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Identification of molecular signatures from different vaccine adjuvants in chicken by integrative analysis of microarray data.

INTRODUCTION

An adjuvant is an agent that stimulates the immune system and increases the host response to an antigen without itself conferring a specific antigenic effect (Bowersock and Martin, 1999). Effective adjuvants utilize multiple compounds and mechanisms to achieve the desired immunological enhancement such as long lasting antigen depots, immunological presentation of vaccine antigens, and induction of T lymphocyte responses (Reed et al., 2009). As of present, much progress has been made to develop novel adjuvants that augment humoral and cell-mediated immunity by enhancing efficacy of vaccines (Bowersock and Martin, 1999; Newman and Powell, 1995), which is particularly crucial for commercial poultry industries in tackling economically important diseases such as Eimeria protozoa-induced avian coccidiosis (Shirley and Lillehoj, 2012).

Examples of adjuvants used with variety of vaccines include Montanide incomplete Seppic adjuvant (ISA) series, ISA 70 VG (ISA 70) and ISA 71 VG (ISA 71), and Quil A, cholesterol, dimethyl dioctadecyl ammonium bromide, and Carbopol (QCDC) adjuvant complex, whereby the former is a water-in-oil emulsion and the latter is composed of Quil A, cholesterol, dimethyl dioctadecyl ammonium bromide (DDA), and Carbopol (Aucouturier et al., 2006; Cox et al., 2003; Dominowski et al., 2009).

ISA 70 and ISA 71 have been successfully applied to enhance immune response against pathogens of poultry, cattle, and small ruminants (Dupuis et al., 2006). Previous studies have shown that either ISA 70 or ISA 71 in conjunction with the recombinant profilin, which is an Eimeria specific antigen, enhances protective immunity against experimental avian coccidiosis in chicken (Jang et al., 2010; Jang et al., 2011; Lee et al., 2011). Also, other than the Montanide ISA series, improvement in vaccine responses against a variety of veterinary pathogens has been demonstrated in the use of QCDC adjuvants (Dominowski et al., 2009). It has been understood that Carbopol, such as dextran, polyethelyne, glycol, or polyacrylic acid that has been added to the QCDC, improves the solubility of DDA and thus makes the final formulation a highly effective adjuvant. Further incorporation of Bay R1005, a synthetic glycolipid analogue, endows the complex with the ability to stimulate both Th1-and Th2-type immunity, giving the QCDCR adjuvant a broad range of desirable immune enhancing characteristics (Dominowski et al., 2009).

Chickens infected with Eimeria spp. commonly develop protective immunity against reinfection by the homologous parasite, which makes immunization with parasite vaccines a viable method to control coccidiosis (Lillehoj et al., 2000a). Basically, profilin in Eimeria stimulates cell-mediated immunity against experimental avian coccidiosis, which is what makes it a promising vaccine candidate (Lillehoj et al., 2000b; Yarovinsky et al., 2005). In addition, the evidence of profilin shown as a potential immunogenic protein has been published in two studies that, when Montanide ISA series and QCDC formulations were used, efficacy of the profilin vaccine was improved and thus lead to more protective immunity against coccidiosis (Jang et al., 2013). However, molecular signatures related to immune-stimulatory activities of these adjuvants have not been analyzed comparatively.

Therefore, in this study, we attempted to comparatively analyze integrated microarray data from two vaccine adjuvants in experimental coccidiosis model, i.e. ISAs and QCDCs, which confer protective immunity in combination with parasitic antigen, and sought to identify common gene ontology (GO) and pathways that are targeted by these adjuvants. The idea of integrating datasets from independent, but related, sources for a comparative analysis for this study stemmed from the increasing amount of research that has used microarray technology based datasets from publicly available repositories such as Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo), ArrayExpress (http://www.ebi.ac.uk/arrayexpress), and Stanford Microarray Database (http ://smd.princeton.edu).

MATERIALS AND METHODS

Associated microarray data

To investigate common immunological effects of four adjuvants, we used the Agilent Chicken Gene Expression Microarray dataset from two previous studies on vaccination effects that were obtained from GEO. In one of the studies, Montanide ISA 70 VG (ISA70) or Montanide ISA 71 VG (ISA71) with an Eimeria recombinant profilin protein was the experimental adjuvants used to immunize chickens subcutaneously, in comparison with only profilin immunization (accession number GSE40743). The other study used two novel adjuvant formulations, which were a combination of QCDC or QCDCR with a recombinant profilin (accession number GSE24966). The dataset used in this study was in reference to these previous studies, which contains details on immunization procedures (Kim et al., 2012; Jang et al., 2013). To briefly describe the procedure, seven-day-old Ross broiler chickens were subcutaneously immunized with profilin emulsified in each adjuvant or profilin alone. At 7 days post immunization, splenic lymphocytes were prepared from the chickens. Total RNAs were isolated from the cells using Trizol (Invitrogen, Carlsbad, CA, USA) and amplified with cyanine 3 (Cy3)- or Cy5-labeled CTP. The labeled RNAs were then hybridized to a Chicken Gene Expession Microarray (Agilent Technologies, Santa Clara, CA, USA).

Microarray image analysis was performed to analyze immunological effects of adjuvants specifically, thus the four adjuvants were categorized into two groups (Table 1). Adjuvants were grouped according to ISA adjuvants (ISA70 and ISA71) and QCDC adjuvants (QCDC and QCDCR).

Identification and functional analysis of differentially expressed genes

R package 'limma' was used to normalize and qualify microarray images. Median signal intensities were corrected by adaptive background correction (Ritchie et al., 2007) and normalized by locally-weighted scatterplot smoothing method. The log2-transformed fold changes and standard errors were estimated by fitting a linear model and empirical Bayes statistics was applied for smoothing standard errors. Differentially expressed genes (DEG) were filtered by cutoff 0.1 of false discovery rate (FDR), with adjusted p-value of two-sample t-test. Annotation of DEGs and biological function analysis were performed using DAVID Bioinformatics Resources (http://david.abcc. ncifcrf.gov/).

RESULTS

Identification and annotation of differentially expressed genes

In the present study, microarray datasets from two independent studies were combined to analyze the discriminative effects of two different types of adjuvants, Montanide ISA series and QCDC formulations. All adjuvants were administered with recombinant profilin antigens. In the results, ISA adjuvants modified greater number of genes than those of QCDC adjuvants. The numbers of DEGs were 8,932 and 1,761 in the immunization of ISA adjuvants and QCDC adjuvants, respectively. The common DEGs altered by both ISA and QCDC adjuvants were 489 (Figure 1A).

In DEGs annotation analysis, DAVID annotation analysis tool was used. Among 8,932 DEGs from the ISA adjuvants immunization, 3,030 genes (33.9%) were mapped to the chicken gene names in DAVID database. In the treatment of QCDC adjuvants, 439 (24.9%) out of 1,761 DEGs were mapped to the chicken genes in DAVID database. Among the 489 genes, which were altered commonly by both adjuvants, 136 genes (27.8%) were annotated in DAVID (Figure 1B). The annotated DEGs were used to identify their biological functions and the pathways in which the genes are involved.

Biological function analysis

In the GO analysis, 165 and 27 terms of biological process (BPs) were significantly identified from the DEGs by the treatment of ISA and QCDC adjuvants, respectively. The GO terms of BP modified by ISA or QCDC adjuvants were grouped into 23 and 8 upper categories (Table 2). Among the upper categories, six terms, i.e., "Anatomical structure development", "Cell death", "Regulation of metabolic process", "Response to stimulus", "Signal transduction", and "Single organism cellular process" were common to both adjuvant groups. The significant BPs related with the terms of "Response to stimulus" and "Cell death" were shown in Table 3 and Figure 2, respectively. All significant BPs identified were listed in Supplementary Table S1.

Pathway analysis

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway terms enriched by DEGs from the treatment of two different adjuvant groups are listed in Table 4. Both adjuvant groups showed common effects on the pathway for "Regulation of actin cytoskeleton". QCDC adjuvants altered genes involved in "Cell adhesion molecules (CAMs)" pathway and ISA adjuvants modified the pathways related with "Spliceosome", "Ubiquitin mediated proteolysis", "Adherens junction", "Lysosome", "Natural killer (NK) cell mediated cytotoxicity", "Cell cycle", "Endocytosis", and "Focal adhesion".

DISCUSSION

For the comparative analysis of the effects of two different adjuvant types, we collected microarray datasets from the independent trials for each adjuvant groups, ISAs and QCDCs, and identified GO terms and pathways modified by the DEGs.

In the present study, the most enriched categories of BP affected by two adjuvants, showing 6 categories that are commonly affected by two adjuvants. But, further detailed analysis revealed that the categories consisting of the same BPs in both adjuvants are quite specific, rather than common. For example, in the term of "Response to stimulus", no BPs was identified simultaneously in the two different adjuvant groups (Table 3). Generally, the QCDC adjuvants showed effects on innate immune response and immune response to external stimulus, such as "Response to toxin" and "Response to bacterium". For the immune response to toxins, a good humoral immune response is required in the adjuvants. Most adjuvants on the market today mainly activate the humoral immune response. Nevertheless, it is obvious that the introduction of cell-mediated immune response by adjuvants is beneficial to control bacterial infection, where cytotoxic T cells and Th1 cells mediate these responses (Leclerc, 2003). In this sense, ISCOM, a component of QCDC adjuvants, has been known to generate both strong humoral and cellular immune responses in an extensive range of animal species (Drane et al., 2007). The ISCOMATRIX adjuvant has also shown to be safe and well tolerated as well as immunogenic, generating both antibody (Ab) and CD4+ and CD8+ T cell responses (Drane et al., 2007). Also, DDA is reported to promote both strong cell mediated immune responses and humoral immune responses, which are essential for the induction of protective immunity against most diseases (Davidsen et al., 2005).

As for the adjuvants of ISA series, these modified the BPs that regulate cell movement such as cell locomotion, chemotaxis, and cell migration. Although ISA and QCDC adjuvants modified biological functions related to the regulation of cell death and apoptosis, they showed a different manner in mode of action. The effects of ISA adjuvants on cell death were both of positive and negative regulation while the QCDC adjuvants had no function in regulating cell death negatively. Such apoptotic and necrotic effects are reported to be associated with emulsion-type vaccine adjuvants such as the ISA series and QCDC whereby both are composed of various oils and surfactants (Yang et al., 2004). The mechanism behind the surfactants is that their amphiphilic nature adsorbs not only the oil/water interface in the emulsion, but also biological membranes, resulting in an increase in surface pressure that thus lead to apoptosis and necrosis of the cells (Yang et al., 2004). This suggests that the surfactants in ISA adjuvants and Quil A in QCDC adjuvants could induce apoptosis or cell death during immunization. In the results of pathway analysis, "Adherens junctions" and "Focal adhesion" pathways were the two main types of junction that were modified by ISA adjuvants, which are known to mediate adhesion in epithelial cells (Yeatman, 2004). Cell-cell adherens junctions are important for maintaining tissue architecture and cell polarity and can limit cell movement and proliferation. Cell-matrix adhesions, also called focal adhesions, play essential roles in important BPs including cell motility, cell proliferation, cell differentiation, regulation of gene expression and cell survival. Collectively, the ISA adjuvants may have effects on cell mobility and mitigate cytotoxicity through the cellular junction pathways.

In addition, lysosomal and endocytic reactions were also modified by ISA adjuvants. In the case of lysosomes, the lysosomal enzymes' important role in the inflammatory process has been documented in previous studies in arthritic condition induced by injection of Freund's complete adjuvant (FCA) in rats (Anderson, 1970; Reddy and Dhar, 1988; Geetha and Varalakshmi, 1999). When endocytosis takes place, adjuvants first exert their targeting mechanism by binding to antigens and the adjuvant-antigen complex is delivered to antigen-presenting cells (APCs) to form aggregates that are then engulfed by APCs to form endosomes (Cox and Coulter, 1997; Zamze et al., 2002). More effective targeting can be achieved by using adjuvants with residues that are recognized by receptors on APCs such as the mannose receptor that belongs to the endocytic pattern recognition receptors (PRRs) that bind compounds containing mannose, N-acetylglucosamine, or fucose residues and sulfated oligosaccharides (Stahl and Ezekowitz, 1998).

The ISA adjuvants' functions related with cellular response to oxidative stress shown from our result can be used to partly explain the immune-stimulatory properties of adjuvants that induce inflammation and oxidative stress in the host animal (Kumar and Roy, 2007). An example can be drawn from previous studies that showed that intra-articular injection of a well-known adjuvant, i.e., FCA, induced inflammation as well as immune response and produced features that resembled rheumatoid arthritis in humans. Such acute inflammatory response induced by FCA is associated with leukocyte infiltration, mast cell activation, and release of cytokines and free radicals (Nigrovic and Lee, 2005; Yamada et al., 2006). Despite the shared similarity both ISA adjuvants and FCA and FIA (Freund's incomplete adjuvant) have with respect to antibody responses, the fact that less inflammatory response is induced by ISA adjuvants relative to FCA and FIA needs to be taken into account (Johnston et al., 1991; Leenaars et al., 1994; Leenaars et al., 1995; Leenaars et al., 1998).

In the pathway analysis, QCDC adjuvants altered genes involved in "CAMs" pathway. Inflammatory and immune responses involve adhesive interactions that mediate migration of cells to sites of inflammation and the effector functions of cell within the lesions. Therefore, CAMs, as complex proteins expressed on the cell surface and precisely regulated by cytokines and other biologic response modifiers, are versatile mediators of the complex dynamics of cell interactions in the inflammatory/immune response (Crawford and Watanabe, 1994).

In conclusion, the integrative analysis of microarray datasets from the treatment of two different types of adjuvants, ISA series and QCDC formulations, has provided discriminative and common molecular signatures as well as aided the distinction of immunological functions of the two adjuvant groups in chicken. Although much progress has been undertaken in the formulation of novel adjuvants that augment the immunogenicity of protein vaccines, very little information is available in poultry (Baldridge and Ward, 1997; Gupta and Siber, 1995; Richards et al., 1996). Therefore, this study could contribute to the selection of appropriate adjuvants according to the types of vaccines or diseases as well as the development of efficient vaccine adjuvants in poultry industry. Furthermore, the potent immune adjuvants might promise another usefulness of vaccine as therapeutic agents rather than prophylactic agents.

Limitations such as small sample size and limited case numbers are weaknesses of this study; however, to our knowledge, this is the first integrative approach to identify molecular signatures impacted by adjuvant in livestock, especially in chicken. Therefore, this study could serve as an informative framework to integrated microarray data to identify molecular signatures that can be used to develop precise adjuvants based on their immunological functions.

http://dx.doi.org/10.5713/Ajas.15.0923

CONFLICT OF INTEREST

We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.

ACKNOWLEDGMENTS

The authors thank Dr. Song, Ki Duk and Ahn, Hyun Ju for their significant contribution to this research. This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ01104401), Rural

Development Administration, Republic of Korea.

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Supplementary Table S1. Significant biological process (BPs) from
differentially expressed genes by the treatment of ISA and QCDC
adjuvants

Categories        Adjuvants   Biological process           P-value

Anatomical           ISA      Angiogenesis                 7.26E-04
  structure                   Vasculature development      7.36E-04
  development                 Blood vessel morphogenesis   1.24E-03
                              Blood vessel development     1.53E-03
                              Heart development            1.20E-02
                              Cell motion                  1.93E-02
                              In utero embryonic           4.19E-02
                               development
                              Liver development            4.52E-02
                    QCDC      Gut development              1.65E-02

Cell death           ISA      Regulation of programmed     2.49E-05
                               cell death
                              Regulation of cell death     2.96E-05
                              Regulation of apoptosis      4.80E-05
                              Negative regulation of       1.42E-03
                               cell death
                              Negative regulation of       1.42E-03
                               programmed cell death
                              Negative regulation          2.00E-03
                               of apoptosis
                              Programmed cell death        6.40E-03
                              Apoptosis                    7.51E-03
                              Death                        9.84E-03
                              Cell death                   1.32E-02
                              Regulation of neuron         1.55E-02
                               apoptosis
                              Positive regulation          1.93E-02
                               of apoptosis
                              Positive regulation of       1.93E-02
                               programmed cell death
                              Positive regulation of       2.23E-02
                               cell death
                              Anti-apoptosis               3.80E-02

                    QCDC      Regulation of apoptosis      1.16E-02
                              Regulation of programmed     1.35E-02
                               cell death
                              Regulation of cell death     1.40E-02
                              Positive regulation          1.69E-02
                               of apoptosis
                              Positive regulation of       1.69E-02
                               programmed cell death
                              Positive regulation          1.79E-02
                               of cell death

Regulation of        ISA      Positive regulation of       1.26E-03
  metabolic                    macromolecule
  process                      metabolic process
                              Regulation of cellular       2.08E-03
                               protein metabolic process
                              Regulation of                4.62E-03
                               phosphorylation
                              Regulation of kinase         5.19E-03
                               activity
                              Regulation of phosphate      5.30E-03
                               metabolic process
                              Regulation of phosphorus     5.30E-03
                               metabolic process
                              Regulation of protein        7.32E-03
                               kinase activity
                              Regulation of transferase    7.41E-03
                               activity
                              Negative regulation of       7.65E-03
                               macromolecule metabolic
                               process
                              Regulation of translation    9.95E-03
                              Regulation of peptidyl-      1.32E-02
                               tyrosine phosphorylation
                              Negative regulation of       1.50E-02
                               kinase activity
                              Negative regulation of       1.50E-02
                               protein kinase activity
                              Negative regulation of       1.58E-02
                               molecular function
                              Positive regulation of       2.10E-02
                               DNA metabolic process
                              Positive regulation of       2.17E-02
                               nucleobase, nucleoside,
                               nucleotide and
                              nucleic acid metabolic
                               process
                              Negative regulation of       2.42E-02
                               cellular protein
                               metabolic process
                              Negative regulation of       2.42E-02
                               protein metabolic process
                              Negative regulation of       2.45E-02
                               biosynthetic process
                              Positive regulation of       2.54E-02
                               protein kinase cascade
                              Regulation of system         2.72E-02
                               process
                              Positive regulation of       2.74E-02
                               cellular biosynthetic
                               process
                              Negative regulation of       2.84E-02
                               transferase activity
                              Positive regulation of       2.98E-02
                               biosynthetic process
                              Negative regulation of       3.02E-02
                               catalytic activity
                              Positive regulation of       3.20E-02
                               macromolecule
                               biosynthetic process
                              Regulation of protein        3.24E-02
                               kinase cascade
                              Positive regulation of       3.26E-02
                               cellular protein
                               metabolic process
                              Positive regulation of       3.60E-02
                               nitrogen compound
                               metabolic process
                              Positive regulation of       3.86E-02
                               protein metabolic process
                              Negative regulation of       3.93E-02
                               nucleobase, nucleoside,
                               nucleotide and
                              nucleic acid metabolic
                               process
                              Negative regulation of       4.06E-02
                               macromolecule
                               biosynthetic process
                              Negative regulation of       4.32E-02
                               cellular biosynthetic
                               process
                              Negative regulation of       4.80E-02
                               nitrogen compound
                               metabolic process
                              Positive regulation of       4.96E-02
                               catalytic activity

                    QCDC      Regulation of                1.23E-02
                               phosphorylation
                              Regulation of phosphate      1.57E-02
                               metabolic process
                              Regulation of phosphorus     1.57E-02
                               metabolic process
                              Positive regulation of       4.56E-02
                               catalytic activity
                              Positive regulation of       3.34E-02
                               molecular function

Response to          ISA      Cellular response to         2.69E-04
  stimulus                     stress
                              Response to oxidative        2.06E-03
                               stress
                              Positive regulation          4.61E-03
                               of locomotion
                              Positive regulation          7.27E-03
                               of chemotaxis
                              Regulation of chemotaxis     7.27E-03
                              Chemotaxis                   9.95E-03
                              Taxis                        9.95E-03
                              Positive regulation          1.20E-02
                               of behavior
                              Regulation of behavior       1.20E-02

                              Response to tumor            1.74E-02
                               necrosis factor
                              Cellular response to         2.02E-02
                               oxidative stress
                              Positive regulation of       3.17E-02
                               cell migration
                              Response to inorganic        3.34E-02
                               substance
                              Positive regulation of       3.41E-02
                               cell motion
                              Fibroblast growth factor     3.42E-02
                               receptor signaling
                               pathway

                    QCDC      Immune response              9.48E-04
                              Response to toxin            1.78E-02
                              Defense response             2.01E-02
                              Response to bacterium        4.49E-02
                              Innate immune response       4.85E-02

Signal               ISA      Positive regulation of       4.18E-04
  transduction                 signal transduction
                              Positive regulation of       4.49E-04
                               cell communication
                              Small GTPase mediated        1.45E-02
                               signal transduction
                              Intracellular                2.30E-02
                               signaling cascade
                              Positive regulation of       3.42E-02
                               calcium-mediated
                               signaling
                              Integrin-mediated            4.86E-02
                               signaling pathway

                    QCDC      Intracellular signaling      2.21E-02
                               cascade

Single organism      ISA      Cell cycle                   2.53E-02
  cellular                    Cell cycle process           4.44E-02
  process
                    QCDC      Collagen fibril              4.30E-02
                               organization

Categories        Adjuvants   Biological process           Number
                                                           of genes

Anatomical           ISA      Angiogenesis                    21
  structure                   Vasculature development         34
  development                 Blood vessel morphogenesis      28
                              Blood vessel development        32
                              Heart development               30
                              Cell motion                     39
                              In utero embryonic              23
                               development
                              Liver development               7
                    QCDC      Gut development                 4

Cell death           ISA      Regulation of programmed        69
                               cell death
                              Regulation of cell death        69
                              Regulation of apoptosis         67
                              Negative regulation of          36
                               cell death
                              Negative regulation of          36
                               programmed cell death
                              Negative regulation             35
                               of apoptosis
                              Programmed cell death           33
                              Apoptosis                       32
                              Death                           35
                              Cell death                      34
                              Regulation of neuron            12
                               apoptosis
                              Positive regulation             27
                               of apoptosis
                              Positive regulation of          27
                               programmed cell death
                              Positive regulation of          27
                               cell death
                              Anti-apoptosis                  15

                    QCDC      Regulation of apoptosis         14
                              Regulation of programmed        14
                               cell death
                              Regulation of cell death        14
                              Positive regulation             8
                               of apoptosis
                              Positive regulation of          8
                               programmed cell death
                              Positive regulation             8
                               of cell death

Regulation of        ISA      Positive regulation of          77
  metabolic                    macromolecule
  process                      metabolic process
                              Regulation of cellular          38
                               protein metabolic process
                              Regulation of                   38
                               phosphorylation
                              Regulation of kinase            28
                               activity
                              Regulation of phosphate         39
                               metabolic process
                              Regulation of phosphorus        39
                               metabolic process
                              Regulation of protein           27
                               kinase activity
                              Regulation of transferase       28
                               activity
                              Negative regulation of          52
                               macromolecule metabolic
                               process
                              Regulation of translation       13
                              Regulation of peptidyl-         11
                               tyrosine phosphorylation
                              Negative regulation of          10
                               kinase activity
                              Negative regulation of          10
                               protein kinase activity
                              Negative regulation of          19
                               molecular function
                              Positive regulation of          10
                               DNA metabolic process
                              Positive regulation of          57
                               nucleobase, nucleoside,
                               nucleotide and
                              nucleic acid metabolic
                               process
                              Negative regulation of          14
                               cellular protein
                               metabolic process
                              Negative regulation of          14
                               protein metabolic process
                              Negative regulation of          42
                               biosynthetic process
                              Positive regulation of          15
                               protein kinase cascade
                              Regulation of system            23
                               process
                              Positive regulation of          60
                               cellular biosynthetic
                               process
                              Negative regulation of          10
                               transferase activity
                              Positive regulation of          60
                               biosynthetic process
                              Negative regulation of          14
                               catalytic activity
                              Positive regulation of          58
                               macromolecule
                               biosynthetic process
                              Regulation of protein           17
                               kinase cascade
                              Positive regulation of          18
                               cellular protein
                               metabolic process
                              Positive regulation of          57
                               nitrogen compound
                               metabolic process
                              Positive regulation of          18
                               protein metabolic process
                              Negative regulation of          37
                               nucleobase, nucleoside,
                               nucleotide and
                              nucleic acid metabolic
                               process
                              Negative regulation of          39
                               macromolecule
                               biosynthetic process
                              Negative regulation of          40
                               cellular biosynthetic
                               process
                              Negative regulation of          37
                               nitrogen compound
                               metabolic process
                              Positive regulation of          30
                               catalytic activity

                    QCDC      Regulation of                   10
                               phosphorylation
                              Regulation of phosphate         10
                               metabolic process
                              Regulation of phosphorus        10
                               metabolic process
                              Positive regulation of          8
                               catalytic activity
                              Positive regulation of          9
                               molecular function

Response to          ISA      Cellular response to            53
  stimulus                     stress
                              Response to oxidative           15
                               stress
                              Positive regulation             14
                               of locomotion
                              Positive regulation             8
                               of chemotaxis
                              Regulation of chemotaxis        8
                              Chemotaxis                      13
                              Taxis                           13
                              Positive regulation             8
                               of behavior
                              Regulation of behavior          8

                              Response to tumor               5
                               necrosis factor
                              Cellular response to            6
                               oxidative stress
                              Positive regulation of          11
                               cell migration
                              Response to inorganic           9
                               substance
                              Positive regulation of          12
                               cell motion
                              Fibroblast growth factor        5
                               receptor signaling
                               pathway

                    QCDC      Immune response                 13
                              Response to toxin               3
                              Defense response                8
                              Response to bacterium           5
                              Innate immune response          4

Signal               ISA      Positive regulation of          32
  transduction                 signal transduction
                              Positive regulation of          33
                               cell communication
                              Small GTPase mediated           36
                               signal transduction
                              Intracellular                   93
                               signaling cascade
                              Positive regulation of          5
                               calcium-mediated
                               signaling
                              Integrin-mediated               10
                               signaling pathway

                    QCDC      Intracellular signaling         20
                               cascade

Single organism      ISA      Cell cycle                      41
  cellular                    Cell cycle process              30
  process
                    QCDC      Collagen fibril                 3
                               organization


Duk Kyung Kim (1), Kyeong Hye Won, Seung Hyun Moon, and Hak-Kyo Lee *

Department of Animal Biotechnology, Chonbuk National University, Jeonju 561-756, Korea

* Corresponding Author: Hak-Kyo Lee. Tel: +82-63-270-2548, Fax: +82-63-270-2614, E-mail: breedlee@empas.com

(1) C&K Genomics, Seoul 151-919, Korea.

Submitted Dec. 8, 2014; Revised Jan. 9, 2015; Accepted Feb. 11, 2015

Table 1. Designs used in microarray image analysis using R
'limma ' package

Adjuvants group    GEO series No.        Case        Control

ISA adjuvants         GSE40743      ISA70+profilin   Profilin
                                    ISA71+profilin

QCDC adjuvants        GSE24966      QCDC+profilin    Profilin
                                    QCDCR+profilin

Adjuvants group        Reference

ISA adjuvants      Jang et al. (2013)

QCDC adjuvants     Kim et al. (2012)

ISA, incomplete Seppic adjuvant; QCDC, Quil A, cholesterol,
dimethyl dioctadecyl ammonium bromide, and Carbopol.

Table 2. Categories for the significant biological processes
identified from DEGs in the treatment of two different adjuvant
groups

ISA adjuvants                        QCDC adjuvants

  Anatomical structure development     Anatomical structure development
  Cell death                           Cell death
  Regulation of metabolic process      Regulation of metabolic process
  Response to stimulus                 Response to stimulus
  Signal transduction                  Signal transduction
  Single organism cellular process     Single-organism cellular process
  Biological regulation                Regulation of cellular process
  Cell adhesion                        Single-multicellular organism
  Cell developmental process            process
  Cellular component organization
  Cellular metabolic process
  Cellular process
  Circulatory system process
  Immune system process
  Macromolecule localization
  Macromolecule metabolic process
  Metabolic process
  Organelle organization
  Protein metabolic process
  Regulation of biological process
  Regulation of cell communication
  Regulation of transport
  RNA metabolic process

DEGs, differentially expressed genes; ISA, incomplete Seppic
adjuvant; QCDC, Quil A, cholesterol, dimethyl dioctadecyl
ammonium bromide, and Carbopol.

Table 3. Significant biological processes for DEGs in the
treatment of adjuvants which were categorized to the term of
"Response to stimulus"

Adjuvants group   Biological process                      p-value

ISA adjuvants     Cellular response to stress             2.69E-04
                  Response to oxidative stress            2.06E-03
                  Positive regulation of locomotion       4.61E-03
                  Positive regulation of chemotaxis       7.27E-03
                  Regulation of chemotaxis                7.27E-03
                  Chemotaxis                              9.95E-03
                  Taxis                                   9.95E-03
                  Positive regulation of behavior         1.20E-02
                  Regulation of behavior                  1.20E-02
                  Response to tumor necrosis factor       1.74E-02
                  Cellular response to oxidative stress   2.02E-02
                  Positive regulation of cell migration   3.17E-02
                  Response to inorganic substance         3.34E-02
                  Positive regulation of cell motion      3.41E-02
                  Fibroblast growth factor receptor       3.42E-02
                   signaling pathway

QCDC adjuvants    Immune response                         9.48E-04
                  Response to toxin                       1.78E-02
                  Defense response                        2.01E-02
                  Response to bacterium                   4.49E-02
                  Innate immune response                  4.85E-02

Adjuvants group   Biological process                      No. of
                                                           genes

ISA adjuvants     Cellular response to stress               53
                  Response to oxidative stress              15
                  Positive regulation of locomotion         14
                  Positive regulation of chemotaxis          8
                  Regulation of chemotaxis                   8
                  Chemotaxis                                13
                  Taxis                                     13
                  Positive regulation of behavior            8
                  Regulation of behavior                     8
                  Response to tumor necrosis factor          5
                  Cellular response to oxidative stress      6
                  Positive regulation of cell migration     11
                  Response to inorganic substance            9
                  Positive regulation of cell motion        12
                  Fibroblast growth factor receptor          5
                   signaling pathway

QCDC adjuvants    Immune response                           13
                  Response to toxin                          3
                  Defense response                           8
                  Response to bacterium                      5
                  Innate immune response                     4

DEGs, differentially expressed genes; ISA, incomplete Seppic
adjuvant; QCDC, Quil A, cholesterol, dimethyl dioctadecyl
ammonium bromide, and Carbopol.

Table 4. Significantly changed pathways from the DEGs in the
treatment of ISA adjuvants or QCDC adjuvants

Adjuvants                   KEGG pathway                     p-value
group

ISA adjuvants    Spliceosome                                 4.34E-04
                 Ubiquitin mediated proteolysis              5.86E-03
                 Adherens junction                           9.53E-03
                 Lysosome                                    1.86E-02
                 Natural killer cell mediated cytotoxicity   1.93E-02
                 Cell cycle                                  2.49E-02
                 Endocytosis                                 2.74E-02
                 Focal adhesion                              2.81E-02
                 Regulation of actin cytoskeleton            3.44E-02

QCDC adjuvants   Cell adhesion molecules (CAMs)              3.61E-02
                 Regulation of actin cytoskeleton            4.91E-02

Adjuvants        KEGG pathway                                Number
group                                                        of genes

ISA adjuvants    Spliceosome                                    38
                 Ubiquitin mediated proteolysis                 38
                 Adherens junction                              24
                 Lysosome                                       30
                 Natural killer cell mediated cytotoxicity      23
                 Cell cycle                                     33
                 Endocytosis                                    47
                 Focal adhesion                                 49
                 Regulation of actin cytoskeleton               48

QCDC adjuvants   Cell adhesion molecules (CAMs)                 8
                 Regulation of actin cytoskeleton               11

DEGs, differentially expressed genes; ISA, incomplete Seppic
adjuvant; QCDC, Quil A, cholesterol, dimethyl dioctadecyl
ammonium bromide, and Carbopol; KEGG, Kyoto Encyclopedia of Genes
and Genomes.

Figure 1. Differentially expressed genes (DEGs) and their
annotation. (A) Venn diagram showing the number of DEGs by the
treatment of two different types of adjuvants. False discovery rate
(FDR) <0.1, (B) DEGs mapped to the chicken gene names in DAVID
database.

(B)

No. of DEGs
             DEGs    Mapped DEGs

ISA          8932       3030

QCDC         1761       439

ISA & QCDC    489       136

Note: Table made from bar graph.
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Author:Kim, Duk Kyung; Won, Kyeong Hye; Moon, Seung Hyun; Lee, Hak-Kyo
Publication:Asian - Australasian Journal of Animal Sciences
Article Type:Report
Date:Jul 1, 2016
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