Restoration of colonic barrier function by tofacitinib in experimental colitis: anti-inflammatory effects and decreased expression of claudins-2 and claudin-15

Humberto Barbosa da Costa Filho; Gerardo Autran Cavalcante Araújo; Thiago Meneses Araújo Leite Sales; Suliana Mesquita Paula; Marco Antonio de Freitas Clementino; Alexandre Havt; Pedro Marcos Gomes Soares; Marcellus Henrique Loiola Ponte Souza

doi:10.5217/ir.2024.00186

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Original Article Restoration of colonic barrier function by tofacitinib in experimental colitis: anti-inflammatory effects and decreased expression of claudins-2 and claudin-15: Humberto Barbosa da Costa Filho¹, Gerardo Autran Cavalcante Araújo², Thiago Meneses Araújo Leite Sales², Suliana Mesquita Paula², Marco Antonio de Freitas Clementino¹, Alexandre Havt¹, Pedro Marcos Gomes Soares³, Marcellus Henrique Loiola Ponte Souza^2,; DOI: https://doi.org/10.5217/ir.2024.00186
Published online: March 31, 2025

¹Department of Physiology and Pharmacology, School of Medicine, Federal University of Ceará, Fortaleza, Brazil

²Department of Medicine, School of Medicine, Federal University of Ceará, Fortaleza, Brazil

³Department of Morphology, Federal University of Ceará, Fortaleza, Brazil

Correspondence to Marcellus Henrique Loiola Ponte Souza, Department of Medicine, School of Medicine, Federal University of Ceará, Coronel Nunes de Melo Street, 1315, Rodolfo Teófilo, 60.430-270, Fortaleza, Brazil. E-mail: souzamar.ufc@gmail.com

• Received: November 18, 2024 • Revised: February 5, 2025 • Accepted: February 9, 2025

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Abstract
INTRODUCTION
METHODS
RESULTS
DISCUSSION
NOTES
REFERENCES

Abstract

Background/Aims
Inflammatory bowel disease can be triggered by disturbances in intestinal mucosal integrity, leading to bacterial transmigration. The treatment of inflammatory bowel diseases must not only aim to reduce inflammation, but also to reverse the damage to mucosal barrier function. Janus kinase (JAK) inhibitors have been used to treat inflammatory diseases, including ulcerative colitis. However, little is known about the ability of this class of drugs to reverse the loss of mucosal integrity. This study evaluated the effects of tofacitinib, a JAK pathway inhibitor, on inflammation and colonic mucosal integrity in a rat model of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis.
Methods
Colitis was induced in Wistar rats via rectal administration of TNBS (20 mg+50% ethanol). The control group received only saline. The animals were pretreated with tofacitinib (15 mg/kg) or saline 30 minutes before induction and twice daily thereafter. Seven days after induction, the animals were euthanized, the colon was removed, and myeloperoxidase activity, baseline transepithelial electrical resistance (TER), TER after 1 hour, and fluorescein permeability were assessed. Tight junction proteins in the colon (claudin-2, claudin-15, and tricellulin) were detected using Western blotting.
Results
Tofacitinib treatment significantly reduced (P<0.05) the inflammatory parameters and preserved the integrity of the intestinal epithelial barrier compared with the colitis group (P<0.05), increased baseline TER, reduced the drop in TER after 1 hour, and decreased paracellular permeability to fluorescein by reducing claudin-2 and claudin-15 expression.
Conclusions
JAK inhibition by tofacitinib restored colonic barrier function through antiinflammatory effects and decreased claudin-2 and claudin-15 expressions.
Keywords: Mucosal barrier function; Colitis; Claudin-2; Claudin-15

INTRODUCTION

Inflammatory bowel disease (IBD) is characterized by the persistent inflammation of the intestinal mucosa and includes Crohn’s disease (CD) and ulcerative colitis (UC). IBD can trigger changes in the gastrointestinal tract, such as fistulas and obstruction of the colon and small intestine [1], as well as extraintestinal problems [2]. IBD is a serious public health concern. A review by Ng et al. [3] showed that although the incidence of IBD has stabilized in the Western world, its prevalence remains high. In contrast, recently industrialized countries are facing increasing incidence. Furthermore, Mak et al. [4] indicate that the aging population in Asia may lead to a significant increase in the number of patients with IBD globally. However, many available treatments only control the symptoms without curing the disease, resulting in relapse. Additionally, adverse reactions to medications can compromise treatment effectiveness, which varies widely among patients [1].

Although the exact cause of IBD is unknown, disruption of the intestinal mucosal barrier is associated with this disease [1,5]. This barrier is maintained by tight junctions (TJs), which function as a “gate and fence,” allowing the paracellular transport of some solutes while blocking the passage of proteins, lipids, and peptides [6]. TJs are composed of proteins such as claudins (including pore-forming claudins such as claudin-2 and claudin-15 and barrier-forming claudins) and tricellulin, which interact with the cytoskeleton to maintain a complex and selective structure [7].

Experimental models have been developed to improve the understanding of IBD, including the induction of colitis in rodents using 2,4,6-trinitrobenzenesulfonic acid (TNBS). This model allows observation of inflammatory changes, loss of colonic mucosal integrity, and increased paracellular permeability and is especially useful for mimicking the clinical aspects of CD in humans [8].

Owing to the complexity of IBD pathogenesis, current therapies typically only alleviate symptoms and have high relapse rates [9]. New approaches are being developed and tested to overcome these limitations. One such example is tofacitinib, a pan-Janus kinase (JAK) inhibitor approved for moderate-to-severe UC. Tofacitinib improves barrier function and TJ regulation in co-culture systems of intestinal epithelial cells and inflammatory macrophages deficient inprotein tyrosine phosphatase non-receptor type-2 [10]; however, no results have been reported in animal models.

Given that the TNBS-induced colitis model causes severe inflammation and damages the colonic mucosa, and that JAK inhibition has emerged as a promising target for treating immune-inflammatory diseases, this study aimed to evaluate the effect of tofacitinib on restoring colonic barrier function in a TNBS-induced colitis animal model and the mechanisms involved in this effect.

METHODS

1. Ethical Approval

All treatments and surgical procedures were carried out in compliance with the “Care Guide for the Use of Laboratory Animals” and the standards set forth by the National Council for the Control of Animal Experimentation. The study was approved by the Animal Use Ethics Committee of the Federal University of Ceará (CEUA-UFC; protocol number 4082010222).

2. Animals

Male Wistar rats (190 ± 10 g; 6–7 weeks old) were provided by the Animal Facility of the Department of Physiology and Pharmacology at the Federal University of Ceará. The animals were housed in cages with free access to food and water, as well as strictly controlled temperature (22 ± 2°C) and light (12-hour light/dark cycle) conditions.

3. TNBS-Induced Colitis and Treatments

First, an enema (10 mL of 0.9% saline solution via the rectum) was administered before the induction of colitis. Subsequently, the animals were anesthetized with ketamine (80 mg/kg) and xylazine (10 mg/kg). The animals were positioned in left lateral decubitus, and a polyethylene catheter n°6 was introduced intracolonically up to 8 cm from the anal margin to administer the colitis induction solution (0.8 mL TNBS [20 mg] diluted in 50% ethanol, intracolonic) [11]. Each animal was then suspended by the tail for 2 minutes to prevent the return of the administered content [8]. The sham group received 0.8 mL of a 0.9% saline solution via the rectum.

The animals were treated 30 minutes before colitis induction and every 12 hours post-induction for 7 days with saline (0.9%, intramuscular) or dexamethasone (Decadron, Aché, Guarulhos, Brazil; 1 mg/kg, intramuscular) or tofacitinib (Xeljanz, Pfizer, New York, NY, USA; 15 mg/kg, orally) [12] dissolved in saline and Tween 20 (0.025%) [13]. Tween 20 (0.025%), a diluent for tofacitinib, was orally administered to the colitis group. Seven days after induction, the animals were euthanized, and edema (wet weight), macroscopic scores of colonic lesions, microscopic scores, neutrophil infiltration (myeloperoxidase [MPO] activity), changes in colonic mucosal integrity (epithelial resistance and permeability to fluorescein), and Western blot analysis of TJs were evaluated.

4. Histopathological Lesion Scores

Histological slides were prepared from colon samples, stained with hematoxylin and eosin, and examined by a skilled pathologist blinded to the sample identities (P.M.G.S.). The histopathological assessment was based on the criteria set forth by Appleyard and Wallace [14], with scores assigned according to the level of colonic damage. The parameters evaluated included loss of mucosal structure (score 0–3), degree of cellular infiltration (score 0–3), thickness of the muscular layer (score 0–3), presence of crypt abscesses (score 0–1), and absence of goblet cells (score 0–1).

5. Wet Weight of the Colon

Colonic samples were weighed using a precision scale, with the results reported in grams per centimeter (g/cm) of the colon [8].

6. Macroscopic Injury Scores

Colonic samples were laid flat on a surface and scored based on the observed level of damage, following the technique described by Morris et al. [11]. This method assesses hyperemia, ulceration, colonic wall thickening, as well as the length and number of lesions. A specific score is assigned to each criterion, which is recorded when the parameter is present in the tissue and not recorded when the parameter is absent. At the end, the scores for all criteria are summed.

7. MPO Activity

Colonic samples were collected, weighed, and macerated in 0.5% hexadecyltrimethylammonium bromide at pH 6.0. The mixture was centrifuged at 1,677 g for 7 minutes at 4°C. Subsequently, 10 mL of the supernatant was transferred to a 96-well plate in duplicates. A solution containing O-dianisidine (200 µL; 5 mg diluted in 3 mL phosphate buffer) was added to each well, followed by 15 µL of 1% H₂O₂. The absorbance was measured at 450 nm using a microplate reader (BMG Labtech, Madrid, Spain). The results were expressed as MPO per milligram of tissue [15].

8. Cytokine Levels

Colon fragments were collected for cytokine analysis, processed in protease inhibitor buffer, and homogenized before centrifugation. The MILLIPLEX Cytokine/Chemokine 4-plex kit was used to measure interleukin (IL)-6 and IL-1β in samples from the sham, colitis, tofacitinib, and dexamethasone groups. Tissue homogenates were diluted and mixed with standards and magnetic beads, then incubated overnight at 4°C. After washing, the detection antibodies and streptavidin-phycoerythrin were added, followed by additional incubation and washing. Cytokine concentrations were measured using the Luminex MAGPIX instrument (Thermo Fisher Scientific Inc., Waltham, MA, USA) with xPONENT software [16].

9. Experimental Protocol in the Ussing Chamber

Colonic samples were positioned on a petri dish containing Krebs solution (145 mM NaCl, 0.4 mM KH₂PO₄, 1.6 mM K₂HPO₄, 5 mM glucose, 1 mM MgCl₂, and 1.2 mM CaCl₂; pH 7.4) and dissected to extract the mucous layer for placement in the Ussing chamber (Mussler Scientific Instruments, Aachen, Germany). The mucosal sections had an exposure area of 0.017 cm², with 3.5 mL of Krebs solution per semi-chamber, and were aerated using a mixture of 95% O₂ and 5% CO₂ at a steady temperature of 37°C [8], transepithelial electrical resistance (TER) and epithelial permeability to fluorescein were assessed.

10. Transepithelial Electrical Resistance

The TER was calculated using Ohm’s law and the methodology outlined by Tobey et al. [17]. After 30 minutes, the electrical system stabilized, and the baseline TER, measured in Ω × cm², was determined. The results were expressed as a percentage (%) of the resistance variation at 0, 15, 30, 45, and 60 minutes. The zero time, corresponding to the peak TER, was defined as 100%, and the other evaluated times were compared with this point, indicating the percentages of decline from this value.

11. Fluorescein Permeability

Next, paracellular epithelial permeability was assessed. Colonic tissues were kept in Ussing chambers, and the luminal-side solution was replaced with a solution containing fluorescein (1 mg/mL, 376 Da, diluted in Krebs pH 7.4), a fluorescent tracer that permeates the mucosal layers [17]. Permeability was measured at 30-minute intervals for 90 minutes, with 100 μL samples taken from the non-luminal side. Fluorescein was quantified using a fluorescence reader (FLUOstar Omega; BMG Labtech). Fluorescein flow values were expressed in relation to fluorescein intensity using a standard curve generated from known concentrations in the reference range of 6–800 ng/mL.

12. Western Blotting

For protein extraction, rat colons were macerated using an electric homogenizer in small tubes containing RIPA buffer and protease inhibitor (Sigma-Aldrich, Burlington, MA, USA; 1 μL of inhibitor per 100 μL of RIPA), submerged in ice. The samples were then centrifuged (17 minutes, 4°C, 13,000 rpm). The protein concentration in the supernatant was determined using the bicinchoninic acid method according to the manufacturer’s instructions (Thermo Fisher Scientific Inc.). Vertical SDS-PAGE was then performed using a 10% polyacrylamide gel and SDS running buffer (25 mM Tris, 192 mM glycine, 1% SDS). After transfer, the membranes were blocked for 1 hour with constant shaking using 5% BSA (Sigma-Aldrich) in Tris-HCl saline buffer supplemented with Tween 20. Membranes were then washed with tris-buffered saline with Tween 20 (TBST) and incubated overnight at 4°C with primary antibodies (claudin-2, claudin-15, and tricellulin) diluted in 1% BSA in TBST, and further incubated with secondary antibodies. After TBST washes, membranes were incubated with Clarity Western ECL Substrate (BioRad, Hercules, CA, USA). Band images were captured using a ChemiDoc XRS+ system (BioRad), and band densities were measured with Image Lab software (BioRad Brasil, Lagoa Santa, Brazil), using Ponceau S staining to track the total protein level, following the protocol by Rivero-Gutiérrez et al. [18]. A molecular weight of 37 kDa for Ponceau was chosen for comparison, as reference proteins within this molecular range facilitate between-sample analysis.

13. Statistical Analysis

The Shapiro-Wilk test was conducted to assess the normality of the samples. For parametric data, both Student t-test and analysis of variance with the Bonferroni post-hoc test were employed, whereas the Kruskal-Wallis test followed by Dunn test was used for non-parametric data. Results are presented as means ± standard error of the mean, with statistical significance determined at P<0.05. Statistical analyses were performed using Jamovi version 2.3.

RESULTS

1. Tofacitinib on Histopathological Damage

Histopathological evaluation showed preservation of the criteria of loss of mucosal architecture, cellular infiltration, muscle thickening, and absence of goblet cells in the tofacitinib and dexamethasone groups compared with the colitis group (Table 1, Fig. 1).

2. Attenuation of Inflammatory Parameters by Tofacitinib

The colitis group had a higher wet weight (0.46 ± 0.08 mg/cm of colon) than the sham group (0.13 ± 0.01 mg/cm of colon). In contrast, both the tofacitinib (0.16 ± 0.01 mg/cm of colon) and dexamethasone (0.18 ± 0.03 mg/cm of colon) groups had a lower wet weight than the colitis group (Fig. 2A). Similarly, the parameters of macroscopic scores (Fig. 2B) and MPO concentration (Fig. 2C) were higher in the colitis group (17.11 ± 0.80 damage scores and 218.1 ± 31.36 U/mg) than in the sham group (0.43 ± 0.20 damage scores and 20.74 ± 3.90 U/mg). Tofacitinib and dexamethasone reduced these parameters both in macroscopic scores (8.43 ± 1.10 and 6.12 ± 0.10 damage scores) and in MPO concentration (144.0 ± 32.87 and 6.48 ± 1.30 U/mg).

3. Reduction of Cytokine Levels by Tofacitinib

The levels of cytokine IL-6 (48.67 ± 19.66 pg/mg of proteins vs. 3.07 ± 0.6 pg/mg of proteins) and IL-1β (500.60 ± 89.13 pg/mg of proteins vs. 19.62 ± 1.64 pg/mg of proteins) were higher in the colitis group than in the sham group. In contrast, the tofacitinib group had attenuated IL-6 (3.53 ± 1.45 pg/mg of proteins) and IL-1β (28.50 ± 3.90 pg/mg of proteins) levels. Similarly, dexamethasone also attenuated levels of IL-6 (6.88 ± 1.10 pg/mg of proteins) and IL-1β (58.10 ± 31.64 pg/mg of proteins) (Fig. 3).

4. Preservation of Basal TER by Tofacitinib

The colitis group had a lower basal TER than the sham group (33.25 ± 2.70 Ω × cm² vs. 45.00 ± 1.20 Ω × cm²). In contrast, tofacitinib prevented the reduction in basal TER (62.30 ± 8.14 Ω ×cm²), which was not observed in the dexamethasone group (39.13 ± 3.17 Ω × cm²) (Fig. 4).

5. Reduction of TER Drop and Fluorescein Permeability by Tofacitinib

The colonic mucosa of the colitis group showed a significant TER decrease at 60 minutes compared with that of the sham group (80.90% ± 2.35% vs. 95.07% ± 2.36%). However, the tofacitinib group had a smaller TER decrease than the colitis group (92.60% ± 2.80% vs. 80.90% ± 2.35%) (Fig. 5A and B). Fluorescein permeability after 90 minutes (Fig. 5C and D) was higher in the colitis group (1,889 ± 701.4 μg/mL) than in the sham group (364.2 ± 127.3 μg/mL). The tofacitinib group had reduced fluorescein permeability (132.40 ± 37.84 μg/mL).

6. Preservation of Junction Proteins by Tofacitinib

The colitis group had higher expression levels of claudin-2 (376.80% ± 52.30%) and claudin-15 (174.70% ± 37.02%) than the sham group (100% ± 14.30% and 81.93% ± 10.90%, respectively) (Fig. 6A and B). The tofacitinib group showed improved expression levels of both claudin-2 (227.20% ± 44.40%) and claudin-15 (40.53% ± 10.20%). Additionally, the colitis group had lower tricellulin expression (21.31% ± 3.80%) than the sham group (109.70% ± 36.30%) (Fig. 6C). The tofacitinib group partially restored tricellulin expression (64.37% ± 15.46%).

DISCUSSION

IBD, including CD and UC, is characterized by persistent intestinal inflammation, with a stable incidence in the Western world and an increasing incidence in recently industrialized countries [3]. The pathogenesis of IBD is associated with the disruption of the intestinal mucosal barrier, which is maintained by TJs composed of proteins such as claudins and tricellulin [7]. Experimental models, such as the induction of colitis in rodents using TNBS acid, help to better understand the disease by allowing the observation of inflammatory changes and loss of mucosal integrity [8]. New approaches such as tofacitinib, a JAK inhibitor approved for moderate-to-severe UC, show promise in improving barrier function and TJs regulation, although the results in animal models are still pending [10]. Our data showed that tofacitinib reduced not only inflammatory damage, but also the loss of mucosal integrity, promoting the recovery of pore-forming proteins such as claudin-2 and claudin-15.

The same parameters were used in this study to select the induction days as in a previous study conducted by our group, which showed that TNBS-induced colitis in rats leads to an inflammatory process with a loss of mucosal integrity within 7 days [8]. However, treatment with tofacitinib effectively reduced these inflammatory parameters and preserved the colonic mucosal integrity.

Microscopic damage was observed in the colon, including loss of mucosal architecture, cellular infiltration, muscular thickening, decreased goblet cell numbers, increased wet weight, changes in macroscopic lesion scores, and elevated MPO levels. Our results corroborate those of other studies that associated these changes with the use of ethanol and TNBS, highlighting that alcohol is crucial for disrupting the intestinal epithelial barrier, allowing TNBS to penetrate the intestinal wall and activate the immune system, resulting in mucosal damage [11,19,20].

Treatment with tofacitinib and dexamethasone both attenuated inflammatory parameters by reducing microscopic lesion scores, wet colon weight, macroscopic lesion scores, and MPO concentrations compared with the colitis group. Our findings are consistent with those of previous studies [20,21].

Analysis of the inflammatory data showed that the colitis group had increased levels of IL-6, a cytokine that regulates immunity and inflammation, stimulates the production of inflammatory cytokines, and contributes to chronic intestinal inflammation, which is elevated in patients with IBD and in experimental models of colitis [22,23]. IL-1β also increased, which is associated with the activation of the nuclear factor-κB (NF-kB) pathway and proinflammatory signals in the intestine [24,25]. However, treatment with tofacitinib reversed these levels, reducing IL-6 and IL-1β, consistent with the drug’s action, which specifically inhibits the phosphorylation of JAK1 and JAK3 and the activation of signal transducer and activator of transcription (STAT), without regulating gene transcription or cytokine production [1,26,27]. Similarly, dexamethasone effectively reduced IL-6 and IL-1β, an effect that can be attributed to the functions of glucocorticoids, such as the reduction of proinflammatory factor production by inhibiting NF-κB activity [28].

When analyzing mucosal integrity, we observed that the basal TER was lower in the colitis group than in the sham group, with this decline persisting at higher levels over a 1-hour period. Previous studies suggest that the increase in proinflammatory cytokines, such as IL-1β and IL-6, and the activation of the immune response are associated with the development of IBD [29,30]. Our results align with those of previous studies, indicating that the reduction in basal TER may result from inflammatory processes related to an increase in these cytokines [8,20,21].

In this study, we observed that tofacitinib maintained the basal TER, preventing its decrease. This is consistent with findings by Sayoc-Becerra et al. [31], who showed that tofacitinib preserved mucosal integrity in intestinal epithelial cells challenged with interferon-γ (IFN-γ). However, dexamethasone did not prevent a reduction in basal TER. Previous studies on Caco-2 intestinal cells indicated that the repair function of dexamethasone and the increase in TER were only evident after more than 10 days of treatment [32], suggesting that the treatment duration in our study may explain our results. Additionally, we noted that fluorescein permeability was higher in the colitis group, indicating increased paracellular permeability, a finding corroborated by previous studies [20,33,34]. Treatment with tofacitinib reduces this paracellular passage, indicating preservation of mucosal integrity [34].

The reduction in TER and increase in fluorescein permeability suggest TJ dysregulation, which are essential for the integrity of the epithelial barrier. We observed that colitis induced an increase in the expression of claudin-2 and claudin-15, both of which are TJs that form pores. Claudin-2, which increases permeability and is highly expressed in colitis, was reduced by treatment with tofacitinib, consistent with studies demonstrating the influence of proinflammatory cytokines on the regulation of these proteins [35]. To date, we have not found any studies reporting alterations in claudin-15 in TNBS-induced colitis models. This protein, which forms pores that allow the selective passage of cations, is important in maintaining intestinal barrier integrity and nutrient absorption, including glucose [36]. Furthermore, the positive regulation of claudin-2 and claudin-15 may be an attempt by the organism to compensate for the reduction in the mucosal surface area, thereby increasing absorption [37]. However, long-term dysregulation may lead to more serious complications, such as bacterial translocation.

Finally, tofacitinib did not reverse the reduction in tricellulin levels but maintained mucosal integrity. This suggests that the maintenance of the mucosal structure relies on multiple components. While tofacitinib has demonstrated efficacy in UC, its effectiveness in CD is still debated [38]. However, our positive results in the TNBS-induced colitis model indicate promising therapeutic possibilities.

A limitation of this study was the lack of a direct investigation into the effect of tofacitinib on the JAK pathway. However, the study by Sayoc-Becerra et al. [31] demonstrated that tofacitinib exerts a direct effect on intestinal epithelial cells by inhibiting the activation of JAK1-STAT1/3 induced by IFN-γ, which supports the possibility that the drug modulates this pathway. Although other limitations were identified, such as the use of a single dose of tofacitinib, application in only one IBD model, and the lack of evaluation of the effect on bacterial translocation, this study significantly contributes to deepening our understanding of the pathophysiology of colitis and the pharmacology of tofacitinib, paving the way for future research.

In conclusion, the TNBS-induced colitis rat model showed a loss of colonic mucosal integrity, which was associated with an increase in proinflammatory cytokines. Treatment with tofacitinib demonstrated efficacy not only in reducing inflammatory damage, but also in recovering mucosal integrity. This was evidenced by restoration of the pore-forming proteins claudin-2 and claudin-15. These results highlight the potential of tofacitinib to restore colonic barrier function under inflammatory conditions.

NOTES

Funding Source

This research was supported by the Fundacao Cearense de Apoio ao Desenvolvimento Cientifico e Tecnologico (FUNCAP; Edital nº 05/2019).

Conflict of Interest

No potential conflict of interest relevant to this article was reported.

Data Availability Statement

All study-related data is included in the publication or provided as supplementary information.

Author Contributions

Conceptualization: Costa Filho HB, Souza MHLP. Methology: Costa Filho HB, Araujo GAC, Paula SM, Sales TMAL, Clementino MAF, Havt A, Soares PMG, Souza MHLP. Project administration: Costa Filho HB, Souza MHLP. Visualization: Costa Filho HB, Soares PMG, Souza MHLP. Writing - original draft: Costa Filho HB, Souza MHLP. Writing - review & editing: Costa Filho HB, Souza MHLP. Approval of final manuscripts: all authors.

Fig. 1.

Tofacitinib attenuates microscopic damage to colonic mucosa in a rat model of TNBS-induced colitis. Representative images of colonic tissue in H&E-stained histological sections. Micrograph (×100) representing. (A) Sham group, (B) colitis group, (C) colitis+dexamethasone group, and (D) colitis+tofacitinib group. Black arrow, loss of mucosal architecture; yellow arrow, cellular infiltration; blue arrow, thickening of the muscularis muscle; green arrow, goblet cells. TNBS, 2,4,6-trinitrobenzenesulfonic acid; H&E, hematoxylin and eosin.

Fig. 2.

Tofacitinib attenuates colonic mucosal wet weight, macroscopic scores and MPO in a rat model of TNBS-induced colitis. Tofacitinib attenuated wet weight, macroscopic scores, and MPO in colonic tissues. (A) Wet weight, (B) macroscopic score, and (C) MPO. Results are expressed as mean±SEM. Sham (n=7), colitis (n=8), tofacitinib (n=8), dexamethasone (n=8). ^aP<0.05 versus sham group; ^bP<0.05 versus colitis group (one-way ANOVA followed by the Bonferroni test). MPO, myeloperoxidase; TNBS, 2,4,6-trinitrobenzenesulfonic acid; SEM, standard error of the mean; ANOVA, analysis of variance.

Fig. 3.

Tofacitinib reduces cytokine levels in a TNBS-induced colitis model in rats. Tofacitinib reduces cytokine levels in a TNBS-induced colitis model in rats. (A) IL-6 and (B) IL-1β. Results are expressed as mean±SEM. Sham (n=7), colitis (n=8), tofacitinib (n=8), dexamethasone (n=8). ^aP<0.05 versus sham group; ^bP<0.05 versus colitis group (one-way ANOVA followed by the Bonferroni test). TNBS, 2,4,6-trinitrobenzenesulfonic acid; IL, interleukin; SEM, standard error of the mean; ANOVA, analysis of variance.

Fig. 4.

Tofacitinib preserves basal TER in a rat model of TNBS-induced colitis. Baseline TER comparison among the sham, colitis, colitis+tofacitinib, and colitis+dexamethasone groups. Values are presented as mean±SEM. ^aP<0.05 versus sham; ^bP<0.05 versus colitis (ANOVA followed by the Bonferroni post-hoc test). TER, transepithelial electrical resistance; TNBS, 2,4,6-trinitrobenzenesulfonic acid; SEM, standard error of the mean; ANOVA, analysis of variance.

Fig. 5.

Tofacitinib reduces TER decline and increased fluorescein permeability in a rat model of TNBS-induced colitis. Comparison of the drop in TER during 60 minutes (A) with representation of time 60 minutes (B) and of the permeability to fluorescein during 90 minutes (C) with representation of time 90 minutes (D) between the sham, colitis, and colitis+tofacitinib groups. Values are presented as mean±SEM. ^aP<0.05 versus sham; ^bP<0.05 versus colitis (ANOVA followed by the Bonferroni post-hoc test). TER, transepithelial electrical resistance; TNBS, 2,4,6-trinitrobenzenesulfonic acid; SEM, standard error of the mean; ANOVA, analysis of variance.

Fig. 6.

Tofacitinib preserves junction proteins in a rat model of TNBS-induced colitis using Western blotting. Expression of claudin-2 (A), claudin-15 (B), and tricellulin (C) in sham, colitis and colitis+tofacitinib groups. Values are presented as mean±SEM. ^aP<0.05 versus sham; ^bP<0.05 versus colitis (ANOVA followed by the Bonferroni post-hoc test). TNBS, 2,4,6-trinitrobenzenesulfonic acid; SEM, standard error of the mean; ANOVA, analysis of variance.

Table 1.

Microscopic Damage in TNBS-Induced Colitis

Experimental group	Criteria
Experimental group	Loss of mucsal architecure (0–3)	Cellular infiltration (0–3)	Muscle thickening (0–3)	Crypt abscess (0–1)	Goblet cell depletion (0–1)
Sham (n = 5)	0 (0–1)	0 (0–1)	0 (0–0)	0 (0–1)	0 (0–0)
Colitis (n = 8)	3 (3–3)^a	3 (3–3)^a	3 (2–3)^a	1 (1–1)^a	1 (0–1)^a
Tofacitinib (n = 8)	0.5 (0–1)^b	1 (1–2)^b	1 (0–2)^b	0.5 (0–1)	0 (0–0)^b
Dexamethasone (n = 8)	0 (0–1)^b	1 (0–1)^b	0 (0–1)^b	0 (0–0)^b	0 (0–0)^b

Values are presented as median (range).

^a P<0.05 versus sham group;

^b P<0.05 versus colitis group 7th day. Kruskal-Wallis test followed by Dunn’s test.

TNBS, 2,4,6-trinitrobenzenesulfonic acid.

REFERENCES

1. Yeshi K, Ruscher R, Hunter L, Daly NL, Loukas A, Wangchuk P. Revisiting inflammatory bowel disease: pathology, treatments, challenges and emerging therapeutics including drug leads from natural products. J Clin Med 2020;9:1273.Article PubMed PMC
2. Rogler G, Singh A, Kavanaugh A, Rubin DT. Extraintestinal manifestations of inflammatory bowel disease: current concepts, treatment, and implications for disease management. Gastroenterology 2021;161:1118–1132.Article PubMed
3. Ng SC, Shi HY, Hamidi N, et al. Worldwide incidence and prevalence of inflammatory bowel disease in the 21st century: a systematic review of population-based studies. Lancet 2017;390:2769–2778.Article PubMed
4. Mak WY, Zhao M, Ng SC, Burisch J. The epidemiology of inflammatory bowel disease: east meets west. J Gastroenterol Hepatol 2020;35:380–389.Article PubMed PDF
5. Martel J, Chang SH, Ko YF, Hwang TL, Young JD, Ojcius DM. Gut barrier disruption and chronic disease. Trends Endocrinol Metab 2022;33:247–265.Article PubMed
6. Chelakkot C, Ghim J, Ryu SH. Mechanisms regulating intestinal barrier integrity and its pathological implications. Exp Mol Med 2018;50:1–9.Article PDF
7. Tsukita S, Tanaka H, Tamura A. The claudins: from tight junctions to biological systems. Trends Biochem Sci 2019;44:141–152.Article PubMed
8. Costa-Filho HB, Sales TM, Paula SM, et al. Role of cyclooxygenases 1 and 2 in the maintenance of colonic mucosal integrity in an experimental colitis model. Braz J Med Biol Res 2023;56:e12946.Article PubMed PMC
9. Petagna L, Antonelli A, Ganini C, et al. Pathophysiology of Crohn’s disease inflammation and recurrence. Biol Direct 2020;15:23.Article PubMed PMC PDF
10. Spalinger MR, Sayoc-Becerra A, Ordookhanian C, et al. The JAK inhibitor tofacitinib rescues intestinal barrier defects caused by disrupted epithelial-macrophage interactions. J Crohns Colitis 2021;15:471–484.Article PubMed PDF
11. Morris GP, Beck PL, Herridge MS, Depew WT, Szewczuk MR, Wallace JL. Hapten-induced model of chronic inflammation and ulceration in the rat colon. Gastroenterology 1989;96:795–803.Article PubMed
12. Lee SN, Stork C, Wahl C, Singh S, Chuang E. Targeted delivery of soluble tofacitinib citrate to the site of inflammation to improve efficacy and safety. Gastroenterology 2021;160:S328.
13. Iglesias M, Khalifian S, Oh BC, et al. A short course of tofacitinib sustains the immunoregulatory effect of CTLA4-Ig in the presence of inflammatory cytokines and promotes long-term survival of murine cardiac allografts. Am J Transplant 2021;21:2675–2687.Article PubMed PDF
14. Appleyard CB, Wallace JL. Reactivation of hapten-induced colitis and its prevention by anti-inflammatory drugs. Am J Physiol 1995;269(1 Pt 1): G119–G125.Article PubMed
15. Bradley PP, Priebat DA, Christensen RD, Rothstein G. Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker. J Invest Dermatol 1982;78:206–209.Article PubMed
16. Richens JL, Urbanowicz RA, Metcalf R, Corne J, O’Shea P, Fairclough L. Quantitative validation and comparison of multiplex cytokine kits. J Biomol Screen 2010;15:562–568.Article PubMed PDF
17. Tobey NA, Hosseini SS, Argote CM, Dobrucali AM, Awayda MS, Orlando RC. Dilated intercellular spaces and shunt permeability in nonerosive acid-damaged esophageal epithelium. Am J Gastroenterol 2004;99:13–22.Article PubMed
18. Rivero-Gutiérrez B, Anzola A, Martínez-Augustin O, de Medina FS. Stain-free detection as loading control alternative to Ponceau and housekeeping protein immunodetection in Western blotting. Anal Biochem 2014;467:1–3.Article PubMed
19. Rodrigues de Carvalho L, de Brito TV, Simião da C Júnior J, et al. Epiisopiloturine, an imidazole alkaloid, reverses inflammation and lipid peroxidation parameters in the Crohn disease model induced by trinitrobenzenosulfonic acid in Wistar rats. Biomed Pharmacother 2018;102:278–285.Article PubMed
20. Monteiro CE, Filho HB, Silva FG, et al. Euterpe oleracea Mart. (Açaí) attenuates experimental colitis in rats: involvement of TLR4/COX-2/NF-ĸB. Inflammopharmacology 2021;29:193–204.Article PubMed PDF
21. Dutra NL, de Brito TV, de Aguiar Magalhães D, et al. Sulfated polysaccharide extracted from seaweed Gracilaria caudata attenuates acetic acid-induced ulcerative colitis. Food Hydrocoll 2021;111:106221.Article
22. Atreya R, Mudter J, Finotto S, et al. Blockade of interleukin 6 trans signaling suppresses T-cell resistance against apoptosis in chronic intestinal inflammation: evidence in Crohn disease and experimental colitis in vivo. Nat Med 2000;6:583–588.Article PubMed PDF
23. Kai Y, Takahashi I, Ishikawa H, et al. Colitis in mice lacking the common cytokine receptor gamma chain is mediated by IL-6-producing CD4+ T cells. Gastroenterology 2005;128:922–934.Article PubMed
24. Akhtar M, Shaukat A, Zahoor A, et al. Anti-inflammatory effects of Hederacoside-C on Staphylococcus aureus induced inflammation via TLRs and their downstream signal pathway in vivo and in vitro. Microb Pathog 2019;137:103767.Article PubMed
25. Zha ZX, Lin Y, Wang KX, et al. Hederacoside C ameliorates colitis via restoring impaired intestinal barrier through moderating S100A9/MAPK and neutrophil recruitment inactivation. Acta Pharmacol Sin 2023;44:105–119.Article PubMed PDF
26. Karaman MW, Herrgard S, Treiber DK, et al. A quantitative analysis of kinase inhibitor selectivity. Nat Biotechnol 2008;26:127–132.Article PubMed PDF
27. Ghoreschi K, Jesson MI, Li X, et al. Modulation of innate and adaptive immune responses by tofacitinib (CP-690,550). J Immunol 2011;186:4234–4243.Article PubMed PDF
28. Dong K, Deng SJ, He BY, et al. Mucoadhesive nanoparticles enhance the therapeutic effect of dexamethasone on experimental ulcerative colitis by the local administration as an enema. Drug Des Devel Ther 2023;17:191–207.Article PubMed PMC PDF
29. Lundgren D, Widbom L, Hultdin J, Karling P. Preclinical markers in inflammatory bowel disease: a nested case-control study. Crohns Colitis 360 2021;3–otab072.Article PDF
30. Voshagh Q, Anoshiravani A, Karimpour A, et al. Investigating the association between the tissue expression of miRNA-101, JAK2/STAT3 with TNF-α, IL-6, IL-1β, and IL-10 cytokines in the ulcerative colitis patients. Immun Inflamm Dis 2024;12:e1224.Article PubMed PMC
31. Sayoc-Becerra A, Krishnan M, Fan S, et al. The JAK-inhibitor tofacitinib rescues human intestinal epithelial cells and colonoids from cytokine-induced barrier dysfunction. Inflamm Bowel Dis 2020;26:407–422.Article PubMed PDF
32. Robinson JM, Turkington S, Abey SA, Kenea N, Henderson WA. Differential gene expression and gene-set enrichment analysis in Caco-2 monolayers during a 30-day timeline with dexamethasone exposure. Tissue Barriers 2019;7:e1651597.Article PubMed PMC
33. Nighot M, Liao PL, Morris N, et al. Long-term use of proton pump inhibitors disrupts intestinal tight junction barrier and exaggerates experimental colitis. J Crohns Colitis 2023;17:565–579.Article PubMed PDF
34. Olivier S, Diounou H, Pochard C, et al. Intestinal epithelial AMPK deficiency causes delayed colonic epithelial repair in DSS-induced colitis. Cells 2022;11:590.Article PubMed PMC
35. Suzuki T, Yoshinaga N, Tanabe S. Interleukin-6 (IL-6) regulates claudin-2 expression and tight junction permeability in intestinal epithelium. J Biol Chem 2011;286:31263–31271.Article PubMed PMC
36. Tamura A, Hayashi H, Imasato M, et al. Loss of claudin-15, but not claudin-2, causes Na+ deficiency and glucose malabsorption in mouse small intestine. Gastroenterology 2011;140:913–923.Article PubMed
37. Ong ML, Yeruva S, Sailer A, Nilsen SP, Turner JR. Differential regulation of claudin-2 and claudin-15 expression in children and adults with malabsorptive disease. Lab Invest 2020;100:483–490.Article PubMed PDF
38. Panés J, Sandborn WJ, Schreiber S, et al. Tofacitinib for induction and maintenance therapy of Crohn’s disease: results of two phase IIb randomized placebo-controlled trials. Gut 2017;66:1049–1059.Article PubMed

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Restoration of colonic barrier function by tofacitinib in experimental colitis: anti-inflammatory effects and decreased expression of claudins-2 and claudin-15

DOI: https://doi.org/10.5217/ir.2024.00186

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Restoration of colonic barrier function by tofacitinib in experimental colitis: anti-inflammatory effects and decreased expression of claudins-2 and claudin-15

Fig. 1. Tofacitinib attenuates microscopic damage to colonic mucosa in a rat model of TNBS-induced colitis. Representative images of colonic tissue in H&E-stained histological sections. Micrograph (×100) representing. (A) Sham group, (B) colitis group, (C) colitis+dexamethasone group, and (D) colitis+tofacitinib group. Black arrow, loss of mucosal architecture; yellow arrow, cellular infiltration; blue arrow, thickening of the muscularis muscle; green arrow, goblet cells. TNBS, 2,4,6-trinitrobenzenesulfonic acid; H&E, hematoxylin and eosin.

Fig. 2. Tofacitinib attenuates colonic mucosal wet weight, macroscopic scores and MPO in a rat model of TNBS-induced colitis. Tofacitinib attenuated wet weight, macroscopic scores, and MPO in colonic tissues. (A) Wet weight, (B) macroscopic score, and (C) MPO. Results are expressed as mean±SEM. Sham (n=7), colitis (n=8), tofacitinib (n=8), dexamethasone (n=8). aP<0.05 versus sham group; bP<0.05 versus colitis group (one-way ANOVA followed by the Bonferroni test). MPO, myeloperoxidase; TNBS, 2,4,6-trinitrobenzenesulfonic acid; SEM, standard error of the mean; ANOVA, analysis of variance.

Fig. 3. Tofacitinib reduces cytokine levels in a TNBS-induced colitis model in rats. Tofacitinib reduces cytokine levels in a TNBS-induced colitis model in rats. (A) IL-6 and (B) IL-1β. Results are expressed as mean±SEM. Sham (n=7), colitis (n=8), tofacitinib (n=8), dexamethasone (n=8). aP<0.05 versus sham group; bP<0.05 versus colitis group (one-way ANOVA followed by the Bonferroni test). TNBS, 2,4,6-trinitrobenzenesulfonic acid; IL, interleukin; SEM, standard error of the mean; ANOVA, analysis of variance.

Fig. 4. Tofacitinib preserves basal TER in a rat model of TNBS-induced colitis. Baseline TER comparison among the sham, colitis, colitis+tofacitinib, and colitis+dexamethasone groups. Values are presented as mean±SEM. aP<0.05 versus sham; bP<0.05 versus colitis (ANOVA followed by the Bonferroni post-hoc test). TER, transepithelial electrical resistance; TNBS, 2,4,6-trinitrobenzenesulfonic acid; SEM, standard error of the mean; ANOVA, analysis of variance.

Fig. 5. Tofacitinib reduces TER decline and increased fluorescein permeability in a rat model of TNBS-induced colitis. Comparison of the drop in TER during 60 minutes (A) with representation of time 60 minutes (B) and of the permeability to fluorescein during 90 minutes (C) with representation of time 90 minutes (D) between the sham, colitis, and colitis+tofacitinib groups. Values are presented as mean±SEM. aP<0.05 versus sham; bP<0.05 versus colitis (ANOVA followed by the Bonferroni post-hoc test). TER, transepithelial electrical resistance; TNBS, 2,4,6-trinitrobenzenesulfonic acid; SEM, standard error of the mean; ANOVA, analysis of variance.

Fig. 6. Tofacitinib preserves junction proteins in a rat model of TNBS-induced colitis using Western blotting. Expression of claudin-2 (A), claudin-15 (B), and tricellulin (C) in sham, colitis and colitis+tofacitinib groups. Values are presented as mean±SEM. aP<0.05 versus sham; bP<0.05 versus colitis (ANOVA followed by the Bonferroni post-hoc test). TNBS, 2,4,6-trinitrobenzenesulfonic acid; SEM, standard error of the mean; ANOVA, analysis of variance.

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Restoration of colonic barrier function by tofacitinib in experimental colitis: anti-inflammatory effects and decreased expression of claudins-2 and claudin-15

Experimental group	Criteria
Experimental group	Loss of mucsal architecure (0–3)	Cellular infiltration (0–3)	Muscle thickening (0–3)	Crypt abscess (0–1)	Goblet cell depletion (0–1)
Sham (n = 5)	0 (0–1)	0 (0–1)	0 (0–0)	0 (0–1)	0 (0–0)
Colitis (n = 8)	3 (3–3)^a	3 (3–3)^a	3 (2–3)^a	1 (1–1)^a	1 (0–1)^a
Tofacitinib (n = 8)	0.5 (0–1)^b	1 (1–2)^b	1 (0–2)^b	0.5 (0–1)	0 (0–0)^b
Dexamethasone (n = 8)	0 (0–1)^b	1 (0–1)^b	0 (0–1)^b	0 (0–0)^b	0 (0–0)^b

Table 1. Microscopic Damage in TNBS-Induced Colitis

Values are presented as median (range).

P<0.05 versus sham group;

P<0.05 versus colitis group 7th day. Kruskal-Wallis test followed by Dunn’s test.

TNBS, 2,4,6-trinitrobenzenesulfonic acid.