Peer review of: Mesenchymal stem cells offer a drug-tolerant and immune-privileged niche to (2019)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
Summary: This paper investigates mechanisms by which mesenchymal stem cells protect M.tb bacilli 
from antibiotics (transporter pumps) and immune responses (variety of different/unusual responses to 
proinflammatory cytokines that traditionally activate macrophages but have different effects on 
MSCs). The research tools were primarily in vitro using human adipose-derived stem cells with 
additional supporting information from mouse studies and tissues from TB patients. The overall 
findings will be of substantial interest to the TB research and clinical community, as the results provide 
insight and a direction to develop and screen/test therapies that may eliminate M.tb from this cell 
population. Statistical analyses were primarily 2-tailed student's t-tests (which seems OK) but possibly 
the authors should consider if other tests are appropriate as well, for example ANOVAs? 
 
Major Critiques: This is a solid paper with experimental evidence supporting the conclusions. My main 
critique is whether experimental results were reproducible in Fig 3 and 4. Please report how many 
times the experiments were performed. As written, it seems like experiments were performed once 
with 1 lot of cells throughout or 1 set of animals. Twice would be minimum, and three times better. If 
experiments were performed once, please repeat the most critical set. 
 
Minor Critiques: 
1. Recommend an editor to find and correct the minor language, grammar mistakes. A few word 
choices are non-standard. 
 
2. Double check effect versus affect. 
 
3. I may have missed it in the methods. Please clarify how you defined, identified and calculated 
"tolerance" on the Y-axes of many graphs. 
 
4. Suggestions for data presentations: For the mouse studies - consider putting CFUs on a linear 
scale. I know this is unusual but changing the scale will better show the differences that are lost on a 
Log10 transformation. For the IHCs and photomicrographs, consider higher magnification. It is too 
hard to see what the arrows are pointing to. 
 
5. Considering adding to Intro or discussion additional reference on MSCs by last author Campos-Neto 
in AJP. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
In the current study Jain et al. address the Mtb infection of mesenchymal stem cells (MSC) in vitro and 
in vivo. As MSCs have only recently identified and been proposed to play an important role in during 
infection. The detailed functional role of MSC in the context of infection however is not known. Thus 
the authors address a highly relevant issue very likely having an impact on TB vaccinaton and also TB 
therapy strategies. 
 
The authors present a very thorough and very detailed analysis of the infection of MSC by Mtb in vitro 
and in vivo. The data are very convincing and the experiments are well controlled. The paper is well 
written and provides novel and thought provoking mechanistic insights into the role of MSC in the Mtb 
infected host. However a few things should be carefully considered, as the authors state in their 

discussion the limited reliance of ex vivo data. Said that the authors should readdress the histological 
findings in TB patients. 
 
• Fig4 H: The IHC data provided in Figure 4H look specific. However the respective controls should be 
provided by the authors in the supplementary part. 
• Fig4 I: In contrast to Fig4H, Fig4I is not convincing. First larger orange arrows lie on top of smaller 
arrows, if you zoom into the picture. This is misleading and should be avoided. Irrespective of the 
arrows the CD73 positive cells cannot be identified with the blue counterstaining within the pictures of 
the tissue sections provided. This needs to readdressed in detail. 
• The authors do state in their text that Ag85 and CD73 are “in close vicinity” but this is in fact several 
cell diameters away. It appears that in contrast to their Bl6 mouse data, the authors do not see CD73 
cells harbouring Mtb bacteria in human TB lesions. The apparent key question is: Do MSC in the 
human TB patient harbour Mtb bacteria as the mouse data suggest, or does that differ. In order to 
address this the authors may consider e.g. a version of a proximity ligation assays (e.g. CD73 vs 
Ag85) to demonstrate the presence of Mtb within MSC or may use an independent staining protocol, 
where the signals can be undoubtedly visualized and clearly separated from each other. Another way 
to address this may be the use of a Ziehl-Neelsen-stain followed by IHC for CD73. 
 
• The wording used by the authors should be more cautious in some instances. The authors should 
refrain from exaggerating and overstating their findings: e.g. l.121: please use "(strongly) suggest" or 
similar instead of "evident", or l.144: "reduced" instead of "wiped out" because 2-4% are still left. As 
mentioned above (l.341), the authors do state that Ag85 and CD73 are “in close vicinity” but this is in 
fact several cell diameters away. However this point needs to be addressed in further experiments 
anyway. Please check the whole manuscript for these issues, since a more precise wording would 
improves the quality of the manuscript. 
 
 
 
Reviewer #3: 
Remarks to the Author: 
Comments to the Author 
 
In the manuscript,” Mesenchymal stem cells (MSCs) offer a drug-tolerant and immune 
privileged niche to Mycobacterium tuberculosis” the authors present a study where they show that 
MSC provide a favorable environment for Mtb to reside and becomes more drug resistant. They 
hypothesize that there is a role for ABC transporters in drug tolerance and they find that lysosomal 
function rather than efflux activity is responsible to achieve the drug-tolerant phenotype. Authors also 
show a pro bacterial roles for inflammatory cytokines and lipid mediator PGE2 in MSC. 
The authors showed the novel ability of MSCs to provide a niche for Mtb to survive in harsh conditions. 
 
The manuscript is overall interesting, however, the data needs improvement before publication. 
 
Introduction: 
The introduction is nicely written, but authors could provide a little information on why they have used 
and adipose-derived stem cells specifically. 
 
General comments: 
All supplementary figures lack statistical analysis 
 
Specific comments 
Results: 

1st part: Adipose-derived Mesenchymal stem cells (ADSCs) support mycobacterial 
82" growth 
• Authors have characterized ADSCs by using just two markers CD73 and CD271 markers. However, 
CD73 marker is not specific for ADSCs, it is also present in lymphocytes and many of the cancerous 
cell lines. It would be useful to use additional markers like CD90, CD44 or CD105 along with a dump 
channel marker like CD11b. 
• Also, in figure S1A, authors have shown two separate histograms for CD73 and CD271, I would 
suggest showing a bivariate plot and the gating strategy. 
• For characterization of differentiation of ADSCs into chondrocytes, adipocytes, and osteocytes, 
authors could also perform a flow cytometry along with staining to confirm the results. 
• Line 88, fig 1B, authors shown only day 6. Other time points showing the kinetics of growth would 
be really informative showing MFI ranging from lowest to highest. It will provide readers a better idea 
to whether the bacteria are multiplying inside or not. 
• In fig 1C-F, please convert the CFUs to log scale and make the same scale for all plots. Or at least 
use the same scale so it is easier to visualize. Additionally, please show CFUS up to day 12 for all 
conditions and infections. This will make it easier to compare the four graphs. 
• Fig S1C, it’s the same as point 2, authors can do an additional flow cytometry or any other analysis 
to confirm that ADSCs do not differentiate after H37Rv infection. Also, the authors have not 
mentioned what is the right lane in that image. They could write legend on the top. 
2nd part: ADSC resident Mycobacterium tuberculosis shows drug-tolerant phenotype 
• Why authors have selected day 9 to plate CFUs? It would be good to show several timepoints and 
explain why they chose 9 days based on results. 
• Authors have compared the treatment of INH and Rif on ADSCs with THP-1. I understand that 
ADSCs used here are primary cells and THP-1 is a monocytic cell line. The comparison would be more 
realistic they use primary macrophages (minor). 
• Fig 1H, 5 ug.ml INH has the least CFUs, where are the plots for that concentration. 
 
3rd part: Host ABC transporters ABCC1 and ABCG2 play a key role in bacterial drug tolerance 
 
• Microarray data could be presented in a way where authors can highlight and show upregulation and 
downregulation of different genes. And they can point out the specific ones. 
• Fig 1J, I am not convinced of the upregulations of ABCC1 and ABCG2 based on those histograms, 
maybe if they write down the percentage of increase in ABCC1 and ABCG2 receptors? It really does 
not look to be statistically significant. 
• Line 131, authors have not mentioned the timeline and dose of H37Rv infection, cell harvesting, and 
staining 
• Supplementary figure S2G and H, Please show the same scale in y-axis 
 
4th part: Role of lysosomal function in mycobacterial drug tolerance in ADSCs 
• Results still do not provide a valid justification of fig 1, why non-drug resistant Mtb is increasing 
after inhibition of ABCC1 and ABCG2. A better explanation of why BafA1, 3MA, and Chloroquine 
inhibition increase CFUs of non-drug resistant Mtb. It is still confusing. 
5th part: Effect of inflammatory cytokines IFNγ and TNFα on drug tolerance within ADSCs 
 
• Line 175, Could you please provide more references for this statement? 
• Line 180, Is it figure 2D? 
• Fig 2E and 2F, why concentration and units of measurement are different? If this is the case, then 
we cannot compare between these two (Line 184). 
• Line 193-194, How can we say that Mtb is rescued from cytokine-mediated killing? 
6th part: Analysis of intracellular niches of Mtb shows classic phagosome maturation dynamics in 
ADSCs 

 
• Fig S3A, please mention the scale and magnification. 
• Fig 2K, Line 215-216, It is mentioned that BCG is present mainly in Lysotracker-LAMP1 double 
positive compartment, but from the figure, it seems like the highest percentage of BCG is in 
Lysotracker compartment. 
• Line 223, Fig S2E, For ABCC1, Mtb seems to colocalize but I am not convinced with ABCG2. 
• Line 226-227, How it is giving an impression that ABCC1 is directly involved in killing? 
7th part: The lipid mediator PGE2 helps MSCs exhibit pro-bacterial attributes 
• Figure S5C, actually shows the opposite of what the authors mention in 272. Celecoxib reduces CFUs 
in THP1 30% while only 10-15% in MSCs 
8th part: MSCs serve as a niche for Mtb during in vivo infection allowing drug tolerance in PGE2 
dependent manner 
 
• Please show the group with the aerosol challenge (control) 
• When INH+ Celecoxib were given in combination, where the doses of these two 50 and 10mg/kg 
respectively? 
• Fig 4A, line 300-301, it is not clear from the figure that there are differences in CFUs, maybe if 
authors present the figure in log scale, things could be clear. 
• Plot fig 4A and 4B please show the figures with the scale same. 
• Fig 4C, I am not convinced with the markers used for characterizing MSCs and macrophages 
populations. I would suggest using more markers like CD44+, CD90+, CD105+ and CD45- and 
CD11B- for MSCs and Ly6C+ or F4/80+, CD11C-, Ly6G- for macrophages. 
• Line 136, Fig 4F, how did you count cells? 
• Fig 4G, please explain why CFU can decrease at week 8 and then increase again in case of the group 
treated with celecoxib? 
 
Materials and methods: 
• Reagents: Please provide all the necessary information about the antibodies like the fluorophore 
attached, clone etc. Also, few of the antibodies have not been written, please add all. 
• Please explain where did the H37Rv came from or how it was transfected 
• Please mention the strain of BCG used in this study. 
• Why authors have used ADC and not OADC? 
• Line 530, Why 4 hours for macrophage and 12 hours for ADSCs? 
• At what time were plates counted? 
• Which version of FlowJo was used? 
• How many reads were acquired in Illumina? 
• Please mention how mice were euthanized. 
• Tissue processing, it seems like authors have processed lung and spleen in the same way, which is 
probably not the case. Please write the protocol clearly 
• Please mention the dilution used for anti-rabbit IgG antibody (Line 634). 
 
 
Marcela Henao-Tamayo