Identification of the Circulating Langerhans Cell Precursor.
Authors: Merad Miriam , Angeli Veronique , Tacke Franck , Bogunovic Milena , Frenette Paul S. , Randolph Gwendolyn J. , Ginhoux Florent 
Published: October 2019

Abstract
We have recently shown that by contrast to other dendritic cell populations, epidermal dendritic cells also called Langerhans cells (LCs) are maintained by a pool of local radio-resistant precursor cells that persist in quiescent skin throughout life (Merad et al. Nat Immunol (3): 1135. 2002). However, skin LC precursors are replaced by circulating LC precursors after major skin lesions such as exposure to ultraviolet light (UV) but not after minor injuries such as skin sensitization (Merad et al. Nat Immunol (3): 1135. 2002). To identify the developmental stage at which LC precursors seeds quiescent skin, we reconstituted sub-lethally irradiated mice between day 0 and day 2 of life with fetal liver cells isolated from E14.5 congenic mice embryos. No donor LCs were detected over a period of one year despite high level (80%) of blood leukocyte chimerim, suggesting that a first wave of LC precursors seeds the skin during embryonic life and lose their capacity to infiltrate quiescent skin at birth or soon after birth. To explore the homeostasis of LCs recruited in inflamed skin, we devised a model of tandem transplant in which (CD45.1+ BM >CD45.2+ mice) chimeric mice were exposed to UV light to induce major skin lesions and the recruitment of blood-borne CD45.1+ LCs to the skin. Six weeks later, chimeric mice were lethally irradiated again and injected with GFP+ CD45.2+ BM cells. Although more than 90% of B cells, monocytes and granulocytes were GFP+ CD45.2+ CD45.1--, 85% of LCs in the skin was GFP- CD45.2− CD45.1+ up to 6 months after the second transplant. Using Ki-67 staining, BrdU labeling studies and cell cycle analysis, we found that CD45.1+ LCs proliferate in situ at the same rate as LCs in naïve mice. We also found that after minor injury such as skin sensitization, 40% of LCs are lost and are replaced locally by CD45.1+ LC precursors. These results suggest that a second wave of LC precursors is recruited during adult life in inflamed skin and behave as their embryonic counterpart. Indeed, adult LC precursors are radio-resistant, self renew in situ and can repopulate and maintain the LC pool during steady state or minor injuries. To explore the nature of the circulating LC precursor, we labeled monocytes in vivo using intravenous injection of fluorescent latex beads and followed the capacity of beads+ monocytes to differentiate into LC precursors in inflamed skin. We found that a subset of monocytes (CCR2+ Ly6Chi) infiltrate the inflamed skin, proliferate in situ and differentiate into LC precursors in 10 days. Similarly, we found that purified Ly6Chi monocytes injected into a UV treated animal infiltrate inflamed skin and differentiate into LC precursors. Using transfer experiment of cells deficient in the chemokine receptor CCR2 or CCR6 into wild type animals exposed to UV light, we found that recruitment of Ly6Chi monocytes to the skin was dependent on CCR2 and independent of CCR6 but CCR2+ Ly6Chi monocytes require CCR6 to differentiate into LC precursors. To our knowledge this is the first time that circulating monocytes are shown to proliferate in inflamed skin and differentiate into a LC precursor that self-renew in situ throughout life.