Amelioration of Acute Oxazolone-Induced Colitis via Oral Administration of EPICERTIN, a Mucosal Healing Biotherapeutic for Inflammatory Bowel Disease

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled “Amelioration of Acute Oxazolone-induced Colitis via Oral Administration of EPICERTIN, a Mucosal Healing Biotherapeutic for Inflammatory Bowel Disease” presents a useful preclinical evaluation of EPICERTIN in an acute oxazolone-induced colitis model. The study is relevant because mucosal healing and epithelial restitution remain important therapeutic goals in ulcerative colitis. The authors show that oral EPICERTIN, particularly at the 3 µg dose, improves body weight recovery, disease activity, histopathological injury, epithelial repair-associated markers, and selected immune-cell populations.

Overall, I find the manuscript interesting and generally suitable for publication after minor revision. The study is clearly written, and the combination of clinical disease indices, histology, RT-qPCR, IMC, and CyTOF provides a useful descriptive dataset. I have the following comments, which I believe can be addressed without additional major experiments.

1. Please clarify the animal inclusion and exclusion criteria

Lines 129–143: The Methods state that animals with less than 5% body-weight loss one day after oxazolone challenge were excluded. This is understandable for ensuring successful disease induction, but the authors should clarify whether this criterion was defined before treatment allocation. Please also provide the number of excluded animals in each group. A short animal-flow statement would be sufficient.

2. Please clarify the treatment of sex as a biological variable

Lines 130–149: The initial efficacy experiment included both male and female BALB/c mice, whereas the IMC and CyTOF experiments were performed in male cohorts. The manuscript states that no significant sex-dependent differences were observed. Please provide a brief supporting statement or a simple sex-stratified summary for the main outcome measures. If the study was not powered to evaluate sex-dependent effects, this should be stated directly.

3. Please clarify the experimental unit in the IMC analysis

Lines 142–143: The IMC analysis was performed using three animals per group and three ROIs per animal. In the figure legend, the data are presented as n = 9 per group. Please clarify that these are ROI-level measurements rather than independent biological replicates. If the analysis was performed at the ROI level, this should be stated clearly. Ideally, the authors should also indicate whether animal-level averaging or clustering was considered.

4. Please frame the RT-qPCRdata with appropriate caution

Lines 159–164: The RT-qPCR analysis used three animals per group selected to represent the average DAI score of each group. This approach is acceptable for exploratory transcriptional profiling, but the text should avoid implying that these data provide definitive mechanistic proof. Please describe these RT-qPCR results as supportive or exploratory. Please also indicate where primer details are provided.

5. Please clarify multiple-comparison handling

The manuscript evaluates multiple RT-qPCR targets, IMC markers, and CyTOF-defined cell populations. The Statistical Analysis section notes that p-values were not adjusted for family-wise error rates because the work is exploratory. This is acceptable for an early preclinical communication, but this point should be made more visible in the Results or figure legends so that readers interpret the high-dimensional findings appropriately.

6. Please moderate the translational conclusion

The data support continued preclinical development of EPICERTIN. However, the statement that the present findings strongly substantiate a path toward a first-in-human clinical trial is somewhat strong for an acute mouse model. I suggest softening the wording. For example, the authors may state that the findings “support further preclinical development and evaluation of EPICERTIN in additional chronic and translationally relevant models.”

7. Please discuss the limitations of the oxazolone model

Oxazolone colitis is a useful model with several UC-like immunological and histopathological features, but it remains an acute hapten-induced model and does not fully reproduce human UC. A brief limitation statement in the Discussion would improve the balance of the manuscript.

8. Please clarify the non-linear dose response

The 3 µg dose showed the clearest therapeutic effect, whereas the 30 µg dose was less effective and was associated with increased collagen gene expression in the supplementary data. The authors discuss this briefly, but the interpretation should be cautious. Please state more clearly that further dose-ranging and longer-term studies will be needed to define the optimal therapeutic window and tissue-remodeling profile.

In summary, this is a well-written and useful preclinical study. The data support the conclusion that EPICERTIN ameliorates acute oxazolone-induced colitis and promotes epithelial repair-associated changes. The manuscript would benefit from several clarifications in the Methods, figure legends, statistical interpretation, and Discussion, but I do not consider additional major experiments necessary.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This preclinical study assesses the therapeutic efficacy of orally administered EPICERTIN in the acute oxazolone (OXA)-induced colitis model in BALB/c mice, which replicates important Th2-driven adaptive immune and histopathological characteristics of human ulcerative colitis (UC). The authors test three doses (0.3, 3, and 30 µg) with endpoints such as body weight recovery, Disease Activity Index (DAI), histopathology, qRT-PCR for wound-healing/inflammatory genes, Imaging Mass Cytometry (IMC), and CyTOF analysis of colon lamina propria immune populations, building on previous data from DSS colitis and human IBD explants. With enhanced epithelial barrier markers (such as E-cadherin and EpCAM), decreased inflammatory infiltration, and a change toward a healing phenotype (increased γδ T cells) without fibrosis, they claim maximum efficacy at the 3 µg dose. The study concludes that EPICERTIN supports mucosal healing via epithelial restitution and immune modulation, strengthening its translational potential for UC. 

I have some comments: 

The OXA model is a suitable and well-founded choice. However, insights into long-term mucosal repair or the likelihood of chronic fibrosis are limited due to the acute nature (euthanasia soon after induction). This is a limitation that needs to be addressed more clearly. 

In the early cohorts, sex inclusion was beneficial; however, later high-dimensional studies were limited to males "to minimise variability." Explain whether this was predetermined and talk about possible generalisability  

Gene expression: Only specific genes (Cdh1 trend, Il1b downregulation) are emphasised. A more thorough display of the entire panel results, including TGFβ1, WNT5A, etc., should be provided (e.g., heatmap or full supplementary data). For exploratory data, the statistical method for qPCR (Kruskal-Wallis because of normalised control) is appropriate; nevertheless, be aware of its limitations. 

In the discussion: provide a little more detail on the translational next steps (e.g., human dosing regimen, biomarkers). Discuss any possible side effects of the component generated from cholera toxin. 

Language/Typos: There are some OCR artefacts and fragmentation in the given text (e.g., "EPT" perhaps EPICERTIN, unfinished phrases). Proofread carefully (for example, "phocytes" and the repeated Conclusions section). There are a few minor grammatical errors throughout.. 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf