RBP-J regulates homeostasis and function of circulating Ly6C^lo monocytes

Reviewer #1 (Public Review):

Kou and Kang et al. investigated the role of Notch-RBP-J signaling in regulating monocyte homeostasis. Specifically, they examined how a conditional knockout of Rbpj expression in monocytes through a Rbpjfl/fl Lyz2cre/cre mouse affects the homeostasis of Ly6Chi versus Ly6Clo monocytes. They discovered that Rbpj deficiency did not affect the percentage of Ly6Chi monocytes but instead, led to an accumulation of Ly6Clo monocytes in the peripheral blood. Using a comprehensive number of in vivo techniques to investigate the origin of this increase, the authors revealed that the accumulation of Rbpj deficient Ly6Clo monocytes was not due to an increase in bone marrow egress and that this defect was cell intrinsic. However, EdU-labelling and apoptosis assays revealed that this defect was not due to an increase in proliferation nor conversion of Ly6Chi to Ly6Clo monocytes. Interestingly, it was revealed that Rbpj deficient Ly6Clo monocytes had increased expression of CCR2 and ablation of CCR2 expression reversed the accumulation of these cells in the periphery. Lastly, they discovered that Rbpj deficiency also led to downstream effects such as an accumulation of Ly6Clo monocytes in the lung tissue and increased CD16.2+ interstitial macrophages, presumably due to dysregulated monocyte differentiation and function.

Their findings are interesting and highlight a previously unexplored mechanistic link between Notch-RBP-J signaling and CCR2 expression in monocyte homeostasis, providing further insight into the distinct pathways that regulate Ly6Chi vs Ly6Clo monocyte subsets individually.

The conclusions of this paper are mostly well substantiated from the experimental data. The strengths of this paper include the use of multiple conditional genetic knock out mouse models to explore their hypothesis and the use of sophisticated in vivo techniques to study the major mechanisms involved in monocyte homeostasis.

Reviewer #2 (Public Review):

The authors provide compelling data to demonstrate that the Notch-related transcription factor RBP-J can influence the number of circulating and recruited monocytes. The authors first delete the Rbpj gene in the myeloid lineage (Lyz2) and show that, as a proportion, only Ly6Clo monocytes are increased in the blood. The authors then attempted to identify why these cells were increased but ruled out proliferation or reduced apoptosis. Next, they investigated the gene signature of Rbpj null monocytes using RNA-sequencing and identified elevated Ccr2 as a defining feature. Crossing the Rbpj null mice to Ccr2 null mice showed reduced numbers of Ly6Clo monocytes compared with Rbpj null alone. Finally, the authors identify that an increased burden of blood Ly6Clo monocytes is correlated with increased lung recruitment and expansion of lung interstitial macrophages.

The main conclusion of the authors, that there is a 'cell intrinsic requirement of RBP-J for controlling blood Ly6CloCCR2hi monocytes' is strongly supported by the data. However, other claims and aspects of the study require clarification and further analysis of the data generated.

Strengths
The paper is well written and structured logically. The major strength of this study is the multiple technically challenging methods used to reinforce the main finding (e.g. parabiosis, adoptive transfer). The finding reinforces the fact that we still know little about how immune cell subsets are maintained in situ, and this study opens the way for novel future work. Importantly, the authors have generated an RNA-sequencing dataset that will prove invaluable for identifying the mechanism - they have promised public access to this data via GEO.

Weaknesses - The main weakness of the study, is that although the main result is solidly supported, as written it is mostly descriptive in nature. For instance, there is no given mechanism by which RBP-J increases Ly6Clo monocytes. The authors conclude this is dependent on CCR2, however CCR2 deletion has a global effect on monocyte numbers and importantly in this study, it does not remove the Ly6Clo bias of cell proportions, if anything it seems to enhance the difference between the ly6C low and high populations in Rbpj null mice (figure 5C). This oversight in data interpretation likely occurred because this experiment is missing a potentially important control (Lyz2cre/cre Ccr2RFP/RFP or RBP-J variations). In general, there seemed to be a focus on the Ly6C low cells, where the mechanism may be more identifiable in their precursors - likely the Ly6C high monocytes.

Other specific weaknesses were identified:

1. The confirmation of knockout in supplemental figure 1A shows only a two third knockdown when this should be almost totally gone. Perhaps poor primer design, cell sorting error or low Cre penetrance is to blame, but this is below the standard one would expect from a knockout.
2. Many figures (e.g. 1A) only show proportional data (%) when the addition of cell numbers would also be informative
3. Many figures only have an n of 1 or 2 (e.g. 2B, 2C)
4. Sometimes strong statements were based on the lack of statistical significance, when more n number could have changed the interpretation (e.g. 2G, 3E)
5. There is incomplete analysis (e.g. Network analysis) and interpretation of RNA-sequencing results (figure 4), the difference between the genotypes in both monocyte subsets would provide a more complete picture and potentially reveal mechanisms
6. The experiments in Figures 5 and 7 are missing a control (Lyz2cre/cre Ccr2RFP/RFP or the Rbpj+/+ versions) and may have been misinterpreted. For example if the control (RBP-J WT, CCR2 KO) was used then it would almost certainly show falling Ly6C low numbers compared to RBP-J WT CCR2 WT, but RBP-J KO CCR2 KO would still have more Ly6c low monocytes than RBP-J WT, CCR2 KO - meaning that the RBP-J function is independent of CCR2. I.e. Ly6c low numbers are mostly dependent on CCR2 but this is irrespective of RBP-J.
7. Figure 6 was difficult to interpret because of the lack of shown gating strategy. This reviewer assumes that alveolar macrophages were gated out of analysis
8. The statements around Figure 7 are not completely supported by the evidence, i) a significant proportion of CD16.2+ cells were CCR2 independent and therefore potentially not all recently derived from monocytes, and ii) there is nothing to suggest that the source was not Ly6C high monocytes that differentiated - the manuscript in general seems to miss the point that the source of the Ly6C low cells is almost certainly the Ly6C high monocytes - which further emphasises the importance of both cells in the sequencing analysis
9. The authors did not refer to or cite a similar 2020 study that also investigated myeloid deletion of Rbpj (Qin et al. 2020 - https://doi.org/10.1096/fj.201903086RR). Qin et al identified that Ly6Clo alveolar macrophages were decreased in this model - it is intriguing to synthesise these two studies and hypothesise that the ly6c low monocytes steal the lung niche, but this was not discussed

Reviewer #3 (Public Review):

In this study, the authors investigate the role of the Notch signalling regulator RBP-J on Ly6Clow monocyte biology starting with the observation that RBP-J-deficient mice have increased circulating Ly6low monocytes. Using myeloid specific conditional mouse models, the authors investigate how RBP-J deficiency effects circulating monocytes and lung interstitial macrophages.

A major strength of this study is that it describes RBP-J as a novel, critical factor regulating Ly6Clow monocyte cell frequency in the blood. The authors demonstrate that RBP-J deficiency leads to increased Ly6Clow monocytes in the blood and lung and CD16.2+ interstitial macrophages in steady state. The authors use a number of different techniques to confirm this finding including bone marrow transplantation experiments and parabiosis.

There are several critical weaknesses that need to be assessed to improve the manuscript, in summary the data presented in the current manuscript are highly descriptive and without mechanistic insight. The inclusion of more mechanistic insight would greatly improve the manuscript.

The authors begin to explore the potential mechanism underlying why Ly6Clow monocytes are increased in the absence of RBP-J - is it through increased survival, increased conversion from Ly6C+ monocytes, increased proliferation or increased egress from the bone marrow. The majority of the data they present here is negative. Whilst I applaud the authors for including negative data, I think that their exploration into how RBP-J leads to increased monocytes does not go far enough and it is critical to understand the mechanism by which RBP-J increases circulating monocytes. Low n-numbers in multiple figures mean that the claims made are not fully supported.

The current title of the paper "RBP-J regulates homeostasis and function of circulating Ly6Clo monocytes" does not fully reflect the manuscript in its current form - there is no exploration of Ly6Clow monocyte functionality in the paper as it stands.
Given that targeting monocytes and macrophages in a range of inflammatory diseases is an attractive yet elusive therapeutic option, understanding the underlying biology that regulates monocyte biology are critically important. This manuscript has the potential to add to our current knowledge of how Ly6Clow monocyte biology is regulated and potentially opens novel avenues for preferentially enhancing Ly6Clow monocytes without influencing Ly6C+ monocytes. This is an attractive proposition for many inflammatory conditions however, considerably more in-depth analysis is required to understand the role of RBP-J in monocyte biology.