Rab7 dependent regulation of goblet cell protein CLCA1 modulates gastrointestinal homeostasis

Reviewer #1 (Public Review):

Assessment:

The manuscript titled 'Rab7 dependent regulation of goblet cell protein CLCA1 modulates gastrointestinal 1 homeostasis' by Gaur et al discusses the role of Rab7 in the development of ulcerative colitis by regulating the lysosomal degradation of Clca1, a mucin protease. The manuscript presents interesting data and provides a potential molecular mechanism for the pathological alterations observed in ulcerative colitis. Gaur et al demonstrate that Rab7 levels are lowered in UC and CD. However, a similar analysis of Rab7 levels in ulcerative colitis (UC) and Crohn's disease (CD) patient samples was conducted recently (Du et al, Dev Cell, 2020) which showed that Rab7 levels are found to be elevated under these conditions. While Gaur et al have briefly mentioned Du et al's paper in passing in the discussion, they need to discuss these contradictory results in their paper and clarify these differences. Additionally, Du et al are not included in the list of references.

Strengths:

The manuscript used a multi-pronged approach and compares patient samples, mouse models of DSS, and protocols that allow differentiation of goblet cells. They also use a nanogel-based delivery system for siRNAs, which is ideal for the knockdown of specific genes in the gut.

Weaknesses:

Du et al, Dev Cell 2020 (https://doi.org/10.1016/j.devcel.2020.03.002) have previously shown that Rab7 levels are elevated in a similar set of colonic samples (age group, number etc) from UC and CD patients. Gaur et al have not discussed this paper or its findings in detail, which directly contradicts their results. Clarification regarding this should be provided.

Reviewer #2 (Public Review):

Summary:

In this work, the authors report a role for the well-studied GTPase Rab7 in gut homeostasis. The study combines cell culture experiments with mouse models and human ulcerative colitis patient tissues to propose a model where, Rab7 by delivering a key mucous component CLCA1 to lysosomes, regulates its secretion in the goblet cells. This is important for the maintenance of mucous permeability and gut microbiota composition. In the absence of Rab7, CLCA1 protein levels are higher in tissues as well as the mucus layer, corroborating with the anti-correlation of Rab7 (reduced) and CLCA1 (increased) from ulcerative colitis patients. The authors conclude that Rab7 maintains CLCA1 level by controlling its lysosomal degradation, thereby playing a vital role in mucous composition, colon integrity, and gut homeostasis.

Strengths:

The biggest strength of this manuscript is the combination of cell culture, mouse model, and human tissues. The experiments are largely well done and in most cases, the results support their conclusions. The authors go to substantial lengths to find a link, such as alteration in microbiota, or mucus proteomics.

Weaknesses:

There are also some weaknesses that need to be addressed. The association of Rab7 with UC in both mice and humans is clear, however, claims on the underlying mechanisms are less clear. Does Rab7 regulate specifically CLCA1 delivery to lysosomes, or is it an outcome of a generic trafficking defect? CLCA1 is a secretory protein, how does it get routed to lysosomes, i.e. through Golgi-derived vesicles, or by endocytosis of mucous components? Mechanistic details on how CLCA1 is routed to lysosomes will add substantial value.

Why does the level of Rab7 fluctuate during DSS treatment (Fig 1B)? Does the reduction seen in Rab7 levels (by WB) also reflect in reduced Rab7 endosome numbers? Are other late endosomal (and lysosomal) populations also reduced upon DSS treatment and UC? Is there a general defect in lysosomal function?

The evidence for lysosomal delivery of CLCA1 (Fig 7 I, J) is weak. Although used sometimes in combination with antibodies, lysotracker red is not well compatible with permeabilization and immunofluorescence staining. The authors can substantiate this result further using lysosomal antibodies such as Lamp1 and Lamp2. For Fig 7J, it will be good to see a reduction in Rab7 levels upon KD in the same cell. In this connection, Fig S3D is somewhat confusing. While it is clear that the pattern of Muc2 in WT and Rab7-/- cells are different, how this corroborates with the in vivo data on alterations in mucus layer permeability -- as claimed -- is not clear.

Overall, the work shows a role for a well-studied GTPase, Rab7, in gut homeostasis. This is an important finding and could provide scope and testable hypotheses for future studies aimed at understanding in detail the mechanisms involved.

Author Response

Reviewer #1 (Public Review):

Assessment:

The manuscript titled 'Rab7 dependent regulation of goblet cell protein CLCA1 modulates gastrointestinal 1 homeostasis' by Gaur et al discusses the role of Rab7 in the development of ulcerative colitis by regulating the lysosomal degradation of Clca1, a mucin protease. The manuscript presents interesting data and provides a potential molecular mechanism for the pathological alterations observed in ulcerative colitis. Gaur et al demonstrate that Rab7 levels are lowered in UC and CD. However, a similar analysis of Rab7 levels in ulcerative colitis (UC) and Crohn's disease (CD) patient samples was conducted recently (Du et al, Dev Cell, 2020) which showed that Rab7 levels are found to be elevated under these conditions. While Gaur et al have briefly mentioned Du et al's paper in passing in the discussion, they need to discuss these contradictory results in their paper and clarify these differences. Additionally, Du et al are not included in the list of references.

Strengths:

The manuscript used a multi-pronged approach and compares patient samples, mouse models of DSS, and protocols that allow differentiation of goblet cells. They also use a nanogel-based delivery system for siRNAs, which is ideal for the knockdown of specific genes in the gut.

Weaknesses:

Du et al, Dev Cell 2020 (https://doi.org/10.1016/j.devcel.2020.03.002) have previously shown that Rab7 levels are elevated in a similar set of colonic samples (age group, number etc) from UC and CD patients. Gaur et al have not discussed this paper or its findings in detail, which directly contradicts their results. Clarification regarding this should be provided.

We thank and appreciate the reviewer for bringing this point.

The results shown by Du et al, Dev Cell, 2020 depict elevated expression of Rab7 in UC and CD patients compared to controls. In first occurrence, these results appear contradictory, but there may be a few possible explanations for this.

Firstly, Rab7 expression levels may fluctuate in the tissue depending on the degree of the gut inflammation. This can be concluded from our observations in DSS-mice dynamics model and the human patient samples with mild and moderate UC. Furthermore, Du et al provide no information of the severity of the condition among the patients employed in the study. Our motive, in the current work, was to emphasise this aspect. This point was mentioned in the discussion section of the manuscript. However, in view of the reviewer’s concern, we now intend to add a detailed comment on this in the main text of the revised version of the manuscript.

Secondly, the control biopsies in our investigation were acquired from non-IBD patients, and not what was done by Du et al., wherein biopsies from the normal para-carcinoma region of the colorectal cancer patients was used. One can not overlook the fact that physiological and molecular changes are apparent even in non-inflamed regions in the gut of an IBD or CRC patient. It is possible that the observed discrepancy arises due to the differences in the sample type used for comparing the Rab7 expression.

Finally, the main sub-tissue region showing a decrease in Rab7 expression in UC samples, appeared to be the Goblet cells which was not covered by Du et al.

Keeping these points in mind we do not think that there is a contradiction in our findings with that of Du et al., 2020. In the revised submission some of these explanations will be incorporated. Include Du et al in the reference list and add the comment in main text.

This was an oversight from our side. We have actually mentioned Du et al., 2020 in the discussion (line number 338) but somehow the reference was missing in the main list. We will ensure that the reference is included in the revised version and that their findings are included both in main text and in the discussion.

Reviewer #2 (Public Review):

Summary:

In this work, the authors report a role for the well-studied GTPase Rab7 in gut homeostasis. The study combines cell culture experiments with mouse models and human ulcerative colitis patient tissues to propose a model where, Rab7 by delivering a key mucous component CLCA1 to lysosomes, regulates its secretion in the goblet cells. This is important for the maintenance of mucous permeability and gut microbiota composition. In the absence of Rab7, CLCA1 protein levels are higher in tissues as well as the mucus layer, corroborating with the anticorrelation of Rab7 (reduced) and CLCA1 (increased) from ulcerative colitis patients. The authors conclude that Rab7 maintains CLCA1 level by controlling its lysosomal degradation, thereby playing a vital role in mucous composition, colon integrity, and gut homeostasis.

Strengths:

The biggest strength of this manuscript is the combination of cell culture, mouse model, and human tissues. The experiments are largely well done and in most cases, the results support their conclusions. The authors go to substantial lengths to find a link, such as alteration in microbiota, or mucus proteomics.

Weaknesses:

There are also some weaknesses that need to be addressed. The association of Rab7 with UC in both mice and humans is clear, however, claims on the underlying mechanisms are less clear. Does Rab7 regulate specifically CLCA1 delivery to lysosomes, or is it an outcome of a generic trafficking defect? CLCA1 is a secretory protein, how does it get routed to lysosomes, i.e. through Golgi-derived vesicles, or by endocytosis of mucous components? Mechanistic details on how CLCA1 is routed to lysosomes will add substantial value.

We thank the reviewer for the insightful comment. We would like to bring forth the following explanation for each these concerns:

(a) Our immunofluorescence imaging experiments revealed co-localization of Rab7 protein with CLCA1 and the lysosomes (Fig 7I). In addition, the absence of Rab7 affects the transport of CLCA1 to lysosomes (Fig 7J). This demonstrates that Rab7 may be involved in regulation of CLCA1 transport (presumably along with other cargo), to lysosomes selectively. However, we do recognise that the point raised by the reviewer about possible effect of a generic trafficking defect is valid. (b) As mentioned in the manuscript, the trafficking of CLCA1 protein or CLCA1-containing vesicles within the goblet cell is unknown, with no information on the proteins involved in its mobility. The switching of CLCA1 containing vesicles from the secretory route to lysosomes needs extensive investigation involving overall trafficking of the protein. Taken together, the complete answer to both these important questions will need a series of experiments and those may be interesting avenues for future research.

(a) Why does the level of Rab7 fluctuate during DSS treatment (Fig 1B)? (b) Does the reduction seen in Rab7 levels (by WB) also reflect in reduced Rab7 endosome numbers?

This is a very thoughtful point from the reviewer. We detected a distinct pattern of Rab7 expression fluctuation in intestinal epithelial cells after DSS-dynamics treatment in mice. Perhaps, these changes are the result of complex cellular signalling in response to the DSS treatment. Rab7, being a fundamental protein involved in protein sorting pathway, is expected to undergo alteration based on cells requirement. Presently there are no reports suggesting the regulatory mechanisms that govern Rab7 levels in the gut. (b) We observed reduction in Rab7 expression both at RNA and protein levels. To confirm whether this alteration will lead to reduced Rab7 positive endosome numbers may require detailed investigations.

Are other late endosomal (and lysosomal) populations also reduced upon DSS treatment and UC? Is there a general defect in lysosomal function?

There are no direct evidences showing reduction in the late endosomal and lysosomal population during gut inflammation, but few studies link lysosomal dysfunction with risk for colitis (doi: 10.1016/j.immuni.2016.05.007).

The evidence for lysosomal delivery of CLCA1 (Fig 7 I, J) is weak. Although used sometimes in combination with antibodies, lysotracker red is not well compatible with permeabilization and immunofluorescence staining. The authors can substantiate this result further using lysosomal antibodies such as Lamp1 and Lamp2. For Fig 7J, it will be good to see a reduction in Rab7 levels upon KD in the same cell.

We used Lysotracker red in live cells followed by fixation. So, permeabilization issues were resolved. Lamp1, as suggested by the reviewer, is definitely a better marker for lysosomes in immunofluorescence studies, but is also shown to mark late endosomes (doi: 10.1083/jcb.132.4.565). As Rab7 protein also marks the late endosomes, using Lamp1 may leave the ambiguity of CLCA1 in Rab7 positive late endosomes versus lysosomes. Nevertheless, we will be carrying out this experiment and the data will be shared in revised version of the work.

In this connection, Fig S3D is somewhat confusing. While it is clear that the pattern of Muc2 in WT and Rab7-/- cells are different, how this corroborates with the in vivo data on alterations in mucus layer permeability -- as claimed -- is not clear.

The data in Fig. S3D suggest the involvement of Rab7 in packaging of Muc2. The whole idea for doing this experiment was to support our observation in the Rab7KD-mice model where mucus layer was seen to be loose and more permeable in Rab7 deficient mice.

Overall, the work shows a role for a well-studied GTPase, Rab7, in gut homeostasis. This is an important finding and could provide scope and testable hypotheses for future studies aimed at understanding in detail the mechanisms involved.

We thank the reviewer for this comment.