The ClC-2 Chloride Channel Activator, Lubiprostone, Improves Intestinal Barrier Function in Biopsies from Crohn’s Disease but Not Ulcerative Colitis Patients

Round 1

Reviewer 1 Report

The authors have showed the efficiency of Lubiprostone for improving intestinal barrier function in Biopsies from Crohn’s Disease but not Ulcerative Colitis Patients. This is very interesting that the authors found the effect of Lubiprostone on intestinal barrier that can lead to repositioning this drug for consideration of IBD treatment. However, there are some concerns. If the authors can improve that would be very nice.

1. The authors perform TER measurement and 4 kDa FITC dextran flux assay across tissue barriers of Biopsies. The data indicated that lubiprostone can increase TER but not affecting 4 kDa FITC dextran permeability as shown in Figure 4 A - B. Why? There is no explanation. Could you repeat this experiment again or at least provide the strong discussion? Is it possible that lubiprostone inhibit cluadin-2 expression or activity?

2. According to the data from figure 4 - 5, the authors just stained ad used occludin as the marker of tight junction, why don't the authors stain other tight junction proteins including claudin-1, cluadin-4, ZO-1, or even tricellulin? Indeed, low expression of tricellulin was shown to be implicated with IBD pathogenesis as well. On the other hand, there is some evidence suggesting that knocking out occludin was not sufficient to interrupt intestinal barrier. In my opinion, just staining only occludin is not sufficient. 

3. According to the data from figure 4, lubiprostone increased TER but not affecting paracellular flux of 4 kDa FITC dextran. Therefore, this may be due to inhibitory effect of lubiprostone on claudin-2-dependent pore pathway permeability. Since Ussing chamber is available in your lab, why don't the authors perform Bi-ionic potential experiment to investigate the effect of lubiprostone on claudin-2 in Biopsies. 

4. Intracellular signaling molecule related to IBD and barrier loss, for example MLCK, must be investigated to see if lubiprostone interrupt this pathway or others. 

5. Discussion part must be improved since your discussion was not consistent with your data.

Author Response

REVIEWER 1

The authors have showed the efficiency of Lubiprostone for improving intestinal barrier function in Biopsies from Crohn’s Disease but not Ulcerative Colitis Patients. This is very interesting that the authors found the effect of Lubiprostone on intestinal barrier that can lead to repositioning this drug for consideration of IBD treatment. However, there are some concerns. If the authors can improve that would be very nice.

1. The authors perform TER measurement and 4 kDa FITC dextran flux assay across tissue barriers of Biopsies. The data indicated that lubiprostone can increase TER but not affecting 4 kDa FITC dextran permeability as shown in Figure 4 A - B. Why? There is no explanation. Could you repeat this experiment again or at least provide the strong discussion? Is it possible that lubiprostone inhibit cluadin-2 expression or activity?

RESPONSE: This is a very good point. We do not have mechanistic data to explain why there are differing effects on TER vs. FD4 permeability. However, the reviewer’s suggestion that claudin-2 regulation may be important is a very valid one. Studies in a rodent water stress avoidance model (Zong et al. Neurogastroenterol. Motility, 2019) show that in addition to restoring normal levels of occludin, lubiprostone can also prevent the increased protein expression of claudin-2 in this stress model. Of note, a sealing claudin, claudin-1, was unaffected by lubiprostone. The same study also identified that lubiprostone could prevent claudin-2 overexpression induced by glucocorticoid treatment in intestinal epithelial cell lines and in isolated colonic crypts from healthy human subjects. This confirmed that lubiprostone inhibition of claudin-2 expression was applicable across multiple model systems. Therefore, effects of lubiprostone on TER may be mediated, at least in part, by changes in claudin-2 which have been previously demonstrated by other groups. We have now included this evidence in the Discussion section to address the reviewer’s concern.

2. According to the data from figure 4 - 5, the authors just stained ad used occludin as the marker of tight junction, why don't the authors stain other tight junction proteins including claudin-1, cluadin-4, ZO-1, or even tricellulin? Indeed, low expression of tricellulin was shown to be implicated with IBD pathogenesis as well. On the other hand, there is some evidence suggesting that knocking out occludin was not sufficient to interrupt intestinal barrier. In my opinion, just staining only occludin is not sufficient. 

RESPONSE: We appreciate the reviewer’s concern. Unfortunately, we had insufficient tissues (and sections) to perform a more detailed analysis of epithelial junction marker localization as our focus was on utilizing all available tissues for Ussing chamber studies. Therefore, we will not be able to perform additional stainings. The lack of a robust permeability defect in the original occludin-knockout mouse study that the reviewer refers to, is widely believed to reflect a compensatory response to loss of occludin. Moreover, work from the Fromm/Schulzke/Krug group and others indicates that tricellulin and occludin can partly compensate for each other when one is knocked down.

3. According to the data from figure 4, lubiprostone increased TER but not affecting paracellular flux of 4 kDa FITC dextran. Therefore, this may be due to inhibitory effect of lubiprostone on claudin-2-dependent pore pathway permeability. Since Ussing chamber is available in your lab, why don't the authors perform Bi-ionic potential experiment to investigate the effect of lubiprostone on claudin-2 in Biopsies. 

RESPONSE: This is an excellent suggested experiment. However, these experiments were performed while I was a member of the Division of Gastroenterology at UC San Diego and had ready access to patient biopsies. In my current institution, we do not have our own hospital and it has been an ongoing source of frustration to try to establish research collaborations, and establish IRB approvals, with hospitals in the region. In short, we do not have access to fresh patient biopsies. In addition, my collaborators at UCSD who had Ussing chamber equipment have all left that institution. Therefore, we no longer have the capacity to perform these proposed experiments at UCSD either.

4. Intracellular signaling molecule related to IBD and barrier loss, for example MLCK, must be investigated to see if lubiprostone interrupt this pathway or others. 

RESPONSE: This is another interesting set of experiments proposed by the reviewer. However, due to the absence of available freshly isolated human IBD patient tissue, it is not feasible to perform the suite of signaling pathway analyses required in order to thoroughly identify the signaling events leading to tight junction rearrangement in Crohn’s disease vs. ulcerative colitis, and identify which of these pathways are modulated by lubiprostone.

5. Discussion part must be improved since your discussion was not consistent with your data.

RESPONSE: We have utilized the reviewers insightful comments to expand the Discussion section and improve the clarity of the findings.

Reviewer 2 Report

This manuscript by Park et al. reports that the effect of lubiprostone on intestinal barrier function of IBD patients using mucosal biopsy samples. Despite some experimental limitations due to the use of patient samples, this study is well designed and executed. These data are informative when considering improvement in barrier function in IBD patients. I have a few minor points the authors may want to address.

 

1. Lubiprostone have shown different effects on biopsies from CD and UC patients. Is it possible to expand discussion about these results in relation to pathophysiological differences and characteristics of CD and UC patients?

2. Lubiprostone have been reported to activate not only ClC2 but also EP4 receptors. Since EP4 receptors is known to play an important role in maintaining intestinal barrier function, further investigation is expected to determine which of ClC2 and EP4 contributes more to results of this study.

Author Response

REVIEWER 2

This manuscript by Park et al. reports that the effect of lubiprostone on intestinal barrier function of IBD patients using mucosal biopsy samples. Despite some experimental limitations due to the use of patient samples, this study is well designed and executed. These data are informative when considering improvement in barrier function in IBD patients. I have a few minor points the authors may want to address.

 

1. Lubiprostone have shown different effects on biopsies from CD and UC patients. Is it possible to expand discussion about these results in relation to pathophysiological differences and characteristics of CD and UC patients?

 

RESPONSE: We are grateful for this suggestion and have now expanded the discussion to elaborate on the observed differences between CD and UC patients by inserting the following text:

Given the established heterogeneity between CD and UC with respect to location, inflammation penetrance (transmural vs. mucosal), cytokine profiles, unique vs. overlapping genetic risk variants, and most pertinently the differential responses to various treatment approaches (i.e. anti-TNF to treat CD; the pan JAK inhibitor, tofacitinib, to treat UC), it should not be surprising that lubiprostone also displayed a selective effect between IBD subtypes.

2. Lubiprostone have been reported to activate not only ClC2 but also EP4 receptors. Since EP4 receptors is known to play an important role in maintaining intestinal barrier function, further investigation is expected to determine which of ClC2 and EP4 contributes more to results of this study.

RESPONSE: We completely agree with this insightful observation. Future investigations using new patient material will be required to identify if lubiprostone can modulate ClC2 and EP4 receptors in IBD patient biopsies, however this is beyond the scope of the present study.

Reviewer 3 Report

This well-written manuscript investigates the effects of lubiprostone, a pharmaceutical agent directing chloride ions, and thus water, to the intestinal lumen of constipation-predominant irritable bowel syndrome patients, on mucosal function in Crohn’s disease (CD) and ulcerative colitis (UC) bowel from both active cases and cases in remission, as compared to control. Lubiprostone has also been shown to induce hastened repair of the intestinal epithelium and alleviate symptoms of colitis. Therefore, tight junction localization, electrical resistance, ion transport, and 4kD permeability were measured in colonic human biopsies. Lubiprostone was effective in increasing ion transport in control and remission cases, while improving TER in only cases of CD.

Major:

1.       Can the authors comment on how they believe TER increased in biopsies while simultaneously increasing solute transport? TER generally measures ability of small ions (including Cl-) to cross membrane, so an increase in TER and solute transport is very counterintuitive.

2.       Please make the scale bars in Figure 1 visible. Can barely see them in the Normal and CD Remission images, and not at all in the other images. Are the CD active images truly at the same scale as CD remission and UC remission?

3.       In Figure 2, please clarify whether the n’s listed under A and B are per treatment or total? For example, is the n=15 for Normal including the 3 doses of lubiprostone and DMSO, or are there 15 samples for DMSO, 15 for Lubi 0.01, etc.? Same applies to Panel B, Figure 3A&B, and Figure 4A&B.

4.       Authors do not discuss the histological effects of lubiprostone on sigmoid colon, if any, in Figure 1.

5.       Were statistics run on Figure 2C-E and Figure 3B-D and G-H? If not, why not? These trendlines look to have significant differences in some cases.

6.       Could the authors comment on why occluding localization seemed to be significantly reduced in CD cases in remission, yet there was apparently no histological difference concluded from H& E staining?

7.       Don’t understand the correlation between stated sample number in Figure 6 caption and the sample representation (dots) in Figure 6A. If there are 25 tissues for control, why are there not 25 dots representing those samples? Where is the significance denotation in Figure 6A or the caption (supposedly significant per Line 213—though CD samples are comparatively rare). Also, do authors have data on baseline TER in CD in remission? Would be nice to add to Figure 6A for comparison.

8.       Can the authors comment on why they believe CD and UC biopsies behaved differently to effects of lubiprostone?

Minor:

1.       Would suggest rewording title to “Biopsies from Crohn’s Disease, but Not Ulcerative Colitis, Patients Indicates Lubiprostone Improves Intestinal Barrier Function” or similar, for clarity.

2.       Line 24 should read “in animal models of colitis.”

3.       Lines 53-54—define acronyms CFTR and EP4 (and remove acronym definition for CFTR in lines 55-56).

4.       In Figure 2 caption, p<0.001 is missing the 3 asterisks.

5.       End of manuscript mentions a BioRender graphical abstract, but didn’t see a graphical abstract included.

Author Response

REVIEWER 3

This well-written manuscript investigates the effects of lubiprostone, a pharmaceutical agent directing chloride ions, and thus water, to the intestinal lumen of constipation-predominant irritable bowel syndrome patients, on mucosal function in Crohn’s disease (CD) and ulcerative colitis (UC) bowel from both active cases and cases in remission, as compared to control. Lubiprostone has also been shown to induce hastened repair of the intestinal epithelium and alleviate symptoms of colitis. Therefore, tight junction localization, electrical resistance, ion transport, and 4kD permeability were measured in colonic human biopsies. Lubiprostone was effective in increasing ion transport in control and remission cases, while improving TER in only cases of CD.

Major:

1. Can the authors comment on how they believe TER increased in biopsies while simultaneously increasing solute transport? TER generally measures ability of small ions (including Cl-) to cross membrane, so an increase in TER and solute transport is very counterintuitive.

RESPONSE: We are happy to address this question. It is established that ion transport responses require an intact epithelial barrier and consequently, appropriately functioning tight junctions are needed in order to maintain the electrical gradient required for transport processes. Therefore, we were not surprised to observe improved electrogenic ion transport responses concurrent with improved TER prior to the addition of secretagogues.

2. Please make the scale bars in Figure 1 visible. Can barely see them in the Normal and CD Remission images, and not at all in the other images. Are the CD active images truly at the same scale as CD remission and UC remission?

RESPONSE: We have now amended the size of the scale bars.

 

3. In Figure 2, please clarify whether the n’s listed under A and B are per treatment or total? For example, is the n=15 for Normal including the 3 doses of lubiprostone and DMSO, or are there 15 samples for DMSO, 15 for Lubi 0.01, etc.? Same applies to Panel B, Figure 3A&B, and Figure 4A&B.

RESPONSE: The n-numbers represent individual human subjects from each group (normal control subjects, quiescent Crohn’s, and quiescent UC). However, four biopsies were taken from each patient for Ussing chamber experiments and one tissue (from each patient) was used for each treatment condition. Thus, in Figure 2A, the normal subjects group of biopsies has 59 biopsies in total taken from 15 patients. This comprises DMSO (Control) = 15 individual tissues; Lubi 0.01 = 14 individual tissues; Lubi 0.1 = 14 individual tissues; Lubi 1.0 = 15 individual tissues. This has now been clarified in the appropriate figure legends (Figures 2, 3, 4). We did try to generate graphs by plotting individual data points, but the resulting figures did not look well and were difficult to navigate.

4. Authors do not discuss the histological effects of lubiprostone on sigmoid colon, if any, in Figure 1.

RESPONSE: We apologize for the confusion. The histology (Figure 1) was performed on adjacent biopsies from the same subjects as those biopsies subsequently treated ex vivo in Ussing chambers with lubiprostone (or untreated as appropriate). Therefore, the histology images are not a direct comparison of the effects of lubiprostone (vs. no treatment) on histology. Rather, the images in Figure 1 are just meant to convey that tissues subsequently mounted in Ussing chambers for lubiprostone (vs. vehicle) studies showed no major difference in histology. We have now amended the text in the Figure 1 legend to clarify this point.

5. Were statistics run on Figure 2C-E and Figure 3B-D and G-H? If not, why not? These trendlines look to have significant differences in some cases.

RESPONSE: We apologize for any confusion regarding these data. Figures 2C-E, 3B-d and 3 G-H are representative traces from the pooled data in Figure 2B, 3A and 3E respectively. The pooled data (Figures 2A, 2B; 3A, 3E) were subjected to statistical analysis.

6. Could the authors comment on why occluding localization seemed to be significantly reduced in CD cases in remission, yet there was apparently no histological difference concluded from H& E staining?

RESPONSE: We apologize again for the confusion regarding the histology images in Figure 1. As explained in answer to point #4, the images are not from lubiprostone-treated (vs. untreated) tissues. They are all "untreated" images from biopsies adjacent to those subsequently mounted in Ussing chambers that were then subsequently treated with lubiprostone. We have clarified this in the Figure 1 legend. 

7. Don’t understand the correlation between stated sample number in Figure 6 caption and the sample representation (dots) in Figure 6A. If there are 25 tissues for control, why are there not 25 dots representing those samples? Where is the significance denotation in Figure 6A or the caption (supposedly significant per Line 213—though CD samples are comparatively rare). Also, do authors have data on baseline TER in CD in remission? Would be nice to add to Figure 6A for comparison.

RESPONSE: We apologize for this confusion. Rather than plot the individual tissue TER values, we followed recommendations to plot the mean TER values for each human subject. We have also included the significance symbol in Figure 6A. We have now included baseline TER data from CD subjects in remission in the revised Figure 6 as suggested by the reviewer.

8. Can the authors comment on why they believe CD and UC biopsies behaved differently to effects of lubiprostone?

RESPONSE: This is an excellent point and one that we have been puzzled by. We can only speculate that either the signaling mechanisms used by lubiprostone to modify occludin are differentially affected in CD vs. UC, or there may be additional factors required to restore occludin that are more affected in UC than CD, and thus are less responsive to lubiprostone in UC. We have now inserted this text discussing this point in the Discussion section.

 

Minor:

1. Would suggest rewording title to “Biopsies from Crohn’s Disease, but Not Ulcerative Colitis, Patients Indicates Lubiprostone Improves Intestinal Barrier Function” or similar, for clarity.

RESPONSE: We have now amended the title to “ The ClC2 Chloride Channel Activator, Lubiprostone, Improves Intestinal Barrier Function in Biopsies from Crohn’s Disease but Not Ulcerative Colitis Patients”.

 

2. Line 24 should read “in animal models of colitis.”

RESPONSE: Thank you for informing us of this error. We have now made the suggested correction.

3. Lines 53-54—define acronyms CFTR and EP4 (and remove acronym definition for CFTR in lines 55-56).

RESPONSE: We have inserted the appropriate definitions as requested.

4. In Figure 2 caption, p<0.001 is missing the 3 asterisks.

RESPONSE: We apologize for this error. This has now been corrected.

5. End of manuscript mentions a BioRender graphical abstract, but didn’t see a graphical abstract included.

RESPONSE: Thank you for informing us of this omission. We did upload a graphical abstract, but it must not have been included in the final pdf. We have now included the graphical abstract in the main document after the references.

Reviewer 4 Report

In this study, Young Su Park, et al. studied the effects of lubiprostone on barrier function defects associated with IBD by using explanted colonic tissue isolated from control subjects as well as Crohn’s disease (CD) and ulcerative colitis (UC) patients in remission or CD patients with active disease. The topic of this paper is current and clinically relevant in the pathogenesis and therapies for Inflammatory Bowel Diseases (IBD). However, the study is limited by the small sample size as mentioned by authors. Authors may want to mention how did they calculated the sample size to ensure the study has adequate power to validate their results. I would also be interested in knowing why did they not include active UC patients.

Author Response

REVIEWER 4

In this study, Young Su Park, et al. studied the effects of lubiprostone on barrier function defects associated with IBD by using explanted colonic tissue isolated from control subjects as well as Crohn’s disease (CD) and ulcerative colitis (UC) patients in remission or CD patients with active disease. The topic of this paper is current and clinically relevant in the pathogenesis and therapies for Inflammatory Bowel Diseases (IBD). However, the study is limited by the small sample size as mentioned by authors. Authors may want to mention how did they calculated the sample size to ensure the study has adequate power to validate their results. I would also be interested in knowing why did they not include active UC patients.

RESPONSE: We are grateful to the reviewer for their complimentary remarks about this paper. We did initially power the study at n=8-10 based on our prior published work on Ussing chamber studies of intestinal biopsies treated ex vivo with lubiprostone (Kang et al. Pharm Res Perspec, 2015). However, we were unable to reach this target for active Crohn’s disease biopsies due to issues with tissue viability, and restricted access to this particular category of patients, as well as patients with active ulcerative colitis due to their inclusion in unrelated clinical trials under the direction of the co-author (WJS). 

Round 2

Reviewer 3 Report

Much improved, thank you. Please note caption for figure 1 mentions scale bar is in meters--believe this is a typo.