Down-regulation of mechanisms involved in cell transport and maintenance of mucosal integrity in pigs infected with Lawsonia intracellularis
© Smith et al.; licensee BioMed Central Ltd. 2014
Received: 15 October 2013
Accepted: 22 April 2014
Published: 20 May 2014
Lawsonia intracellularis is an obligate intracellular bacterium, responsible for the disease complex known as proliferative enteropathy (PE). L. intracellularis is associated with intestinal crypt epithelial cell proliferation but the mechanisms responsible are yet to be defined. Microarray analysis was used to investigate the host-pathogen interaction in experimentally infected pigs to identify pathways that may be involved. Ileal samples originating from twenty-eight weaner pigs experimentally challenged with a pure culture of L. intracellularis (strain LR189/5/83) were subjected to microarray analysis. Microarray transcriptional signatures were validated using immunohistochemistry and quantitative real time PCR of selected genes at various time points post challenge. At peak of infection (14 days post challenge) 86% of altered transcripts were down regulated, particularly those involved in maintenance of mucosal integrity and regulation of cell transport. Among the up-regulated transcripts, CD163 and CDK1 were novel findings and considered to be important, due to their respective roles in innate immunity and cellular proliferation. Overall, targeted cellular mechanisms included those that are important in epithelial restitution, migration and protection; maintenance of stable inter-epithelial cell relationships; cell transport of nutrients and electrolytes; innate immunity; and cell cycle.
Lawsonia intracellularis is an obligate intracellular, Gram negative bacterial pathogen that causes the disease complex known as proliferative enteropathy (PE). In terms of economic impact this disease is primarily a problem for the pig industry but it also occurs in several other species, though much more sporadically [1–4]. In weaner and grower pigs, clinical signs consist of non-haemorrhagic diarrhoea, ill-thrift and reduced growth rates but in older pigs the consequences of infection can include fulminant intestinal haemorrhage and death [5, 6]. In severe cases, PE is characterised by macroscopically obvious thickening of the intestinal mucosal lining which is due to proliferation of epithelial cells which line intestinal crypts, mainly in the distal ileum, though not restricted to it [7, 8]. There is a consistent close association between this proliferation and the intracytoplasmic presence of L. intracellularis in the hyperplastic crypt epithelial cells .
Koch’s postulates were proven in the 1990s and subsequent research has pursued diverse paths, ranging from the development of molecular-based diagnostic tests to the evaluation of various control and treatment options [10–14]. Despite this progress, important mechanistic questions remain unanswered, including the cause of the pathognomonic proliferative lesion and why there are such distinct clinical manifestations of the disease in two different age-groups. There has been limited exploration of the host/pathogen interaction at the cellular level, probably due to the fact that L. intracellularis has such rigorous growth requirements [15, 16]. The relationship between L. intracellularis and the associated crypt proliferation seems to be unique and its potential value as a model for studying the cell cycle, and even the molecular pathways that ultimately lead to cancer, has been recognised for some time [17, 18]. Initial analyses of host transcriptional responses to infection using microarrays or RNA-seq support the theory that cell cycle proteins and growth factors are altered in cells infected with L. intracellularis[16, 19, 20]. A number of bacterial pathogens have been associated with cellular proliferation and have been discussed in this context previously [16, 21]. One of the main goals of the study reported here was to identify changes in gene expression that might shed some light on host-pathogen interactions occurring in PE. The particular strengths of this work were the analysis of transcript alterations in pigs at several time points following experimental infection with a known strain of cultured L. intracellularis, using a comprehensive microarray platform capable of monitoring 47 000 porcine transcripts.
Materials and methods
Samples of frozen ileum originated from a previous challenge study . Briefly, 28 seven-week-old pigs were randomly selected from a minimal-disease herd, penned in seven groups of four pigs each and tested for various enteric pathogens. Faecal samples from all animals were culture negative for Brachyspira hyodysenteriae, B. pilosicoli, Yersinia spp. and Salmonella spp., and PCR negative for L. intracellularis[22, 23]. All pigs were also serologically negative for L. intracellularis. The pigs were challenged orally with a pure culture of L. intracellularis (isolate LR189/5/83), euthanased and subjected to a full necropsy at 3, 7, 14, 21, 28, 35 or 42 days post challenge (dpc), with four pigs per time point. A full-thickness sample of ileum was collected from each pig, snap frozen at -95 °C using isopentane and dry ice, fixed to a cork disk with optimal cutting temperature compound and stored at -80 °C. The results have been described fully by MacIntyre et al. . For the current study a separate group of three uninfected age-matched pigs was used as controls to provide base line data for the gene expression analyses.
Extraction of total genomic DNA from ileum
Total genomic DNA was extracted from ~100 mg of ileum from 24 pigs using a standard salt extraction method incorporating proteinase K. The quantity of each DNA sample was assessed using a Nanodrop spectrophotometer (NanoDrop Technologies Inc, Wilmington, DE, USA) and the quality was assessed using agarose gel electrophoresis.
Quantification of L. intracellularis-specific genomic DNA
In order to allow measurement of L. intracellularis-specific genomic DNA in the total genomic DNA samples, a standard curve for quantitative real time PCR (qPCR) was constructed using a pGEM®-T Vector plasmid which contained a 322 bp L. intracellularis ribosomal 16S rRNA gene insert . Serial dilutions of the amplified plasmid were analysed by qPCR using the following primers as previously described : Forward primer, 5′-GCGCGCGTAGGTGGTTA-3′; reverse primer, 5′-GCCACCCTCTCCGATACTCA-3′ and platinum SYBR Green PCR SuperMix UDG (Invitrogen, Paisley, UK). The thermocycling profile used on a Stratagene Mx3000 was as follows: 50 °C for 2 min, 95 °C for 2 min, 40 cycles of 95 °C for 15 s and 60 °C for 30 s. The profile of the final cycle was 95 °C for 1 min, 60 °C for 30 s and 95 °C for 15 s. The standard curve was used to estimate the L. intracellularis-specific genomic DNA concentration using DNA of sufficient quantity and quality from the ileal samples (24 of the 28 pigs) and the concentration was expressed as number of 16S rRNA copies per ng of DNA.
Total RNA was extracted from the ileum of 20 pigs using Trizol (Invitrogen, Paisley, UK) according to standard methods . The RNA was cleaned using the Qiagen RNeasy minikit following the manufacturers’ instructions (Qiagen, Crawley, UK). RNA was eluted from the spin column in 30 μL of RNase-free water and stored as aliquots at -80 °C. The quantity and quality of RNA were assessed using a Nanodrop spectrophotometer (NanoDrop Technologies Inc, Wilmington, DE, USA) and Agilent 2100 bioanalyser (Agilent Technologies, Palo Alto, CA, USA).
Quantitative real time PCR validation
The differential expression of several selected genes, as identified from the microarray data, was verified at various time points using qPCR. Reverse transcription was performed as described previously [26, 27]. Briefly, one microgram of total RNA was reverse transcribed using a TaqMan kit (Applied Biosystems, Foster City, CA, USA). For qPCR, Platinum SYBR Green PCR SuperMix UDG was used, as described above. The qPCR was performed with a Stratagene MX3000P (Stratagene, La Jolla, CA, USA). Samples were tested in triplicate, GAPDH served as the housekeeping gene and results were calculated as described previously [26, 27]. Primers used are listed in Additional file 1.
Microarray platform and data analysis
To assess if host transcriptional responses were affected, the L. intracellularis-infected ileal tissues described above (3, 7, 14, 21, 28 and 42 dpc) were analysed using the Affymetrix Snowball GeneChip® . Sense-strand cDNA was generated from total RNA (500 ng) and subjected to two rounds of amplification (Ambion® WT Expression Kit). The resulting cDNA was used for biotin labelling and fragmentation according to the Affymetrix GeneChip® WT Terminal Labelling and Hybridization protocol (Affymetrix UK, High Wycombe). Biotin-labelled fragments of cDNA (5.5 μg) were hybridized to Affymetrix SNOWBALL arrays using the Affymetrix HybWashStain kit and manufacturer’s recommendations. After hybridization, the arrays were washed and stained using the Affymetrix Fluidics Station 450 and then scanned in an Affymetrix 7G scanner. Image generation and the resulting CEL files for analysis were produced in AGCC – Affymetrix GeneChip Command Console Software. Initial QCs were performed in Expression Console. All microarray data used in the analyses herein are freely available from the Array Express repository under the accession number E-MTAB-1396 . The Affymetrix.CEL files were imported into the Genomics Suite software package version 6.13.0213 (Partek software, Partek Inc.) for data analysis. Transcriptional responses were normalised to those from age-matched uninfected pig ileum prior to running an ANOVA analysis of the data. Up-regulated and down-regulated differentially expressed transcripts at each dpc were selected for further consideration if the false discovery rate (FDR) was ≤ 0.1.
Gene ontology and pathway analysis
Sections of formalin fixed ileum from all four pigs euthanased at 14 dpc, three pigs euthanased at 42 dpc and one uninfected control pig were stained immunohistochemically to detect CD163 antigen expression. Briefly, after antigen retrieval with proteinase K (Dako UK Ltd., Ely, UK) for 10 min at room temperature, endogenous peroxidase activity was blocked using a commercial blocking agent for 10 min (REAL™ peroxidase blocking agent, Dako UK Ltd., Ely, UK S202386). Following serial washes, sections were incubated with mouse-anti-pig CD163 monoclonal antibody (Serotec Ab MCA2311) diluted 1:30 in Tris buffered saline Tween at pH 7.5. They were incubated with labelled polymer for 40 min at room temperature (Envision mouse HRP reagent, Dako K4001), treated with 3, 3′-diaminobenzidine and counterstained with haematoxylin. Unrelated porcine ileum containing a known macrophage population as defined by a previous study served as a positive control . For each section, the density of immunopositive cells was calculated using image analysis (Olympus Soft Imaging System, Münster, Germany). For each of six randomly selected high power fields (400×) a field of interest was outlined, consisting only of lamina propria, and its area calculated. The number of CD163-positive cells within that specified area was counted per high power field. An aggregate density was calculated for each pig by dividing the total number of CD163 positive cells by the total area examined. A Kruskal-Wallis test was used to compare the aggregate densities for the pigs in the 14 dpc group with those in the 42 dpc group. Assessment of these particular time points allowed comparison of the density of CD163 positive cells at peak infection with the density when infection had virtually resolved. The CD163-positive cell density at 14 dpc was also tested for evidence of a direct correlation with infection burden, as measured by qPCR of L. intracellularis genomic DNA. This time point was selected as it corresponded with the highest fold alteration in CD163 gene expression in the microarray analysis.
Quantification of L. intracellularis bacterial load
Host transcript regulation during L. intracellularis infection
Summary of transcript changes at various time points post challenge (pc)
Time pc (days)
Number of up-regulated genes
Number of down-regulated genes
Total number of transcripts changed
Validation of differentially regulated transcripts using quantitative real time PCR
Comparison between microarray and quantitative real time PCR assays
Days post infection
Gene ontology (GO) and Ingenuity pathway analysis (IPA)
Altered cellular networks at 14 dpc identified using ingenuity pathway analysis
Cancer, Gastrointestinal Disease, Cellular Function & Maintenance
Lipid Metabolism, Molecular Transport, Small Molecule Biochemistry
Cellular Function & Maintenance, Developmental Disorder, Endocrine System Disorders
Cell Morphology, Organ Morphology, Molecular Transport
Endocrine System Disorders, Reproductive System Disease, Developmental Disorder
Energy Production, Lipid Metabolism, Small Molecule Biochemistry
Gastrointestinal Disease, Inflammatory Disease, Inflammatory Response
Cell-To-Cell Signalling and Interaction, Cell Signalling, Behaviour
CD163 regulation during L. intracellularis infection
Only three previous studies have focused on host gene expression in the context of PE [16, 19, 20]. The first of these studies mapped transcript changes over three time points using cultured mouse McCoy fibroblast-like cells as in vitro hosts . This mesenchymal cell line is superior to others in its capacity to generate large numbers of L. intracellularis bacteria but extrapolation of its expression profile to the porcine crypt enterocyte (the natural target cell) may not be reliable [15, 34, 35]. The three time points were also assessed much earlier than the time of peak lesion development in experimentally infected pigs [21, 36]. Nevertheless, that study provided valuable baseline data pertaining to early bacterial invasion and indicated that, even at this stage of infection, there was altered transcription of genes involved in cell cycle control.
The second study used intestinal tissues from field cases of pigs with diarrhoea to investigate cytokine expression, also using microarray methodology . This provided whole animal information, something that cell culture models cannot, but the study was confounded by co-infection with porcine circovirus type 2 (PCV2) and, since diseased pigs were field cases, the findings were not correlated with stage of infection.
The most recent study introduced the use of laser capture microdissection in combination with RNA-seq analyses, allowing specific analysis of infected intestinal crypts from pigs experimentally challenged with a pure culture of L. intracellularis (strain PHE/MN1-00), negating any risk of confounding enteric infections. This novel approach facilitated the isolation of the cell of interest (crypt enterocyte) from the natural host, enabling a glimpse of the transcriptional alterations that may result from a combination of host and pathogen effects. Compared to that study, in which analysis was performed at a single time point coinciding with peak of infection, our own study examined multiple time points through initiation, peak and resolution of lesions. Specifically, we measured fold changes in gene expression for six weeks following challenge, allowing correlation with the previously described morphological lesions and level of infection in the same cohort of pigs . In contrast to Vannucci et al. , we used a well-characterised comprehensive microarray platform which comprises over 47 000 probesets . We also extended the interrogation to surrounding host tissues, specifically the intestinal lamina propria, where immune responses in particular are likely to be manifest.
Quantitative real time PCR analysis of L. intracellularis-specific genomic DNA detected peak infection at 14 dpc, correlating with previous reports [21, 22, 36]. There were some differences in the estimates of the infection burden between our qPCR data and the immunohistochemistry data reported by MacIntyre et al. . Our qPCR assays detected infection in 80% of pigs tested between 21 and 35 dpc, whilst MacIntyre et al.  confirmed infection in only 25% of pigs tested at similar time points, using IHC. Since we tested tissues from the same cohort of pigs as MacIntyre et al. , the difference in detection rate cannot be due to differences in bacterial strain or source of pig. Rather, it is more likely that qPCR is more sensitive than IHC for detection of infection in frozen tissue. There is also accepted variation between PCR and immunologically based tests that further depends on whether tissue, faeces or blood are tested [37, 38]. Different depths within the tissue block were also used for DNA extraction and IHC, which may have contributed to variability.
We found statistically significant changes in transcript levels for a total of 218 separate genes. The assumption that any cellular changes triggered by L. intracellularis are maximally expressed at time of peak infection burden (14 dpc in this study) was substantiated by fold changes that reached their maximum at this time point in most measured transcripts. At 14 dpc there were changes in 185 transcripts, 86% of which were down-regulated, a trend similar to that observed by Jacobson et al. . This low number of altered transcripts is surprising, particularly as we used a highly comprehensive method that should have enhanced our ability to detect changes in gene expression . One possible reason for this low detection level is individual host variation, intensified by the fact that the disease is unlikely to progress at exactly the same rate in different animals, as indicated in Figure 1, where there is variability in the accumulation of L. intracellularis 16S rRNA. The only way to overcome this using the same model would be to increase the number of pigs in each group, which is not realistic with a large animal model such as the pig. A more speculative reason is that the pathogen has a low impact on the host due to co-evolution or because it acts via a limited number of pathways that still have regulatory consequences for the infected cell. Further investigations of gene expression would help to determine whether this low level of transcriptional alteration and the predominance of down-regulated genes are genuine phenomena. Comparison of gene expression in intestinal tissues from pigs infected with a more aggressive pathogen, such as Salmonella or Clostridium spp., may also improve understanding in this regard.
Comparison of solute carrier family gene expression changes between this study and Vannucci et al.
Vannuci et al.
solute carrier function
tissue-specific expression with BIOGPS
solute carrier family 10 (sodium/bile acid cotransporter family), member 2
solute carrier family 13 (sodium/sulfate symporters), member 1
solute carrier family 15 (oligopeptide transporter), member 1
solute carrier family 26, member 3
solute carrier family 30, member 10
solute carrier family 31 (copper transporters), member 1
solute carrier family 5 (sodium/glucose cotransporter), member 1
early intestine and bladder
solute carrier family 5 (sodium/glucose cotransporter), member 9
solute carrier family 6 (neurotransmitter transporter, serotonin), member 4
solute carrier family 7 (cationic amino acid transporter, y + system), member 9
solute carrier family 7 (amino acid transporter)
solute carrier family 2 (glucose tranporter)
The apparent ability of L. intracellularis to impede the maturation of crypt enterocytes is one of the most intriguing aspects of PE, more so because it is reversible . However, underlying mechanisms of pathogenesis and resolution remain undefined. McOrist et al.  speculated that L. intracellularis could influence genes controlling differentiation and the first support for this hypothesis was provided by Oh et al.  who reported differential expression of several genes involved in cell cycle, cellular differentiation, apoptosis and signal transduction. Up-regulation of one such gene, IGFBP3 (insulin-like growth factor binding protein 3), has been described by two separate and quite different PE studies [19, 20]. In our study, cellular proliferation was one of the functional classes where transcriptional alterations were infrequent, with only one gene that is directly involved in cell cycle regulation, CDK1, up-regulated. CDK1 drives the cell through the G2 phase of the cell cycle, which immediately precedes mitosis . A link between altered CDK1 expression and PE has not been previously reported. Vannucci et al.  described up-regulation of CDK2-associated protein 1 (CDK2AP1), a growth suppressor that down-regulates CDK2 . Since CDK2 has an effect on the cell cycle that is broadly similar to CDK1, it seems counter-intuitive that a CDK2 suppressor protein is up-regulated in PE. This merits further exploration. We detected 5-fold down-regulation in the transcription of HEPACAM2, a gene encoding hepacam which is an immunoglobulin-like cell adhesion molecule purported to be a tumour suppressor . HEPACAM down-regulation has been reported in several human cancers and is believed to function by down-regulating c-myc and cyclin D1 [47–50]. Thus, reduced transcription of HEPACAM2 associated with L. intracellularis infection could effectively “release the brakes” on c-myc and cyclin D1, leading to increased transcriptional activation and cellular proliferation. This is also an area worthy of deeper investigation.
In the context of local immunity, some of our findings are comparable with previous studies, such as the up-regulation of SLA-3 (encodes MHCI), and the down-regulation of genes encoding elements of the CD3 T-cell receptor [16, 22]. This apparent immunosuppression has been borne out by Jacobson et al.  who found limited expression of serum and tissue cytokines in natural cases of PE. Several previous studies have described macrophages in the lamina propria of infected pigs, often containing L. intracellularis organisms and sometimes peaking with maximum proliferation and burden of infection [10, 22, 50–53]. In our study, CD163 up-regulation at 14 dpc correlated with these previous results, although we could not convincingly confirm this immunohistochemically in tissues, possibly due to low pig numbers.
To conclude, this microarray-based study progresses the growing literature base that has more recently focused on the pathogenesis of PE, particularly at the molecular level. It provides evidence to support disruption of cell transport and mucosal integrity in pigs infected with L. intracellularis. Perhaps most interestingly, it has identified CDK1 and hepacam as potentially important molecules capable of influencing cellular proliferation in infected pigs.
SHS, NM and IVE were supported by funding from the Edinburgh Campaign and the ISG. ADW, ALA and TAA were supported by BBSRC Institute Strategic Programme grants. We are indebted for the help provided by ARK-Genomics (now part of Edinburgh Genomics ) for the microarray hybridizations and support for the data analysis using Partek Software. We are also grateful to Professor David G.E. Smith (University of Glasgow & Moredun Research Institute) and Dr Michelle Sait (Moredun Research Institute) for the 16S rRNA pGEM®-T plasmid and to Dr Ian Handel of the Royal (Dick) School of Veterinary Studies for his contributions to the statistical analysis. Finally, we also thank Professor David G.E. Smith for critical reading of the manuscript.
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