Human enteroendocrine cell responses to infection with Chlamydia trachomatis: a microarray study

Enteroendocrine cells (EEC) are present in the gastrointestinal (GI) tract (mucosa of the stomach, small intestine, colon, and rectum) and are highly specialized cells that produce hormones and other signalling substances that are vital to the normal function of the gut. Due to their diffuse localisation, EEC are difficult to study and so far, their role in the pathogenesis of bowel disorders has been only little explored. EEC play a pivotal role in sensory signalling for the enteric reflex regulating intestinal propulsive motility, secretion, and visceral hyperalgesia [1]. Alteration of endocrine cell function, particularly in the context of serotonin (5-HT), has been shown to be associated in a number of GI diseases including inflammatory bowel disease (IBD), coeliac disease and irritable bowel syndrome (IBS) [2‐4]. The association between alterations in the production of gut hormones from EEC and various GI diseases emphasizes the significance of these cells in intestinal homeostasis.

EEC can be activated by bacterial ligands like lipopolysaccharide LPS [5] and may play a role in immune activation and in the regulation of gut inflammation. Altered endocrine function of the gut accompanies an inflammatory immune response [6]. Recently, our group found altered serotonin and chromogranin-A distribution in EEC infected with Chlamydia trachomatis[7]. In line with the microscopical findings, we found a significant down-regulation of the gene coding for the vesicular monoamine transporter (VMAT1) in infected compared with non-infected EEC (P < 0.05). Bacteria of the genus Chlamydia are gram-negative, obligate intracellular pathogens that can infect a wide range of eukaryotic cells and cause chronic and acute diseases in humans and animals. All Chlamydia species share a common biphasic developmental cycle in which the organism alternates between two developmental forms, the elementary body (EB), the infectious form of the organism and the reticulate body (RB), the metabolically active but non-infectious form of the bacterium. Upon attachment and internalization, EBs differentiate into RBs, that multiply inside a membrane-bound vacuole termed inclusion. RBs eventually differentiate back into EBs, which upon their exit from the host cell can start a new round of infection. In addition to such acute chlamydial infection, Chlamydia can persist within infected host cells, a condition that has been associated with a number of chronic diseases. Chlamydial persistence has been described as a viable but non-cultivable growth stage resulting in a long-term relationship with the infected host cell. Such relationships can be established in vitro through exposure to antibiotics [8].

The aim of this study was to evaluate the response pattern of enteroendocrine cells to intracellular bacteria in vitro using microarray technology. Based on previous results from our laboratory we used C. trachomatis as the infectious agent for studying gene expression alterations upon bacterial infection of EEC (2). Analysis of genome-wide expression patterns on the host during infection provides an insight into how the host recognizes and process a pathogen [9]. We assessed transcription profiles of EEC lines from both the large bowel and the small bowel after infection with C. trachomatis.

With TEM we confirmed active and persistent infection caused by C. trachomatis in enteroendocrine cells, originating from both large and small bowel. As shown in Figure 1, growth of C. trachomatis, manifested through inclusions containing both elementary bodies (EB) and reticulate bodies (RB) at 24 h post infection (p.i.), could be observed for both cell lines. When EECs were treated with penG we observed enlarged, aberrant bacteria reminiscent of persistent growth forms.

In order to screen host-cell gene regulation in response to C. trachomatis infection we used microarrays representing 30 000 human genes. We studied gene changes (up or down regulation) at the same time point (24 h post infection) in both active and persistent infection. The time point at 24 h was chosen as it represents a period of active metabolism and binary fission of C. trachomatis reticulate bodies within the host cell.

Several studies have described Chlamydia-induced transcriptome changes in epithelial cells [10‐12]. Our study is the first to describe such changes in enteroendocrine cells. Studying EEC response to infection seems to be very important since the adult human intestine is home to between 1000 and 1150 prevalent bacterial species [13]. The role of EEC in immune activation and promotion of inflammation in the gut has been postulated before [14‐16]. A recent study showed that regenerating islet-derived gene REG4 coding for protein associated with epithelial inflammation was expressed in serotonin-producing enteroendocrine cells and expanded to epithelial cells of the upper colonic crypts during inflammation [17]. Alterations in the number of EEC have been observed in response to different bacterial, viral and parasitic infections of the GI tract [18, 19] but the impact of a bacterial infection on EEC themselves has never been studied. Previous studies on Chlamydia infection of epithelial cells showed alterations in genes encoding apoptosis inhibitors, regulators of cell differentiation, components of the cytoskeleton, transcription factors, proinflammatory cytokines and cellular growth factors and cell cycle [18, 20, 21]. Our results showed that the impact of Chlamydia infection on apoptosis (BIRC3, EMP1, CHAC1), immune response, host cell structure and cell cycle in EEC is similar to what has been observed in epithelial cells [21, 22].

The expression of BIRC3 (also known as cIAP2 or HIAP) [23], an apoptotic suppressor, was up-regulated in both cell lines during active and persistent infection. BIRC 3 is a component of the tumour necrosis factor receptor 2 (TNFR2) complex and, therefore, is a constituent of the TNF alpha (TNFα) signalling pathway [24]. BIRC3 has been demonstrated to inhibit cell death by directly repressing the proapoptotic activity of a family of cysteine proteases, caspases, as well as targeting proapoptotic components of the TNFα signalling pathway for ubiquitin degradation [25]. It was suggested that infection with Chlamydia protects mammalian host cells against apoptosis via BIRC3 up-regulation [26, 27]. Inhibition of apoptosis and cytoskeleton modifications possibly accommodate chlamydial growth [9].

The ability of Chlamydiae to alter host cell proliferation and differentiation was manifested by up-regulation of growth factors: growth differentiation factor 15 (GDF15), vascular endothelial growth factor A (VEGFA), amphiregulin (AREG) and transforming growth factor beta 2 (TGFB2). GDF15 was the most up-regulated gene in persistently infected LCC-18 cells (>10 fold change). GDF15 is a member of the TGFB family with a capacity to inhibit LPS-induced macrophage activation. GDF15 also provide significant neuroprotection with prominent effects on dopaminergic and serotonergic neurons [28].

The innate response upon Chlamydia infection of EEC is mediated by Toll-like receptor 4 (TLR4). TLR4 was up-regulated in LCC-18 and lymphocyte antigen 96 (LY96) was up-regulated in CNDT-2. LY96 encodes a protein, which associates with TLR 4 on the cell surface and confers responsiveness to LPS, thus providing a link between the receptor and LPS signalling.

Besides similarities in host-pathogen interaction between EEC and epithelial cells we found changes in gene expression in EEC that have never been described in epithelial cells. We found significant differences in gene transcription between persistently infected and non-infected cells in 10 genes coding for different solute carrier transporters (SLC) (Table 5). Transporters are the gatekeepers for all cells and organelles, controlling uptake and efflux of crucial compounds such as sugars, amino acids, nucleotides, inorganic ions and drugs. The SLC family include genes encoding passive transporters, ion coupled transporters and exchangers [29]. The biggest expression change was found for SLC7A11 in CNDT-2 cells (>13-fold). SLC7A11 is responsible for a sodium-independent, high-affinity exchange of anionic amino acids with high specificity for anionic forms of cysteine and glutamate. Up-regulation of SLC7A11 was previously reported in small bowel mucosa during cholera infection [30].

In addition to up-regulation of genes coding for two transporters involved in glutamate transport (SLC7A11 and SLC1A4) we also found significant differences in the expression of other genes related to glutamate signalling (argininosuccinate synthetase 1 (ASS1) and glutamate receptor interacting protein 1(GRIP 1)) in persistently infected LCC-18 cells compared to non-infected cells. GABAA receptor-associated protein like 1 (GABARAPL1) was up-regulated during persistent infection in both cell lines. Glutamate and GABA (γ-aminobutyric acid) are the major neurotransmitters acting on specific G-protein-coupled receptors (the metabotropic glutamate (mGlu) and GABAB receptors) to modulate synaptic transport.

EEC provide an important link in transfer of information between gut and brain. Primary afferent neurons are excited indirectly by serotonine or hormones released from EEC. Sensory information from the gastrointestinal tract is transmitted via a glutamatergic synapse to neurons of the nucleus tractus solitarius, which integrate this sensory information to regulate autonomic functions and homeostasis. The integrated response is conveyed to the preganglionic neurons of the dorsal motor nucleus of the vagus using mainly GABA, glutamate and catecholamines as neurotransmitters [31]. Glutamate is involved in the regulation of gut motility and secretion by acting as an excitatory neurotransmitter in the gut [32]. Changes in glutamate related gene expression could be beneficial to Chlamydia. It was shown that glutamate could support chlamydial growth [33]. Ojcius et al. [34] demonstrated the stimulation of glutamate synthesis in cells infected with Chlamydia psittaci.

Genes associated with glutamate transport (SLC7A11, SLC1A4) were up-regulated in CNDT-2 cells and ASS1, which is involved in glutamate synthesis [35] and GRIP1 were up-regulated in LCC-18 cells. GRIP1 is a synaptic protein interacting with glutamate receptors, playing an important role in their trafficking, synaptic targeting and recycling. GABARAP involves GRIP1 in the regulation of GABAA receptor function at inhibitory synapses [36].

GABA acts through a paracrine or autocrine mechanism via GABAA receptor activation [37]. This agent may play a similar role as a signalling molecule in endocrine organs and can for example inhibit the serotonin secretion [38]. The role of GABARAP binding proteins is not restricted to GABAA receptor transport but they participate in multiple biological processes, such as general vesicular transport and fusion events, autophagy and apoptosis. GABARAP appears to be associated with transport vesicles rather than the cell surface [39].

DRD2 gene, encodes the D2 subtype of the dopamine receptor (another G-protein-coupled receptor), was up-regulated in persistent infection in LCC-18 cells. Dopamine (DA) is an enteric neurotransmitter and D2 seems to be an important mediator of neuronal responses to DA. Li et al. [40] showed that propulsive motility is increased in the gut of mice that lack D2 receptors suggesting that D2 is a major mediator of the effects of endogenous DA in the enteric nervous system (ENS).

Synaptotagmins SYT5 and SYT7 are other endocrine related genes that were differentially transcribed in persistent infection of LCC-18. Neurotransmitters, neuropeptides and hormones are released through the regulated exocytosis of SVs (synaptic vesicles) and LDCVs (large dense-core vesicles), a process that is controlled by calcium [41]. Ca²⁺ triggers many forms of exocytosis in different types of eukaryotic cells, for example synaptic vesicle exocytosis in neurons, granule exocytosis in mast cells, and hormone exocytosis in endocrine cells [42]. Synaptotagmins are a family of type-1-membrane proteins that share a common domain structure. Most synaptotagmins are located in brain and endocrine cells, and some of these synaptotagmins bind to phospholipids and calcium at levels that trigger regulated exocytosis of SVs and LDCVs [41]. Synaptotagmin 5 is a neuroendocrine cell-specific synaptotagmin responsible for phospholipid binding. Synaptotagmin 7 is a major Ca²⁺-sensor in endocrine LDCV exocytosis [42].

Our study provides new insights into the molecular events taking place in Chlamydia infection of EEC. We found that Chlamydia infected EEC may share some gene expression patterns with infected epithelial cells but they also seem to have some cell type-specific patterns related to vesicular transport and secretion, and neurotransmitters. We also observed differences in gene expression changes caused by Chlamydia infection between our two cell lines. It is well known that there are important differences in the luminal environments between the small and large intestines and we cannot exclude the influence of cell origins on obtained results. We found many differences in basal gene transcriptions (non-infected cells) between the two cell lines but infected cells seem to share the same genetic pattern independent of their origin.

Given the functions of EEC, it can be anticipated that altered function, in the presence of disease, might underpin symptom development. EEC clearly have multiple important functions in the control of appetite, GI motility and mucosal immunity. At present, EEC dysfunction related to various GI diseases is acknowledged, but remains poorly understood [43].

We do not have evidence that Chlamydia is the causative agent for motility disorders but we have shown that such an infection can influence EEC function. We studied the response pattern of enteroendocrine cells to intracellular bacteria in vitro using C. trachomatis as the infectious agent. Our findings can serve as a model of bacterial impact on EEC function but we cannot determine if our findings are Chlamydia specific or represent a general response to intracellular infection. Infection of mouse STC-1 enteroendocrine cells with Salmonella typhimurium was recently reported [44]. We successfully infected LCC18 with Salmonella typhimurium obtaining intracellular growth of these bacteria in EEC, but we have not analysed the effects of this infection on gene expression.

Based on our findings that infected EEC exhibit cell-type specific gene expression patterns related to vesicular transport, secretion and neurotransmitter regulation our study sheds some light on the potential impact of bacteria on the exocrine function of EEC. Since EEC play a pivotal role in the regulation of gut motility any impairment of enteroendocrine function could potentially contribute to motility disorders.