Messenger RNA expression of chicken CLOCK gene in the responseto Campylobacter jejuni inoculation

Xiaoyi Liu,∗,1 Liying Liu,†,1 Maozhi Zhang,∗ Ning Yang,§ Yukai Qi,∗ Yu Sun,∗ and Xianyao Li∗,‡,2

∗College of Animal Science, Shandong Agricultural University, Taian 271018, China; †College of Life Science,Shandong Agricultural University, Taian 271018, China; ‡Shandong Provincial Key Laboratory of Animal

Biotechnology and Disease Control and Prevention; Tai’an 271018, China; and §College of Animal Science andTechnology, China Agricultural University, Beijing 100193, China

ABSTRACT Campylobacter jejuni (C. jejuni) is a lead-ing cause of human bacterial gastroenteritis worldwide.Previous research has shown that circadian rhythmplays a critical role in host response to C. jejuni col-onization. The CLOCK gene is one of the core genesregulating circadian rhythms and shows significant ex-pression on 7 d post-C. jejuni inoculation. The ob-jective of this study was to investigate temporal andspatial expression of chicken CLOCK gene post-C. je-juni inoculation. Cecal and splenic RNA were isolatedfrom 2 distinct chicken breeds and used to compare themRNA expression of CLOCK gene between inoculated

and noninoculated chickens within each breed and be-tween breeds within each of inoculated and noninocu-lated groups. Our results showed that the CLOCK genewas significantly down-regulated at 20 h postinocula-tion (hpi) in cecum and spleen in Jiningbairi chicken.CLOCK gene was significantly down-regulated at 4and 16 hpi and up-regulated at 8 hpi in cecum andspleen in specific pathogen free white leghorn noninoc-ulated chicken. The findings suggested that expressionof CLOCK gene was significantly changed post C. je-juin inoculation. This change was affected by geneticbackground, tissue, and time points postinoculation.

Key words: chicken, Campylobacter jejuni, CLOCK, expression2015 Poultry Science 94:2124–2130

http://dx.doi.org/10.3382/ps/pev203

INTRODUCTION

Campylobacter jejuni (C. jejuni), a Gram-negative,spiral-shaped bacterium, is one of the most commoncauses of human bacterial enteritis worldwide (Zilbaueret al., 2008; van Putten et al., 2009; Gruntar et al.,2010), primarily due to contaminated poultry products(Altekruse et al., 1999). It represents a major publichealth and economic burden, and commonly linked topostinfectious onset of autoimmune diseases, such asGuillain–Barre syndrome (Tam et al., 2007) and MillerFisher syndrome (Koga et al., 2005).

The chicken is the natural reservoir of C. jejuni(Altekruse et al., 1999) where bacteria colonize the di-gestive tract, but rarely cause disease symptoms (Beeryet al., 1988). Cecum is the primary colonizing site ofC jejuni in chicken (Beery et al., 1988; Meade et al.,2009). Moreover, several previous studies have shownthat host genetic background plays an important role inthe response to C jejuni inoculation. Significantly dif-ferent susceptibility to C. jejuni inoculation has been

C© 2015 Poultry Science Association Inc.Received February 3, 2015.Accepted May 13, 2015.1These authors contributed equally to this study.2Corresponding author: xyli@sdau.edu.cn

observed between different chicken lines (Stern et al.,1990; Boyd et al., 2005; Li et al., 2008a).

Genes induced by C. jejuni inoculation in the chickencecum are associated with Toll-like receptors (TLR;i.e., TLR4, TLR5, and TLR21) (de Zoete et al., 2010),proinflammatory response (IL-8, IL-6, IL-1β, CXCLi1,and CXCLi2) (Byrne et al., 2007; Larson et al., 2008;Smith et al., 2008), lymphocyte involvement (TNFSF6and TNFSF8) (Li et al., 2010; Li et al., 2011), andcircadian rhythm function (CLOCK) (Li et al., 2010).Studies have shown that a set of cytokines of IL-6, IL-1β, IL-18, IL-2, TGF-β4, K60, and IL-8, and circadianclock genes (cry1/2, per2/3, Bmal1/2, and CLOCK)have a 24-h different expression in the response of thebacterial colonization (Li et al., 2008b; Naidu et al.,2010).

Circadian rhythms are daily oscillations of multi-ple biological processes driven by endogenous clocks,which play important roles in local and systemic phys-iology and pathology (Lamia et al., 2008; Sahar andSassone-Corsi, 2009). It has been reported that circa-dian rhythms can influence mammal immune responsethrough regulating the blood circulation during diurnalsleeping/waking cycles (Born et al., 1997; Liu et al.,2006).

In poultry, circadian rhythms are generated bya transcription/translation-based oscillatory loop

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including Per2, Per3, CLOCK, and Bmal1 (Young andKay, 2001; Reppert and Weaver, 2002; Ueda et al.,2005). The CLOCK gene is one of the core genesregulating circadian rhythms and plays an importantrole in maintaining the normal circadian rhythms(Harrisingh and Nitabach, 2008). Chicken CLOCKgene locates on Chromosome 4 (Noakes et al., 2000). Ithas been reported that chicken CLOCK gene expressesnot only in pineal gland (Kommedal et al., 2011) andbrain (Helfer et al., 2006), but also in peripheral tissue(Naidu et al., 2010). It plays an important role in earlychicken development (Goncalves et al., 2012) and inhost response to bacterial colonization (Li et al., 2010;Bellet et al., 2013).

The detailed mRNA expression of chicken CLOCKgene responding to C. jejuni colonization is still un-clear. Therefore, the objective of the present study wasto investigate the temporal and spatial expression ofCLOCK gene post-C. jejuni inoculation in 2 differentgenetic chicken lines.

MATERIALS AND METHODS

Chickens, C. jejuni Inoculation, and SampleCollection

Jiningbairi, a China local chicken breed, and specificpathogen free (SPF) white leghorn were used in thecurrent study. The meconium was collected from eachindividual chicken and checked for C. jejuni negativethrough the plating method. C. jejuni free chickens wereused for further inoculation. C. jejuni NCTC11168 wasenriched in Bolton broth at 37◦C for 48 h, pelleted,and diluted with PBS to make the inoculant. Withineach breed, fifty-six 3-day-old C. jejuni free chickenswere randomly divided into 2 groups; 28 chickens wereorally inoculated with 0.5 mL inoculants of 1.0 × 108

cfu/chicken as inoculated group [inoculated group (I),Jiningbairi inoculated (JI), and SPF white leghorn in-oculated (SI)], and 28 chickens were mock inoculatedwith 0.5 mL PBS as control group (noninoculated group(N), Jiningbairi noninoculated (JN), and SPF whiteleghorn noninoculated (SN)].

Four birds in each group were randomly selected andsacrificed by cervical dislocation at 4, 8, 12, 16, 20, 24,and 48 h postinoculation (hpi). Cecal contents werecollected for C. jejuni enumeration. Spleen and cecum(including cecal tonsil) were aseptically harvested fromeach chicken, frozen in liquid nitrogen, and stored at−80◦C until further RNA isolation. All animal proce-dures were approved by Shandong Agricultural Univer-sity Animal Care and Use Committee (SDAUA-2014-010).

Total RNA Isolation, Reverse Transcription,and Quantitative Real-Time PCR

Total RNA was isolated from each homogenizedspleen and cecum sample using TRIZOL reagent

(Invitrogen, Grand Island, NY) following manufac-turer’s instruction. Fifty-six RNA samples were iso-lated from each inoculated and noninoculated group,respectively. In total, 224 individual RNA sam-ples were isolated. The RNA quantity and qual-ity was checked by Nanodrop (Thermo Scien-tific, Grand Island, NY) and gel electrophoresis,respectively.

Total RNA of each sample was reverse transcribedinto the cDNA using the Prime Script RT Reagent Kit(Perfect Real Time) (Takara, Dalian, China) accord-ing to the manufacturer’s instructions. The 20-μL reac-tion system was used, which contains 5 × Primer ScriptBuffer (for Real Time) 4 μL, Primer Script RT EnzymeMix I 1 μL, Random 6 mers (100 uM) 1 μL, total RNA1 μg, and add RNase free double-distilled H2O to 20μL. The reaction condition is 37◦C, 15 min; 85◦C, 5s.

Quantitative real-time PCR was performed usingthe Stratagene MX3000P with chicken CLOCK gene(GenBank Accession: NM 204174) specific primers(forward: 5′-TTCTGTCTTCTCATCTGCTGGA-3′,reverse: 5′-GGTTGCTCTTTTGGGTCTATTG-3′). Chicken β-actin gene (forward:5′-TGCTGTGTTCCCATCTATCG-3′, reverse: 5′-TGCTGTGTTCCCATCTATCG-3′) was used as aninternal standard to correct the input of cDNA.PCRmixtures (20 μL) contained SYBR Primer Ex Taq(2×) 10 μL, forward primer (10 μM) 0.4 μL, reverseprimer (10 uM) 0.4 μL, ROX Reference Dye II (50×)0.4 μL, cDNA 2 μl, and double-distilled H2O 6.8 μL.The conditions of the PCR were as follows: 94◦C for5 min, followed by 40 cycles of 95◦C 5s, 59◦C 30s, andthen 95◦C 1 min, 61◦C 30s, 95◦C 30s. Triplicate wasperformed for each cDNA sample.

Statistical Analysis

The relative of mRNA level was calculated using the2−ΔΔCt method (Livak and Schmittgen, 2001). The datawere analyzed using the general linear model procedureof SAS 9.1 software (SAS Institute, Cary, NC). Themain effects of line, infection, and time were tested inthe statistical model. A Bonferroni test was used formultiple mean comparisons. P < 0.05 was consideredas statistical significance.

RESULTS

Relative Expression of CLOCK GeneBetween I and N Groups in Cecum andSpleen

Relative expression of CLOCK gene in the com-parison of JI and JN. In the cecum, expression levelsof CLOCK gene were induced from 4 to 16 hpi and sup-pressed from 20 to 48 hpi. The expression of CLOCK

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Figure 1. The relative expression of CLOCK gene between inoculated and noninoculated chickens. A: The relative expression of CLOCKgene in cecum between SI and SN group. B: The relative expression of CLOCK gene in spleen between SI and SN group. C: The relative mRNAexpression of CLOCK gene in cecum between JI and JN group. D: The relative mRNA expression of CLOCK gene in spleen between JI andJN groups. Note that two asterisks indicates that the values differ significantly (P < 0.05), ∗∗means differ significantly (P < 0.01). The y axisrepresents the relative expression of CLOCK gene, relative fold-difference in expression was calculated by normalizing to SN or JN at 4 hpi.(cecum = 1, spleen = 1; SE are from experimental triplicates on one plate). SN: SPF white leghorn non-inoculated. SI: SPF white leghorninoculated. JN: Jiningbairi noninoculated. JI: Jiningbairi inoculated. hpi: hours postinoculation.

gene was significantly down-regulated at 20 hpi (P <0.05) with a fold-change of 1.45 (Figure 1A).

In the spleen, the mRNA expression level of CLOCKwas down-regulated at 4, 12, 20, 24, and 48 hpi. Itwas significantly down-regulated at 20 and 24 hpi(P < 0.05), with the fold-change of 1.32 and 1.39,respectively. The CLOCK gene was suppressed from 20to 48 hpi (Figure 1B).

Relative expression of CLOCK gene in the com-parison of SI and SN. In the cecum, CLOCK genewas up-regulated at 8, 12, 24, and 48 hpi, and down-regulated at 4, 16, and 20 hpi, following C. jejuni inocu-lation (Figure 1C). It was significantly down-regulatedat 4 and 16 hpi (P < 0.05). It was significantly up-regulated at 8 and 24 hpi (P < 0.05). The highest fold-change (1.55) of CLOCK gene was found at 16 hpi.

In the spleen, CLOCK gene was significantly down-regulated at 4 and 16 hpi (P < 0.05). It was significantlyup-regulated at 8 and 24 hpi (P < 0.05). The highest

fold-change (1.54) of the CLOCK gene was observed at4 hpi (Figure 1D).

Relative expression of CLOCK genebetween SPF and Jiningbairi chicken incecum and spleen

To investigate the genetic effect on the expressionof CLOCK gene in cecum and spleen, the different ex-pression of CLOCK gene between SPF and Jiningbairichicken within noninoculated or inoculated cecum andspleen was analyzed at different time points post-C. je-juni inoculation (Figure 2).

Different expression of CLOCK gene between SNand JN. For the cecum, the CLOCK gene had a lowerexpression level in JN than that in SN at all the timepoints post C. jejuni inoculation excluding 16 hpi. The

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CHICKEN CLOCK GENE EXPRESSION FOLLOWING C. JEJUNI INOCULATION 2127

Figure 2. Comparison of the mRNA expression of CLOCK gene between SPF and Jiningbairi chickens. A: The relative expression of CLOCKgene in cecum in the comparison of SN and JN. B: The relative expression of CLOCK gene in spleen in the comparison of SN and JN.C: Therelative expression of CLOCK gene in cecum between SI and JI. D: The relative expression of CLOCK gene in spleen between SI and JI. Notethat one asterisk indicates that the values differ significantly (P < 0.05), and two asterisks indicates that the values differ significantly (P <0.01). The y axis represents the relative expression of CLOCK gene. Relative fold-difference in expression was calculated by normalizing to SNor SI at 4 hpi. (cecum = 1, spleen = 1; SE are from experimental triplicates on one plate). SN: SPF white leghorn noninoculated. SI: SPF whiteleghorn inoculated. JN: Jiningbairi noninoculated. JI: Jiningbairi inoculated. hpi: hours postinoculation.

CLOCK gene had significantly lower expression in JNthan that in SN at 8, 12, 24, and 48 hpi (Figure 2A).

For the spleen, the CLOCK gene had higher expres-sion in JN than that in SN at all the time points post-C.jejuni inoculation, and significantly higher at 8, 24, and48 hpi, respectively (Figure 2B) (P < 0.05).

Different expression of CLOCK gene between SIand JI. For the cecum, the CLOCK gene had lowerexpression in JI than that in SI at all the time pointspost-C. jejuni inoculation excluding 24 hpi (Figure 2C).The difference of CLOCK gene expression betweenJI and SI was significant at 4, 8, 12, and 16 hpi(P < 0.05).

For the spleen, the CLOCK gene had lower expres-sion in JI than that in SI at all the time points post-C. jejuni inoculation excluding 48 hpi (Figure 2D).The difference of CLOCK gene expression betweenJI and SI was significant at 4, 8, 12, and 16 hpi(P < 0.05).

DISCUSSION

In the present study, the expression profile of thechicken CLOCK gene, one of the core genes regulat-ing circadian rhythms between inoculated and non-inoculated group within each line and between 2 dis-tinct chicken lines within inoculated and noninoculatedgroups, were measured and analyzed.

Genetic Effect on the Chicken CLOCKGene Expression

Gene expression in the response to bacterial inocula-tion can be affected by many factors, and these factorsmay differ among individuals and genetic lines.

Jiningbairi chicken is a China native chicken breedwith good ability of disease and stress resistance. SPFchicken originates from commonly used white leghorn.

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The genetic background between these 2 breeds is sig-nificantly different.

Several previous studies have shown that the host ge-netic background plays an important role in responseto C. jejuni inoculation (Stern et al., 1990; Boyd et al.,2005; Li et al., 2008a). Moreover, circadian rhythmplays a critical role in host response of the more re-sistant line to C jejuni colonization (Li et al., 2010).The CLOCK gene is related to the immunity (Curtiset al., 2014); when the host encounters bacteria inoc-ulation, it contributes to systemic and local immunesystem responses against bacteria inoculation. Differ-ent response of CLOCK gene expression following C.jejuni inoculation was observed between these 3 distinctlines. The expression of CLOCK gene was significantlydown-regulated at 20 hpi in Jiningbairi chicken; how-ever, it was significantly different at 4, 8, 16, and 24hpi in SPF chicken. The different expression pattern ofCLOCK gene may contribute to different response toC. jejuni inoculation between these 2 distinct geneticlines.

It has been reported most of the immune relatedgenes were lower expressed in resistant line than thatin susceptible line following C. jejuni inoculation (Liet al., 2010). The expression of the CLOCK gene in ce-cum and spleen of Jiningbari was lower than that inSPF post-C. jejuni inoculation (Figure 2C, D). Thiscould be the evidence that Jiningbairi is more resistantto C. jejuni inoculation than SPF chicken.

Tissue Specificity of Chicken CLOCKGene Expression

In animals, each tissue has uniquely physiologicaland metabolic functions. Different tissue-specific phys-iological and metabolic rhythms are regulated by theCLOCK gene (Menet and Hardin, 2014). Cecum andspleen have different functions. Cecum, one of the gutassociated lymphoid tissues, includes a lymphoid tissueof the cecal tonsil and plays important regulation rolesin responding to C. jejuni inoculation between 2 differ-ent lines and different individuals within each line (Liet al., 2010; Li et al., 2011). Spleen, a major periph-eral lymphoid organ in the chicken, is one of the majorsites involved in initiating lymphocyte activation, pro-liferation, and differentiation, which are all essential toinitiate the immune response against C. jejuni (Li et al.,2012).

C. jejuni can colonize not only the cecum (Joneset al., 2004), but also spleen, liver, thymus. and bursa(Cox et al., 2006). Studies have shown that the gene ex-pression profiling between cecum and spleen was signif-icantly different in the response to C. jejuni inoculation(Li et al., 2010; Li et al., 2012). The regulatory directionof the CLOCK gene was more complicated in spleenthan that in cecum from 4 to 12 h post-C. jejuni inoc-ulation in Jiningbairi. CLOCK had significantly higherexpression level in cecum than that in spleen in SPF

chicken or Jininbairi chicken regardless of C. jejuni col-onization (Supplemental Figure S1). This is consistentwith that higher expression of CLOCK gene cecum thanthat in spleen in Hy-line chicken (Liu et al., 2015).

Effect of C. jejuni Inoculation on CLOCKGene Expression

C. jejuni inoculation greatly influences host geneexpression. Thousands of genes were significantly ex-pressed at 7 d following C. jejuni inoculation (Li et al.,2010; Li et al., 2012). The CLOCK gene was signifi-cantly up-regulated in chicken cecum on d 7 post-C. je-juni inoculation in both resistant and susceptible lines(Li et al., 2010). Early response of CLOCK gene to C.jejuni inoculation has been investigated in the currentstudy. Dynamic expression of CLOCK gene at differ-ent time points post-C. jejuni inoculation has been ob-served in the current study. The expression of CLOCKgene was more complicated from 4 to 16 hpi than thatfrom 20 to 48 hpi (Figure 1B-D). It has been reportedthat Lipopolysaccharide-induced circadian clock genes(cry1/2, per2/3, Bmal1/2, and CLOCK) show a dailyrhythm of inflammation in the avian spleen (Naiduet al., 2010).

The CLOCK gene may regulate C. jejuni inoculationthrough cross-talking with immune related genes. Stud-ies have shown that the core clock proteins BMAL1,CLOCK, and REV-ERB α control fundamental aspectsof the immune response, and the BMAL1:CLOCK het-erodimer regulates TLR9 expression (Silver et al., 2012)and represses expression of the inflammatory mono-cyte chemokine ligand (i.e., CCL2) (Nguyen et al.,2013), and REV-ERB α suppressed the induction ofIL-6 (Gibbs et al., 2012). Further study is warrantedto confirm the cross-talking between the CLOCK geneand immune-related genes.

CONCLUSIONS

In summary, expression of the CLOCK gene wassignificantly changed post-C. jejuin inoculation. Thischange was affected by the genetic background andtissue. The result herein will pave the foundation forfurther understanding molecular mechanisms of theCLOCK gene contributing to the response to C jejunicolonization.

ACKNOWLEDGMENTS

This study was supported by the National NaturalScience Foundation of China (31172204), the NationalHigh Technology Research and Development Programof China (863 Program) (2011AA100305), the Shan-dong Modern Agricultural Industry and TechnologySystem (SDAIT-13-011-02), and a Shandong Provinceagricultural seed project.

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CHICKEN CLOCK GENE EXPRESSION FOLLOWING C. JEJUNI INOCULATION 2129

SUPPLEMENTARY DATA

Figure S1. The relative expression of CLOCK genebetween cecum and spleen. A: The relative expressionof CLOCK gene between cecum and spleen in SN. B:The relative expression of CLOCK gene between cecumand spleen in SI. C: The relative expression of CLOCKgene between cecum and spleen in JN. D: The relativeexpression of CLOCK gene between cecum and spleenin JI. Note that two asterisks indicates that the valuesdiffer significantly (P < 0.01). The y axis representsthe relative expression of CLOCK gene; relative fold-change was calculated by normalizing to cecum at 4hpi in SPF or Jiningbairi chicken (cecum = 1; SE arefrom experimental triplicates on one plate). SN: SPFwhite leghorn noninoculated. SI: SPF white leghorn in-oculated. JN: Jiningbairi noninoculated. JI: Jiningbairiinoculated. hpi: hours postinoculation.

Supplementary data is available at PSA Journal on-line.

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