Immunity Article Stroma-Derived Interleukin-34 Controls the Development and Maintenance of Langerhans Cells and the Maintenance of Microglia Melanie Greter,1,9,* Iva Lelios,1,9 Pawel Pelczar,2 Guillaume Hoeffel,3 Jeremy Price,4,5 Marylene Leboeuf,4,5 Thomas M. Ku¨ndig,7 Karl Frei,8 Florent Ginhoux,3 Miriam Merad,4,5,6,10,* and Burkhard Becher1,10 1Institute of Experimental Immunology, Neuroimmunology 2Institute of Laboratory Animal Science University of Zu¨ rich, CH 8006 Zu¨ rich, Switzerland 3Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (ASTAR), BIOPOLIS, 138648, Singapore 4Department of Oncological Sciences 5The Immunology Institute 6Tisch Cancer Institute Mount Sinai School of Medicine, New York, NY 10029, USA 7Clinical Tumor Biology & Immunotherapy Unit 8Department of Neurosurgery University Hospital Zu¨ rich, 8091 Zu¨ rich, Switzerland 9These authors contributed equally to this work 10These authors contributed equally to this work *Correspondence: [email protected] (M.G.), [email protected] (M.M.) http://dx.doi.org/10.1016/j.immuni.2012.11.001 SUMMARY the first immune barrier to foreign antigens that breach the skin. Microglia represent the resident macrophages of the Colony stimulating factor-1 (Csf-1) receptor and its CNS and are considered the first line of defense in many CNS ligand Csf-1 control macrophage development, main- pathologies. Most lymphoid and nonlymphoid tissue DCs and tenance, and function. The development of both macrophages are constantly repopulated by blood-derived Langerhans cells (LCs) and microglia is highly depen- circulating myeloid precursors. In contrast, epidermal LCs and dent on Csf-1 receptor signaling but independent microglia derive from embryonic precursors that take residence of Csf-1. Here we show that in both mice and in the skin and brain prior to birth and are maintained locally throughout adult life in the steady state (Ginhoux et al., 2010; humans, interleukin-34 (IL-34), an alternative ligand Hoeffel et al., 2012; Merad et al., 2002). Apart from the cytokine for Csf-1 receptor, is produced by keratinocytes in transforming growth factor b (TGF-b), which is essential for the the epidermis and by neurons in the brain. Mice lack- maintenance of LCs (Kaplan et al., 2007), the local cutaneous ing IL-34 displayed a marked reduction of LCs and growth factors that control LC homeostasis are only now being a decrease of microglia, whereas monocytes, dermal, discovered (Wang et al., 2012). As for microglia, the factors that and lymphoid tissue macrophages and DCs were govern their development and maintenance remain largely unaffected. We identified IL-34 as a nonredundant unclear. cytokine for the development of LCs during embryo- Csf-1 is a critical growth factor for the generation, differentia- genesis as well as for their homeostasis in the adult tion, and survival of macrophages and monocytes (Dai et al., skin. Whereas inflammation-induced repopulation 2002; Wiktor-Jedrzejczak et al., 1990). Mice lacking Csf-1 of LCs appears to be dependent on Csf-1, once receptor (Csf-1r) or bearing a spontaneous null mutation in Csf-1 (Csf1op/op) display greatly reduced macrophage popula- inflammation is resolved, LC survival is again IL-34- tions in various tissues including osteoclasts, leading to multiple dependent. In contrast, microglia and their yolk sac developmental defects such as impaired bone remodeling, precursors develop independently of IL-34 but rely osteopetrosis, and tooth eruption (Dai et al., 2002). In particular, on it for their maintenance in the adult brain. mice deficient in Csf-1r also lack epidermal LCs and microglia (Ginhoux et al., 2006, 2010). Interestingly however, in contrast to Csf1rÀ/À mice, Csf1op/op mice display relatively normal LC INTRODUCTION and microglia development (Ginhoux et al., 2006), suggesting that an alternative ligand for Csf-1r controls the maintenance In both mice and humans, the epidermis and the central of these two cell populations in vivo. Interleukin-34 (IL-34) nervous system (CNS) are colonized by homogenous and onto- has been identified as an additional ligand for Csf-1r and was genically unique populations of the mononuclear phagocyte shown to bind Csf-1r with high affinity in vitro (Garceau et al., system (MPS), namely Langerhans cells (LCs) and microglia. 2010; Lin et al., 2008). IL-34 is more conserved in mammalian LCs are the dendritic cells (DCs) of the epidermis and form and avian species than Csf-1 (Garceau et al., 2010). IL-34 and 1050 Immunity 37, 1050–1060, December 14, 2012 ª2012 Elsevier Inc. Immunity IL-34 Controls Langerhans Cells and Microglia -/- P1 - + - D ) A Csf1r ) x Csf1r x Csf1r Control LangCre LangCre 2 x Csf1r 2 250 120 LangCre *** Control + 5 5 105 105 LangCre x Csf1r 10 10 Csf1r-/- 100 47.5 2.36 200 54.8 9.1 10.458.1 4 4 104 104 10 10 80 150 60 3 3 103 103 100 10 10 R2R1 40 2 2 CD11b CD11b 102 102 50 10 10 20 0 0 0 0 0 number (x10 Cell 0 0102 103 104 105 0102 103 104 105 0102 103 104 105 0102 103 104 105 R1 R2 MHCII Langerin op/op P5 - + ) Csf1 ) number (x 10 Cell x Csf1r x Csf1r - 2 2 LangCre LangCre B Control Control 200 LangCre x Csf1r 5 5 + 10 10 60 Csf1op/op 105 105 LangCre x Csf1r 55.8 53.7 66.8 38.3 150 4 4 4 4 10 10 40 10 10 100 3 3 10 10 103 103 20 2 2 CD11b CD11b 2 2 50 10 10 10 10 0 0 0 0 0 number (x10 Cell 2 3 4 5 2 3 4 5 0 010 10 10 10 010 10 10 10 0102 103 104 105 0102 103 104 105 MHCII Langerin + P10 - + C x Csf1r ) number (x 10 Cell LangCre x Csf1r LangCre x Csf1r 2 Control LangCre 60 ) ** *** 2 - 5 5 Control 5 5 LangCre x Csf1r 10 10 + 10 10 60 30.8 0.91 LangCre x Csf1r 40.4 8.25 LangCre+x Csf1r 4 4 104 104 40 10 10 40 3 3 103 103 10 10 20 20 2 2 2 2 CD11b CD11b 10 10 10 10 0 0 0 0 Cell number (x 10 Cell 0 number (x10 Cell 0 2 3 4 5 2 3 4 5 0102 103 104 105 0102 103 104 105 010 10 10 10 010 10 10 10 MHCII Langerin E F T2- x Csf1r T2+ x Csf1r ) Isotype αCsf-1r ) CreER CreER 15 60 2 2 *** T2- 5 5 Isotype 5 5 Csf1r 10 10 10 30.5 10 1.01 CreER x 14 2.84 αCsf-1r CreERT2+ x Csf1r 4 4 10 10 40 104 104 10 3 3 10 10 103 103 20 5 2 2 CD11b 10 10 CD11b 102 102 0 0 0 0 Cell number (x10 Cell Cell number (x10 Cell 2 3 4 5 2 3 4 5 0 0 010 10 10 10 010 10 10 10 0102 103 104 105 0102 103 104 105 MHCII MHCII Figure 1. Csf-1r Signaling Is Critical for LC Homeostasis in Adult Skin (A–F) Plots show the percentage and total cell number (±SEM) of LCs among CD45+ epidermal cells in adult mice (A–C, E and F) or neonates on postnatal day (p)1, p5, and p10 (D). Graphs represent data of pooled experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test, unpaired). (A) Csf1rÀ/À and control mice (Csf1r+/À or Csf1r+/+)(nR 3). (B) Csf1op/op and control mice (Csf1op/+ or Csf1+/+) (n = 5). (C) Langerin (Lang) Cre+ 3 Csf1rfl/fl or control mice (LangCre+ 3 Csf1rfl/+ or LangCreÀ 3 Csf1rfl/fl) (n = 4). (D) LangCre+ 3 Csf1rfl/fl or LangCreÀ 3 Csf1rfl/fl neonates. On p1, R1 represents LC precursors (LangerinÀ) and R2 represents LCs (Langerin+). p2: n = 6, p5: n R 3, p10: n R 6. (E) Anti-(a)Csf-1r treated or isotype treated WT mice (n = 6). One representative of at least three individual experiments is shown. (F) Rosa26-CreERT2+ (CreERT2+) 3 Csf1rfl/fl and CreERT2À 3 Csf1rfl/fl mice were treated with tamoxifen and the skin was analyzed 7 days post treatment (n = 2). One representative of two individual experiments is shown. Csf-1 were shown to bind on different regions of the Csf-1r RESULTS and share no overt sequence homology (Chihara et al., 2010). IL-34 can promote the survival and proliferation of human Csf-1r Signaling Is Critical for LC Homeostasis monocytes and stimulate macrophage colony formation of As previously shown, Csf1op/op mice display only slightly human bone marrow (BM) cells (Lin et al., 2008). However, the reduced numbers of LCs whereas Csf1rÀ/À mice are completely role of IL-34 in the development and maintenance of LCs and devoid of LCs (Figures 1A and 1B) (Ginhoux et al., 2006). microglia in vivo is only now becoming clearer (Wang et al., However, in Csf1rÀ/À embryos, LC precursors are lacking 2012). Here, by generating IL-34 deficient reporter mice, we already in the embryonic skin (Hoeffel et al., 2012). Thus, in order demonstrated that IL-34 is crucial for the embryonic develop- to circumvent such developmental defect and to determine ment of LCs and their homeostasis in the adult in the steady whether Csf-1r signaling also controls LC homeostasis in the state. During skin inflammation, IL-34 is not required for the adult skin, we generated mice in which Csf-1r was specifically recruitment of monocytes and their subsequent differentiation deleted in Langerin+ cells. Within the epidermis, Langerin into LCs as was observed by Wang et al.