Transcription factor Zeb2 regulates commitment to plasmacytoid dendritic cell and monocyte fate Xiaodi Wua, Carlos G. Briseñoa, Gary E. Grajales-Reyesa, Malay Haldara, Arifumi Iwataa, Nicole M. Kretzera, Wumesh KCa, Roxane Tussiwanda, Yujiro Higashib, Theresa L. Murphya, and Kenneth M. Murphya,c,1 aDepartment of Pathology and Immunology, School of Medicine, Washington University, St. Louis, MO 63110; bDepartment of Perinatology, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Aichi 480-0392, Japan; and cHoward Hughes Medical Institute, School of Medicine, Washington University, St. Louis, MO 63110 Contributed by Kenneth M. Murphy, November 14, 2016 (sent for review July 20, 2016; reviewed by Christophe Benoist and Richard A. Flavell) Dendritic cells (DCs) and monocytes develop from a series of bone- have impaired migration from tissues (12, 13). Notch2 is required for marrow–resident progenitors in which lineage potential is regu- cDC2s in the spleen and mesenteric lymph node (LN) to acquire lated by distinct transcription factors. Zeb2 is an E-box–binding expression of CD4 and ESAM and produce IL-23 in response to protein associated with epithelial–mesenchymal transition and is pathogens (14–16). Klf4 expression in cDC2s is required to induce widely expressed among hematopoietic lineages. Previously, we protective TH2 responses to Schistosoma mansoni infection (17). observed that Zeb2 expression is differentially regulated in pro- A recent study has argued that the transcription factor Zeb2 genitors committed to classical DC (cDC) subsets in vivo. Using (Sip1, Zfhx1b) regulates commitment to the cDC2 lineage by re- systems for inducible gene deletion, we uncover a requirement pression of Id2 (18). Zeb2 interacts with Smad proteins and contains for Zeb2 in the development of Ly-6Chi monocytes but not neutro- N- and C-terminal zinc finger domains flanking a Smad-binding phils, and we show a corresponding requirement for Zeb2 in ex- domain, homeodomain, and a C-terminal–binding protein in- pression of the M-CSF receptor in the bone marrow. In addition, teraction domain (19). Zeb2 represses E-cadherin and other com- we confirm a requirement for Zeb2 in development of plasmacy- ponents of cell junctions during epithelial–mesenchymal transition toid DCs but find that Zeb2 is not required for cDC2 development. (20, 21), and germline deletion of Zeb2 leads to embryonic lethality Instead, Zeb2 may act to repress cDC1 progenitor specification in in mice (22, 23). Heterozygous Zeb2 defects in humans are associ- the context of inflammatory signals. ated with Hirschprung’s disease and Mowat–Wilson syndrome, and Zeb2 expression is dysregulated in several human cancers (19). In monocyte | plasmacytoid dendritic cell | transcription factor the nervous system, Zeb2 controls myelination by modulating the activity of Smads activated by bone morphogenetic proteins, mem- endritic cells (DCs) comprise several related lineages that bers of the TGF-β superfamily (24). In oligodendrocyte precursors, Dinitiate and regulate immune responses (1). Classical DCs where Zeb2 expression is low in abundance, activated Smads bind (cDCs) present antigens to prime naive T cells and produce cyto- the coactivator histone acetyltransferase p300 and activate the ex- kines to activate T cells and innate lymphoid cells. They can be pression of negative regulatory genes such as Id2 and Hes1;by categorized into two distinct lineages, termed cDC1 and cDC2 (2), contrast, in differentiating oligodendrocytes, expression of Olig1 and that rely on different transcription factors for their development and Olig2 induces Zeb2, which binds Smad–p300 complexes and re- + function. The cDC1 subset includes lymphoid-resident CD8α presses expression of Id2 and Hes1 (24). Within the hematopoietic + system, Zeb2 cooperates with Tbx21 (T-bet) to promote terminal cDCs and tissue-resident CD103 cDCs that function in cross- + presentation of viral antigen and defense against intracellular patho- maturation of natural killer (NK) cells and CD8 T cells (25–27), gens. The cDC2 subset includes heterogeneous populations of and its inactivation results in broadly dysregulated hematopoiesis + CD172a (Sirp-α) cDCs that promote TH17-type responses to bac- with prominent neutrophilia and loss of B cells and monocytes (28). teria and fungi and TH2-type responses to parasites. Plasmacytoid DCs INFLAMMATION (pDCs) are a lineage distinct from cDCs identified by surface ex- Significance IMMUNOLOGY AND pression of CD45R (B220), Siglec-H, and CD317 (Bst2). They do not function directly in T-cell priming (3) but are specialized for produc- Distinct transcription factors regulate the development of im- tion of large quantities of type I IFN in response to infection (4–6). mune cell lineages, and changes in their expression can alter the DCs arise from a series of progenitors with progressively restricted balance of cell types responding to infection. Recent studies − + − − potential (1). Within lineage (Lin) Kit Sca-1 IL-7Rα bone marrow have identified Zeb2 as a transcription factor important for the + + (BM) cells, CD16/32 (FcγRII/III)loCD34 common myeloid pro- final maturation of natural killer cells and effector CD8 T cells. + genitors give rise to all myeloid lineages through FcγRII/IIIhiCD34 In this study, we show that Zeb2 is required for the development − granulocyte–macrophage progenitors (GMPs) and FcγRII/IIIloCD34 of two myeloid cell types, the monocyte and the plasmacytoid megakaryocyte–erythrocyte progenitors. Macrophage–DC pro- dendritic cell, and clarify that this factor is not required for the genitors (MDPs) differ from GMPs by decreased expression of Kit development of classical dendritic cells. and increased expression of the chemokine receptor CX3CR1. MDPs express the receptors M-CSFR and Flt3 and give rise Author contributions: X.W., W.K., T.L.M., and K.M.M. designed research; X.W., C.G.B., + + − + to Kit M-CSFR Flt3 Ly-6C –committed monocyte progenitors G.E.G.-R., M.H., A.I., N.M.K., R.T., and T.L.M. performed research; Y.H. contributed new int + + reagents/analytic tools; X.W., C.G.B., A.I., R.T., T.L.M., and K.M.M. analyzed data; and (7) and to Kit M-CSFR Flt3 common DC progenitors (CDPs). X.W. and K.M.M. wrote the paper. int − α− + From CDPs, pDCs develop via Kit M-CSFR IL-7R Flt3 Reviewers: C.B., Harvard Medical School; and R.A.F., Yale School of Medicine, Howard progenitors (8). Committed progenitors of cDCs also develop Hughes Medical Institute. from CDPs, and progenitors committed to either the cDC1 or the The authors declare no conflict of interest. cDC2 lineage have been identified in the BM and blood (9, 10). Freely available online through the PNAS open access option. Several transcription factors are required for development of DCs Data deposition: Gene expression microarray data have been deposited in the Gene (11). cDC1 development requires Irf8, Nfil3, Id2, and Batf3, whereas Expression Omnibus (accession nos. GSE87882, GSE87883, and GSE87884). pDC development requires Irf8 and Tcf4 (E2-2). cDC2 development 1To whom correspondence should be addressed. Email: [email protected]. was thought to require Irf4; however, recent analysis has shown that This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cDC2s develop in the absence of Irf4 but lack CD4 expression and 1073/pnas.1611408114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1611408114 PNAS | December 20, 2016 | vol. 113 | no. 51 | 14775–14780 Downloaded by guest on September 26, 2021 Previously, we and others have observed that Zeb2 is down- regulated upon specification of the CDP to the cDC1 lineage (9, 29). Id2 is induced by TGF-β and is required for development of cDC1s but is not required for development of cDC2s (30, 31). Furthermore, the balance between Id2 and E2-2 influences cDC1 and pDC development (32–34), and exogenous TGF-β applied to BM progenitors accelerates differentiation to cDCs rather than pDCs (35). Modest decreases in pDC and cDC2 frequency have been observed in mice with conditional deletion of Zeb2 in + CD11c cells, leading to the interpretation that Zeb2 regulates commitment of pDC and cDC2 lineages by controlling Id2 ex- pression (18). However, expression of CD11c occurs coordinately with lineage specification or, in the case of the committed cDC1 progenitor, actually occurs after specification (9). Thus, condi- + tional deletion of Zeb2 in CD11c cells may not fully eliminate the actions of that transcription factor during lineage specification. To address these issues, we used several systems to control the timing of Zeb2 deletion during DC development, and we find that, in contrast to Scott et al. (18), deletion in early progenitors regulates specification to the pDC lineage but not to the cDC2 lineage. This finding is consistent with reports that Id2 is required for the de- velopment of cDC1s but not cDC2s (30, 36). Finally, we found that loss of Zeb2 impaired both the expression of M-CSFR and the development of Ly-6Chi monocytes, implicating Zeb2 activity in the diversification of multiple myeloid lineages. Results We generated mice in which Zeb2 is conditionally deleted in cells expressing Cre recombinase driven by the Itgax promoter (CD11c- Cre) (14). Compared with Zeb2-sufficient (Zeb2fl/fl)mice,CD11c- Cre–driven Zeb2-deficient [Zeb2fl/fl;CD11c-Cre(tg)] mice showed substantially decreased pDC frequency in the spleen and skin- draining LN but not in the BM (Fig. 1A). Within the cDC com- partment, Zeb2-deficient mice showed an increased ratio of splenic cDC1s to cDC2s (identified by expression of CD24 and Sirp-α, respectively) and an increased ratio of equivalent cell types in other organs (Fig. 1 B and C). Neither spleen nor BM cellularity was significantly different between the two groups (Fig. S1). Using an inducible model of Zeb2 deficiency driven by the type I IFN- Fig. 1. Zeb2 is required for pDC development in vivo. (A)Samplesprepared inducible Mx1-Cre, pDCs were ablated in vivo 7–9 d after two from the spleen, skin-draining LN (SLN), and BM, harvested from mice of the treatments with poly(I:C) (Fig.