LECT2 drives haematopoietic stem cell expansion and mobilization via regulating the macrophages and osteolineage cells

Haematopoietic stem cells (HSCs) can differentiate into cells of all lineages in the blood. However, the mechanisms by which cytokines in the blood affect HSC homeostasis remain largely unknown. Here we show that leukocyte cell-derived chemotaxin 2 (LECT2), a multifunctional cytokine, induces HSC expansion and mobilization. Recombinant LECT2 administration results in HSC expansion in the bone marrow and mobilization to the blood via CD209a. The effect of LECT2 on HSCs is reduced after specific depletion of macrophages or reduction of osteolineage cells. LECT2 treatment reduces the tumour necrosis factor (TNF) expression in macrophages and osteolineage cells. In TNF knockout mice, the effect of LECT2 on HSCs is reduced. Moreover, LECT2 induces HSC mobilization in irradiated mice, while granulocyte colony-stimulating factor does not. Our results illustrate that LECT2 is an extramedullar cytokine that contributes to HSC homeostasis and may be useful to induce HSC mobilization.

. The claim that HSC function is increased by LECT2 is not convincing. Figure 1m measures short-term repopulating activity-not HSCs. Figure 1n is presumably a competitive repopulation assay. To assess HSC function, need to show multilineage time-dependent engraftment. N = 3 is not sufficient.    Editorial Note: Parts of this peer review file have been redacted as we could not obtain permission to publish the reports of Reviewer #3 . This manuscript is interesting and the first to report that the chemokine LECT2 mobilizes hematopoietic stem cells (HSC) and progenitor cells (HPC) into blood. Generally well performed experiments show that daily administration of LECT2 protein in mice causes mobilization of colonyforming units and phenotypic HSPC (Lin-Kit+ Sca1+). However demonstration that long-termreconstituting HSCs in transplantation setting are mobilized is lacking.
The mechanistic aspect of this work is well developed. Using blocking antibodies, inhibitors and KO mice, the authors convincingly demonstrate that the mobilizing effect of LETC2 is mediated by CD209a (or DC-SIGN) but not by its second receptor c-MET. Furthermore, LETC2 administration accelerates leukocyte recovery following cytotoxic challenge with 5-fluorouracil whereas CD209a gene deletion in KO mice compromises recovery. As CD209a+ sorted cells are found to express CD169 (macrophage restricted antigen) and RUNX2 (essential to osteolinage cells and their maturation) and the protein co-localized with CD169 and osteopontin, this suggests that the effect of LETC2 is indirectly mediated by macrophages and/or osteolineage cells. Using CD169-DTR mice and clodronate loaded liposomes to deplete macrophages in vivo and biglycan KO mice which have defective osteoblasts and SrCl2 to stimulate bone formation, the authors show that depletion of macrophages partially reduces the mobilizing effect of Lect2. As biglycan gene deletion partially reduces mobilizing effect of LECT2 and SrCl2 increases it, one may conclude that the effect is mediated by both macrophages and osteolineage cells. To further dissect the mechanisms, the authors show that LECT2 treatment reduces TNF expression and that administration of recombinant TNF abolishes the mobilizing effect of TNF whereas deletion of the TNF gene causes a small but significant leakage of HSPC into the blood and important loss of mobilization in response to LECT2. Together these data suggest that down-regulation of TNF secretion is an important step of HSPC mobilization in response to LECT2. As disruption of the chemotactic interaction between the chemokine SDF-1 and its receptor CXCR4 is known to play a major role in the mobilizing effect of many cytokines, the authors investigated this aspect by showing that LECT2 increases CD26 enzymatic activity in the marrow, reducing the amount of intact (active) SDF-1 in the marrow whereas injection of TNF reduces CD26 activity. These experiments support the model describe in figure 10. Finally the authors finish their manuscript by showing convincingly in GCSF receptor KO mice and CD209a KO mice that the mechanisms leading to mobilization in response G-CSF and LECT2 are independent as GCSFR KO mice mobilize in response to LETC2 whereas CD209a KO mice mobilize in response to G-CSF.
Despite the novelty and originality, well performed experiments providing novel mechanisms that support the model, detailed Material and Methods section, etc. several issues must be addressed by the authors before publication: 1) The authors' claim in title, abstract results and particularly the discussion that LECT2 expands HSCs in the BM is the weakest. It is based on the finding of higher number of Lin-Kit+Sca1+Flt3-CD34-cells in the BM following LETC2 treatment. While this phenotype is OK to detect long-term reconstituting HSC in steady-state, this phenotype is not reliable when mice are stimulated by an exogenous agent. Sca1 and CD34 are two activation antigens in the mouse: Sca1 is up-regulated in . Therefore, the authors must confirm their claim with a functional competitive repopulation assay to quantify the number of repopulating units in the BM of mice LETC2 treated mice versus saline treated mice. In the absence of this functional assay, expansion of LT-HSC is not definitively established and this claim should be toned down.
2) The authors claim that in humans, number of circulating Lin CD34+CD38-CD90+ HSCs is correlated with plasma concentration of LECT2 (fig 1a). This should be substantiated by giving Pearson correlation coefficient and p value. Also the authors should clarify whether this is in steadystate.
3) In figure 1, the authors make several transplantation assays on sorted mobilized LSK cells. While this is fine to show that the engraftment potential of equivalent number of LSK cells is increased following LECT2 treatment, it does not quantify the number of reconstituting HSCs mobilized per ml of blood. It would be good to quantify this in a competitive transplantation assay in which 20uL whole mobilized blood is transplanted in competition with 200,000 congenic BM cells and compare blood content in competitive repopulating units in mice mobilized with LECT2 versus G-CSF. Fig 1m, the survival curve should also be plotted as number of transplanted LSK cells on the X axis versus percentage of mice that did not engraft on the Y axis. This would enable to calculate by Poisson statistic the frequency of reconstituting HSCs within the LSK populations from the control and LECT2 treated mice and determine whether these differences are statistically different. This calculation can be performed using the L-calc software that can be downloaded from the Stem Cell Technologies website. Fig 1d,1e,1g, 1h and immunohistofluorescence images in Fig 2i and 4a are too small to see anything and be of any use. Their size must be increased.

Major concerns
The major concern of this study is readability. By this reviewer's count, there are 77 figure components (not including subpanels and supplementary material). Many of the experiments are not adequately outlined, leaving the reader guessing as to the experimental design. This reviewer suggests that the authors consider condensing their manuscript to focus on key experiments. For example, there is no need to show WBC counts in every figure. The CCL3 experiments are preliminary and could be eliminated. The mitomycin C experiments are difficult to interpret and should be eliminated. By reducing the number of figures, the authors can provide a more clear rationale and design for each experiment. Response: We thank the reviewer for this comment which helps us to improve our study. We removed the WBC data in Fig. 2 (Now Fig. 3), Fig. 6, Fig. 7. The data of CCL3 expression after LECT2 treatment were removed in previous Fig. 5 (now supplementary Fig. 7b,c.). Mitomycin C experiements were removed in previous Fig. 5 (now supplementary Fig. 7). Figure 1n. The claim that HSC function is increased by LECT2 is not convincing. Figure 1m measures short-term repopulating activity-not HSCs. Figure 1n is presumably a competitive repopulation assay. To assess HSC function, need to show multilineage time-dependent engraftment. N = 3 is not sufficient. Response: We thank the reviewer for his most helpful review and comments. We showed the multilineage time-dependent engraftment in Fig. 2d. The data were existed in this paper last year when submitted to other Journal. We removed them when the paper was submitted to Nature Communications this year. We also added new data in previous Fig. 1n (now Fig. 2c, n = 5). We also added the description of these data in Results in line 75-97, and the protocols in Methods in 535-537.
Minor concerns Figure 1b. Need to verify that the LECT2 preparation does not contain LPS Response: This is a very good point. Endotoxin in the recombinant proteins was less than 0.1 EU/mg after toxin removal with an endotoxin-removal column (Pierce). We also added the description in line 403. We also found that LECT2 treatment also enhanced the CFU-Cs, WBCs and LSK cells in the blood of C3H/HeJ mice, a strain that is relatively insensitive to endotoxin. We also added the experiment data in supplementary Fig. 1 and description in Results in line 63-65. Figure 1g. The % of KLS cells in G0 appears low compared to published data. The authors may want to check their assay. The dot plots are too small to interpret. Response: We agree with the reviewer's comment. Indeed, our data is low compared with published data (about 90% of published data). We repeated this experiment, and found that the average percentage of LSK cells in G0 was 39.4% in WT mice. We also put the bigger dot plots in Fig. 1g. It is similar with published data, 39% (Blood, 2012, 120: 1843-1855), 38.7% (Blood, 2013, 121: 5158-5166), about 45% (Immunity, 2015, 42:1021-1032). Figure 2d. The LSK number in the BM is down in LECT2 KO mice but normal in CD209a KO mice. Why? Response: We agree with the reviewer's comment. The LSK numbers have about 10% fluctuation performed at same conditions in different time according to our experimental experience. We repeated the experiment with WT control in Fig. 2d (Now Fig. 3d). The LSK numbers in the BM of CD209a KO mice are about 70.8% of WT mice.

Reviewer #2 (Expert in HSC, macrophage) (Remarks to the Author):
1) The authors' claim in title, abstract results and particularly the discussion that LECT2 expands HSCs in the BM is the weakest. It is based on the finding of higher number of Lin-Kit+Sca1+Flt3-CD34-cells in the BM following LETC2 treatment. While this phenotype is OK to detect long-term reconstituting HSC in steady-state, this phenotype is not reliable . Therefore, the authors must confirm their claim with a functional competitive repopulation assay to quantify the number of repopulating units in the BM of mice LETC2 treated mice versus saline treated mice. In the absence of this functional assay, expansion of LT-HSC is not definitively established and this claim should be toned down. Response: We thank the reviewer for this comment which helps us to improve our study. We indeed added more description about HSC expansion in results (in line 75-97) and discussion (in line 265-280). The HSC expansion is mentioned in title and abstract. The word limitation prevents the further description of HSC expansion in title and abstract.
We showed the number of repopulating units in the 1 × 10 3 LSK cells from the BM of LECT2 treated and PBS treated mice in Fig. 2e. We also showed the number of repopulating units in the 1 × 10 5 BM cells from the BM of LECT2 treated and PBS treated mice in Fig. 2e. The data were existed in this paper last year when submitted to other Journal. We removed them when the paper was submitted to Nature Communications this year. We also cited the papers (Essers MAG et  2) The authors claim that in humans, number of circulating Lin CD34+CD38-CD90+ HSCs is correlated with plasma concentration of LECT2 (fig 1a). This should be substantiated by giving Pearson correlation coefficient and p value. Also the authors should clarify whether this is in steady-state. Response: We agree with the reviewer's comment. We added the Pearson correlation coefficient, p value, and in steady state in line 57-59.
3) In figure 1, the authors make several transplantation assays on sorted mobilized LSK cells. While this is fine to show that the engraftment potential of equivalent number of LSK cells is increased following LECT2 treatment, it does not quantify the number of reconstituting HSCs mobilized per ml of blood. It would be good to quantify this in a competitive transplantation assay in which 20uL whole mobilized blood is transplanted in competition with 200,000 congenic BM cells and compare blood content in competitive repopulating units in mice mobilized with LECT2 versus G-CSF.