Peer review of: Alterations of gut microbiota contribute to the progression of unruptured intracranial (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
A recent study has proposed an association between alterations in gut microbioal composition and 
the incidence of intracranial aneurysms (IAs). This is analgous to studies showing an association 
between alterations in gut microbioal composition and the incidence of intracranial cavernous 
malformations. In the present study, the authors performed a metagenome-wide association study 
between 140 Chinese patients with UIAs and 140 controls. The authors note 17 species, including 
Parabacteroides sp. and Odoribacter splanchnicus,revealing microbial changes in the deteriorating 
gut microbiota of UIA patients. They identified UIA-associated species linked to changes in 
circulating amino acids,including taurine. By fecal transplantation from UIA patients and control 
donors into mice, they note that UIA gut microbiota contributed to the formation and rupture of 
intracranial aneurysms. Taurine supplementation partly reversed the progression of intracranial 
aneurysms. 
 
A recent study reported that depletion of the gut microbiota by antibiotics reduces the incidence of 
intracranial aneurysms in mice (1). Nevertheless, direct evidence for altered gut microbial 
composition in UIA patients and the precise underlying mechanisms are still lacking. 
 
 
How did the authors come to the total number of human patients and mice? Were power analysis 
done before experiments? 
 
 
In the human cohort, total of 140 UIA patients and 140 age-, sex- and blood pressure-matched 
controls were collected from 2 cohorts. Were other factors similarly distributed between cases and 
controls such as smoking (2)? I believe they said that none of the patients had a familial history of 
IA? 
 
 
 
Interesting that a prior study report that guy bacteria may transmigrate to cerebral arteries and 
play a direct role in the pathogenesis of UIA, independent of a metabolite-based mechanism (3). 
However, the current authors did not detect bacterial DNA in the cerebral arteries in UIA patients 
or mice with UIAs. 
 
 
 
Why did the authors ultimately focus on Tuarine? They performed some many experiments with 
sequencing in humans and mice, but did not use this other than to show differential regulation. 
Ultimately they also performed metabolomics profiling of fatty acids and amino acids in mice after 
fecal transplantation. In total, the serum concentrations of 2 of 8 fatty acids and 8 of 38 amino 
acids differed substantially between mice transplanted with feces from UIA patients and controls 
(Supplementary Table 12). Specifically, among these altered metabolites, taurine, L-histidine, and 
linoleic acid also corroborated the lower abundance in UIA patients than in controls. Did the 
authors assess L-histidine or LA for their effects on aneurysms? Did they assess alterations in 
taurine in human UIA patients. This would make for a much stronger argument and the potential 
that taurine supplementation may have an impact in human UIA patients. 
 
 
Is there any way to downregulate taurine to see if it leads to an increase incidence of aneurysm 
formation, progression, rupture? 
 
 

The authors found that taurine altered macrophages, neurophils, TNF, IL, MMPs. These have all 
been shown to have an impact on aneurysm formation and rupture in vivo via genetic inactivation 
or pharmacological inhibition. TNF and MMPs have also been found to be upregulated in human 
aneurysms. These sources could be further referenced to provide support for their mechanistic 
studies (2, 4-9). 
 
 
At an elastase dose of 35 mU no mice died either following surgery or from aneurysm rupture? 
 
 
I must have missed it somewhere in the supplement but when were samples collected in patients 
with UIA in relation to treatment of their UIAs? How were controls selected? 
 
REFERENCES 
 
1. Shikata, F. et al. Potential Influences of Gut Microbiota on the Formation of 13 Intracranial 
Aneurysm. Hypertension 73, 491-496 (2019). 
2. Starke, RM et al. Cigarette Smoke Initiates Oxidative Stress-Induced Cellular Phenotypic 
Modulation Leading to Cerebral Aneurysm Pathogenesis. Arterioscler Thromb Vasc Biol. 2018 
Mar;38(3):610-621. 
3. Pyysalo, M. J. et al. Bacterial DNA findings in ruptured and unruptured 30 intracranial 
aneurysms. Acta Odontol. Scand. 74, 315-320 (2016). 
4. Starke RM et al. Critical role of TNF-α in cerebral aneurysm formation and progression to 
rupture. J Neuroinflammation. 2014 Apr 16;11:77 
5. Kanematsu. Critical roles of macrophages in the formation of intracranial aneurysm. Stroke. 
2011 Jan;42(1):173-8 
6. Ali et al. TNF-α induces phenotypic modulation in cerebral vascular smooth muscle cells: 
implications for cerebral aneurysm pathology. 
J Cereb Blood Flow Metab. 2013 Oct;33(10):1564-73. 
7. Hasan et al. Macrophage imbalance (M1 vs. M2) and upregulation of mast cells in wall of 
ruptured human cerebral aneurysms: preliminary results. J Neuroinflammation. 2012 Sep 
21;9:222. 
8. Jayaraman et al. TNF-alpha-mediated inflammation in cerebral aneurysms: a potential link to 
growth and rupture. Vasc Health Risk Manag. 2008;4(4):805-17. 
9. Kim et al. Matrix metalloproteinase-9 in cerebral aneurysms. Neurosurgery. 1997 
Sep;41(3):642-66; 
 
 
 
Reviewer #2: 
Remarks to the Author: 
Li et al. performed metagenome-wide association analysis on unruptured intracranial aneurysms 
(UIA), demonstrated causal role of the gut microbiome by transplanting UIA feces to mice and also 
shown that Taurine supplementation could partly reverse the progression of UIA. This is a 
comprehensive study, as authors have tried to: 1) identify microbial difference between UIA and 
controls; 2) establish the link between UIA microbes to circulating levels of fatty acids and amino 
acids; 3) prove causality via faecal transplantion; 4) investigate the impact of UIA microbiome on 
transcriptional profile of cerebral vessels; and 5) the impact of Taurine supplement on the 
progression of UIA. The findings are relevant to the field. However, I have several concerns: 
 
The authors qualified and quantified taxa and KEGG pathways based on MLGs (metagenomic 
linkage groups). The biological interpretation of these MLGs is difficult as they are based on co-
abundance clustering. Nowadays, MetaPhlan and HUMAnN2 are two commonly used tools for taxa 
and functional assignments based on metagenomics sequencing data. Why did authors still choose 
to use MLG? Author should also compare their taxa and pathway assignments to the results of 

MetaPhlan and HUMAnN2. 
 
Authors did not observe significant difference in a-diversity at the genus level. How about the a-
diversity at the species level? 
 
The fecal transplantation experiment is nice as it demonstrated the impact of gut microbiome on 
IA. However, it is not clear how these donors (2 UIA and 2 controls) were selected? To what extent 
they represented the identified UIA microbial composition. The comparison of 4 genera in Sup Fig 
6 is not informative. Why were only 4 genera detected? To my surprise, the genera abundances 
between two UIA donors were very different. The same was observed between two control donors. 
For instance: the abundance of Bacteroides in UIA2 donor (80%) was nearly 2 times higher than 
that in UIA1 donor (40%). Were mice single-housed? The authors need to add the link between 
donors and receivers and perform statistical analysis to demonstrate that mice receivers did have 
more similar microbial profiles as the human donors. 
 
Which microbial difference in fecal transplanted mice could explain the observed difference in IA 
phenotype? Were they in line with the associated microbial features identified in metagenome 
association in human? 
 
It is difficult to interpret the co-abundance network analysis. It is unclear how this part can aid a 
better understanding on the microbial difference between UIAs and controls. It is unclear how the 
spearman correlation coefficient 0.6 was chosen for network construction. Moreover, it is well 
known that spearman correlation is not an ideal method for co-abundance analysis on 
compositional data. Several tools, like SparCC has been proposed for this purpose. Comparison of 
Spearman to other network construction methods have been extensively assessed in 
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4918442/ 
 
 
Page 8-9 show patients can be classified based on 17 MLS. The cross-validation did not make any 
sense. These 17 MLS were selected as their associations were shared in both cohorts. Thus both 
cohorts have been used for discovery. The cross-validation using the same (or sub) cohorts cannot 
be considered as independent validation. There is an overfitting problem. 
 
The authors did not fill in the Report Summary accurately. For instance, graphic software 
(Graphpad, ImageJ and Adobe illustrators) was mentioned as data analysis software. 
 
Fig 1: Authors should rarefy the number of reads to assess the impact of sequencing depth. 
 
Minor: 
 
Sup Fig 9: L-histidine association in human and mice were not consistent. Were colors of mouse 
experiment swapped in figure? 
 
Page 6: subtitle “microbial strains associated with UIAs”. There is no strain analysis. 
 
Page 10: we investigated the microbe-host interactions in UIA patients. There was no interaction 
analysis. 
 
Page 22: patients and controls were enrolled through different hospitals and from different areas. 
Authors should also discuss other potential confounding effects. It is not true that Han population 
share similar dietary habits (page 23). 
 
Authors should provide accurate P values for all statistical analyses. 
 
 

 
Reviewer #3: 
Remarks to the Author: 
The authors present a novel body of work examining the fecal microbiome of individuals with 
intracranial aneurysms. Major strengths of the study include the second cohorts that validate 
findings, corroborating metabolomic analyses, an in vivo murine model demonstrating causality, 
and furthermore identification of a potentially translatable strategy that mediates a meaningful 
benefit in the pre-clinical model. 
 
From the methods, it seems that some of the control patients were enrolled at different sites from 
the cases - could this be a potential confounder? 
 
Figure 1E - PCA indicates that the authors utilized a Euclidean distance matrix to generate these 
plots. Typically, microbiome studies will instead utilize a Bray-curtis dissimilarity (already 
calculated in 1D) which is more suited for abundance data, or even more commonly a weighted 
UniFrac distance which incorporates abundance and phylogenetic information. The authors can also 
quantify if the samples cluster separately with statistical significance; commonly used tools 
included ANOSIM and ADONIS ("permutational manova"), which are available in the vegan R 
package. 
 
Figure 1F - are any of these significant? 
 
SFig 6 - a known limitation of human-into-mouse FMT experiments is that large prominent groups 
of human bacteria fail to take in mice, in particular Clostridia. Can the authors more clearly convey 
which bacteria did and did not take? SFig6A indicates that PC1 clearly distinguishes human 
samples from mouse samples. At what time point were mouse samples assayed? Did the authors 
monitor for stability of take of human bacteria? How much of the microbiome in these mice were 
residual mouse flora that returned after antibiotics? 
 
Are the authors able to identify which bacteria in the control subjects, and in the mice, are 
responsible for taurine production? Can they show that these bacteria are differentially abundance 
in the human cohorts and in the mice? 
 
Did the authors assay to see if their taurine intervention in the mice normalized serum levels? 
 
The authors could consider citing a recent study where taurine was found to be reduced in mice 
receiving FMT from autistic individuals, and taurine supplementation improved autistic-like 
behavior in mice. Is autism known to be associated with intracranial aneurysms?