Bcl6, Irf2, and Notch2 promote nonclassical monocyte development

Abstract

Ly6C^lo monocytes are a myeloid subset that specializes in the surveillance of vascular endothelium. Ly6C^lo monocytes have been shown to derive from Ly6C^hi monocytes. NOTCH2 signaling has been implicated as a trigger for Ly6C^lo monocyte development, but the basis for this effect is unclear. Here, we examined the impact of NOTCH2 signaling of myeloid progenitors on the development of Ly6C^lo monocytes in vitro. NOTCH2 signaling induced by delta-like ligand 1 (DLL1) efficiently induced the transition of Ly6C^hi TREML4^- monocytes into Ly6C^lo TREML4⁺ monocytes. We further identified two additional transcriptional requirements for development of Ly6C^lo monocytes. Deletion of BCL6 from myeloid progenitors abrogated development of Ly6C^lo monocytes. IRF2 was also required for Ly6C^lo monocyte development in a cell-intrinsic manner. DLL1-induced in vitro transition into Ly6C^lo TREML4⁺ monocytes required IRF2 but unexpectedly could occur in the absence of NUR77 or BCL6. These results imply a transcriptional hierarchy for these factors in controlling Ly6C^lo monocyte development.

Keywords: Bcl6; IRF2; Notch2; nonclassical monocytes.

Fig. 1.

Notch stimulation induces Ly6C^– TREML4⁺ CD11c⁺ nonclassical monocytes. (A) Peripheral blood was analyzed from wild-type or Nr4a1^–/– (Nr4a1^KO) mice. Shown analysis is pregated on CD45⁺ single cells. (B) Total monocytes (gated as CD45⁺ CD11b⁺ CD115⁺ as in A) are shown as a fraction of CD45⁺ single cells. (C) Peripheral blood was analyzed from wild-type or Nr4a1^KO mice. Shown analysis is pregated on CD45⁺ CD11b⁺ CD115⁺ single cells. (D) Ly6C^hi classical monocytes (gated as Ly6C⁺ TREML4^– CD45⁺ CD11b⁺ CD115⁺) are shown as a fraction of CD45⁺ single cells. (E) Ly6C^– TREML4⁺ cells are shown as a fraction of CD45⁺ CD11b⁺ CD115⁺ cells. (F) Peripheral blood was analyzed from wild-type or Nr4a1^KO mice. Shown analysis is pregated on CD45⁺ CD11b⁺ CD115⁺ single cells. (G) Ly6C^– CD11c⁺ cells are shown as a fraction of CD45⁺ CD11b⁺ CD115⁺ cells. (H) MDPs were sorted as Lin⁻ CD45⁺ CD115⁺ CD117⁺ CD135⁺ and cocultured with OP9 or OP9-DLL1 stromal cells in SCF, IL-3, and IL-6 for 2 days. Shown is FACS analysis of CD11b⁺ CD115⁺ MerTK^lo CD64^lo cells. (I) Ly6C^– TREML4⁺ cells are shown as a fraction of cultured monocytes (gated as CD11b⁺ CD115⁺ MerTK^lo CD64^lo). (J) MDPs were sorted as Lin⁻ CD45⁺ CD115⁺ CD117⁺ CD135⁺ and cocultured with OP9 or OP9-DLL1 stromal cells in SCF, IL-3, and IL-6 for 2 days. Shown is FACS analysis of CD11b⁺ CD115⁺ MerTK^lo CD64^lo cells. (K) Ly6C^– CD11c⁺ cells are shown as a fraction of cultured monocytes (gated as CD11b⁺ CD115⁺ MerTK^lo CD64^lo). Bars represent average values ± SEM. **P < 0.01, ****P < 0.0001, n.s. not significant (Student’s t test).

Fig. 2.

Characterization of differentially expressed transcription factors in nonclassical monocytes. (A) cMoPs (sorted as Lin⁻ CD45⁺ CD115⁺ CD117⁺ CD135⁻) and Ly6C^hi monocytes (sorted as Lin⁻ CD45⁺ CD115⁺ CD117⁻ CD135⁻ Ly6C⁺ TremL4⁻) were isolated from bone marrow and cocultured with OP9 or OP9-DLL1 stromal cells in SCF, IL-3, and IL-6 for 6 h before isolation and bulk RNA sequencing. Shown are 20 genes with the greatest fold increase in OP9-DLL1-cocultured Ly6C^hi monocytes relative to OP9-cocultured Ly6C^hi monocytes among significantly differentially expressed genes (Benjamini–Hochberg adjusted P-value < 0.05). (B) Volcano plot comparing gene expression in OP9-DLL1-cocultured Ly6C^hi monocytes to gene expression in OP9-cocultured Ly6C^hi monocytes. (C and D) Lin^– (CD3ε, CD19, CD105, Ly6G, Ter119) CD117⁺ progenitor cells were sorted and transduced with plasmids expressing a GFP reporter and either no gene (Empty Vector), the intracellular domain of Notch2 (NICD), or Hes1 (HES1). Cells were cultured in SCF, IL-3, and IL-6 for 2 d, then subjected to FACS analysis. Shown populations are pregated on CD11b⁺ CD115⁺ MerTK^lo CD64^lo single cells. (E and F) Ly6C^– TREML4⁺ or Ly6C⁻ CD11c⁺ cells, respectively, are shown as a fraction of GFP⁺ CD11b⁺ CD115⁺ MerTK^lo CD64^lo cells. (G) Gene expression microarray analysis of MDPs, cMoPs, Ly6C^hi monocytes, and Ly6C^lo monocytes sorted from bone marrow. Shown are select transcription factors known to influence myeloid development. Bars represent average values ± SEM. ****P < 0.0001, n.s. not significant (Student’s t test).

Fig. 3.

BCL6 is required for nonclassical monocytes. (A) Peripheral blood was analyzed from Bcl6^flox/flox (Bcl6^flox) or Bcl6^flox/flox Csf1r-cre⁺ (Bcl6^cKO) mice for total monocytes. Shown analysis is pregated on CD45⁺ single cells. (B) Quantification of Ly6C^hi classical monocytes (gated as Ly6C⁺ TREML4⁻ CD11b⁺ CD115⁺ CD45⁺ single cells) per 100 µL peripheral blood. (C) Peripheral blood was analyzed as in A for Ly6C^lo monocytes. Shown analysis is pregated on CD11b⁺ CD115⁺ CD45⁺ single cells. (D) Quantification of Ly6C^lo nonclassical monocytes (gated as Ly6C⁻ TREML4⁺ CD11b⁺ CD115⁺ CD45⁺ single cells) per 100 µL peripheral blood. (E) Peripheral blood was analyzed as in A for peripheral B cells. Shown analysis is pregated on CD45⁺ single cells. (F) Quantification of peripheral B cells (gated as CD45R⁺ MHCII⁺ CD45⁺ single cells) per 100 µL peripheral blood. (G) Peripheral blood was analyzed as in A for neutrophils. Shown analysis is pregated on CD45⁺ single cells. (H) Quantification of neutrophils (gated as CD11b⁺ Ly6G⁺ CD45⁺ single cells) per 100 µL peripheral blood. (I) Bone marrow was analyzed from Bcl6^flox or Bcl6^cKO mice for Ly6C^lo monocytes. Shown analysis is pregated on Lin⁻ (CD3ε, CD19, CD105, Ly6G, Ter119) CD45⁺ CD115⁺ CD117⁻ CD135⁻ single cells. (J and K) Quantification of Ly6C^lo monocytes (gated as Lin⁻ CD45⁺ CD115⁺ CD117⁻ CD135⁻ Ly6C⁻ TREML4⁺) or Ly6C^hi monocytes (gated as Lin⁻ CD45⁺ CD115⁺ CD117⁻ CD135⁻ Ly6C⁺ TREML4⁻) as a function of total bone marrow CD45⁺ cells. (L) Bone marrow was analyzed as in I for MDPs and cMoPs. Shown analysis is pregated on Lin⁻ CD45⁺ CD115⁺ single cells. (M) Quantification of MDPs (gated as Lin⁻ CD45⁺ CD115⁺ CD117⁺ CD135⁺) or cMoPs (gated as Lin⁻ CD45⁺ CD115⁺ CD117⁺ CD135⁻) as a function of total bone marrow CD45⁺ cells. Bars represent average values ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s. not significant (Student’s t test).

Fig. 4.

IRF2 is required for nonclassical monocytes. (A) Peripheral blood was analyzed from Irf2^+/− (Irf2^het) or Irf2^–/– (Irf2^KO) mice for total monocytes. Shown analysis is pregated on CD45⁺ single cells. (B) Quantification of Ly6C^hi classical monocytes (gated as Ly6C⁺ TREML4⁻ CD11b⁺ CD115⁺ CD45⁺ single cells) per 100 µL peripheral blood. (C) Peripheral blood was analyzed as in A for Ly6C^lo monocytes. Shown analysis is pregated on CD11b⁺ CD115⁺ CD45⁺ single cells. (D) Quantification of Ly6C^lo nonclassical monocytes (gated as Ly6C⁻ TREML4⁺ CD11b⁺ CD115⁺ CD45⁺ single cells) per 100 µL peripheral blood. (E) Peripheral blood was analyzed as in A for peripheral B cells. Shown analysis is pregated on CD45⁺ single cells. (F) Quantification of peripheral B cells (gated as CD45R⁺ MHCII⁺ CD45⁺ single cells) per 100 µL peripheral blood. (G) Peripheral blood was analyzed as in A for neutrophils. Shown analysis is pregated on CD45⁺ single cells. (H) Quantification of neutrophils (gated as CD11b⁺ Ly6G⁺ CD45⁺ single cells) per 100 µL peripheral blood. (I) Bone marrow was analyzed from Irf2^het or Irf2^KO mice for Ly6C^lo monocytes. Shown analysis is pregated on Lin⁻ (CD3ε, CD19, CD105, Ly6G, Ter119) CD45⁺ CD115⁺ CD117⁻ CD135⁻ single cells. (J and K) Quantification of Ly6C^lo monocytes (gated as Lin⁻ CD45⁺ CD115⁺ CD117⁻ CD135⁻ Ly6C⁻ TREML4⁺) or Ly6C^hi monocytes (gated as Lin⁻ CD45⁺ CD115⁺ CD117⁻ CD135⁻ Ly6C⁺ TREML4⁻) as a function of total bone marrow CD45⁺ cells. (L) Bone marrow was analyzed as in I for MDPs and cMoPs. Shown analysis is pregated on Lin⁻ CD45⁺ CD115⁺ single cells. (M) Quantification of MDPs (gated as Lin⁻ CD45⁺ CD115⁺ CD117⁺ CD135⁺) or cMoPs (gated as Lin⁻ CD45⁺ CD115⁺ CD117⁺ CD135⁻) as a function of total bone marrow CD45⁺ cells. Bars represent average values ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, n.s. not significant (Student’s t test).

Fig. 5.

The requirement for IRF2 in nonclassical monocytes is cell-intrinsic. (A) CD45.1-expressing wild-type mice were lethally irradiated and injected i.v. with congenically marked CD45.2⁺ wild-type or Irf2^KO bulk bone marrow. Spleen was analyzed 8 wk later via FACS for Ly6C^lo monocytes. Shown analysis is pregated on CD45.2⁺ CD11b⁺ CD115⁺ cells. (B) Quantification of spleen Ly6C^lo monocytes (gated as CD45.2⁺ Ly6C⁻ TREML4⁺ CD11b⁺ CD115⁺) as a function of total monocytes (gated as CD45.2⁺ CD11b⁺ CD115⁺). (C) Bone marrow chimeras were set up as in A and bone marrow was analyzed 8 wk later via FACS for Ly6C^lo monocytes. Shown analysis is pregated on CD45.2⁺ Lin⁻ (CD3ε, CD19, CD105, Ly6G, Ter119) CD115⁺ CD117⁻ CD135⁻. (D) Quantification of bone marrow Ly6C^lo monocytes (gated as CD45.2⁺ Ly6C⁻ TREML4⁺ Lin⁻ CD115⁺ CD117⁻ CD135⁻) as a function of total monocytes (gated as CD45.2⁺ Lin⁻ CD115⁺ CD117⁻ CD135⁻). (E) Bone marrow chimeras were set up as in A and peripheral blood was analyzed 8 wk later via FACS for Ly6C^lo monocytes. Shown analysis is pregated on CD45.2⁺ CD11b⁺ CD115⁺ single cells. (F) Quantification of peripheral blood Ly6C^lo monocytes (gated as CD45.2⁺ Ly6C⁻ TREML4⁺ CD11b⁺ CD115⁺) as a function of total monocytes (gated as CD45.2⁺ CD11b⁺ CD115⁺). Bars represent average values ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001 (Student’s t test).

Fig. 6.

IRF2 but not NUR77 or BCL6 are required for Notch-induced nonclassical monocyte development. (A) Sort-purified wild-type or Nr4a1^KO MDPs (sorted as Lin⁻ CD45⁺ CD115⁺ CD117⁺ CD135⁺) were cocultured with OP9 or OP9-DLL1 stromal cells in SCF, IL-3, and IL-6 for 2 d. Shown is FACS analysis of CD11b⁺ CD115⁺ MerTK^lo CD64^lo cells. (B) Ly6C⁻ TREML4⁺ cells are shown as a fraction of cultured monocytes (gated as CD11b⁺ CD115⁺ MerTK^lo CD64^lo). (C) Sort-purified Bcl6^flox or Bcl6^cKO MDPs were cocultured with OP9 or OP9-DLL1 stromal cells in SCF, IL-3, and IL-6 for 2 d. Shown is FACS analysis of CD11b⁺ CD115⁺ MerTK^lo CD64^lo cells. (D) Ly6C⁻ TREML4⁺ cells are shown as a fraction of cultured monocytes (gated as in B). (E) Sort-purified Irf2^het or Irf2^KO MDPs were cocultured with OP9 or OP9-DLL1 stromal cells in SCF, IL-3, and IL-6 for 2 d. Shown is FACS analysis of CD11b⁺ CD115⁺ MerTK^lo CD64^lo cells. (F and G) Ly6C⁻ TREML4⁺ and Ly6C⁺ TREML4⁺ cells, respectively, are shown as a fraction of cultured monocytes (gated as in B). Bars represent average values ± SEM. **P < 0.01, n.s. not significant (Student’s t test).

Fig. 7.

Loss of IRF2 reduces Bcl6 and Nr4a1 expression in Ly6C^hi monocytes. (A) Ly6C^hi monocytes were obtained from WT, Nr4a1^KO Bcl6^cKO, and Irf2^KO BM and subjected to RNA sequencing. Shown is a heat map displaying differentially expressed genes organized by hierarchical clustering. (B) Volcano plot comparing gene expression in WT BM Ly6C^hi monocytes to gene expression in Nr4a1^KO BM Ly6C^hi monocytes, as determined by RNA-Seq. (C) Volcano plot comparing gene expression in WT BM Ly6C^hi monocytes to gene expression in Bcl6^cKO BM Ly6C^hi monocytes, as determined by RNA-Seq. (D) Volcano plot comparing gene expression in WT BM Ly6C^hi monocytes to gene expression in IRF2^KO BM Ly6C^hi monocytes, as determined by RNA-Seq.