Digestive Diseases and Sciences https://doi.org/10.1007/s10620-018-5002-y

ORIGINAL ARTICLE

Assessment of Serum sTREM‑1 as a Marker of Subclinical Inflammation in Diarrhea‑Predominant Patients with Irritable Bowel Syndrome Chao Du1,3 · Lijun Peng1 · Guanjun Kou2,3 · Peng Wang2,3 · Lin Lu1 · Yanqing Li2,3 Received: 9 January 2017 / Accepted: 23 February 2018 © Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract Background  Irritable bowel disease (IBS) is viewed upon as a functional disorder of subclinical inflammatory changes in recent years, and there is no reliable biomarker. Triggering receptor expressed on myeloid cells 1 (TREM-1), also produced in a soluble form (sTREM-1), is involved in the activation of inflammatory cascades of intracellular events and may play a role in pathogenesis of IBS. Aim  To investigate whether serum sTREM-1 level can be used as a marker of subclinical inflammation in D-IBS. Methods  Abdominal pain was quantified by a validated questionnaire. Expression level of TREM-1 in colonic mucosa as well as sTREM-1 level in serum was also detected. Furthermore, we investigated the involvement of TREM-1-associated macrophage activation in IBS-like visceral hypersensitivity. Results  No evidence for obvious inflammation was found in D-IBS patients. Serum sTREM-1 level in D-IBS patients was significantly higher than that in HCs, which was also significantly correlated with abdominal pain scores. We showed a marked increase in the proportion of TREM-1-expressing macrophages in D-IBS, which was significantly correlated with abdominal pain scores. Functionally, gadolinium chloride ­(GdCl3), a macrophage selective inhibitor, or LP17, the TREM1-specific peptide, significantly suppressed the visceral hypersensitivity in trinitrobenzene sulfonic acid (TNBS)-treated mice with IBS-like visceral hypersensitivity. Conclusions  Serum sTREM-1 level is significantly higher in D-IBS patients and positively correlates with abdominal pain, which may be initiated by TREM-1-associated macrophage activation, indicating the existence of subclinical inflammation in D-IBS. Therefore, serum sTREM-1 is a potential marker of subclinical inflammation in D-IBS. Keywords  Diarrhea-predominant irritable bowel disease · Soluble triggering receptor expressed on myeloid cells 1 · Subclinical inflammation · Macrophage activation · Abdominal pain

Introduction

* Lin Lu [email protected] * Yanqing Li [email protected] 1



Department of Gastroenterology, Linyi People’s Hospital, Shandong University, Linyi 276000, Shandong, People’s Republic of China

2



Department of Gastroenterology, Qilu Hospital, Shandong University, Jinan 250012, Shandong, People’s Republic of China

3

Laboratory of Translational Gastroenterology, Qilu Hospital, Shandong University, Jinan 250012, Shandong, People’s Republic of China



Irritable bowel disease (IBS) is a common functional intestinal disorder characterized by the presence of recurrent abdominal pain or discomfort associated with altered bowel movements and the absence of reliable biomarkers [1–4]. A recurrent sense of abdominal pain or discomfort in IBS is the most likely complaint to result in a large general practitioner visits. The pathophysiology of IBS is complex and still incompletely understood. Current therapies for IBS are suboptimal, targeting the symptoms and providing inconsistent and temporary relief, while the lack of a defined pathophysiology limits the therapies to cure these conditions [5, 6]. Traditional pathophysiological factors in IBS include dysregulation of the brain–gut axis, visceral hypersensitivity, altered gastrointestinal motility, as well as psychological

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factors [7, 8]. Currently, IBS is also viewed upon as a disorder of subclinical inflammatory changes supported by the increased number of mast cells and T cells in colonic mucosa, presence of some cytokines in peripheral blood and at tissue level, and high TNF-α and low IL-10 secretion genotypes [1, 7–9]. Hence, subclinical inflammation may play a role in D-IBS, but to date no serum inflammatory marker reflecting this putative inflammation has been found. Mucosal macrophages play an essential role in the homeostasis of the intestine [10, 11]. However, they also play important roles in intestinal inflammation, when they switch from the peaceful regulators to the powerful aggressors [11–13]. In inflammatory bowel disease (IBD) and experimental colitis, resident macrophages in colonic mucosa switch to activated ones that produce proinflammatory cytokines, such as TNF-α, IL-1, and IL-6, contributing to the dysregulation of mucosal immune response in IBD [12–14]. There are studies reporting that the number of mucosal macrophages is decreased in IBS patients [15, 16], but the role of macrophage in IBS and its possible mechanisms are not further investigated. Triggering receptor expressed on myeloid cells 1 (TREM-1), a member of the immunoglobulin superfamily, is expressed on a variety of innate immune cells including neutrophils, monocytes, dendritic cells, macrophage subsets, microglia and et al. [17–19]. Engagement of TREM-1 on macrophage cell surface results in activation of an inflammatory cascade of intracellular events, such as cytokine production [19]. TREM-1 has been indicated as an amplifier of the immune response in the context of infection [18]. Upregulation of TREM-1 in septic shock and other infections appears to be a significant step during the initiation of the cytokine production [18]. In experimental colitis and patients with IBD, TREM-1-expressing intestinal macrophages crucially amplify chronic inflammation in the intestine and correlates with the disease activity [20]. Release of the soluble receptor (sTREM-1) is one of the most intriguing features of TREM-1 [17]. The origin of sTREM-1 is complex. It is suggested that sTREM-1 may result from alternative splicing of mRNAs producing soluble secreted receptor isoforms, or from cleaving the extracellular domains off the cell surface [17]. Augmented sTREM-1 levels have been detected in the biological fluids (serum, bronchoalveolar lavage fluid, and gastric fluid) of patients suffering from a variety of diseases, including chronic obstructive pulmonary disease, cardiac arrest, rheumatoid arthritis, acute pancreatitis, and peptic ulcer disease [17, 21]. In patients with IBD, it is indicated that elevated serum sTREM-1 level is not only observed in patients with active disease, but also sensitive enough to be detected in patients with quiescent disease, which always showed IBSlike symptoms [17, 21, 22]. However, serum sTREM-1 levels have not been evaluated in patients with IBS.

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Our study aimed at evaluating the serum sTREM-1 level in patients with D-IBS and its correlation with the degree of abdominal pain in patients, thus assessing whether serum sTREM-1 can serve as a marker of subclinical inflammation in D-IBS.

Methods Subjects A total of 112 patients with D-IBS (59 women and 53 men; age 45.6 ± 12.7 years) and 46 healthy controls (24 women and 22 men; age 43.1 ± 11.4 years) were recruited. This study was approved by the clinical ethical committee of the Qilu Hospital of Shandong University and the clinical ethical committee of the Linyi People’s Hospital of Shandong University, and written informed consent in agreement with the Declaration of Helsinki was obtained from each patient. The diagnosis of D-IBS was based on the Rome III criteria [3]. Controls were selected from patients undergoing colonoscopy for polyps and cancer surveillance; all received negative results. Subjects were excluded if they had bile acid malabsorption; received nonsteroidal anti-inflammatory drugs or other anti-inflammatory drugs (including mast cell stabilisers, histamine antagonists, probiotics, immunosuppressants and steroids) or on pain medications; had undergone major abdominal surgery; or had any organic syndrome, including celiac disease, allergic diseases, and psychiatric disorders as assessed by history taking, appropriate consultations, and laboratory tests [2]. Sera were isolated from all subjects. All sera were analyzed for the concentration of sTREM-1. Participants underwent colonoscopy following a standard bowel preparation with polyethylene glycol, and water enema was used to cleanse stool if necessary. Specimens were taken from the rectosigmoid junction to standardize the site of sampling. The biopsy was used for western blot and real-time RT PCR for detection of TREM-1 in colonic mucosa, as well as for immunofluorescence studies.

Questionnaires Patients were asked to score the frequency and severity of their abdominal symptoms over the last 3 months by using a validated questionnaire [2, 23]. The severity of abdominal pain/discomfort was graded 0–4 according to the impact on patients’ daily activities: (0) absent; (1) mild (not influencing activities); (2) relevant (diverting from but not urging modification of activities); (3) severe (influencing activities markedly enough to urge modifications); and (4) extremely severe (precluding daily activities). The frequency of abdominal pain/discomfort was graded 0–4 according to the following

Digestive Diseases and Sciences

scale: (0) absent; (1) up to 1 day/week; (2) 2 or 3 days/week; (3) 4–6 days/week; and (4) daily.

ELISA Detection After collection, peripheral blood was centrifuged for 5 min at 1500g, and serum was collected. Human serum sTREM-1 levels were determined by an in-house optimized ELISA using the Human TREM-1 Duoset (R&D) for detection, while anti-TREM-1 monoclonal antibody (Hycult Biotechnology, Uden, The Netherlands) was used as an alternative capture antibody [21, 24]. The anti-TREM-1 monoclonal antibody was coated at 10 μg/ml in PBS, and then the other procedures were the same as the manufacturers’ instruction of the Human TREM-1 Duoset (R&D). Colonic mucosa was cut into pieces and was initially homogenized in a prepared ice-cold 100 mm Tris–HCl buffer, pH 7.0, containing a cocktail of protease inhibitors (Beyotime, Shanghai, China) supplemented with 1 mm phenylmethanesulfonyl fluoride. Levels of TNF-α, IL-1β, and IL-6 in serum and colonic mucosa were measured by ELISA kits (KYM, Beijing, China) according to the manufacturers’ recommendations.

Histological and Western Blot Analysis Histopathological studies were performed on paraffinembedded, 4-mm-thick mucosa sections, stained with hematoxylin and eosin. Histology was evaluated microscopically in a blinded fashion. Total protein was extracted from biopsy samples in radioimmunoprecipitation assay (RIPA) buffer (Beyotime Institute of Biotechnology, Shanghai). Protein was quantified by using a BCA protein quantification kit (Beyotime). An amount of 20 μg total protein from each sample was separated by SDS-PAGE and transferred to a PVDF membrane (0.22 μm pore; Millipore, Bedford, MA, USA). After being blocked with 5% skim milk powder diluted in TBS containing 0.1% Tween-20 for 1 h, the membrane was incubated with the primary antibody (anti-TREM-1 monoclonal antibody, Hycult Biotechnology, Uden, the Netherlands) at 4 °C overnight. Horseradish peroxidase-conjugated secondary antibodies (Zhongshan Gold Bridge, Beijing, China) were probed the next day, and an enhanced chemiluminescent substrate (Millipore) was used to detect the protein bands.

Real‑Time RT PCR Total RNA was isolated by Trizol reagent method (Invitrogen, San Diego, CA, USA). RNA from each sample was reverse-transcribed into cDNA. For real-time PCR, cDNA was amplified with SYBY Green (Takara, Japan) in a fluorescence thermocycler (LightCycler; Roche Diagnostics). The primers for TRME-1 were: forward 5′ TGG​TCT​TCT​

CTG​TCC​TGT​ TTG 3′ and reverse 5′ ACT​CCC​TGC​CTT​ TTA​CCT​ C 3′. The housekeeping gene β-actin was used for normalization of TREM-1 mRNA expression [25].

Immunofluorescence Biopsies were immediately fixed in 10% buffered formalin. For immunofluorescence, paraffin-embedded tissues were cut into 4-µm-thick sections and were incubated overnight with a mixture of CD68 mouse antibody (1/100; Millipore) and TREM-1 rabbit antibody (1/200 dilution; Santa, Cruz, CA, USA). Rhodamine-conjugated goat anti-mouse IgG antibody (1:100; Zhongshan Gold Bridge, Beijing, China) and a FITC-conjugated goat anti-rabbit antibody (1:100; Zhongshan Gold Bridge) were used to detect the above primary antibodies, respectively. Additional controls (PBS in place of the primary antibodies) were conducted to exclude the cross reaction during double labeling. Slides were then detected using a fluorescence microscope (Olympus IX-70). As for each tissue section, we chose five 40× magnification fields at random and two independent blinded observers got the mean area values of positive signals for final analysis by using the Image-Pro Plus 6.0 software.

Animals Male C57BL/6J mice weighing 20–25  g (aged about 8–10 weeks) were used in this study. All mice were purchased from Beijing HFK Bioscience Company. Animals were housed on a temperature- (20 ± 1 °C) and light-controlled cycle (12 h) with free access to standard laboratory chow and tap water. All procedures were approved by the Animal Care and Use Committee of Shandong University and the Animal Care and Use Committee of Linyi People’s Hospital and were performed in accordance with the Animal Management Rules of the Chinese Ministry of Health. As for electromyographic (EMG) recordings, silver electrode-implanted mice were prepared before all mice experiments according to Christianson’s methods [26]. A sterile tube (a 12- to 15-cm-long polyethylene catheter with two sets of sterilized silver wires passing through) was inserted into a neck incision and passed subcutaneously (cranial to caudal) until it emerged at the abdominal incision. Remove the polyethylene catheter and tape the silver wires. Implant the electrode wires into the abdominal musculature. The other two ends were exteriorised from the back of the mice neck via a subcutaneous tunnel for connection to an amplifier. Suture the neck incision and the abdominal incision. After surgery, mice were allowed to recover for at least 4 days before performing CRD and TNBS administration. The mice model with IBS-like visceral hypersensitivity was induced by rectal administration of TNBS (2 mg in 50% ethanol, 0.1 ml in total) (Sigma-Aldrich, St. Louis, MO) via

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a polyethylene catheter inserted 2–3 cm from the anus. An equivalent volume of PBS was instilled into control mice. Mice were sacrificed on day 28 after induction of colitis. ­GdCl3 (10 mg/kg body weight; dissolved in 0.1 ml PBS as a vehicle) was administrated to mice on day 3 of TNBS application through intravenous route. The PBS vehicle (0.1 ml) was administrated as a control. As for the LP17-treated TNBS group, mice were treated with either LP17, an antagonistic TREM-1 peptide, or a sequence-scrambled control peptide as previously described [27, 28]. The peptides were chemically synthesized by the Pepscan Systems. Mice were treated once daily with 200 μg peptide after TNBS administration for the next 5 days, injected i.p. in 200 μl saline. CRD started on day 28 after TNBS administration under pressures of 15, 30, 45, and 60 mmHg [26]. Mice were kept in a plastic chamber (4.5 cm in diameter and 10 cm in length) to restrict their movement. A polyethylene catheter with 1 cm polyethylene balloon secured to one end was inserted into the colon, 1 cm proximal to the anus. The catheter was then taped to the tail to hold the balloon in position. After the mice were fully awake and recovered from anesthesia, the balloon was progressively inflated from 0 to 60 mmHg in 15 mmHg steps (15, 30, 45, and 60 mmHg), each step lasting 10 s with a 5 min non-distension interval and repeated three times. The EMG activity was used to evaluate the VMR of mice and was recorded by the Powerlab BL-420E system. Data as expressed by the area under the curve were then analyzed by Labchart v5.0 software. EMG activity was finally corrected for the baseline activity and expressed as “% of baseline.”

Statistical Analysis All data given were expressed as mean values ± SD or medians (IQR). Univariate analysis of the characteristics of the population involved the Chi-square test, independent Student t test, or Manne–Whitney nonparametric test. Two-group differences were determined by an independent Student’s t test and multiple comparisons by a one-way ANOVA test. Correlations between two parameters were assessed by Spearman rank correlation. All analyses involved use SPSS

version 22.0. Differences were considered significant at P < 0.05.

Results Group Characteristics The clinical characteristics of healthy controls and patients with D-IBS are described in Table 1. The groups were not statistically different for sex (P = 0.29) or age (P = 0.36). Among the included 112 patients with D-IBS, the mean pain intensity was moderate for patients with D-IBS and significantly higher than that for controls (severity: D-IBS: 2.3 ± 0.8 vs HCs: 0.5 ± 0.7, P < 0.0001; frequency: D-IBS: 3.0 ± 1.1 vs HCs: 0.6 ± 0.8, P < 0.0001). No significant difference was found between male and female patients with IBS in terms of abdominal pain/discomfort severity and frequency scores (severity: male: 2.3 ± 0.8 vs female: 2.2 ± 0.7, P = 0.94; frequency: male: 3.0 ± 1.2 vs female: 3.1 ± 1.1, P = 0. 91) (Table 2).

Analysis of Inflammation and ELISA Detection of Cytokines No evidence for inflammation was found in D-IBS according to the H&E-stained sections. No differences were detected between D-IBS patients and healthy controls regarding cytokine levels of TNF-α, IL-1β, and IL-6 in serum and colonic mucosa (data not shown).

Unaltered Protein and mRNA Levels of TREM‑1 in Colonic Mucosa in D‑IBS Patients with HCs Western blot analysis revealed that there was a relatively low expression of TREM-1 in the intestine of patients with D-IBS. Relative protein level of TREM-1 in colonic mucosa was not significantly different between HCs and D-IBS patients (P = 0.71) (Fig. 1a). Thus, the protein level of TREM-1 in colonic mucosa is not altered in D-IBS patients compared to HCs. Similarly, there was no significant

Table 1  Clinical characteristics of the study subjects Sex (F/M) Age (years) IBS duration (years) Severity of abdominal pain/discomfort Frequency of abdominal pain/discomfort

HCs (n = 46)

D-IBS (n = 112)

P value

24/22 43.1 ± 11.4 – 0.5 ± 0.7 0.6 ± 0.8

59/53 45.6 ± 12.7 6.7 ± 8.9 2.3 ± 0.8 3.0 ± 1.1

0.29 0.36 – <0.0001 <0.0001

Quantitative data are expressed as mean (SD) or median (range) D-IBS diarrhea-predominant irritable bowel disease, F female, HCs healthy controls, M male

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Digestive Diseases and Sciences Table 2  Clinical characteristics of D-IBS patients

D-IBS

Age (years) IBS duration (years) Severity of abdominal pain/discomfort Frequency of abdominal pain/discomfort

P value

Female (n = 59)

Male (n = 53)

48.7 ± 14.7 5.8 ± 9.3 2.2 ± 0.7 3.1 ± 1.1

43.0 ± 10.0 7.5 ± 8.5 2.3 ± 0.8 3.0 ± 1.2

0.053 0.41 0.94 0.91

Quantitative data are expressed as mean (SD) or median (range) D-IBS diarrhea-predominant irritable bowel disease Fig. 1  Expression of protein and mRNA TREM-1 in colonic mucosa (a, b) and level of sTREM-1 in serum (c, d) in patients with D-IBS and healthy controls. D-IBS diarrheapredominant irritable bowel disease, HCs healthy controls, sTREM-1 soluble triggering receptor expressed on myeloid cells 1, TREM-1 triggering receptor expressed on myeloid cells 1

difference in the mRNA level of TREM-1 between HCs and D-IBS patients (Fig. 1b).

Augmented Serum sTREM‑1 Levels in Patients with D‑IBS

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Compared with HCs, exhibiting median serum sTREM-1 levels of 130.7 pg/ml, serum sTREM-1 levels were significantly increased in patients with D-IBS with median basal serum levels of 139.6 ± 1.8 pg/ml (P = 0.0052) (Fig. 1c). The serum levels of sTREM-1 were similar between male and female participants in the control group or the D-IBS group (Fig. 1d). However, the elevation of serum sTREM-1 levels in female patients with D-IBS was significant as compared with female HCs (female D-IBS: 139.7 ± 2.9 pg/ml vs female HCs: 129.1 ± 1.9 pg/ml, P = 0.020).

Digestive Diseases and Sciences

It is known that immunological dysregulation participates in the pathogenesis of IBS and may be responsible for IBS-like

visceral hypersensitivity [1, 8, 16]. Macrophage in colonic mucosa plays an important role in colonic inflammation in intestinal inflammatory diseases [12–14], but its role in the pathogenesis of IBS is still unclear. Previous studies have shown that the number of macrophages is decreased in IBS patients [15, 16]. In this study, we also found that the relative number of macrophages was significantly decreased in colonic mucosa in D-IBS patients compared to HCs through immunofluorescence assay (P < 0.05; Fig. 2). To investigate whether macrophages in colonic mucosa have the capacity of expressing pain-related molecules in D-IBS patients, we further detected the expression of TREM-1 in mucosa. While the results showed that although the expression of TREM-1 in colonic mucosa was not statistically different between D-IBS patients and HCs, the proportion of TREM-1-expressing macrophages was significantly

Fig. 2  Detection of macrophages and TREM-1 expression on macrophages in colon of D-IBS patients. a, b The number of macrophages was significantly decreased in colonic mucosa in D-IBS patients compared to HCs through immunofluorescence assay. The

expression of TREM-1 in colonic mucosa was not statistically different between D-IBS patients and HCs. However, the proportion of TREM-1-expressing macrophages was significantly higher in colonic mucosa in D-IBS patients than that in HCs

Increased Expression Level of TREM‑1 on Macrophage in D‑IBS

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Digestive Diseases and Sciences

higher in colonic mucosa in D-IBS patients than that in HCs (P < 0.05; Fig. 2). Therefore, mucosal macrophages in D-IBS patients showed a higher expression level of TREM1. Next, we sought to investigate the relationship between macrophage and pain generation in D-IBS patients.

Correlation Analysis In patients with D-IBS, there was no significant alteration in protein and mRNA TREM-1 levels in colonic mucosa, but a significant elevation of sTREM-1 in serum. It was revealed that mucosal protein and mRNA TREM-1 levels did not correlate with serum sTREM-1 level (data not shown). However, a significant correlation was found between serum sTREM-1 level and both severity and frequency of abdominal pain/discomfort (r = 0.55, P < 0.001 and r = 0.34, P < 0.002, respectively) (Fig. 3a, b). Therefore, the level of sTREM-1 in serum significantly correlated with the degree of abdominal pain in patients with D-IBS. Previous studies found that decreased number of macrophages in colonic mucosa is not associated with the

presence of visceral hypersensitivity in IBS [16]. Our study also showed that the decreased number of macrophages in colonic mucosa did not correlate with abdominal pain severity and frequency scores in D-IBS patients (data not shown). Therefore, we propose a question whether macrophage in colonic mucosa plays a role in visceral hypersensitivity in D-IBS. Further investigation indicated that TREM-1 expression on macrophages in colonic mucosa correlated significantly with abdominal pain severity and frequency scores in D-IBS patients (Fig. 3c, d). Accordingly, macrophages in colonic mucosa in D-IBS patients have the capacity of expressing pain-related molecules, such as TREM-1, which plays an essential role in visceral hypersensitivity in D-IBS. Together, these data provide a foundation for the direct participation of TREM-1-expressing macrophages in the process of pain generation in D-IBS patients. Inhibition of macrophage activation or blockade of TREM-1 improved visceral hypersensitivity in TNBS mice model. For further study, TNBS-induced colitis in mice, one of the animal models of IBS that likewise results in delayed maintained visceral hypersensitivity, was used in

Fig. 3  Correlation of serum sTREM-1 with severity of abdominal pain (a, b). Increased TREM-1 expression on macrophages in colon correlates with abdominal pain scores in D-IBS patients

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Digestive Diseases and Sciences

our study. It was shown that the visceromotor response (VMR) evoked by colorectal distention (CRD) increased according to the increase in ballon pressure that mice presented IBS-like visceral hypersensitivity to CRD after TNBS administration for 28  days in this TNBS model (Fig. 4). To further investigate the effect of macrophage on visceral hypersensitivity in TNBS model, we inhibited the activity of macrophage through using G ­ dCl 3 intravenously. It was shown that administration of ­G dCl 3 in mice significantly decreased the VMR scores under CRD

pressures of 30, 45, and 60 mmHg in TNBS-treated mice with IBS-like visceral hypersensitivity (Fig. 4a, b). These results suggest that ­GdCl3, a selective macrophage inhibitor, suppress the TNBS-induced visceral hypersensitivity to CRD. Next, we evaluated the potential of TREM-1 by using LP17 in pain generation in the TNBS model. The results displayed that the elevated VMR scores under CRD pressures of 30, 45, and 60 mmHg were significantly decreased after the blockade of TREM-1 in TNBS-treated mice(Fig. 4a, c). Together, mucosal macrophage may be

Fig. 4  GdCl3 or LP17 significantly suppressed the visceral hypersensitivity in mice. a, b ­GdCl3-mediated inhibition of macrophages significantly decreased VMR scores under CRD pressures of 30, 45,

and 60  mmHg. a, c Blockade of TREM-1 using LP17 significantly decreased the elevated VMR scores under CRD pressures of 30, 45, and 60 mmHg

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involved in IBS-like visceral hypersensitivity, and this process is likely initiated by TREM-1-associated activation.

Discussion IBS is an intestinal disorder characterized by abdominal pain or discomfort and an alteration in bowel habits. A group body of evidence has shown that subclinical inflammation plays a role in the pathogenesis of D-IBS, but until now there is no serum marker of subclinical inflammation. To the best of our knowledge, TREM-1 has never been investigated in D-IBS patients. In the present study, we investigated the potential of serum sTREM-1 to serve as a marker of subclinical inflammation in D-IBS. In our study, histological analysis showed that no evidence for inflammation was found in D-IBS patients. Also, no differences were detected between D-IBS patients and HCs regarding cytokine levels of TNF-α, IL-1β, and IL-6 in serum and colonic mucosa. Therefore, there was no obvious inflammation in D-IBS patients. Biopsies from patients with D-IBS revealed no significant difference in protein and mRNA levels of TREM-1 as compared with HCs. However, our findings demonstrated a significant augmented level of serum sTREM-1 in patients with D-IBS, and it was closely correlated with the degree of abdominal pain in D-IBS. It was reported that TREM-1 mRNA expression in intestinal mucosa was not increased in patients with quiescent IBD, while significantly elevated serum sTREM-1 levels were observed compared to normal controls [21]. Therefore, intestinal TREM-1 mRNA level and serum sTREM-1 level did not correlate in IBD patients with quiescent disease. And it was hypothesized that the elevated serum sTREM-1 level preceded an enhanced expression of intestinal TREM-1 in IBD patients with quiescent disease, and subclinical mucosal inflammation might be sufficient to cause minor intestinal epithelial injury and translocation of bacterial products into the circulation in patients with D-IBS, leading to the release of sTREM-1 into serum distantly from the intestinal mucosa [21]. In our study, we also found that the proportion of macrophages was significantly decreased in colonic mucosa in D-IBS patients compared to HCs, which was in accordance with previous studies [15, 16]. However, the decreased number of macrophages in colonic mucosa did not correlate with abdominal pain severity and frequency scores in D-IBS patients. Next, we investigated the expression of TREM-1 on macrophages in colon, and the results showed that the expression of TREM-1 in colonic mucosa was not statistically different between D-IBS patients and HCs, but the proportion of TREM-\1-expressing macrophages was significantly higher in colonic mucosa in D-IBS patients than that in HCs. Also, TREM-1 expression on macrophages in

colonic mucosa correlated significantly with abdominal pain severity and frequency scores in D-IBS patients. TNBS-induced colitis in mice likewise results in delayed maintained visceral hypersensitivity, which is regarded as a model of some aspects of IBS [29]. In our study, it was shown that mice presented IBS-like visceral hypersensitivity after TNBS administration for 28 days. To further investigate the role of macrophages in the TNBS model, we inhibited the activity of macrophage through using G ­ dCl3 intravenously to confirm the role of macrophages in visceral hypersensitivity. It was shown that administration of ­GdCl3 in mice significantly decreased the elevated VMR scores under CRD pressures of 30, 45, and 60 mmHg in TNBStreated mice with IBS-like visceral hypersensitivity. Also, we blocked TREM-1 using LP17 to investigate the role of TREM-1 in pain generation in the TNBS model. The results displayed that the elevated VMR scores under CRD pressures of 30, 45, and 60 mmHg were significantly decreased after the blockade of TREM-1 in TNBS model. Therefore, macrophages in colon play a significant role in pain generation in TNBS-treated mice with IBS-like visceral hypersensitivity, and TREM-1 may participate in this process. The level of serum sTREM-1 was significantly elevated in D-IBS patients, which may be initiated by TREM-1-associated macrophage activation, and this elevation closely correlated with the degree of abdominal pain/discomfort in D-IBS patients, indicating a potential to serve as a marker of subclinical inflammation in D-IBS. Current therapies for IBS are suboptimal, targeting the symptoms and providing inconsistent and temporary relief, and TREM-1-associated macrophage activation may be a potential treatment target in IBS. Therefore, subclinical inflammation may contribute to the pathophysiology of pain in IBS and provide useful percepts into potential treatments for this condition, which deserves further studies.

Conclusions The increased level of serum sTREM-1 was demonstrated in patients with D-IBS, and it may be initiated by TREM-1-associated macrophage activation. Therefore, serum sTREM-1 has a potential to serve as a marker of subclinical inflammation in D-IBS for better diagnosis and TREM-1-associated macrophage activation may be a possible treatment target. Acknowledgments  This research was supported by the National Natural Science Foundations of China (No. 81500425), the Shandong Provincial Natural Science Foundation, China (No. ZR2014HL018), and the Project of Medical and Health Technology Development Program in Shandong Province (No. 2015WS0370). The authors appreciate the considerable assistance from the Key Laboratory of Cardiovascular Remodeling and Function Research in the Qilu Hospital of Shandong

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University and the Central Laboratory in Linyi People’s Hospital of Shandong University.

Compliance with ethical standards  Conflict of interest  There is no financial conflict of interest to declare.

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