Enhancement of efferocytosis through biased FPR2 signaling attenuates intestinal inflammation

Abstract Efficient clearance of dying cells (efferocytosis) is an evolutionarily conserved process for tissue homeostasis. Genetic enhancement of efferocytosis exhibits therapeutic potential for inflammation resolution and tissue repair. However, pharmacological approaches to enhance efferocytosis remain sparse due to a lack of targets for modulation. Here, we report the identification of columbamine (COL) which enhances macrophage‐mediated efferocytosis and attenuates intestinal inflammation in a murine colitis model. COL enhances efferocytosis by promoting LC3‐associated phagocytosis (LAP), a non‐canonical form of autophagy. Transcriptome analysis and pharmacological characterization revealed that COL is a biased agonist that occupies a part of the ligand binding pocket of formyl peptide receptor 2 (FPR2), a G‐protein coupled receptor involved in inflammation regulation. Genetic ablation of the Fpr2 gene or treatment with an FPR2 antagonist abolishes COL‐induced efferocytosis, anti‐colitis activity and LAP. Taken together, our study identifies FPR2 as a potential target for modulating LC3‐associated efferocytosis to alleviate intestinal inflammation and highlights the therapeutic value of COL, a natural and biased agonist of FPR2, in the treatment of inflammatory bowel disease.

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Referee #1 (Remarks for Author): The authors report studies investigating the effects of a novel natural product, columbamine (COL), on macrophage efferocytosis and show that COL enhances efferocytosis.COL also improves the outcome of DSS-induced colitis through biased agonism of FPR2, which is an interesting finding.This report would benefit from minor revisions.Specific Comments 1.The authors show in Figure 1 that several compound enhance macrophage efferocytosis, some to a similar level as columbamine.It would be of interest to explain what those other compounds were and why COL was chosen for further analysis.Are the other active compounds structurally related to columbamine? 2. The Figure 1 legend and the text needs to include the dose of COL used to treat the mice.Was the concentration achieved in vivo similar to that used to treat macrophages in vitro?This should be provided or discussed.
3. The authors show that COL affects macrophage functions.Was there any cytotoxicity associated with this compound?4. The authors show that COL affects FPR2 responses and suggest it is an FPR2 biased agonist.Does COL also have effects at FPR1?This should be addressed since many FPR agonists hit both receptors.5.This study was performed on murine cells.However, it is well documented that some FPR ligands that impact mouse cells have no effect on human macrophages, and vice versus.This issue should be addressed or at least a discussion of this issue should be provided to enhance medical implications of this work.Was COL tested with any human macrophages or macrophage cell lines?Referee #2 (Remarks for Author): In this manuscript, Wu et al. report on the identification of columbamine (COL) as a biased FPR2 agonist that enhances efferocytosis and the efficacy of COL in attenuating intestinal inflammation in a murine colitis model.COL was first identified as a potential efferocytosis enhancers from a small library of natural products.The authors subsequently showed that COL enhances LC3-associated phagocytosis of mouse BMDMs, and they used a murine colitis model to demonstrate the anti-inflammatory function of COL.Stemmed from these findings, the authors have identified FPR2 as the molecular target of COL in enhancing efferocytosis and promoting the resolution of inflammation.Furthermore, the authors have conducted docking and mutagenesis studies to investigate the binding mechanism of COL on FPR2 and mechanistic studies to prove the involvement of RAC1 in COL-mediated enhancement of efferocytosis.Overall, this study is of high quality.The identified novel biased FPR2 agonist as well as the mechanism of FPR2-mediated enhancement of efferocytosis may offer new therapeutic opportunities for inflammatory diseases such as colitis.The paper is expected to have a high impact on the research fields of GPCR pharmacology, inflammation, and IBD.However, there are some issues that I believe need to be addressed or at least discussed.
1.The intriguing observation that COL does not induce Ca2+ immobilization raises interesting questions.Previous research (https://doi.org/10.1038/ncomms14232)has shown that another FPR2 agonist, compound 17b, also appears to be biased away from Ca2+.Interestingly, compared to the FPR2 agonist compound 43, which induces strong Ca2+ responses, compound 17b has been shown to exhibit superior anti-inflammatory and cardioprotective effects in cardiomyocytes.To gain a better understanding of the functional role of biased signaling in efferocytosis, it may be worth investigating the W-peptide, which is typically considered a non-biased agonist of FPR2.Testing the W-peptide in efferocytosis could serve as an important control experiment to help establish a link between the biased signaling properties of COL and its functional role in efferocytosis.
2. Related to 1, is the functional role of COL in promoting efferocytosis exclusive to this compound, or can other synthetic FPR2 agonists, such as compound 43 and BMS compounds, also induce efferocytosis as effectively?Understanding the mechanism of action and efficacy of various FPR2 agonists in promoting efferocytosis could lead to the development of novel therapeutic agents.
3. Another study (https://doi.org/10.1021/acsptsci.2c00042)comparing two clinical stage FPR2 agonists, BMS-986235 and ACT-389949, showed that while both agonists induced strong receptor internalization, only BMS-986235 effectively recycled the receptors back to the plasma membrane.Does COL induce strong receptor internalization?Would prolonged treatment of COL cause receptor desensitization?This may be related to the therapeutic efficacy of COL.Investigating these questions could provide valuable insights into the mechanism of action of COL and its potential as a therapeutic agent.
4. There is a published structure of FPR2 bound to compound 43.How does the binding of COL to the receptor compare to that of compound 43? 5. Humans and mice have very different formyl peptide receptor systems (https://doi.org/10.1111/bcpt.12903).Does COL act on human FPR2?Also, for experiments using heterologous expression systems, e.g.HEK293 over-expressing FPR2, it would be helpful to specify human or mouse FPR2 in figure legend.
Referee #3 (Comments on Novelty/Model System for Author): The authors claim columbamine could promote inflammation resolution by enhancing macrophage efferocytosis.This is an interesting area of research with immense translational implications.However, the study is limited by its lack of data on specific mechanisms (such as efferocytosis which is the central claim) and lack of PK/PD data for columbamine in in-vivo studies.
Referee #3 (Remarks for Author): In this manuscript, Wu et al show that columbamine is a biased FPR2 agonist that increases macrophage efferocytosis in a LAPdependent fashion.These effects are claimed to promote resolution of inflammation in a murine model of colitis.The characterization of the biased signaling on FPR2 is rigorous and well conducted.Although the cell biological and signaling findings are interesting, the data to support the central claim that columbamine-mediated increase in macrophage efferocytosis is critical for the in vivo phenotype is generally lacking.

Major comments:
Screening assay: 1) The authors use the quantification of the numbers of uncleared ACs as an indirect measure of macrophage efferocytosis efficiency.While this is a counter-intuitive but interesting strategy, the authors do not present data for validation of this screening model.For example, the robustness of screening assays is defined by Z-factor (PMID: 10838414).Such data are unavailable to test the validity and usefulness of this novel screening strategy for discovery of efferocytosis modulators.
2) Similarly, how this screening strategy performs with known macrophage efferocytosis modulators such as dexamethasone, PMA, and Annexin A1 are not shown.These data will be critical to test the validity of the assay.
3) Lastly, screening strategies often use Z-scores to detect "hits" (For eg.PMID: 21515799).These are typically those that fall 2 or even 3 standard deviations above or below the population mean.However, such an approach was not taken here.Instead, the authors used a standard calculation of intensity to identify efferocytosis modulators.While I agree that the authors identified columbamine as an efferocytosis enhancer using this strategy, the robustness and usefulness of this approach for future use has not been well established.
Columbamine as an efferocytosis enhancer 4) Columbamine treated mice show lower numbers of remaining ACs in liver and spleen (Figure 1 H and I).The authors interpret that this is due to enhanced macrophage efferocytosis efficiency.However, an alternate interpretation would be that columbamine treatment alters the trafficking of injected ACs and therefore less of the injected ACs reach liver and spleen.It would be best if the authors directly measure efferocytosis efficiency of macrophages by analysing the percent macrophages that have engulfed an injected AC.Alternatively, the authors can conduct in-situ efferocytosis assays to monitor free and engulfed ACs (such as PMID: 30291029).5) Similarly, in the DSS-colitis model, the decrease in the numbers of ACs could simply represent decreased intestinal cell death (instead of accelerated clearance).How do the authors exclude that possibility?As above, quantification of macrophage efferocytosis efficiency in the colonic tissue will facilitate direct interpretation of the effect of columbamine on efferocytosis.
6) The authors claim that macrophages and macrophage-mediated efferocytosis are critical for inflammation resolution in the DSS-colitis model.In this context, it is intriguing that macrophage depletion by itself did not affect colon length or disease activity (Figure 4).I would have predicted that that the mere depletion of macrophages would have made the disease worse.Can the authors explain?
7) The authors deplete macrophages and show that the protective effects of columbamine on intestinal inflammation are lost.These data argue a role for macrophages in eliciting the protective effects of columbamine.However, these do not represent evidence to support the claim that columbamine-induced efferocytosis enhancement mediates the beneficial effects.Can the authors block LAP in vivo or other such relevant pathways to show that columbamine indeed protects against intestinal inflammation via enhancement of efferocytosis?Additionally, can the authors directly test the efferocytosis efficiency of macrophages in the colonic sections?
8) It would be important to present TUNEL data for experiments in Figure 4. Can the authors also directly measure macrophage efferocytosis efficiency in the colon?Similar comment for data presented in Figure 6. 9) The authors could discuss the PK/PD of columbamine and whether they could detect columbamine or its metabolic products in their in vivo experiments.These data will be critical for translating these findings into clinical scenario.

Dear Editor:
Thank you very much for your kind consideration on our manuscript.We are also grateful to all the reviewers' insightful comments.We have revised the manuscript according to the comments and hopefully the reviewer will find our paper meet the publication standards.This is a really nice report describing the discovery of a natural compound that enhances efferocytosis and improves the outcome of DSS-induced colitis.The investigators show that this is du to effects on FPR2.This is an important discovery and links FPR2 function and efferocytosis.
Referee #1 (Remarks for Author): The authors report studies investigating the effects of a novel natural product, columbamine (COL), on macrophage efferocytosis and show that COL enhances efferocytosis.COL also improves the outcome of DSS-induced colitis through biased agonism of FPR2, which is an interesting finding.This report would benefit from minor revisions.Specific Comments 1.The authors show in Figure 1 that several compounds enhance macrophage efferocytosis, some to a similar level as columbamine.It would be of interest to explain what those other compounds were and why COL was chosen for further analysis.Are the other active compounds structurally related to columbamine?
Response: We thank reviewer for raising the question.
The top 10 compounds identified through the screening were shown in supplementary table 1. Comparing the structures, we didn't find any shared characteristics among them compare to COL.We select COL because of two reasons: 1, no obvious toxicity was observed at the COL working concentration; 2, COL can be isolated from herbal plant traditionally used to treat colitis and we speculate that COL may be the active compound of this plant to treat colitis.We will characterize other positive compounds in the future.
19th Sep 2023 1st Authors' Response to Reviewers 2. The Figure 1 legend and the text needs to include the dose of COL used to treat the mice.Was the concentration achieved in vivo similar to that used to treat macrophages in vitro?This should be provided or discussed.
Response: Thanks for the valuable suggestions.
We have added the dose of COL used in the Figure 1 legend (words in blue).
The PK/PD studies of columbamine has been published in a Chinese journal, "Chinese Journal of Health Laboratory Technology" (1).In the study, the half-life of columbamine t 1/2z was (10 ± 7.9) h or (7.7 ± 4.8) h after intravenous injection and (5.2 ± 2.8) h after oral administration after treating rat with columbamine (10 mg/kg).According to our results, COL at 1 μM is effective to trigger efferocytosis, indicating that the concentration of 0.338 ng/ml in blood is enough to work.Based on the medication time curve, COL can be effective for at least 12 h in vivo.The analysis has been added into discussion part (words in blue).
3. The authors show that COL affects macrophage functions.Was there any cytotoxicity associated with this compound?
Response: Thanks for the question.
To clarify the toxicity of COL, we have tested the cell viability of macrophage after COL treatment for 24 hours using CCK-8 kit.The data shows that COL did not trigger obvious cell toxicity at working concentration (1 μM).Actually, even at concentration up to 100 μM, macrophages still remain more than 80% viability.The data indicates that COL did not trigger obvious toxicity on macrophages.The data has been added in the Fig. S1A.
Response: We conducted additional experiments to address these questions.
In RBL-FPR1 cells, COL did not induce Ca 2+ mobilization (A), which is consistent with data from FPR2-expressing cells (C).In cAMP inhibition assay, the EC 50 value of COL on FPR1 (about 44.3 μM) was 10 times higher than that on FPR2 (3.4 μM).These results suggest that COL activates biased signaling both on FPR2 and FPR1, though with 10 times higher selectivity on FPR2. 5.This study was performed on murine cells.However, it is well documented that some FPR ligands that impact mouse cells have no effect on human macrophages, and vice versus.This issue should be addressed or at least a discussion of this issue should be provided to enhance medical implications of this work.Was COL tested with any human macrophages or macrophage cell lines?
Response: Thanks for raising the important question.
Function of FPR2 in different species is a concerned point in many reports.We have evaluated FPR2 signaling on cells expressing human FPR2 (Fig. 7 and Figure S3 (B-H)), the results showed that COL activates human FPR2.
Referee #2 (Remarks for Author): In this manuscript, Wu et al. report on the identification of columbamine (COL) as a biased FPR2 agonist that enhances efferocytosis and the efficacy of COL in attenuating intestinal inflammation in a murine colitis model.COL was first identified as a potential efferocytosis enhancers from a small library of natural products.The authors subsequently showed that COL enhances LC3-associated phagocytosis of mouse BMDMs, and they used a murine colitis model to demonstrate the anti-inflammatory function of COL.Stemmed from these findings, the authors have identified FPR2 as the molecular target of COL in enhancing efferocytosis and promoting the resolution of inflammation.Furthermore, the authors have conducted docking and mutagenesis studies to investigate the binding mechanism of COL on FPR2 and mechanistic studies to prove the involvement of RAC1 in COL-mediated enhancement of efferocytosis.Overall, this study is of high quality.The identified novel biased FPR2 agonist as well as the mechanism of FPR2-mediated enhancement of efferocytosis may offer new therapeutic opportunities for inflammatory diseases such as colitis.The paper is expected to have a high impact on the research fields of GPCR pharmacology, inflammation, and IBD.However, there are some issues that I believe need to be addressed or at least discussed.
1.The intriguing observation that COL does not induce Ca2+ immobilization raises interesting questions.Previous research (https://doi.org/10.1038/ncomms14232)has shown that another FPR2 agonist, compound 17b, also appears to be biased away from Ca2+.Interestingly, compared to the FPR2 agonist compound 43, which induces strong Ca2+ responses, compound 17b has been shown to exhibit superior anti-inflammatory and cardioprotective effects in cardiomyocytes.To gain a better understanding of the functional role of biased signaling in efferocytosis, it may be worth investigating the W-peptide, which is typically considered a non-biased agonist of FPR2.Testing the W-peptide in efferocytosis could serve as an important control experiment to help establish a link between the biased signaling properties of COL and its functional role in efferocytosis.
Response: Thanks for the valuable suggestions.
We performed the experiment as suggested and found W-peptide at 1 μM can also induce efferocytosis.The data indicates that FPR2 agonists, either biased or unbaised, are capable of inducing efferocytosis in macrophages.
2. Related to 1, is the functional role of COL in promoting efferocytosis exclusive to this compound, or can other synthetic FPR2 agonists, such as compound 43 and BMS compounds, also induce efferocytosis as effectively?Understanding the mechanism of action and efficacy of various FPR2 agonists in promoting efferocytosis could lead to the development of novel therapeutic agents.
Response: Thanks for raising this important concern.
We have tested the efferocytosis-promotion activity of Cp43 at different concentrations.We found Cp43 can also induce efferocytosis in a dose dependent manner (data was shown as below).According to the results, it seems that efferocytosis induction is a general response of FPR2 activation.
3. Another study (https://doi.org/10.1021/acsptsci.2c00042)comparing two clinical stage FPR2 agonists, BMS-986235 and ACT-389949, showed that while both agonists induced strong receptor internalization, only BMS-986235 effectively recycled the receptors back to the plasma membrane.Does COL induce strong receptor internalization?Would prolonged treatment of COL cause receptor desensitization ?This may be related to the therapeutic efficacy of COL.Investigating these questions could provide valuable insights into the mechanism of action of COL and its potential as a therapeutic agent.
Response: Thanks for raising the interesting question.
COL induced a mild and transient internalization of FPR2 compared with W-pep.The data shown in the original submission was from a 60 min-stimulation experiments (Figure 6E and 6F).We now extended the stimulation time to 120 min, but no further increase in FPR2 internalization was observed.In comparison, W-pep induced strong internalization of FPR2 after 60 min stimulation.As for receptor desensitization, since COL did not induce Ca 2+ mobilization, the desensitization effect could not be determined.We conclude that prolonged incubation with COL did not cause significant loss of cell surface FPR2 due to receptor internalization.Since COL does not induce Response: Thanks for posing the interesting question.
We have compared the binding features of these two compounds to FPR2 (The docking figure and figure legend are shown as below).
Compound 43 (Cpd43) is a dual agonist of FPR1 and FPR2.This pharmacological property is different from COL which is a biased agonist with preference for FPR2.In docking model, Cpd43 inserts into the binding pocket wedge-like with a longitudinal angle (A in Figure below).Cpd43 interacts with V113 but not S288, in addition to key amino acids such as R201, R205 and D106.In comparison, both COL (B) and LXA4 (C) interacts with S288 but not V113.We conclude that COL is closer to LXA4 than Cpd43 in the FPR2 binding pocket.(right).Hydrophobic connections between LXA4 and FPR2 are formed by L81, H102, V105, L109, F110, W254, F257, F292.Besides the hydrogen network formed by D106-R201-R205, the residue S288 of FPR2 is important in the recognition of LXA4.The polar interactions were indicated in red dash lines.
5. Humans and mice have very different formyl peptide receptor systems (https://doi.org/10.1111/bcpt.12903).Does COL act on human FPR2?Also, for experiments using heterologous expression systems, e.g.HEK293 over-expressing FPR2, it would be helpful to specify human or mouse FPR2 in figure legend.
Response: Thanks for raising the important question.
According to our observation, we found COL acted both on human and mouse FPR2.To determine the downstream of human FPR2 and mouse FPR2 after COL treatment, we have done calcium mobilization test on human or mouse FPR2 overexpressing RBL cells (Figure S3B-E).The results showed that COL did not affect calcium mobilization both on human and mouse FPR2-RBL cells.There is no obvious specie difference on FPR2 signal.In other experiments, such as cAMP response, membrane recruitment and internalization, we have used cells with human FPR2 construct expressing in Figure 7 as recommendation.These data indicates that COL acts on human FPR2.To avoid confusion, we have specified the FPR2 into hFPR2 in the revised MS.
Referee #3 (Comments on Novelty/Model System for Author): The authors claim columbamine could promote inflammation resolution by enhancing macrophage efferocytosis.This is an interesting area of research with immense translational implications.However, the study is limited by its lack of data on specific mechanisms (such as efferocytosis which is the central claim) and lack of PK/PD data for columbamine in in-vivo studies.
Referee #3 (Remarks for Author): In this manuscript, Wu et al show that columbamine is a biased FPR2 agonist that increases macrophage efferocytosis in a LAP-dependent fashion.These effects are claimed to promote resolution of inflammation in a murine model of colitis.The characterization of the biased signaling on FPR2 is rigorous and well conducted.Although the cell biological and signaling findings are interesting, the data to support the central claim that columbamine-mediated increase in macrophage efferocytosis is critical for the in vivo phenotype is generally lacking.

Major comments:
Screening assay: 1) The authors use the quantification of the numbers of uncleared ACs as an indirect measure of macrophage efferocytosis efficiency.While this is a counter-intuitive but interesting strategy, the authors do not present data for validation of this screening model.For example, the robustness of screening assays is defined by Z-factor (PMID: 10838414).Such data are unavailable to test the validity and usefulness of this novel screening strategy for discovery of efferocytosis modulators.
Response: Thanks for raising the concern.
As suggestion, we have repeated and re-evaluated our data by adding positive control.Dexamethasone (Dex) (1 μM) and phorbol 12-myristate 13-acetate (PMA) (100 nM) are two positive molecules to stimulate efferocytosis.6 biological replicates were assigned for each group, and edge wells were excluded.We pretreated the BMDMs in 96-wells with Vehicles, Dex, and PMA for 24 h, followed by adding CMFDA-stained apoptotic cells for another 24 h.We finally obtained and recorded the fluorescent intensity value remained in medium in each well.The results were shown as below.There is a significant difference between vehicle and positive control groups.The data indicates that our method is a reliable measurement of macrophage efferocytosis efficiency.
However, the calculated S/N is about 2-3 and the Z factor is about -1 to -0.5 (shown as below).These data indicate that our screening strategy may not be so robust for a high throughput screening application.We speculate that the high heterogeneity of macrophages and the small difference in fluorescent intensity change make the robustness of assay not so high.Since our focus is the compound itself, we move the high content screening result to the supplementary figure (Fig. S1A-B).

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2) Similarly, how this screening strategy performs with known macrophage efferocytosis modulators such as dexamethasone, PMA, and Annexin A1 are not shown.These data will be critical to test the validity of the assay.
Response: According to reviewer's question, we have determined the efferocytosis capacities of known efferocytosis modulators by using our methods.As shown in the data below, dexamethasone, PMA and Ac2-26 (the active peptide of annexin A1) treatment reduced the apoptotic cell fluorescent intensity value significantly.This data supports the reliability of our assay in identifying efferocytosis inducers.
3) Lastly, screening strategies often use Z-scores to detect "hits" (For eg.PMID: 21515799).These are typically those that fall 2 or even 3 standard deviations above or below the population mean.However, such an approach was not taken here.Instead, the authors used a standard calculation of intensity to identify efferocytosis modulators.While I agree that the authors identified columbamine as an efferocytosis enhancer using this strategy, the robustness and usefulness of this approach for future use has not been well established.
Response: Thanks for providing the valuable suggestion.According to author's the suggestion, we have tried to transfer the value into Z-score.We found the graph become more intuitive to indicate the increase and decrease of efferocytosis.We have changed the Data in Fig. 1B into a Z-Score analysis in new version.
Response: Thanks for raising the important concern.Clodronate-liposome induced macrophage depletion has been repeatedly applied in DSS-induced colitis mouse model to figure determine the role of macrophage function in IBD (2-4).However, the disease severity after macrophage depletion is inconsistent in different studies.
Macrophages are regarded as a major source of cytokines that can trigger inflammatory storm and aggravate IBD syndrome (5).Reduction of inflammation by depletion macrophage has been regarded as a strategy for treating IBD (4)( 6).On the contrary, macrophages can act as immunosuppressors and aid in tissue repairment after being polarized to M2 states (7).Regulation of macrophage to anti-inflammatory state is an emerging approach for IBD treatment (8)(9).We noticed that similar to our observation, several studies didn't show obvious attenuation effects in colitis model after depletion macrophage (3)(10).
A possible explanation of the inconsistent results after macrophage depletion in DSS-induced colitis model may be the limitation of the macrophages depletion method.The clodronate-liposome macrophages depletion assay is a "knock-down" rather than "knock-out" strategy.In our case, clodronate-liposome depleted roughly 60% peritoneal macrophages, and the remained macrophage may still functional.While in another experiment, we applied a stronger clodronate-liposome administration protocol in which more than 70% macrophages are depleted.In this condition, a more serious damage was observed in the macrophage depletion group (shown as below).These results indicate that the macrophage depletion rates may affect the disease activity in DSS-induced colitis model.
7) The authors deplete macrophages and show that the protective effects of columbamine on intestinal inflammation are lost.These data argue a role for macrophages in eliciting the protective effects of columbamine.However, these do not represent evidence to support the claim that columbamine-induced efferocytosis enhancement mediates the beneficial effects.Can the authors block LAP in vivo or other such relevant pathways to show that columbamine indeed protects against intestinal inflammation via enhancement of efferocytosis?Additionally, can the authors directly test the efferocytosis efficiency of macrophages in the colonic sections?
Response: Thanks for the valuable suggestion.As suggested, we used 3-methyladenine (3-MA) to block LAP in vivo ( 11), and treated mice with DSS to induce colitis mouse model.We found the protective effects of COL in colitis mouse model can be blocked by treating 3-MA (supplementary figure 3A-F).These data demonstrated that COL potentially induced a LAP-dependent protective effects on intestinal inflammation.We have also tested the in-situ efferocytosis efficiency in colon, the results showed a higher macrophage-associated ACs ratio (Figure S3G-H).
8) It would be important to present TUNEL data for experiments in Figure 4. Can the authors also directly measure macrophage efferocytosis efficiency in the colon?Similar comment for data presented in Figure 6.
Response: According to reviewer's suggestion, we have counted and analyzed the efferocytosis efficiency in colon as shown in Fig. 4 and Fig. 6.We found depletion of macrophage partially inhibited the COL-induced enhancement of efferocytosis efficiency in colon (A).COL also failed to trigger increase of efferocytosis in the mice colon without FPR2 (B).These results were consistent with our conclusion and the results have been added in the Fig. S4A and Fig. S5B in the revised MS.
9) The authors could discuss the PK/PD of columbamine and whether they could detect columbamine or its metabolic products in their in vivo experiments.These data will be critical for translating these findings into clinical scenario.
Response: Thanks for reviewer's suggestion.
PK/PD studies of columbamine has been published in a Chinese journal, Chinese Journal of Health Laboratory Technology (1).In this study, rats were oral administrated (10 mg/kg) or intravenous injected (0.5 mg/kg and 1.0 mg/kg) with columbamine, and bloods were collected after 0.08 h, 0.25 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h for pharmacokinetics study (the result were shown as the figure below).The absolute bioavailability of columbamine was about 15.1%.The half-life period of columbamine t 1/2z was (10 ± 7.9) h or (7.7 ± 4.8) h after intravenous injection and (5.2 ± 2.8) h after oral administration.
Although the bioavailability isn't high, COL can still work at a low concentration level for a long period in blood.According to our results, COL at 1 μM is effective to trigger efferocytosis, indicating that the concentration of 0.338 ng/ml in blood is enough to work.Based on the medication time curve, COL can be effective for at least 12 h.Taken together, COL shows potential application value on clinical scenario.Thank you for the submission of your revised manuscript to EMBO Molecular Medicine.I am pleased to inform you that we will be able to accept your manuscript pending the following final amendments: 1) Source data: -Please complete the Source Data Checklist.
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In case you performed RNA seq experiment please deposit the raw data in an appropriate public repository and use the following format to report the accession number of your data: The datasets produced in this study are available in the following databases: [data type]: [full name of the resource] [accession number/identifier] ([doi or URL or identifiers.org/DATABASE:ACCESSION]) Please check "Author Guidelines" for more information.https://www.embopress.org/page/journal/17574684/authorguide#availabilityofpublishedmaterial 3) Appendix: Please move supplementary materials and methods to M&M section in the main manuscript file.Add table of content and page numbers and correct nomenclature to "Appendix Figure S1" etc and "Appendix Table S1" etc, also in the main manuscript text.4) Movies: Renamed the files to Movie EV1, etc. Remove their legends form the main manuscript file and zipp each legend as a readme.txtfile with its corresponding movie.
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10) We replaced Supplementary Information with Expanded View (EV) Figures and Tables that are collapsible/expandable online.A maximum of 5 EV Figures can be typeset.EV Figures should be citedas 'Figure EV1, Figure EV2" etc... in the text and their respective legends should be included in the main text after the legends of regular figures.
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Figure legends :
Figure legends: Comparison of COL-induced FRP1 signaling with FPR2 signaling.(A) Cacium-response curves of COL and fMLF.Calcium responses in FPR1-expressing cells were stimulated by the ligands at the indicated concentrations (n=3).The experimental procedure was similar to that for FPR2.(B) cAMP-response curves of COL and fMLF.The inhibition of forskolin-stimulated cAMP accumulation in FPR1-expressing cells was stimulated by the ligands at the indicated concentrations (n=3).The cAMP responses of FPR1 were detected with FRET-based Epac sensors as described in a published paper (https://doi.org/10.1073/pnas.2214324120).Data of (C) and (D) were the same with those of figure 6A and 6B, respectively.
Figure legends: Comparison of different ligands binding to FPR2 with COL.Molecular docking analysis of columbamine binding to FPR2.The chemical structure of the ligands were obtained from ChemSpider database.Docking analysis was performed by MOE software.A. Overall structure (left, PDB ID: 7T63) and interaction sites (right) of compound 43 (cpd43) binding to FPR2.The cpd43 (sphere and sticks with carbon in purple) occupied a wedge-pose with its chlorophenyl group insertion to FPR2.The polar connections between cpd43 and FPR2 are composed of R201 and R205.The hydrophobic interactions involve L81, H102, L109, F110, V113, W254, F257, V284, F292, and the carbonyl group of D106.B. Ligand binding poses in FPR2 of columbamine (COL) generated from docking analysis.The overall conformation and binding sites viewed from bundle and extracellular side is shown in the left and right panel, respectively.The polar connections between columbamine and FPR2 are composed of D106, R205, and S288.The hydrophobic interactions involve H102, V105, L109, F110, W254, F257, F292, and the guanidino group of R205.C. Docking model of FPR2 bound with lipoxin A4 (LXA4).The detialed

I
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