Notch and TLR signaling coordinate monocyte cell fate and inflammation
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Abstract
Conventional Ly6C hi monocytes have developmental plasticity for a spectrum of differentiated phagocytes. Here we show, using conditional deletion strategies in a mouse model of Toll-like receptor (TLR) 7-induced inflammation, that the spectrum of developmental cell fates of Ly6C hi monocytes, and the resultant inflammation, is coordinately regulated by TLR and Notch signaling. Cell-intrinsic Notch2 and TLR7-Myd88 pathways independently and synergistically promote Ly6C lo patrolling monocyte development from Ly6C hi monocytes under inflammatory conditions, while impairment in either signaling axis impairs Ly6C lo monocyte development. At the same time, TLR7 stimulation in the absence of functional Notch2 signaling promotes resident tissue macrophage gene expression signatures in monocytes in the blood and ectopic differentiation of Ly6C hi monocytes into macrophages and dendritic cells, which infiltrate the spleen and major blood vessels and are accompanied by aberrant systemic inflammation. Thus, Notch2 is a master regulator of Ly6C hi monocyte cell fate and inflammation in response to TLR signaling.
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###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on April 12, 2020, follows.
Summary
The authors explored the processes involved in the fate of LY6Chi monocytes and the factors influencing this process under TLR7-induced inflammation. They show that the Ly6Chi monocyte conversion to Ly6Clo monocytes is dependent on cell intrinsic Notch2 and Myd88 pathway as selective invalidation of Notch2 bias the differentiation of monocytes in steady state and more extensively in inflammatory condition toward circulating macrophages. This is an interesting paper and the presented experiments are of good quality. This study uses a variety of conditional genetic deletions, cell transfers and cell culture techniques to track these cells in vivo and in vitro.
Essential Revisions
While the editors and …
###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on April 12, 2020, follows.
Summary
The authors explored the processes involved in the fate of LY6Chi monocytes and the factors influencing this process under TLR7-induced inflammation. They show that the Ly6Chi monocyte conversion to Ly6Clo monocytes is dependent on cell intrinsic Notch2 and Myd88 pathway as selective invalidation of Notch2 bias the differentiation of monocytes in steady state and more extensively in inflammatory condition toward circulating macrophages. This is an interesting paper and the presented experiments are of good quality. This study uses a variety of conditional genetic deletions, cell transfers and cell culture techniques to track these cells in vivo and in vitro.
Essential Revisions
While the editors and reviewers all agreed on the interest of the study, several points need to be addressed to strengthen the study.
The transcriptome analysis presented in Fig. 4 is important, but missing Ly6Clow monocytes from wt and myeloid Notch deficient cells under steady state conditions. It would be important to compare the profiles of untreated and IMQ-treated Notch2-deficient cells to dissect the treatment effects from the steady state condition caused by Notch2 deletion.
The authors should provide deletion efficiency in both monocyte subsets, for instance by qRT-PCR for the deleted exon. There is experimental proofs that the Cre lox system is not fully efficient in classical monocytes, usually associated to their short life span and likely partial in the ncMo (as confirmed in Gamrekelashvili et al Nat com 2016) and others. This could explain why the PCA analysis detects minimal difference in the Ly6Chigh subset as well as no impact on numbers in Notch2-mutant mice. Hence it is dificult to support the main conclusion that Notch2-mediated decision occurs at the Ly6Chigh level as it is still mostly present. Notch2 could rather regulate the survival of Ly6Clow Mo. This point is slightly discussed on p13. The efficiency of Notch recombination in Mo subsets after the different treatments must be presented (even if previously published at steady state) to better apprehend this limit.
Lineage tracing is always challenging, and it is difficult to assess whether notch signaling is requested in the differentiation toward a specific lineage or is requested for cell survival. Notch regulated monocyte survival is a well taken point, since also Bcl2 is strongly decreased in notch2-/- monocytes. Therefore providing absolute numbers for the in vivo and in vitro experiments (cell numbers instead of %) is absolutely required. In fact, most of the study is based on % (often unclear among which population) which could lead to misinterpretation. The authors should provide absolute numbers per organs along with per mg, whenever possible. For example "By comparison, the TLR4 ligand LPS also increased Ly6Clo cell numbers and expression levels of Nr4a1and Pou2f2. However, the absolute conversion rate was lower under LPS and there was no synergy with DLL1 (Figure 1D and E)". The numbers are not evaluated here neither the conversion rate as long as survival difference cannot be excluded.
The 'unrestrained inflammation' part either should be experimentally proven our should be completely rephrased (or deleted). The authors state in the abstract that "the absence of functional Notch2 signaling promotes resident tissue macrophage gene expression (...) resulting in unrestrained systemic inflammation" that could be interpreted as an overstatement. The inflammatory response shows perturbation in Notch-deficient mice, but not a clear pro-inflammatory shift (see also point below). Accordingly, it is not clear, if the splenomegaly or the "unrestrained systemic inflammation" is directly caused by monocytes. LysM-Cre is also active in neutrophils, which similarly express high levels of Notch2 according to immgen and can contribute to the observed phenotype. If the authors want to keep the link of 'Notch2-deficient monocytes cause unrestrained systemic inflammation' then the authors should perform monocyte (anti-CCR2 treatment) and neutrophil (anti-Ly6G treatment) depletion experiments in IMQ-treated wt and Notch-deficient mice. If the observed splenomegaly in Notch2-deficient mice is reduced to wt levels when treated with CCR2 (but not after Ly6G treatment), it is likely that monocytes are the direct cause.
T0 purity and flow analysis (F4/80, Ly6C,CD43, CD11c CD11b and GFP) of the sorted monocyte from all mouse strains should be provided. Working with bone marrow monocytes can be precarious, as the bone marrow may be contaminated with progenitors (and should be mentioned in the text).
In Fig. 3 the authors perform t-SNE analysis based on the Ly6C, CD43, MHCII, F4/80 and CD11c marker set and according to this identified 5 monocyte 'subsets' (Fig. 2c). Please also show the corresponding flow cytometry analysis (dot plots) (especially for PB) to identify these 5 subsets by regular gating and to see intensity of especially F4/80 and MHCII staining in Ly6Chigh and Ly6Clow monocytes in all conditions. In addition, in the gating strategy Fig. S1c the authors used F4/80 to discriminate CD115+ MF from monocytes. Rose et al., 2011 in Cytometry A showed that splenic monocytes also express F4/80 and that this antigen can be used to identify monocytes. Therefore it is possible that MF cells in the authors' gating strategy are contaminated by (probably aged) Ly6C low monocytes that up-regulated F4/80. To counter argue this please show their gated MF in a FACS plot with Ly6C vs CD43.
Consistent with the working hypothesis: Notch deficient LY6Clo monocyte phenotype drastically changes following IMQ Figure 3. Why were Ly6Clo wt and N2 deficient monocytes not examined without IMQ Figure 4? It is important to know how these cells alter from baseline and help distinguish if the effects are IMQ alone, Notch alone or IMQ and Notch.
The conclusion from these studies is in the presence of IMQ and absence of Notch2 LY6Chi cells become more of a "macrophage" compared to the natural progression towards LY6Clo monocytes Figure 5a/b. In Figure 5b, c and F what is the phenotype without IMQ. At present, without the controls, it is hard to comment on these experiments.
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