Aliment Pharmacol Ther 2011; 33: 1045–1052
Background Irritable bowel syndrome (IBS) is a stress-related disorder with disturbed brain-gut communication, gastrointestinal homeostasis and, based on recent evidence, low grade inflammation and an altered microbiota. The immune system is a critical regulator of the brain-gut axis. Toll-like receptors (TLRs) are pattern recognition molecules regulating innate immunity.
Aim To characterise toll-like receptor activity in IBS.
Methods Thirty IBS patients and 30 healthy controls (HC) were recruited. Venous blood was collected, and cultured with a panel of toll-like receptor agonists for 24 h. Cell supernatants were analysed using a multiplex ELISA approach to measure IL1β, IL6, IL8 and TNFα. Plasma was analysed for levels of inflammatory cytokines and cortisol.
Results Toll-like receptor agonist-induced cytokine (IL1β, IL6, IL8 and TNFα) release was markedly enhanced in stimulated whole blood from IBS (n = 30) patients compared with healthy controls (n = 30). An exaggerated response to the TLR8 agonist for all cytokines investigated was seen in IBS patients. In addition, enhanced TLR2-induced TNFα release, TLR3-induced IL-8 release, TLR4-induced IL1β and TNFα release, TLR5-induced IL1β and TNFα release and TLR7-induced IL-8 release were also observed in IBS patients. No differences in TLR1, TLR6 or TLR9 activity were detected. In addition, plasma levels of cortisol, IL-6 and IL-8 were significantly increased in IBS patients.
Conclusion Taken together, these data demonstrate elevated cytokine levels and toll-like receptor activity in the periphery of patients with the irritable bowel syndrome, indicating some immune dysregulation in these patients.
Irritable bowel syndrome (IBS) is a highly prevalent functional disorder of the gastrointestinal tract characterised by the presence of abdominal pain, an alteration in bowel habit and the absence of reproducible biomarkers.1–3 Altered visceral perception and gut dysmotility are important contributors to symptom expression.4, 5 IBS is a disorder of the brain-gut axis with stress being a frequently invoked precipitant of symptoms among sufferers.6, 7 IBS subjects have been shown to demonstrate exaggerated motor and sensory response to a variety of stressors.8–10 Patients also display an over activation of the hypothalamic-pituitary adrenal (HPA) axis.11–13 Immune dysfunction in IBS patients in both mucosal and systemic compartments has been reported.11, 14–16 We have shown that plasma levels of the inflammatory cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8) are elevated in IBS patients.11, 16 Others have reported that levels of tumour necrosis factor α (TNFα), interleukin-1β (IL-1β) and IL-6 were elevated in diarrhoea predominant IBS patients (IBS-D).14
Toll-like receptors (TLRs) are activated by various bacterial and viral cell components.17 Receptor binding results in transcription of inflammatory cytokines.18–20 TLRs are found in high numbers on mucosal surfaces (e.g. the colonic epithelium) and on monocytic cells.21 Recently, we have also shown that the mRNA expression of certain TLRs is increased in the colonic mucosa of rat models of visceral hypersensitivity22 and have demonstrated increased expression of TLR-4 in mucosal biopsies from IBS patients.23 However, the relevance of these mucosal findings to the immune activation observed in the systemic circulation is unclear. In these studies, we sought to characterise the activity of various TLRs on peripheral blood cells to explore the possibility that, as in the colonic mucosa, IBS patients may be primed for an inflammatory response via certain TLR ligands.
Materials and methods
The study protocol (APC020 2009) was approved by the University College Cork Clinical Research Ethics committee of the Cork University Hospital. IBS patients were recruited from gastroenterology and rheumatology clinics at Cork University Hospital, while healthy controls were recruited from the laboratory, institute or hospital staff. Each healthy control and patient donated 20 mL of venous blood between 11:00 and 13:00 hours to avoid diurnal variations. Thirty IBS patients aged between 18 and 65 years of age who satisfied Rome II criteria for the diagnosis of IBS were studied. No patient was categorised as postinfectious IBS (PI-IBS). Individuals with inflammatory diseases, coeliac disease, lactose intolerance, immunodeficiency, individuals who had undergone any abdominal surgery, with the exception of hernia repair and appendectomy, and those with a psychiatric history were excluded. No attempt was made to select IBS patients on the basis of predominant bowel habit. Each potentially eligible patient was evaluated by a full review of clinical history including current health status, vitals, BMI, family history and current medication. Bowel habit was defined as constipation-dependent (IBS-C), diarrhoea-predominant (IBS-D) or alternating (IBS-A) according to Rome II subclassifications. In addition, a questionnaire that assessed IBS severity was given to participants. The IBS severity scoring system (IBS-SS)24 assesses abdominal pain, abdominal distension (bloating), bowel habit (including frequency and stool consistency) and the effect of IBS on quality of life. A cumulative score (maximum is 500) was calculated and symptom severity was assigned as mild (75–175), moderate (175–300) or severe (>300). Current mood was assessed using the patient health questionnaire (PHQ-9)25 and interview with a psychiatrist.
Plasma isolation and whole blood culture
Collected whole blood (15 mL) was added to an equal volume of Histopaque 1077 (Sigma, St. Louis, MO, USA) in a sterile 50 mL tube and centrifuged at 400 × g for 30 min at room temperature. Plasma on the upper layer was transferred to a separate tube and stored at −80 °C for future use. Collected whole blood (2 mL) was diluted 1:10 in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Dublin, Ireland). Blood was aliquoted into 24-well plates and cultured in 37 °C incubator with 5% CO2. Each blood sample was cultured in DMEM cell culture medium supplemented with 10% Fetal Calf Serum (Sigma, Dublin, Ireland) with or without the following Toll-like receptor ligands from Human TLR agonist kit (InvivoGen, San Diego, CA, USA) for 24 h: TLR1/2 – Palmitoyl-3-cysteine-serine-lysine 4 (Pam3Cys); TLR2 – Heat-killed Listeria Monocytogenes (HKLM); TLR3 – Polyriboinosinic polyribocytidylic acid (Poly I:C); TLR4 – lipopolysaccharide (LPS); TLR5 –Salmonella typhimurium Flagellin; TLR6/2: FSL-1; TLR7 – Imiquimod; TLR8 – ssRNA40. Agonists were reconstituted in endotoxin-free water (supplied) with a final concentration of 1 μg/mL except for HKLM (108 cells) and Poly I:C 10 μg/mL. Each treatment was carried out in duplicate. After the indicated culture period, supernatant from both untreated and stimulated cells was aspirated and stored at −80 °C.
Enzyme-linked immunosorbent assay
Measurements of IL-1β, IL-6, IL-8 and TNFα in both plasma and following TLR agonist stimulation of whole blood was carried out using an electrochemiluminescence multiplex system with the Mesoscale Discovery (MSD, Gaithersburg, MD, USA) 4-plex Human Proinflammatory Kit II. Measurements of IL-2, IL-4, IL-5, IL-10, IL-12p70, IL-13, IFN-γ in plasma were carried out in duplicate using the MSD 7-plex Human Th1/Th2 kit. ELISA plates were analysed using the Sector 2400 imager from Mesoscale Discovery. This is an ultra-sensitive method which has a detection limit for IL-1β of 0.3 pg/mL, IL-6 of 0.3 pg/mL, IL-8 of 1.0 pg/mL, IL-10 of 1.9 pg/mL, IL-12p70 of 1.1 pg/mL, IL-13 of 2.8 pg/mL, TNFα of 0.3 pg/mL and IFNγ of 0.8 pg/mL. Plasma cortisol was measured using the Cortisol Enzyme Immunoassay Kit (Assay Designs, Ann Arbor, MI, USA) with a detection limit of 26.99 pg/mL.
The sample size was determined by a power calculation based on our previous data and aimed at detecting differences between IBS patients and healthy controls at the 0.05 level. ELISA data were expressed as mean ± S.E.M. All statistical analyses were carried out using GraphPad Prism for Windows (Version 4). Plasma cortisol cytokine levels were compared using an unpaired two-tailed Student’s t-test, differences were considered significant at P < 0.05. Differences between the IBS and control groups for TLR-agonist induced release of cytokines were determined using two-way anova with a Bonferroni post hoc test.
Demographic data for IBS patients and control subjects are presented in Table 1. There were no significant differences between the groups in terms of age, gender distribution or body mass index (BMI). According to Rome II subclassification in terms of predominant bowel habit, this cohort had 11 alternating (IBS-A), 10 constipation-predominant (IBS-C) and nine diarrhoea-predominant (IBS-D) patients. IBS symptoms were assessed using the IBS-SS, it was found that the average cumulative score for the IBS patients was 258.8 (moderate) compared to 4.83 (out of 500) for the control subjects; as expected, the difference was highly significant (P < 0.0001) (Table 1). Additionally, two of the control group had a family history of IBS compared to 13 of the IBS group (Table 1).
|Age||36.24 ± 1.835||40.9 ± 2.034||0.09|
|Gender||10 M, 20 F||5 M, 25 F|
|Height||1.71 ± 0.017||1.67 ± 0.017||0.0876|
|Weight||68.4 ± 2.558||69.74 ± 2.673||0.7192|
|BMI||23.19 ± 0. 6084||24.89 ± 0.7459||0.0827|
|Family history IBS||2||13|
|IBS subtype||N/A||11A, 10C, 9D|
|IBS-SS||4.83 ± 4.83||258.8 ± 13.58||P < 0.0001|
Plasma cytokine and cortisol levels
Plasma from both healthy controls and IBS patients was analysed for eleven cytokines and cortisol. Student’s t-tests (with correction for multiple comparisons) confirmed that there were significantly elevated levels of IL-6 (P = 0.008), IL-8 (P = 0.028) and cortisol (P = 0.048) in the plasma of IBS patients compared with healthy controls (Figure 1). Levels of other cytokines tested (IL-1β, TNFα, IL-2, IL-4, IL5, IL-10, IL-12, IL-13 and IFNγ) were not found to be significantly different between groups (Table 2). No gender differences were found for plasma cortisol or cytokines measured.
|Cortisol||78220 ± 7590||107000 ± 12340*||0.0488|
|IL-1β||0.7751 ± 0.09349||0.9111 ± 0.1270||0.3922|
|IL-6||1.581 ± 0.1462||2.875 ± 0.3350**||0.0008|
|IL-8||7.474 ± 0.3745||9.397 ± 0.7667*||0.0282|
|TNFα||7.487 ± 0.4606||7.500 ± 0.2472||0.9808|
|IL-2||0.7668 ± 0.09624||0.9327 ± 0.2535||0.5430|
|IL-12||8.227 ± 2.990||13.84 ± 4.923||0.3342|
|IFNγ||4.381 ± 0.7624||5.484 ± 0.9830||0.3789|
|IL-4||2.114 ± 0.6566||1.120 ± 0.2286||0.1585|
|IL-5||3.708 ± 0.7518||5.121 ± 1.387||0.3745|
|IL-10||16.62 ± 2.327||20.50 ± 3.762||0.3840|
|IL-13||14.32 ± 4.894||9.615 ± 3.226||0.4257|
TLR-agonist cytokine release
Supernatants from whole blood treatments with TLR agonists of both healthy controls and IBS patients were analysed for levels of four different pro-inflammatory cytokines IL-1β, IL-6, IL-8 and TNFα. Two-way anova demonstrated a significant effect of both disease state (F1, 1684 = 38.10, P < 0.001) and TLR agonist treatment (F8, 1684 = 115.7, P < 0.001) with a significant interaction (F8, 1684 = 5.98, P < 0.001) between the two factors for IL-1β levels. A significant effect of both disease state (F1, 1717 = 10.41, P = 0.0013) and treatment (F8, 1717 = 119.7, P < 0.001) with a significant interaction (F8, 1717 = 2.142, P = 0.0293) between the two factors was found for IL-6 levels. Two-way anova demonstrated a significant effect of both disease state (F1, 1725 = 66.93, P < 0.001) and treatment (F8, 1725 = 31.50, P < 0.001) with a significant interaction (F8, 1725 = 8.321, P < 0.001) between the two factors for IL-8 levels. A significant effect of both disease state (F1, 1709 = 51.71, P < 0.001) and treatment (F8, 1709 = 124.3, P < 0.001) with a significant interaction (F8, 1709 = 6.515, P < 0.001) between the two factors for TNFα levels also. When age was entered as a co-variate the differences between the groups are still significant.
Post hoc analysis of individual treatments revealed significant differences between IBS patients and healthy controls. In the untreated blood, levels of IL8 were found to be significantly higher in IBS patients compared with healthy controls (HC) (1287 ± 119.7 vs. 2135 ± 107.4 pg/mL; P < 0.001) (Figure 2a). No significant differences were found in cytokine release following treatment with the TLR1/2 agonist Pam3Csk (Figure 2b). TNFα release was found to be significantly elevated in IBS compared with HC blood (1462 ± 127.9 vs. 934.6 ± 94.93 pg/mL; P < 0.001) following treatment with the TLR2 agonist HKLM (Figure 2c). IL-8 release was found to be significantly elevated in IBS compared with HC blood (2582 ± 122.7 vs. 2015 ± 168.3 pg/mL; P < 0.05) following treatment with the TLR3 agonist Poly I:C (Figure 2d). IL-1β was found to be significantly elevated in IBS compared with HC (1715 ± 100.8 vs. 1157 ± 95.4 pg/mL; P < 0.001) following treatment with the TLR4 agonist LPS as well as TNFα levels (2427 ± 175.5 vs. 1723 ± 118.5 pg/mL; P < 0.001) (Figure 2e).
Interleukin-1β release was found to be significantly elevated in IBS compared with HC (947.8 ± 91.27 vs. 570.8 ± 65.95 pg/mL; P < 0.001) as well as TNFα (1354 ± 128.8 vs. 921.7 ± 85.61 pg/mL; P < 0.01) following treatment with the TLR5 agonist Flagellin (Figure 3a). No significant differences were found following treatment with the TLR6/1 agonist FSL1 (Figure 3b). IL-8 release was found to be significantly elevated in IBS compared with HC blood (3408 ± 112.2 vs. 2128 ± 147.9 pg/mL; P < 0.001) following treatment with the TLR7 agonist Imiquimod (Figure 3c). IL-1β release was found to be significantly elevated following treatment with the TLR8 agonist ssRNA40 (1404 ± 118.5 vs. 913.8 ± 88.15 pg/mL; P < 0.001), IL6 release was found to be significantly elevated (2037 ± 144.3 vs. 1326 ± 137.6 pg/mL; P < 0.01); in addition IL-8 release was found to be significantly elevated (3438 ± 126.9 vs. 2477 ± 146.8 pg/mL; P < 0.001); finally, TNFα release was found to be significantly elevated in IBS compared with HC blood following treatment with the ssRNA40 (1944 ± 161.2 vs. 1076 ± 110.3 pg/mL; P < 0.001) (Figure 3d). No significant changes in cytokine levels were detected following treatment with the TLR9 agonist ODN2006 (Figure 3e). A separate analysis was carried out in male and female participants. There was no significant difference between these groups with the exception of LPS (TLR4) induced cytokine release, where there was significantly more IL1β, IL6 and TNFα released in female IBS compared with male IBS patients (data not shown).
The exact pathophysiology of IBS is still unknown. It has been suggested to have an immune component,26 with low level immune activation reported.11, 14, 15, 27–29 These studies have shown increases in proinflammatory cytokines,11, 14, 15, as well as alterations in certain immune cell populations in IBS patients.15, 27–29 Given the key role of toll-like receptors in the activation of cytokine release, we hypothesised that TLR activity may be altered in IBS patients. IBS patients were shown to have elevated plasma cortisol levels, in agreement with previous reports from this laboratory.11 Cortisol is a product of the hypothalamic–pituitary-adrenal (HPA) axis and an important hormonal indicator of stress. The HPA axis has been demonstrated to be dysregulated in IBS patients by a number of groups including our own.11–13 Stress is known to disrupt the function of the immune system, including cytokine levels.30 Of cytokines measured, only IL-6 and IL-8 were significantly elevated in IBS patients, in agreement with previous studies.11, 30
The effects of elevated cytokine levels on the pathophysiology of IBS remain to be defined. Cytokines play a role in central sensitization. IL-1β, IL-6 and TNFα have been shown to affect transmission in the spinal cord resulting in hyperalgesia.31 This finding is relevant to IBS, in which both elevations in cytokines and hypersensitivity to pain have been demonstrated.32 Furthermore, cytokines are known to have an effect on the HPA-axis and stress response.33 IL-1β, IL-6 and TNFα have all been reported to stimulate secretion of corticotrophin-releasing hormone (CRH) in rat as well as in humans.34, 35 Cytokines can cross the blood brain barrier and stimulate cells at the barrier to release more cytokines within the brain.35 However, it is not clear whether the elevations in cytokine levels observed in IBS patients are responsible for the alteration in HPA-axis activity or vice versa.
Whole blood stimulations revealed a distinct pattern of peripheral TLR activity in IBS patients. Accordingly, we demonstrated an association between increased TLR4, 5 and 8 activities and IL1β production; these receptors recognise bacterial lipopolysaccharide (LPS), bacterial flagellin and viral nucleic acid. Increased TLR8 activity was associated with IL-6 release. IL-8 release was greater following activation with agonists for TLR3, 7 and 8. All of these receptors recognise viral nucleic acids. Finally, increased TNFα release was related to increased TLR2, 4, 5 and 8 activities. Enhanced TLR8-mediated cytokine release was the common factor in relation to each of these cytokines. At present, viral nucleic acids are the only known ligand for TLR8,17 although a yet undiscovered endogenous or exogenous ligand(s) may also activate this receptor. In addition, there were some differences between male and female patients for LPS stimulation, indicating changes in TLR4 activity may be linked not only to the condition but also sex, and further studies are needed to investigate this matter.
Irritable bowel syndrome patients have been shown to have a different microbiota to that of healthy controls.36 The gut microbiota is known to have an influence on a number of physiological parameters including the immune system and may prime immune cells that enter the peripheral circulation.37 This may impact on the TLR expression profile of immune cells and result in altered TLR activity. Indeed, our group has recently reported increased expression of TLR4 in mucosal biopsies from IBS patients.23 Secondly, IBS patients appear to be particularly susceptible to stress11 and this seems to have effect on gut motility.9, 10 It is apparent from both animal models and clinical studies that chronic stress may affect the immune response.30 Repeated social defeat stress in mice enhanced the bactericidal activity of splenic macrophages, and led to an increase in the expression of TLR2 and TLR4 on these cells.38 Social defeat can also activate splenic dendritic cells and enhance TLR activity on these cells as assessed by cytokine output in response to ligand stimulation.39 Furthermore, chronic restraint stress resulted in immune suppression which was mediated by TLR4.40, 41 Elevations in the expression of TLR mRNA was observed in the colonic mucosa of two different rat models of stress-induced visceral hypersensitivity when compared with normosensitive rats.22 These rats develop visceral hypersensitivity, changes in immune response and differences in the gut microbial populations.42 Thirdly, cytokines and glucocorticoids may synergise to alter TLR expression.43, 44 Plasma cytokines and cortisol have been shown to be elevated in this cohort of patients and this potentially may explain why there are increases in TLR activity in these patients.
A limitation of the current study is that the TLR receptor activity in the periphery may not resemble that of the gut mucosa. This study has provided novel insights into relationships between TLR activation, plasma cytokine levels and clinical phenotype among a well-characterised group of IBS patients. We have described for the very first time, distinctive patterns of TLR activity in the periphery. Based on observed relationships between certain phenotypic features and demonstrated associations between stress and TLR expression and activity in animal models, it is plausible to suggest that there maybe a link between stress, TLR activation and cytokine profiles in IBS patients.
Declaration of personal interests: None. Declaration of funding interests: The Alimentary Pharmabiotic Centre is a Centre for Science and Technology (CSET) funded by Science Foundation Ireland (SFI), through the Irish Government’s National Development Plan. The authors and their work were supported by SFI and GlaxoSmithKline through grant 07/CE/B1368.