 72°C and a final
   extension at 72°C for 5 min. The PCR products showed a clear band in 2%
   agarose gels (Figure [60]S1) and were purified using Agarose Gel DNA
   Purification Kit (DV805A; Takara). Then the purified DNA fragments were
   subcloned using a pMD19-T Vector and transformed into chemically
   competent Escherichia coli. Fifteen DNA samples were performed in 2-W
   and 40-W group, respectively. And 5–7 positive clones of each sample
   were sequenced (BGI, Shenzhen, China). The sequencing results of BSP
   were statistically analyzed and plotted using an online Quantification
   tool for Methylation Analysis software ([61]http://quma.cdb.riken.jp/)
   ([62]20).

Statistical analysis

   Statistical analyses were analyzed by using SPSS 19.0 software. To
   determine the thymus differences between 40-W group and 2-W group,
   Student's t-test was adopted to analyze the data of thymus weight,
   genes transcription, and overall CD40 promotor methylation. And
   chi-square test was adopted to analyze the data of single CpG site
   methylation. Results were presented as the mean ± SE, and a P-value <
   0.05 was considered statistically significant.

Results

Thymus weight and morphology differences between 2-W and 40-W group

   It showed that the thymus weight of 40-W group was highly significantly
   heavier (p < 0.01) than that of 2-W group (Figure [63]1A). Whereas,
   when it comes to thymus index, 40-W group was significantly smaller (p
   < 0.01) than that of 2-W group (Figure [64]1C). Compared with the 2-W
   group, thymus morphology of 40-week-old chickens would tend to be
   thinner, and much more oval and elongate (Figure [65]1B).

Figure 1.

   [66]Figure 1
   [67]Open in a new tab

   Weight and morphology of thymus. Thymus weight (A), thymus index (C),
   and thymus morphology (B) of 40-W group were highly different from
   those of 2-W group. Data shown as mean ± SD, **P < 0.01.

Overview of RNA-Seq data

   All the 30 thymus samples' RNA quality could meet the need of
   transcriptome sequencing (RNA-Seq) library construction (Table [68]S4).
   And then, we assessed the quality of RNA-Seq, the related indexes
   including clean reads number, base percentage composition along reads,
   and so on. After filtration, illumina paired-end sequencing generated a
   total of 1,442,358,732 clean raw reads, and more than 46 million clean
   reads from each sample (Table [69]S5). All 30 samples' base composition
   of raw reads was of high quality, adenine curve nearly be overlapped
   with thymine curve while guanine curve overlapped with cytosine curve,
   and there was no abnormal nucleotide base (Figure [70]S2). Thirty
   samples' quality distribution of bases along reads was also been good,
   we can see that the percentage of the bases with low quality (< 20) was
   low (Figure [71]S3). All these indexes indicated that these samples'
   reads could be used for further analyzation. Alignment statistics
   showed that about 70 percent of the total clean raw reads could be
   matched to the reference genome. When it comes to the reference gene,
   the percent of total matched reads was about 52 (Table [72]S5). And
   then we calculated the distribution of reads on reference genes and the
   gene coverage distribution. The results showed that reads were evenly
   distributed on reference genes (Figure [73]S4). These data suggested
   that the RNA-seq could well represent the expression level of unigenes.

It showed a clear separation between 2-W and 40-W group based on genes
transcriptional pattern

   We took an unsupervised approach to partition the 30 analyzed samples
   into different populations based on their transcriptomic similarity.
   Result of Pearson correlation coefficients (PCC) showed high
   heterogeneity between 2-W and 40-W group, supporting the idea that
   genes transcription patterns in chicken thymus were significantly
   different between early life and adulthood stages (Figure [74]2A). And
   then, principal component analysis (PCA) was applied to further dissect
   the two main groups. As shown in Figure [75]2B, the results showed a
   clear separation between 2-W and 40-W group. To characterize the
   functional state of the two subpopulations, we screened the genes
   differentially expressed on the basis of RPKM. From Figure [76]2C, we
   could see that 1949 DEGs (1722 up-regulated and 127 down-regulated)
   were screened between 40-W and 2-W group. Hierarchical cluster analysis
   of DEGs was also in accordance with the results of Pearson correlation
   coefficients analysis and PCA, and showed distinct transcriptional
   patterns between 2-W and 40-W group.

Figure 2.

   [77]Figure 2
   [78]Open in a new tab

   Transcriptional pattern of all the 30 samples. Results of pearson
   correlation coefficients (A) and principal component analysis (B)
   showed a clear separation between 2-W and 40-W group. And 1949 DEGs
   (1722 up-regulated and 127 down-regulated) were screened between 40-W
   and 2-W group (C).

Gene ontology (GO) and KEGG pathway enrichment analysis of DEGs

   On the basis of DEGs, GO, and KEGG enrichment analysis were conducted
   to investigate the effects of developmental stages on thymus immunity
   of chickens, and the results of enrichment analysis were shown in
   Figures [79]3, 4, respectively. The 20 top canonical GO terms
   significantly (p < 0.05) enriched were shown in Figure [80]3A, 7 terms
   (response to stimulus, biological regulation, cell communication,
   single organism signaling, signaling, regulation of biological process,
   signal transduction) of these 20 enriched GO terms were in relation to
   signal transduction and biological regulation (Figure [81]3B). To learn
   the canonical pathways related to thymus developmental stages of
   chicken, we found out the relevant pathways within DEGs, and the top 20
   significantly enriched KEGG pathways were showed in Figure [82]4A.
   Among these 20 enriched pathways, 4 pathways (Cytokine-cytokine
   receptor interaction, Intestinal immune network for IgA production,
   Toll-like receptor signaling pathway, AGE-RAGE signaling pathway in
   diabetic complications) were closely associated with immune regulation
   (Figure [83]4B). Through GO and KEGG pathway enrichment analysis, we
   further confirmed that thymus immunity of chickens had a strong
   age-related correlation.

Figure 3.

   [84]Figure 3
   [85]Open in a new tab

   Gene ontology enrichment analysis of DEGs screened from 40-W group and
   2-W group. The 20 top gene ontology terms significantly enriched were
   shown in Figure [86]3A, and 7 of these 20 terms were related to signal
   transduction and biological regulation (B).

Figure 4.

   [87]Figure 4
   [88]Open in a new tab

   KEGG pathway enrichment analysis of DEGs. The 20 top KEGG pathways
   significantly enriched were shown in (A). Four (including statistical
   significance and DEGs of the 4 pathways) of these 20 pathways were
   associated with immune regulation (B).

Expression data validation using quantitative real time PCR (qRT-PCR)

   According to the results of GO and KEGG pathway enrichment analysis,
   eight DEGs related to immunoregulation, including TLR4, TLR1, TLR5,
   CD40, AP-1, IL-8, TACI, and PIGR [89](Figure 5A), were selected and
   tested using RT-qPCR method to validate the accuracy of RNA-seq data.
   The results showed that the relative expression levels of the eight
   DEGs gotten from qRT-PCR [90](Figure 5C) were largely in line with that
   of RNA-Seq [91](Figure 5B). This demonstrated that the transcription
   data of RNA-Seq was accurate. Meanwhile, compared with 2-W group,
   transcription of these eight DEGs were all significantly (p < 0.01)
   up-regulated in 40-W group. It meant that the thymus immunity of 40-W
   grown chickens was up-regulated, in contrast to the 2-W young chicks.

Figure 5.

   Figure 5
   [92]Open in a new tab

   Expression data validation using quantitative real time PCR (qRT-PCR).
   The 8 analyzed DEGs (IL-8, CD40, AP-1, TLR1, TLR4, TLR5, TACI, and
   PIGR) were closely associated to the 4 enriched immune-related pathway
   (A). It showed that the relative expression levels of the eight DEGs
   gotten from qRT-PCR (C) were in line with that of RNA-Seq (B), and
   transcription of the 8 genes were all up-regulated in 40-W group
   compared to the 2-W group. Data shown as mean ± SD, **P < 0.01.

Negative correlation between CD40 transcription and promoter methylation

   To explore the transcriptional regulatory mechanism responsible for
   activation of the gene, we analyzed the methylation level of promoter
   region of CD40. The promoter region analyzed was composed of 201
   nucleotides, and contained 17 CpGs (Figure [93]6A). It showed that
   appropriate level of methylation occurred in CD40 promoter of thymus,
   and the promoter methylation level of CD40 was decreased in 40-W group
   compared to 2-W group (Figure [94]6B). T-test and histogram of the
   results indicated that the methylation level was significantly (p <
   0.01) decreased from 79.77% in 2-W group to 65.52% in 40-W group
   (Figure [95]6C). Of all the 17 CpG sites within the promoter region
   analyzed, compared to the 2-W group, the methylation level of 11
   promoter CpG sites were significantly (p < 0.05, −824, −816, −784,
   −763, −751, −740, −725, and −716) or highly significantly (p < 0.01,
   −758, −743, and −731) decreased in the promoter region between −824 and
   −716 (Figure [96]6E). Our study suggested there might be a correlation
   between developmental stages and promoter CpG island methylation of
   CD40.

Figure 6.

   [97]Figure 6
   [98]Open in a new tab

   Developmental stages' (2 and 40-weeks-age) effect on promotor
   methylation of CD40 in thymus. Primer sequences for CD40 promotor
   methylation were shown as (A), and the promotor CpG island of CD40
   contained 201 bases and 17 CpG sites. Results of CD40 promotor
   methylation were shown in (B). A single raw means a cloned sequence,
   and a circle means a CpG site. A filled circle means a methylated
   cytosine, and an open circles means an un-methylated cytosine. T-test
   and histogram of the overall CD40 promotor CpG methylation showed
   highly significant effects between 2-W and 40-W group (C).
   Transcription factor binding site prediction showed that the
   transcription factors with high-affinity binding activity in the region
   analyzed were all transcription activators (D). Chi-square test and
   histogram of single CpG site showed that the methylation level of 40-W
   group was significantly down-regulated in transcription
   activators-binding domain (−840 ~ −745; E). Data shown as mean ± SD, *P
   < 0.05, **P < 0.01.

Discussion

   Thymus plays a pivotal role in the development of the immune system,
   and it is the primary donor of lymphocyte for the immune system
   ([99]2). Nevertheless, morphology and immunity of mammals' thymus are
   varied with age. This is also true for chicken thymus, herein, thymus
   weight and morphology of chick (2-W) was quite different from that of
   grown chicken (40-W). According to the thymus' weight and morphology,
   thymus weight of 2-weeks old chick was much smaller than that of grown
   chicken, and its thymus morphology was much rounder than the grown
   chicken's. We can conclude that thymus of the chick undergoes rapid
   growth. Whereas, as to grown chicken, its thymus index was highly
   significantly smaller than young chick's, and its thymus morphology was
   thinner, and much more oval and elongate. And so, thymus of grown
   chicken has already been in a state of degeneration. Considering these
   differences, immune-function of thymus would also inevitably be
   affected. Indeed, researches before indicated that compared to the
   adult animals, the developing immune system of young animals was
   characterized by suppressed immune system, blunted inflammatory
   cytokine production and skewed T and B cell development in favor of
   regulatory responses ([100]21). However, the detailed effects of
   developmental stages on chicken thymus immunity are remains to be fully
   elucidated. And so, transcriptome analysis of thymus was conducted to
   get a better handle on this problem.

   The results of PCC and PCA led us to believe that the whole
   transcriptional pattern of 2-W group was distinct from that of 40-W
   group. Considering thymus is an immune organ, these results also proved
   that thymuses of young chick and grown chicken were of vary different
   immunity. The detailed biological differences between chick and grown
   chicken arise from the DEGs. In comparison to the 2-W group, we got
   1949 DEGs (1722 up-regulated and 127 down-regulated) from the 40-W
   group. To understand the possible immune functions associated with
   these DEGs observed in the 40-W group vs. the 2-W group, GO, and KEGG
   enrichment analysis was adopted. We found 4 significantly enriched KEGG
   signaling pathways which were crucial for immune regulation. These
   screened pathways, including cytokine-cytokine receptor interaction,
   intestinal immune network for IgA production, Toll-like receptor
   signaling pathway, AGE-RAGE signaling pathway in diabetic
   complications, could partly explain the correspondence between
   developmental stages and thymus immunity. Toll-like receptors are the
   main receptor proteins located on the surface of immune cells, and
   tasked with recognizing conserved structures of intruders ([101]22).
   This suggests, Toll-like receptor signaling pathway is crucial for
   modulating innate and adaptive immune responses to infected pathogens,
   as well as endogenous antigens ([102]23–[103]25). While,
   cytokine-cytokine receptor interaction is involved in recognition of
   cytokine and chemotactic factor, and further controlling the
   hematopoietic cells' differentiation, proliferation, migration, or to
   perform bio-functions such as phagocytosis or activate inflammatory
   response ([104]26, [105]27). Another significantly enriched pathway,
   AGE-RAGE signaling pathway in diabetic complications, as well, has a
   close relation with inflammatory response ([106]28). Last but not
   least, intestinal immune network for IgA production, is mainly
   implicated in the intestinal adaptive immune response to gut
   microbiota, and specifically reducing the growth of some microbes
   ([107]29–[108]31). Transcription of 8 screened DEGs (as well as core
   regulators of these significantly enriched immune pathways) in 40-W
   group were up-regulated compared with that of 2-W group. Other DEGs in
   these 4 signaling pathway exhibited consistency change between 2-W and
   40-W group. This means that, in contrast to 2-W group, DEGs enriched in
   these 4 screened KEGG pathways were all hyper-activated in 40-W group.
   It indicated that thymus immunity of 2-weeks-age chick, mainly about
   immune recognition and signaling transduction, intestinal adaptive
   immune response to gut microbiota, and regulation of inflammatory
   response, was kept in a status of hypo-activation. A possible
   explanation to these results is that thymus immunity is varied with
   age, and this variance is related to the co-adaptation between
   chicken's thymus immunity and intestinal microbiota.

   Age is one of the major drivers of changes in gut microbiota ([109]32,
   [110]33). For chicken, its intestine contained significantly different
   microbiome composition through the life span, but the intestinal
   microbiome showed clear successional changes as the bird matured
   ([111]34–[112]36). The host immune system is frequently described as
   being “tolerant” to the high numbers of bacteria that symbiotic with
   the host ([113]32, [114]37). While, for young animals, the neonate
   innate immune system can't successfully deal with trillions of
   intestinal microbiotas. To survive from various intestinal pathogens,
   young animals must have more “patient” than adult animals. For example,
   in the intestine of young animals, more mucosal mucin must be secreted
   to shield host from intestinal microbiota ([115]37, [116]38). Even if,
   some intestinal microorganisms can be directly in contact with
   intestinal epithelial cells and recognized by pattern-recognition
   receptors (PRR, such as TLRs), the induced immune response to these
   microorganisms is highly distinct from that of adult animals, with
   notable impairment in the production of inflammatory mediators such as
   pro-inflammatory cytokines and oxygen radicals, but heightened
   production of regulatory cytokines such as IL-10 ([117]32, [118]39).
   For young animals, the primary fraction of the immune system's
   constitutive function is to control the interaction between animals and
   intestinal microbiota, and have the characteristics of a reduced number
   of T cells and intestinal IgA-production ([119]32). In our research, as
   well, thymus immunity, including antigen recognition, intestinal
   IgA-production, and inflammation of 2-weeks-age chick, was
   down-regulated compared to those of 40-weeks-age chicken.

   Hypo-activation of chick thymus immunity in young age is modulated by
   the corresponding DEGs, and this transcriptional pattern could sustain
   a rather long time (at least 2 weeks). Among all kinds of regulatory
   mechanisms of gene expression, only epigenetics modification can meet
   this requirement. Epigenetic modifications, including DNA methylation,
   histone covalent modifications, chromosome reconstruction, and
   micro-RNA, have a common characteristic, high in stability, and the
   status of epigenetic modification can be maintained for a long time.
   DNA methylation is a kind of gradient mechanism in regulating gene
   expression by a time-dependent manner, and it is an important factor
   that responsible for establishment, maintenance, and reversal of
   metastable transcriptional states ([120]40). Many researches before
   found that DNA methylation is also critical in the coadaptation between
   symbiotic microorganisms and the host ([121]41–[122]45), and modulation
   of immune cell differentiation ([123]46–[124]48). It means that there
   exist microbiota-dependent epigenetic programming of genes related to
   intestinal mucosal immunity and systematic immunity, which is critical
   for animal health and intestinal function. So we hypothesized that the
   thymus immunity varied with age was modulated by promotor methylation
   of core regulators.

   CD40 is one of the 8 core regulators, and it closely related to 3
   (cytokine-cytokine receptor interaction, intestinal immune network for
   IgA production, and Toll-like receptor signaling pathway) of the 4
   screened pathways. In addition, compared with the other 7 DEGs, the
   promoter CpG island of CD40 contains a maximum amount of CpG sites (17
   CpG sites in the region analyzed). CD40 is an important transmembrane
   receptor which is widely expressed in almost all kinds of immune cells,
   including T/B cells, dendritic cells, monocytes, and macrophages, and
   it plays a critical role in immunity and inflammation
   ([125]49–[126]51). In the process of host immune-regulation, CD40
   pathway is involved in both cellular immunity and humoral immunity. By
   interaction with CD40L, CD40 could modulate the crosstalk between
   antigen presenting cells (macrophages and dendritic cell) and T/B cell,
   and further induce the expression of pro-inflammatory cytokines and
   activation of B cell response ([127]52), as well as synthesis of IgG/A
   ([128]50, [129]53). As a key co-stimulatory molecule in T cell, CD40 is
   critical for body's defense against invading pathogens, and plays
   essential role in adaptive immunity by eliciting survival,
   proliferation, differentiation and/or cell death signaling pathways
   ([130]54, [131]55), and so, contributes much to immune response against
   pathogen infection ([132]56), however, these immune responses often
   accompany inflammatory response ([133]56–[134]58). CD40 and CD40 ligand
   (CD40L) participate in numerous inflammatory pathways, as well as
   oxidative stress ([135]50). Studies in monocytes and macrophages
   revealed that activation of CD40 would result in a primarily
   pro-inflammatory response, and efficiently activate the synthesis of
   pro-inflammatory cytokines (e.g., IL-1, IL-8, TNF-α, IL-12, IL-6, CCL2,
   and so on) ([136]59). Above all, CD40 is critical for host immunity,
   and down-regulation of CD40 had a certain correlation with suppression
   of thymus immunity.

   So we analyzed the promoter methylation of CD40 by using BSP method to
   learn if it existed differences in CD40 promoter methylation between
   2-W group and 40-W group. The results showed that the CD40 promoter
   methylation was significant different between 2-W group and 40-W group,
   and it was significantly down-regulated in the 40-W group. Previous
   studies on DNA methylation have found that methylated CpG dinucleotide
   can be recognized and bound by the proteins with a methyl-CpG-binding
   domain (MBD), such as methyl-CpG-binding protein 2, MBD1, MBD2, and
   MBD4 ([137]16, [138]60, [139]61). These MBD-containing proteins can
   compete with transcription factors for non-sequence-specific DNA
   binding, and controlling the genes' expression at the transcriptional
   level ([140]15). Results of transcription factor binding site
   prediction showed that the transcription factors with high-affinity
   binding activity in CD40 gene promoter region (−840 ~ −735) were all
   transcription activators (Figure [141]6D). Methylation level of 11 CpG
   sites in the region between −824 and −716 was significantly increased
   in 2-W group. And so, hyper-methylation of CD40 promotor in 2-W group
   can inhibit the recruit of transcription activators, and stabilize and
   suppress the transcription of CD40. Given that DNA (de)methylation
   modification is a time-consuming biochemical reaction.
   Hyper-methylation of CD40 promotor in chick thymus can stabilize the
   low expression of CD40, and further contribute to the hypo-activation
   of thymus immunity.

   From the discussion above, we can draw the conclusion that thymus
   immunity of chicken is varied with age. Thymus immunity of chick,
   including antigen recognition and presentation, intestinal
   IgA-production, and inflammation, was inhibited in comparison to that
   of grown chicken. Suppression of CD40, which is induced by promotor
   hyper-methylation, might be critical for the maintenance of
   hypo-activated immune status in chick thymus. But more research should
   be conducted to further analyze the internal relation between gut
   microbiota and thymus immunity, and the regulatory effects of CD40 in
   chicken's immune response to gut microbiota.

Data availability

   The authors confirm that all data underlying the findings are fully
   available without restriction.

Author contributions

   YL, XL, ZY, QS, and XY conceived and designed the experiments. YL, XL,
   WG, and SW performed the experiments. YL and XL analyzed the data. XY
   contributed reagents, materials, analysis tools. YL wrote the
   manuscript. XY and HL provided editorial suggestions and revisions.

Conflict of interest statement

   The authors declare that the research was conducted in the absence of
   any commercial or financial relationships that could be construed as a
   potential conflict of interest.

Acknowledgments