The “6B” Strategy: Build Back a Better Blood–Brain Barrier
Round 1
Reviewer 1 Report
The manuscript by Calvier et al. presents a review highlighting the role of the compromised blood-brain barrier compartment during MS. The paper explains the infiltration of leukocytes into the CNS. This review explains further that the BBB offers a potential therapeutic approach emphasizing Reelin, a plasma protein. They further explain the regulation of Reelin leads to the modulation of expression of adhesion marker on the endothelial surface resulting in the influx of inflammatory mediators and cells causing inflammation. The paper is well written and can be accepted in the same
Minor:
Abstract
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Introduction
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To identify ------ Identifying
Human -----------humans
Author Response
REPLY: We thank the reviewer for her/his positive appreciation, and we have fixed the minor modifications as suggested.
Reviewer 2 Report
In this manuscript the authors have discussed the therapeutic properties of anti-Reelin in the context of MS. The commentary is an interesting read but raises some questions
1. Which cells express/ secrete Reelin?
2. What doses of Reelin are safe for prescription?
3. How does administration of Reelin help w building a better blood brain barrier, all its doing is preventing immune cells from infiltrating. Please re-write this part
4. What in vitro and in vivo systems were used to illustrate the beneficial effects of Reelin. Can the authors please elaborate on this?
5. Have there been any clinical studies to test the effects of Reelin? The authors just state GWAS studies which are at best correlations.
6. Small point- when the JC virus for the first time in the review, it is a good idea to expand the name of the virus.
7. The authors have discussed the pitfalls of using Natalizumab – how about the other existing drugs and what are the associated side effects? Can the authors draft a table listing the side effects? Figure 1 lists the other drugs but doesn’t point out the side effects.
Author Response
In this manuscript the authors have discussed the therapeutic properties of anti-Reelin in the context of MS. The commentary is an interesting read but raises some questions
- Which cells express/ secrete Reelin?
REPLY: We thank the reviewer for this important comment, and it is now discussed in the manuscript as followed:
The discrimination of Reelin’s functions in the CNS versus the circulation is a relatively new finding and some uncertainties remain, requiring additional studies. For example, the source of plasma Reelin expression is not well understood. Reelin is expressed in the CNS by the neurons, but it is unlikely that it leaks from there (or the cerebrospinal fluid) into the circulation in significant amounts. To confirm this, we have previously shown that we can deplete Reelin from plasma by injecting Reelinfl/fl mice through the tail vein with an adenovirus expressing Cre recombinase (Ad-Cre) 30. Immunoblot analysis demonstrated efficient and specific ablation of Reelin from plasma, but not from the brain. In the literature, the liver and especially the hepatic stellate cells are believed to be the main source of peripheral Reelin 30,37–40, but we challenge this paradigm as we haven’t observed an increased expression in hepatic Reelin mRNA expression matching plasma Reelin accumulation during EAE (data not shown). Therefore, plasma Reelin secretory cells during neuroinflammation remain to be determined.
- What doses of Reelin are safe for prescription?
REPLY: We believe that there is a misunderstanding as there is no recommendation for Reelin prescription, rather anti-reelin therapies (via antibodies or ASO). In mice, the amount of anti-Reelin antibody (= CR-50) injected i.p. was 100µg/mice 29.
- How does administration of Reelin help w building a better blood brain barrier, all its doing is preventing immune cells from infiltrating. Please re-write this part
REPLY: Here as well, we believe that there is a misunderstanding as Reelin was not administered in the studies cited in our review, but rather anti-Reelin antibody CR-50. As detailed in the section “Reelin inhibition protects the endothelial function and presents a clinical potential”, plasma Reelin promotes the expression of adhesion proteins on the endothelial cell surface, thus promoting adhesion and infiltration of leukocytes into the CNS. By depleting Reelin with an anti-Reelin antibody or using KO model, the expression of these adhesion proteins is reduced, thus decreasing the infiltration of leukocytes, neuroinflammation, and neurodegeneration.
- What in vitro and in vivo systems were used to illustrate the beneficial effects of Reelin. Can the authors please elaborate on this?
REPLY: We have clarified this important point in the section “Reelin inhibition protects the endothelial function and presents a clinical potential”. The characterization of the cellular pathway was performed in human aortic endothelial cells and the expression of target genes (adhesion proteins such as E-selectin and ICAM1) was confirmed in mouse aorta. The manuscript has been modified as followed:
“In human aortic endothelial cells, adhesion and permeability are largely regulated via NF-κB pathway activation26. NF-κB target genes include adhesion molecules such as E-selectin, ICAM-1 or VCAM-1, but also cytokines and chemokines. Interestingly, it has been demonstrated in human aortic endothelial cells that Reelin activates NF-κB and thereby controls the expression of its target genes, especially adhesion molecules27–29, via its membrane receptor apolipoprotein E receptor 2 (ApoER2)27 member of the LDLR family30. Accordingly, it has been shown that Reelin promotes leukocyte adhesion to endothelial cells and that Reelin blocking antibodies can prevent this adhesion, in vitro using an adhesion assay with human aortic endothelial cells and human monocyte U937 and in vivo by intravital microscopy on mesenteric vessels27–29.”
- Have there been any clinical studies to test the effects of Reelin? The authors just state GWAS studies which are at best correlations.
REPLY: This is an interesting question, unfortunately, no clinical studies have tested the effect of Reelin to date.
- Small point- when the JC virus for the first time in the review, it is a good idea to expand the name of the virus.
REPLY: We have expanded the full name: John Cunningham (JC)
- The authors have discussed the pitfalls of using Natalizumab – how about the other existing drugs and what are the associated side effects? Can the authors draft a table listing the side effects? Figure 1 lists the other drugs but doesn’t point out the side effects.
REPLY: We have focused on Natalizumab among all MS disease-modifying therapies because its target and mechanism are the most related to the anti-Reelin strategy. The other drugs are less relevant for leukocyte adhesion to endothelial cells and therefore are not detailed in this review. To take into consideration this comment, we now refer in the manuscript to two reviews on MS therapies which present in a table the side effects associated with these drugs, as suggested by the reviewer.
- Rommer, P. S. et al. Immunological Aspects of Approved MS Therapeutics. Front Immunol 10, 1564 (2019).
- Callegari, I., Derfuss, T. & Galli, E. Update on treatment in multiple sclerosis. La Presse Médicale 50, 104068 (2021).
Reviewer 3 Report
In the review titled, “The 6B strategy: Build back a better blood-brain barrier”, authors discuss the current therapeutic approaches to treat MS while suggesting a novel strategy which consists of using
anti-Reelin therapy.
Strengths-
· Authors discuss an important and under studied approach for treating MS.
· They shed light on some benchmark studies which demonstrate positive results surrounding anti-Reelin therapy and the significance of Reelin in MS disease pathogenesis.
Weakness-
· Review is very short.
· Authors could have described more studies and results to support their theory especially in humans.
· It’s hard to decipher where does the word “6B” come from? – they have used the word in the title, abstract and conclusion but haven’t really described the abbreviation. Does “B” stand for blood brain barrier? What is 6?
Author Response
In the review titled, “The 6B strategy: Build back a better blood-brain barrier”, authors discuss the current therapeutic approaches to treat MS while suggesting a novel strategy which consists of using anti-Reelin therapy.
Strengths-
- Authors discuss an important and under studied approach for treating MS.
- They shed light on some benchmark studies which demonstrate positive results surrounding anti-Reelin therapy and the significance of Reelin in MS disease pathogenesis.
REPLY: We thank the reviewer for her/his positive appreciation.
Weakness-
- Review is very short.
REPLY: We have extended the text as we addressed the comments from all the reviewers.
- Authors could have described more studies and results to support their theory especially in humans.
REPLY: We agree with the reviewer, and we have added the following paragraph accordingly:
This BBB breakdown hypothesis is supported by a substantial body of radiological and pathological clinical evidence. BBB leakage has been observed in MS patients by Gadolinium-enhancing lesions on MRI as well as by fibrinogen (a plasma protein) deposition in developing lesions19,20. On the cellular level, tight-junctions abnormalities are also seen in endothelial cells around active lesions, which is a hallmark of a permeable endothelium20. This results in increased infiltration of leukocytes into the CNS as demonstrated in postmortem MS brain20. Based on this new paradigm, restoring this blood-brain barrier would prevent leukocyte infiltration into the CNS without targeting and dampening the immune system.
- It’s hard to decipher where does the word “6B” come from? – they have used the word in the title, abstract and conclusion but haven’t really described the abbreviation. Does “B” stand for blood brain barrier? What is 6?
REPLY: We have clarified this acronym in the conclusion as followed: The “6B” strategy - an acronym for Building Back a Better Blood-Brain Barrier – that we are proposing here […]
Round 2
Reviewer 2 Report
The authors have satisfactorily answered my questions
Author Response
We thank the reviewer for her/his positive appreciation.
