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Expression of the TRPV1 receptor differs in quiescent inflammatory bowel disease with or without abdominal pain
  1. Ayesha Akbar1,
  2. Yiangos Yiangou2,
  3. Paul Facer2,
  4. W G Brydon3,
  5. Julian R F Walters1,
  6. Praveen Anand2,
  7. Subrata Ghosh4
  1. 1Department of Gastroenterology, Imperial College London, UK
  2. 2Department of Clinical Neuroscience, Imperial College London, UK
  3. 3Department of Clinical Biochemistry, Western General Hospital, Edinburgh, UK
  4. 4Division of Gastroenterology, University of Calgary, Alberta, Canada
  1. Correspondence to Professor Subrata Ghosh, Division of Gastroenterology, University of Calgary, Alberta, Canada; ghosh{at}ucalgary.ca

Abstract

Objective Transient receptor potential vanilloid type 1 (TRPV1) has been shown to play an important role in visceral hypersensitivity. A significant proportion of patients with inflammatory bowel disease (IBD) continue to complain of abdominal pain despite their disease being otherwise quiescent. We investigated TRPV1-immunoreactive fibres in rectosigmoid biopsies taken from such patients with correlation to abdominal pain severity.

Methods Rectosigmoid biopsies were collected from 20 patients with quiescent IBD fulfilling Rome II criteria for irritable bowel syndrome (IBS), from 20 asymptomatic patients with quiescent IBD and from 28 controls. Abdominal pain scores were recorded using a validated questionnaire. TRPV1- and neuronal marker protein gene product 9.5 (PGP 9.5)-expressing nerve fibres, and lymphocytes (CD3 and CD4) were quantified, following immunohistochemistry with specific antibodies. The biopsy findings were related to abdominal pain scores.

Results A significant 3.9-fold increase in median number of TRPV1-immunoreactive fibres was found in biopsies from patients with quiescent IBD with IBS symptoms when compared with controls (p<0.0001) and a 5-fold increase when compared with the asymptomatic quiescent IBD group (p=0.0003). In the IBD with IBS symptoms group, the total nerve fibres (PGP 9.5) did not differ from those in the asymptomatic IBD group (p=0.10) or the control group (p=0.33) and neither did the CD3 lymphocyte (asymptomatic IBD group p=0.17; controls p=0.88) or CD4 lymphocyte (asymptomatic IBD group p=0.39; controls p=0.97) counts. In stepwise multivariate linear regression analysis, only TRPV1-immunoreactive nerve fibres (R2=0.8; p<0.0001) were significantly related to the abdominal pain score.

Conclusions Increased TRPV1 nerve fibres are seen in quiescent IBD with IBS-like symptoms together with a correlation to pain severity. TRPV1 may contribute to the pathophysiology of ongoing pain and visceral hypersensitivity in this group of patients, providing a potential therapeutic target.

  • Abdominal pain
  • health-related quality of life
  • inflammatory bowel disease (IBD)
  • pain
  • transient receptor potential vanilloid type-1 (TRPV-1)
  • visceral hypersensitivity
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Introduction

Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), is a chronic incurable illness which results in gut inflammation, affecting 1 in 400 of the UK population. The cause is still unknown but involves genetic, environmental and immunological factors.1 Treatment is aimed at symptom control of abdominal pain, diarrhoea and bloating; however, a significant number continue to suffer from debilitating pain despite successful treatment of the active disease. These patients complain of irritable bowel syndrome (IBS)-like symptoms, with one-third of patients with UC2 3 and 42–57% of patients with CD in remission being affected.2 3 The cause of these symptoms in this substantial proportion of patients with quiescent IBD is probably secondary to inflammatory-induced visceral hypersensitivity. Their persistent pain impacts their daily life, leading to misery and depression, limitation of daily activities, impact on social life and time off work. Further knowledge of the mechanisms involved in the perpetuation of the pain may provide considerable benefit by providing new insights into potential treatments.

The transient receptor potential vanilloid type 1 (TRPV1 or VR1) receptor plays an important role in visceral hypersensitivity. First cloned in 1997,4 the polymodal TRPV1 receptor is expressed mainly by primary afferent neurons sensing noxious stimuli.5 6 TRPV1 is activated by capsaicin, lipids, resinferatoxin, endocannabinoids including anandamide4 7 and noxious heat (>43°C), and is potentiated by protons. Upon activation, a burning pain sensation is evoked, along with release of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CRGP) which initiate the process of neurogenic inflammation. Inflammatory mediators have been shown to lower the threshold for activation of TRPV1.

TPRV1 has been implicated in the mechanism of pain produced in gastro-oesophageal reflux disease (GORD),8 Hirschprung disease9 and rectal hypersensitivity, where levels correlated with a decrease in threshold to rectal heat and distension.10 In patients with painful IBD, the numbers of TRPV1 (VR1)-expressing neurons were significantly increased in colonic mucosa.11 Recently our group also reported that there was an upregulation of TRPV1-immunoreactive nerve fibres in colonic biopsies from patients with IBS,12 correlating with severity of abdominal pain. This was associated with elevation of immune cell markers suggestive of subtle inflammation. In the present study we have investigated the potential role of TRPV1 in the pathophysiology of persistent pain in patients with quiescent IBD.

Materials and methods

Patients

The study was approved by Hammersmith Hospitals NHS Trust ethics committee and all participants gave fully informed consent. Patients with IBD in remission for at least 12 months and without previous surgery were included. Information collected included type of IBD (UC/CD), disease extent, disease duration, current medication, symptoms and smoking status. Sixty-eight patients (40 IBD, 28 controls) were to be recruited into three groups as outlined below (groups 1, 2 and 3). Group 1 (n=20) were symptomatic patients with IBD (complaining of abdominal pain) with quiescent disease meeting all of the following criteria: symptomatic (ileo-) colonic CD or UC; CDAI (Crohn's Disease Activity Index) <150 or UCDAI (Ulcerative Colitis Disease Activity Index) ≤1; C-reactive protein (CRP) <10 mg/l; erythrocyte sedimentation rate (ESR) <20 mm/h; platelets <450×1012/l; white cell count <11×109/l; faecal calprotectin <50 mg/g; and macroscopically normal mucosa at colonoscopy (including the terminal ileum in CD). Patients currently complained of abdominal pain and IBS-type symptoms (bloating, varying bowel habit with diarrhoea and/or constipation) as defined by Rome II criteria. Group 2 (n=20) were asymptomatic patients with quiescent IBD, undergoing surveillance colonoscopy for dysplasia. The same criteria as for group 1 were applied except that they did not fulfil Rome II criteria and denied abdominal pain. The control group 3 (n=28) consisted of asymptomatic patients without IBD or IBS having a colonoscopy for an indication other than IBD, for example for colorectal cancer or polyp surveillance. As the CDAI includes pain as a component, our definition of quiescent IBD may exclude some patients with severe IBS-like symptoms who will also have an elevated CDAI; this has been reported by us previously.13 See tables 1–4 for further patient details.

Table 1

General characteristics of the three patient groups

Table 2

Characterisation of the two quiescent IBD groups with and without pain

Table 3

Symptoms reported by the quiescent IBD group with pain

Table 4

Indications for colonoscopy in the control group

All patients had a colonoscopy with standard bowel preparation (4 litres of Kleanprep). Due to the patchy nature of colonic involvement in CD, patients with previous rectal involvement were selected. None of the patients was currently using topical rectal treatment, and all had macroscopically normal colonoscopies. For all groups (1, 2 and 3), four mucosal colonic biopsies were collected using standard biopsy forceps at the rectosigmoid junction to ensure uniform sampling. This is similar to biopsies we took in our previous publication.12

Questionnaires

Pain severity, anxiety and depression

All subjects completed a pain diary for the 7 days prior to commencing their bowel preparation. The validated Short Form McGill Pain Questionnaire (SF-MPQ) was used.14 This uses a 0–10 cm visual analogue scale (VAS) for patients to record pain severity. The pain scores were collected and the maximum recorded VAS over the 7 days, or VASmax, was noted for each subject. As patients with IBD with IBS-like symptoms typically experience episodic pain which lasts 1–2 days, the VASmax was thought to be more representative of the impact of pain on the patients than the mean pain score. The mean pain score was also calculated as VAS average (VASav).

Depression was measured using the Beck Depression Inventory (BDI).15 Participants were asked to complete the BDI based on how they had been feeling over the past 2 weeks. A score of >13 indicates a degree of depression.

The Hospital Anxiety and Depression Scale (HADS)16 has two subscales (anxiety and depression) with each subscale giving maximum scores of 21, and total scores range from 0 to 42. A score of ≥8 on a subscale signifies a degree of anxiety/depression.

Quality of life

Quality of life was assessed using an IBDQ (inflammatory bowel disease questionnaire) and the short form SF-36 questionnaire for groups 1 and 2.

The SF-3617 is a validated health survey questionnaire with 36 questions. The scoring creates a physical composite score (PCS) and a mental health composite score (MCS). A higher score implies better functioning, with a maximum of 100.

The IBDQ18 19 is a validated questionnaire to measure health-related quality of life (HRQoL) in adult patients with IBD. Each of the 32 questions is scored on a 7-point Likert scale to give a total ranging from 32 to 224. A lower score indicates a better HRQoL.

Disease activity

For groups 1 and 2, CDAI or UCDAI scores were also calculated. Markers of inflammation as outlined above were recorded.

Patients were asked to complete the questionnaires at home so they had time to comprehend and complete them and then return them by post.

Immunohistochemistry and histology

Two biopsies from the rectosigmoid junction were fixed in buffered 10% formalin and processed routinely for H&E histology. The histological sections were all evaluated by an experienced gastrointestinal pathologist.

A further two biopsies from each site were used for immunohistochemistry using antibodies for TRPV1 and the pan-neuronal structural nerve marker PGP9.5 (protein gene product 9.5). All biopsies collected were also used for immunohistochemistry using antibodies for CD3 and CD4 lymphocytes to exclude inflammation or other pathology.

The primary antibodies used in the study are listed in table 5, and they were used as previously described.10 The specificity of the TRPV1 antibody (C22) has been previously characterised in the gut10 and other tissues including dorsal root ganglia20 using preabsorption and western blot studies. Tissues were snap-frozen and stored at –70°C or immersed in fixative (4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS: 0.1 M phosphate; 0.9% (w/v) saline; pH 7.3)) then washed in PBS containing 15% (w/v) sucrose and azide for 1 h before snap-freezing in embedding medium (Tissue-Tek OCT compound, Sakura Finetek, Torrance, California, USA). Frozen tissue sections (15 μm) were collected onto poly-l-lysine-coated (Sigma, Poole, Dorset, UK) glass slides. Unfixed tissue sections were postfixed in 4% (w/v) paraformaldehyde, while tissue sections from immersion-fixed biopsies were allowed to dry on the slide (for PGP 9.5 antibodies only). Endogenous peroxidase was blocked by incubation in 0.3% (w/v) hydrogen peroxide in industrial methylated spirit. After rehydration in PBS, sections were incubated overnight with primary antibodies (table 5). Controls included omission of primary antibodies, or their replacement with preimmune serum. Sites of antibody attachment were revealed using the nickel-enhanced, immunoperoxidase method (avidin–biotin complex—ABC elite; Vector Laboratories, High Wycombe, Bucks, UK). Nuclei were counterstained with 0.1% (w/v) aqueous neutral red (except for CD3 and CD4 staining).

Table 5

Primary antibodies

Analysis of immunoreactive fibres

Immunoreactive nerve fibres and cell markers were quantified by computerised image analysis (Olympus Analysis Five DP Soft, UK). Analogue images were captured via video link to an Olympus BX50 microscope and converted into digital monochrome images by the computer. The grey-shade detection threshold was set at a constant level to allow detection of positive immunostaining, and the area of highlighted immunoreactivity in the mucosa was obtained as a percentage (% area) of the field scanned. Five fields (×40 objective magnification) per tissue section, chosen at random, were scanned, and the mean values of readings obtained by two independent blinded observers were used for final analysis. For TRPV1 analysis, as the fibres showed up as fine linear fibres, the total numbers of fibres were counted per section and results were expressed as the mean number of fibres/mm2. Again five random fields and two independent blinded observers were used. Further details of methods are provided in the previous papers of Chan et al10 and Akbar et al.12

Statistical analysis

Data were compared using Mann–Whitney U test using Winstat for EXCEL software or GraphPad Prism. p Values <0.05 were considered as statistically significant. Correlations between two parameters were performed using the Pearson correlation. Univariate and multivariate linear regression models were used to assess the associations between primary antibody variables, patient variables, disease variables and the average pain score (VASav) or maximum pain score (VASmax). All data were reported as median values and IQR unless otherwise stated.

Results

All the biopsies were reported as normal or showing changes consistent with quiescent IBD.

Neuronal markers

TRPV1-immunoreactive fine fibres were seen scattered throughout the mucosa in biopsies from all three groups (see figure 1). These were more abundant in the quiescent IBD with pain group 1 than in the IBD without pain group 2 or the control group. Quantification of these fibres revealed that there were a significantly (3.9-fold) higher number of median TRPV1 fibres in the painful IBD group 1 when compared with controls (p<0.0001, table 6). There was also a significantly (greater than fivefold) higher number of median TRPV1 fibres in the painful group 1 compared with the no pain IBD group 2 (p=0.0003, table 6). Figure 2 illustrates the TRPV1 levels in individual subjects in each of the three groups.

Figure 1

Immunohistochemistry staining (×40 magnification) in quiescent inflammatory bowel disease (IBD) without pain (top row) and quiescent IBD with pain (bottom row). The first column shows TRPV1 (transient receptor potential vanilloid type 1) fine fibres (arrowed); the second column shows CD3 brown staining and the third column shows PGP 9.5 (protein gene product 9.5) staining.

Table 6

Quantification of immunoreactivities in colonic biopsies from IBD and control subjects

Figure 2

Transient receptor potential vanilloid type 1 (TRPV1) levels in the individual groups. IBD, inflammatory bowel disease.

PGP 9.5 staining was similar in all three groups when quantified (table 6, figure 1) and there was no significant difference in the median values of PGP 9.5% staining area between the pain group 1 and the no pain group 2 (p=0.10), or between the pain group 1 and the control group (p=0.33).

To compare any differences arising from disease type, the pain group 1 and no pain group 2 were subdivided into UC or CD with and without pain (UC no pain, n=12; UC pain n=10; CD no pain n=8; CD pain n=10). When these subgroups were compared, there was still a significant difference between the median TRPV1 fibres in the pain versus no pain subgroups; UC pain (fibres/mm2) median 3.15, IQR 1.89–8.88; UC no pain 0.7 (0.5–1.95); p=0.007. Similarly for CD there was a significant difference in the pain versus no pain subgroups of median TRPV1 fibres; CD pain 4.1 (1.9–5.56) vs CD no pain 0.58 (0.26–4.58); p=0.04.

The ratio of TRPV1 to total nerve fibres, in the form of PGP 9.5-positive fibres, was calculated for all three groups (table 7). The TRPV1:PGP 9.5 ratio was significantly different between the IBD group 1 with pain and controls (p=0.001). Similarly, the ratio was significantly different between the IBD group 1 with pain, and the IBD group 2 without pain (p=0.02).

Table 7

TRPV1 staining normalised for total nerve expression (PGP 9.5 staining)

Univariate linear regression analyses for all the different variables revealed that average pain severity, VASav (r=0.66, p<0.001), patient group, that is, pain/no pain (r=−0.6, p<0.001), UCDAI score (r=0.39, p=0.04) and CRP level (r=0.33, p=0.02) were statistically significantly related to TRPV1 levels. When introduced into a stepwise multivariate linear regression model, only VASav was a significant predictor of TRPV1 level. Multiple linear regression analysis revealed that sex, age, disease duration, disease extent, disease type (ie, UC/CD) and smoking status were not significant independent predictors of TRPV1 levels.

Inflammatory markers

CD3-positive T cells were seen scattered throughout the mucosa in the biopsy specimens of all three groups (see figure 1). There was no significant difference between the three groups in the median percentage immunoreactive areas (see table 6); IBD with pain group 1 vs control p=0.88 and IBD pain group 1 vs IBD no pain group 2 p=0.17.

CD4-positive T cells were also seen in the mucosa in all the biopsies from the three groups, and there was no significant difference in the median percentage immunoreactive areas (see table 6 and figure 1); IBD with pain group 1 vs control p=0.97; IBD with pain group 1 vs IBD no pain group 2 p=0.39.

Faecal calprotectin

All patients had a faecal calprotectin of <50 mg/g. The painful IBD group 1 had values ranging from 18 to 40 (mean 28) mg/g. The no pain group 2 had a range of 15–45 (mean 25) mg/g.

Blood markers of inflammation

All subjects had a CRP of <10 mg/l, an ESR of <20 mm/h, platelets <450×1012/l and white cell count <11×109/l.

The painful IBD group 1 had a CRP range of 2–10 (mean 5) mg/l and ESR range of 4–20 (mean 12) mm/h. The no pain group 2 had a CRP range of 1–7 (mean of 4) mg/l; the ESR value ranged from 2 to 18 (average 10) mm/h.

Pain severity

For the painful IBD group 1, the VASav had a range from 1 to 8; the median value was 3.25 (IQR 2–4.5). The VASmax ranged from 2 to 10 with a median of 5 (3.25–7).

As the no pain group 2 and the controls did not complain of pain, they had an average and maximum pain score of zero.

Univariate linear regression analyses for all the immunoreactive species and other factors showed that only TRPV1 levels (r=0.7, p<0.001), duration of disease (r=−0.3, p=0.02) and CDAI (r=0.5, p=0.01) data were statistically significantly related to VASav. When these variables were introduced into a stepwise multivariate linear regression model, only TRPV1 (p<0.0001) was a significant predictor of VASav.

Univariate linear regression analyses for the quality of life and anxiety and depression showed that the HADS score (r=0.3, p=0.007), BDI score (r=0.3, p=0.02) and IBDQ score (r=0.5, p=0.0002) data were statistically significantly related to VASav. When these variables were introduced into a stepwise multivariate linear regression model, only the IBDQ score (p=0.001) was a significant predictor of VASav.

When all the variables including the immunoreactive species, patient variables, quality of life scores, disease variables and markers of inflammation were included in a stepwise multivariate linear regression model, only TRPV1 level (p=0.001; R2=0.8) was a significant predictor of VASav.

The VASav in the IBD with pain group 1 significantly correlated with TRPV1; Pearson correlation r=0.7, p=0.0008 and Spearman correlation r=0.6, p=0.003 (see figure 3). There was no correlation seen with pain score and CD3, CD4 or PGP staining.

Figure 3

Scatter plot to show the correlation between transient receptor potential vanilloid type 1 (TRPV1) levels and average pain scores (VASav) in subjects with inflammatory bowel disease (IBD) with pain. Pearson correlation r=0.7, p=0.0008. Spearman correlation r=0.6, p=0.003. VAS, visual analogue scale.

Anxiety and depression

Total HADS scores in the IBD group 1 ranged from 1 to 28 with a median of 13.5 (IQR 8–19.5). This was significantly higher than in the control group where the range was 1–25, median 8 (3.25–11); p=0.008. The HADS score was significantly greater in the IBD group 1 with pain than the IBD group 2 without pain; range 1–29, median 3 (2–10.5); p=0.003. When separated into the anxiety HADS subscore, again the IBD with pain group had a significantly greater median score of 8 (6–10) versus the no pain group median of 2 (1–7.5). There was no difference in the painful IBD versus control group, with median 9 (5–11); p=0.6. The depression HADS subscore in the painful IBD group with median 5 (3–8) was also significantly greater (p=0.007) than the no pain group median of 2 (1–4.5); there was no difference versus control group, median 3 (1–4); p=0.05.

Total BDI scores in the IBD with pain group 1 ranged from 0 to 31 with a median of 10.5 (5.5–22.5). This was not significantly different from the control group where the range was 0–21, median 7.5 (2.5–14); p=0.12. There was a significant difference between the BDI median scores in the IBD with pain group 1 compared with the IBD without pain group 2; range 0–27, median 4.5 (1–12); p=0.02.

Quality of life: IBDQ and SF-36

The total IBDQ score in the IBD with pain group 1 ranged from 54 to 156 with a median of 115 (IQR 86.5–139). This median score was significantly higher, representing a poorer quality of life, than the IBD without pain group 2 where the IBDQ scores ranged from 32 to 146 with a median of 55 (44–61.5); p<0.0001.

The SF-36 PCS scores in the IBD with pain group 1 ranged from 9.6 to 55.5 with a median value of 45 (IQR 37.4–49.1). This median score was significantly lower than the IBD without pain group 2 where the range was 25.3–58.6 with a median of 52.1 (42.3–55.5); p=0.006.

The SF-36 MCS scores in the IBD group with pain group 1 ranged from 19.2 to 63.1 with a median of 45.7 (IQR 32.4–55.1). In the IBD group 2 without pain there was not a significant difference in the median value compared with the pain group; the MCS scores ranged from 20.7 to 61.3, with a median value of 52.7 (45.1–58.1); p=0.06.

Discussion

The increased TRPV1 fibres in the colonic biopsies from patients with quiescent IBD with IBS-type symptoms and abdominal pain may help to explain the mechanism of these symptoms. This was found in the quiescent IBD group with pain when compared with both the control (3.9-fold greater) and quiescent IBD without pain (fivefold greater) groups. There was no difference in the PGP 9.5 staining between the IBD groups and the controls, suggesting that this is selective upregulation of TRPV1 fibres. As a CDAI score of <150 was an inclusion criterion, there will be a subgroup of patients with severe abdominal pain scores and no active inflammation that will be excluded, as abdominal pain is a component of CDAI, leading to a potential underestimate of the degree of upregulation of TRPV1. This is a highly selected group of patients and it is likely that with less rigorous inclusion criteria we would have seen even more striking changes of inflammation-associated TRPV1 nerve fibres, as previously reported by our group in active IBD.11 The IBD groups consisted of patients with CD and UC; the difference held when the groups were subdivided into patients with CD or UC with and without pain. This suggests there may be a common triggering pathway for sensitisation and TRPV1 upregulation, probably triggered by the initial inflammatory insult but persisting in the symptomatic patients. At the time of assessment all mucosal, faecal or systemic markers of inflammation were similar in patients with IBD in remission with or without pain. Nerve growth factor (NGF) is likely to be the main candidate driving the upregulation (see figure 4). NGF production is increased during inflammation in peripheral tissues and then transported in a retrograde manner to the cell bodies of TRPV1-expressing neurons.21 NGF acts via the p38 mitogen-activated protein kinase (MAPK) pathway, and via the RAC1–nicotinamide adenine dinucleotide phosphate (NADPH) oxidase pathway22 to increase TRPV1 protein23 in the dorsal root ganglia. NGF itself may also directly sensitise the TRPV1 receptor.24 Immunostaining for NGF is unfortunately difficult in gut mucosal biopsies as we have used.

Figure 4

Possible mechanisms leading to persistent hypersensitivity and pain. IBD, inflammatory bowel disease; NGF, nerve growth factor; TRPV1, transient receptor potential vanilloid type 1.

Isgar et al25 reported that 33% of the patients with UC compared with 7% of controls met the Manning criteria for IBS26; p<0.01. Minderhoud et al,2 using Rome II criteria, found IBS-type symptoms in one-third of patients with UC and 42% of patients with CD, associated with an impaired quality of life. Simren et al,3 using symptom criteria (moderate abdominal pain/discomfort and disturbed bowel habit in the preceding 1 week), found that 33% of patients with UC and 57% of patients with CD had IBS-like symptoms. Similarly, patients with IBS-like symptoms reported a reduced quality of life, and had higher anxiety and depression levels. Ansari et al27 found, using Rome II criteria, that 46% of patients with UC in remission had IBS-like symptoms (compared with 13% in the control group, p<0.001) and HRQoL was significantly reduced in patients with UC both in remission and with active disease. This literature suggests that IBS-like symptoms are present in a significant proportion of patients with quiescent IBD with a negative impact on HRQoL.

Mittermaier et al28 recruited inactive patients with IBD after a recent flare-up in an 18-month prospective study. A significant correlation between baseline BDI scores and number of relapses, along with the time to relapse, was observed. In addition, low HRQoL and anxiety were related to an increased frequency of relapses. They surmised that psychological factors and low HRQoL may negatively influence the course of IBD with increased relapse frequency.

In this cohort of patients, the quality of life was impaired in patients with IBD with pain/IBS-like symptoms. The total IBDQ score was significantly higher (p<0.0001) and SF-36 PCS lower (p=0.006) in the pain group when compared with the no pain IBD group; p<0.0001. As outlined above, the lower HRQoL experienced by the patients with IBD with pain may lead to problems with increased healthcare resource usage and more frequent presentation to physicians. The symptom of pain may lead to unnecessary investigations which may be invasive and have healthcare cost implications. Active disease may be mistakenly diagnosed and the patient treated with drugs which have potential serious side effects. The patient may become frustrated that they are not improving despite medication. As suggested by Mittermaier et al,28 the low quality of life may negatively impact on their IBD disease course and contribute to more frequent relapses.

Active inflammation may drive up TRPV1 levels and account for abdominal pain symptoms, along with poorer HRQoL. However, all patients had a normal colonoscopy (to the terminal ileum in CD), coupled with histologically quiescent disease, normal inflammatory markers, CDAI/UCDAI scores suggestive of inactive disease and similar levels of CD3 and CD4 immunohistochemical staining to controls. Patients with CD with ileal resections and hence a possibility of bile salt malabsorption contributing to symptoms were excluded.

TRPV1 upregulation has previously been reported in painful IBD. Yiangou et al11 looked at immunohistochemical expression of TRPV1 using full-thickness surgically resected samples in acute painful IBD. There were significantly greater numbers of TRPV1-immunoreactive nerve fibres in inflamed IBD specimens (median 6.0) than in controls (median 2.7; p=0.0025). NGF was proposed as the potential driver for the upregulation, and possibly the eicosanoids and hydrogen ions produced during inflammation.11 Other inflammatory conditions of the gut such as coeliac disease and acute gastrointestinal infections have also been reported to be associated with alterations in gastrointestinal gut motility and increased perception of sensory stimuli.29 Many animal studies support a role for mucosal inflammation resulting in sensorimotor dysfunction29 which may persist even after the mucosal inflammation has subsided. Various mechanisms have been proposed including a role for cytokine mediators such as interleukin-1β (IL-1 β) and IL-6 as well as proinflammatory neuropeptides such as SP.29 An increase in vasoactive intestinal peptide (VIP)- and calcitionin gene-related peptide (CGRP)-positive nerve fibres has been reported in IBD.30 31 The cause of TRPV1 remaining upregulated in a subset of patients with quiescent IBD is likely to involve a combination of central and peripheral factors culminating in increased NGF. The painful quiescent IBD group had significantly higher HADS and BDI scores compared with the no pain group. NGF levels have been shown to alter with psychosocial influences,32 33 with higher levels found in stress.

The role of TRPV1 in inflammation and colitis remains uncertain as conflicting data come from animal studies. Some suggest a protective role for TRPV1, for example Goso et al,34 where they gave topical capsaicin via an enema in a rat TNBS (trinitrobenzenesulfonic acid) colitis model and found a reduction in ulceration after 1 h. Other groups suggest that TRPV1 is not protective. For example, Nguyen et al35 used an intracolonic proteinase-activated receptor-2 (PAR2) agonist-induced mouse model of colitis and found that pretreatment with capsaicin attenuated the colitis. Kihara et al,36 in a dextran sulfate sodium (DSS) model of colitis, found that rats chemically denervated in the neonatal period using capsaicin, and adult rats given the TRPV1 antagonist capsazepine (CPZ), developed an attenuated colitis compared with placebo. However, these studies were carried out in animal models, with colitis generated by differing agents so translation to humans is difficult, where not only the physiological and homeostatic mechanisms, but also the disease-causing process are very different.

TRPV1 upregulation has been revealed in patients with quiescent IBD and IBS-like symptoms. Although the TRPV1 antibody has been used in previous studies and shown to be robust and specific,10 12 20 future studies using different scientific techniques such as western blot and in situ hybridisation may serve to reinforce these findings. A multivariate stepwise linear regression analysis showed TRPV1 levels as the sole significant predictor of the average pain score, and hence peripheral factors appear to be more important than central factors in predicting pain in patients with IBD. The severity of abdominal pain correlated positively with TRPV1 levels, suggesting that TRPV1 plays a role in the pathophysiology of ongoing pain in this group of patients. Furthermore there is evidence that TRPV1 may play an important role in intestinal inflammation. Therefore, TRPV1 provides a very attractive molecular drug target, not only for symptom control, but potentially also for disease modification in patients with IBD. Clinical trials with TRPV1 antagonists are needed to assess this potential.

Significance of this study

What is already known about this subject?

  • TRPV1 is an important polymodal receptor involved in inflammation and nociception, and plays a key role in visceral hypersensitivity.

  • TRPV1 has been implicated in various gastrointestinal disorders including oesophageal reflux pain, irritable bowel syndrome, active inflammatory bowel disease, Hirschprung disease and rectal hypersensitivity.

  • A significant proportion of patients with quiescent inflammatory bowel disease (up to 57% with Crohn's disease and 33% with ulcerative colitis) continue to complain of abdominal pain and irritable bowel syndrome-like symptoms despite resolution of inflammation.

What are the new findings?

  • We reported a significant increase of TRPV1-immunoreactive fibres in colonic biopsies from patients with quiescent inflammatory bowel disease with no inflammation and irritable bowel syndrome-like symptoms when compared with controls (3.9-fold greater; p<0.0001) and asymptomatic patients with quiescent inflammatory bowel disease (fivefold greater; p=0.0003).

  • TRPV1 expression correlated with abdominal pain severity.

  • HRQoL was significantly impaired in the quiescent inflammatory bowel disease group with irritable bowel syndrome-like symptoms.

How might it impact on clinical practice in the foreseeable future?

  • The findings suggest that TRPV1 plays a role in the pathophysiology of ongoing pain in this group of patients with quiescent IBD and IBS-like symptoms. Currently treatment options for this group of patients are limited and often disappointing in efficacy, with some being managed with opiates. Hence TRPV1 may be a potential therapeutic target for pain in these patients. Targeting TRPV1 would potentially be very attractive as the action is to block pain modulation rather than inflammatory mediators as is the case with many current analgesics.

Acknowledgments

We thank GlaxoSmithKline, Harlow, UK for support and Drs John B Davis, Iain Chessell and Chas Bountra for TRPV1 antibodies and advice. Dr Duolao Wang (London School of Hygiene and Tropical Medicine) is thanked for statistical help.

References

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Footnotes

  • Funding National Association of Colitis and Crohn's disease, GSK via Imperial College ADI.

  • Competing interests None.

  • Patient consent Obtained.

  • Ethics approval This study was conducted with the approval of the Hammersmith Research Ethics Committee.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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