Ruxolitinib Alleviates Inflammation, Apoptosis, and Intestinal Barrier Leakage in Ulcerative Colitis via STAT3

Abstract

Background

Ulcerative colitis (UC) is an idiopathic, chronic inflammatory disorder of the colonic mucosa with increasing prevalence and limited management. Ruxolitinib is a new anti- JAK/STAT3 biologic agent that has shown potential in protecting against colitis.

Methods

We first constructed an in vivo UC model and an in vitro colonic epithelial cell inflammation model. Ruxolitinib was administered via gavage in mice. After treatment, colon tissues, cells, and cell lysates were collected and prepared for histological evaluation, immunohistochemistry, immunofluorescence staining, quantitative reverse-transcriptase polymerase chain reaction, Western blotting, terminal deoxynucleotidyl transferase mediated dUTP nick end labeling staining, and cytokine analysis. STAT3 expression was silenced and overexpressed via small interfering RNA and overexpression plasmid transfection, respectively, and quantitative reverse-transcriptase polymerase chain reaction was used to examine the downstream effects.

Results

Ruxolitinib administration significantly alleviated colitis both in vivo and in vitro, as manifested by reduced body weight loss, shortened colon lengths, relieved disease activity (measured by the disease activity index), and prolonged survival. A mechanistic study showed that ruxolitinib attenuated nuclear factor kappa B–induced inflammation, reduced apoptosis, and ameliorated epithelial barrier leakage, and thereby reduced colitis activity in vivo. STAT3 knockdown partially reversed the protective effect of ruxolitinib against colitis, while STAT3 overexpression exaggerated the reductions in proinflammatory cytokine levels upon ruxolitinib treatment.

Conclusions

We demonstrate that ruxolitinib alleviates colitis by inhibiting nuclear factor kappa B–related inflammation and apoptosis in addition to restoring epithelial barrier function via STAT3, providing a new strategy for UC treatment.

Introduction

Ulcerative colitis (UC) is a chronic inflammatory bowel disease of unknown etiology that involves the whole length of the colon, starting with mucosal inflammation in the rectum and extending proximally in a continuous pattern.^1,2 The prevalence of UC has been increasing globally in recent decades, especially in Asian countries, particularly China; thus, the burden of UC is rising substantially.^3,4 The current treatment goal of UC is to achieve resolution of symptoms, including rectal bleeding and diarrhea, as well as healing of mucosal friability and ulceration within 3 months after initiation of management. Achieving this goal with currently available therapy remains challenging mainly because of incontinence due to intolerance or adverse events and limited efficacy.⁵

A better understanding of UC pathogenesis would aid in the development of effective new agents with improved tolerance and reduced side effects. Although the exact cause and pathogenesis of UC are unclear, potential influencing factors include genetic background, environmental and luminal factors, and mucosal immune dysregulation.⁶ Apoptosis, barrier leakage, and inflammation are 3 key components of UC pathogenesis. UC is an intestinal barrier dysfunction disease driven initially by epithelial cell defects, such as apoptosis; after these defects occur, the barrier can be disrupted by strong inflammatory mediators or inflammatory cell attack in the lamina propria, resulting in barrier leakage and ultimately leading to disease chronicity.^2,7,8

A basic approach to cure UC is to inhibit apoptosis and inflammation and restore barrier function. Currently, conventional management agents include mesalazine, steroids, and thiopurines.⁹ Since the early 2000s, several biological agents and small molecules have emerged, providing new insight into the management of inflammatory bowel disease; however, the clinical application of these drugs has been limited because of a lack of tolerance and patient compliance.¹⁰ Therefore, orally administered small molecules have been under intense development in recent years, and these agents are likely to substantially shift the way this chronic disease is treated.¹¹

The JAK-STAT pathway is an intracellular kinase pathway involved in cytokine signaling, and JAK inhibitors are a promising new class of drugs that can interrupt inflammation by modulating adaptive and innate immune responses in UC.¹² Multiple JAK inhibitors, especially tofacitinib, have been widely investigated for their effects on UC and have shown potential efficacy through twice-daily doses in both induction and maintenance therapy.¹³ Ruxolitinib is a first-generation pan-JAK inhibitor that blocks a wide spectrum of cytokines and has been shown to be safe. Ruxolitinib has already been approved for a range of diseases, including polycythemia rubra vera and myelofibrosis, and is already approved by the Food and Drug Administration (FDA) for treatment of graft-vs-host disease.¹⁴ A recent study has shown the potential of ruxolitinib in protecting against immune-related colitis¹⁵; however, the effect of ruxolitinib in UC and the exact mechanism have not yet been discussed.

Therefore, in this study, we first tested the effect of ruxolitinib on UC and discovered that ruxolitinib administration significantly alleviated murine colitis by relieving disease activity and prolonged survival. Furthermore, a mechanistic study demonstrated that ruxolitinib attenuated intestinal epithelial cell (IEC) nuclear factor kappa B (NF-κB)–induced inflammation, reduced apoptosis, and ameliorated epithelial barrier leakage via STAT3.

Methods

Mice

Six- to 8-week-old male C57BL/6 mice were purchased from Shanghai SLAC Laboratory Animal Co, Ltd and were bred and maintained in the experimental animal center of Ningbo University under a 12-hour light cycle with a regular chow diet and water ad libitum. The mice were acclimated for 7 days before each experiment. All animal experiments were approved by the Animal Care and Use Committee of Ningbo University and were performed in accordance with the Guidelines for Animal Care of Kiel University and the University of Cambridge.

Colitis models and ruxolitinib treatment in vivo

Colitis was induced by addition of 3.5% dextran sulfate sodium (DSS) (36-50 kDa; MP Biomedicals) to drinking water or administration of 2.5% TNBS (Ark Pharm), as shown in Figure 1A and 1I and Supplementary Figure 1A and according to the methods in a previous study.¹⁶ Ruxolitinib was administered via gavage at a dose of 60 mg/kg/d.^17-19 In the 12 days of the ruxolitinib treatment model, DSS was administered for 7 days, followed by 5 days of treatment with ruxolitinib beginning on the final day of DSS administration, with a total course of 12 days to establish the therapeutic acute DSS-induced colitis model according to a previous study.¹⁶ The severity of colitis was scored daily according to standard parameters such as body weight and the presence of diarrhea and bloody stools. Colonic tissues were collected from the mice after sacrifice, fixed in 10% paraformaldehyde overnight, embedded in paraffin, and stained with hematoxylin and eosin or prepared for immunohistochemistry. An inflammatory score and disease activity index were assigned after histological evaluation and used to evaluate disease severity according to the methods in previous reports.^20,21

Cell culture

Heterogeneous human epithelial colorectal adenocarcinoma cells (HCT116 cells and Caco-2 cells) (Institute of Biochemistry and Cell Biology, China Academy of Sciences) were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen) and 100 U/mL penicillin‒streptomycin (Sigma-Aldrich) at 37 °C in a humidified 5% CO₂ atmosphere.

In vitro colonic epithelial cell colitis model

HCT116 cells were exposed to 100 ng/mL tumor necrosis factor α (TNF-α) (PeproTech) for 24 hours, and Caco-2 cells were exposed to 1 μg/mL lipopolysaccharide (Sigma-Aldrich), 50 ng/mL recombinant human (hr) TNF-α, 50 ng/mL hr interferon γ (IFN-γ), and 25 ng/mL hr interleukin (IL)-1β (PeproTech) for 24 hours according to a previous study.¹⁶ The cells were then treated with different concentrations of ruxolitinib. After treatment, the cell lysates were prepared for further quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) or Western blot analysis.

Cell viability assay

HCT116 cells and Caco-2 cells were plated at a density of 8000 cells/well in 96-well plates and cultured with ruxolitinib at various concentrations, respectively. After treatment for 24 hours, 10 μL/well CCK-8 solution was added to the medium, the cells were incubated in the dark for another 4 hours, and then the absorbance was measured using a microplate reader at 450 nm.

Mouse epithelial cell isolation and dissociation

Colons were collected from mice and washed in ice-cold phosphate-buffered saline (PBS) until the luminal contents were removed and then digested on ice in dissociation reagent containing EDTA (Sigma-Aldrich), DTT (Sigma-Aldrich), and Y27632 (Sigma-Aldrich). Then, the intestine samples were treated with another dissociation reagent (EDTA and Y27632) and incubated at 37 °C for 10 minutes. The tube containing each intestine sample was shaken, and the removed epithelium was resuspended in Hank’s Balanced Salt Solution (Sigma-Aldrich) containing dispase (Sigma-Aldrich) and incubated for 10 minutes at 37 °C. After the addition of 10 mg/mL DNase (Roche), the solution was passed through 70-μm filters and colonic epithelial cells were obtained.

Intestinal permeability assay

Mice were fasted for 6 hours and then given FITC-Dextran 4000 (600 mg/kg) through intragastric administration, followed by cardiac aspiration for blood collection 1 hour later. Next, 100 μL of the sample, standard substance, and mouse plasma were added to a 96-well plate, followed by detection using a fluorescence spectrophotometer. Then, the concentration of FITC-Dextran 4000 was analyzed and the permeability was evaluated.

Immunohistochemistry

Cell lysates were immunoprecipitated with beads prelinked with anti-phosphorylated (p)-p65 antibodies (Abcam) for 3 hours at 4 °C. After the immunoprecipitate was washed, the immunoprecipitated complexes were eluted with 0.1 M glycine buffer and then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Immunofluorescence staining and confocal microscopy

First, cells were fixed with 4% formaldehyde for 30 minutes. After the cells were washed 3 times with PBS, they were incubated in PBS with fetal calf serum to block nonspecific antibody adsorption. Then, the cells were incubated with the corresponding primary and secondary antibodies in blocking buffer. Images were captured with an Olympus confocal microscope.

Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay

The terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method was used to evaluate apoptosis in paraffin-embedded thin tissue sections (5-μm thick). A commercially available TUNEL apoptosis detection kit (Roche) was used according to the manufacturer’s instructions, and images were captured with a light microscope (Leica).

Western blot analysis

To measure the expression levels of certain proteins, target cells or distal colon tissues were collected, washed with cold PBS, and then lysed in RIPA buffer (Pulilai BioTech) containing a protease inhibitor cocktail (Sigma-Aldrich). Proteins (30 μg/sample) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (0.45-mm pore; Millipore). After antigens were blocked with 5% skim milk powder diluted in Tris-buffered saline containing 0.5% Tween 20 for 1 hour, each membrane was incubated with JAK2, p-JAK1/2, STAT3, p38, phosphorylated IKKα/β (p-IKKα/β) (Ser176/180), IKKα, IKKβ, occludin, cleaved caspase-3 (Asp175) (Cell Signaling Technology), zonulin-1, NF-κB p65 (Abcam), JAK1, p-STAT3, p-p38, p-NF-κB p65 (Ser536), Bcl-xl (Santa Cruz Biotechnology), Bid, and GAPDH (Proteintech) primary antibodies at 4 °C overnight and then with secondary antibodies at room temperature for 2 hours. Proteins were then detected using an enhanced chemiluminescence light detection kit (Lianke Multi Sciences). GAPDH was used as the loading control. Each experiment was conducted in triplicate for each of the conditions.

Quantitative RT-PCR

RNAiso Plus (Takara) was used to extract total RNA from cells or tissues. PrimeScript RT Master Mix (Takara) was used to generate complementary DNA. Quantitative PCR was performed with SYBR Green Premix Dimer Eraser (Takara) on a 7900HT Fast Real-Time PCR system (Applied Biosystems). The cycle threshold values for all triplicate samples were averaged and further analyzed using the ΔΔCT method. All data were normalized to β-actin or GAPDH expression. The primer sequences are presented in Supplementary Table 6, and all primers were synthesized by Sangon Biotech.

Statistical analysis

All statistical analyses were performed with SPSS 22 (IBM). The values are presented as the mean ± SD. Statistical analysis was conducted using Student’s 2-tailed t test or Pearson’s correlation analysis. A P value <.05 was considered to indicate statistical significance.

Ethical considerations

The final protocol and any amendments were reviewed and approved by the Animal Care and Use Committee of Ningbo University. The study was conducted in compliance with the Declaration of Helsinki and with all International Council for Harmonization Good Clinical Practice guidelines.

Results

Ruxolitinib alleviated colitis in a rodent model

To investigate the effect of ruxolitinib on UC, a 12-day ruxolitinib treatment model (Figure 1A), a prophylactic ruxolitinib model (Figure 1I), and a simultaneous use of ruxolitinib in acute DSS model (Supplementary Figure 1A) were used. The results showed that ruxolitinib treatment significantly reversed DSS-induced acute colitis in the 12-day ruxolitinib treatment model, as indicated by the reduced body weight loss (Figure 1B), shortened colon length (Figure 1C and 1D), alleviation of distorted epithelial crypt architecture accompanied by significant immune cell infiltration (Figure 1E and 1F), improvement of survival (Figure 1G), and reduction in disease activity as measured by the disease activity index (Figure 1H). To further test the effect of ruxolitinib in other classic colitis models, a prophylactic ruxolitinib model (Figure 1I-1P) was adopted, in which similar effectiveness was found. However, ruxolitinib was not robust enough to improve colitis when used simultaneously for DSS models (Supplementary Figure 1B-1H)

Ruxolitinib alleviated colonic inflammation

To further clarify the mechanism by which ruxolitinib affects colitis, 3 key elements in colitis pathogenesis, colonic inflammation, intestinal barrier leakage, and apoptosis, were examined. Ruxolitinib significantly inhibited the expression of proinflammatory cytokines (TNF-α, IL-6, IL-1β, and IFN-γ) in the 12-day ruxolitinib treatment model (Figure 2A) and the prophylactic ruxolitinib model (Figure 2B); it also inhibited the expression of chemokines (IL-8, CXCL1, CXCL2, and CCL2) in the 12-day ruxolitinib treatment model (Figure 2C) and the prophylactic ruxolitinib model (Figure 2D). Ruxolitinib exhibited similar inhibitory effects on proinflammatory cytokines and chemokines in the simultaneous use of ruxolitinib in acute DSS model (Supplementary Figure 2A and 2B). Moreover, ruxolitinib reduced TNF-α and IFN-γ mRNA levels in the in vitro colonic epithelial HCT116 cell colitis model, and TNF-α and IL-6 mRNA levels in the in vitro colonic epithelial Caco-2 cell colitis model in a concentration-dependent manner, respectively (Supplementary Figure 3A-3F).

Ruxolitinib ameliorated intestinal barrier leakage in colitis

To examine intestinal barrier leakage in the context of colitis, a FITC-Dextran experiment was performed. The results showed that colonic permeability was increased in colitis but decreased by ruxolitinib treatment in the 12-day ruxolitinib treatment model, the prophylactic ruxolitinib model and the simultaneous use of ruxolitinib in acute DSS model (Figure 3A and 3C and Supplementary Figure 4A). Furthermore, zonulin-1 and occludin, 2 classic protein markers of epithelial barrier function, were significantly downregulated in IECs upon colitis in the 3 models, but their expression was restored by ruxolitinib treatment (Figure 3B and 3D, Supplementary Figure 4B-4D, and Supplementary Figure 5A and 5B).

Ruxolitinib inhibited apoptosis in colitis

Furthermore, to evaluate apoptosis, we performed TUNEL assays and examined the expression of cleaved caspase 3, Bcl-xl, and Bid. Apoptosis induction was observed, as shown by the increased sizes of dots in the TUNEL assay, and this outcome was ameliorated by ruxolitinib treatment in all of the models mentioned previously (Figure 4A and 4B and Supplementary Figure 6A). Similarly, the inhibition of Bcl-xl and Bid expression and transcription and the increased cleavage of caspase 3 observed in IECs upon colitis were all reversed by ruxolitinib treatment (Figure 4C-4J and Supplementary Figure 6B-6D).

Ruxolitinib alleviated colitis via STAT3

As a JAK inhibitor, ruxolitinib might protect against colitis via JAK/STAT3; thus, the downstream NF-κB p65 and MAPK p38 pathways were also analyzed. JAK1/2 and STAT3 phosphorylation was induced in colitis, but this effect was attenuated by treatment with ruxolitinib both in vivo and in vitro in IECs (Figure 5A and 5B, Supplementary Figure 7A, and Supplementary Figure 8A). Similarly, ruxolitinib inhibited the downstream NF-κB p65 pathway and MAPK p38 activation in IECs in colitis (Figure 5, Supplementary Figure 7, and Supplementary Figure 8). To investigate whether ruxolitinib can alleviate colitis through STAT3, STAT3 expression in HCT116 and Caco-2 cells was selectively knocked down via small interfering RNA transfection and overexpressed through plasmid transfection (Figure 6). STAT3 knockdown partially blocked the ability of ruxolitinib to ameliorate proinflammatory cytokine upregulation in cellular colitis models. In contrast, overexpression of STAT3 exaggerated the reduction in proinflammatory cytokine levels upon ruxolitinib treatment in both HCT116 cells and Caco-2 cells. Taken together, these findings verify that the protective effect of ruxolitinib against colitis relies on STAT3.

Discussion

This study revealed the protective effect of the new pan-JAK inhibitor ruxolitinib against UC. Ruxolitinib administration significantly alleviated murine colitis by reducing disease activity, restoring body weight and prolonging survival in 3 patterns of ruxolitinib-treated models with 2 chemical-induced colitis models. Concerning the mechanism, ruxolitinib attenuated inflammation, reduced apoptosis, and restored epithelial barrier leakage in IECs, which alleviated colitis. STAT3 knockdown partially reversed the protective effect of ruxolitinib against colitis in an in vitro colonic epithelial cell colitis model. This study reveals the mechanism by which ruxolitinib affects UC and provides a new target for drug development.

The current therapies for UC have limitations of mild effects, slow onset of action, and intolerable adverse events.⁹ Since the early 21st century, several biological agents and small molecules have shown efficacy in UC,^22-24 including newly developed JAK inhibitors.^13,25 According to current clinical experience, JAK inhibitors, which are targeted oral therapies, have potential benefits including ease of use, which is likely to improve patient compliance; reduced cost; convenient storage; low transport cost; a shortened half-life; the option of a stop-start strategy; minimal adverse effects upon discontinuation; and reduced immunogenicity for patients who already exhibit immunogenic responses to protein molecules.²⁶ The development of JAK inhibitors for UC treatment is therefore of great clinical value.

JAKs are intracellular tyrosine kinases that play crucial roles in the signaling pathways of many cytokines involved in immunity, acting as key targets of immune-related diseases, including UC.¹² JAK is activated via the phosphorylation of receptor-associated tyrosine residues to provide docking sites for STAT proteins, and phosphorylated STAT molecules then dimerize and translocate to the nucleus, where they act as potent regulators of gene expression.²⁷ Inhibiting JAK signaling therefore offers a novel strategy by which to block a range of cytokines using small molecule drugs, including first-generation pan-JAK inhibitors (tofacitinib, baricitinib, ruxolitinib, peficitinib) and second-generation selective JAK inhibitors (decernotinib, filgotinib, upadacitinib), which have been widely explored in the fields of dermatology, rheumatology, hematology, and, recently, inflammatory bowel disease treatment.^28-31 Itaconate and itaconate derivatives inhibit JAK1 to suppress M2 macrophage activation.³² The JAK2 inhibitor pacritinib significantly alleviates alcoholic and nonalcoholic liver fibrosis.³³ Another example is tofacitinib, which the U.S. Food and Drug Administration (FDA) approved for the treatment of rheumatoid arthritis in 2014.³⁴ The highly JAK3-selective inhibitor Z583 has been found to suppress γc cytokine signaling and inhibit the inflammatory response in a rheumatoid arthritis model.³⁵ In addition, treatment with ruxolitinib improves quality of life in myeloproliferative neoplasm patients.³⁴ All of the above JAK inhibitors show striking performance and good tolerance in clinical circumstances, including ruxolitinib, as shown in our study.

Ruxolitinib has been approved by the FDA for use in patients with polycythemia vera and myelofibrosis in 2011, hydroxyurea resistance or intolerance in 2014, and atopic dermatitis in 2021.^36,37 What’s more, many clinical trials tested the efficacy and safety of ruxolitinib. In a phase 2 study of ruxolitinib in chronic neutrophilic leukemia and atypical chronic myeloid leukemia patients, ruxolitinib showed an estimated response rate of 32%, and no serious adverse events due to ruxolitinib were observed.³⁸ In 2 phase 3 trials, greater repigmentation of vitiligo lesions appeared in the group applied ruxolitinib, although along with acne and pruritus at the application site.³⁹ With regard to clinical ethics,⁴⁰ larger trails are required to determine the effect and safety of ruxolitinib. Additionally, JAK antagonists, tofacitinib and upadacitinib, are used to treat UC and rheumatoid arthritis.⁴¹ Tofacitinib reduced remission of UC at the accepted dose and also improved health-related quality of life.⁴² Here, we demonstrate that ruxolitinib could alleviate murine colitis, so it may pass clinical trials eventually and be applied to treat UC patients in the future.

In the current study, we used colitis models to mimic UC pathogenesis and investigate the effects of ruxolitinib treatment. A large number of mouse colitis models have been introduced that exhibit histological and/or immunological features characteristic of inflammatory bowel diseases in humans. The most classic colitis models are colitis induced by DSS, TNBS, and oxazolone. Administration of TNBS or oxazolone results in T cell– or natural killer T cell–mediated^43,44 immunity. To generate DSS colitis models, mice are administered DSS, which causes epithelial cell death and subsequent inflammation.⁴³ We provide evidence in this study that ruxolitinib inhibits and reverses DSS-induced acute colitis and prevents TNBS-induced acute colitis, providing solid evidence for the use of ruxolitinib in UC treatment.

Importantly, we found that ruxolitinib attenuated IEC NF-κB–induced inflammation, reduced apoptosis, and ameliorated epithelial barrier leakage, but the effect of ruxolitinib on immune response of lamina propria is lack of research and it might require further research. Furthermore, our findings indicate that STAT3 knockdown partially blocked the ability of ruxolitinib to ameliorate proinflammatory cytokine upregulation and overexpression of STAT3 exaggerated the reduction in proinflammatory cytokine levels upon ruxolitinib in cellular colitis models. However, whether ruxolitinib alleviates colitis via STAT3 in murine colitis models is unclear; therefore, the role of ruxolitinib on STAT3 knockout colitis mice worth further exploring. What’s more, ruxolitinib needs to be validated for UC treatment in clinical trials as well as in real clinical circumstances in the future.

Conclusions

This study identified ruxolitinib as a potential therapeutic agent in UC and determined the mechanism by which ruxolitinib protects against colitis. Specifically, ruxolitinib attenuates NF-κB–induced inflammation, reduces apoptosis occurrence, and ameliorates epithelial barrier leakage, and thereby reduces colitis activity via STAT3 in IECs. These findings identify new strategies for UC treatment (Supplementary Figure 9).

Author Contribution

C.L., Y.X., and T.G. proposed the idea and designed the study procedures. C.L. conducted the animal and cellular experiments. Y.X. provided the experimental materials. T.G. performed qRT-PCR and S.S., Zhe.L., S.G., Y.F., X.Y., and S.Y. collected colonic tissues from mice. Q.J. and X.D. guided the manuscript writing. Zho.L. provided expertise regarding manuscript writing. J.Z., Q.W., and M.G. contributed new reagents/analytic tools. X.Z. guided the histological evaluations of inflammation. J.S. guided revisions to the article. Y.C. supervised and provided consultation during the study. All authors have reviewed and approved this manuscript.

Funding

This study was supported by funding from the National Natural Science Foundation of China (No. 82170582 to Y.C. and No. 82200578 to C.L.), the Natural Science Foundation of Zhejiang Province (No. LQ22H030001 to C.L. and No. LY21H160035 to X.Z.), the Medical and Health Plan of Zhejiang (No. 2021KY300 to S.G. and No. 2019KY580 to Q.W.), the Ningbo Medical Science and Technology Program (No. 2019Y32 to S.Y.), and the Medical and Health Plan of Zhejiang (No. 2019KY154 to Q.J.). The funding agency did not play any role in the study design, data collection and analysis, decisions regarding data release, or manuscript preparation.

Conflict of Interest

None of the authors have any conflicts of interest to claim.

References

Author notes