Notch-RBP-J signaling plays an essential role in maintenance of myeloid homeostasis. However, its role in monocyte cell fate decisions is not fully understood. Here we showed that conditional deletion of transcription factor RBP-J in myeloid cells resulted in marked accumulation of blood Ly6Clo monocytes that highly expressed chemokine receptor CCR2. Bone marrow transplantation and parabiosis experiments revealed a cell intrinsic requirement of RBP-J for controlling blood Ly6CloCCR2hi monocytes. RBP-J-deficient Ly6Clo monocytes exhibited enhanced capacity competing with wildtype counterparts in blood circulation. In accordance with alterations of circulating monocytes, RBP-J deficiency led to markedly increased population of lung tissues with Ly6Clo monocytes and CD16.2+ interstitial macrophages. Furthermore, RBP-J deficiency-associated phenotypes could be genetically corrected by further deleting Ccr2 in myeloid cells. These results demonstrate that RBP-J functions as a crucial regulator of blood Ly6Clo monocytes and thus derived lung-resident myeloid populations, at least in part through regulation of CCR2.
This study presents a valuable examination into the role Notch-RBP-J signaling in regulating monocyte subset homeostasis. The data were collected and analyzed using solid and validated methodology and can be used as a starting point for exploring the mechanisms involved in RBP-J signaling in non-classical monocytes.
Monocytes are integral components of the mononuclear phagocyte system (MPS) that develop from monocyte precursors in the bone marrow1. Several functionally and phenotypically distinct subsets of blood monocytes have been defined on the basis of expression of surface markers2-5. In mice, Ly6Chi monocytes (also called inflammatory or classical monocytes) characterized by Ly6ChiCCR2hiCX3CR1lo exhibit a short half-life, and are recruited to tissue and differentiate into macrophages and dendritic cells6. Ly6Chi monocytes are analogous to CD14+ human monocytes based on gene expression profiling7. A second subtype of monocytes is called Ly6Clo monocytes (also termed patrolling monocytes or non-classical monocytes), which express high level of CX3CR1 and low level of CCR2, equivalent to CD14loCD16+ human monocytes. Ly6Clo monocytes are regarded as blood-resident macrophages that patrol blood vessels and scavenge microparticles attached to the endothelium under physiological conditions, and exhibit a long half-life in the steady-state8-10. Ly6Clo monocytes may exert a protective effect by suppressing atherosclerosis in mice, while high levels of nonclassical monocytes have been associated with more advanced vascular dysfunction and oxidative stress in patients with coronary artery disease11-13. In the inflammatory settings, Ly6Clo monocytes often show anti-inflammatory properties, yet also exhibit a proinflammatory role under certain circumstances5,9,14-18. In addition, Ly6Clo monocytes have demonstrated protective functions in tumorigenesis, such as engulfing tumor materials to prevent cancer metastasis, activating and recruiting NK cells to the lungs19,20. While the functions of Ly6Clo monocytes are complex and sometimes contradictory depending on the animal models and experimental approaches, it is clear that they play a crucial role in both health and disease.
The generation and maintenance of Ly6Clo monocytes are regulated by several transcription factors, including Nr4a1 and CEBPβ21,22. The colony-stimulating factor 1 receptor (CSF1R) signaling pathway is important for the generation and survival of Ly6Clo monocytes, and neutralizing antibodies against CSF1R can reduce their numbers23. Interestingly, recent studies have suggested that different subsets of monocytes may be supported by distinct cellular sources of CSF-1 within bone marrow niches, in which targeted deletion of Csf1 from sinusoidal endothelial cells selectively reduces Ly6Clo monocytes but not Ly6Chi monocytes24. LAIR1 has been shown to be activated by stromal protein Colec12, and LAIR1 deficiency leads to aberrant proliferation and apoptosis in bone marrow non-classical monocytes25. These findings further underscore the complexity of the mechanisms that regulate Ly6Clo monocyte, and suggest that multiple factors are involved in this process.
Recombination signal-binding protein for immunoglobulin kappa J region (RBP-J; also named CSL in humans) is commonly known as the master nuclear mediator of canonical Notch signaling26. In the absence of Notch intracellular domain, RBP-J associates with co-repressor proteins to repress transcription of downstream target genes. In the immune system, the best studied functions of Notch signaling are its roles in regulating the development of lymphocytes, such as T cells and marginal zone B cells26. Notch signaling also has been reported to regulate the differentiation and function of myeloid cells including granulocyte/monocyte progenitors, osteoclasts and dendritic cells 27-30. In osteoclasts, RBP-J represses osteoclastogenesis, particularly under inflammatory conditions30. In dendritic cells, Notch-RBP-J signaling controls the maintenance of CD8- DC28. In addition, Notch2-RBP-J pathway is essential for development of CD8-ESAM+ DC in the spleen and CD103+CD11b+ DC in the lamina propria of the intestine29. However, the role of Notch-RBP-J signaling pathway in regulating monocyte subsets remains elusive. Here, we demonstrated that RBP-J controlled homeostasis of blood Ly6Clo monocytes in a cell intrinsic manner. RBP-J deficiency led to a drastic increase in blood Ly6Clo monocytes, lung Ly6Clo monocytes and CD16.2+ IM. Our results suggest that RBP-J plays a critical role in regulating the population and characteristics of Ly6Clo monocytes in the blood and lung.
RBP-J is essential for maintenance of blood Ly6Clo monocytes
To investigate the role of RBP-J in monocyte subsets, we utilized mice with RBP-J specific deletion in the myeloid cells (Rbpjfl/flLyz2cre/cre mice). Efficient deletion of Rbpj in blood monocytes was confirmed by quantitative real-time PCR (qPCR) (Figure 1 - figure supplement 1A). Flow cytometric analysis revealed that RBP-J deficient mice had a significant increase in the proportion of Ly6Clo monocytes, but not Ly6Chi monocytes, in blood, compared to age-matched control mice with the genotype Rbpj+/+Lyz2cre/cre (Figure 1A). In contrast to circulating monocytes, RBP-J deficient mice exhibited minimal alterations in the percentages of monocyte subsets in bone marrow (BM) and spleen (Figure 1B; Figure 1 - figure supplement 1C). Additionally, RBP-J deficiency in myeloid cells did not appear to have an effect on neutrophils (Figure 1 - figure supplement 1B). Therefore, among circulating myeloid populations, RBP-J selectively controlled the subset of Ly6Clo monocytes.
BM progenitors that give rise to circulating monocytes are monocyte-dendritic cell progenitors (MDPs) and common monocyte progenitors (cMoPs)21,31-33. Next, we analyzed BM progenitors, and found that the percentages of MDPs and cMoPs were equivalent in control and RBP-J deficient mice (Figure 1C). These results in conjunction with the observations of normal BM monocyte populations suggest that alterations of peripheral blood Ly6Clo monocytes may not originate from BM. Next, we examined whether RBP-J may influence the egress of Ly6Clo monocytes from BM, and measured BM exit rate of Ly6Clo monocytes using in vivo labeling of sinusoidal cells as previously described (Figure 1D)34. The results showed that RBP-J deficiency did not affect egress of Ly6Clo monocytes from BM (Figure 1E). Taken together, these data revealed RBP-J as a critical regulator controlling homeostasis of peripheral blood Ly6Clo monocytes.
RBP-J deficiency does not affect Ly6Chi monocyte conversion or Ly6Clo monocyte survival and proliferation
Next, we aimed to determine whether the increase of blood Ly6Clo monocytes in RBP-J deficient mice was due to decreased cell death or enhanced proliferation. We first stained monocytes with Annexin V and 7-AAD to identify apoptotic cells and observed comparable percentages of apoptotic blood Ly6Clo monocytes in control and RBP-J deficient mice (Figure 2A). Given the crucial role of Nr4a1 in the survival of Ly6Clo monocytes21, we detected the expression of Nr4a1, which was similar in Ly6Clo monocytes from control and RBP-J deficient mice (Figure 2 - figure supplement 1A). We then assessed the proliferative capacity by analyzing Ki-67 expression as well as in vivo EdU incorporation. Ki-67 levels in blood monocytes displayed no differences between control and RBP-J deficient mice (Figure 2B). The percentage of EdU+ monocytes did not significantly differ between control and RBP-J deficient mice at the indicated time points, implying that the turnover of Ly6Clo monocytes was normal in RBP-J deficient mice (Figure 2C). Fluorescent latex beads as particulate tracers could be phagocytosed by monocytes after intravenous injection, and stably label Ly6Clo monocytes35. Thus, we intravenously injected fluorescent latex beads into control and RBP-J deficient mice to track circulating monocytes (Figure 2D). By day 7 post injection, only Ly6Clo monocytes were latex+ as previously reported35, whereas control and RBP-J deficient mice groups presented a similar frequency of latex+ monocytes (Figure 2E). Together, these data indicated that RBP-J did not influence monocyte survival and proliferation.
Previous studies have shown that Ly6Clo monocytes are observed in recipient mice following the adoptive transfer of Ly6Chi monocytes, indicating that Ly6Chi monocytes can convert into Ly6Clo monocytes10,31. We next wished to evaluate whether conversion of Ly6Chi monocytes was regulated by RBP-J by isolating bone marrow GFP+Ly6Chi monocytes from Lyz2cre/creCx3cr1gfp/+control or Rbpjfl/flLyz2cre/creCx3cr1gfp/+mice and adoptively transferring them into Ccr2RFP/RFP recipients (Figure 2F). Sixty hours after transfer, a subset of cells from donors were converted into Ly6Clo monocytes (Figure 2 - figure supplement 1B), and equal percentages of Ly6Clo monocytes were derived from control and Rbpjfl/flLyz2cre/creCx3cr1gfp/+donors (Figure 2G). Thus, the conversion of Ly6Chi monocytes into Ly6Clo monocytes was not affected by RBP-J deficiency.
RBP-J regulates blood Ly6Clo monocytes in a cell intrinsic manner
We next wondered whether the increase of Ly6Clo monocytes in RBP-J deficient mice was bone marrow-derived and cell intrinsic. We performed bone marrow transplantation by engrafting lethally irradiated mice with a 1:4 mixture of Rbpj+/+Lyz2cre/cre control and Rbpjfl/flLyz2cre/cre BM cells (CD45.2) and Cx3cr1gfp/+ BM cells (CD45.1) (Figure 3A). Eight weeks after transplantation, we analyzed the frequencies of CD45.2+ donor cells in the bone marrow and blood of recipient mice and found that the frequencies of CD45.2+ cells within total cells were similar to the mixture ratio of bone marrow cells for Ly6Chi monocytes and neutrophils in both BM and blood (Figure 3B; Figure 3 - figure supplement 1A-B). Specifically, more blood Ly6Clo monocytes were derived from RBP-J deficient donors than control donors (Figure 3B), reflecting that the contribution of RBP-J deficient cells in blood Ly6Clo cells was significantly higher than that of control cells. These results implied that RBP-J deficient BM cells were highly efficient in generating blood Ly6Clo monocytes.
Given that RBP-J deficient mice had more Ly6Clo monocytes in blood than did control mice, we performed parabiosis experiments, a surgical union of two organisms, which allowed parabiotic mice to share their blood circulation. The contribution of circulating cells from one animal in another can be estimated by measuring the percentage of blood cells that originated from each animal36. We joined a CD45.1, Cx3cr1gfp/+ mouse with an age and sex-matched Rbpj+/+Lyz2cre/cre control or Rbpjfl/flLyz2cre/cre mouse (CD45.2) (Figure 3C). Four weeks after the procedure, about 50% of B cells and T cells in parabiotic mice displayed efficient exchange of their circulation (Figure 3 - figure supplement 1C). As expected, RBP-J deficient mice exhibited higher percentages of Ly6Clo monocytes than control animals (Figure 3D). Intriguingly, in the parabiotic Cx3cr1gfp/+mice, RBP-J deficient cells still constituted significantly higher proportion of circulating Ly6Clo, but not Ly6Chi monocytes, than RBP-J sufficient counterparts as indicated by the percentages of the GFP-negative population (Figure 3E). These results implicated that the enhanced ability of Ly6Clo monocytes to circulate in the peripheral blood as a result of RBP-J deficiency was cell intrinsic.
RBP-J regulates phenotypical marker genes in blood Ly6Clo monocytes
To further study the consequences of RBP-J loss of function in blood Ly6Clo monocytes, we performed gene expression profiling by RNA-seq, which revealed that Ly6Clo monocytes in RBP-J deficient mice exhibited low expression of Itgax but high expression of Ccr2, in comparison to those in control mice (Figure 4A). These differential expression patterns of phenotypic marker genes were also confirmed by qPCR and flow cytometry analysis (Figure 4B). However, Ly6Chi blood monocytes and BM monocytes displayed normal levels of CD11c and CCR2 (Figure 4B; Figure 4 - figure supplement 1A). Moreover, principal component analysis showed distinct expression pattern of monocytes between control and RBP-J deficient mice (Figure 4C). To determine whether the regulation of phenotypic markers by RBP-J in blood Ly6Clo monocytes was cell-intrinsic, we performed BM transplantation as described above (Figure 3A). The findings indicated that Ly6Clo monocytes derived from RBP-J deficient BM cells expressed high levels of CCR2 but low levels of CD11c, in comparison to those derived from control BM cells, whereas no significant changes were observed in blood Ly6Chi and BM monocytes, as expected (Figure 4D; Figure 4 - figure supplement 1B). These results collectively suggested that RBP-J regulated the expression of CCR2/CD11c in a cell-intrinsic manner.
RBP-J-mediated control of blood Ly6Clo monocytes is CCR2 dependent
Given that RBP-J deficiency led to enhanced CCR2 expression in Ly6Clo monocytes and that CCR2 is essential for monocyte functionality under various inflammatory and non-inflammatory conditions37, we wished to genetically investigate the role of CCR2 in the RBP-J deficient background. We generated RBP-J/CCR2 double deficient (DKO) mice with the genotype Rbpjfl/flLyz2cre/creCcr2RFP/RFPwith Lyz2cre/creCcr2RFP/+ and Rbpjfl/flLyz2cre/creCcr2RFP/+mice serving as controls. As expected, the expression of CCR2 in Ly6Clo monocytes was reduced in DKO mice compared to RBP-J deficient mice (Figure 5A), confirming the successful deletion of the Ccr2 gene. DKO mice showed a lower percentage of both Ly6Chi and Ly6Clo monocytes than RBP-J deficient mice (Figure 5B-C). Notably, the percentage of Ly6Clo monocytes in DKO mice was comparable to that observed in the control mice (Figure 5B-C), implicating that deletion of CCR2 corrected RBP-J deficiency-associated phenotype of increased Ly6Clo monocytes. Whereas, both RBP-J deficient and DKO mice exhibited lower expression levels of CD11c in their Ly6Clo monocyte than control mice, suggesting that the regulation of CD11c expression was independent of CCR2 (Figure 5D). These results suggested that RBP-J regulated Ly6Clo monocytes, at least in part, through CCR2.
Lung Ly6Clo monocytes are accumulated in RBP-J deficient mice
In mice, alveolar macrophages (AM) are maintained by local self-renewal, and arise from fetal liver derived precursors, whereas interstitial macrophages (IM) are probably originated from monocytes 38-40. Joey et al identified 3 subpopulations of IM according to expression of CD206 and CD16.2 and Ly6Clo monocytes are proposed to give rise to CD64+CD16.2+ IM41. We next compared the populations of lung monocytes and IM between control and RBP-J deficient mice. The conditional deletion of RBP-J resulted in a significant increase in the absolute and relative numbers of Ly6Clo monocytes and CD16.2+ IM, while the numbers of Ly6Chi monocytes, CD206- IM, and CD206+ IM remained unchanged (Figure 6A-B). To confirm these findings, lung sections from Lyz2cre/creCx3cr1gfp/+ control and Rbpjfl/flLyz2cre/creCx3cr1gfp/+ mice were stained with an anti-GFP antibody, which revealed a significant increase in the number of GFP+ cells in RBP-J deficient mice (Figure 6C). In addition, we evaluated the proliferative capacity of Ly6Clo monocytes and CD16.2+ IM, but did not observe enhanced EdU incorporation in Ly6Clo monocytes and CD16.2+ IM from RBP-J deficient mice (Figure 6 - figure supplement 1A-B). These findings suggested that the augmented population of these cells might not be resulted from increased in situ proliferation. Previous reports have indicated that LPS can increase the population of IM in a CCR2-dependent manner40. We thus challenged mice with LPS, and analyzed monocytes and IM at day 0 and day 4. LPS exposure induced an increase in numbers of Ly6Clo monocytes, CD16.2+ IM and CD206- IM compared to baseline both in control and RBP-J deficient mice (Figure 6D-E). There was a trend toward higher Ly6Chi monocyte after LPS treatment, although this observation was not statistically significant. Of note, RBP-J deficient mice exhibited robustly elevated numbers of Ly6Clo monocytes and CD16.2+ IM compared with control mice after LPS treatment (Figure 6D-E), suggesting that RBP-J deficient Ly6Clo monocytes were recruited to inflamed tissue in large numbers and differentiated into CD16.2+ IM.
To investigate whether the elevated levels of lung monocytes and CD16.2+ IM in RBP-J deficient mice were derived from blood Ly6Clo monocytes, we examined monocytes and IM in Lyz2cre/creCcr2RFP/+control, Lyz2cre/creCcr2RFP/RFP, Rbpjfl/flLyz2cre/creCcr2RFP/+, and Rbpjfl/flLyz2cre/creCcr2RFP/RFP(DKO) mice. The deletion of CCR2 led to a decrease in Ly6Chi monocytes, and the Ly6Clo monocytes in DKO mice were reduced to a level similar to that of control mice (Figure 7A, B). While the DKO mice had more CD16.2+ IM compared to control mice, these cells were severely reduced compared to RBP-J deficient mice (Figure 7A, C). The above data supported the notion that increased lung Ly6Clo monocytes and CD16.2+ IM in RBP-J deficient mice were derived from Ly6Clo blood monocytes. In summary, these results suggested that in RBP-J deficient mice, recruitment of blood Ly6Clo monocytes to the lung was markedly facilitated by increased cell number as well as heightened expression of CCR2, leading to the increase of lung Ly6Clo monocytes and CD16.2+ IM.
In this study, we identified RBP-J as a pivotal factor in regulating blood Ly6Clo monocyte cell fate. Our results showed that mice with conditional deletion of RBP-J in myeloid cells exhibited a robust increase in blood Ly6Clo monocytes, and subsequently accumulated lung Ly6Clo monocytes and CD16.2+ IM under steady-state conditions. Bone marrow transplantation experiments in which RBP-J deficient cells were transplanted into recipient mice as well as the parabiosis experiment showed similar increase in blood Ly6Clo monocytes, demonstrating that RBP-J was an intrinsic factor required for the regulation of Ly6Clo monocytes. Further analysis revealed that RBP-J regulated the expression of CCR2 and CD11c on the surface of Ly6Clo monocytes. Moreover, the phenotype of elevated blood Ly6Clo monocytes and their progeny was further ameliorated by deleting Ccr2 in an RBP-J deficient background.
Notch-RBP-J signaling have been shown to play a role in regulating functional polarization and activation of macrophages, and regulated formation of Kupffer cells and macrophage differentiation from Ly6Chi monocytes in ischemia42-48. At steady-state, interaction of DLL1 with Notch 2 regulates conversion of Ly6Chi monocytes into Ly6Clo monocytes in special niches of the BM and spleen49. Under inflammatory conditions, Notch2 and TLR7 pathways independently and synergistically promote conversion of Ly6Chi monocytes into Ly6Clo monocytes50. However, in our study, the percentage of blood Ly6Clo monocytes increased in RBP-J deficient mice. These results somewhat differ from what was observed in mice with conditional deletion of Notch249. Actually, 4 members of the Notch family have been identified in mammals26. Thus, Notch receptors seem to be non-redundant in regulating monocyte cell fate, and distinct Notch receptors are likely to act in a concerted manner to coordinate the monocyte differentiation program.
Colony-stimulating factor 1 receptor (CSF1R) signal, CX3CR1, C/EBPβ and Nr4a1 have been suggested to be involved in the generation and survival of Ly6Clo monocytes. The lifespan of Ly6Clo monocytes is acutely shortened after blockade of CSF1R, and the numbers of these cells are reduced, and the percentage of dead cells increased in mice with endothelial cell-specific depletion of Csf123,24. CX3CR1 and C/EBPβ-knockout mice show accelerated death of Ly6Clo monocyte, display the decreased level of Ly6Clo monocytes22,51. Nr4a1 knockout mice had significantly fewer monocytes in blood, bone marrow, and spleen, due to differentiation deficiency in MDP and accelerated apoptosis of Ly6Clo monocytes in bone marrow21. RBP-J deficient mice had normal MDP and Ly6Clo monocytes, expressed normal level of Nr4a1 and Ki-67, exhibited normal half-life and turnover rate, which suggested that RBP-J may not regulate Ly6Clo monocytes through these factors. Further studies are required to elucidate the precise molecular mechanisms of RBP-J in Ly6Clo monocytes under steady state.
At the steady state, Ly6Clo monocytes patrol endothelium of blood vessel. After R848 induced endothelial injury, Ly6Clo monocytes recruit neutrophils to mediate focal endothelial necrosis and fuel vascular inflammation, and subsequently, the Ly6Clo monocytes remove cellular debris9,52. In response to Listeria monocytogenes infection, Ly6Clo monocytes can extravasate but do not give rise to macrophages or DCs8. Additional evidence shows that Ly6Clo monocytes do not merely act as luminal blood macrophages. In lung, exposure to unmethylated CpG DNA expands CD16.2+ IM, which originate from Ly6Clo monocytes and spontaneously produce IL-10, thereby preventing allergic inflammation41. A recent study shows that Ly6Clo monocytes give rise to CD9+ macrophages, which provide an intracellular replication niche for Salmonella Typhimurium in spleen, and Ly6Clo-depleted mice are more resistant to Salmonella Typhimurium infection, suggesting that Ly6Clo monocytes exert certain functions in systemic infection53. Our data provide evidence for RBP-J in the function of blood Ly6Clo monocytes, as RBP-J deficient Ly6Clo monocytes exhibited enhanced competition in blood circulation, as well as gave rise to increased numbers of lung CD16.2+ IM. In summary, we showed that RBP-J acted as a fundamental regulator of the maintenance of blood Ly6Clo monocytes and their descendent, at least in part through regulation of CCR2. These results provide insights into understanding the mechanisms that regulate monocyte homeostasis and function.
We thank the core facility at Institute for Immunology, Tsinghua University for valuable technical assistance.
This research was supported by National Natural Science Foundation of China grants (31821003, 31991174, 32030037 and 82150105 to X. Hu), a Ministry of Science and Technology of China grant (2020YFA0509100 to X. Hu), and funds from Tsinghua-Peking Center for Life Sciences and Institute for Immunology at Tsinghua University (to X. Hu).
T. Kou designed and performed experiments, analyzed and interpreted data, and wrote the manuscript. L. Kang designed and performed experiments, analyzed and interpreted data. B. Zhang provided advice on RNA-seq analyses. J. Li performed experiments. B. Zhao provided advice on experiments. W. Zeng provided advice on experiments. X. Hu conceptualized the project, supervised experiments, interpreted data, and wrote the manuscript.
Declaration of interests
The authors declare no competing interests.
Sequencing data sets are deposited in the Genome Expression Omnibus with assigned accession numbers as follows: RNA-seq (GEO accession no. GSE208772).
Materials and methods
Cx3cr1gfp/gfpmice (JAX stock 005582; Jung et al., 2000) and Ccr2RFP/RFP mice (JAX stock 017586) were purchased from the Jackson Laboratory. Mice with a myeloid-specific deletion of the Rbpj were generated by crossing Rbpjfl/fl mice to Lyz2-Cre mice as described previously (Hu et al., 2008). Rbpjfl/flLyz2cre/cre were crossed to Cx3cr1gfp/gfpmice to obtain Lyz2cre/creCx3cr1gfp/+and Rbpjfl/flLyz2cre/creCx3cr1gfp/+ mice. Cx3cr1gfp/+ mice were obtained by crossing Cx3cr1gfp/gfpwith C57/BL6 CD45.1+ mice. Rbpjfl/flLyz2cre/cre mice were crossed with Ccr2RFP/RFP mice to obtain Lyz2cre/creCcr2RFP/+, Lyz2cre/creCcr2RFP/RFP, Rbpjfl/flLyz2cre/creCcr2RFP/+ and Rbpjfl/flLyz2cre/creCcr2RFP/RFP mice. All mice were maintained under specific pathogen-free conditions. All animal experimental protocols were approved by the Institutional Animal Care and Use Committees of Tsinghua University. Gender- and age-matched mice were used at 7–12 weeks old for experiments.
Blood monocytes were sorted by flow cytometry. The total RNA was extracted using total RNA purification kit (GeneMarkbio) and reversely transcribed to cDNA by M-MLV Reverse Transcriptase (Takara). qPCR was performed on a real-time PCR system (StepOnePlus; Applied Biosystems) using FastSYBR mixture (CWBIO). Gapdh messenger RNA was used as internal control to normalize the expression of target genes. Primer sequences are provided in supplemental Table 1.
Ly6Chi and Ly6Clo blood monocytes were isolated from Rbpj+/+Lyz2cre/cre and Rbpjfl/flLyz2cre/cre mice, and total RNA was extracted using total RNA purification kit (GeneMarkbio). RNA was converted into RNA-seq libraries, which were sequenced with the pair-end option using an Illumina-HiSeq2500 platform at Beijing Genomics Institute (BGI), China. The significantly down-regulated genes were identified with P value <0.05 and (FPKM + 1) fold changes ≤0.2, and significantly up-regulated genes were identified with P value <0.05 and (FPKM + 1) fold changes ≥5.7. The RNA-seq data are deposited in Gene Expression Omnibus under accession number GSE208772
Annexin V staining
Annexin V and 7-amino-actinomycin D (7-AAD) were used for identification of apoptotic monocytes by flow cytometry. Blood cells were stained with annexin V (eBioscience) and 7-AAD (Biolegend) according to the manufacturers’ protocols.
EdU pulsing and latex beads labeling
Mice were injected intravenously with a single 1mg EdU (Thermo Scientific). BM and blood cells were collected and stained with Fluorescence-conjugated mAb against CD45, CD11b, Ly6G, CD115 and Ly6C. Cells were then fixed, permeabilized and stained with reaction cocktail using EdU Assay Kit. Labeled cells were analyzed by flow cytometry.
Mice were injected intravenously with a single 10 µL Latex beads (Polysciences) in 250 µl PBS. Blood cells were harvested at indicated time. Cells were then stained for CD45, CD11b, Ly6G, CD115, Ly6C, and analyzed by flow cytometry.
In vivo labeling of sinusoidal leukocytes
Mice were injected intravenously with 1 μg of PE-conjugated anti-CD45 antibodies. Two minutes after antibody injection, mice were sacrificed, and bone marrow were harvested. BM cells were then stained and analyzed by flow cytometry.
Generation of BM chimera
C57/BL6 CD45.2+ mice were lethally irradiated in two doses of 5.5Gy 2 h apart. 0.8 × 105 BM cells from Rbpj+/+Lyz2cre/cre mice (CD45.2+) or Rbpjfl/flLyz2cre/cre mice (CD45.2+) were mixed with 3.2× 105 BM cells from Cx3cr1gfp/+ mice (CD45.1+), and injected intravenously into recipient mice. Mice were used for experiments 8 weeks after irradiation.
BM GFP+Ly6Chi monocytes were sorted from Lyz2cre/creCx3cr1gfp/+or Rbpjfl/flLyz2cre/creCx3cr1gfp/+ mice, and transferred to Ccr2RFP/RFP mice. Blood cells were collected 60 hours later for flow cytometry analyses.
Cx3cr1gfp/+CD45.1+ mice were surgically joined with age-matched female Rbpjfl/flLyz2cre/cre or Rbpj+/+Lyz2cre/cre CD45.2+ mice at the age of 6 weeks. Bloods were obtained via cardiac puncture, and cell populations were analyzed by flow cytometry at 4 weeks after the surgery.
Intranasal instillations of LPS
Rbpj+/+Lyz2cre/cre or Rbpjfl/flLyz2cre/cre mice were anesthetized with isoflurane and intranasally instilled with 10 μg LPS in 25 μl of PBS. Lungs were harvested 4 days later for flow cytometry analyses.
Lungs from Rbpjfl/flLyz2cre/creCx3cr1gfp/+ and Lyz2cre/creCx3cr1gfp/+ mice were fixed in 1% paraformaldehyde and incubated in 30% sucrose separately overnight at 4℃. The samples were then incubated in the mixture of 30% sucrose and OTC compound (Sakura Finetek) overnight at 4℃. The tissues were embedded and frozen in OCT compound and then cut at 10-µm thickness. Tissue sections were dried for 10 min at 50℃ and then fixed in 1% paraformaldehyde at room temperature for 10 min, permeabilized in PBS/0.5% Triton X-100/0.3 M glycine for 10 min and blocked in PBS/5% goat serum for 1 h at room temperature. Sections were stained with rabbit anti-mouse GFP antibodies (1:1000; EASYBIO) overnight at 4℃, and washed with PBS/0.1% Tween-20 for 30 min three times at room temperature. Sections were then incubated with AF488-conjugated goat anti-rabbit antibodies (1:200; proteintech) for 2 h at room temperature, and washed with PBS/0.1% Tween-20 for 30 min three times at room temperature. Sections were stained with DAPI (Solarbio) for 7 min, washed in PBS for 8 min two times at room temperature and mounted with SlowFade Diamond Antifade Mountant (Life Technologies).
Cell isolation and Flow cytometry
Peripheral blood was sampled by eyeball extirpating, spleens were mashed through a 70-μm strainer, and bone marrow cells were collected from femurs. Lungs were perfused with 5ml of HBSS (MACGENE) through the right ventricle, excised, and digested in HBSS containing 5% FBS, 1 mg/ml collagenase type I (Sigma-Aldrich) and 0.05 mg/ml DNase I (Sigma-Aldrich) for 1 h at 37 °C. The digested tissues were homogenized by shaking, passed through a 70-µm cell strainer to create a single cell suspension. The suspension was enriched in mononuclear cells and harvested from the 1.080:1.038 g ml-1 interface using a density gradient (Percoll from GE Healthcare). BM, Spleen, PB and lung cells were stained with fluorescently conjugated antibodies. The absolute number of cells was counted by using CountBright™ Absolute Counting Beads (Invitrogen).
Antibodies against CD45 (30-F11), Ly6C (HK1.4), CD64 (X54-5/7.1), CD206 (C068C2), CD16.2 (9E9), CD45.1 (A20), F4/80 (BM8), Biotin, CCR2 (SA203G11) and CD3ε (145-2C11) were purchased from BioLegend. Antibodies against Ly6G (1A8-Ly6g), CD11b (M1/70), CD115(AFS98), CD117(2B8), CD135 (A2F10), CD11c (N418), CD206 (MR6F3), CD45.2 (104), Nur77 (12.14), Ki-67 (SolA15) and lineage maker were purchased from eBioscience. Fluorescence-conjugated mAb against Ly6C (AL-21) were purchased from BD Biosciences. Isotype-matched antibodies (eBioscience) were used for control staining. All antibodies were used in 1:400 dilutions, and surface antigens were stained on ice for 30 min.
For intracellular staining, cells were stained with antibodies to surface antigens, fixed and permeabilized with the Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences), and stained with antibodies or isotype control diluted in permeabilization buffer separately for 30 min at room temperature.
Cells were analyzed on FACSFortessa or FACSAria III flow cytometer (BD Biosciences) using FlowJo software.
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