Evaluation of host and bacterial gene modulation during Lawsonia intracellularis infection in immunocompetent C57BL/6 mouse model.

BACKGROUND: Proliferative enteritis caused by Lawsonia intracellularis undermines the economic stability of the swine industry worldwide. The development of cost-effective animal models to study the pathophysiology of the disease will help develop strategies to counter this bacterium.
OBJECTIVES: This study focused on establishing a model of gastrointestinal (GI) infection of L. intracellularis in C57BL/6 mice to evaluate the disease progression and lesions of proliferative enteropathy (PE) in murine GI tissue.
METHODS: We assessed the murine mucosal and cell-mediated immune responses generated in response to inoculation with L. intracellularis.
RESULTS: The mice developed characteristic lesions of the disease and shed L. intracellularis in the feces following oral inoculation with 5 × l07 bacteria. An increase in L. intracellularis 16s rRNA and groEL copies in the intestine of infected mice indicated intestinal dissemination of the bacteria. The C57BL/6 mice appeared capable of modulating humoral and cell-mediated immune responses to L. intracellularis infection. Notably, the expression of genes for the vitamin B12 receptor and for secreted and membrane-bound mucins were downregulated in L. intracellularis -infected mice. Furthermore, L. intracellularis colonization of the mouse intestine was confirmed by the immunohistochemistry and western blot analyses.
CONCLUSIONS: This is the first study demonstrating the contributions of bacterial chaperonin and host nutrient genes to PE using an immunocompetent mouse model. This mouse infection model may serve as a platform from which to study L. intracellularis infection and develop potential vaccination and therapeutic strategies to treat PE.

INTRODUCTION

Proliferative enteropathy (PE) is an intestinal disease with a wide host range [123], and is commonly diagnosed in pigs [45]. The bacterium responsible for PE multiplies in the cytoplasm of immature enterocytes, predominantly within those of the ileum and colon [67]. In horses, PE has been classified as an emerging disease as this disease has remained largely unaddressed [8]. Lawsonia intracellularis is thought to be transmitted by the fecal-oral route. After the successful entry into the intestinal lumen, L. intracellularis exhibit tropism for epithelial cell crypts, where they preferentially invade rapidly dividing immature enterocytes [9]. The gross pathological lesions of PE are often restricted to the intestinal epithelium and the distribution of active infection in other organs is yet to be elucidated. In infected pigs, L. intracellularis antigens have been detected in mesenteric lymph nodes, tonsillar crypt cells, and the peripheral circulation; these findings have been attributed to the distribution of bacterial antigens by infected macrophages [1011]. The economic impact of PE on the swine and equine industries is the result of prolonged recovery and/or severely reduced growth performance of infected animals [12].

The pathogenesis of PE has been studied after challenging experimental pigs and foals using L. intracellularis isolates grown in vitro [13] or mouse enteroids [14]. There have been only a few studies reporting successful L. intracellularis infection of laboratory rodents and chickens, and the infection produced different disease outcomes [15]; however, hamsters have been reported to be both naturally and experimentally infected with L. intracellularis. Infected hamsters manifest profound weight loss with severe hyperplasia of the ileum, a syndrome similar to that observed in pigs [16]. Therefore, the hamster is used as an animal model of PE following infection with ileal homogenates harvested from naturally diseased pigs or with bacteria grown in pure cell culture.

The use of experimentally challenged pigs and foals to study PE increases the cost of research by manifold. Thus, we urgently need animal models of PE that are cost-effective and allow the analysis of the critical aspects of L. intracellularis infection, multiplication, pathogenesis, and transmission, and more importantly, support therapeutic testing and identification of vaccine candidates. Mice are the most commonly used animal models in research due to their small size, short reproduction time, high fecundity, and low maintenance cost [17]. Although INF-γ is shown to be required for intestinal epithelial hyperplasia in knockout mice infected with L. intracellularis, data regarding host-bacterial interactions in immunocompetent mice are limited.

In the current study, we evaluated the ability of L. intracellularis to infect immunocompetent C57BL/6 mice. The outcomes of this study may provide a better understanding of host-pathogen interactions in the C57BL/6 mouse model. To the best of our knowledge, there have been no previous studies of the ability of L. intracellularis to infect immunocompetent mice and compromise the intestinal barrier.

MATERIALS AND METHODS

Mice and ethics statement

Seven-week-old female specific-pathogen-free C57BL/6 mice (n = 50) were procured from Koatech Laboratory Animals, Inc. (Korea). All animal experiments were approved by the Jeonbuk National University Animal Ethics Committee (CBNU2015-00085). Animals were provided antibiotic-free deionized water and fed ad libitum. The mice were assigned to either the test (n = 30) or the control (n = 20) group.

Bacterial strain and experimental infection

The test group mice were experimentally infected with about 5 × 107 L. intracellularis (Enterisol Ileitis; Boehringer Ingelheim, Germany) administered by gavage. The control group received phosphate-buffered saline (PBS). For 5 consecutive weeks, the mice were weighed and their feces were collected. Blood samples were collected once weekly and stored at −20°C. Additionally, the ileum and spleen (n = 5/group) were collected for further analyses. The ileal tissue was processed for quantification of the L. intracellularis 16S rRNA gene. Further, the expression of genes encoding for mucin, pro- and anti-inflammatory cytokines, the vitamin B12 transporter and Lawsonia chaperonin GroEL. The spleen was processed for profiling of the T-cell population.

Evaluation of L. intracellularis-specific genomic DNA

DNA was extracted from feces with Exgene Stool DNA Mini Kit (GeneAll Biotechnology, Korea), following the manufacturer’s instructions. The L. intracellularis 16S rRNA gene was amplified using the primers listed in Table 1. The PCR mixture was calibrated using a known number of L. intracellularis and performed as described elsewhere [18]. A negative result was assigned if no amplification ensued or for a threshold cycle greater than 36. The reactions were performed in triplicate for each sample.

Table 1

The primers used in this study

Gene	Primer sequence	Reference
16S rRNA	FP: 5′-GCGCGCGTAGGTGGTTA-3′	This study
	RP: 5′-ATGCTTGGTTGTATTCTCT-3′
GroEL (Hsp60)	FP: 5′-ACTAAGCGACATTACAAAGCC-3′	This study
	RP: 5′-CAAACTTCATTCCTTCAACCAC-3′
Cubilin (intrinsic factor cobalamin receptor)	FP: 5′-CCGTGTTCTATTCCTCAG-3′	This study
	RP: 5′-ATGCTTGGTTGTATTCTCT-3′
Mucin 2	FP: 5′-GCTGACGAGTGGTTGGTGAATG-3′	This study
	RP: 5′-GATGAGGTGGCAGACAGGAGAC-3′
Mucin 5AC	FP: 5′-CACACACAACCACTCAACC-3′	This study
	RP: 5′-TCACACTTCACCACCTGAC-3′
Mucin 1	FP: 5′-GCAGTCCTCAGTGGCACCTC-3′	This study
	RP: 5′-CACCGTGGGCTACTGGAGAG-3′
Mucin 4	FP: 5′-AACCTCAAACCACCACAAC-3′	This study
	RP: 5′-TGTGTGTCCTGAGTCTACTG-3′
Mucin 12	FP: 5′-TCATCACCATAGAAACCCCAG-3′	This study
	RP: 5′-GTCACACCCCAATAAGCAAG-3′
Mucin 13	FP: 5′-CGGGAACAGGAAGTGTGAAG-3′	This study
	RP: 5′-CAGCAAGATGAGGATGAGGG-3′
IL-2	FP: 5′-CCTGAGCAGGATGGAGAATTACA-3′	[18]
	RP: 5′-TCCAGAACATGCCGCAGAG-3′
TNF-α	FP: 5′-CATCTTCTCAAAATTCGAGTGACAA-3′	This study
	RP: 5′-TGGGAGTAGACAAGGTACAACCC-3′
IL-6	FP: 5′-GAGGATACCACTCCCAACAGACC-3′	This study
	RP: 5′-AAGTGCATCATCGTTGTTCATACA-3′
IFN-γ	FP: 5′-TCAAGTGGCATAGATGTGGAAGAA-3′	This study
	RP: 5′-TGGCTCTGCAGGATTTTCATG-3′
IL-4	FP: 5′-ACAGGAGAAGGGACGCCAT-3′	This study
	RP: 5′-GAAGCCCTACAGACGAGCTCA-3′
CXCL1	FP: 5′-GCTTGAAGGTGTTGCCCTCAG-3′	[19]
	RP: 5′-AAGCCTCGCGACCATTCTTG-3′
IL-10	FP: 5′-GGTTGCCAAGCCTTATCGGA-3′	This study
	RP: 5′-ACCTGCTCCACTGCCTTGCT-3′
TGF-β	FP: 5′-CCGCATCTCCTGCTAATGTTG-3′	This study
	RP: 5′-AATAGGCGGCATCCAAAGC-3′

FP, forward primer; RP, reverse primer.

Anti-L. intracellularis antibody

Antibody against L. intracellularis was developed in-house using the bacterial whole cell lysate (WCL). The antibody was raised in New Zealand white rabbit. Rabbit was injected subcutaneously with WCL mixed with equal quantity of Freund’s complete adjuvant. A booster dose was administered 15 later using Freund’s incomplete adjuvant. Two weeks post-booster, sera sample was collected from the rabbit and used in the subsequent experiments.

Western blot analysis

Total protein (20 µg) extracted from mouse ileal tissue was separated on a 10% sodium dodecyl sulfate polyacrylamide gel, transferred to 0.45 µm pore size nitrocellulose membranes (Bio-Rad, USA) The primary anti- L. intracellularis antibody at a dilution of 1:500 was used to detect the immunoreactivity. An horseradish peroxidase (HRP)-conjugated anti-rabbit-IgA antibody (1:6,000; Southern Biotech, USA) was used as secondary antibody. The membranes were developed with DAB substrate and images were documented.

Protein digestion and mass spectrometric analysis

The uppermost immunodominant band was excised from the gel and digested as previously described [19]. Briefly, the excised gels were destained in 30 mM potassium ferricyanide and 50 μL of 100 mM sodium thiosulfate, dehydrated with acetonitrile (ACN), and finally dried in a vacuum centrifuge for 30 min. The gels were reswollen with 5 μL of 25 mM ammonium bicarbonate containing 10 ng of trypsin at 4°C for 30 min and then digested overnight at 37°C. After tryptic digestion, peptides were extracted three times with 50% ACN containing 5% formic acid. The extracted solutions were pooled and lyophilized. The dried peptide was dissolved in 2 μL 0.1% (vol/vol) trifluoroacetic acid and used for matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analyses. Mass spectra were prepared using an Ettan MALDI-TOF Pro mass spectrometer (GE Healthcare, USA).

Immunohistochemistry assay

The intestinal tissue samples collected and processed by Swiss rolling method followed by fixation for formalin fixation and paraffin embedding [20]. The sections were incubated overnight at 4°C with the primary anti-L. intracellularis antibody at a dilution of 1:500. The sections were then incubated for 1 h with HRP conjugated secondary antibody followed by 10 min with metal enhanced DAB substrate working solution. Percent positive area was quantified by Image J analysis (National Institutes of Health, USA).

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

Expression of mucin, cytokine and virulence-associated gene transcripts in control and infected ileal tissues was measured using qRT-PCR [21]. The quantification of target genes was assessed relative to β-actin expression using the 2−ΔΔCT method [22]. The relative expression of the target genes was quantified and compared at each time point using the primers listed in Table 1.

Serological evaluation of the immune response

An enzyme-linked immunosorbent assay (ELISA) was employed to determine the L. intracellularis-specific IgA and IgG immunoglobulin response, as described previously [23]. Briefly, L. intracellularis bacteria were heated for 5 min at 95°C and centrifuged at 1.2 × 104 g for 60 sec. The supernatant was discarded and the pellet was resuspended in PBS. The wells of ELISA plates (Greiner, Germany) were coated with 100 μL of heat-killed L. intracellularis bacteria (4 mg/mL) in 100 mM sodium carbonate (pH 9.7). The L. intracellularis-specific IgA was detected using serum from control and test mice and HRP-conjugated goat-anti-mouse antibodies (Southern Biotech) and measured using an automated ELISA spectrophotometer (Tecan, Austria) at 492 nm.

Flow cytometry immunophenotyping of T cell populations

Splenocytes harvested from the mice were analyzed for viability by diluting the samples in 0.4% trypan blue solution at a 1:1 ratio. Then, 3 × 106 cells per tube were stained for cell surface CD3, CD4, and CD8 markers using the following monoclonal antibodies: PE-labeled anti-CD3e, PerCPVio700-labeled anti-CD4, and FITC-labeled anti-CD8a (Miltenyi Biotec, Germany). Cells were gated on the basis of their forward and side scatter profiles.

Statistical analyses

The data are presented as the mean of the results obtained from 4 or 5 mice per group ± standard deviations. The statistical analyses were performed using a two-tailed paired t-test for weight loss and an unpaired t-test for T cell responses and mRNA transcript expression in ileal tissue.

RESULTS

Assessment of an in vivo infection model using C57BL/6 mice

The average body weight of C57BL/6 mice administered 5 × 107 L. intracellularis by gavage was monitored and recorded compared to control mice receiving PBS. A weekly gain in body weight was recorded in the control group. In contrast, L. intracellularis infection experienced reduced weight gain in the infected group (Fig. 1A). Furthermore, to confirm L. intracellularis colonization of the intestine, the copy number of the L. intracellularis-specific 16S rRNA gene in feces was evaluated using quantitative PCR. The presence of L. intracellularis 16S rRNA gene copies in the ileal tissue of the infected mice (Fig. 1B) confirmed the ability of the bacteria to invade the host ileum. Furthermore, the infected mice were found to shed up to 50 L. intracellularis in feces on Day 3 post-infection, and by Day 15 post-infection up to 1,600 L. intracellularis 16S rRNA gene copies/g feces were recovered from the mice (Fig. 1C).

Fig. 1

Preliminary assessment of an in vivo L. intracellularis infection model in C57BL/6 mice. (A) The average body weight was recorded daily within 5 min of first contact for 4 or 5 mice per group. (B) Each week post-infection, mice (n = 3/group) were sacrificed and the ileum was collected to evaluate the average number of L. intracellularis 16S rRNA genes per gram of ileal tissue. (C) The feces were collected from post-infection Day 0 up to Day 32. The data presented are quantitative polymerase chain reaction analyses of the average number of L. intracellularis 16S rRNA genes per gram of feces for 4 or 5 mice per group per day. The data represent one of two independent experiments.

ns, not significant.

Significant differences are depicted as follows: nsp > 0.05; *p < 0.05; **p < 0.01.

Differential regulation of bacterial and host genes in the infected ileum

The expression of the genes for the Lawsonia heat shock protein 60 (Hsp60) and the host vitamin B12 receptor called cubilin was evaluated in ileal samples from infected mice; Hsp60 expression revealed that L. intracellularis was successful in establishing infection in C57BL/6 mice intestine. A gradual increase in mRNA transcripts for the Hsp60 was evident at 1 wk post-infection that continued up to 5 wk (Fig. 2A). After infection with L. intracellularis, the host cubilin gene was downregulated (Fig. 2B). Reduction of the vitamin B12 receptor may contribute to poor absorption of this essential vitamin during L. intracellularis infection. The Fig. 3 provides representative images of healthy control and diseased intestinal tissue with hemorrhagic congestion following L. intracellularis infection. Further, analysis of infected mouse intestine by H & E staining revealed crypt hyperplasia and damage to villi architecture (Fig. 3C and D).

Fig. 2

Determination of bacterial and host gene expression in the ileum. Every week post-infection, mice (n = 3/group) were sacrificed and the ileum was collected to evaluate the expression of mRNA transcripts for (A) GroEL and (B) cubilin in the infected mice, and to compare the results with the outcomes in the control group. The data represent one of two independent experiments and are depicted as means ± SE.

ns, not significant.

Significant differences are depicted as follows: nsp > 0.05; *p < 0.05; **p < 0.01.

Fig. 3

The L. intracellularis infected intestine compared to the control. C57BL/6 mice infected with L. intracellularis causes hemorrhagic lesions throughout the intestine with pronounced damage to the caecum (B). The gross pathological lesions are shown in the representative image for L. intracellularis infected mice intestine (B) compared to the control (A). H & E stained intestinal tissue from control (C) and infected (D) mice have been shown. Note the crypt hyperplasia (yellow arrows) and loss of villi architecture (black arrowheads) in L. intracellularis infected mice intestine.

Detection of L. intracellularis in ileal tissue by immunohistochemistry

The presence of L. intracellularis in paraffin-wax-embedded ileal tissue sections was detected using antibody specific for L. intracellularis. The immunohistochemistry assay demonstrated the presence of L. intracellularis bacteria in the apical cytoplasm of hyperplastic immature enterocytes (Fig. 4). Ileal sample isolated from mice at 1 wk post-infection revealed L. intracellularis localization. Greater immunoreactivity corresponding to L. intracellularis presence was observed in the ileal samples collected 2 and 3-wk post-infection.

Fig. 4

Immunohistochemical staining of L. intracellularis in the ileum of 7-wk-old mice. The representative images demonstrate the L. intracellularis localization in the apical cytoplasm as visualized with peroxidase staining. The tissues were counterstained with methyl green. The ileal samples with dark brown positive staining (pink arrowheads) illustrate the presence of L. intracellularis in the tissue. Percent positive area from Image J analysis has been shown in the right panel as means ± SE. The data representative of 5 mice per group from two independent experiments. Data was analyzed by analysis of variance with Tukey’s multiple comparisons test.

****p < 0.0001.

Western blot analysis detected L. intracellularis proteins in the mouse intestine

To substantiate the presence of L. intracellularis in the intestines of infected mice, total proteins collected from the ileum of control and inoculated mice were separated on a polyacrylamide gel and blotted against L. intracellularis-antibody. Fig. 5A demonstrates the protein components of different molecular weights as determined using a wide-range Biofact marker kit. The antibody recognized three distinctive protein bands of L. intracellularis. The recognition of the proteins at approximately 25, 35, and 55 kDa was taken as a demonstration of L. intracellularis infection in the inoculated mice. Western blot analyses revealed that all mice receiving L. intracellularis expressed all three immunodominant proteins. The L. intracellularis proteins were detected at 1 wk post-infection and remained detectable until 3 wk. The L. intracellularis proteins were not identified in control animals at any time point.

Fig. 5

Western blot and MALDI-TOF analyses of the L. intracellularis proteome. Protein samples were collected from the intestinal tissue of control and infected mice every week for 4 wk post-infection. (A) The presence of L. intracellularis in the intestinal tissue was confirmed by the identification of three immunoreactive bands at 25, 35 and 55 kDa. The representative images show positive immunoreactivity compared to the control group. (B) Identification results of immunodominant protein band. Protein scores (n = 126) that are greater than 57 are significant (p < 0.05). Protein scores were derived from ion scores on a non-probabilistic basis for ranking protein hits. The amino acid sequence of GroEL (a Hsp60 homologue) has been presented with matched peptides depicted in bold.

MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; M, molecular protein marker; PBS, phosphate-buffered saline.

Identification of immunoreactive proteins

The immunoreactive protein bands were excised from the polyacrylamide gel by in-gel digestion (Supplementary Fig. 1) and identified using MALDI-TOF-MS based on peptide mass matching (Supplementary Data 1). A search of the NCBI database revealed that the protein, GroEL (was homologous with L. intracellularis Hsp60, a protein with a molecular mass of approximately 60 kDa (Fig. 5B; protein scores greater than 57 were considered significant, p < 0.05).

Expression of mucin genes during L. intracellularis infection

The mRNA transcripts encoding secreted mucins 2 and 5AC, and membrane-bound mucins 1, 4, 12 and 13, in the ileum of healthy and inoculated mice, were measured using qRT-PCR (Fig. 6, < 0.05). Upregulation of all the mucin genes was recorded for 2 wks post-infection. Interestingly, significant downregulation of the transcripts for secretory mucins was observed at 3 wk post-infection (Fig. 6). This downregulation may have been due to the increased number of invading bacteria within the enterocytes. On the other hand, the mRNA expression of membrane-bound mucins was upregulated for up to 5 wk post-infection (Fig. 6).

Fig. 6

Successful infection of the mice with L. intracellularis resulted in an altered mucin barrier. The mRNA levels of muc1, muc2, muc4, muc5AC, muc12 and muc13 in ileum of healthy mice and mice infected orally with L. intracellularis for 0, 1, 2, 3, 4 and 5 wk (n = 3 mice/group). Expression of mRNA encoding secreted and mRNA membrane-bound mucins (β-actin mRNA was used as an internal standard). The data represent one of two independent experiments and are depicted as means ± SE.

ns, not significant.

Significant differences are depicted as follows: nsp > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001.

Antibody-mediated immune response

The L. intracellularis-specific IgA and IgG antibody levels within the serum were evaluated for 2 wk post-infection in the mice receiving the bacteria orally. The detection of circulating antibodies demonstrated the ability of L. intracellularis to elicit the B-cell mediated immune response in the infected mice. The production of IgA antibodies was greater than that of IgG (Fig. 7A).

Fig. 7

Humoral and cell-mediated immune responses in infected mice. (A) Evaluation of induced IgA and IgG induction in mouse sera 2 wk post-infection with 5 × 107 L. intracellularis. The results are represented as the optical density at 492 nm (OD492). The data represent one of two independent experiments and are depicted as means ± SE for 4 mice per group. (B) T-cell proliferation was evaluated in freshly isolated splenocytes incubated with fluorochrome-conjugated antibodies against T-cell surface markers (PE-CD3, FITC-CD8 and PE-Cy7-CD4) and data collected for 10,000 cells per sample. The data are presented as the mean ± SD percentage of splenocytes for 3 or 4 mice per group. The p values were determined using a two-tailed unpaired t-test.

ns, not significant.

Significant differences are depicted as follows: *p < 0.05; **p < 0.01.

Proliferation of CD4+ T-cells

Following infection with 5 × 107 bacteria, the splenocytes derived from the infected mice were subjected to flow cytometric analyses and the outcome was compared with the control group that received only PBS. The flow cytometric analyses showed an expansion of the T cell population (Fig. 7B) and a significant increase in the CD4+ subpopulation together with a reduction in the CD8+ T subpopulation.

Varied production of cytokines post-infection

The transcripts of pro-inflammatory cytokines IL-2, TNF-α, IL-6, and IFN-γ were upregulated at 7 d post-infection and continued to increase until the termination of the experiment at 5 wk post-infection (Fig. 8). The increase in the amount of transcript of pro-inflammatory cytokines appeared to be correlated with the degree of local inflammation and the severity of the enteritis [2425]. In contrast, the expression of anti-inflammatory cytokines, including IL-4, IL-10, and TGF-β, increased at 4 wk post-infection (Fig. 8).

Fig. 8

Expression of pro-inflammatory cytokines in L. intracellularis-infected ileum. The Kinetics of cytokines mRNA expression in the ileal tissues of healthy and infected mice (n = 3 mice/group) was measured by semi-quantitative real time RT-PCR by using gene specific primers. β-actin mRNA was used as an internal standard. The data represent one of two independent experiments and are depicted as the means ± SE. The p values were determined using an unpaired t-test.

ns, not significant; RT-PCR, reverse transcription-polymerase chain reaction.

Significant differences are depicted as follows: nsp > 0.05, *p < 0.05, **p < 0.01 and ***p < 0.001.

DISCUSSION

We established an experimental model of porcine L. intracellularis infection in immunocompetent C57BL/6 mice. The development of a mouse model is advantageous as it offers genetic and immunological tools to study the pathophysiology of PE in a cost-effective host. In this study, infection of seven-week-old immunocompetent C57BL/6 mice with L. intracellularis led to successful colonization of the intestine that caused severe weight loss (Fig. 1A). The invasion was confirmed by detection of L. intracellularis 16S rRNA gene copies in the ileal tissue and feces of the infected mice (Fig. 1B and C). Shedding of L. intracellularis bacteria along with significant loss of body weight from 7 d post-infection until the termination of the study indicated colonization of the intestine. The features of L. intracellularis infection in C57BL/6 mice were similar to those seen in porcine PE, wherein PCR detection of L. intracellularis in feces occurs from 2 to 38 d post-infection [26]. As a potential reservoir, mice may play a vital role in the epidemiology of PE [27]. In certain strains, such as ICR mice, the L. intracellularis-induced lesions were more severe than in other strains of mice [28]. Although extensive research has indicated L. intracellularis infection of mice occurs, there is limited information regarding the pathogenesis of infection, the interactions between bacterial and mouse tissues, or regarding the immune response during infection [15]. The upregulated expression of L. intracellularis groEL (Fig. 2A) in the ileum of infected mice indicated that L. intracellularis bacteria regulate the expression of chaperonin during the initial stages of colonization. GroEL (also known as Hsp60) is a stress response protein that promotes refolding and proper assembly of polypeptides and undergoes changes in cellular localization from cytosol to cell surface making it possible immunogen [2930]. The upregulation of groEL expression was associated with the presence of multiple copies of the L. intracellularis 16S rRNA gene in the mouse ileum. Excessive bacterial colonization of the small intestine, including the ileum, leads to impaired absorption of vitamin B12 [31]. Downregulation of the vitamin B12 receptor gene, cubilin in infected mice may have contributed to the severe weight loss due to malabsorption of vitamin B12 (Fig. 2B). The precise localization of L. intracellularis in ileum was confirmed using an immunohistochemistry assay (Fig. 4). The degree of positive immunoreactivity was consistent with the expression of cubilin mRNA. Additionally, western blot and MALDI-TOF analyses confirmed the presence of the L. intracellularis proteome in the ileal region of the small intestine (Fig. 5).

Bacteria using GroEL binds to host mucin to initiate colonization [30]. The pathogenic bacteria in the intestine are shown to alter the protective mucin barrier to enable better invasion and dissemination [32]. In this study, L. intracellularis-infection-mediated regulation of mucin genes was evident as early as at the first week post-infection. The mRNA expression of secreted mucins 2 and 5AC peaked at 2 wk post-infection (Fig. 6) as a result of an inflammatory response to the invading bacteria. Any insult to the epithelium causes upregulation of mucin genes and increased secretion of mucus to counter the pathogen [33]. Although expression of all the membrane-bound mucin genes (i.e., 1, 4, 12 and 13) continued to increase throughout the period of infection, significant downregulation of mucin 2 and 5AC was observed beginning at 3 wk post-infection. This downregulation of expression of the genes for secreted mucins may have been due to a specific inhibitory effect exerted by L. intracellularis [21]. In addition to hypersecretion of mucus, the host secretes immunoglobulins to inhibit pathogen invasion. Increased serum IgA and IgG in the infected mice two weeks post-infection was consistent with an L. intracellularis-specific acquired humoral immune response in the mice (Fig. 7A) [34]. The comparatively lower IgG titers revealed that the production of IgA was essential during L. intracellularis infection and IgG plays a minor role during the development of enteropathy [35]. Bacterial-infection-associated damage to the gut epithelium elicits a T-cell mediated immune response [3637]. Here, the flow cytometry results suggested the cellular expansion was due to CD3+/CD4+ cells (Fig. 7B). The expansion of CD4+ cells is essential to induce early inflammatory responses in tissues and contributes to upregulation of cytokines (Fig. 8). The increased levels of TNF-α, IL-6 and IFN-γ in the infected mice suggested the potential for L. intracellularis bacteria to induce mucosal proliferation and necrosis. A similar response is seen in natural hosts during L. intracellularis infection, wherein acute intestinal hemorrhage or acute to chronic diarrhea with enterocyte hyperplasia are evident [38]. The upregulation of anti-inflammatory cytokines was observed during the later stages of the infection in the C57Bl/6 mice. Thus, tight regulation of the Th1- and Th2-mediated immune response against L. intracellularis seems crucial as it may determine the outcome of the disease. An increased Th1 response may lead to an acute form of the infection, whereas a predominantly Th2 response may lead to a chronic form of enteritis [5]. The ability of L. intracellularis to modulate the expression of its surface antigen may also influence the early stages of PE pathogenesis. The results of this study indicated L. intracellularis infection altered the expression of nutrient uptake genes, altered the intestinal protective barrier and elicited B- and T-cell mediated immune responses to cause the disease.

In conclusion, we showed that the C57BL/6 mice may serve as an in vivo model of infection to study L. intracellularis pathogenesis. The mice inoculated with the porcine variant of the bacteria showed signs of infection, and increased numbers of bacteria within ileal tissue and in the feces. The upregulation of groEL significantly contributed to the colonization of bacteria in the small intestine of infected mice. Bacterial proliferation in the small intestine caused downregulation of cubilin and altered the protective mucin barriers. Furthermore, the infection elicited B- and T- cell mediated immune responses in the host mice, contributing to inflammatory cytokine production. Overall, this study shows that the C57BL/6 mice are susceptible to L. intracellularis infection and provides a plausible mechanism of the pathogenesis of PE. The successful establishment of L. intracellularis infection in mice provides a cost-effective system to continue investigation of the regulatory mechanisms involved in PE and to develop novel therapeutic targets to treat PE and the associated intestinal hyperplasia.