Etanercept attenuates TNBS-induced experimental colitis - Springer Link

J Mol Hist (2011) 42:443–450 DOI 10.1007/s10735-011-9349-z

ORIGINAL PAPER

Etanercept attenuates TNBS-induced experimental colitis: role of TNF-a expression Ana Paula Ribeiro Paiotti • Sender Jankiel Miszputen • Celina Tizuko Fujiyama Oshima • Ricardo Artigiani Neto Daniel Araki Ribeiro • Marcello Franco

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Received: 7 July 2011 / Accepted: 29 July 2011 / Published online: 24 August 2011 Ó Springer Science+Business Media B.V. 2011

Abstract Crohn0 s disease (CD) is associated with gut barrier dysfunction. Tumour necrosis factor-a (TNF-a) plays an important role into the pathogenesis of several inflammatory diseases because its expression is increased in inflamed mucosa of CD patients. Anti-TNF therapy improves significantly mucosal inflammation. Thus, this study aimed to evaluate the effect of Etanercept (ETC), a tumour necrosis factor alpha (TNF-a) antagonist on the 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced experimental colitis. A total of 18 Wistar rats were randomized into four groups, as follows: (1) Sham: sham induced-colitis; (2) TNBS: non-treated induced-colitis; (3) ETC control; (4) ETC-treated induced-colitis. Rats from group 4 presented significant improvement either of macroscopic or of histopathological damage in the distal colon. The gene expression of TNF-a mRNA, decreased significantly in this group compared to the TNBS non-treated group. The treatment with etanercept attenuated the colonic damages and reduced the inflammation caused by TNBS. Taken together, our results suggest that ETC attenuates

A. P. R. Paiotti (&) C. T. F. Oshima R. A. Neto D. A. Ribeiro M. Franco Department of Pathology, Universidade Federal de Saõ Paulo— Escola Paulista de Medicina, UNIFESP, Saõ Paulo, SP, Brazil e-mail: [email protected] S. J. Miszputen Division of Gastroenterology, Universidade Federal de Saõ Paulo—Escola Paulista de Medicina, UNIFESP, Saõ Paulo, SP, Brazil D. A. Ribeiro Department of Biosciences, Universidade Federal de Saõ Paulo—Escola Paulista de Medicina, UNIFESP, Saõ Paulo, SP, Brazil

intestinal colitis induced by TNBS in Wistar rats by TNF-a downregulation. Keywords Inflammatory bowel disease TNBS-colitis TNF-a Etanercept RT-PCR

Introduction Inflammatory bowel disease (IBD) is a common chronic gastrointestinal disorder characterized by alternating periods of remission and active intestinal inflammation. The precise etiology of IBD, including Crohn0 s disease (CD) and ulcerative colitis (UC), remains unclear. However, environmental factors, immunological disturbances, genetic influences and the presence of certain chemical mediators (cytokines) have been established as putative participants in the pathogenesis of the disease (Lashner 1995; Barbieri 2000; Podolsky 2002). In the last few decades, the development of experimental models for studying IBD has greatly contributed to enhance understanding of the immunological mechanisms involved, such as changes in the gut epithelial barrier (Shorter et al. 1972; Colpaert et al. 2001). IBD seems to occur when luminal antigens from the bacterial flora stimulate the immune system in the gut barrier towards an exacerbated, genetically defined response. Patients present an increase in the amount of intestinal bacterial antigen compared to healthy individuals (Bonen and Cho 2003). In particular, some human and animal studies have shown the prime importance of gut epithelial barrier integrity and changes that lead to deregulation of the immune system as a result of the loss of intestinal homeostasis (E´lson et al. 1995; Somasundaram et al. 1997).

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Tumour necrosis factor-a (TNF-a) is a proinflammatory cytokine secreted predominantly by monocytes/macrophages but also by T cells, B cells, NK cells and mast cells. Increased expression of TNF-a has been observed in the inflamed mucosa of IBD and in murine colitis models (Braegger and MacDonald 1994; Neurath et al. 1997). It has been shown that TNF-a-induced apoptosis contributes significantly the loss of ions and water, and the passage of small antigens (Schulzke et al. 2006). The anti-TNF-a treatment improves mucosal inflammation. Etanercept (ETC) is a dimeric, soluble form of the 75-kDa TNF receptor. The anti-inflammatory effects of ETC are owing to its ability to bind to TNF, preventing it from interacting with cell-surface receptors and rendering it biologically inactive. ETC can also modulate biological responses that are induced or regulated by TNF (Di Paola et al. 2007). As a result and because of limited evidence, the goal of this study was to evaluate the effects of ETC, the TNF-a antagonist on the 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced experimental colitis. Such data should also contribute to improving current understanding of the effect of experimental colitis on the cellular system.

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was induced by the administration of 50 mg/kg of TNBS (Sigma, St. Louis, MO) in ethanol (50% v/v) in a total volume of 1 mL, via rubber catheter inserted 8 cm into the colon from the anus. The sham group received 1 mL of 0.9% saline solution by the same technique. The rats were previously randomized into four groups, as follows: (1) Sham group: sham induced-colitis rats; (2) TNBS group: nontreated induced-colitis; (3) ETC control; (4) ETCtreated induced-colitis. Etanercept (WhyethÒ, Brazil) (5 mg/kg/day) was administered subcutaneously once a day for 9 days, beginning on day 7 after colitis induction or on the same day in the control group. All the rats were checked daily for behavior and general health conditions and body mass was recorded weekly. Macroscopic evaluation of colon lesions All rats were sacrificed on day 16 of the experiment. The colon was removed from cecum to anus and opened by a longitudinal incision. The tissue was rinsed with saline and tissue damage was examined. The criteria of the macroscopic score used a previously validated scoring system (Table 1) (Peran et al. 2005).

Materials and methods General procedures All experimental protocols involving animals conformed to procedures described in the Guiding Principles for the Use of Laboratory Animals and the study approved by the Animal Committee of the Universidade Federal de Sao Paulo, UNIFESP. A total of 18 adult male Wistar rats (Rattus norvegicus albinus), weighing 250–300 g, provided by the Center for the Development of Experimental Models for Medicine and Biology (Centro de Desenvolvimento de Modelos Experimentais para Medicina e Biologia, CEDEME) of the Universidade Federal de Sao Paulo, were maintained in restricted-access rooms at a controlled temperature (23°C) and on a 12 h light–dark cycle. Standard laboratory chow and drinking water were provided ad libitum. Before initiating the experimental procedures, all the rats were treated with PanacurÒ to eliminate intestinal parasites; 2 doses were administered orally with an interval of 3 days, 5 days before the experiment. Induction of colitis and treatment protocols Colitis was induced by TNBS acid using the modified method described by Morris et al. (1989). Briefly, rats were submitted to fasting 1 day before the experiment. Each rat was anesthetized with ketamine and xylazine and colitis

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Histological evaluation of colonic lesions After macroscopic assessment of the mucosa, samples were fixed in 10% buffered formalin, embedded in paraffin and

Table 1 Criteria for the assessment of macroscopic colonic damage (Modified from Peran et al. 2005) Criteria 1st Score 0

No damage

1

Hyperemia, no ulcers

2

Linear ulcer with no significant inflammation

3

Linear ulcer with inflammation at one site

4

Two or more sites of ulceration/inflammation

5

Two or more major sites of ulceration and inflammation or one site of ulceration/inflammation, extending [ 1 cm along the length of the colon

6–10

If damage covers [ 2 cm along the length of the colon, the score is increased by one, for each additional centimeter of involvement

2nd Score 0

No adhesions

1

Mild adhesions (easily separable from other tissues)

2

Severe adhesions

Total

J Mol Hist (2011) 42:443–450 Table 2 Criteria for the assessment of microscopic colonic damage (Modified from Appleyard and Wallace 1995) Parameters

Score

Edema

0 or 1 (absent or present)

Ulceration


Cellular infiltration

0, 1, 2 or 3 (absent, mild ? extensive)

Muscle thickening

0, 1, 2 or 3 (absent, mild ? extensive)

Neoangiogenesis


Crypt abscess formation


Loss of mucosal architecture

0, 1, 2 or 3 (absent, mild ? severe)

Cellular turn over

0 or 1 (absent–extensive)

Total

tissue sections were stained with hematoxylin and eosin for histopathological evaluation of colonic damage by light microscopy. The assessment criteria of the histological score was according to previous literature (Table 2) (Appleyard and Wallace 1995). Preparation of total RNA Frozen colonic tissue was homogenized and total RNA was isolated using cold Trizol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Total RNA was determined using a NanoDropÒ ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). RNA samples were treated with DNAse (Invitrogen, Carlsbad, CA, USA) to avoid contamination with genomic DNA. cDNA synthesis and quantitative real time PCR cDNA synthesis was performed using reverse transcriptase M-MVL (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Real-time PCR was performed in an ABI Prism 17500 Real-Time PCR system using the SYBR Green Kit (Applied Biosystems, Foster City, CA, USA). Primers for the specific amplification of each cDNA were designed using the Primer Express software (Applied Biosystems), considering established criteria, such as product length, optimal PCR annealing temperature and the likelihood of primer self-annealing. The following primers were used for each gene: TNF-a, forward: 50 -CCC AGA AAA GCA AGC AAC CA-30 and reverse: 50 -GCC TCG GGC CAG TGT ATG-30 and GAPDH, forward: 5’-GCT CTC TGC TCC TCC CTG TTC-3’ and reverse: 5’-GAC GCT GGC ACT GCA CAA-3’. PCR reactions were performed in triplicate containing 12 ll final volume using 3.0 ll of a 1:5 (v/v) dilution of cDNA, 3.0 ll of primer

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mix—600 nM (forward and reverse) and 6.0 ll of SYBR Green master mix 29 (Applied Biosystems). The reactions were performed in MicroAmp 96-well plates (Applied Biosystems) covered with optical adhesive (Applied Biosystems). Samples were submitted to forty cycles of 95°C for 10 min, 95°C for 15 s and 60°C for 1 min. An amplification efficiency curve using different cDNA dilutions was also performed for each gene tested. Analysis of gene expression data To normalize the data for the control and experimental groups, arbitrary units were calculated as: arbitrary unit = 2 - DDCT, and DDCT = sample DCT - control DCT, where CT is the threshold cycle. Immunohistochemistry TNF-a immunoexpression was determined using the streptavidin–biotin-peroxidase method. 3 lm thick sections were deparaffinized in xylene and hydrated. Antigen retrieval was performed in a steamer in citrate buffer, pH 6.0, for 40 min. Endogenous peroxidase activity was blocked by the addition of 3% hydrogen peroxide for 20 min at room temperature. After washing in phosphate buffered saline (PBS), the sections were incubated with polyclonal antibody anti-TNF-a (diluted to 1:400, Santa Cruz Biotechnology). The sections were then washed in PBS and incubated with the biotinylated secondary antibody for 15 min, followed by incubation with the streptavidin–biotin-peroxidase complex (Dako) for 15 min at room temperature. Finally, the reaction was developed with 3.30 -diaminobenzidine (DAB-Sigma) as chromogen and 1% hydrogen peroxide, and the slides were counterstained with Harris hematoxylin. Sections known to express high levels of TNF-a were included as positive controls, while negative control slides omitted the primary antibody. Evaluation of the staining The cytoplasmic brown granule was marked as a positive expression. The results were evaluated semi-quantitatively according to the percentage of positive cells in the randomly selected fields under high-power microscope (400-fold magnification) for each sample (Zhou et al. 2006). Statistical analysis All data are expressed as the median ± semi-quartile. For analysis of macroscopic damage, histological damage and mRNA expression, the Kruskal–Wallis test was used, followed by Mann–Whitney test. For the immunohistochemistry analysis, the Kruskal–Wallis test was used, followed

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by Fisher’s exact test. The relation between two variables was evaluated by Spearman’s correlation. A value of P \ 0.05 was considered for statistical significance.

Results Clinical evaluation All rats exposed to TNBS showed a significant decrease in body mass from day 1 to day 5 after colitis induction, after which time they recovered (Fig. 1). These rats developed severe diarrhea on day 2 after colitis induction and rectal bleeding was occasionally observed. Nevertheless, the ETC-treated induced-colitis group showed clinic remission on day 10 after the onset of treatment and presented marked clinical improvement. In some rats from the ETC control group, pasty stools were observed, while in the sham group, normal stools were observed throughout the experiment. In both groups, an increase of 15–20% in body mass was verified. The macroscopic assessment included hyperemia, ulceration, inflammation and adhesion between colon and other tissues. The TNBS induced-colitis showed higher tissue damage in the distal colon (77.1 ± 19.3). The majority of the rats showed greater thickening of the colon.

Fig. 2 Macroscopic scoring of rat colonic tissue of sham, TNBS nontreated induced-colitis, etanercept (5 mg/kg/day) control and etanercept-treated (5 mg/kg/day) induced-colitis groups. Values are expressed as the median ± sq. *P = 0.038 as compared with sham group; #P = 0.010 as compared with etanercept-treated induced colitis group (Kruskal–Wallis; Mann–Whitney) (n = 6–8 per group)

The treatment with ETC significantly reduced the intensity of the macroscopic score and the incidence of adhesion (33.3 ± 0.0). In the sham group, no macroscopic damage was observed (Table 3; Figs. 2, 3). Histological analysis Statistically significant differences (P \ 0.05) occurred in the histological analysis (Table 3; Figs. 4, 5). Rats from the

Fig. 1 Body mass alterations in the different groups

Table 3 Overview of parameters assessed and their modulation by the medication Group

Macroscopic score

Histological score

mRNA TNF-a

TNF-a protein expression

Sham

0.0 ± 0.0

0.0 ± 3.9

1.4 ± 0.7

22.2 ± 5.6

TNBS

77.1 ± 19.3*#

84.6 ± 6.8*#

5.1 ± 2.9*#

33.3 ± 20.9

ETC ctrl TNBS ? ETC

0.0 ± 4.2 33.3 ± 0.0

0.0 ± 3.9 53.8 ± 4.9

0.2 ± 0.0 0.2 ± 0.1

27.8 ± 22.2 0.0 ± 4.2

Values are expressed as the median ± sq. Macroscopic score: * P = 0.038 as compared with sham group; etanercept-treated induced colitis group (Kruskal–Wallis; Mann–Whitney) (n = 6 to 8 per group) Histological score: * P \ 0.001 as compared with sham; Wallis; Mann–Whitney) (n = 6 to 8 per group)

#

P = 0.010 as compared with

P = 0.020 as compared with etanercept-treated induced colitis group (Kruskal–

mRNA TNF expression: * P = 0.038 as compared with sham control group; group (Kruskal–Wallis; Mann–Whitney) (n = 4 to 6 per group)

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#

#

P = 0.010 as compared with etanercept-treated induced colitis

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Analysis of TNF-a mRNA expression by qRT-PCR Weak expression of TNF-a mRNA was detected in normal colon cells from the negative control group. By contrast, higher levels were observed in the TNBS induced-colitis group. Interestingly, we observed that treatment with ETC suppressed the increased TNF-a mRNA expression (Table 3; Fig. 6). Immunohistochemical analysis

Fig. 3 Macroscopic observations of colon. a Sham control group, normal colon; b etanercept (5 mg/kg/day) control group, hyperemia; c TNBS nontreated induced-colitis group, severe colon thickening, ulcers and necrosis; and d etanercept-treated (5 mg/kg/day) inducedcolitis group, slight colon thickening, hyperemia and ulcers

Fig. 4 Histologic scoring of rat colonic tissue of sham, TNBS nontreated induced-colitis, etanercept (5 mg/kg/day) control and etanercept-treated (5 mg/kg/day) induced-colitis groups. Values are expressed as the median ± sq. *P \ 0.001 as compared with sham; #P = 0.020 as compared with etanercept-treated induced colitis group (Kruskal–Wallis; Mann–Whitney) (n = 6–8 per group)

TNBS induced-colitis group presented histological damages significantly high when compared to the other groups (84.6 ± 6.8). We observed that the treatment with ETC reduced the intensity of the histological score (53.8 ± 4.9). In addittion, some rats from sham and ETC control groups presented edema.

The expression of TNF-a in the TNBS induced-colitis group significantly increased compared with the ETCtreated induced-colitis group. The TNF-a immunopositive cells predominantly located within the mucosa and lamina propria with a brown-yellow pattern. Administration of ETC resulted in a reduction of colon TNF-a levels Such findings are summarized in Table 3 and Fig. 7.

Discussion TNBS-induced colitis is accompanied by marked thickening of the colon wall, infiltration of polymorphonuclear leukocytes and ulceration, resembling human Crohn0 s disease. Inflammation plays an important role in modulating tissue repair. However, depending on its intensity, this can cause discomfort and pain to the patient, becoming aggressive in relation to the surrounding tissues due to the release of chemical mediators. TNF-a plays an important role in the pathogenesis of several inflammatory diseases. Its expression is increased in inflamed mucosa of CD patients. Anti-TNF-a treatment improves mucosal inflammation (Shen et al. 2007). We evaluated the therapeutic effects of etanercept on the TNBS-induced experimental colitis. Etanercept (ETC) is a dimeric, soluble form of the 75-kDa TNF receptor. The anti-inflammatory effects of ETC are owing to its ability to bind to TNF, preventing it from interacting with cell-surface receptors and rendering it biologically inactive. ETC can also modulate biological responses that are induced or regulated by TNF (Di Paola et al. 2007). To best of our knowledge, the approach has not been addressed before. Our results demonstrate that the therapeutic drug was able to reduce the severity and extension of damage caused by TNBS. In fact, a decrease of the extent of colitis was noticed. Specifically, rats treated with etanercept had significantly macroscopic and histopathological improvement. A study by Fries et al. demonstrated the similar efficacy of both etanercept and infliximab by the decrease of macroscopic damage on DNBS-induced colitis in mouse (Fries et al. 2008). The infliximab is a chimeric anti-tumour necrosis factor alpha

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Fig. 5 Photomicrograph of the haematoxylin and eosin stained section of rat colons in the different groups. a Sham control group and b etanercept (5 mg/ kg/day) control group: the mucosa appears intact; c TNBS nontreated induced-colitis group: areas of ulceration, inflammatory cellular infiltration, severe transmural colitis and cellular architecture distortion and d etanercepttreated induced-colitis group: focal ulcers, mild inflammatory cellular infiltration and architecture distortion. Bar 56 lm

Fig. 6 Analysis of mRNA TNF-a expression in the different groups by qRT-PCR. Values are expressed as the median ± sq. *P = 0.038 as compared with sham control group; #P = 0.010 as compared with etanercept-treated induced colitis group (Kruskal–Wallis; Mann– Whitney) (n = 4–6 per group)

monoclonal antibody with potent anti-inflammatory effect. It represents an effective treatment in patients with severe IBD. Suenaert showed that infliximab decreased the

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inflammation and intestinal permeability suggesting a modulatory process in turn intestinal barrier repair (Suenaert et al. 2002). Our results are fully in line with these studies. To further elucidate the role of TNF-a on TNBS induced experimental colitis, TNF-a expression was evaluated in this setting. The results showed that mRNA levels of TNF-a decreased significantly compared with TNBS inducedcolitis group. In addition, the immunoexpression of TNF-a was significantly different among groups. Colpaert et al. demonstrated that polyclonal anti- TNF-a therapeutic advance improvement of macroscopic and microscopic damages on indometacin-induced small bowel inflammation (Colpaert et al. 2001). Shen et al. showed that anti-TNF downregulates proinflammatory cytokines and decreases cell infiltration in the bowel diases after TNBS application (Shen et al. 2007). By comparison, a previous study conduced by our group has revealed that treatment with the selective COX-2 inhibitor lumiracoxib did not reduce inflammation-associated colonic injury in TNBS-induced experimental colitis (Gibson 2004). These studies reinforce the interrelation between TNF-a and intestinal barrier, where it commands the whole inflammation cascade, resulting in increase of the intestinal lumen, and permeability increased (Paiotti et al. 2009).

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Fig. 7 Photomicrograph of the TNF-a protein expression in the different groups by immunohistochemistry. Cytoplasmic staining in epithelial cells. a Sham control group and b TNBS nontreated induced-colitis group: c etanercept (5 mg/kg/day) control group and d etanercepttreated induced-colitis group. Bar 20 lm

In summary, our results suggest that ETC attenuated the colonic damage and reduced the inflammation caused by TNBS as a result of TNF-a downregulation. Further studies are necessary to elucidate the issue. Acknowledgments The authors wish to express their gratitude to Profa. Dra. Silvia Regina Caminada de Toledo, Indhira Dias Oliveira of the Pediatric Oncology Institute IOP/GRAACC of the Universidade Federal de Sao Paulo, UNIFESP, SP, Brazil and Profa. Dra. Nora Manoukian Forones of the Division of Gastroenterology, Universidade Federal de Saõ Paulo, Escola Paulista de Medicina, UNIFESP, SP, Brazil, for the technical assistance.

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