Peer review of: Micro-environmental sensing by bone marrow stroma identifies IL-6 and TGFβ1 as (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

In this manuscript, Valletta et al. analyzed the stromal cells of the bone marrow to address the decline 
of erythropoiesis and lymphopoiesis upon ageing. They found upregulation of IL-6 and TGFb signaling 
induced gene expression in BM stromal cells of aged mice, and could subsequently demonstrate that 
both cytokines play an important, but distinct role in this process. The authors demonstrate HSC-
intrinsic and extrinsic mechanisms that drive hematopoietic decline during ageing, and associate the 
BM stroma in this process. The amount of work presented is substantial, and the fact that the 
involvement of not one, but two cytokines is investigated, provides extra value to this manuscript. 
This study is important for the field, as it helps us to understand how vital physiological processes 
deteriorate during ageing, and what could be done to counteract this. 
The manuscript is very well written, and the experiments are of a high technical level and well-
performed. Data is generally well displayed and conclusions are in line with the findings. Although 
most of the findings and claims are novel, it should be noted that the contribution of IL-6 to erythroid 
decline during ageing has been shown before in several studies (some of them also referenced by the 
authors; Refs 21-24). 
Eventhough not all of the observations are experimentally addressed and still remain unclear (eg. 
what is the contribution of the other inflammatory networks, beyond IL-6 and TGFb, that are also 
observed in BM stromal cells of aged mice?), it is obvious that gene expression analysis evokes new 
questions for future studies. Hence, I restrict my feedback to the questions that the authors 
experimentally addressed and the conclusions they drew from their experiments. 

Major comments: 
A. Based on the stromal cell analysis, the authors hypothesized that increased IL-6 signaling could 
underly (part of) the ageing phenotype. Indeed, based on blocking experiments with anti-IL-6 
antibodies (Figure 3), the authors conclude that IL-6 is an important driver of the age-mediated 
decline in erythropoiesis, which agrees with several other studies. However, it is not clear whether the 
authors now conclude that IL-6 affects erythropoiesis through the BM stroma or that it acts directly on 
the erythroid progenitor cells? It could well be that the observed IL-6 mediated changes in BM stroma 
are an epiphenomenon and not causally related to the erythroid decline, but this should be made clear, 
given the important focus in the manuscript on the stromal cells in the BM. 
B. The authors argue that TGFb acts independently of IL-6, and plays a different role in the ageing of 
HSCs, LSK and myelo-erythroid progenitor compartments; here, it is made likely that the stroma itself 
is actively contributing, as the authors found more TGF-b1 in ageing compared to young stroma. 
However, it has been well established that TGFb is a major determinant of HSC quiescence, which is 
neither referred to nor experimentally addressed here. It is conceivable that the effects observed with 
the TGFbR-inhibitor in Figures 4 and 5 are simply due to a loss of HSC quiescence, which subsequently 
resulted in enhanced/increased differentiation of the HSCs and downstream progenitors. It is 
important that the authors adequately address this possibility and, if necessary, adjust their 
conclusions accordingly. 

Minor comments: 
C. The authors show a lot of GSEA graphs, but it is unclear for the reader which genes are part of the 
indicated enrichment. For instance, in Figure 2h there is an increase in so-called “preGM-associated 
genes” in CFU-Es from old mice, but the identity of these genes is not revealed and it is also not clear 
which genes within these gene-sets are indeed higher or lower. It would be highly valuable if the 
GSEA analyses that are shown throughout this manuscript would be expanded by including the related 
genes in a supplemental figure or table. 
D. In figure 2, the authors describe that purified MPP2, preCFU-Es and CD71+ CFU-Es from aged mice 

all have decreased erythroid differentiation (Figure 2f). The prediction is that they all show a gain in 
myeloid differentiation, but this is only shown for the preCFU-E population in Figure 2i. It would be 
worth to also show the myeloid potential of the MPP2 population, as this would reveal how far up in 
the hematopoietic hierarchy the switch from erythroid to myeloid occurs. 
E. In Figure 3g&h, the authors show in a GSEA that the loss of preCFU-E genes upon ageing is 
prevented by depletion of IL-6. It is important to also show whether the enrichment of preGM genes 
upon ageing (Fig 2h) is also lost by the IL-6 depletion. This would denote whether IL-6 truly affects a 
shift in the balance between erythroid and myeloid commitment, or only affects erythroid 
differentiation. 
F. At the bottom of page 5, the authors refer to Suppl Fig 3e, but this does not exist and should be 
Suppl Fig 4a. 

Reviewer #2 (Remarks to the Author): 

In the manuscript, “Using bone marrow stromal cells as micro-environmental sensors identifies IL-6 
and TGFβ signaling as regulators of declining erythropoiesis and lymphopoiesis during hematopoietic 
ageing,” Valletta et al. utilized bone marrow (BM) stromal cells to determine consistent differences in 
cellular signaling across multiple cell types between young and aged BM and find enhanced IL-6 and 
TGF beta gene programs in aged BM. Next, hematopoietic stem cells (HSC) were assessed functionally 
by transplantation to determine if age related defects in erythropoiesis and lymphopoiesis are cell-
autonomous, and the author observed small non-significant changes in the intrinsic ability for aged 
HSC to initiate erythropoiesis while declining lymphopoiesis was a cell-autonomous effect. Taken 
together these data indicated both cell-autonomous and non-cell-autonomous effects on the ability of 
aged HSC to produce normal lineage output. 

Overall, the work shows some important findings relating to cell-extrinsic signaling networks in aging 
impacting platelet and erythroid output, which are often ignored in favor of myeloid vs. lymphoid 
lineage alterations only. 

Major concerns: 

Inflammatory gene signatures in aging HSC are well described in prior work (Chambers, Plos Biology 
2007) Here, RNA-seq analysis of stromal cells identified a similarly inflammatory signature that 
impacts hematopoiesis. Thus, the value of analyzing gene expression in the stroma or calling them 
'sensors' is not clear, given gene expression analysis of HSC/HSPC would yield similar results. Value 
would be added to this analysis if the impact of the inflammatory signature on HSC maintenance 
function of the stromal cells themselves is addressed. Indeed a larger issue with the work as a whole 
is that a preliminary level of mechanistic insight with respect to the impact of TGF-beta and/or IL-6 on 
aged hematopoietic function is provided. 

Increased levels of IL-6/TGF (as opposed to increased signaling activity in these pathways due to cell-
intrinsic changes) in the BM are not shown. Indeed other previous analysis of aged BM (Ergen et al, 
Blood 2012) show decreased IL-6 levels in BM fluid by direct interrogation, whereas the study cited by 
the authors (Mei et al, 2018) only shows protein levels in serum and not BM (indirect measurement of 
IL-6 was performed by qRT-PCR in this study). Echoing the point above relating to mechanism, it will 
be important for the authors to distinguish between the alternative hypotheses of increased levels of 
these factors in situ in the BM vs. increased signaling activity due to intrinsic deregulations. 

Reviewer #3 (Remarks to the Author): 

In this paper from Valetta et al., the authors attempt to find an answer to the molecular features 
underlying ageing hematopoiesis/aging HSC behavior. The studies try to argue for a role for IL6 and 
Tgfb signaling in controlling hematopoiesis at two levels (erythroid progenitor cells and HSCs, 
respectively). The interpretations appear for the most part to be supported by the data provided and 
statistics (where applied) seem adequate. 

The authors first describe the general features of ageing hematopoiesis, focusing on decreased 
erythropoiesis and enhanced platelet (biogenesis?). They validate previous findings and apply a 
transgenic mouse model that permit tracing of these two lineages (demonstrating that also this 
transgenic strain exhibits these well-known age-associated features) (Figure 1). In essence, the 
authors conclude that while there is a general reduction of the contribution of aged HSCs to 
multilineage hematopoiesis, this is more pronounced for the lymphoid lineages. 

Not sure if there is anything wrong with the resolution in my figures, but I cannot really see any cells 
in the gates connected to Figure 1b (plots to far right) and Figure 1d (CD11b vs Gr1 plot; no myeloid 
cells?). 

Having verified/established these basic features, the authors next investigate myeloerythroid 
progenitor cells in young and aged mice. They demonstrate an increase in megakaryocyte progenitors 
and a decrease of early erythroid progenitors (including MPP2s – it could benefit the general reader to 
explain a bit more why these cells were investigated). Finally, authors conduct some gene expression 
studies and culture experiments to demonstrate that the myeloid activity of aged preCFU-Es is higher 
from aged cells. These latter experiments seem to indicate that the myeloid (as opposed to erythroid) 
activity/cell goes up with age. This raises some concerns as to the way the prospective isolation was 
made – if f.i. CD55 had been included (positivity) as a marker for candidate preCFU-Es, would the 
authors obtain the same results? 

The authors next go on to use the rather clever approach of using stromal/microenvironmental cells as 
probes for what might underlie the hematopoietic phenotypes of aging. This lead to the identification 
of two candidate signaling pathways; IL6 and Tgfb. Further experimentation is focusing on the 
neutralisation of the two pathways, followed by re-evaluation of hematopoiesis. From these lines of 
studies, the conclusions are: 1) that IL6 exerts negative effects of erythropoiesis (rather than having 
direct effects on HSCs) while 2) Tgfb-inhibition appears to restore the lineage (or rather progenitor) 
output from aged HSCs (including the overproduction of megakaryocyte progenitors; what about 
platelets?). They provide some molecular evidence (several types of genome-wide RNA expression 
experiments) to support their conclusions. 

Finally, the investigators conduct transplantation experiment using purified HSCs from vehicle or Tgfb-
inhibited treated mice. They try to demonstrate that this treatment ”normalizes” the output from HSCs 
with enhanced relative output of non-platelet lineages. 

It appears that the overall reconstitution of Tgfbi-inhibited cells is dramatically lower than that from 
vehicle-treated. What is going on here? It appears to this reviewer that Tgfb-inhibition is causing an 
overall decline in HSC function, that perhaps has some selectivity for HSCs with a ”myeloid-
bias”/”compromised lymphopoiesis”. But this is very difficult to conclude if the variation in overall 
reconstitution is very compromised – f.i. low-level chimeras tend to be skewed towards “lymphoid-
biased” cells (which is most likely reflecting more on the longevity of mature lymphoid cells). 

Some emphasis is on the “rescue” of B lymphopoiesis (Figure 5e) as a consequence of Tgfb-inhibition, 
but details on whether there is restoration of B lymphopoiesis in the bone marrow is lacking. It would 
have been very interesting for the authors to provide some information specifically on IL7-signaling, 
as compromised IL7 signaling in the past has been proposed to be a major mechanism for the reduced 
B (and/or T) lymphopoiesis with age.