Bryostatin‐1 ameliorated experimental colitis in Il‐10 −/− Mice by protecting the intestinal barrier and limiting immune dysfunction

Abstract Bryostatin‐1 (Bry‐1) has been proven to be effective and safe in clinical trials of a variety of immune‐related diseases. However, little is known about its effect on Crohn's disease (CD). We aimed to investigate the impact of Bry‐1 on CD‐like colitis and determine the mechanism underlying this effect. In the present study, 15‐week‐old male Il‐10 −/− mice with spontaneous colitis were divided into positive control and Bry‐1‐treated (Bry‐1, 30 μg/kg every other day, injected intraperitoneally for 4 weeks) groups. Age‐matched, male wild‐type (WT) mice were used as a negative control. The effects of Bry‐1 on colitis, intestinal barrier function and T cell responses as well as the potential regulatory mechanisms were evaluated. We found that the systemic delivery of Bry‐1 significantly ameliorated colitis in Il‐10 −/− mice, as demonstrated by decreases in the disease activity index (DAI), inflammatory score and proinflammatory mediator levels. The protective effects of Bry‐1 on CD‐like colitis included the maintenance of intestinal barrier integrity and the helper T cell (Th)/regulatory T cell (Treg) balance. These effects of Bry‐1 may act in part through nuclear factor erythroid 2‐related factor 2 (Nrf2) signalling activation and STAT3/4 signalling inhibition. The protective effect of Bry‐1 on CD‐like colitis suggests Bry‐1 has therapeutic potential in human CD, particularly given the established clinical safety of Bry‐1.


| INTRODUC TI ON
Crohn's disease (CD) is a chronic, relapsing, progressive disease characterized by immune-mediated inflammation in the gastrointestinal tract. 1 The incidence and prevalence of CD is increasing worldwidely. 1,2 In the absence of aetiological treatment, medical therapy with the purpose of inducing and maintaining remission plays a key role in the management of CD. 3 Safer and more effective drugs need to be found to compensate for the shortcomings of current medical treatments. 1,3 Although the pathogenesis of CD remains largely unknown, CD is considered a disease arising from an interaction between genetic and environmental factors. 1 Over activated T helper cell type 1 (Th1) and T helper cell type 17 (Th17) immune responses contribute to intestinal damage in CD. 4 Regulatory T cells (Tregs), which are required for controlling excessive inflammation and maintaining immune homeostasis, also play an essential role in the pathogenesis of CD. 5 Undoubtedly, intestinal mucosal inflammation can indirectly or directly damage the intestinal barrier. 6 The intestinal epithelium at the interface between the intestinal microbiome/other antigens and the lymphoid tissue associated with the gastrointestinal system plays a critical role in shaping the mucosal immune response. 7,8 In addition, the inflammatory response often results in continued epithelial injury, which causes erosions and ulcerations. Indirect data suggest that a barrier defect might precede the onset of disease. 7 This conclusion stems from the fact that even patients with quiescent CD exhibit increased paracellular permeability. 9 However, there is no answer to the question of whether barrier dysfunction is a cause or consequence of CD. It seems that cross-talk between the impaired intestinal barrier and abnormal intestinal mucosal immune responses plays an important role in the occurrence and development of CD.
The diseased bowel could be removed by surgery, however, recurrence would still occur after surgery. In fact, CD is a disease with a tendency to relapse for life. However, protecting the intestinal mucosal barrier or regulating intestinal mucosal immunity should be beneficial to CD patients. 10 Interleukin (IL)-10 gene-deficient (Il-10 −/− ) mice are recognized as a CD animal model with intestinal barrier defects and abnormal intestinal mucosal immunity similar to those in human CD. 11, 12 We have long been committed to the study of how to improve the intestinal barrier and intestinal mucosal immunity in Il-10 −/− mice from multiple perspectives, including inhibiting Th1/Th17 immune responses, 13 promoting Treg activities, 14,15 limiting intestinal mucosal epithelial cell apoptosis, 14,16 protecting intestinal epithelial tight junction (TJ) proteins 17 and regulating autophagy. 15 However, we still think more agents, particularly drugs with the potential for rapid clinical application, should be developed to meet CD treatment needs.
Bryostatin-1 (Bry-1) is a naturally occurring macrocyclic lactone obtained from the marine bryozoan Bugula neritina. 18 Previous studies have shown that Bry-1 has a variety of biological activities, including protecting cell TJs, anti-inflammatory functions and immune regulation. 19 A recent study indicated that Bry-1 could promote the differentiation of CD4+ T helper (Th) lymphocytes into Th2 effector cells over Th1 and Th17 effector cells in experimental multiple sclerosis mice. 20 Furthermore, recent phase IIa clinical trials confirmed that Bry-1 not only showed good efficacy but also exhibited high safety in the treatment of Alzheimer's disease. 21,22 These findings suggest Bry-1 has potential as a therapeutic agent in CD, particularly given its established clinical safety.
In the present study, we investigated the effects of Bry-1 on spontaneous CD-like colitis in Il-10 −/− mice and the possible mechanism of these effects with the hope of providing a new therapeutic option for CD.

| Mice
Our experimental procedures were approved by the Animal Ethics Committee of Bengbu Medical College (Bengbu, China). Wild-type (WT) mice (C57BL/6J) and Il-10 −/− mice on the C57BL/6J background were obtained from the Jackson Laboratory and were maintained in a specific pathogen-free (SPF) environment at the Animal Center of Bengbu Medical College (Bengbu, China). All mice were housed in plastic-bottomed, wire-lidded cages and kept at 25°C with a 12-hour light/dark cycle. The mice had free access to water and were fed regular mouse chow. As reported previously, the Il-10 −/− mice consistently developed colitis at 15 weeks of age when housed in the SPF environment. 23

| Drug administration protocol
Fifteen-week-old male Il-10 −/− mice with spontaneous enteritis were divided into positive control (IL-10 −/− ) and Bry-1-treated groups (Bry-1). Age-matched male WT mice were used as negative controls (WT). Each group in this study contained eight mice.

| Colitis symptom assessment
Each Il-10 −/− mouse was scored weekly with the inflammatory bowel disease activity index (DAI) using the numerical system described by Spencer et al 23 Briefly, the DAI was calculated by scoring 1 point for the appearance of each of the following characteristics: ruffled fur, occult faecal blood, rectal prolapse <1 mm and soft stool, with an additional point for diarrhoea or severe rectal prolapse >1 mm. The DAI was calculated on a 6-point (0-5) scale.

| Histological analysis
Tissue from the colon was routinely stained with haematoxylin and eosin (H&E) and analysed for morphological changes. The intestinal inflammation grading was performed as previously described. 24 Briefly, intestinal inflammation was scored on a scale of 0-4 as follows: 0, no inflammation; 1, infiltration of a modest number of cells into the lamina propria; 2, infiltration of mononuclear cells leading to the separation of the crypts and mild mucosal hyperplasia; 3, massive infiltration of inflammatory cells accompanied by disrupted mucosal architecture, loss of goblet cells and marked mucosal hyperplasia; and 4, all of the above features plus crypt abscesses or ulceration. All sections were scored by two independent histologists who were blinded to the treatment group.

| Intestinal permeability in vivo
As we previously described, 25 the mice were fasted for 4 hours and then administered fluorescein isothiocyanate (FITC)-dextran (4 kDa; Sigma-Aldrich, Cat#: FD4, St. Louis, MO) by gavage at a dose of 600 mg/kg. After 4 hours, the mice were killed and bled by cardiac puncture. The serum was isolated using centrifugation, and the serum FITC levels were evaluated using fluorometry.

| Bacterial translocation
As we previously described, 25 using aseptic techniques, tissue samples from the mesenteric lymph nodes (MLN) and liver were taken for bacteriological cultures. Two samples for each histologic type were taken for culture respectively. The collected tissue samples were weighed, and 0.1 g of each sample was homogenized in a tissue grinder with 0.9 mL of sterile saline. The homogenates were diluted, and 100 μL dilutions were taken and cultured on MacConkey's agar (Sigma-Aldrich, Cat#: M7408, St. Louis, MO) at 37°C for 24 hours.
Bacterial growth on the plates was expressed as colony-forming units/g of tissue. The culture result was considered to be positive when more than 10 2 colony-forming units/g of tissue were found. 26

| Transmission electron microscopy (TEM) of TJs
Consistent with our procedures in previous report, 25 sections of colon (2 mm) were fixed for 2 hours in 4% buffered glutaraldehyde.
The sections were cut into smaller pieces, after fixed in 1% osmium

| Western blot analysis
Whole-cell and nuclear protein extracts were prepared from tissue homogenates as previously described, 27 and Western blot analysis was conducted as previously described. 28 Briefly, the protein extracts from the tissue homogenate were separated by SDS-PAGE and transferred to PVDF membranes for immunoblotting.

| Flow cytometry
T cell responses were analysed by flow cytometry as previously described. 29 Antibodies specific for Foxp3 (intracellular staining,

| Immunofluorescence
Immunofluorescence was assessed as described previously. 27 Colonic segments were immediately removed, washed with PBS, mounted in embedding medium, and stored at −80°C until use. Frozen sections were cut at 10 μm and mounted on slides. Nonspecific background signals were blocked by incubation with 5% bovine serum albumin plus 5% newborn bovine serum in PBS for 30 minutes at room temperature. The sections were incubated with rabbit polyclonal antibodies against occludin (Abcam, Cat#: ab216327, Cambridge, MA, UK) and claudin-1 (Abcam, Cat#: ab15098, Cambridge, MA, UK) at 4°C overnight. The sections were probed with the appropriate FITC-conjugated secondary IgG antibodies. The nuclei were counterstained with DAPI. Slides incubated without any primary antibody were used as negative controls. Confocal analysis was performed with a confocal scanning microscope (Leica Microsystems, Heidelberg GmbH, Mannheim, Germany).

| Immunohistochemical analysis
The intestinal levels of p-STAT3 and p-STAT4 were determined by immunohistochemical analysis as previously described. 32 Briefly, 5 µm paraffin sections were deparaffinized, rehydrated, sub-

| Statistical analysis
The data analyses were performed using Statistical Package for Social Sciences (SPSS; SPSS Inc, Chicago, IL) version 23.0.
Continuous, normally distributed data are presented as the mean ± standard deviation (SD). Unpaired t tests were used to compare data between two groups. The binary and categorical data were compared using chi-squared tests to produce the contingency tables. Fisher's exact test was performed when the sample number was ≤5. All tests were two-sided. A P value <0.05 was considered statistically significant.

Il-10 −/− mice
The Bry-1-treated Il-10 −/− mice showed lower mean DAI values than the untreated Il-10 −/− mice beginning at the third week after drug administration ( Figure 1A). In addition, the tissue histological inflammation score for the Bry-1-treated mice was significantly decreased compared with that for the untreated Il-10 −/− mice ( Figure 1B).

| Bry-1 treated Il-10 −/− mice show decreased intestinal permeability than the untreated Il-10 −/− mice
We evaluated intestinal permeability in vivo. As shown in Figure 3A, Therefore, we examined intestinal epithelial cell death by TUNEL staining. As shown in Figure 4A D); however, these proportions were still higher than those in WT mice. (E and F) A significant decrease in the proportion of IL-17A + CD4 + T cells was observed in Bry-1-treated mice compared with untreated Il-10 −/− mice in both the spleen (E and F) and MLN (G and H), but these T lymphocyte proportions were still higher than those in WT mice. Bry-1, Bryostatin-1; WT, wild-type; MLN, mesenteric lymph nodes; and NS, no significance. At least three independent experiments with six to eight mice in each group were performed, with one representative experiment is shown. The data are expressed as the mean ± SD. ▼ P < 0.05 in the WT mice ( Figure 4C,D). In contrast, compared to the untreated Il-10 −/− mice, the Bry-1-treated Il-10 −/− mice showed significant decreases in the Bax and cleaved caspase-3 levels, but the levels in the Bry-1-treated mice were still higher than those in the WT mice ( Figure 4C,D). These results indicated that the protective effect of Bry-1 on Il-10 −/− mice was at least partly mediated through preventing intestinal epithelial cell apoptosis.

| Antiapoptotic effect of Bry-1 on Il-10 −/− mice may be partly mediated by activating Nrf2 signalling
We are interested in the antiapoptotic mechanism of Bry-1, and recently published research showing that the Nrf2 signal is an important antiapoptotic signal. [34][35][36] As shown in Figure 5, the levels of both Nrf2 and its downstream factor HO-1 were significantly increased in the Bry-1-treated Il-10 −/− mice compared to the untreated Il-10 −/− mice, and the levels in Bry-1-treated Il-10 −/− mice were similar to those in the WT mice ( Figure 5A,B). These results were confirmed by PCR ( Figure 5C). These data indicated that Nrf2 signalling activation may be one of the antiapoptotic mechanisms of Bry-1.

| Bry-1 treated Il-10 −/− mice show lower Th1 and Th17 responses than the untreated Il-10 −/− mice
The inhibition of Th1 and Th17 immune responses has long been the approach to treat human CD. Therefore, we performed intracellular cytokine staining to confirm the effect of Bry-1 on Th1 and Th17 cells in Il-10 −/− mice. As shown in Figure 6 To the best of our knowledge, our study provides the first evidence that the administration of Bry-1 could significantly ameliorate spontaneous CD-like enteritis in Il-10 −/− mice, as demonstrated by decreases in the DAI, inflammatory score and proinflammatory mediator levels. To explore the possible mechanism underlying the antienteritis effects of Bry-1, we analysed the changes in the intestinal barrier, as these changes are important in human CD pathogenesis. We found that Bry-1 administration was associated with increased expression of intestinal mucosal epithelial TJ proteins (claudin-1 and occludin).
Using TEM, we found more direct evidence that Bry-1 protected the TJ structures of intestinal mucosal epithelial cells in Il-10 −/− mice.
Increased intestinal permeability is an important factor in causing or maintaining intestinal inflammation in CD. 6 Our results indicated that Representative immunohistochemical staining for p-STAT3 and p-STAT4 is shown. Western blot analysis showed that p-STAT3 and p-STAT4 levels were significantly decreased in Bry-1-treated Il-10 −/− mice compared to untreated Il-10 −/− mice, although the levels in the Bry-1-treated mice were higher than those in WT mice. Bry-1, Bryostatin-1; WT, wildtype; and NS, no significance. At least three independent experiments with six to eight mice in each group were performed, with one representative experiment is shown. The data are expressed as the mean ± SD. ▼ P < 0.05 the changes in apoptosis-related proteins help us to better understand the antiapoptotic effect of Bry-1, we still wanted to discover a possible in-depth mechanism. Activated Nrf2 signalling shows strong antiapoptotic and antioxidant effects, especially in alleviating intestinal epithelial cell apoptosis during intestinal injury in recent studies. [34][35][36] In the present work, the increased expression of Nrf2 and its downstream factor HO-1 in the Bry-1-treated Il-10 −/− mice may partly explain the protective role of Bry-1 in Il-10 −/− mice.
The therapeutic effects of Bry-1 on colitis and intestinal barrier injury in Il-10 −/− mice were very encouraging and suggested that Bry-1 has potential clinical application value. These findings drove us to continue to explore the potential curative mechanisms. We evaluated the effects of Bry-1 on the intestinal mucosal immune response and found that Bry-1 treatment decreased the Th17 and Th1 responses and increased the Treg response in Il-10 −/− mice.
Next, we focused on STAT signalling, as this pathway has a key role in regulating T cell-mediated immune responses in human CD. 38 We found that p-STAT3 and p-STAT4 expression decreased with Bry-1 treatment in Il-10 −/− mice. Activated STAT4 is important for the differentiation of Th1 cells, and activated STAT3 is important for the differentiation, functions and amplification of Th17 cells. 39,40 In addition, the inhibition of STAT3 contributes to the activation of Tregs. 41 These findings may partly explain the effects of Bry-1 on the intestinal mucosal immune response in CD-like colitis.
The findings of our study have potential clinical implications.
Given the rising incidence of CD and the importance of drug therapy, research teams, including our team, are looking for possible new drugs. However, drugs that have the potential to be applied in the clinic soon may be more valuable than those that will still require extensive research. In fact, to the best of our knowledge, Bry-1 has been studied for more than 30 years. 42 Previous studies have shown that Bry-1 has immunomodulatory, anti-oxidant and anti-inflammatory effects, suggesting its potential clinical application value. 19 Although the biological functions of Bry-1 have not been fully revealed, in the past 10 years, more than 20 clinical trials have been conducted with Bry-1 as a monotherapy or in combination with clinically used cytotoxic drugs. 43 A recent clinical trial confirmed the efficacy and safety of Bry-1 in the treatment of Alzheimer's disease. 21 Even more exciting findings in recent research have shown that Bry-1 has therapeutic potential in progressive forms of multiple sclerosis, which have immune response types similar to those of CD. 20 We hope that Bry-1 may benefit CD patients in the future.
Our study has some limitations. For example, our results showed that Bry-1 protects against CD-like colitis by improving the intestinal barrier and the abnormal intestinal mucosal immune response; however, Bry-1 could also improve colitis through other means.
The changes in Nrf2 and STAT3/4 signalling may partly explain the mechanism underlying the treatment effects of Bry-1, but we may have ignored other signalling pathways. It seems likely that Bry-1 has multiple biological functions, as previously reported. 19 In conclusion, this study provides initial evidence that the systemic delivery of Bry-1 ameliorates spontaneous colitis in Il-10 −/− mice, and this effect is associated with the attenuation of intestinal barrier injury and the abnormal intestinal mucosal immune response.
The protective effect of Bry-1 on CD-like colitis, particularly given the established clinical safety of Bry-1, suggests Bry-1 will have therapeutic potential in human CD.

ACK N OWLED G EM ENTS
This work was supported partly by funding from the National Natural