TIMP-1 As a Promising Therapeutic Indicator for the Intestinal Inflammation and Fibrosis.

doi:10.21203/rs.3.rs-530142/v1

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TIMP-1 As a Promising Therapeutic Indicator for the Intestinal Inflammation and Fibrosis.

https://doi.org/10.21203/rs.3.rs-530142/v1

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Background: Intestinal fibrosis is a major complication of inflammatory bowel disease (IBD), which can be prevented with anti-inflammatory therapies. However, inflammatory biomarkers do not necessarily reflect the progression of fibrosis, and hence, an indicator that reflects the therapeutic effects of available anti-inflammatory drugs on fibrosis is needed. This study shows that tissue inhibitor of metalloproteinase-1 (TIMP-1) is a potential therapeutic indicator for ongoing intestinal fibrosis in IBD patients.

Methods: TIMP-1 mRNA levels in human myofibroblasts were measured, and TIMP-1 expression in the intestine of 10 Crohn’s disease (CD) patients was examined by immunohistochemistry. Colitis was induced in C57BL/6 mice by administration of 1.5% dextran sodium sulfate for 7 days; then, the mice were treated with anti-TNF-α antibody or phosphate-buffered saline for 14 days. Intestinal fibrosis was evaluated by Masson’s trichrome staining, and TIMP-1 mRNA levels in the intestinal tissue and plasma TIMP-1 levels were measured.

Results: TIMP-1 expression in human fibroblasts increased after differentiation into myofibroblasts. TIMP-1 expression in CD intestinal tissue was significantly higher in the inflammation area than in the fibrotic or normal areas. In a murine colitis mode in which TIMP-1 mRNA levels were increased, anti-TNF-α antibody administration significantly attenuated inflammation and fibrosis assessed by a reduction in TIMP-1 mRNA levels in intestinal tissue and plasma TIMP-1 levels.

Conclusions: The results in this study show that TIMP-1 is a potential new biomarker that could be used to assess the effectivity of different therapeutic strategies for intestinal inflammation and fibrosis in IBD patients.

Gastroenterology & Hepatology

Inflammatory bowel disease

intestinal fibrosis

tissue inhibitor of metalloproteinase-1

anti-TNF-α antibody

Inflammatory bowel diseases (IBD) such as ulcerative colitis or Crohn’s disease (CD) are chronic, progressive, and relapsing inflammatory disorders of unknown etiology that can lead to gastrointestinal disorders like abdominal pain and diarrhea. The cause of IBD is thought to be related with genetic, environmental and intestinal microbial factors, although the exact etiology of IBD still remains to be fully elucidated [1]. Fibrosis is a major complication of IBD, and is especially common in CD patients. Approximately 75% of patients with CD eventually develop intestinal fibrosis after at least one surgical procedure [2]. Transmural inflammation involving all layers of the intestinal wall, which leads to dysfunctional wound healing and abnormal collagen accumulation, is considered a major cause of excessive fibrosis and stricture formation [3]. Despite the recent advances in the understanding of the pathophysiological mechanism of IBD, as well as in the anti-inflammatory therapies, a medical treatment that can effectively reverse the fibrosis in IBD patients is not available yet [4].

Several studies have shown that anti-tumor necrosis factor (TNF) agents, such as infliximab and adalimumab, can reduce inflammation and consequently prevent the progression of intestinal fibrosis [5–7]. Infliximab has shown to have a preventive effect on the recurrence of CD after an endoscopic procedure, including preventing the luminal narrowing 18–24 months after surgery for CD patients [8, 9]. However, these compounds do not reduce the incidence of intestinal strictures in CD [2, 10–13], and therefore, developing new therapies that prevent intestinal fibrosis and reduce the incidence of intestinal strictures is still necessary. When both inflammatory biomarkers and clinical symptoms are considered to manage the inflammation alterations in CD patients, outcomes are better than when decisions are only symptom-driven [14]. Similarly, treatment decisions for preventing intestinal fibrosis should also be based on objective indicators like biomarkers levels. However, existing inflammatory biomarkers such as C-reactive protein, erythrocyte sedimentation, serum amyloid A, immunological fecal occult blood test, and fecal calprotectin are not suitable biomarkers to be used in this context. As there is a complex interface between inflammation and fibrosis, including cytokine networks; the anti-inflammatory effect of a certain therapy and the inflammatory biomarkers profile do not necessarily reflect the therapeutic effect of the compound. Hence, inflammatory markers cannot be used as indicators of the prevention of intestinal fibrosis, and it is necessary to find new, different indicators.

Fibrosis refers to the accumulation of collagen-rich extracellular matrix (ECM) in response to tissue damage, and it is caused by repeated epithelial injury due to inflammation. The epithelial injury causes the accumulation and activation of mesenchymal cells, such as fibroblasts and myofibroblasts, as well as the infiltration of inflammatory cells [15–17]. Excessive accumulation of ECM occurs due to an aberrant restoration of the tissue architecture [18]. ECM accumulation is regulated by two protein families: matrix metalloproteinases (MMPs), which degrade the ECM, and tissue inhibitors of metalloproteinases (TIMPs), which inhibit the MMPs function. Hence, the balance between fibrogenesis and fibrolysis is controlled by the reciprocal relationship between MMPs and TIMPs. During fibrogenesis, an excess of TIMPs over MMPs occurs, which results in the inhibition of matrix degradation [19]. TIMP-1, a member of the TIMP family, is a potent inhibitor of many MMPs [17]. Since several studies have shown TIMP-1 overexpression in profibrotic environments, such as scar tissue formation, pulmonary fibrosis, and diabetic nephropathy, TIMP-1 is considered to play an important role in restricting ECM proteolysis [20]. In addition, serum TIMP-1 is one of the index factors included in in the enhanced liver fibrosis score, which is used to evaluate liver fibrosis [21]. Increased expression of TIMP-1 has also been shown in fibrotic lesions in CD patients and in a murine model of colitis [22–25]. Since TIMP-1 is strongly associated with organ fibrosis and inhibits the degradation of ECM, it is possible that TIMP-1 is a suitable biomarker to predict the therapeutic effect of anti-inflammatory agents on ongoing intestinal fibrosis.

In the present study, we show that the expression levels of TIMP-1 reflect the prospective effect of anti-inflammatory therapies that aim to prevent intestinal fibrosis progression using a murine colitis model. These results suggest that TIMP-1 has potential applications as an indicator of ongoing intestinal fibrosis in IBD.

Isolation of fibroblasts from human colon tissues and TGF-β treatment

Human colonic fibroblasts were isolated from the resected colon tissue of patients undergoing endoscopic submucosal dissection for transverse colon adenoma, as described previously [26]. The fibroblasts were maintained in Dulbecco’s modified essential medium and Ham’ s F-12 (Wako, Osaka, Japan) supplemented with 10% heat-inactivated fetal bovine serum and 1% antibiotic-antimycotic at 37 °C in an atmosphere containing 5% CO₂. The fibroblasts were grown in serum-free media for 24 h and then exposed to recombinant human transforming growth factor beta (TGF-β) (10 ng/mL, R&D Systems, Minneapolis, MN, USA) for 72 h. This study was approved by the Tohoku University ethics committee (no. 2020-1-482), and informed consent was obtained from the patients prior to the study. This study was performed in accordance with the standards of the Declaration of Helsinki and the current ethical guidelines.

Patients and tissues

This study group consisted of 10 patients with established CD diagnoses who consecutively underwent surgery for intestinal strictures from 2019 to 2020 at Tohoku University Hospital. Of these patients, six were female and four were male, with ages ranging from 19 to 60 years (mean 37.6 years, Table 1). Informed consent was obtained from all of the patients, and the Tohoku University ethics committee approved the research protocols for this study (no. 2020-4-064). This study was performed in accordance with the standards of the Declaration of Helsinki and the current ethical guidelines.

Table 1. Patient characteristics

Case	Age	Sex	Location	Disease Duration (months)	Biological agent
1	41	F	colon	372	Infliximab
2	59	M	small bowel	28	Infliximab
3	45	F	small bowel	324	Adalimumab
4	33	F	colon	216	Adalimumab
5	60	F	colon	132	Ustekinumab
6	27	F	small bowel	156	Ustekinumab
7	31	M	colon	3	-
8	25	M	small bowel	1	-
9	38	F	small bowel	252	-
10	19	M	colon	27	-

Immunohistochemistry for TIMP-1

Formalin-fixed, paraffin-embedded sections were cut from the tissue to a thickness of 3 μm. Paraffin sections were deparaffinized in xylene and rehydrated with graded ethanol and distilled water. To visualize the TIMP-1 antigens, sections were heated in a microwave oven for 15 min in citrate buffer at pH 6. The nonspecific binding sites were blocked with 1% bovine serum albumin (Invitrogen, Carlsbad, CA, USA) in phosphate-buffered saline (PBS) for 30 min. The samples were then incubated overnight at 4ºC with the primary antibody anti-TIMP-1 rabbit monoclonal antibody (Abcam, Cambridge, UK; dilution 1:300). After blocking the endogenous peroxidase activity with methanol containing 0.3% hydrogen peroxide (H₂O₂), the sections were sequentially incubated with EnVision+ System‐HRP (Dako, Glostrup, Denmark), and the immune complexes were visualized with 3,3-diaminobenzidine (DAB; Dojindo, Kumamoto, Japan) solution (1 mmol/L DAB, 50 mmol/L Tris-HCl buffer [pH 7.6], and 0.006% H₂O₂). All sections were counterstained with hematoxylin. For the negative controls, the incubation with the primary antibody step was omitted. As expected, no detectable staining was evident. Images were captured using a BZX-800 fluorescence microscope (Keyence, Osaka, Japan). To evaluate TIMP-1 expression, the percentage of TIMP-1 positive cells was calculated as follows: the number of TIMP-1 positive cells/the number of all cells in a field of view.

Establishment of a murine colitis model

All animal experiments were approved by the Tohoku University Animal Care Committee. The study was carried out in compliance with the ARRIVE guidelines (http://www.nc3rs.org.uk/page.asp?id=1357). Nine week old male C57BL/6 mice weighing 22–25 g were purchased from SLC Japan, Inc. (Shizuoka, Japan). Mice were fed with common commercial pellet diets and ordinary tap water, and housed in an air-conditioned room at a temperature of 24 °C. Intestinal fibrosis was induced in the models by oral administration of 1.5% dextran sodium sulfate (DSS) (MP Biomedicals, Santa Ana, CA, USA) dissolved in the drinking water. The mice were acclimatized for the first week. At 10 weeks of age, mice were randomly divided into two groups: DSS mice treated with PBS (PBS group, n = 15) and DSS mice treated with anti-TNF-α antibody (anti-TNF-α group, n = 15). Mice received 1.5% DSS drinking water on days 0–7 and regular drinking water on days 7–21. One hundred microliters of PBS or 20 μg of mouse anti-TNF-α monoclonal antibody (Clone MP6-XT22, R&D Systems) in 100 μL PBS were injected intraperitoneally twice a week, from day 7 onwards. Mice body weight, stool consistency, and stool bleeding were monitored twice a week. Colitis severity was scored by evaluating the clinical disease activities. The disease activity index (DAI) was determined as previously described [24]: change in body weight loss (no weight loss or weight gain = 0; 5%–10% weight loss = 1; 11%–15% weight loss = 2; 16%–20% weight loss = 3; >21% weight loss = 4), stool consistency (normal and well-formed = 0; very soft and unformed = 2; watery stool = 4), and stool bleeding (normal color stool = 0, reddish color stool = 2, bloody stool = 4). The DAI score was calculated as the total of these scores, and ranged from 0 (healthy) to 12 (severe colitis). Mice were sacrificed by an overdose of sevoflurane on days 7, 14, and 21 (n = 5). The distal colon was excised longitudinally. Colon tissues were stored in liquid nitrogen for quantitative real-time PCR, fixed in 10% buffered formalin, and embedded in paraffin for histological examination.

Histological examinationof murine colitis model

Murine colon tissue was fixed in 10% buffered formalin, embedded in paraffin, and stained with Masson’s trichrome stain (MTS). To evaluate fibrosis of the colon, area positive for MTS staining was quantified by measuring five randomly non-overlapping fields from the distal colon using a BZX-800 fluorescence microscope (Keyence).

Reverse transcription and quantitative real-time PCR

Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. cDNA was synthesized using the PrimeScript RT Reagent Kit (TaKaRa Bio, Shiga, Japan). Quantitative real-time PCR was performed using SYBR Premix Ex Taq II, ROX Plus (Takara Bio) according to the manufacturer’s instructions using a StepOnePlus Real Time PCR System (Applied Biosystems, Foster City, CA, USA). The cycle threshold (Ct) was calculated using the comparative CT method. The relative mRNA expression was normalized to that of GAPDH. The primers used in this study are listed in Supplemental Table 1.

Enzyme-linked immunosorbent assay (ELISA)

Blood samples were collected via cardiac puncture at sacrifice. Plasma was collected by centrifugation at 1300 × g for 10 min using heparin as an anticoagulant, and plasma samples were stored at − 80 °C until analysis. Commercial IL-6 and TIMP-1 ELISA kits (Abcam) were used to measure plasma levels according to the manufacturer’s instructions.

Statistical analyses

All data are presented as the mean ± standard error of the mean (SEM). Statistical analysis was performed using the Wilcoxon signed-rank test and Wilcoxon rank-sum test. Statistical significance was set at p < 0.05. JMP Pro software version 14 (SAS Institute Inc., Cary, NC, USA) was used for statistical analyses.

TIMP-1 expression was increased after TGF-β stimulation in human colonic normal fibroblasts.

Human fibroblasts were cultured and stimulated with TGF-β to observe the differentiation of fibroblasts into myofibroblasts. The fibroblasts showed no morphological changes after TGF-β stimulation (Fig. 1a). However, mRNA expression levels of fibrotic markers α-SMA and Col1a1 were significantly increased 72 h after TGF-β stimulation, which suggested that the fibroblasts had differentiated into myofibroblasts (Fig. 1b). The mRNA level of TIMP-1 was also significantly increased after TGF-β stimulation, in agreement with the elevated expression levels of other fibrotic markers (Fig. 1b).

TIMP-1 expression in CD intestinal tissue was higher in inflammation areas than in fibrosis areas.

Immunohistochemical analysis using clinical samples from CD patients clarified the features and patterns of TIMP-1 expression in the intestinal tissue. Regardless of the case, TIMP-1 positive cells were observed most frequently in the stroma; however, TIMP-1 expression levels differed according to the area in the same patient (Fig. 2a). In most cases, the inflammation area showed the highest level of TIMP-1 expression, whereas that in the fibrotic area was relatively low. The percentage of TIMP-1 positive cells in the inflammation area was significantly higher than that in the fibrotic area (Fig. 2b). In contrast, TIMP-1 positive cells were rarely observed in normal areas.

Anti-TNF-α antibody treatment attenuated inflammation and fibrosis in murine colitis model.

A DSS-induced murine colitis model was established as summarized in Fig. 3a, and subsequently, the animals were treated with anti-TNF-α antibody or PBS. Mice treated with DSS showed severe symptoms of colitis indicated by the change in the DAI score, including body weight loss (Fig. 3b, c).

Anti-TNF-α antibody treatment showed significant improvement effects on body weight loss on day 14 and the severity of colitis on days 11 and 14 (Fig. 3b, c). Masson’s trichrome staining revealed that deposition of collagen fibers in the intestinal submucosa was induced in DSS-treated mice in accordance with severe colitis, and anti-TNF-α antibody treatment reduced the thickness of submucosal fibrosis (Fig. 4a), indicating that anti-TNF-α antibody treatment also showed an inhibitory effect on the induced fibrosis. The ratio of the area containing collagen fibers was significantly reduced by anti-TNF-α antibody treatment on day 21 (Fig. 4b).

TIMP-1 gene expression in murine colon tissue was increased after DSS administration and was reduced by anti-TNF-α antibody treatment, as with inflammatory markers.

The mRNA levels of inflammatory and fibrosis markers in the colon tissue of the murine colitis model changed over time. The expression of TIMP-1 in the PBS group was consistently high after DSS administration (Fig. 5), similarly to the expression levels of the representative inflammatory markers IL-1β and IL-6. Anti-TNF-α treatment significantly reduced gene expression levels of the inflammatory markers. In addition, the expression levels of TIMP-1 were also significantly reduced by anti-TNF-α treatment on day 21 (Fig. 5), in concordance with the tissue fibrosis evaluation by Masson’s trichrome staining (Fig. 4b). In contrast, the expression level of Col1a1, another representative fibrosis marker, was not increased in day 7 and there was no significant difference in Col1A1 expression between the PBS and anti-TNF-α groups (Fig. 5).

Plasma TIMP-1 level in murine colitis model reflected effects of anti-TNF-α antibody.

Plasma levels of TIMP-1 also reflected the effects of anti-TNF-α antibody treatment in a murine colitis model. Anti-TNF-α antibody treatment drastically reduced the plasma levels of IL-6 (Fig. 6), indicating that the systemic administration of the anti-cytokine agent has a powerful effect. In addition, anti-TNF-α treatment significantly reduced the plasma level of TIMP-1 on day 21 (Fig. 6). These results were consistent with the histological evaluation of intestinal fibrosis (Fig. 4b) and mRNA levels in colon tissue (Fig. 5).

Advances in our understanding of the pathogenesis of fibrosis have led to the identification of many molecular factors associated with the progression of intestinal fibrosis, such as Col1a1 and α-SMA. Although many fibrotic markers that can detect intestinal fibrosis are known, few reports on the application of fibrotic markers to assess therapeutic strategies for IBD are available. Considering that current drug therapies for IBD are only able to prevent intestinal fibrosis as a consequence of suppressing inflammation, but are unable to reverse established fibrosis [28], it is necessary to identify an indicator to investigate if potential new treatments can prevent fibrosis. The results presented in this study suggest that TIMP-1 could be used as a clinical indicator to assess the effectivity of therapeutic strategies for intestinal fibrosis associated with inflammatory response in IBD.

TIMP-1 is a key molecule in fibrogenesis, and it has been previously used as a marker for fibrosis. In vitro studies using myofibroblasts suggest that TIMP-1 appears is a fibrotic marker. In an in vitro study using human fibroblasts, mRNA expression of TIMP-1 increased during TGF-β-stimulated myofibroblast differentiation. TGF-β is a key factor in the development of fibrosis and a central mediator of fibrogenesis by modulating fibroblast phenotype and function, inducing myofibroblast differentiation, and promoting matrix accumulation [29, 30]. Another in vitro study showed that TIMP-1 significantly enhanced the migratory potential of colonic myofibroblasts isolated from patients with CD [31]. As these and other studies show, TIMP-1 can be considered a representative marker of intestinal fibrosis.

In contrast with the previously described in vivo studies, TIMP-1 appears to behave as an inflammatory marker in human and murine intestinal tissues. In this study, we found that TIMP-1 expression was significantly higher in the inflamed area than in the fibrotic area of CD intestinal tissue. Moreover, in the murine colitis model, the expression levels of TIMP-1 in the colon tissue increased immediately following DSS administration, similarly to the inflammatory markers. Another study using a DSS-induced murine colitis model reported that TIMP-1 knock-out mice recovered faster after acute colitis with a lower disease activity index than wild-type mice [32]. TIMP-1 has not only an MMP inhibitory function, but also an independent immunomodulatory function [32, 33]. Thus, TIMP-1 seems to be a molecule suitable as an inflammatory marker and a fibrotic marker, suggesting that it could be used to evaluate ongoing processes of developing intestinal fibrosis from inflammation. In contrast to TIMP-1, Col1A1, another representative fibrosis marker, had neither the characteristics of an inflammatory marker or of an ongoing fibrosis marker.

In the present study, the plasma TIMP-1 levels in a murine colitis model reflected the therapeutic effect of anti-TNF-α administration. Blood samples are more suitable to examine biomarkers of fibrosis than tissue samples due to their minimal invasiveness and availability. Additionally, plasma levels of the inflammatory marker IL-6 in the murine colitis model showed different changes than the IL-6 gene expression changes analyzed in the colon tissue, suggesting that the systemic administration of certain biological agents can have profound effects on the plasma levels of inflammatory cytokines. At the same time, it also suggests that the plasma levels of inflammatory markers do not necessarily reflect the therapeutic effect on intestinal fibrosis. After anti-TNF-α administration, TIMP-1 corresponded with the tissue fibrosis evaluated by Masson’s trichrome staining, but plasma IL-6 were extremely low compared with the results of tissue fibrosis and IL-6 gene expression in the colon tissue. As it is possible that plasma levels of inflammatory markers undergo exacerbated changes upon the systemic administration of biological agents, inflammatory markers such as IL-6 are not suitable indicators to evaluate the therapeutic effect of those biological agents on ongoing intestinal fibrosis.

Further discussion is needed on using mice exposed to one cycle of DSS as a model for chronic intestinal inflammation and fibrosis. Although repeated DSS administration, usually during 7–10 days, is known to induce acute intestinal inflammation [34, 35], it has been reported that C57BL/6 mice develop chronic colitis and intestinal fibrosis even after a single DSS cycle [35, 36]. In contrast to C57BL/6 mice, BALB/c mice do not develop chronic colitis subsequent to the acute response, possibly as a result of a different cytokine response [35]. Hence, C57BL/6 mice treated with one cycle of DSS can be considered simple and suitable for assessing the effects of anti-TNF-α antibody administration to prevent intestinal fibrosis as a consequence of suppressing inflammation.

The present study has some limitations. First, the plasma levels of TIMP-1 were evaluated only in the murine colitis model, and not in IBD patients. Demonstrating that TIMP-1 changes in a similar manner in human patients is necessary to implement its application in the clinic. In addition, anti-TNF-α administration was used to treat inflammation in this animal study. Recently, new treatments for IBD have been developed, and various drugs are currently available for the treatment of IBD. To apply TIMP-1 as an indicator of the therapeutic effects of various anti-inflammatory drugs on intestinal fibrosis, TIMP-1 expression changes in response to these anti-inflammatory therapies needs to be examined. Therefore, further experiments are necessary to confirm the clinical utility, validity, and versatility of TIMP-1 as an effective indicator for different drugs.

the present study suggests that TIMP-1 could potentially be applied in a clinical setting as a therapeutic indicator of intestinal inflammation and ongoing fibrosis in IBD patients. TIMP-1 expression may be suitable for evaluating the therapeutic effects of the different treatments available for IBD and determining the appropriate treatments to prevent the progression of intestinal fibrosis in IBD patients.

IBD: inflammatory bowel disease; CD: Crohn’s disease; TNF: tumor necrosis factor; ECM: extracellular matrix; MMP: matrix metalloproteinases; TIMP: tissue inhibitor of metalloproteinase; PBS: phosphate-buffered saline; DAB: diaminobenzidine; DSS: dextran sodium sulfate; DAI: disease activity index; MTS: Masson’s trichrome stain; Ct: cycle threshold; SEM: standard error of the mean

Ethics approval and consent to participate

Studies involving human participants were reviewed and approved by the Tohoku University Ethics Committee (2021-1-091). All animal experiments were approved by Tohoku University Animal Care Committee. The experimental protocols and animal care were performed according to the guidelines for animal experiments at Tohoku University.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by JSPS KAKENHI Grant Numbers JP18K16339 and JP18K08669.

Authors' contributions

K.S. contributed to acquisition of the data, analysis and interpretation of the data, drafting of the manuscript, and statistical analysis. H.S. and H.K. contributed to the study design, supervised and supported the study, and critically revised the manuscript. A.Y., T.K., M.K., and Y.H. interpreted the results and revised the manuscript. T.H. and M.K. contributed to the provision of human fibroblasts. A.M. contributed to the provision of human fibroblasts and critical revision of the manuscript. T.K., S.O., and M.U. contributed to critical revision. All authors approved the final version of the manuscript.

Acknowledgments

We would like to thank Editage (www.editage.com) for English language editing.

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No competing interests reported.

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TIMP-1 As a Promising Therapeutic Indicator for the Intestinal Inflammation and Fibrosis.

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