Endothelial cell‐derived oxysterol ablation attenuates experimental autoimmune encephalomyelitis

Abstract The vasculature is a key regulator of leukocyte trafficking into the central nervous system (CNS) during inflammatory diseases including multiple sclerosis (MS). However, the impact of endothelial‐derived factors on CNS immune responses remains unknown. Bioactive lipids, in particular oxysterols downstream of Cholesterol‐25‐hydroxylase (Ch25h), promote neuroinflammation but their functions in the CNS are not well‐understood. Using floxed‐reporter Ch25h knock‐in mice, we trace Ch25h expression to CNS endothelial cells (ECs) and myeloid cells and demonstrate that Ch25h ablation specifically from ECs attenuates experimental autoimmune encephalomyelitis (EAE). Mechanistically, inflamed Ch25h‐deficient CNS ECs display altered lipid metabolism favoring polymorphonuclear myeloid‐derived suppressor cell (PMN‐MDSC) expansion, which suppresses encephalitogenic T lymphocyte proliferation. Additionally, endothelial Ch25h‐deficiency combined with immature neutrophil mobilization into the blood circulation nearly completely protects mice from EAE. Our findings reveal a central role for CNS endothelial Ch25h in promoting neuroinflammation by inhibiting the expansion of immunosuppressive myeloid cell populations.


8th Jun 2022 1st Editorial Decision
Dear Prof. Pot, Thank you for the submission of your research manuscript to our journal, which was now seen by three referees, whose reports are copied below.
We concur with the referees that the proposed role of endothelial-derived oxysterols downstream of Ch25h in regulation of myeloid-derived suppressor cells at the blood-brain barrier is in principle very interesting. However, referees also raise some concerns that need to be addressed to consider publication here. I find the reports informed and constructive, and believe that addressing the concerns raised will significantly strengthen the manuscript. As the reports are below, and I think all points need to be addressed, I will not detail them here.
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11) The journal requires a statement specifying whether or not authors have competing interests (defined as all potential or actual interests that could be perceived to influence the presentation or interpretation of an article). In case of competing interests, this must be specified in your disclosure statement. TO THE AUTHORS This certainly is a very interesting manuscript linking a mouse model of multiple sclerosis with Ch25h, 25-hydroxycholesterol and PGE2. I have a few suggestions the authors may like to consider. Major Suggestions. 1. As this paper covers a rather wide scientific area, a scheme could be added showing the interactions of the different enzymes, metabolites, cells and immunological processes. Few readers will be expert in each of the different areas. 2. Being familiar with the work of Cyster on EAE and Ch25h I was forever wondering when it would be mentioned. I think some mention of these conflicting results could come in the Introduction. 3. It would be nice to have a better feel of how much oxysterol is actually being secreted by cells. Figure 3 has concentrations of eicosanoids in nM. Could the same units be used for oxysterols. In this regard it is also important to know the cell number. This is important as the concentration of 7-keto-25-hydroxycholesterol appears to be an order of magnitude greater than that of 25hydroxycholesterol. 4. Following on from the last point, the authors present good evidence for Ch25h expression in BEC, but I wonder how important Ch25h expression in these cells is compared to macrophages expression. Both Diczfalusy and Bauman have shown activated macrophages generate high concentrations of 25-hydroxycholesterol in man and mouse. Again the use of concentration units would give the reader some idea. 5. With respect to experiments with IL-1β, Cyster and Russell have shown that 25-hydroxycholesterol attenuates IL-1β formation. This should be considered in the context of the experiments and their interpretation.
Other Suggestions. 1. Figure 3 and Experimental. More information should be given with respect to metabolites and cell cultures. The medium used and the cell number should be given. 2. Figure S2A and page 8. It is interesting that 24S-hydroxycholesterol is found in pMBMECs incubations. 24S-HC is thought to be a metabolite produced by Cyp46a1 in neurons. Similarly, 7α-hydroxycholestenone is generated by Cyp7a1 which is a hepatic enzyme. Some comment is needed. 3. Figure S2. Again it would be useful to have an idea of the concentrations of oxysterol so the reader can get a feel of the levels being discussed. Are these very minor oxysterols compared to 25-hydroxycholeasterol or more abundant? I would recommend a chromatogram being shown in the Supplemental. 4. Page 8. Could the authors provide a list of the 49 eicosanoids consistently detected above threshold. It would be nice if a chromatogram was included in the supplemental. 5. Page 9 and elsewhere. Perhaps some discussion of SREBP-1a is require. From the classic paper by Horton (PNAS 2003) it seems that γ-linolenic acid formation is regulated by this transcription factor. 6. Figure 4. What were the incubation concentrations of PGE2 and 25-OHC? 7 Experimental. It would be good to indicate the deuterated standards used for quantification of metabolites. 8. Oxysterol measurements. What was the volume of cell supernatant added to extraction solvent?
In summary a very interesting paper, but could be improved by providing the reader with a little more quantitative and experimental data.
Referee #2: The manuscript by Ruiz et al explores the role of the enzyme Ch25h and its downstream metabolites in brain endothelial cells (ECs) during neuroinflammation. The authors have previously demonstrated that Ch25h full knockout mice develop less severe EAE, a finding that was then linked to Ch25h expression in hematopoietic cells (Chalmin et al 2015). In the current manuscript the authors explore the role of Ch25h in endothelial cells, first demonstrating that Ch25h expression is upregulated in brain ECs during EAE. Using several mouse strains to remove Ch25h expression from ECs (including BBB ECs), the authors demonstrate a partial protection from developing EAE in the absence of Ch25h. This is the major finding of the manuscript and represents a novel and interesting discovery. The authors then go on to explore the transcriptional and lipidomic profile of Ch25h deficient ECs in the presence or absence of IL1b. Finally the authors attempt to link the diminished EAE severity observed in Ch25h knockout mice with an accumulation of suppressive neutrophils in the CNS. The neutrophil-related findings are potentially interesting but would have to be addressed by additional experiments in order to better link the attenuated EAE with the accumulation of neutrophils in the CNS.
Major points: 1. The results in Figure 1 would be considerably strengthened by showing Ch25h-eGFP expression in tissue sections. The authors claim that increased expression during EAE is restricted to the EC compartment, however the comparison is made with CNS resident and CNS hematopoietic cells, which each represent pools of several different cell types.
2. Figure 4C: The suppressive capacity of the identified CNS-infiltrating neutrophils relies entirely on this figure. However, it lacks a proper control. Simply showing that the addition of CNS-derived neutrophils limits proliferation of anti-CD3/CD28-stimulated Tcells could have multiple explanations. If the authors claim that PMN-MDSCs isolated from the EAE CNS are indeed suppressive they should compare them to "naive" neutrophils isolated for example from blood or bone marrow of a naive mouse. If authors think the suppression is mediated by soluble factors, an alternative approach could be to use conditioned media.
The manuscript by Florian Ruiz et al. "Endothelial-derived oxysterols regulate myeloid-derived suppressor cells at the bloodbrain barrier" describes a new role for the enzyme Ch25h and its product 25-OHC in EAE, namely the inhibition of myeloid suppressor cell expansion in the inflammatory process at the blood brain barrier. Using a new mouse strain, made in the lab, which reports expression of Ch25h and which can be used for tissue-specific deletion of the enzyme, they show that endothelial cells of the CNS are the main cell type expressing Ch25h in EAE and that its deletion in endothelial cells and importantly in CNS-ECs leads to a delayed and lowered EAE, similar to mice lacking CH25h body-wide as shown in a previous paper by the lab. From that point they show that the lack of Ch25h in inflamed endothelial cells changes expression of lipid biosynthetic pathways leading finally to a shift from 25-OHC to PGE2 in mice lacking Ch25h in ECs. In vitro data shows that PGE2 promotes differentiation of MDSCs whereas 25-OHC strongly inhibits their differentiation. These findings could explain the found expansion of MDSCs in the CNS of CH25hEC-KO mice with EAE compared to control mice. Finally, depletion of mature neutrophils at DPI 14 led to a near complete resistance of EAE in CH25hEC-KO mice and a further expansion of MDSCs in the CNS. Additionally, one figure shows analyses of data from human glioblastoma ECs in context to Ch25h expression and its similarity to ECs of mice lacking Ch25h in EAE CNS.
This manuscript is very interesting, well written and the experiments are thoroughly performed. Nevertheless, some points are not clear enough yet for the main claim to be made and further experiments should be performed to make the main conclusions more solid.
1. The main claim of the paper is that EAE is lower in CH25hEC-KO due to expansion of MDSCs in the CNS. Several points are unclear here. a) Expansion: in all experiments showing MDSCs in the CNS only percentages of live cells are shown. Since EAE is lower in CH25hEC-KO mice, also infiltration of T cells and of monocytes should be reduced. Hence, the higher percentage of MDSCs in the CNS in EAE in these mice might only be relative. Therefore, it is necessary to determine the absolute cell numbers and to show them next to the percentages. This accounts also for data showing T cell-infiltrates. b) MDSCs in the CNS: As also discussed by the authors, it is difficult to distinguish PMN-MDSCs from normal PMNs in the mouse and an alternative scenario of the effectiveness of the PMN depletion might be their pathogenic effect in EAE as claimed by several others previously combined with a lack of depletion of PMNs in the CNS. To be sure that the CNS PMNs are indeed MDSCs, one should perform an Arginase 1 staining (e.g. intracellular with clone A1exf5) and compare this to PMNs in the blood, spleen and BM. c) How do 25-OHC/PGE2 act on immature PMN? Unfortunately, PMN/ MDSCs of controls and CH25hEC-KO were only compared in the CNS and not in blood, spleen and BM (eg. Figure 4f). This should at least clarify whether 25-OHC/PGE2 have a more systemic or local impact at the CNS. d) Are MDSCs also expanded in the CNS of CH25h-BBBKO mice? Since CH25hEC-KO mice may show an activation of ECs in the whole body and an increased level of PGE2 over 25-OHC, it might be possible that the increase of PMN/MDSCs occurs already at the level of the BM and due to an expansion there, more of these cells may migrate to the CNS during inflammation. Therefore, it is also important to know whether also CH25h-BBBKO mice, which show the same EAE phenotype, also show an enhanced level of PMN/MDSCs in the CNS.

Since EAE is mediated by T helper cells, an analysis of CNS-infiltrating T cells is lacking in respect to
Th17/Th1/ThGMCSF/Tregs. These should also be analyzed in spleen/dLNs and blood. All in % and in absolute numbers.
3. Ch25h is important in generating the EBI2 ligand 7a,25-OHC. This ligand was previously shown to be upregulated in EAE in the CNS. Since your data show that EC are the main cells expressing Ch25h in the CNS in EAE one would expect a lack of 7a,25-OHC upregulation in the CNS of CH25h-BBBKO mice. Showing this would make your findings using the reporter mouse even more convincing. In the same line a western blot against CH25h of full CNS vs. CNS-ECs of healthy vs. EAE of control vs. CH25hEC-KO would be a great confirmation of your reporter data.
4. Ch25h expression in EAE: Microglia cells should be properly stained with CD11b and CD45 and then gated on CD11b (int) and CD45(int) for analysis of reporter expression. Ter119 staining to exclude erythrocytes should be performed on a separate channel. In addition, the FACS staining should also be shown reversed by gating on all live cells expressing Ch25h-eGFP and then checking in which population these positive cells fall. By such a gating a staining of CNS from CH25hEC-KO mice should reveal a near complete absence of eGFP in the live cell population. Since FACS shows only a part of CNS resident cells, histology against eGFP in Ch25h reporter mice with EAE should reveal a more complete picture of Ch25h expression in the CNS in EAE.
Minor Points: 5. Although very interesting, the data shown on GBM ECs is rather confounding the overall story and not necessary for the main points to make. I would suggest to omit this and to put this rather in another small publication. 6. Some histology of infiltration and demyelination of controls versus CH25hEC-KO in EAE would be nice to have to see whether there might be also other qualitative changes. 7. For EAE experiments it would be nice to see for each group shown the exact N number of the mice directly in the figure. 8. Mean max score: Was this calculated only of diseased mice? 9. Figure 2e seems to show the averaged data of 3 measurements for each condition. I would suggest to show them individually. 10. Figure 4c shows that PMN-MDSCs can inhibit T cell proliferation. A control showing the specificity of these cells by using PMN from BM would strengthen that point. 11. Correlation should be rather -0.68, what do the ** mean here? 12. Figure 6a: There seems to be at least some impact of the combo treatment also in controls. 13. In Figure 6e and f the comparison of controls vs. CH25hEC-KO in EAE should also be shown. 14. For Figure 6f one should rather show bars than lines since the loss of PMNs occurs most likely only on day 14 and not before. 15. Figure 6f right: axes labels were forgotten 16. Page 16 mentions deletion of Ch25h in GBM ECs although it is rather likely "not expressed" what was meant. 17. Although in general well written the grammar needs to be checked.

Major Suggestions
1. As this paper covers a rather wide scientific area, a scheme could be added showing the interactions of the different enzymes, metabolites, cells and immunological processes. Few readers will be expert in each of the different areas.
We are now providing a graphical abstract that summarizes our model and is included as a synopsis.
2. Being familiar with the work of Cyster on EAE and Ch25h I was forever wondering when it would be mentioned. I think some mention of these conflicting results could come in the Introduction.
We are now mentioning these contrasting results in the introduction on page 3 -Lines: 71-75.

It would be nice to have a better feel of how much oxysterol is actually being secreted by cells. Figure 3 has concentrations of eicosanoids in nM. Could the same units be used for oxysterols. In this regard it is also important to know the cell number. This is important as the concentration of 7-keto-25hydroxycholesterol appears to be an order of magnitude greater than that of 25-hydroxycholesterol.
We now show all the oxysterols, including 25-hydroxycholesterol, in concentration in nM (rebuttal Concerning the comparison of 7-keto-25-hydroxycholesterol with 25-hydroxycholesterol, we could previously not perform a direct comparison between these two oxysterols when represented in the form of a ratio oxysterol/ d-oxysterol. Indeed the intensity of the peaks measured by HPLC-MS is not necessarily directly proportional to the compound concentration as it also depends on the intrinsic ionizability of the molecule. We are now showing the concentrations of the oxysterols that enables us a direct comparison. The level of 25-hydroxycholesterol is in the same range as of the levels of 7-keto 25-hydroxycholesterol with 25-OHC being however at a higher concentration in all conditions. We have now included those new results in the new manuscript (new Figure 3 and new Figure EV1   replicates/group except for Ch25 fl/fl IL-1β were n = 5 biological replicates. ns= non-significant, * P < 0.05, ** P ≤ 0.005, **** P ≤ 0.00005. p values were determined by two-way ANOVA with Sidak's post hoc test.

14th Nov 2022 1st Authors' Response to Reviewers
Concerning the question of the cell number, pMBMECs are cultivated in 96 well plates and plated at a concentration of 51 000 digested capillaries/cm 2 onto matrigel-coated wells. The purpose of the digestion step of the vascular pellet prior to pMBMECs seeding is not to obtain single cells but capillary fragment, which is the approach that gives the best results for microvascular ECs culture. Therefore, the seeding density is not based on the number of single cells but on the number of capillary fragments. Oxysterol measurements were performed when cells were confluent to avoid a potential proliferation bias induced by IL-1β stimulation. Furthermore IL-1β is only added in the culture when cells are confluent for 24hours. Thus, the cell count was not performed at the time of supernatant collection.

Following on from the last point, the authors present good evidence for Ch25h expression in BEC, but I wonder how important Ch25h expression in these cells is compared to macrophages expression. Both Diczfalusy and Bauman have shown activated macrophages generate high concentrations of 25hydroxycholesterol in man and mouse. Again the use of concentration units would give the reader some idea.
We agree that 25-OHC concentration is an important determinant of its biological activity and we have adapted the manuscript accordingly. Please also see point 3 above and referee 2 major point 1. Comparison of 25-hydroxycholesterol in vitro in two different subsets of primary cells might not reflect the in vivo biological effect. In order to answer this question and question 1 of referee 2, we have now performed additional experiments and quantified Ch25h expression by RNA scope in phagocytes (macrophages/microglia) and endothelial cells (ECs) in the CNS of non-immunized and EAE mice (rebuttal Figure 3 below and new Figure 1). We found that Ch25h was significantly more increased in ECs compared to phagocytes (macrophages/microglia) in the CNS both in non-immunized and EAE conditions. Despite increased expression of Ch25h in both phagocytes and ECs during EAE, our results show that the sole deletion of Ch25h in CNS ECs is sufficient to attenuate EAE.

With respect to experiments with IL-1β, Cyster and Russell have shown that 25-hydroxycholesterol attenuates IL-1β formation. This should be considered in the context of the experiments and their interpretation.
We agree that activated innate immune cells are a major source of IL-1β and within this context; this has to be taken in consideration. However, all the IL-1β-stimulation experiments were performed on pMBMECs and our RNAseq analysis indicates either low or absence of IL-1β mRNA in these cells. In light with this, IL-1β was not identified as a differentially expressed gene in Ch25h ECKO versus Ch25h fl/fl pMBMECs. This suggests that IL-1β is either expressed at very low levels or not expressed by brain endothelial cells. We also refer the referee to the vascular single cell database from the mouse brain from Christer Betsholtz laboratory (http://betsholtzlab.org/VascularSingleCells/database.html) where it can be seen that IL-1β transcripts are only detectable in Microglia at steady state. Similarly, the RNAseq analysis of CNS ECs published by Munji et al shows that IL-1β mRNA expression in CNS ECs is very low both at steady state and during EAE (Munji et al, 2019). Therefore, we consider that a bias of the interpretation of our results, resulting from increased production of IL-1β by CNS ECs in absence of 25-OHC, is unlikely.

Other Suggestions
1. Figure 3 and Experimental. More information should be given with respect to metabolites and cell cultures. The medium used and the cell number should be given.
The medium used in culture is now included in the Material and Method section: isolation and culture of primary brain microvascular cells, page 20 -lines: 588-592. Regarding the cell number please refer to the rebuttal response of major point 3 above. Figure S2A and page 8. It is interesting that 24S-hydroxycholesterol is found in pMBMECs incubations. 24S-HC is thought to be a metabolite produced by Cyp46a1 in neurons. Similarly, 7αhydroxycholestenone is generated by Cyp7a1, which is a hepatic enzyme. Some comment is needed.

2.
Concerning the 24(S)-hydroxycholesterol in the pMBMECs supernatant, its concentration is 10 times lower than the levels of 25-hydroxycholesterol and 7-keto-25-hydroxycholesterol. We did not detect Cyp46a1 in our pMBMECs on the RNAseq data. We further performed RT-qPCR on pMBMECs under all conditions (Ch25h ECKO and Ch25h fl/fl pMBMECs treated or not with IL-1) and confirmed the absence of Cyp46a1 mRNA detection (Rebuttal Fig 2a). We propose that Cyp46a1 is not expressed or at undetectable levels in our setting. Cyp46a1 has however been described in endothelial cells in the retina (Saadane et al, 2019) and in a protein atlas (https://www.proteinatlas.org/ENSG00000036530-CYP46A1/single+cell+type). Furthermore, it is not excluded that 24(S)-hydroxycholesterol is present in the Fetal Bovine Serum used in pMBMECs culture media. Its modest and just significant (p.value = 0.0281) increase upon IL-1β stimulation in Ch25h ECKO pMBMECs could be an indirect results of a reduced Cyp39a1 activity, the enzyme that catalyzes its hydroxylation in 7α,24(S)-OHC. However, in our RNAseq, Cyp39a1 mRNA expression was increased just reaching significance upon IL-1β stimulation (adjusted p.value = 0.053 and 0.045, FC 1.29 and 1.3 for Ch25h ECKO and Ch25h fl/fl pMBMECs respectively) but did not statistically differ between Ch25h ECKO and Ch25h fl/fl pMBEMCs. However, Cyp39a1 mRNA expression is not necessarily correlated with its activity at this specific time point (24 hours of IL-1β stimulation). 24S-HC and concentration measurement in cell-free culture medium and in Cy46a1 K.O pMBMECs-derived supernatant might help address this specific point, however, we consider that this out of the scope of our manuscript.
The enzyme responsible for 7α-hydroxycholestenone synthesis is HSD3B7 that synthesizes 7αhydroxycholestenone from 7α-OHC. 7α-OHC is produced by Cyp7a1 or in a ROS dependent manner. It could also be present in Fetal Bovine Serum used in pMBMECs culture media. We therefore performed RT-qPCR on Ch25h fl/fl and Ch25h ECKO pMBMECs and found that Cyp7a1 is expressed by pMBMECs at low levels compared to the liver control and its expression is increased upon IL-1β stimulation in Ch25h fl/fl pMBMECs (Rebuttal Fig 2b). This contrasts with the results obtained in the supernatant showing that 7α-OHC levels are not impacted by the deletion or IL-1β stimulation but does suggest that Cyp7a1 expression is not restricted to the liver. In light with this, 7α-hydroxycholestenone reduction observed in Ch25h fl/fl pMBMECs supernatant could be due to a reduction of HSD3B7 activity induced by IL-1β stimulation. Furthermore, we did not detected significant changes in HDS3B7 mRNA levels in our RNAseq when comparing either Ch25h fl/fl NS and Ch25h fl/fl IL-1β (adjusted p.value = 0.33, Table S2) or Ch25h fl/fl IL-1β and Ch25h ECKO IL-1β (adjusted p.value = 0.7, Table S3).
3. Figure S2. Again it would be useful to have an idea of the concentrations of oxysterol so the reader can get a feel of the levels being discussed. Are these very minor oxysterols compared to 25hydroxycholesterol or more abundant? I would recommend a chromatogram being shown in the Supplemental.
We now show all oxysterols with their concentrations in the revised manuscript and Figures (rebuttal  Figure 3). This now enables a direct comparison of oxysterol concentrations in the pMBMECs supernatants. 25-hydroxycholesterol and 7-keto-25hydroxycholesterol are the most abundant oxysterols measured. For example, the concentration of 24-(S)OHC and 27-OHC are ten times lower than the one of 25-hydroxycholesterol and 7-keto-25hydroxycholesterol in-vitro. This is in clear contrast with the levels of 24(S)-OHC and 25-OHC in the total spinal cord (new Figure 3D) showing that, as previously described in many studies, 24(S)-OHC is the most abundant oxysterol. This further emphasize our hypothesis that local concentrations of 25-OHC at the level of the microvascular endothelium is different from the concentrations at the tissue scale. We changed the text of the manuscript accordingly.
Rebuttal Figure 4 is showing the chromatograms of the eicosanoids displayed in the manuscript. As the intensity of the peaks measured by HPLC-MS it is not directly proportional to the compound concentration, we prefer to show only the concentrations of the eicosanoids in the manuscript.
5. Page 9 and elsewhere. Perhaps some discussion of SREBP-1a is require. From the classic paper by (Horton (PNAS 2003) it seems that γ-linolenic acid formation is regulated by this transcription factor.
We have now added a discussion about the potential involvement of SREBP-1a in increased production of γ-linolenic acid and FADS2 synthesis in absence of 25-OHC on pages 16 -lines: 457-460.
The concentration of PGE2 and 25-OHC were 20nM and 1µM respectively. They are provided in the Material and Methods section of the manuscript in the "Bone marrow derived cell culture and MDSC polarization" at page 23-lines: 692-693. We have now added the concentration in the Figure 4 legend.
7 Experimental. It would be good to indicate the deuterated standards used for quantification of metabolites.

Oxysterol measurements. What was the volume of cell supernatant added to extraction solvent?
The volume of the supernatant for oxysterol measurements was 200µL, it was added to the extraction solvent (8ml dichloromethane, 4 ml methanol with BHT and 2ml of water with EDTA). We have now included this information in Material and Methods section page 21 -lines: 630.

In summary a very interesting paper, but could be improved by providing the reader with a little more quantitative and experimental data. This certainly is a very interesting manuscript linking a mouse model of multiple sclerosis with Ch25h, 25-hydroxycholesterol and PGE2. I have a few suggestions the authors may like to consider.
We thank the referee for his suggestions, we believe that with the proposed modifications and addition of new results in the manuscript and Figures according to his comments have strengthened our manuscript.

The manuscript by Ruiz et al explores the role of the enzyme Ch25h and its downstream metabolites in brain endothelial cells (ECs) during neuroinflammation. The authors have previously demonstrated that Ch25h full knockout mice develop less severe EAE, a finding that was then linked to Ch25h expression in hematopoietic cells (Chalmin et al 2015). In the current manuscript the authors explore the role of Ch25h in endothelial cells, first demonstrating that Ch25h expression is upregulated in brain ECs during EAE. Using several mouse strains to remove Ch25h expression from ECs (including BBB ECs), the authors demonstrate a partial protection from developing EAE in the absence of Ch25h. This is the major finding of the manuscript and represents a novel and interesting discovery. The authors then go on to explore the transcriptional and lipidomic profile of Ch25h deficient ECs in the presence or absence of IL1b. Finally the authors attempt to link the diminished EAE severity observed in Ch25h knockout mice with an accumulation of suppressive neutrophils in the CNS. The neutrophilrelated findings are potentially interesting but would have to be addressed by additional experiments in order to better link the attenuated EAE with the accumulation of neutrophils in the CNS.
Major points:

The results in Figure 1 would be considerably strengthened by showing Ch25h-eGFP expression in tissue sections. The authors claim that increased expression during EAE is restricted to the EC compartment, however the comparison is made with CNS resident and CNS hematopoietic cells, which each represent pools of several different cell types.
We thank the referee for this important comment. We initially focused on ECs and only evaluated Ch25h-eGFP expression by flow cytometry on CNS CD45 + cells on a bulk cellular extraction dedicated to ECs and not on leukocyte extraction. Those results suggested that Ch25h-eGFP expression was restricted to ECs. We now properly evaluated Ch25h-eGFP expression on dedicated leukocyte extraction by flow cytometry and on tissue sections. We show that microglia also express Ch25h-eGFP as suggested by the referee. Flow cytometry results are discussed in detail on referee 3 major point 4.
We were not able to visualize eGFP expression in CNS tissue section, as eGFP expression is sensitive to fixation. In line with this observation, Ch25h-eGFP was only detectable in flow cytometry analysis in unfixed cells that were acquired immediately after staining which might explain that eGFP expression is below detection threshold in fixed tissues. To overcome this, we performed RNA in-situ hybridization of Ch25h in CNS tissue sections, comparing non immunized mice and mice at the peak of EAE (Score 2,5-3). Isolectin B4 and IBA1 were used to identify ECs and activated macrophages (phagocytes)/microglia respectively. We used IBA1 as increased Ch25h expression in microglia had been described in EAE (Wanke et al, 2017). We identified Ch25h expression in ECs and macrophages/microglia; its expression increased during EAE in both cell types (Rebuttal Fig. 5a and new Fig. 1A). Ch25h expression was significantly higher in ECs both at steady state and during EAE (Rebuttal Fig. 5b and new Fig. 1B). These results, together with the attenuated EAE observed in Ch25h ECKO , Ch25h BBBKO and Ch25h BECKO confirm the importance of endothelial cells as a source of Ch25h and nuances the previous studies suggesting that activated myeloid cells are the main source of 25-OHC in inflamed tissue. Please also refer to the responses of Referee 3 major points 3 and 4 and new Fig. 3 D. However, we do not rule out that Ch25h deletion in macrophages/microglia can modulate EAE but this was not the scope of our manuscript.
These new results unambiguously demonstrate that Ch25h expression is not restricted to the EC compartment. We have now adapted the manuscript accordingly. The results of the RNA in-situ hybridization are now shown in new Fig. 1 A and B of the manuscript and the discussion have been adapted in accordance to the new results. Despite increased expression of Ch25h in both phagocytes and ECs during EAE, our results show that the sole deletion of Ch25h in CNS ECs is sufficient to attenuate EAE.

Figure 4C: The suppressive capacity of the identified CNS-infiltrating neutrophils relies entirely on this figure. However, it lacks a proper control. Simply showing that the addition of CNS-derived neutrophils limits proliferation of anti-CD3/CD28-stimulated T-cells could have multiple explanations. If the authors claim that PMN-MDSCs isolated from the EAE CNS are indeed suppressive they should compare them to "naive" neutrophils isolated for example from blood or bone marrow of a naive mouse. If authors think the suppression is mediated by soluble factors, an alternative approach could be to use conditioned media.
To address this important question, we performed additional experiments to evaluate the suppressive activity of granulocytes within different organs. Live cells CD45 + CD11b + Ly6G + Ly6C int were sorted from the bone marrow of naïve and EAE mice and co-cultured with CD4 + T cell isolated from the spleen, labelled with CSFE and stimulated with anti-CD3/28 beads in the exact same conditions as Live cells CD45 + CD11b + Ly6G + Ly6C int sorted from the CNS at the peak of EAE in former Figure 4C (Rebuttal Fig.  6 and new Fig. 4C). We did not observed a reduction in the percentage of CFSE low CD4 T cells with BMderived Live cells CD45 + CD11b + Ly6G + Ly6C int . These results, combined with the experiment for Arginase-1 staining proposed by referee 3 at point 1B (see below) suggest that CNS derived live cells CD45 + CD11b + Ly6G + Ly6C int are indeed PMN-MDSC while the same population derived from either the blood, the spleen or the BM rather correspond to Bona-fide neutrophils. This is line with the current model of PMN-MDSC development proposed by the Contamide et al. suggesting that the suppressive capacity is acquired in peripheral tissues (Condamine et al, 2015), more specifically in the CNS in our model. This is also in accordance with what was previously proposed by Knier et al (Knier et al, 2018).

The authors need to better link the accumulation of neutrophils in the CNS with the abrogation of disease. Depletion of circulating neutrophils appears to, counterintuitively, cause accumulation of neutrophils in the CNS. Could the authors instead attempt to boost accumulation of suppressive neutrophils using for example G-CSF or PGE2 administration (or other means)?
We thank the referee for these suggestions. Knier et al showed that G-CSF administration partially attenuates EAE disease course, linking accumulation of suppressive neutrophils and EAE attenuation (Knier et al., 2018). Furthermore, Zehntner et al. also demonstrated that GR1 High cells sorted from the CNS could supress CD4 + T cell proliferation (Zehntner et al, 2005). Finally, Arg1 expression in CNS CD45 + CD11b + Ly6G + Ly6C int cells at the peak of the disease that we describe in referee 3 major point 1b combined the proliferation assay that we performed with PMN-MDSC sorted from the CNS in Fig.4C further indicate that this population shows a suppressive phenotype. We thus consider that there is a substantial amount of evidence showing that a subset of CD45 + CD11b + Ly6G + Ly6C int cells infiltrating the CNS during EAE display a suppressive phenotype.
Administration of a stable form of PGE2 (dm-PGE2) has also been shown to attenuate EAE (Esaki et al, 2010). Given that CNS endothelial-secreted PGE2 is increased in Ch25h ECKO pMBMECs, we asked if PGE2 administration favors CNS PMN-MDSC accumulation. We reproduced the experiment initially performed by Esaki et al. We confirmed that subcutaneous injections of a stable form of dm-PGE2, starting from day 7 after immunization attenuates EAE in WT mice (Rebuttal Fig. 6a). However, this phenotype seems to be independent of CNS-infiltrating PMN-MDSC, as neither the percentage nor the absolute number of these cells differed between controls and dm-PGE2-injected mice (Rebuttal Figure  6b right upper panel). Moreover, absolute numbers and percentage of Ki67 + CD4 + T cells was also similar between these two conditions. We believe that an elevation of the systemic levels of PGE2 is not sufficient to recapitulate our model and hypothesize that systemic injection of PGE2 induces PMN-MDSC outside the CNS, but we have not evaluated this hypothesis due to the time allocated to perform the revisions. Knier et al.(Knier et al., 2018) previously suggested that the acquisition of the PMN-MDSC phenotype by granulocytes is restricted to the CNS during EAE. The experiment that we performed to address referee 2, major point 2 and referee 3 major point 1b confirm these results. Additionally, as already mentioned, this is highly consistent with model of PMN-MDSC development proposed by Gabrilovitch. Here, we propose that the increased expression of Ch25h by microvascular endothelial cells during EAE results in increased levels of 25-OHC and decreased levels of PGE2 which restrain PMN-

Rebuttal Figure 6. Proliferation assay of CD4 + T cells co-cultured Live cells CD45 + CD11b + Ly6G + Ly6C int sorted from the bone marrow of non-immunized (NI) and EAE mice. (A)
Impact of BM-PMN on CD4 T cell proliferation (anti CD3/CD28) assessed by CFSE dilution using flow cytometry. PMN-MDSC were FACS-sorted from the BM of WT mice in non-immunized (NI) mice and at the peak of EAE. NS= non stimulated. n= 6 biological replicates/group. ns= non significant, * : p<0.05. p values were determined by two tailed unpaired t-test (B) Illustrative images of CFSE analysis.
MDSC expansion at the level of their extravasation site within the CNS which are microvascular endothelial cells and thus not be modelled by systemic injections of PGE2. This is again, consistent, with mode of action of 25-OHC which is believed to be autocrine and paracrine.

Minor points:
1. It would be interesting to understand where the neutrophils accumulate inside the CNS. Are they mainly located in subarachnoid/meningeal/perivascular spaces or do they infiltrate the brain parenchyma?
Single cell RNA analysis of CNS myeloid cells during EAE revealed the presence neutrophils in perivascular space, parenchyma, leptomeninges and choroid plexus (Jordao et al, 2019). Knier et al., have also identified that Ly6G + cells interact with B cells in the leptomeninges (Knier et al., 2018). We performed IHC on CNS tissues section of mice during EAE. Briefly, after deparaffination, antigen retrieval was performed using a Pascal Citrate buffer at pH 6.0. To inactivate endogenous peroxidases, slides were incubated with Dako REAL peroxidase-blocking solution (Dako, K0672) and subsequently blocked with Fab Fragment Goat anti-mouse IgG (Jackson ImmunoResearch, 115-007-003) and 2.5% Goat Serum in PBS (Vector Laboratories, S-100). Tissue sections were stained with a rat anti-Ly6G antibody (Biolegend, 127622) diluted in Dako REAL antibody diluent (Dako, S2022) at RT, 1 hour. To visualize the specific signal, anti-rat HRP (Vector Laboratories, MP-7444) together with TSA Vivid 650 (TOCRIS, 7536) was used as secondary antibody and amplification system. After washing, slides were incubated goat serum to avoid subsequent unspecific binding. To perform the vessels staining, sections were incubated with a rabbit anti-van Willebrand Factor antibody (Abcam, ab6994). Conjugated secondary antibodies corresponding to the species was used to visualize specific staining. Nuclei were stained with DAPI (Invitrogen, D1306). Slides were mounted with Fluoromount aqueous mounting medium (Sigma-Aldrich, F4680). For image acquisition, slides were scanned with the Pannoramic 250 FLASH II (3DHISTECH) Digital Slide Scanner at 20× magnification. We identified Ly6G + cells in the perivascular space (Rebuttal Fig. 8a), leptomeninges (Rebuttal Fig. 8b), and the parenchyma (Rebuttal Fig. 8c).

The authors should report counts instead of % of live cells as the latter can be misleading. See Fig 4f, 6d-f.
We agree that this point was unclear in the initial submission. One of our main finding is increased percentage of PMN-MDSC relative to total live cells and decreased percentage of proliferating CD4 + T cells in the CNS of Ch25h ECKO mice compared with Ch25h fl/fl mice during EAE. We did not observe significant differences in the abundance of these two populations using absolute numbers as readout (Rebuttal Fig. A, B and new Fig. 4). However, we observed an increased variance of both live cells CD45 + CD4 + CD44 + Ki67 + and live cells CD45 + CD11b + Ly6G + Ly6C int (PMN-MDSC) in Ch25h ECKO mice compared with Ch25h fl/fl mice (F test, P values = 0.0053 and 0.0236 respectively). Furthermore, the ratio PMN-MDSC/proliferating CD4 + T cells was increased while the ratio CD11b/proliferating CD4 + T cell is unchanged (Rebuttal Fig. 9C and D). These data suggests a remodeling of CNS infiltrating leukocytes in Ch25h ECKO mice in favor of PMN-MDSC expansion. Furthermore, results shown in new Fig. 4 and the additional experiments performed to address the major point 2 of referee 2 and major point 1b of referee 3 increase our confidence that CD45 + CD11b + Ly6G + Ly6C int suppress CD4 + T cell proliferation. Moreover, expression of ARG1 (Rebuttal Fig.11 and new Fig. 4E) is reminiscent of pro-regenerative neutrophils (Sas et al, 2020) described in a mouse model of optic nerve injury.
Therefore, we propose, that PMN-MDSC expansion relative to other immune cell populations contributes to attenuated EAE and that the phenotype that we observe is explained by a qualitative rather than a quantitative change in leukocyte infiltrates. We suggest that a relative increase in the fraction of cells displaying immunosuppressive and potentially wound healing functions is sufficient to attenuate EAE clinical scores. However, we do not exclude that this phenomenon could be transient, as in some experiments Ch25h ECKO mice displayed a delayed EAE disease course kinetic (Fig.  5A). In summary, we consider that absolute numbers of infiltrating leukocytes might not be the most appropriate readout in our settings. For these reasons, we are using the percentages as our main readout throughout the paper. However, we added absolute numbers in new Fig. 4H and a discussion regarding these relative changes in our manuscript.

The GBM data is interesting but is mostly correlative and not backed up by any experimental data in for example a mouse model. Taking this into account, it is my opinon that the authors should revise their discussion on the GBM data accordingly.
We have now removed the GBM data from the manuscript.

For figure 3 it would be helpful to guide the reader by including a schematic figure of cholesterol metabolism including enzymes and metabolite steps.
We have now added a schematic figure of cholesterol metabolism (Rebuttal Figure 10) in the new Figure EV1.

also infiltration of T cells and of monocytes should be reduced. Hence, the higher percentage of MDSCs in the CNS in EAE in these mice might only be relative. Therefore, it is necessary to determine the absolute cell numbers and to show them next to the percentages. This accounts also for data showing T cell-infiltrates.
We propose that the expansion of PMN-MDSC is relative to the other leukocyte populations. We now show the absolute numbers in new Fig. 4H and discussed our reasoning in the manuscript. Please refer to Referee 2 minor point 2 for the discussion.
b) MDSCs in the CNS: As also discussed by the authors, it is difficult to distinguish PMN-MDSCs from normal PMNs in the mouse and an alternative scenario of the effectiveness of the PMN depletion might be their pathogenic effect in EAE as claimed by several others previously combined with a lack of depletion of PMNs in the CNS. To be sure that the CNS PMNs are indeed MDSCs, one should perform an Arginase 1 staining (e.g. intracellular with clone A1exf5) and compare this to PMNs in the blood, spleen and BM.
We thank the referee for this advice. We performed additional EAE experiment and show that, in agreement with the data obtained from the suppression assays for the major point 2 of referee 2, Arg1 expression is restricted to CNS Live cells CD45 + CD11b + Ly6C int Ly6G + during EAE (Rebuttal Fig. 11 and new Fig. 4E and F). Therefore, we propose that CD45 + CD11b + Ly6C int Ly6G + acquire their suppressive properties in the CNS in our model, as already suggested (Knier et al., 2018). Those results are now included in new Fig. 4 in addition to the suppression assay.  Figure 4f). This should at least clarify whether 25-OHC/PGE2 have a more systemic or local impact at the CNS.
We showed in former Figure S5B, the percentage of Live cells, CD45 + CD11b + Ly6C int Ly6G + (neutrophils) in the blood of Ch25h fl/fl and Ch25h ECKO in isotype and combo treated mice. We did not observe differences in the percentage of neutrophils in the blood of the isotype treated mice suggesting that Rebuttal Figure 11. ARG1 expression in Live cells CD45 + CD11b + Ly6C int Ly6G + in the bone marrow, blood, spleen and CNS during EAE. A) representative contour plots of ARG1 staining in the bone marrow (BM), blood, spleen and CNS assessed by flow cytometry and gated of live cells CD45 + CD11b + Ly6C int Ly6G + .B) as in a except that quantitative analysis is shown. n= 11 biological replicates. ns= non significant, * : p<0.05, ***: p<0.0005 ****: p<0.00005. p values were determined by Kruskal-Wallis test with Dunn's post test.
the expansion of CD45 + CD11b + Ly6G + Ly6C int cells is CNS restricted. This result is now discussed in the new manuscript and included in new Fig. 5E  We thank the reviewer for this important question. We repeated the experiment in Ch25h fl/fl and Ch25h BBKO and showed that Ch25h BBKO mice phenocopied Ch25h ECKO for both EAE attenuation and CNS PMN-MDSC expansion (Rebuttal Fig. 12A and B and new Fig. EV2). We now show this result in the manuscript together with the corresponding disease course.

Since EAE is mediated by T helper cells, an analysis of CNS-infiltrating T cells is lacking in respect to
Th17/Th1/ThGMCSF/Tregs. These should also be analyzed in spleen/dLNs and blood. All in % and in absolute numbers.
We now evaluated different subsets of T cells both in percentage and absolute numbers of Th17 (CD4 + CD44 + RORgt + IL-17 + ), Th1 (CD4 + CD44 + Tbet + IFN- + ), ThGMCSF (CD4 + CD44 + GM-CSF + ), Tregs (CD4 + CD44 + Foxp3 + ) in the CNS, blood, LN and spleen at the peak of EAE disease. While we observed different percentages and absolute numbers of different T cell subsets between organs, we did not observe significant alteration in the CD4 + T cell subsets within the different organs analysed in Ch25h ECKO mice compared with Ch25h fl/fl mice (Rebuttal Fig. 19). Overall, those results are in accordance with the data from major point 2 of Referee 2. This suggest that the impact of PMN-MDSC expansion in our model does not target a specific CD4 + T cell subset. However, we do not exclude that, FACS analysis performed at different time points of EAE, especially at early time points of the disease, could differ.   To answer the question of the referee, we now measured oxysterols from spinal cords collected from Ch25h fl/fl and Ch25h ECKO /Ch25h BBKO female mice during EAE (16 days after immunization). We observed a significant reduced production of both 25-OHC and 7-keto-25OHC in the mice lacking Ch25h in ECs (Ch25h ECKO /Ch25hB BBKO ) compared to the controls (Ch25h fl/fl ) (rebuttal Figure 14 left and middle panel and new Figure EV1). 7,25-OHC was not detected. 24(S)-OHC levels, the main oxysterol of the CNS, was higher than 25-OHC and 7-keto-OHC but did not differ between the two mouse groups (Rebuttal Fig. 14 and new Fig. 3D). As Ch25h remains expressed in other cell types than ECs and as our mouse model is a Knockdown and not a full KO, we did not expect a higher decrease in 25-OHC and 7-keto-25-OHC levels. This is in accordance with the results shown in Rebuttal Fig. 5 and Rebuttal Fig. 15 b showing Ch25h expression in microglia. We thank the referee for this important question. We have now performed the experiment (rebuttal Fig. 15) as suggested with a FACS staining for Ch25h-eGFP on leukocytes extracted with a different protocol than for ECs to properly assess microglia. Briefly, CNS tissue was digested in a DMEM containing collagenase D (2.5 mg/ml Sigma) and Dnase 1 (1 mg/ml Sigma) to give a single-cell suspension. Of note, for ECs, brain enzymatic digestion is performed with Collagenase/Dispase (2mg/ml), DNAse I (10µg/ml) and Nα-Tosyl-L-Lysin-chlormethyl keton hydrochlorid (TLCK, 0.147µg/ml). Using this extraction protocol, we detected a Ch25h-eGFP signal in microglia in the CNS of non-immunized (NI) mice at low levels with an almost significant increase of Ch25h expression in the CNS of EAE mice (Rebuttal Fig. 15). We have now adapted the text of our manuscript accordingly, included those new results in Appendix Fig. S1 and discussed them in the manuscript on page 5 -lines: 132-137. For a detailed discussion of that specific point, please refer to referee 2 major point 1. The reverse gating strategy is a very good suggestion. However the necessity to use two different extraction methods (for the leukocytes and the ECs) impairs the comparison of the different cell type. Indeed, in the extraction for the leukocytes, ECs are not detected as they necessitate specific digestion protocols (collagenase/dispase) to be released from their entrapment in the basement membrane. We however performed an RNA-scope and additional FACS analysis that confirm and compare the expression of Ch25h in ECs and also in microglia (Rebuttal Fig. 5 and new Fig. 1A and B).

Rebuttal
Minor Points: 5. Although very interesting, the data shown on GBM ECs is rather confounding the overall story and not necessary for the main points to make. I would suggest to omit this and to put this rather in another small publication.
We have now removed the GBM data from the manuscript as it was proposed by 2 referees.

Some histology of infiltration and demyelination of controls versus CH25hEC-KO in EAE would be nice to have to see whether there might be also other qualitative changes.
We performed H&E staining in Ch25h ECKO mice and Ch25h fl/fl mice during EAE and found a significant reduced number of lesions Ch25h ECKO compared to Ch25h fl/fl (rebuttal Fig. 16 and new Fig. EV2).
Rebuttal Figure 15: Analysis of Ch25h-eGFP expression in microglia. A) Gating strategy for flow cytometry analysis of Ch25h-eGFP expression in TER119 -CD45 int CD11b int microglial cells. B) Flow cytometry analysis of Ch25h-eGFP expression in TER119 -CD45 + CD11b int microglia in non-immunized (NI) Ch25h fl/fl mice (n=3 biological replicates) and at day 17 post-immunization (EAE, n= 6 biological replicates). P value was determined by unpaired Student's t test. C) Representative FACS plot of Ch25h-eGFP in NI and EAE Ch25h fl/fl mice.

For EAE experiments it would be nice to see for each group shown the exact N number of the mice directly in the figure.
We have now included the number of mice for each EAE graphs on the Figures.

Mean max score: Was this calculated only of diseased mice?
Mean and max scores are calculated for all the EAE mice.

Figure 2e seems to show the averaged data of 3 measurements for each condition. I would suggest to show them individually.
We have modified Fig. 2E accordingly and are now showing the z-score of CPM for each replicate.

Figure 4c shows that PMN-MDSCs can inhibit T cell proliferation. A control showing the specificity of these cells by using PMN from BM would strengthen that point.
We have now performed additional experiment and have included a suppressive test with PMN-MDSCs from the BM showing that they are not able to inhibit T cell proliferation (Rebuttal Fig. 5 and new Fig. 4). See referee 2, question 2. Fig. 4d Correlation should be rather -0.68, what do the ** mean here?

11.
The former figure 4D now 4F has been modified accordingly.
12. Figure 6a: There seems to be at least some impact of the combo treatment also in controls. Figure 16. Histopathological A) quantifications and B) representatives staining of spinal cord sections of immunized Ch25h fl/fl mice (n=7) and Ch25h ECKO (n=10) at day 21 post-immunization for cellular infiltration (H&E). Five sections per mouse were quantified. Scale bars 200 µm (top panels), 100 µm (bottom panels). P values were determined by unpaired Student's t test This is correct, there is a trend towards a protective impact of the combo treatment in controls that did however not reach statistical significance in our setting. Figure 6e and f the comparison of controls vs. CH25hEC-KO in EAE should also be shown.

In
We have now modified the new Fig. 5E and 5F. 14. For Figure 6f one should rather show bars than lines since the loss of PMNs occurs most likely only on , day 14 and not before.
We now show the results on bar graph and not with lines in new Fig. 5F.

Figure 6f right: axes labels were forgotten
This has been corrected in new Figure 5.

Page 16 mentions deletion of Ch25h in GBM ECs although it is rather likely "not expressed" what was meant.
We have now removed the section on GBM as proposed by referees 1 and 2.
17. Although in general well written the grammar needs to be checked.
The grammar has been checked. Thank you for the submission of your revised manuscript. We have now received the enclosed reports from the referees. Referee 1 still has a few minor suggestions that I would like you to address and incorporate before we can proceed with the official acceptance of your manuscript.
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I would like to suggest some minor changes to the title and abstract. Please let me know whether you agree with the following (and please clarify my questions): Endothelial-derived oxysterols inhibit myeloid-derived suppressor cell expansion at the blood-brain barrier or Endothelial cell-derived oxysterol ablation attenuates experimental autoimmune encephalomyelitis The vasculature is a key regulator of leukocyte trafficking into the central nervous system (CNS) during inflammatory diseases including multiple sclerosis (MS). However, the impact of endothelial-derived factors on CNS immune responses remains unknown. Bioactive lipids, in particular oxysterols downstream [downstream of? or: products of?] Cholesterol-25-hydroxylase (Ch25h), promote neuroinflammation but their functions in the CNS are not well understood. Using a floxed-reporter Ch25h knock-in mice, we trace Ch25h expression to CNS endothelial cells (ECs) and myeloid cells and demonstrate that Ch25h ablation specifically from ECs attenuates experimental autoimmune encephalomyelitis (EAE). Mechanistically, inflamed Ch25h-deficient CNS ECs display altered lipid metabolism favoring polymorphonuclear myeloidderived suppressor cell (PMN-MDSC) expansion, which suppresses encephalitogenic T lymphocyte proliferation. Additionally, endothelial Ch25h-deficiency combined with the mobilization of immature neutrophils in the circulation [??] nearly completely protects mice from EAE. Our findings reveal a central role for CNS endothelial Ch25h in promoting neuroinflammation by inhibiting the expansion of immunosuppressive myeloid cell populations.
factors on other aspects of CNS immune responses remains unknown. Bioactive lipids, in particular oxysterols downstream of the enzyme Cholesterol-25-hydroxylase (Ch25h), promote neuroinflammation. Nevertheless their CNS functions are largely unravelled. Using a floxed-reporter Ch25h knock-in mice, we traced Ch25h expression to CNS endothelial cells (ECs) and myeloid cells and demonstrated that Ch25hspecific ablation in ECs attenuates experimental autoimmune encephalomyelitis (EAE). Mechanistically, inflamed Ch25h-deficient CNS ECs displayed altered lipid metabolism favoring polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) expansion that suppresses encephalitogenic T lymphocyte proliferation. Additionally, endothelial Ch25h-deficiency combined with immature neutrophils mobilization into the blood circulation resulted in nearly complete EAE protection. Our findings reveal a central role for CNS endothelial Ch25h in promoting neuroinflammation by regulating the expansion of immunosuppressive myeloid cell populations." 5th Jan 2023 2nd Revision -Editorial Decision Prof. Caroline Pot Lausanne University Hospital Switzerland Dear Prof. Pot, I am very pleased to accept your manuscript for publication in the next available issue of EMBO reports. Thank you for your contribution to our journal.
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