Review Reports - MFG-E8-Derived Oligopeptide MOP3 Facilitates Anti-Inflammatory M2-like Macrophage Polarization in Gut Ischemia/Reperfusion

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Overall Assessment

This manuscript addresses an important and clinically relevant problem immune modulation in gut ischemia reperfusion injury and presents a conceptually interesting strategy centered on MOP3-mediated clearance of eCIRP to promote an anti-inflammatory macrophage phenotype. The study builds logically upon the authors’ prior work and integrates in vivo and in vitro approaches to support the proposed mechanism. The topic is timely, and the translational implications are meaningful, particularly in the context of sterile inflammation and DAMP-driven immune responses. Overall, with moderate revisions aimed at strengthening mechanistic interpretation and expanding phenotypic validation, this work has strong potential for publication and would be of interest to the readership of this journal focused on inflammation, immunology, or translational medicine.

Comments

Page 2: The introduction appropriately frames gut ischemia–reperfusion (I/R) injury as a sterile inflammatory condition; however, the mechanistic positioning of MOP3 remains somewhat descriptive. The authors should more clearly articulate whether the central hypothesis is that DAMP clearance actively induces macrophage reprogramming or merely attenuates inflammatory amplification. This distinction is critical for mechanistic novelty of this manuscript.

Page 5: Macrophage polarization is defined primarily using CD86 and CD163. This limited marker panel is insufficient to establish functional phenotype. High-impact standards require multiparametric validation, including additional surface and intracellular markers (e.g., CD206, CD80, iNOS protein, Arg-1 protein). The absence of protein-level confirmation of iNOS and Arg-1 is a significant limitation.

Page 6: Details regarding doublet exclusion, fluorescence-minus-one (FMO) controls, and compensation strategy are not described. Full gating hierarchies should be included in supplementary materials to ensure reproducibility.

Page 4: The rationale for selecting 20 mg/kg MOP3 in vivo and 10 μg/ml in vitro is not sufficiently justified. Dose response experiments would significantly strengthen translational interpretation and mechanistic robustness.

Page 8: While MOP3 reduces eCIRP levels and shifts macrophage markers, direct mechanistic causality is not established. It remains unclear whether intracellular signaling downstream of eCIRP uptake drives M2-like polarization or whether reduced extracellular TLR4/TREM-1 stimulation indirectly permits repolarization. Downstream pathway interrogation (e.g., STAT3, NF-κB suppression, PI3K/Akt) would significantly strengthen mechanistic claims.

Page 10: Peritoneal macrophages are used for mechanistic experiments, yet intestinal lamina propria macrophages are phenotypically and developmentally distinct. The translational extrapolation should be discussed explicitly, or validation performed using tissue-resident intestinal macrophages.

Page 14: The discussion reiterates M1/M2 polarization without acknowledging the spectrum nature of macrophage activation. Incorporating contemporary macrophage biology literature would enhance scholarly depth.

Author Response

Reviewer 1

Overall Assessment

Response: We thank the reviewer for critically appraising our manuscript and for his/her valuable suggestions. Our point-by-point responses are provided below:

Comments

Response: We thank the reviewer for highlighting this distinction in the mechanism of MOP3, which is indeed critical to the novelty of this manuscript. We hypothesize that the act of clearing the DAMP induces macrophage reprogramming, analogous to the reprogramming that occurs after phagocytosis of an apoptotic cell. Previous studies evaluating the effect of efferocytosis on macrophage polarization have demonstrated a reprogramming toward the M2-link, anti-inflammatory phenotype. The mechanism behind this reprogramming involved MFG-E8 linking phosphatidyl serine signals to α_vβ₃ integrin on macrophages (Ji et al. 2025; Hirayama et al. 2018; Soki et al., 2014). We have now included these references to explain our hypothesis of actively reprogramming (page 3, lines 94-97). Furthermore, we have adjusted the phrasing of our hypothesis to clarify our central hypothesis (page 3, lines 99-101):

“Previous studies have evaluated the role of efferocytosis on polarization, which is in part facilitated by MFG-E8 [19–21]. Efferocytosis, they reported, may initiate a cascade of intra-cellular events that promotes M2 polarization [19-21].” (page 3, lines 94-97)

“Given our current understanding of the peptide, we hypothesized that MOP3-facilitated phagocytosis of eCIRP polarizes macrophages toward an M2-like phenotype in gut I/R to suppress inflammation and promote healing.” (page 3, lines 99-101)

Response: We appreciate the reviewer’s comments regarding our classification of M1 and M2 macrophages. Our focus was to evaluate both phenotype and functional differences between the untreated macrophage populations and those treated with MOP3. It is true that multiple markers can define M1 and M2 populations, but CD163 is a defining marker of the M2c subset, which also produces IL-10 and plays a pivotal role in phagocytosis, which coincides with our mechanism of MOP3-facilitated phagocytosis. We further support the evidence of increased CD163 and IL-10 expression with Arg-1 mRNA. In addition, we show the decrease in M1-associated cytokines, namely TNFα and IL-6. We believe these data provide sufficient evidence of macrophage polarization to M2c and have discussed this in the limitations. We further expanded on our interpretation of M2c as the predominant subtype that is expanded (page 14, lines 469-474):

“M2c is also the most likely subset of macrophages affected by MOP3 due to its association with increased phagocytic activity [15]. Even though several surface and intracellular markers are widely used for M1 and M2 macrophage detection, our strategy of using select markers, although it does detect macrophage polarization, has the limitation of not including additional markers in this study.”

Response: We thank the reviewer for bringing this need for further description of flow cytometry methods to our attention. We used FSC-H by FSC-A gating to exclude doublets. We realized in our review that the gating diagrams for SSC-A by FSC-A and FSC-H by FSC-A were mislabeled in Figure 1. We have now corrected this error. In addition, we used fluorescence-minus-one controls to determine positive thresholds for our fluorophore-conjugated antibodies indicating M1 and M2 macrophages. Lastly, we used single stained compensation beads for each fluorophore to calibrate the cytometer for compensation. We have added descriptions of doublet exclusion, fluorescence-minus-one (FMO) controls, and compensation strategy to our methods. We have made the following revisions to our methods section (page 5, lines 192-197):

“The gating strategy for flow cytometry experiments included exclusion of doublets by gating forward scatter height (FSC-H) by forward scatter area (FSC-A) and removing ob-servations with a high area to height ratio from our analysis. Fluorescence-minus-one (FMO) controls were used to determine the threshold of positivity for M1 and M2 markers. Compensation of fluorophores was performed using single-stained compensation beads for each fluorophore.”

Response: We thank the reviewer for pointing out the need for justification of these doses. We have chosen these doses based on previously published dose response experiments from our lab and from prior studies validating these doses (Nofi, Prince et al., 2024; Nofi, Tan et al., 2024; Hollis, Tenet et al., 2025). We have provided explanations in the following sections for in vivo and in vitro doses, respectively (page 4, lines 139-143; page 4 lines 162-164):

“This dose of MOP3 was previously established as an effective dose in sepsis, necrotizing enterocolitis, and gut and hepatic I/R models [10,11,22]. At that single optimized dose, we observed statistically significant attenuation of gut I/R and induction of M2 macrophage polarization. Consequently, we established this as the optimized dose for our study.

“In vitro dosing was determined by previously published dose response experiments using RAW 264.7 cells, which show 10 µg/ml as an effective therapeutic dose [15].”

Response: Thank you for your recommendations regarding our mechanistic data. We have found evidence in the literature supporting the direct effect of MFG-E8, the parent protein of MOP3, on M2 polarization rather than suppression of M1 polarization (Soki et al., 2014). Furthermore, our in vitro data shows significant elevation of M2 polarization with MOP3 in the presence of eCIRP compared to either MOP3 or eCIRP alone. In this way, the clearance of eCIRP by macrophages seems to have a direct effect on M2 reprogramming. If the absence of eCIRP in circulation leading to suppressed M1 polarization was responsible for permitting repolarization, we would expect to see significant M2 percent increases in the MOP3 alone group. Regarding downstream pathway interrogation, our focus was to establish the phenomenon of macrophage reprogramming. Absence of further pathway interrogation is a limitation of this study, and evaluation of intracellular mechanisms would be an interesting subject of future studies. We have included this discussion in the discussion section of our reviewed manuscript (page 14, lines 480-484).

“In addition, we did not determine the downstream, intracellular pathway directly responsible for M2-like polarization after phagocytosis. However, previous literature has associated efferocytosis directly with M2 reprogramming, such as via MOP3’s parent pro-tein, MFG-E8 [21,39]. Our data also shows MOP3 with eCIRP results in higher M2 polar-izaiton rates than either MOP3 or eCIRP alone.”

Response:

We appreciate the distinction between peritoneal and intestinal resident macrophages, such as resident lamina propria macrophages and peritoneal macrophages. Since acute inflammation from reperfusion injury involves both a local and systemic response, we sought to evaluate the response of different macrophage subsets after gut I/R and eCIRP stimulation with MOP3 treatment. We annotated which results came from which macrophage subtypes in our discussion by adding “intestinal and peritoneal” and “peritoneal” on page 13, lines 433 and 435, respectively. We also added an explanation regarding the translational application in the discussion (page 14, lines 458-460):

“In addition, our in vitro studies evaluated peritoneal macrophage response, which have different characteristics than intestinal macrophages, but both response to systemic and local inflammation from reperfusion injury.”

Response:

Thank you for providing this critique. We agree that the spectrum nature of macrophage activation should be emphasized, since recent literature has supported the plasticity of macrophages and their ability to express multiple phenotypes across time. We have included an additional explanation as well as the following contemporary reviews (page 12-13, lines 411-423):

“At the same time, we must acknowledge recent literature has challenged the dichotomous nature of macrophage polarization in favor of a more dynamic model [31,32] Macrophages have demonstrated plasticity in their ability to display both M1 and M2 characteristics simultaneously and at different time points [31,32]. However, a shift towards anti-inflammation, referred to in this study as M2-like, can still prove beneficial in disease recovery.”

Citation: Yan L, Wang J, Cai X, Liou YC, Shen HM, Hao J, Huang C, Luo G, He W. Macrophage plasticity: signaling pathways, tissue repair, and regeneration. MedComm (2020). 2024 Aug 1;5(8):e658. doi: 10.1002/mco2.658.

Wei Y, Wang M, Ma Y, Que Z, Yao D. Classical Dichotomy of Macrophages and Alternative Activation Models Proposed with Technological Progress. Biomed Res Int. 2021 Oct 21;2021:9910596. doi: 10.1155/2021/9910596.

Reviewer 2 Report

Comments and Suggestions for Authors

This interesting manuscript provides convincing data supporting the efficacy of the MOP3 oligopeptide in reducing I/R-induced gut damage in mice by shifting the macrophagic response toward an anti-inflammatory M2-like phenotype. The experiments are well-designed and well-performed, and the findings are properly illustrated and discussed. The relevant literature has been reported.

This study is not an absolute novelty, as the same authors have already published articles on the efficacy of MOP3 in alleviating I/R gut injury in a similar animal model (see refs. 12, 13). However, the data presented here extend existing knowledge on this matter, particularly concerning the cellular and molecular mechanisms involved.

Some minor points should be clarified, as detailed below

Besides SMA thromboembolism, it could be mentioned that gut I/R also occurs upon incarceration of a gut loop in an abdominal hernia, one of the most common surgical emergencies.
Please add a note in the Methods (lines 109-113) to disclose that the rationale for using a single dose of 1 mg of MOP3, instead of exploring dose-response effects as is usual for pharmacological studies, relies on the prior literature.
Methods, page 4, line 5: was pure DMSO the control vehicle given by retro-orbital injection? Or rather, was it dissolved in PBS at the same concentration as the test MOP3 solution?
Was the purity of the isolated macrophage preparations assessed, e.g. by flow cytometry, before the in vitro treatments? Peritoneal mast cells can also be retrieved by a similar method. Anyhow, since the further flow cytometry analyses were performed upon gating on macrophage markers, the presence of other cell types should not have biased the results.
Legend to fig. 2: correct F480 into F4/80.

Author Response

Reviewer 2

Overall Assessment

Some minor points should be clarified, as detailed below

Response: We thank the reviewer for a thoughtful interpretation and evaluation of our work and for the feedback to strengthen the manuscript. Our point-by-point responses are provided below:

1. Besides SMA thromboembolism, it could be mentioned that gut I/R also occurs upon incarceration of a gut loop in an abdominal hernia, one of the most common surgical emergencies.

Response: We thank the reviewer for providing this suggestion about the translatability of our work. We have made the following changes to our introduction to include this relevant pathophysiology with additional references (Yang et al., 2014; Schick et al., 2026) (page 2, lines 55-57):

“I/R injury can also occur after a strangulated segment of small intestine is surgically or manually reduced from an incarcerated hernia of the abdominal wall [8,9].”

2. Please add a note in the Methods (lines 109-113) to disclose that the rationale for using a single dose of 1 mg of MOP3, instead of exploring dose-response effects as is usual for pharmacological studies, relies on the prior literature.

Response: We thank the reviewer for this important comment and apologize for the lack of clarity on the dose of MOP3. The 1 mg referenced in “Materials and Methods: Protein and Peptide Synthesis and Preparation” refers to the quantity we received per tube in lyophilized powder as stock. We have made corrections to clarify this process and add the volume delivered to the mice via retro orbital injection (page 3, line 116 and page 4, line 137). We also provide rationale for where we received our optimal dose (Prince et al. 2024)(page 4, lines 139-143; page 4 lines 162-164):

3. Methods, page 4, line 5: was pure DMSO the control vehicle given by retro-orbital injection? Or rather, was it dissolved in PBS at the same concentration as the test MOP3 solution?

Response: We thank the reviewer for raising this question. The vehicle was given as DMSO dissolved in normal saline (NS) for in vivo experiments. We have made clarification in the methods section (line page 4, lines 138).

4. Was the purity of the isolated macrophage preparations assessed, e.g. by flow cytometry, before the in vitro treatments? Peritoneal mast cells can also be retrieved by a similar method. Anyhow, since the further flow cytometry analyses were performed upon gating on macrophage markers, the presence of other cell types should not have biased the results.

Response: We thank the reviewer for raising this question. Although we did perform gating for macrophages in the flow cytometry experiments as you indicated, we did not conduct further assessments of macrophage purity, which we consider to be one of the limitations of our study.

5. Legend to fig. 2: correct F480 into F4/80.

Response: Corrected