Myeloid‐resident neuropilin‐1 promotes choroidal neovascularization while mitigating inflammation

Abstract Age‐related macular degeneration (AMD) in its various forms is a leading cause of blindness in industrialized countries. Here, we provide evidence that ligands for neuropilin‐1 (NRP1), such as Semaphorin 3A and VEGF‐A, are elevated in the vitreous of patients with AMD at times of active choroidal neovascularization (CNV). We further demonstrate that NRP1‐expressing myeloid cells promote and maintain CNV. Expression of NRP1 on cells of myeloid lineage is critical for mitigating production of inflammatory factors such as IL6 and IL1β. Therapeutically trapping ligands of NRP1 with an NRP1‐derived trap reduces CNV. Collectively, our findings identify a role for NRP1‐expressing myeloid cells in promoting pathological angiogenesis during CNV and introduce a therapeutic approach to counter neovascular AMD.

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Please do not share this URL as it will give anyone who clicks it access to your account. ***** Reviewer's comments ***** Referee #1 (Comments on Novelty/Model System for Author): State of the art experimental models to study retinal immunity and choroidal neovascularization. The depth of data analysis from RNA Seq analyses could be improved, see comments.
Referee #1 (Remarks for Author): Andriessen et al present a very interesting short report on the role of neuropilin-1 in retinal mononuclear phagocytes and the effects in the laser-induced model of choroidal neovascularization. As key findings, the group showed that cell-specific knockout in phagocytes or injection of NRP1 trap molecules significantly reduced late stage phagocyte recruitment and CNV formation. These data are especially interesting as further angiogenesis-related molecules in addition to VEGF could be targets to treat neovascular retinal diseases such as age-related macular degeneration of diabetic macular edema.
The paper is well written and many conclusions are supported by the data. The findings fit very well in the scope of EMM and should not only be important for vision research but also for angiogenesis researcher in general.
I have a few minor comments that could improve the quality of the paper further: 1. Introduction and following text. I understand that the total word count for reports is limited but the abbreviation MP´s for mononuclear phagocytes is not very elegant. I would recomment to use the terms "mononuclear phagocytes", "phagoctes", "macrophages" etc. where appropriate at the relevant text passages.
2. Fig. 1 shows ELISA data for VEGF, SEMA3A, TGFb and PDGFb in ocular fluids from patients/controls and mRNA data from the laser CNV model. It could be interesting to include placental growth factor (PGF) in the mRNA anylses as recent data in the same mouse model showed a prominent PGF expression in phagocytes and a differential regulation by anti-VEGF injections.It would be nice to see whether NRP1 has an influence on PGF levels.
3. Figure 2 H etc. I inderstand that the absolute numbers and Iba1+ arease are not significantly different in NRP1-/-vs NRP1 ++ laser spots at day 7. I suggest that the authors could reanalyze their flat mount image data using grid cross analyses or other tools to determine the ramification changes as potential early events before cell numbers may change at later time points. 4. Figure 3 A-H denotes choroidal mRNA expression. I wonder whether the RPE/choroid complex (it is hard to dissect RPE from choroidal tissue) or indeed the choroid alone was analyzed here. If the first assumption is the casse, the the Y-axis labels should be changed to RPE/choroid. 5. The group presents preliminary bioinformatic analyses from NRP1 deficient and control peritoneal macrophages. I would suggest a) to deposit the raw data in a repository such as GEO and b) to perform a more detailed analysis of the transcriptomic changes to better define the proposed phagocyte polarization. I noticed that the authors were very carefully handling the M1/M2 macrophage paradigm, which is indeed a problematic topic when analyzing tissues and not cells, but the more sophisticated analysis of RNAseq may provide further insights into the cellular events in the absence or presence of NRP1.
Referee #2 (Remarks for Author): The present study from Przemyslaw Sapieha's lab investigated the function of Nrp1+ macrophages in the progression of the human AMD and mouse CNV. The experiments using LysM-Cre mediated Nrp1 deletion in mice revealed that Nrp1 expressed on macrophages promotes perfusion of pathologically expanding neovessels. Interestingly the Nrp1-Trap injection showed therapeutic effects on such perfusion. Overall, the data promote our understanding of retinal neovascular diseases and may lead to new therapeutic options. However, this reviewer find several points needed to be addressed as listed below: 1. (Fig. 1A) In this reviewer's opinion, this scheme is unnecessary as we find everywhere.
2. (Fig. 2G) In the panels of LysM-Cre/Nrp1+/+, Nrp1 does not merge with Iba1 in some places (upper right and upper left area). As such immunoreactivity diminished in CKO mice, it may not be nonspecific. Could authors comment on this unexplained staining?
3. (Fig. 2O, Fig 4B) What is the FITC+IB4-substances? Does not IB4 stain all blood vessels in this model? Supple Fig. 1D looks differently. Also, the FITC positive cells in Fig. 4B do not look like ECs. In any case, the section immunostaining or orthogonal view might be helpful to demonstrate FITC is indeed located in the endothelial lumen. 4. The section title "NRP1-expressing MP's display a pro-angiogenic alternatively activated phenotype" is not correct. Perhaps it should be "NRP1-deficient MP..."? 5. The molecular mechanism how loss of Nrp1 leads to M1 polarization is totally unclear. In this reviewer's opinion, authors should address by additional experiments investigating the intra-cellular signaling, or discuss much referring to the relevant literature. 6. The data do not support "Graphical Summary", in particular "proliferation" and "fibrosis" have not been substantially analyzed. The scheme should reflect the actual data, but not speculation from the expression profiles.
Referee #3 (Remarks for Author): NRP1 binds several pro-angiogenic ligands in association with various co-receptors. In this manuscript, Andriessen et al. describe a pro-angiogenic role for NRP1+ retinal myeloid cells in a model of wet age-related macular degeneration (AMD). The authors show that NRP1-expressing phagocytes, which express a pro-angiogenic/alternatively-activated phenotype, accumulate in the retina after laser-induced damage in the Brunch membrane and therein promote choroidal neovascularization (CNV), which ultimately results in AMD. In LysM.Cre/Nrpfl/fl mice, myeloid cellspecific inactivation of Nrp1 skews the polarization of retinal myeloid cells toward a more pronounced pro-inflammatory phenotype and reduces CNV. In order to determine the therapeutic potential of blocking the interaction between NRP1 and its ligands (including VEGFA), the authors used an NRP1-derived extracellular trap. When injected in the vitreus, the NRP1-derived trap reduced the laser-induced CNV area. Because the NRP1 trap can potentially block a number of proangiogenic ligands, the authors conclude that this approach may represent an effective strategy to reduce pathological CNV also in wet AMD patients who fail on VEGFA blockade.
The study is overall well performed. However, I have some concerns with conceptual flow, translational advance, as well as mechanistic insight.
Mechanistic insight: 1. A premise of the study is that NRP1 binds several pro-angiogenic factors; so, interfering with NRP1 may potentially block multiple pro-angiogenic pathways in wet AMD. The authors should investigate whether the NRP1-derived extracellular trap indeed sequesters VEGFA along with other pro-angiogenic factors. Is SEMA3A neutralized?
2. More extensive characterization of he retinas of mice that received the NRP1 trap should be provided. Indeed, the only readout used here is CNV. What about IL1B and IL6 levels? Also, it is unclear whether NRP1-expressing myeloid cells were affected. 9. It is unclear (at least to me) what the NRP1 trap experiment was performed. Indeed, the first part of the study focused on the role of NRP1 in myeloid cell-mediated CNV, whereas the NRP1 trap experiment addresses the potential role of NRP1 ligands in CNV. Because NRP1 is expressed by vascular endothelial cells and, potentially, other cell types, the NRP1 trap experiment does not in any way address the contribution of NRP1-expressing myeloid cells to CNV. The NRP1 trap should also be tested in mice with Nrp1-deficient myeloid cells in order to formulate a relevant mechanistic hypothesis.

How does
Other points: 10: The proficiency of Nrp1 deletion should also be tested in microglial cells and not only in BMDMs.
11. The authors refer to Table 1, which was not present in this version of the manuscript.
12. The gating strategy in Supplemental Figure 1A lacks of crucial controls. As the main message of this paper is based on the presence of NRP1+ cells, the authors should provide control flow cytometry dot plots (FMOs in particular). The NRP1+ population presented in this figure appears questionable and not convincing based on the data shown.
13: Figures 3P and 3Q are not described in the text.

Detailed response to reviewers
Reviewer #1: We thank the reviewer for their thoughtful comments and positive assessment of our study and thank them for acknowledging that this is a ' very interesting short report that uses state of the art experimental models'. We would also like to thank them for suggesting that "the findings should not only be important for vision research but also for angiogenesis researcher in general." Based on the recommendations and queries of the reviewer, we have performed a series of new experiments that have permitted us to provide additional data.
Query 1: Introduction and following text. I understand that the total word count for reports is limited but the abbreviation MP´s for mononuclear phagocytes is not very elegant. I would recomment to use the terms "mononuclear phagocytes", "phagoctes", "macrophages" etc. where appropriate at the relevant text passages. Answer: We thank the reviewer for this suggestion and we agree with them. We have made all the requested changes.
Query 2: Fig. 1 shows ELISA data for VEGF, SEMA3A, TGFb and PDGFb in ocular fluids from patients/controls and mRNA data from the laser CNV model. It could be interesting to include placental growth factor (PGF) in the mRNA analyses as recent data in the same mouse model showed a prominent PGF expression in phagocytes and a differential regulation by anti-VEGF injections. It would be nice to see whether NRP1 has an influence on PGF levels.  I understand that the absolute numbers and Iba1+ areas are not significantly different in NRP1-/-vs NRP1 ++ laser spots at day 7. I suggest that the authors could reanalyze their flat mount image data using grid cross analyses or other tools to determine the ramification changes as potential early events before cell numbers may change at later time points. Answer: We thank the reviewer for this suggestion and we have now performed grid-cross analysis and findings are provided to the reviewer. We found morphological data consistent with mononuclear phagocytes in LysM-Cre/Nrp1fl/fl mice initially more ramified and then slightly less suggesting a state resembling M2 and then M1. While interesting, we believe that we would need to further characterize the sate of these cells on a molecular levels to draw definitive conclusions. This is unfortunately not possible in the current frame of the study.
Query 4: Figure 3 A-H denotes choroidal mRNA expression. I wonder whether the RPE/choroid complex (it is hard to dissect RPE from choroidal tissue) or indeed the choroid alone was analyzed here. If the first assumption is the ca se, the Y-axis labels should be changed to RPE/choroid. Answer: The reviewer is correct. We have made changes to the labeling of the graphs.  Query 5: The group presents preliminary bioinformatic analyses from NRP1 deficient and control peritoneal macrophages. I would suggest a) to deposit the raw data in a repository such as GEO and b) to perform a more detailed analysis. Answer: We agree with the reviewer and we have deposited the transcriptomic files into GEO Database as GSE110447. Furthermore, in figure 3Z, we now provide an individual gene expression data as a heat map.

Reviewer #2
We are grateful to the reviewer for their insightful evaluation, elegant suggestions and positive appraisal of our study. We thank them for acknowledging that the "data promote our understanding of retinal neovascular diseases and may lead to new therapeutic options". The suggestions of the reviewer have led to several additional experiments and we believe we have addressed their concerns. Query 1: 1. (Fig. 1A) In this reviewer's opinion, this scheme is unnecessary as we find everywhere.
Answer: Thank you for this comment. We have now removed the scheme of NRP1 from figure 1A. Query 2: (Fig. 2G) In the panels of LysM-Cre/Nrp1+/+, Nrp1 does not merge with Iba1 in some places (upper right and upper left area). As such immunoreactivity diminished in CKO mice, it may not be non-specific. Could authors comment on this unexplained staining?
Answer: The reviewer is right and we have changed the images. We must add that NRP1 is only knocked out on the Lys expressing cells and as a consequence mice still express NRP1 on other cells of non myeloid origin. The light staining of NRP1 in LysM-Cre/Nrp1fl/fl is a consequence of the presence of other NRP1 expressing cells, likely endothelial and neuronal.
Query 3: (Fig. 2O, Fig 4B) What is the FITC+IB4-substances? Does not IB4 stain all blood vessels in this model? Supple Fig. 1D looks differently. Also, the FITC positive cells in Fig. 4B do not look like ECs. In any case, the section immunostaining or orthogonal view might be helpful to demonstrate FITC is indeed located in the endothelial lumen.
Answer: We thank the reviewer for this comment. We have used the FITC perfusion model where we inject a detxran-FITC solution into the left arterial chamber before we sacrifice the mouse. This allows for dextran-FITC to perfuse the neovessels. Isolectin IB4 stains not only endothelial cells but also certain immune cells, and some neurons. The underlying choroid is often visible after staining with IB4 because the laser impact has damaged all RPE cells in the center of the burn, exposing IB4 positive choroid and scar tissue. In our experience IB4-staining gives a gross overestimation of the neovascularization. We used FITC as an additional technique to measure CNV. Since only vessels with a lumen will be perfused with FITC the IB4 staining is also necessary. We use the ratio of IB4/FITC to give an idea of both the laser impact (IB4 positive) and the formation of CNV (dextran-FITC perfused). Query 4: The section title "NRP1-expressing MP's display a pro-angiogenic alternatively activated phenotype" is not correct. Perhaps it should be "NRP1-deficient MP..."?
Answer: We believe the data in the section suggests that NRP1 on mononuclear phagocytes renders them pro-angiogenic and in line with an alternatively activated phenotype. Query 5: The molecular mechanism how loss of Nrp1 leads to M1 polarization is totally unclear.
In this reviewer's opinion, authors should address by additional experiments investigating the intra-cellular signaling, or discuss much referring to the relevant literature.
Answer: We agree. We have in the current study limited this section to demonstrating that NRP1 deficiency leads to Nf-kB p65 activation as well as production of cytokines typically associated with M1 such as IL-1b and Tnf-a Query 6: The data do not support "Graphical Summary", in particular "proliferation" and "fibrosis" have not been substantially analyzed. The scheme should reflect the actual data, but not speculation from the expression profiles.
Query 1: A premise of the study is that NRP1 binds several pro-angiogenic factors; so, interfering with NRP1 may potentially block multiple pro-angiogenic pathways in wet AMD. The authors should investigate whether the NRP1-derived extracellular trap indeed sequesters VEGFA along with other pro-angiogenic factors. Is SEMA3A neutralized?

Answer: We thank the reviewer for this comment. The novelty of our study lies in the fact that we demonstrate that NRP1 on myeloid cells prevents an the cells from triggering release of proinflammatory cytokines. Furthermore, we link heightened inflammation due to absence of NRP1 on myeloid cells to lower CNV. Lastly we provide a therapeutic entity (NRP1-derived trap) for CNV. Hence we believe all 3 points provide novelty.
Query 7: It is unclear (at least to me) what the NRP1 trap experiment was performed. Indeed, the first part of the study focused on the role of NRP1 in myeloid cell-mediated CNV, whereas the NRP1 trap experiment addresses the potential role of NRP1 ligands in CNV. Because NRP1 is expressed by vascular endothelial cells and, potentially, other cell types, the NRP1 trap experiment does not in any way address the contribution of NRP1-expressing myeloid cells to CNV. The NRP1 trap should also be tested in mice with Nrp1-deficient myeloid cells in order to formulate a relevant mechanistic hypothesis.
Answer: We have now tested the NRP-1-derived trap in LysM-Cre/Nrp1fl/fl mice. The trap suppresed CNV to levels observed in C57BL6 mice. However, the LysM-Cre/Nrp1fl/fl already show significantly reduced levels of CNV. Hence the magnitude of reduction of CNV by NRP1-derived traps in LysM-Cre/Nrp1fl/fl was not statistically significant when compared to C57BL6 mice suggesting convergent mechanisms of action.

Query 8:
The proficiency of Nrp1 deletion should also be tested in microglial cells and not only in BMDMs.
Answer: We have previously published this data in PMID: 27035626 and found that roughly 30% of retinal microglia are depleted for NRP1 in this model.

Query 9:
The authors refer to Table 1, which was not present in this version of the manuscript.

Answer: We thank the reviewer for this comment. We had omitted to submit the table. It is now included in the submission.
Query 10: The gating strategy in Supplemental Figure 1A lacks of crucial controls. As the main message of this paper is based on the presence of NRP1+ cells, the authors should provide control flow cytometry dot plots (FMOs in particular). The NRP1+ population presented in this figure appears questionable and not convincing based on the data shown.

Answer: Thank you for this comment. We conducted these experiments and published the NRP1 signal and isotype control in supplemental S4 panel B of Wilson et al. (PMID: 29549139).
Query 11: Figures 3P and 3Q are not described in the text.
Answer: We thank the reviewer for bringing this to our attention. We have added the description for this section.
30th Oct 2020 1st Revision -Editorial Decision 30th Oct 2020 Dear Prof. Sapieha, Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. We have now received feedback from the two reviewers who were asked to re-evaluate your manuscript. As you will see from the reports below, both referees acknowledge your efforts to address their initial concerns, and recognize that the manuscript has significantly improved. However, referee #2 also mentions issues that remain unanswered and finds additional experiments and further discussion necessary to support the claims. Therefore, we would like you to address this referee's concerns in a revised version of your manuscript. Please be aware that this will be the last chance for you to address these points.
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We note that you provided 3 images as synopsis. Are some of them suggestions for cover? 17) As part of the EMBO Publications transparent editorial process initiative (see our Editorial at http://embomolmed.embopress.org/content/2/9/329), EMBO Molecular Medicine will publish online a Review Process File (RPF) to accompany accepted manuscripts. In the event of acceptance, this file will be published in conjunction with your paper and will include the anonymous referee reports, your point-by-point response and all pertinent correspondence relating to the manuscript. Let us know whether you agree with the publication of the RPF and as here, if you want to remove or not any figures from it prior to publication. Please note that the Authors checklist will be published at the end of the RPF.
EMBO Molecular Medicine has a "scooping protection" policy, whereby similar findings that are published by others during review or revision are not a criterion for rejection. Should you decide to submit a revised version, I do ask that you get in touch after three months if you have not completed it, to update us on the status. In this revised paper, authors have adequately addressed my previous concerns and strengthened the data. Now the paper is acceptable.
Referee #3 (Remarks for Author): The authors have addressed some of the initial concerns of this reviewer. However, my general concerns with conceptual flow, translational advance, as well as mechanistic insight, unfortunately remain. The authors have provided very brief responses to the reviewer's comments (generally a couple of sentences), often without describing and explaining new data and their significance.
In general, I remain confused about the conclusions that can be drawn from the NRP1 deletion studies and the NRP1 trap studies, as the two approaches target different mechanisms. The author's response to point 7 is not clear and the conclusion that there are "convergent mechanisms of action" is not compelling. Rather, the data in Fig 4I seem to support the notion that different mechanisms (and targets) are involved. In fact, the trap seems to decrease CNV in the NRP1deficient setting, although the data are not statistically different. (lack of statistical significance can be due to high variation and low numerosity, not necessarily to the lack of biological effects.) Points 8 and 10 should be addressed with pertinent new data (key controls), because results may differ from those obtained in previous studies. Also, it is concerning that NRP1 was deleted in only about 30% of the retinal microglia (response to point 8). This point should be discussed.
The corresponding author is listed as affiliated to (employed by?) SemaThera, but in the COI section they are listed as consultant. The COI of the corresponding author should be better defined.
Even with the key limitations mentioned above, the ms could be published after the remaining points have been addressed and discussed in the paper.

Itemized list of corrections
Queries from October 30 th Referee #2: Query 1: In this revised paper, authors have adequately addressed my previous concerns and strengthened the data. Now the paper is acceptable. Answer: We thank the reviewer for their time and for providing helpful comments that have altogether improved the quality of the study.
Referee #3: Query 1: In general, I remain confused about the conclusions that can be drawn from the NRP1 deletion studies and the NRP1 trap studies, as the two approaches target different mechanisms. Conclusion that there are "convergent mechanisms of action" is not compelling. Rather, the data in Fig 4I seem to support the notion that different mechanisms (and targets) are involved. Answer: We agree with the reviewer. We have modified the text to include the notion that only part of the effects of the trap are mediated through myeloid cells. Moreover, we explain that NRP1 is expressed by several other cells in the retina and sclera-choroid-RPE complex. This suggests that a portion of the therapeutic effect of the NRP1 trap is mediated through myeloid cells. We have modified and clarified this in the revised text to include these changes.
Query 2: NRP1 was deleted in only about 30% of the retinal microglia. This point should be discussed. Answer: We agree with the reviewer and have added data to the supplemental section of the text. Concerning expression levels of NRP1 in myeloid cells and microglia, we had obtained data during this study but not yet added in this manuscript, since we performed these experiments for the purpose of assuring ourselves that our results are in line with those obtained in previous studies from our lab (PMID: 27035626 and PMID: 29549139). They are now included in the supplementals. We have not performed the experiments with publication of the results in mind and the number of animals we sacrificed for this experiment were low yet the number of cells we obtained in our FACS samples were sufficient enough to confirm 12th Jan 2021 2nd Authors' Response to Reviewers 18th Jan 2021 2nd Revision -Editorial Decision 18th Jan 2021 Dear Prof. Sapieha, Thank you for the submission of your revised manuscript to EMBO Molecular Medicine, and please accept my apologies for the delay in getting back to you, which is due to the limited staff and increased submitted manuscripts during the holiday season. We have now received the enclosed reports from referee #3 who re-reviewed your manuscript. As you will see, this referee is supportive of publication, and we will be able to accept your manuscript pending the following final minor amendments: 1) Main manuscript text: -Please answer/correct the changes suggested by our data editors in the main manuscript file (in track changes mode). This file will be sent to you in the next couple of days. Please use this file for any further modification. -Please remove the Graphical Summary -Please replace "Competing interests" by "Conflict of interest" -Thank you for updating the reference format, however, we note that some references are still incorrect. Please update all references so as to have 10 authors listed before et al.
2) Checklist: Section C/7: Please indicate whether the cells were tested for mycoplasma contamination. Section D/8 and 9: please provide the requested information (including housing and husbandry conditions). Section E: Please fill in section 11 and 12. Section F/18: this section should list the accession codes for data generated in this study. The accession number provided refers to RNAseq data generated in a previous study (Wilson et al, 2018). Please clarify.
3) Thank you for providing a synopsis. I slightly edited it to match our style and format. Please let me know if you agree with the following: A population of innate immune myeloid cells expressing the NRP1 receptor invades the retina and drives pathological neovascularization during age-related macular degeneration (AMD). A recombinant NRP1-derived trap prevents pathological angiogenesis associated with choroidal neovascularization.
-NRP1 ligands were elevated in patients with neovascular AMD and in a mouse model of choroidal neovascularization (CNV).
-NRP1-expressing mononuclear phagocytes rose in the retina upon injury and promoted CNV.
-CNV was reduced in mice by therapeutic intravitreal administration of soluble NRP1.
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B-Statistics and general methods
the assay(s) and method(s) used to carry out the reported observations and measurements an explicit mention of the biological and chemical entity(ies) that are being measured. an explicit mention of the biological and chemical entity(ies) that are altered/varied/perturbed in a controlled manner.
a statement of how many times the experiment shown was independently replicated in the laboratory.
Any descriptions too long for the figure legend should be included in the methods section and/or with the source data.
In the pink boxes below, please ensure that the answers to the following questions are reported in the manuscript itself. Every question should be answered. If the question is not relevant to your research, please write NA (non applicable). We encourage you to include a specific subsection in the methods section for statistics, reagents, animal models and human subjects.
definitions of statistical methods and measures: a description of the sample collection allowing the reader to understand whether the samples represent technical or biological replicates (including how many animals, litters, cultures, etc.).
The data shown in figures should satisfy the following conditions: Source Data should be included to report the data underlying graphs. Please follow the guidelines set out in the author ship guidelines on Data Presentation.
Please fill out these boxes ê (Do not worry if you cannot see all your text once you press return) a specification of the experimental system investigated (eg cell line, species name).
The effect sizes of the respective methods was determined from pilot experiments or earlier publications. We maintained a minimum of n=3 for all experiments.
graphs include clearly labeled error bars for independent experiments and sample sizes. Unless justified, error bars should not be shown for technical replicates. if n< 5, the individual data points from each experiment should be plotted and any statistical test employed should be justified the exact sample size (n) for each experimental group/condition, given as a number, not a range; Each figure caption should contain the following information, for each panel where they are relevant:

See 1a
Mice were weighed weekly and their general health was monitored. Mice that were not in good health (weight loss or other measures of morbidity) were excluded. If mice developed any complications during or post laser burn such as (sub)retinal bleed or cataract, they were excluded. RNA quality was analyzed before qPCR, and meltcurves were analyzed after qPCR. If the samples did not meet criteria of standard PCR method, they were excluded.
No randomization was used.

Manuscript Number: EMM-2019-11754
Appropriate statistical tests are included in the manuscript.
Statistical analysis was performed using GraphPad software. Before proceeding to the T-test or ANOVA we identified and excluded outliers using the PRISM GraphPad software.
Experimental groups were started at the same time with mice of the same age, from several litters. The control and experimental group use a different mouse strain and can therefor not be randomized. Mice were randomly asigned to the control or experimental group when they belonged to the same mouse strain.
Confocal pictures of the choroidal burns were saved using a randomly assigned number to each mouse unknown to the evaluator. This allowed for a blinding of the investigator for picture analysis. During FACS, ELISA, qPCR samples were randomly assigned a number and did not indicate their group or treatment.
Evaluation of extent of choroidal neovascularization was performed in a blinded manner where the investigator scoring the burns was blinded to the treatement.

Data
the data were obtained and processed according to the field's best practice and are presented to reflect the results of the experiments in an accurate and unbiased manner. figure panels include only data points, measurements or observations that can be compared to each other in a scientifically meaningful way.

E-Human Subjects
BMDM were cultured from bone marrow with M-CSF as described in the manuscript. Cells were not tested for mycoplasma contamination.
Variations are included in all graphs. Scatter blots showing every single data point and the mean plus/minus standard error of the mean were choosen to depict the spreading of the individual data points.
There was normal variation in the data as every single animal reacts individually to the laser damage or the respective treatment. Variation within the experiments was reduced by using disease-free animals of similar age. Scatter blots showing every single data point and the mean plus/minus standard error of the mean were choosen to depict the spreading of the individual data points.
All antibody catalog numbers are provided in the manuscript.