Pathogens and Disease Advance Access published April 15, 2015
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Host gene expression for Mycobacterium avium subsp. paratuberculosis
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infection in human THP-1 macrophages
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Running title: Potential host-response for MAP infection in THP-1 cell
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Min-Kyoung Shin1,2, Seung Won Shin1, Myunghwan Jung1, Hongtae Park1, Hyun-Eui Park1,
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& Han Sang Yoo1, 3
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Department of Infectious Diseases, College of Veterinary Medicine, Seoul National
University, Seoul, 151-742 Korea 2
Dairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada,
Sherbrooke, QC J1M 1Z3, Canada; 3
Institute of Green Bio Science and Technology, Seoul National University, Pyeongchang,
232-916, Korea
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Correspondence: Han Sang Yoo
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Department of Infectious Diseases, College of Veterinary Medicine, Seoul National
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University, Seoul, 151-742 Korea
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Phone: +82-2-880-1263
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Fax: +82-2-874-2738
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E-mail: [email protected]
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Abstract
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Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s
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disease, which causes considerable economic loss in the dairy industry and has a possible
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relationship with Crohn’s disease (CD) in humans. As MAP has been detected in retail
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pasteurized milk samples, its transmission via milk is of concern. Despite its possible role in
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the etiology of CD, there have been few studies examining the interactions between MAP and
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human cells. In the current study, we applied Ingenuity Pathway Analysis to the transcription
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profiles generated from a murine model with MAP infection as part of a previously
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conducted study. Twenty one genes were selected as potential host immune responses,
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compared to the transcriptional profiles in naturally MAP-infected cattle, and validated in
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MAP-infected human monocyte-derived macrophage THP-1 cells. Of these, the potential
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host responses including up-regulation of genes related to immune response (CD14, S100A8,
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S100A9, LTF, HP and CHCIL3), up-regulation of Th 1-polarizing factor (CCL4, CCL5,
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CXCL9 and CXCL10), down-regulation of genes related to metabolism (ELANE, IGF1,
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TCF7L2, and MPO), and no significant response of other genes (GADD45a, GPNMB,
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HMOX1, IFNG, and NQO1) in THP-1 cell infected with MAP.
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Keywords: Mycobacterium avium subsp. paratuberculosis, host responses, THP-1,
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Introduction
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Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne’s disease (JD), a
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chronic granulomatous enteropathy of ruminants such as cattle, sheep, deer, bison and elk
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(Motiwala et al., 2006). Concerns have been raised about JD over economic losses in the
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dairy industry as well as the possible relationship with a chronic inflammatory bowel disease
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(IBD) in humans called Crohn’s disease (CD) (Thayer et al., 1984, Naser et al., 2004, Yoo &
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Shin, 2012). It has been suggested that human IBD may be caused by an autoimmune
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dysregulation and imbalance of T cells or host responsiveness against a microbial trigger
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(Mizoguchi et al., 2003, Singh et al., 2007). In addition, many studies report that MAP or
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MAP-specific serum antibodies are detected in the intestine, blood and breast milk of Crohn’s
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disease patients (Naser et al., 2000, Naser et al., 2000, Schwartz et al., 2000, Naser et al.,
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2004, Sechi et al., 2005). MAP DNA has been detected in retail pasteurized milk samples,
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even commercially pasteurized milk in the United Kingdom. As transmission could occur
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through milk and other dairy products, further research into the mechanisms underlying MAP
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pathogenesis in human is needed (Millar et al., 1996, Ellingson et al., 2005). Despite its
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possible role in the etiology of CD, there have been few studies examining the interactions
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between MAP and human cells. Different gene expression patterns or cytokine responses
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(TNF-α, IL-1β, and IL-10) were observed in human monocyte-derived macrophage THP-1
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cells by different MAP strains (Motiwala et al., 2006, Borrmann et al., 2011).
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In the current study, we applied a microarray-based approach to discover potential immune
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response markers in a model of murine MAP infection and naturally MAP-infected cattle
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followed by validation of the genes in human cells. In our previous study, the host responses
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to MAP infection in mice and cattle were characterized by transcriptional analysis. 3
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Significantly regulated genes were filtered and selected via the Ingenuity Pathways Analysis
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(IPA) and evaluated in vitro using MAP-infected THP-1 cells.
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Materials and Methods
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Selection of potential host response genes using transcriptional profiles in MAP-
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infected mice and naturally MAP-infected cattle
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We previously submitted a dataset of MAP infection to the GEO database (GSE62836) for
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murine study (submmitted to another journal) and GEO database (GSE62835) for bovine
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study (Shin et al., 2015). For murine study, briefly, five-week-old C57BL/6 female mice
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were inoculated intraperitoneally with 300 μL PBS (control group) or a suspension of MAP
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ATCC 19698 (3 × 107 cells/mouse, MAP-infected group). Spleen samples were taken from
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five mice in each group, sacrificed at 3 and 6 weeks post infection (p.i.). Microarray analysis
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was performed using total RNA from harvested spleen cells and the data were used for
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identification of differentially expressed genes in MAP-infected mice compared to control
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mice. Differentially expressed genes were filtered for a log2-fold change >2 with p < 0.05 by
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Biomarker Filter and then analyzed by Biomarker Discovery both of IPA (Ingenuity Systems
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Inc., Redwood, CA). The genes were further clarified in the IPA biomarker filter module
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based on the following criteria: fluid – 〈〈all〉〉, disease – 〈〈Infectious Disease, Inflammatory
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response, Immunological disease〉〉, and species – 〈〈mouse〉〉. Genes selected based on the
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above criteria were compared with bovine transcriptional profiles and verified in THP-1 cells
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following infection with MAP using quantitative real-time RT PCR.
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Bacterial strain, cell line culture, and infection
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MAP ATCC 19698 was cultured as previously described (Cha et al., 2013). For infection of
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cells, MAP ATCC 19698 was suspended in phosphate-buffered saline (PBS) and used at a
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dilution of 1 × 109 cells/mL.
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The THP-1 human leukemic monocyte cell line was obtained from KCLB (Korea Cell Line
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Bank, Seoul, Korea). THP-1 cells were cultured in RPMI 1640, supplemented with 10% heat-
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inactivated fetal bovine serum (FBS), and Antibiotic-Antimycotic (all from Gibco Invitrogen,
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Karlsruhe, Germany) agent at 37°C in humidified air with 5% CO2. THP-1 cells were
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differentiated into macrophages by stimulation with phorbol-12-myristate-13-acetate (PMA;
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Sigma Co., St. Louis, MO, USA) (50ng/ml) for 72h, washed with FBS-free RPMI 1640
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medium and incubated with 5% FBS-RPMI 1640 medium without antibiotics for 24h before
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the experiments. Differentiated THP-1 macrophage cells (1 × 106 cells/ml) were inoculated
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with MAP 19698 at a multiplicity of infection (MOI) of 10:1 and incubated for 0, 6, 24, and
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48h. Total RNA was extracted from cells collected at 0, 6, 24, and 48h post infection (p.i.).
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Quantitative real-time PCR
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The total RNA was isolated with an RNeasy Mini Kit (Qiagen, Valencia, USA) according to
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the manufacturer's instructions. Two micrograms of total RNA from each sample were
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reverse-transcribed into cDNA using the Quantitect® Reverse Transcription kit (Qiagen).
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Real time qRT-PCR reactions were performed with 2 μl of cDNA using the Rotor-Gene
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SYBR Green PCR kit (Qiagen) and Rotor-Gene Q real time PCR cycler (Qiagen). Cycling
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parameters were as follows: 95°C for 5 minutes for 1 cycle, 95°C for 20 seconds, and then
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60°C for 45 seconds for 40 cycles. Primers used in real-time PCR are shown in Table 1. The
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gene expression level was normalized by the 2-ΔΔCt method using β-actin as an internal
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control gene. The relative expression level was compared to the zero-time control to
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determine the changes in expression-fold of each gene.
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Statistical analysis
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The data were expressed as mean ± standard error of the mean (SEM), and statistical
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significance was analyzed by student’s t-test using IBM Statistical Package for Social
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Sciences software (SPSS, version 21, SPSS Inc., Chicago, IL, USA). Differences were
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considered significant if a value of p ≤ 0.05 was obtained.
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Results
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Host gene expression in MAP-infection mice and naturally MAP-infected cattle
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In our previous study, C57BL/6 mice infected with MAP showed most severe
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immunopathologic changes at 3 and 6 weeks p.i. by transcriptional profiling and 6
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histopathological analysis. As the first step of data mining process, the “up-regulated” genes
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were selected using the cut-off value (log2-fold change > 2 and p < 0.05). In the second, the
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genes were imported to the Biomarker Discovery module of IPA and filtered by the criteria,
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like: excreted in all biological fluids, related to infectious disease, inflammatory response, or
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immunological disease. The twenty one eligible markers selected by IPA were related to
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immune system processes (IFNG, CCL4, CCL5, CD14, CXCL9, CXCL10, HP, S100A8,
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S100A9, MPO), metabolic processes (ELANE, CD68, CHI3L1, HP, S100A8, S100A9, IGF1,
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TCF7L2, MPO, LTF, HMOX1), localization (CD68, IGF1, LTF, TFRC), and others
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(GADD45, GPNMB, NQO1) as classified by GO biological processes (Table 1). These genes
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were compared with the transcriptional profiles in naturally MAP-infected cattle (Table 1).
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Naturally MAP-infected cattle were divided into three groups as follows: Test1, fecal PCR
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positive; Test2, MAP-ELISA positive; and Test3, fecal PCR positive and MAP-ELISA
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positive as previously described (Shin et al., 2015). The selected genes showed various
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expression according to the groups in cattle, while all of the genes were up-regulated in
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MAP-infected mice. The selected genes were divided into three groups based on comparison
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of the results of mice and cattle. Group A was occupied by up-regulated genes in cows with
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MAP-specific antibodies (Test2 and Test3). Group B was occupied by down-regulated genes
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in cows as fecal shedder (Test1). The genes, which were up-regulated in MAP-infected mice,
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but no significant response in MAP-affected cows, were Group C.
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Validation of biomarker candidates in THP-1 by qPCR
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The twenty one selected candidates were evaluated in human THP-1 cells at various time
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points following MAP infection. No significant change in expression was observed in 7 of
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these genes (CD68, GADD45, GPNMB, HMOX1, IFNG, NQO1 and TCF7L2) at any time 7
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point relative to the uninfected control. The expression of CCL4 (3.5-fold up-regulation),
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CCL5 (2.4-fod up-regulation), CXCL9 (3.5-fold up-regulation), and TFRC (2.1-fold up-
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regulation) genes were increased at 6h p.i. but not at later time points and the expression of
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CD14 (4.1- and 4.8-fold up-regulation at 24h and 48h p.i.), CXCL10 (1.8- and 3.4-fold up-
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regulation at 24h and 48h p.i.), LTF (1.7- and 1.4-fold up-regulation at 24h and 48h p.i.),
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S100A8 (6.2- and 6.7-fold up-regulation at 24h and 48h p.i.), and S100A9 (2.7- and 3.9-fold
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up-regulation at 24h and 48h p.i.) genes were increased at 24h and 48h p.i. (Figure 1).
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CHI3L1 (2.6-fold up-regulation), and HP (4.3-fold up-regulation) genes showed increased
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expression only at 48h p.i. after MAP infection (Figure 1), while ELANE (1.7- and 1.8-fold
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down-regulation at 24h and 48h p.i.), IGF1 (2.3- and 1.5-fold down-regulation at 24h and
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48h p.i.), and MPO (2.1- and 1.8-fold down-regulation at 6h and 48h p.i.) genes showed
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down-regulation. Therefore, among the selected genes from the transcriptomes of MAP-
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infected mice, the expression of fourteen of these genes were also significantly up- or down-
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regulated in human THP-1 cells following MAP infection.
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Discussion
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Although the oral route is the natural infection route of MAP in cattle, the intraperitoneal
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(IP) injection has been frequently utilized in MAP infection of mice and our previous murine
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study also was challenged MAP intraperitoneally, because earlier studies demonstrated that
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low infectivity rates are the main problem of the oral administration of MAP in mouse model
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(Mutwiri et al., 1992; Veazey et al., 1995). In the previous study, we analyzed transcriptional
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profiles induced in the murine spleen, wherein the innate and adaptive immune systems
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combined, following MAP infection. The expression patterns of twenty one candidate genes
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were analyzed at various time points post infection. CD68 and CD14, both macrophage 8
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markers, showed different expression patterns. CD68 was not significantly altered by MAP
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infection in THP-1 cells. However, in these experiments PMA-stimulated THP-1 macrophage
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cells were used and CD68 was already expressed in these cells prior to infection which may
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explain why no further change was observed. The expression of CD14 increased at 24h and
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48h post infection. A role for CD14 has been reported in the regulation of
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monocyte/macrophage apoptosis so up-regulation of CD14 may have an effect on levels of
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apoptosis during infection (McClure et al., 1987).
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Increased expression of S100A8 and S100A9 (also termed myeloid-related proteins (MRP)8
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and MRP14) was observed, consistent with data demonstrating release of these proteins by
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activated phagocytes during mycobacterial infection both in vitro and in vivo (Pechkovsky et
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al., 2000, Frosch et al., 2004). Cows with MAP-specific antibodies showed up-regulation of
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MRP8 and MRP14 (Shin et al., 2015). They form stable complexes (MRP8/14) and are found
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in a variety of inflammatory conditions, including rheumatoid arthritis, allograft rejections,
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and inflammatory bowel and lung diseases (Kerkhoff et al., 1998). Therefore, their potential
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role in inflammatory disorders including CD has already been highlighted (Lugering et al.,
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1995, Foell et al., 2004). LTF (lactoferrin) gene was also slightly up-regulated in THP-1 cell
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infected with MAP. Serum lactoferrin was strongly and inversely associated with increasing
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bacterial index of M. leprae in leprosy patients (Muruganand et al., 2004). Of group A,
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TCF7L2 gene was down-regulated at 6 h p.i., but there was not statistical significant
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responses. TCF7L2 was suggested to modulate hepatic glucose metabolism in mammals, thus
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contributing to the development of type 2 diabetes (Oh et al., 2012).
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Although expression of th1 type chemokines and IFNG was not observed in naturally MAP-
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infected cows, exception of down-regulation of CCL4 and CCL5 in Test1 of cows, th1 type
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chemokines were increased at 6 h p.i. in THP-1 cell infected with MAP, especially 9
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expression of CXCL10 was gradually increased in course of time. Th1 type chemokines and
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chemokine receptors (CCL5 and CXCR3) show increased infiltration in granulomas of CD
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patients (Oki et al., 2005), and a role for CCL5 and related chemokine receptors was
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suggested in the pathogenesis of JD in THP-1 cells stimulated with MAP isolates (Motiwala
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et al., 2006). Our results which indicate up-regulation of Th1 type chemokines (CCL4, CCL5,
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CXCL9, and CXCL10) in MAP-infected THP-1 cells are consistent with previous data and
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suggest a relationship between Th1 type chemokines and their receptors and the host
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response to MAP infection. Interferon-inducible protein 10 (IP-10, CXCL10) has been
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reported to be up-regulated in some Th1-mediated inflammatory disorders in humans
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including type 1 diabetes mellitus (Cossu et al., 2013), rheumatoid arthritis (Patel et al.,
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2001), multiple sclerosis (Balashov et al., 1999), sarcoid granulomatous disease (Beard et al.,
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1999) and IBD (Zwick et al., 2002). CXCL10 has been described as an alternative or adjunct
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biomarker to IFN-γ, showing high expression in serum of tuberculosis and tuberculosis-HIV
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co-infected patients (Jordao et al., 2008). Detection of CXCL9 and CXCL10 by qPCR was
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also reported as a potential method to enhance diagnostic sensitivity in human tuberculosis
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(Flynn & Chan, 2003, Magee et al., 2012). Monokine induced by interferon-γ (MIG, CXCL9)
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was also induced in a murine model of IBD (McKinney et al., 2000, Ito et al., 2006). This
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suggests that Th1 type chemokines may be valuable biomarkers for MAP infection.
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Haptoglobin (HP), an acute phase protein, has been demonstrated to chemoattract
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monocytes, to modulate the immune response, and to have anti-inflammatory activities
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(Beard et al., 2000, Madsen et al., 2001, Moura et al., 2012). HP was suggested as an obesity
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and inflammatory marker in its role as a novel monocyte chemoattractant (Beard et al.,
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2000). Furthermore, serum HP was used within a disease activity index in a model of murine
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IBD (Ito et al., 2006) and proposed as an independent prognostic factor in epithelial ovarian 10
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cancer patients (Hines et al., 2014). Elevated serum levels of Chitinase 3-Like-1 (CHI3L1)
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have been correlated with increased disease severity and a poorer prognosis for many
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diseases, including cancers, autoimmune diseases, and chronic inflammatory conditions
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(Ravva & Stanker, 2005). Our findings of up-regulation of HP and CHI3L1 at 48h p.i. in
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MAP-infected THP-1 also suggest that these genes could provide a useful measure in
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progression of MAP infection. Expression of TFRC gene was increased at 6h p.i. and
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maintained at 24h and 48h post infection. TFRC is known as an early phagosome marker, and
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its expression may be affected by the interference of MAP in the phagosome maturation
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process (Ruhl & Collins, 1995). TFRC was also up-regulated at 6 h p.i. in Raw 264.7 cell
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infected with MAP (Cha et al., 2013). In addition, fecal shedder of naturally infected cows
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showed down-regulation of MPO (Myeloperoxidase) gene (Test1 and Test3, Table1), and the
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gene was also down-regulated in THP-1 infected with MAP. MPO gene was known to
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generates reactive oxidants and that has been implicated in mycobacterial killing (Borelli et
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al., 1999). Down-regulation of MPO gene could be suspected to be associated with resistance
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of MAP against host defense response. In the present study, THP-1 cell treated with PMA for
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differentiation before infection with MAP. Even though phenotype of THP-1 differentiated
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using modified protocol, which was incubated for further 5 days in fresh media after
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treatment with PMA, but further 1 day incubation in our case, shared characteristics of
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primary monocyte-derived macrophages (Daigneault et al., 2010), the responses of natural
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MAP infection in human macrophages could be different from those in THP-1.
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In the present study, we can see the potential host responses including up-regulation of
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genes related to immune response (CD14, S100A8, S100A9, LTF, HP and CHCIL3), up-
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regulation of Th 1-polarizing factor (CCL4, CCL5, CXCL9 and CXCL10), down-regulation
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of genes related to metabolism (ELANE, IGF1, TCF7L2, and MPO), and no significant 11
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response of other genes (GADD45a, GPNMB, HMOX1, IFNG, and NQO1) in human
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macrophage cells infected with MAP, which were selected by an in silico genomic
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technique (Biomarker Discovery of IPA) in MAP-infected mice.
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Supporting information
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Supplementary table S1. Primers used for qRT-PCR
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Acknowledgements This study was supported by the Rural Development Administration (PJ00897001) and the Research Institute for Veterinary Science, Seoul National University, South Korea.
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Madsen M, Graversen JH & Moestrup SK (2001) Haptoglobin and CD163: captor and receptor gating hemoglobin to macrophage lysosomes. Redox Rep 6: 386-388. Magee DA, Taraktsoglou M, Killick KE, et al. (2012) Global gene expression and systems biology analysis of bovine monocyte-derived macrophages in response to in vitro challenge with Mycobacterium bovis. PLoS One 7: e32034. McClure HM, Chiodini RJ, Anderson DC, Swenson RB, Thayer WR & Coutu JA (1987) Mycobacterium paratuberculosis infection in a colony of stumptail macaques (Macaca arctoides). J Infect Dis 155: 1011-1019. McKinney JD, Honer zu Bentrup K, Munoz-Elias EJ, Miczak A, Chen B, Chan WT, Swenson D, Sacchettini JC, Jacobs WR, Jr. & Russell DG (2000) Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature 406: 735-738. Millar D, Ford J, Sanderson J, Withey S, Tizard M, Doran T & HermonTaylor J (1996) IS900 PCR to detect Mycobacterium paratuberculosis in retail supplies of whole pasteurized cows' milk in England and Wales. Appl Environ Microbiol 62: 3446-3452. Mizoguchi A, Mizoguchi E & Bhan AK (2003) Immune networks in animal models of inflammatory bowel disease. Inflamm Bowel Dis 9: 246-259. Motiwala AS, Janagama HK, Paustian ML, Zhu X, Bannantine JP, Kapur V & Sreevatsan S (2006) Comparative transcriptional analysis of human macrophages exposed to animal and human isolates of Mycobacterium avium subspecies paratuberculosis with diverse genotypes. Infect Immun 74: 60466056. Moura DF, de Mattos KA, Amadeu TP, Andrade PR, Sales JS, Schmitz V, Nery JA, Pinheiro RO & Sarno EN (2012) CD163 favors Mycobacterium leprae survival and persistence by promoting antiinflammatory pathways in lepromatous macrophages. Eur J Immunol 42: 2925-2936. Muruganand D, Daniel E, Ebenezer GJ, Rabboni SE, Segar P & Job CK (2004) Mycobacterium leprae infection and serum lactoferrin levels. Lepr Rev 75: 282-288. Mutwiri GK, Butler DG, Rosendal S, Yager J (1992) Experimental infection of severe combined immunodeficient beige mice with Mycobacterium paratuberculosis of bovine origin. Infect Immun 60:4074-4079. Naser SA, Schwartz D & Shafran I (2000) Isolation of Mycobacterium avium subsp paratuberculosis from breast milk of Crohn's disease patients. Am J Gastroenterol 95: 1094-1095. Naser SA, Ghobrial G, Romero C & Valentine JF (2004) Culture of Mycobacterium avium subspecies paratuberculosis from the blood of patients with Crohn's disease. Lancet 364: 1039-1044. Naser SA, Hulten K, Shafran I, Graham DY & El-Zaatari FA (2000) Specific seroreactivity of Crohn's disease patients against p35 and p36 antigens of M. avium subsp. paratuberculosis. Vet Microbiol 77: 497-504. Oh KJ, Park J, Kim SS, Oh H, Choi CS & Koo SH (2012) TCF7L2 modulates glucose homeostasis by regulating CREB- and FoxO1-dependent transcriptional pathway in the liver. PLoS Genet 8: e1002986. Oki M, Ohtani H, Kinouchi Y, Sato E, Nakamura S, Matsumoto T, Nagura H, Yoshie O & Shimosegawa T (2005) Accumulation of CCR5+ T cells around RANTES+ granulomas in Crohn's disease: a pivotal site of Th1-shifted immune response? Lab Invest 85: 137-145. Patel DD, Zachariah JP & Whichard LP (2001) CXCR3 and CCR5 ligands in rheumatoid arthritis synovium. Clin Immunol 98: 39-45. Pechkovsky DV, Zalutskaya OM, Ivanov GI & Misuno NI (2000) Calprotectin (MRP8/14 protein complex) release during mycobacterial infection in vitro and in vivo. FEMS Immunol Med Microbiol 29: 27-33. Ravva SV & Stanker LH (2005) Real-time quantitative PCR detection of Mycobacterium avium subsp paratuberculosis and differentiation from other mycobacteria using SYBR Green and TaqMan assays. J Microbiol Methods 63: 305-317. Ruhl A & Collins SM (1995) Enteroglial Cells (Egc) Are an Integral-Part of the Neuroimmune Axis in the Enteric Nervous-System (Ens). Gastroenterology 108: A680-A680. Schwartz D, Shafran I, Romero C, Piromalli C, Biggerstaff J, Naser N, Chamberlain W & Naser SA 14
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
(2000) Use of short-term culture for identification of Mycobacterium avium subsp paratuberculosis in tissue from Crohn's disease patients. Clin Microbiol Infec 6: 303-307. Sechi LA, Scanu AM, Molicotti P, Cannas S, Mura M, Dettori G, Fadda G & Zanetti S (2005) Detection and isolation of Mycobacterium avium subspecies paratuberculosis from intestinal mucosal biopsies of patients with and without Crohn's disease in Sardinia. Am J Gastroenterol 100: 1529-1536. Shin MK, Park HT, Shin SW, Jung M, Im YB, Park HE, Cho YI & Yoo HS (2015) Whole-Blood Gene-Expression Profiles of Cows Infected with Mycobacterium avium subsp. paratuberculosis Reveal Changes in Immune Response and Lipid Metabolism. J Microbiol Biotechnol 25: 255-267. Singh UP, Singh S, Singh R, Karls RK, Quinn FD, Potter ME & Lillard JW, Jr. (2007) Influence of Mycobacterium avium subsp. paratuberculosis on colitis development and specific immune responses during disease. Infect Immun 75: 3722-3728. Thayer WR, Coutu JA, Chiodini RJ, Vankruiningen HJ & Merkal RS (1984) Possible Role of Mycobacteria in Inflammatory Bowel-Disease .2. Mycobacterial Antibodies in Crohns-Disease. Digest Dis Sci 29: 1080-1085. Veazey RS, Taylor HW, Horohov DW, Krahenbuhl JL, Oliver JL, 3rd, Snider TG, 3rd (1995) Histopathology of C57BL/6 mice inoculated orally with Mycobacterium paratuberculosis. J Comp Pathol 113:75-80. Yoo HS & Shin SJ (2012) Recent research on bovine paratuberculosis in South Korea. Vet Immunol Immunopathol 148: 23-28. Zwick LS, Walsh TF, Barbiers R, Collins MT, Kinsel MJ & Murnane RD (2002) Paratuberculosis in a mandrill (Papio sphinx). J Vet Diag Invest 14: 326-328.
15
1
Table 1. Expression levels of selected genes in MAP-infected mice and naturally MAP-infected cattle
Group3
A
Mouse spleen1
Gene Symbol
Gene Name
CD14
CD14 molecule
CD68
CD68 molecule
S100A8
S100 calcium binding protein A8
S100A9
S100 calcium binding protein A9
ELANE
elastase, neutrophil expressed
HMOX1
heme oxygenase (decycling 1)
LTF
lactotransferrin
TCF7L2
transcription factor 7-like 2 (T cell specific, HMG-box)
CCL4
chemokine (C-C motif) ligand 4
CCL5
chemokine (C-C motif) ligand 5
HP
haptoglobin
MPO
myeloperoxidase
CXCL9
Chemokine (C-X-C motif) ligand 9
CXCL10
Chemokine (C-X-C motif) ligand 10
IFNG
interferon, gamma
B
C
Location
Cow whole blood2
Family
3 weeks p.i.
6 weeks p.i.
Test1
Test2
Test3
transporter
5.04
2.05
-
2.04
2.5
other
2.3
2.61
-
Cytoplasm
other
64.76
5
-
1.6
1.9
Cytoplasm Extracellular Space Cytoplasm Extracellular Space
other
46.77
4.65
-
1.6
1.9
peptidase
12.99
5.61
-
2.0
1.8
enzyme
6.9
2.85
-
2.3
2.8
peptidase
64.27
7.68
-
3.8
2.9
transcription regulator
3.29
2.12
-
2.0
2.0
cytokine
2.03
2.6
-2.3
-
-
cytokine
2.84
3.02
-2.3
-
-
peptidase
23.85
11.88
-2.8
-
-
enzyme
39.33
7.85
-1.8
-
-1.6
cytokine
4.65
8.4
-
-
-
cytokine
4.94
4.58
-
-
-
cytokine
2.62
3.22
-
-
-
Plasma Membrane Plasma Membrane
Nucleus Extracellular Space Extracellular Space Extracellular Space Cytoplasm Extracellular Space Extracellular Space Extracellular Space 16
2.3
TFRC
transferrin receptor (p90, CD71)
GPNMB
glycoprotein (transmembrane)nmb
GADD45 CHI3L1 IGF1 NQO1
Plasma membrane Plasma Membrane
growth arrest and DNA-damageinducible protein chitinase 3-like 1 (cartilage glycoprotein- 39) insulin-like growth factor1 (somatomedin C)
Extracellular Space Extracellular Space
NAD(P)H dehydrogenase, quione1
Cytoplasm
Nucleus
transporter
11.13
9.12
-
-
-
enzyme
5.49
14.22
-
-
-
other
5.0
6.6
-
-
-
enzyme
12
7.36
-
-
-
growth Factor
2.47
2.35
-
-
-
enzyme
2.14
2.21
-
-
-
1
1
2
2
expression data from microarray after naturally MAP-infected cattle in previous study (Shin et al., 2015). The groups were divided into three groups as follows: Test1,
3
cows shed MAP by feces, Test2, cows with MAP-specific antibodies, and Test3, cows shed MAP by feces with MAP-specific antibodies. 3The selected genes were divided
4
into three groups based on comparison of the results of mice and cattle, A; up-regulated genes in both mice and cattle, B; up- and down-regulated genes in mice and cattle,
5
respectively,
These are gene expression data from microarray at 3 and 6 weeks after infection of mice with MAP in previous study (submitted to another journal). These are gene
C;
up-regulated
and
no
significant
17
changes
in
mice
and
cattle,
respectively.
1
18
1 2
Figure legend
3 4
Fig. 1. Gene expression changes between Map-infected THP-1 cells and uninfected 19
1
control. The selected genes were grouped into three groups as follows: A, up-regulated genes
2
in both mice and cattle; B, up- and down-regulated genes in mice and cattle, respectively; C,
3
up-regulated and no significant changes in mice and cattle, respectively. The relative
4
expression level was normalized with the
5
control by the 2-ΔΔCT method. Significance level set at p ≤ 0.05 (*, p ≤ 0.05; **, p ≤ 0.01; ***,
6
p ≤ 0.001).
-actin expression level relative to zero-time
7
20
1
Host gene expression for Mycobacterium avium subsp. paratuberculosis
2
infection in human THP-1 macrophages
3
Running title: Potential host-response for MAP infection in THP-1 cell
4 5 6
Min-Kyoung Shin1,2, Seung Won Shin1, Myunghwan Jung1, Hongtae Park1, Hyun-Eui Park1,
7
& Han Sang Yoo1, 3
8 9 10 11 12 13 14
1
Department of Infectious Diseases, College of Veterinary Medicine, Seoul National
University, Seoul, 151-742 Korea 2
Dairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada,
Sherbrooke, QC J1M 1Z3, Canada; 3
Institute of Green Bio Science and Technology, Seoul National University, Pyeongchang,
232-916, Korea
15 16
Correspondence: Han Sang Yoo
17
Department of Infectious Diseases, College of Veterinary Medicine, Seoul National
18
University, Seoul, 151-742 Korea
19
Phone: +82-2-880-1263
20
Fax: +82-2-874-2738
21
E-mail: [email protected]
22
1
1 2
Abstract
3
Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s
4
disease, which causes considerable economic loss in the dairy industry and has a possible
5
relationship with Crohn’s disease (CD) in humans. As MAP has been detected in retail
6
pasteurized milk samples, its transmission via milk is of concern. Despite its possible role in
7
the etiology of CD, there have been few studies examining the interactions between MAP and
8
human cells. In the current study, we applied Ingenuity Pathway Analysis to the transcription
9
profiles generated from a murine model with MAP infection as part of a previously
10
conducted study. Twenty one genes were selected as potential host immune responses,
11
compared to the transcriptional profiles in naturally MAP-infected cattle, and validated in
12
MAP-infected human monocyte-derived macrophage THP-1 cells. Of these, the potential
13
host responses including up-regulation of genes related to immune response (CD14, S100A8,
14
S100A9, LTF, HP and CHCIL3), up-regulation of Th 1-polarizing factor (CCL4, CCL5,
15
CXCL9 and CXCL10), down-regulation of genes related to metabolism (ELANE, IGF1,
16
TCF7L2, and MPO), and no significant response of other genes (GADD45a, GPNMB,
17
HMOX1, IFNG, and NQO1) in THP-1 cell infected with MAP.
18
Keywords: Mycobacterium avium subsp. paratuberculosis, host responses, THP-1,
19
2
1 2
Introduction
3
Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne’s disease (JD), a
4
chronic granulomatous enteropathy of ruminants such as cattle, sheep, deer, bison and elk
5
(Motiwala et al., 2006). Concerns have been raised about JD over economic losses in the
6
dairy industry as well as the possible relationship with a chronic inflammatory bowel disease
7
(IBD) in humans called Crohn’s disease (CD) (Thayer et al., 1984, Naser et al., 2004, Yoo &
8
Shin, 2012). It has been suggested that human IBD may be caused by an autoimmune
9
dysregulation and imbalance of T cells or host responsiveness against a microbial trigger
10
(Mizoguchi et al., 2003, Singh et al., 2007). In addition, many studies report that MAP or
11
MAP-specific serum antibodies are detected in the intestine, blood and breast milk of Crohn’s
12
disease patients (Naser et al., 2000, Naser et al., 2000, Schwartz et al., 2000, Naser et al.,
13
2004, Sechi et al., 2005). MAP DNA has been detected in retail pasteurized milk samples,
14
even commercially pasteurized milk in the United Kingdom. As transmission could occur
15
through milk and other dairy products, further research into the mechanisms underlying MAP
16
pathogenesis in human is needed (Millar et al., 1996, Ellingson et al., 2005). Despite its
17
possible role in the etiology of CD, there have been few studies examining the interactions
18
between MAP and human cells. Different gene expression patterns or cytokine responses
19
(TNF-α, IL-1β, and IL-10) were observed in human monocyte-derived macrophage THP-1
20
cells by different MAP strains (Motiwala et al., 2006, Borrmann et al., 2011).
21
In the current study, we applied a microarray-based approach to discover potential immune
22
response markers in a model of murine MAP infection and naturally MAP-infected cattle
23
followed by validation of the genes in human cells. In our previous study, the host responses
24
to MAP infection in mice and cattle were characterized by transcriptional analysis. 3
1
Significantly regulated genes were filtered and selected via the Ingenuity Pathways Analysis
2
(IPA) and evaluated in vitro using MAP-infected THP-1 cells.
3 4
Materials and Methods
5
Selection of potential host response genes using transcriptional profiles in MAP-
6
infected mice and naturally MAP-infected cattle
7
We previously submitted a dataset of MAP infection to the GEO database (GSE62836) for
8
murine study (submmitted to another journal) and GEO database (GSE62835) for bovine
9
study (Shin et al., 2015). For murine study, briefly, five-week-old C57BL/6 female mice
10
were inoculated intraperitoneally with 300 μL PBS (control group) or a suspension of MAP
11
ATCC 19698 (3 × 107 cells/mouse, MAP-infected group). Spleen samples were taken from
12
five mice in each group, sacrificed at 3 and 6 weeks post infection (p.i.). Microarray analysis
13
was performed using total RNA from harvested spleen cells and the data were used for
14
identification of differentially expressed genes in MAP-infected mice compared to control
15
mice. Differentially expressed genes were filtered for a log2-fold change >2 with p < 0.05 by
16
Biomarker Filter and then analyzed by Biomarker Discovery both of IPA (Ingenuity Systems
17
Inc., Redwood, CA). The genes were further clarified in the IPA biomarker filter module
18
based on the following criteria: fluid – 〈〈all〉〉, disease – 〈〈Infectious Disease, Inflammatory
4
1
response, Immunological disease〉〉, and species – 〈〈mouse〉〉. Genes selected based on the
2
above criteria were compared with bovine transcriptional profiles and verified in THP-1 cells
3
following infection with MAP using quantitative real-time RT PCR.
4 5
Bacterial strain, cell line culture, and infection
6
MAP ATCC 19698 was cultured as previously described (Cha et al., 2013). For infection of
7
cells, MAP ATCC 19698 was suspended in phosphate-buffered saline (PBS) and used at a
8
dilution of 1 × 109 cells/mL.
9
The THP-1 human leukemic monocyte cell line was obtained from KCLB (Korea Cell Line
10
Bank, Seoul, Korea). THP-1 cells were cultured in RPMI 1640, supplemented with 10% heat-
11
inactivated fetal bovine serum (FBS), and Antibiotic-Antimycotic (all from Gibco Invitrogen,
12
Karlsruhe, Germany) agent at 37°C in humidified air with 5% CO2. THP-1 cells were
13
differentiated into macrophages by stimulation with phorbol-12-myristate-13-acetate (PMA;
14
Sigma Co., St. Louis, MO, USA) (50ng/ml) for 72h, washed with FBS-free RPMI 1640
15
medium and incubated with 5% FBS-RPMI 1640 medium without antibiotics for 24h before
16
the experiments. Differentiated THP-1 macrophage cells (1 × 106 cells/ml) were inoculated
17
with MAP 19698 at a multiplicity of infection (MOI) of 10:1 and incubated for 0, 6, 24, and
18
48h. Total RNA was extracted from cells collected at 0, 6, 24, and 48h post infection (p.i.).
19 5
1
Quantitative real-time PCR
2
The total RNA was isolated with an RNeasy Mini Kit (Qiagen, Valencia, USA) according to
3
the manufacturer's instructions. Two micrograms of total RNA from each sample were
4
reverse-transcribed into cDNA using the Quantitect® Reverse Transcription kit (Qiagen).
5
Real time qRT-PCR reactions were performed with 2 μl of cDNA using the Rotor-Gene
6
SYBR Green PCR kit (Qiagen) and Rotor-Gene Q real time PCR cycler (Qiagen). Cycling
7
parameters were as follows: 95°C for 5 minutes for 1 cycle, 95°C for 20 seconds, and then
8
60°C for 45 seconds for 40 cycles. Primers used in real-time PCR are shown in Table 1. The
9
gene expression level was normalized by the 2-ΔΔCt method using β-actin as an internal
10
control gene. The relative expression level was compared to the zero-time control to
11
determine the changes in expression-fold of each gene.
12 13
Statistical analysis
14
The data were expressed as mean ± standard error of the mean (SEM), and statistical
15
significance was analyzed by student’s t-test using IBM Statistical Package for Social
16
Sciences software (SPSS, version 21, SPSS Inc., Chicago, IL, USA). Differences were
17
considered significant if a value of p ≤ 0.05 was obtained.
18
19
Results
20
Host gene expression in MAP-infection mice and naturally MAP-infected cattle
21
In our previous study, C57BL/6 mice infected with MAP showed most severe
22
immunopathologic changes at 3 and 6 weeks p.i. by transcriptional profiling and 6
1
histopathological analysis. As the first step of data mining process, the “up-regulated” genes
2
were selected using the cut-off value (log2-fold change > 2 and p < 0.05). In the second, the
3
genes were imported to the Biomarker Discovery module of IPA and filtered by the criteria,
4
like: excreted in all biological fluids, related to infectious disease, inflammatory response, or
5
immunological disease. The twenty one eligible markers selected by IPA were related to
6
immune system processes (IFNG, CCL4, CCL5, CD14, CXCL9, CXCL10, HP, S100A8,
7
S100A9, MPO), metabolic processes (ELANE, CD68, CHI3L1, HP, S100A8, S100A9, IGF1,
8
TCF7L2, MPO, LTF, HMOX1), localization (CD68, IGF1, LTF, TFRC), and others
9
(GADD45, GPNMB, NQO1) as classified by GO biological processes (Table 1). These genes
10
were compared with the transcriptional profiles in naturally MAP-infected cattle (Table 1).
11
Naturally MAP-infected cattle were divided into three groups as follows: Test1, fecal PCR
12
positive; Test2, MAP-ELISA positive; and Test3, fecal PCR positive and MAP-ELISA
13
positive as previously described (Shin et al., 2015). The selected genes showed various
14
expression according to the groups in cattle, while all of the genes were up-regulated in
15
MAP-infected mice. The selected genes were divided into three groups based on comparison
16
of the results of mice and cattle. Group A was occupied by up-regulated genes in cows with
17
MAP-specific antibodies (Test2 and Test3). Group B was occupied by down-regulated genes
18
in cows as fecal shedder (Test1). The genes, which were up-regulated in MAP-infected mice,
19
but no significant response in MAP-affected cows, were Group C.
20 21
Validation of biomarker candidates in THP-1 by qPCR
22
The twenty one selected candidates were evaluated in human THP-1 cells at various time
23
points following MAP infection. No significant change in expression was observed in 7 of
24
these genes (CD68, GADD45, GPNMB, HMOX1, IFNG, NQO1 and TCF7L2) at any time 7
1
point relative to the uninfected control. The expression of CCL4 (3.5-fold up-regulation),
2
CCL5 (2.4-fod up-regulation), CXCL9 (3.5-fold up-regulation), and TFRC (2.1-fold up-
3
regulation) genes were increased at 6h p.i. but not at later time points and the expression of
4
CD14 (4.1- and 4.8-fold up-regulation at 24h and 48h p.i.), CXCL10 (1.8- and 3.4-fold up-
5
regulation at 24h and 48h p.i.), LTF (1.7- and 1.4-fold up-regulation at 24h and 48h p.i.),
6
S100A8 (6.2- and 6.7-fold up-regulation at 24h and 48h p.i.), and S100A9 (2.7- and 3.9-fold
7
up-regulation at 24h and 48h p.i.) genes were increased at 24h and 48h p.i. (Figure 1).
8
CHI3L1 (2.6-fold up-regulation), and HP (4.3-fold up-regulation) genes showed increased
9
expression only at 48h p.i. after MAP infection (Figure 1), while ELANE (1.7- and 1.8-fold
10
down-regulation at 24h and 48h p.i.), IGF1 (2.3- and 1.5-fold down-regulation at 24h and
11
48h p.i.), and MPO (2.1- and 1.8-fold down-regulation at 6h and 48h p.i.) genes showed
12
down-regulation. Therefore, among the selected genes from the transcriptomes of MAP-
13
infected mice, the expression of fourteen of these genes were also significantly up- or down-
14
regulated in human THP-1 cells following MAP infection.
15 16
Discussion
17
Although the oral route is the natural infection route of MAP in cattle, the intraperitoneal
18
(IP) injection has been frequently utilized in MAP infection of mice and our previous murine
19
study also was challenged MAP intraperitoneally, because earlier studies demonstrated that
20
low infectivity rates are the main problem of the oral administration of MAP in mouse model
21
(Mutwiri et al., 1992; Veazey et al., 1995). In the previous study, we analyzed transcriptional
22
profiles induced in the murine spleen, wherein the innate and adaptive immune systems
23
combined, following MAP infection. The expression patterns of twenty one candidate genes
24
were analyzed at various time points post infection. CD68 and CD14, both macrophage 8
1
markers, showed different expression patterns. CD68 was not significantly altered by MAP
2
infection in THP-1 cells. However, in these experiments PMA-stimulated THP-1 macrophage
3
cells were used and CD68 was already expressed in these cells prior to infection which may
4
explain why no further change was observed. The expression of CD14 increased at 24h and
5
48h post infection. A role for CD14 has been reported in the regulation of
6
monocyte/macrophage apoptosis so up-regulation of CD14 may have an effect on levels of
7
apoptosis during infection (McClure et al., 1987).
8
Increased expression of S100A8 and S100A9 (also termed myeloid-related proteins (MRP)8
9
and MRP14) was observed, consistent with data demonstrating release of these proteins by
10
activated phagocytes during mycobacterial infection both in vitro and in vivo (Pechkovsky et
11
al., 2000, Frosch et al., 2004). Cows with MAP-specific antibodies showed up-regulation of
12
MRP8 and MRP14 (Shin et al., 2015). They form stable complexes (MRP8/14) and are found
13
in a variety of inflammatory conditions, including rheumatoid arthritis, allograft rejections,
14
and inflammatory bowel and lung diseases (Kerkhoff et al., 1998). Therefore, their potential
15
role in inflammatory disorders including CD has already been highlighted (Lugering et al.,
16
1995, Foell et al., 2004). LTF (lactoferrin) gene was also slightly up-regulated in THP-1 cell
17
infected with MAP. Serum lactoferrin was strongly and inversely associated with increasing
18
bacterial index of M. leprae in leprosy patients (Muruganand et al., 2004). Of group A,
19
TCF7L2 gene was down-regulated at 6 h p.i., but there was not statistical significant
20
responses. TCF7L2 was suggested to modulate hepatic glucose metabolism in mammals, thus
21
contributing to the development of type 2 diabetes (Oh et al., 2012).
22
Although expression of th1 type chemokines and IFNG was not observed in naturally MAP-
23
infected cows, exception of down-regulation of CCL4 and CCL5 in Test1 of cows, th1 type
24
chemokines were increased at 6 h p.i. in THP-1 cell infected with MAP, especially 9
1
expression of CXCL10 was gradually increased in course of time. Th1 type chemokines and
2
chemokine receptors (CCL5 and CXCR3) show increased infiltration in granulomas of CD
3
patients (Oki et al., 2005), and a role for CCL5 and related chemokine receptors was
4
suggested in the pathogenesis of JD in THP-1 cells stimulated with MAP isolates (Motiwala
5
et al., 2006). Our results which indicate up-regulation of Th1 type chemokines (CCL4, CCL5,
6
CXCL9, and CXCL10) in MAP-infected THP-1 cells are consistent with previous data and
7
suggest a relationship between Th1 type chemokines and their receptors and the host
8
response to MAP infection. Interferon-inducible protein 10 (IP-10, CXCL10) has been
9
reported to be up-regulated in some Th1-mediated inflammatory disorders in humans
10
including type 1 diabetes mellitus (Cossu et al., 2013), rheumatoid arthritis (Patel et al.,
11
2001), multiple sclerosis (Balashov et al., 1999), sarcoid granulomatous disease (Beard et al.,
12
1999) and IBD (Zwick et al., 2002). CXCL10 has been described as an alternative or adjunct
13
biomarker to IFN-γ, showing high expression in serum of tuberculosis and tuberculosis-HIV
14
co-infected patients (Jordao et al., 2008). Detection of CXCL9 and CXCL10 by qPCR was
15
also reported as a potential method to enhance diagnostic sensitivity in human tuberculosis
16
(Flynn & Chan, 2003, Magee et al., 2012). Monokine induced by interferon-γ (MIG, CXCL9)
17
was also induced in a murine model of IBD (McKinney et al., 2000, Ito et al., 2006). This
18
suggests that Th1 type chemokines may be valuable biomarkers for MAP infection.
19
Haptoglobin (HP), an acute phase protein, has been demonstrated to chemoattract
20
monocytes, to modulate the immune response, and to have anti-inflammatory activities
21
(Beard et al., 2000, Madsen et al., 2001, Moura et al., 2012). HP was suggested as an obesity
22
and inflammatory marker in its role as a novel monocyte chemoattractant (Beard et al.,
23
2000). Furthermore, serum HP was used within a disease activity index in a model of murine
24
IBD (Ito et al., 2006) and proposed as an independent prognostic factor in epithelial ovarian 10
1
cancer patients (Hines et al., 2014). Elevated serum levels of Chitinase 3-Like-1 (CHI3L1)
2
have been correlated with increased disease severity and a poorer prognosis for many
3
diseases, including cancers, autoimmune diseases, and chronic inflammatory conditions
4
(Ravva & Stanker, 2005). Our findings of up-regulation of HP and CHI3L1 at 48h p.i. in
5
MAP-infected THP-1 also suggest that these genes could provide a useful measure in
6
progression of MAP infection. Expression of TFRC gene was increased at 6h p.i. and
7
maintained at 24h and 48h post infection. TFRC is known as an early phagosome marker, and
8
its expression may be affected by the interference of MAP in the phagosome maturation
9
process (Ruhl & Collins, 1995). TFRC was also up-regulated at 6 h p.i. in Raw 264.7 cell
10
infected with MAP (Cha et al., 2013). In addition, fecal shedder of naturally infected cows
11
showed down-regulation of MPO (Myeloperoxidase) gene (Test1 and Test3, Table1), and the
12
gene was also down-regulated in THP-1 infected with MAP. MPO gene was known to
13
generates reactive oxidants and that has been implicated in mycobacterial killing (Borelli et
14
al., 1999). Down-regulation of MPO gene could be suspected to be associated with resistance
15
of MAP against host defense response. In the present study, THP-1 cell treated with PMA for
16
differentiation before infection with MAP. Even though phenotype of THP-1 differentiated
17
using modified protocol, which was incubated for further 5 days in fresh media after
18
treatment with PMA, but further 1 day incubation in our case, shared characteristics of
19
primary monocyte-derived macrophages (Daigneault et al., 2010), the responses of natural
20
MAP infection in human macrophages could be different from those in THP-1.
21
In the present study, we can see the potential host responses including up-regulation of
22
genes related to immune response (CD14, S100A8, S100A9, LTF, HP and CHCIL3), up-
23
regulation of Th 1-polarizing factor (CCL4, CCL5, CXCL9 and CXCL10), down-regulation
24
of genes related to metabolism (ELANE, IGF1, TCF7L2, and MPO), and no significant 11
1
response of other genes (GADD45a, GPNMB, HMOX1, IFNG, and NQO1) in human
2
macrophage cells infected with MAP, which were selected by an in silico genomic
3
technique (Biomarker Discovery of IPA) in MAP-infected mice.
4 5
Supporting information
6
Supplementary table S1. Primers used for qRT-PCR
7
8
9 10
Acknowledgements This study was supported by the Rural Development Administration (PJ00897001) and the Research Institute for Veterinary Science, Seoul National University, South Korea.
11
12
1 2
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49
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Madsen M, Graversen JH & Moestrup SK (2001) Haptoglobin and CD163: captor and receptor gating hemoglobin to macrophage lysosomes. Redox Rep 6: 386-388. Magee DA, Taraktsoglou M, Killick KE, et al. (2012) Global gene expression and systems biology analysis of bovine monocyte-derived macrophages in response to in vitro challenge with Mycobacterium bovis. PLoS One 7: e32034. McClure HM, Chiodini RJ, Anderson DC, Swenson RB, Thayer WR & Coutu JA (1987) Mycobacterium paratuberculosis infection in a colony of stumptail macaques (Macaca arctoides). J Infect Dis 155: 1011-1019. McKinney JD, Honer zu Bentrup K, Munoz-Elias EJ, Miczak A, Chen B, Chan WT, Swenson D, Sacchettini JC, Jacobs WR, Jr. & Russell DG (2000) Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature 406: 735-738. Millar D, Ford J, Sanderson J, Withey S, Tizard M, Doran T & HermonTaylor J (1996) IS900 PCR to detect Mycobacterium paratuberculosis in retail supplies of whole pasteurized cows' milk in England and Wales. Appl Environ Microbiol 62: 3446-3452. Mizoguchi A, Mizoguchi E & Bhan AK (2003) Immune networks in animal models of inflammatory bowel disease. Inflamm Bowel Dis 9: 246-259. Motiwala AS, Janagama HK, Paustian ML, Zhu X, Bannantine JP, Kapur V & Sreevatsan S (2006) Comparative transcriptional analysis of human macrophages exposed to animal and human isolates of Mycobacterium avium subspecies paratuberculosis with diverse genotypes. Infect Immun 74: 60466056. Moura DF, de Mattos KA, Amadeu TP, Andrade PR, Sales JS, Schmitz V, Nery JA, Pinheiro RO & Sarno EN (2012) CD163 favors Mycobacterium leprae survival and persistence by promoting antiinflammatory pathways in lepromatous macrophages. Eur J Immunol 42: 2925-2936. Muruganand D, Daniel E, Ebenezer GJ, Rabboni SE, Segar P & Job CK (2004) Mycobacterium leprae infection and serum lactoferrin levels. Lepr Rev 75: 282-288. Mutwiri GK, Butler DG, Rosendal S, Yager J (1992) Experimental infection of severe combined immunodeficient beige mice with Mycobacterium paratuberculosis of bovine origin. Infect Immun 60:4074-4079. Naser SA, Schwartz D & Shafran I (2000) Isolation of Mycobacterium avium subsp paratuberculosis from breast milk of Crohn's disease patients. Am J Gastroenterol 95: 1094-1095. Naser SA, Ghobrial G, Romero C & Valentine JF (2004) Culture of Mycobacterium avium subspecies paratuberculosis from the blood of patients with Crohn's disease. Lancet 364: 1039-1044. Naser SA, Hulten K, Shafran I, Graham DY & El-Zaatari FA (2000) Specific seroreactivity of Crohn's disease patients against p35 and p36 antigens of M. avium subsp. paratuberculosis. Vet Microbiol 77: 497-504. Oh KJ, Park J, Kim SS, Oh H, Choi CS & Koo SH (2012) TCF7L2 modulates glucose homeostasis by regulating CREB- and FoxO1-dependent transcriptional pathway in the liver. PLoS Genet 8: e1002986. Oki M, Ohtani H, Kinouchi Y, Sato E, Nakamura S, Matsumoto T, Nagura H, Yoshie O & Shimosegawa T (2005) Accumulation of CCR5+ T cells around RANTES+ granulomas in Crohn's disease: a pivotal site of Th1-shifted immune response? Lab Invest 85: 137-145. Patel DD, Zachariah JP & Whichard LP (2001) CXCR3 and CCR5 ligands in rheumatoid arthritis synovium. Clin Immunol 98: 39-45. Pechkovsky DV, Zalutskaya OM, Ivanov GI & Misuno NI (2000) Calprotectin (MRP8/14 protein complex) release during mycobacterial infection in vitro and in vivo. FEMS Immunol Med Microbiol 29: 27-33. Ravva SV & Stanker LH (2005) Real-time quantitative PCR detection of Mycobacterium avium subsp paratuberculosis and differentiation from other mycobacteria using SYBR Green and TaqMan assays. J Microbiol Methods 63: 305-317. Ruhl A & Collins SM (1995) Enteroglial Cells (Egc) Are an Integral-Part of the Neuroimmune Axis in the Enteric Nervous-System (Ens). Gastroenterology 108: A680-A680. Schwartz D, Shafran I, Romero C, Piromalli C, Biggerstaff J, Naser N, Chamberlain W & Naser SA 14
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(2000) Use of short-term culture for identification of Mycobacterium avium subsp paratuberculosis in tissue from Crohn's disease patients. Clin Microbiol Infec 6: 303-307. Sechi LA, Scanu AM, Molicotti P, Cannas S, Mura M, Dettori G, Fadda G & Zanetti S (2005) Detection and isolation of Mycobacterium avium subspecies paratuberculosis from intestinal mucosal biopsies of patients with and without Crohn's disease in Sardinia. Am J Gastroenterol 100: 1529-1536. Shin MK, Park HT, Shin SW, Jung M, Im YB, Park HE, Cho YI & Yoo HS (2015) Whole-Blood Gene-Expression Profiles of Cows Infected with Mycobacterium avium subsp. paratuberculosis Reveal Changes in Immune Response and Lipid Metabolism. J Microbiol Biotechnol 25: 255-267. Singh UP, Singh S, Singh R, Karls RK, Quinn FD, Potter ME & Lillard JW, Jr. (2007) Influence of Mycobacterium avium subsp. paratuberculosis on colitis development and specific immune responses during disease. Infect Immun 75: 3722-3728. Thayer WR, Coutu JA, Chiodini RJ, Vankruiningen HJ & Merkal RS (1984) Possible Role of Mycobacteria in Inflammatory Bowel-Disease .2. Mycobacterial Antibodies in Crohns-Disease. Digest Dis Sci 29: 1080-1085. Veazey RS, Taylor HW, Horohov DW, Krahenbuhl JL, Oliver JL, 3rd, Snider TG, 3rd (1995) Histopathology of C57BL/6 mice inoculated orally with Mycobacterium paratuberculosis. J Comp Pathol 113:75-80. Yoo HS & Shin SJ (2012) Recent research on bovine paratuberculosis in South Korea. Vet Immunol Immunopathol 148: 23-28. Zwick LS, Walsh TF, Barbiers R, Collins MT, Kinsel MJ & Murnane RD (2002) Paratuberculosis in a mandrill (Papio sphinx). J Vet Diag Invest 14: 326-328.
15
1
Table 1. Expression levels of selected genes in MAP-infected mice and naturally MAP-infected cattle
Group3
A
Mouse spleen1
Gene Symbol
Gene Name
CD14
CD14 molecule
CD68
CD68 molecule
S100A8
S100 calcium binding protein A8
S100A9
S100 calcium binding protein A9
ELANE
elastase, neutrophil expressed
HMOX1
heme oxygenase (decycling 1)
LTF
lactotransferrin
TCF7L2
transcription factor 7-like 2 (T cell specific, HMG-box)
CCL4
chemokine (C-C motif) ligand 4
CCL5
chemokine (C-C motif) ligand 5
HP
haptoglobin
MPO
myeloperoxidase
CXCL9
Chemokine (C-X-C motif) ligand 9
CXCL10
Chemokine (C-X-C motif) ligand 10
IFNG
interferon, gamma
B
C
Location
Cow whole blood2
Family
3 weeks p.i.
6 weeks p.i.
Test1
Test2
Test3
transporter
5.04
2.05
-
2.04
2.5
other
2.3
2.61
-
Cytoplasm
other
64.76
5
-
1.6
1.9
Cytoplasm Extracellular Space Cytoplasm Extracellular Space
other
46.77
4.65
-
1.6
1.9
peptidase
12.99
5.61
-
2.0
1.8
enzyme
6.9
2.85
-
2.3
2.8
peptidase
64.27
7.68
-
3.8
2.9
transcription regulator
3.29
2.12
-
2.0
2.0
cytokine
2.03
2.6
-2.3
-
-
cytokine
2.84
3.02
-2.3
-
-
peptidase
23.85
11.88
-2.8
-
-
enzyme
39.33
7.85
-1.8
-
-1.6
cytokine
4.65
8.4
-
-
-
cytokine
4.94
4.58
-
-
-
cytokine
2.62
3.22
-
-
-
Plasma Membrane Plasma Membrane
Nucleus Extracellular Space Extracellular Space Extracellular Space Cytoplasm Extracellular Space Extracellular Space Extracellular Space 16
2.3
TFRC
transferrin receptor (p90, CD71)
GPNMB
glycoprotein (transmembrane)nmb
GADD45 CHI3L1 IGF1 NQO1
Plasma membrane Plasma Membrane
growth arrest and DNA-damageinducible protein chitinase 3-like 1 (cartilage glycoprotein- 39) insulin-like growth factor1 (somatomedin C)
Extracellular Space Extracellular Space
NAD(P)H dehydrogenase, quione1
Cytoplasm
Nucleus
transporter
11.13
9.12
-
-
-
enzyme
5.49
14.22
-
-
-
other
5.0
6.6
-
-
-
enzyme
12
7.36
-
-
-
growth Factor
2.47
2.35
-
-
-
enzyme
2.14
2.21
-
-
-
1
1
2
2
expression data from microarray after naturally MAP-infected cattle in previous study (Shin et al., 2015). The groups were divided into three groups as follows: Test1,
3
cows shed MAP by feces, Test2, cows with MAP-specific antibodies, and Test3, cows shed MAP by feces with MAP-specific antibodies. 3The selected genes were divided
4
into three groups based on comparison of the results of mice and cattle, A; up-regulated genes in both mice and cattle, B; up- and down-regulated genes in mice and cattle,
5
respectively,
These are gene expression data from microarray at 3 and 6 weeks after infection of mice with MAP in previous study (submitted to another journal). These are gene
C;
up-regulated
and
no
significant
17
changes
in
mice
and
cattle,
respectively.
1
18
1 2
Figure legend
3 4
Fig. 1. Gene expression changes between Map-infected THP-1 cells and uninfected 19
1
control. The selected genes were grouped into three groups as follows: A, up-regulated genes
2
in both mice and cattle; B, up- and down-regulated genes in mice and cattle, respectively; C,
3
up-regulated and no significant changes in mice and cattle, respectively. The relative
4
expression level was normalized with the
5
control by the 2-ΔΔCT method. Significance level set at p ≤ 0.05 (*, p ≤ 0.05; **, p ≤ 0.01; ***,
6
p ≤ 0.001).
-actin expression level relative to zero-time
7
20
