# Unveiling skin macrophage dynamics explains both tattoo persistence and strenuous removal

Anna Baranska, Alaa Shawket, Mabel Jouve, Myriam Baratin, Camille Malosse, Odessa Voluzan, Thien-Phong Vu Manh, Frédéric Fiore, Marc Bajénoff, Philippe Benaroch, et al.

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► To cite this version:

Anna Baranska, Alaa Shawket, Mabel Jouve, Myriam Baratin, Camille Malosse, et al. Unveiling skin macrophage dynamics explains both tattoo persistence and strenuous removal. Journal of Experimental Medicine, 2018, 215 (4), pp.1115-1133. 10.1084/jem.20171608. inserm-04871484

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# Unveiling skin macrophage dynamics explains both tattoo persistence and strenuous removal

Anna Baranska<fnref n="1" /> Ⓓ, Alaa Shawket<fnref n="1" />*, Mabel Jouve³*, Myriam Baratin<fnref n="1" />*, Ⓓ, Camille Malosse<fnref n="1" />, Odessa Voluzan<fnref n="1" />, Thien-Phong Vu Manh<fnref n="1" />, Frédéric Fiore² Ⓓ, Marc Bajénoff<fnref n="1" />, Philippe Benaroch⁴, Marc Dalod<fnref n="1" />, Marie Malissen<fnref n="1" />,² Ⓓ, Sandrine Henri<fnref n="1" />**, and Bernard Malissen<fnref n="1" />,²** Ⓓ

Here we describe a new mouse model that exploits the pattern of expression of the high-affinity IgG receptor (CD64) and allows diphtheria toxin (DT)-mediated ablation of tissue-resident macrophages and monocyte-derived cells. We found that the myeloid cells of the ear skin dermis are dominated by DT-sensitive, melanin-laden cells that have been missed in previous studies and correspond to macrophages that have ingested melanosomes from neighboring melanocytes. Those cells have been referred to as melanophages in humans. We also identified melanophages in melanocytic melanoma. Benefiting of our knowledge on melanophage dynamics, we determined the identity, origin, and dynamics of the skin myeloid cells that capture and retain tattoo pigment particles. We showed that they are exclusively made of dermal macrophages. Using the possibility to delete them, we further demonstrated that tattoo pigment particles can undergo successive cycles of capture-release-recapture without any tattoo vanishing. Therefore, congruent with dermal macrophage dynamics, long-term tattoo persistence likely relies on macrophage renewal rather than on macrophage longevity.

## Introduction

Recent studies, based on ontogeny and transcriptomic analysis, have succeeded in discriminating conventional dendritic cells (cDCs) from monocyte-derived cells. cDCs comprise two lineages known as cDC1 and cDC2 (Guilliams et al., 2016), have a short life span, and are continually replenished from committed cDC precursors that originate in the bone marrow (BM). Under steady-state conditions, BM-derived blood Ly-6Chigh monocytes extravasate at a slow pace into nonlymphoid tissues, such as the intestinal lamina propria and the skin dermis, where they give rise to two developmental streams (Tamoutounour et al., 2012, 2013; Bain et al., 2013; Jakubzick et al., 2013). Progression through the first developmental stream is associated with the preservation of CC-chemokine receptor 2 (CCR2) expression, the acquisition of MHCII molecules, and a diminution of Ly-6C expression. It can be visualized in Ly-6C-MHCII dot plots as a “monocyte waterfall,” the final product of which share features with cDC2 and is called monocyte-derived DC (mo-DCs; Tamoutounour et al., 2013). Blood Ly-6Chi monocytes extravasate at a higher pace into inflamed tissues (Tamoutounour et al., 2012, 2013; Bain et al., 2013) and give rise to cells that, because of their ability to produce TNFα and inducible NO synthase, are denoted as TIP-DCs (Serbina et al., 2003; De Trez et al., 2009; Tamoutounour et al., 2012). Under steady-state conditions, extravasated Ly-6Chigh

monocytes also proceed through a second developmental stream yielding tissue-resident macrophages (mo-Mac) that have lost expression of CCR2 and Ly-6C and express or not MHCII molecules (Jakubzick et al., 2013; Tamoutounour et al., 2013; Yona et al., 2013; Bain et al., 2014; McGovern et al., 2014). In the intestine and dermis, such mo-Mac replace in part or in totality a pool of macrophages that develops before the establishment of definitive hematopoiesis in a CCR2-independent manner (Tamoutounour et al., 2013; Bain et al., 2014; Sheng et al., 2015). Monocyte fate toward mo-Mac or mo-DCs is specified by microenvironmental cues (Goudot et al., 2017).

The possibility of discriminating cDCs from mo-DCs and macrophages emphasized their distinct functional specialization. Upon infection, antigen-laden cDCs migrate to draining LNs and trigger the conversion of antigen-specific naive T cells into effector T cells. In contrast, most mo-DCs remain in tissue parenchyma where they contribute to local immune responses and provide potent antimicrobial function during infection. Macrophages contribute to tissue homeostasis and wound repair because of their capacity to phagocyte cellular debris, invading organisms, and apoptotic cells. Owing to their strategic positioning at body barriers, macrophages capture a wide range of exogenous particulates. They also ingest self-derived particulates, as

<footnote n="1">¹Centre d'Immunologie de Marseille-Luminy, Aix Marseille Université, INSERM, CNRS UMR, Marseille, France; ²Centre d'Immunophénomique, Aix Marseille Université, INSERM, CNRS, Marseille, France; ³UMR3215, Institut Curie, Paris, France; ⁴Institut Curie, PSL Research University, INSERM U932, Paris, France.</footnote>

<footnote>*A. Shawket, M. Jouve, and M. Baratin contributed equally to this paper; **S. Henri and B. Malissen contributed equally to this paper; Correspondence to Bernard Malissen: bernardm@ciml.univ-mrs.fr; Sandrine Henri: henri@ciml.univ-mrs.fr; A. Baranska's present address is Miltenyi Biotec GmbH, Bergisch Gladbach, Germany.</footnote>

<footnote>© 2018 Baranska et al. This article is distributed under the terms of an Attribution-Noncommercial-Share Alike-No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution-Noncommercial-Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).</footnote>
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<figure><img src="image_4.png" /><figcaption>**Figure 1.** Replenishment kinetics of monocytes, mo-DCs, and macrophages in the dermis of CD64<sup>dtr</sup> mice after DT treatment. CD64<sup>dtr</sup> mice were left untreated (-DT) or treated twice and 24 h apart with DT. Analysis of the CD64<sup>+</sup>CCR2<sup>+</sup> monocytes and mo-DCs and CD64<sup>+</sup>CCR2<sup>-</sup> macrophages found in the ear dermis (Fig. S1A) was performed before (-DT) and 1, 2, 3, 4, 5, 10, 20, and 90 d after the last DT injection. (A) CD64<sup>+</sup>CCR2<sup>+</sup> dermal cells were analyzed at the specified time points by using Ly-6C<sup>-</sup>MHCII<sup>-</sup> dot plots to define the percentages of Ly-6C<sup>+</sup>MHCII<sup>-</sup> (P1) dermal monocytes, Ly-6C<sup>+</sup>MHCII<sup>+</sup> (P2) mo-DCs, and Ly-6C<sup>+</sup>MHCII<sup>+</sup> (P3) mo-DCs (Tamoutounour et al., 2013). (B) Quantification of the results in A showing the absolute number of cells obtained from two ears of each mouse. Each symbol corresponds to a mouse and the mean (horizontal bar) is indicated. (C) CD64<sup>+</sup>CCR2<sup>-</sup> dermal cells were analyzed at the specified time points by using Ly-6C<sup>-</sup>MHCII<sup>-</sup> dot plots to define the percentages of Ly-6C<sup>+</sup>MHCII<sup>-</sup> (P4) dermal macrophages and Ly-6C<sup>+</sup>MHCII<sup>+</sup> (P5) dermal macrophages (Tamoutounour et al., 2013). A Ly-6C<sup>+</sup>MHCII<sup>-</sup> to high cell population transiently developed 2–5 d after the last DT treatment and is denoted as Px cells. (D)</figcaption></figure>
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exemplified by the melanin-laden macrophages that have been
sporadically observed in the normal human dermis and referred
to as “melanophages” (Haniffa et al., 2009, 2012).

To study the functional specialization that exists among
cells of the mononuclear phagocyte system, several strains of
gene-targeted mice have been developed to ablate a given cell
subset by placing the human diphtheria toxin receptor (hDTR)
under the control of a gene promoter specific for the subset of
interest. Those models are sometimes associated with drawbacks
(van Blijswijk et al., 2015; Durai and Murphy, 2016). For instance,
in CD11b-DTR mice, cDC2s, macrophages, and mo-DCs were
depleted, and because of off-target DTR expression, those mice
died after a few diphtheria toxin (DT) injections (Duffield et al.,
2005). Although CCR2-DTR mice were not prone to DT-induced
lethality (Hohl et al., 2009), some of the phenotypes observed
by using those mice likely resulted from the depletion of CCR2+
cDC2s rather than of monocytes and monocyte-derived cells as
generally assumed (Malissen et al., 2014).

In contrast to cDCs, both the mo-DCs and mo-Mac, includ-
ing their precursors, and the macrophages of embryonic origin
that are found in tissues such as the skin, the lung, and the gut
express the high-affinity IgG receptor FcgrI (CD64; Bain et al.,
2013; Plantinga et al., 2013; Tamoutounour et al., 2013). Here, we
developed mice in which a hDTR gene was placed under the con-
trol of the *Fcgr1* gene that codes for CD64. This model has a tighter
ablation specificity compared with previous models in that it
permitted specifically ablating macrophages, monocytes, and
monocyte-derived cells in vivo. It allowed us to identify in the ear
skin dermis of C57BL/6 (B6) mice a population of melanophages
that constitutes a quantitatively major subset of dermal myeloid
cells and has been missed in previous studies. By characterizing
their phenotype, origin, and dynamics, we showed that they cor-
responded to dermal macrophages that have ingested melanin
granules. Benefiting from the knowledge gained on melanophage
dynamics, we further elucidated the identity, origin, and dynam-
ics of the myeloid cells that are found in the mouse skin and capa-
ble of capturing and retaining tattoo pigment particles.

**Results**

Mice permitting ablation of macrophages and
monocyte-derived cells

To assess the dynamics and functional role of CD64⁺ macro-
phages and monocyte-derived cells in vivo, we generated a line
of gene-targeted mice containing in the 3'-UTR of the *Fcgr1*
gene a cassette composed of an internal ribosomal entry site,
an enhanced green fluorescent protein (EGFP), a 2A cleavage
sequence, and an hDTR. To demonstrate that the mo-Mac and
monocyte-derived cells found in mice homozygous for the *Fcgr1-
IRES-EGFP-hDTR* allele (also known as *B6-Fcgr1tm1Ciphe* and called
CD64dtr mice here) developed normally and expressed normal

levels of CD64, we analyzed their skin dermis because it contains
a full complement of such cells (Fig. S1 A; Tamoutounour et al.,
2013). Among dermal CD11b⁺ cells, cDC2s can be distinguished
from non-cDC2s cells by their Ly-6C⁻CD64⁻ phenotype. Dermal
CD11b⁺CD64⁺ non-cDC2 cells can be further resolved into CCR2⁺
and CCR2⁻ cells. CCR2⁺ cells comprise Ly-6ChighMHCII⁻ dermal
monocytes (P1), Ly-6Chigh (P2), and Ly-6C⁻ (P3) MHCII⁺ mo-DCs
that constitute a P1 → P2 → P3 developmental series. CCR2⁻ cells
comprise MHCIIlow (P4) and MHCIIhigh (P5) dermal macrophages.
The ear skin dermis of DT-untreated CD64dtr mice showed ratios
and numbers of monocytes, mo-DCs, and macrophages compa-
rable to those of WT mice (Fig. S1 B), and all those cells expressed
normal levels of CD64 (Fig. S1 C). Similar results were obtained
when CD64⁺ cells found in the lamina propria of the large intes-
tine were analyzed (not depicted).

CD64^dtr^ mice were treated with DT twice and 24 h apart without lethality, and the myeloid cells present in the ear skin were analyzed at various time points after the last DT injection. DT treatment was not associated with overt inflammation as documented by the lack of skin infiltrating neutrophils (not depicted). CD64⁺CCR2⁺ cells were all ablated within 24 h (Fig. 1, A and B), demonstrating that the hDTR was functional. 48 h after the last DT injection, Ly-6C^high^MHCII⁻ dermal monocytes started to return, and Ly-6C^high^ and Ly-6C⁻ MHCII⁺ mo-DCs were replenished in sequence over the next 5 d. A transient rebound in their numbers occurred around day 4, suggesting that their rate of generation can increase in response to acute, DT-induced loss. Both MHCII^low^ and MHCII^high^ CCR2⁻CD64⁺ dermal macrophages showed protracted reconstitution kinetics compared with CD64⁺CCR2⁺ cells in that it took up to 10 d to fully reconstitute their pools (Fig. 1, C and D). Analysis of the gate corresponding to CCR2⁻CD64⁺ cells at days 2-3 revealed a population of Ly-6C^high^MHCII^low^ to high cells (denoted as Px cells in Fig. 1, C and D) that started losing Ly-6C expression on day 4 and thus progressively merged within gates corresponding to MHCII^low^ and MHCII^high^ dermal macrophages. The phenotype, kinetics of reappearance of Px cells, and the occurrence at days 4 and 5 of cells straddling the Px and P4/P5 gates suggest that they correspond to intermediaries between Ly-6C^high^ blood monocytes and MHCII^low^ and MHCII^high^ dermal macrophages. The percentage of Px cells among CCR2⁻CD64⁺ cells reached pre-DT-treatment levels upon replenishment of the MHCII^low^ and MHCII^high^ dermal macrophage compartments (Fig. 1, C and D).

In the lamina propria of the large intestine of CD64dtr mice, CD64⁺ cells were also ablated within 24 h after the last DT injection and reconstituted from 48 h onward akin to dermal CD64⁺ cells (Fig. S2). Therefore, by allowing to conditionally empty the niche occupied in the skin dermis and intestinal lamina propria by CD64⁺ myeloid cells, the CD64dtr mouse model demonstrated that all the mo-DC and macrophage subsets that were present in adult skin and intestine before DT treatment can be readily

Quantification of the results in C showing the absolute number of cells obtained from two ears of each mouse. Each symbol corresponds to a mouse and the mean (horizontal bar) is indicated. Data are representative of three independent experiments involving three to six animals per group. \*, P ≤ 0.05; \*,\*, P ≤ 0.01; \*,\*,\*, P ≤ 0.001; unpaired Student's t test.
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<figure><img src="image_15.png" /><figcaption>**Figure 2.** Replenishment kinetics of blood monocytes of CD64<sup>dtr</sup> mice after DT treatment. (A) Gating strategy used to identify CD11b<sup>+</sup>Lin<sup>-</sup> cell subsets within the blood. Red blood cells were lysed before analysis, and the absolute number of a given cell type present in a given blood volume was determined by staining samples in Trucount Absolute Counting Tubes. Beads were gated out on the basis of their FSC-A–SSC-A profile, and after excluding CD5<sup>+</sup> T cells, CD19<sup>+</sup> B cells, CD161<sup>+</sup> NK cells, as well as SSC-A<sup>int</sup> eosinophils, and Ly-6G<sup>+</sup> neutrophils, the majority of the remaining CD11b<sup>+</sup>Lin<sup>-</sup> cells were CD45<sup>+</sup>CD115<sup>+</sup>; when analyzed on CD115-CD43<sup>−</sup> dot plots, they comprise CD115<sup>+</sup>CD43<sup>−</sup> and CD115<sup>+</sup>CD43<sup>+</sup> subsets. As expected, CD115<sup>+</sup>CD43<sup>−</sup> cells corresponded to Ly-6G<sup>high</sup> blood monocytes, whereas most CD115<sup>+</sup>CD43<sup>+</sup> corresponded to Ly-6C<sup>low</sup> blood monocytes. (B) Absolute number (mean values ± SD) of the specified cells in the</figcaption></figure>
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reconstituted in normal ratios and numbers and with kinetics
consistent with their expected precursor-product relationship.

Kinetics of replenishment of blood monocytes
after DT treatment

Adult steady-state monopoiesis occurs in BM, where mono-
cyte-macrophage DC progenitors (MDPs) give rise to common
monocyte progenitors (cMoP) that generate two main blood
monocyte subsets that are both CD115 (CSF1R)⁺ and can be dis-
tinguished on the basis of CD43 expression (Fig. 2 A). CD43⁻ cells
are CX3CR1intCCR2⁺CD64⁺Ly-6Chigh and CD43⁺ cells are mostly
CX3CR1highCCR2⁻CD64⁺Ly-6Clow (Sunderkötter et al., 2004). They
are denoted here as Ly-6Chigh and Ly-6Clow monocytes, respec-
tively. Ly-6Clow monocytes remain within blood vessels (Carlin et
al., 2013) whereas Ly-6Chigh monocytes contribute to the replen-
ishment of mo-DCs and mo-Mac in tissues, including the skin
and the intestine (Jakubzick et al., 2013; Tamoutounour et al.,
2013; Yona et al., 2013; Bain et al., 2014; McGovern et al., 2014).
Intraperitoneal injection of DT twice and 24 h apart resulted
within 24 h in the complete ablation of the MDPs and cMoPs
and Ly-6Chigh, Ly-6Cint, and Ly-6Clow monocytes found in the
BM (Fig. S3) and of the Ly-6Chigh and Ly-6Clow blood monocytes
(Fig. 2, B-D). Analysis of the blood and BM of CD64dtr mice at
various time points after the last DT injection showed that blood
Ly-6Chigh and Ly-6Clow monocytes were rapidly replenished and
that a transient rebound in their numbers occurred (Fig. 2, B-D;
and Fig. S3). Consistent with the observation that CD115 is dra-
matically down-regulated in response to CSF-1 (Yu et al., 2012),
the earliest BM-derived monocytes reappearing in the blood
expressed lower levels of CD115 than CD115⁺ steady-state mono-
cytes and are denoted here as CD115int cells (Fig. 2, A, B, and E).
Because of their CD115int phenotype, those Ly-6Chigh monocytes
were not recorded in the CD115⁺CD43⁻ gate used in Fig. 2 C. Their
numbers peaked at day 3 after DT treatment (Fig. 2, B and E),
and when blood monocytes were fully replenished, their relative
representation dramatically decreased. Congruent with the view
that circulating CD43⁻Ly-6Chigh monocytes give rise to CD43⁺
Ly-6Clow monocytes (Varol et al., 2007; Thomas et al., 2016;
Mildner et al., 2017), the first CD43⁺ monocytes reappeared at days
3-4 after the last DT treatment and gradually lost Ly-6C expression
(Fig. 2 D). An approximately fourfold increase in blood neutro-
phils occurred in CD64dtr mice and peaked 3 d after DT treatment
(Fig. 2 B). It occurred, however, with a far lower magnitude and
shorter duration than that observed upon DT administration in
CD11c-DTR mice (Autenrieth et al., 2012; Tittel et al., 2012).

DT treatment of CD64<sup>dtr</sup> mice spares skin cDCs

We analyzed next whether DT treatment of CD64<sup>dtr</sup> mice affects
the cDCs found in the skin. Langerhans cells (LCs; CD24<sup>hi</sup>CD11b<sup>+</sup>),

cDC1s (CD11b⁻CD24⁺), cDC2s (CD24<sup>low</sup>CD11b⁺), and double-nega-
tive (DN) cDCs (CD11b⁻CD24⁻) were identified as described in Fig.
S4 A. Although the absolute numbers of LCs, cDC1s, and DN cells
were not affected by DT treatment, a 1.3-fold reduction in cDC2
absolute number was observed 1 d after DT treatment (Fig. S4 B).
Therefore, our regimen of DT administration had a rather mini-
mal impact on the cDC subsets found in the skin of CD64<sup>dtr</sup> mice.
CD64<sup>dtr</sup> mice can thus be used to monitor the dynamics and func-
tion of macrophages and monocyte-derived cells without major
collateral effects or lethality.

Melanophages predominate among dermal myeloid
cells of B6 ear skin

Former analyses of CD45⁺Lin⁻ myeloid cells present in the ear skin dermis focused on SSC-A<sup>low</sup>FSC-A<sup>low</sup> to high (SSC-A<sup>low</sup> in short) cells, leaving aside an uncharacterized population of SSC-A<sup>high</sup>FSC-A<sup>low</sup> to int (SSC-A<sup>high</sup> in short) cells (Tamoutounour et al., 2013). After treatment of CD64<sup>dtr</sup> mice with DT twice and 24 h apart, all those SSC-A<sup>high</sup> cells were unexpectedly ablated (Fig. 3 A). Further characterization of such DT-sensitive, SSC-A<sup>high</sup> cells showed that they had a CD24<sup>low</sup>CD11b<sup>+</sup>CD64<sup>+</sup>CCR2<sup>−</sup> MHCII<sup>+</sup> or <sup>−</sup> phenotype identical to that of SSC-A<sup>low</sup> dermal macrophages and that CD64 expression accounted for their DT sensitivity (Fig. 3 B).

In the almost hairless skin of mouse ears, melanocytes are primarily found in the dermis (Fig. 3 C; Aoki et al., 2009) and synthetize melanin pigment within specialized lysosome-related structures known as melanosomes. Considering that the SSC-A parameter used in cytometry is proportional to cell granularity and that CD64<sup>dtr</sup> mice were of B6 origin and have a black coat, we surmised that the high granularity of the SSC-A<sup>high</sup> myeloid cells found in the ear dermis was due to their capture of melanin granules from neighboring melanocytes. Hematoxylin and eosin staining of cytospin preparations showed that the SSC-A<sup>low</sup>CCR2<sup>−</sup>CD64<sup>+</sup> cells and the SSC-A<sup>high</sup> cells sorted from the ear dermis have typical features of tissue macrophages; they were large with prominent cytoplasmic vacuoles (Fig. 3 D). The vacuoles of SSC-A<sup>high</sup> cells differed, however, from those of SSC-A<sup>low</sup> CCR2<sup>−</sup>CD64<sup>+</sup> cells in that they were loaded with dark material (Fig. 3 D). Upon analysis by electron microscopy, such dark vacuolar material had an extremely electron-dense appearance and an ellipsoidal shape (~0.2 µm × 0.7 µm), two hallmarks of melanosomes (Fig. 3 E). All the ingested melanosomes were in a mature stage, showing a uniformly electron-dense granule without discernable internal structure (Fig. 3 E).

In contrast to their precursors, dermal macrophages do not
express CX3CR1 (Tamoutounour et al., 2012). Consistent with
our hypothesis that melanophages correspond to macrophages
that ingested melanosomes, they were CX3CR1⁻ (not depicted)

blood of CD64dtr mice before and 1, 2, 3, 4, 5, 10, and 20 d after DT treatment. (C) CD115+CD43- monocytes were analyzed at the specified time points by using Ly-6C-MHCII dot plots to define the percentages of Ly-6ChighMHCII- and Ly-6ChighMHCII+ monocytes. (D) CD115+CD43+ cells were analyzed at the specified time points by using Ly-6C-MHCII dot plots to define the percentages of Ly-6C+MHCII- and Ly-6ClowMHCII- monocytes. (E) Ly-6G-SSC-Alow cells were analyzed at the specified time points by using CD115-CD45 dot plots to define the percentages of CD115int and CD115+ blood monocytes. Data are representative of at least three experiments involving three to six animals per group.
<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /><figcaption>Figure 3. CD64<sup>dtr</sup> mice unveil the existence of a novel CD64<sup>+</sup> cell population in the skin that is highly granular and DT sensitive and corresponds to melanophages. (A) Cell suspensions from the ear skin of CD64<sup>dtr</sup> mice were analyzed by flow cytometry before (–DT) or 48 h after (DT + 48 h) DT treatment. After excluding CD3<sup>+</sup> T cells, CD19<sup>+</sup> B cells, CD161<sup>+</sup> NK cells, and Ly-6G<sup>+</sup> neutrophils, the remaining lineage-negative (Lin<sup>-</sup>) CD45<sup>+</sup> live cells were analyzed by using FSC-A–SSC-A dot plots to define the percentages of SSC-A<sup>low</sup> and SSC-A<sup>high</sup> cells. (B) Comparative analysis of the SSC-A<sup>low</sup> and SSC-A<sup>high</sup> cells found in the ear skin by using a gating strategy resolving the cDC2s, monocytes, mo-DCs and macrophages present among CD45<sup>+</sup>Lin<sup>-</sup>CD11b<sup>+</sup> cells (Fig. S1A). The percentage of cells found in each of the specified gate is indicated. (C) Transversal tissue sections of B6 ear or trunk skin (at telogen and anagen phases) were stained with</figcaption></figure>
<figure><img src="image_19.png" /></figure>

and identified as melanin⁺tdTomato⁺CD64⁺ cells in the ear sec-
tion of CX3CR1<sup>Cre/+</sup> Rosa<sup>sl/-tdt/+</sup> mice that fate map cells that have
expressed CX3CR1 during their development (Fig. 3 F). In the
densely-haired mouse trunk, melanocytes are absent from the
dermis and confined to the hair follicles where melanosomes are
transferred from melanocytes to matrix keratinocytes to give
rise to melanized hairs (Fig. 3 C). Such transfer is strictly coupled
to the anagen phase of hair growth, and melanin is not formed
during the telogen phase (Fig. 3 C; Slominski et al., 2005). Consis-
tent with these anatomical features, the CD11b⁺CD64⁺CCR2⁻ mac-
rophages isolated from the trunk skin during both the anagen
and telogen phases lacked melanophages (Fig. 3 G). Likewise,
the ear skin of B6-albino and BALB/c mice, which is deprived
of melanic pigment, also lacked SSC-A<sup>high</sup> melanophages (Fig. 4,
A and B). Therefore, the presence of dermal melanophages cor-
relates with the presence of dermal melanocytes. Although they
represent 55 ± 10% of the monocyte-derived cells present in the
B6 ear dermis (Fig. 4, A and B), it is likely that melanophages
have been missed in previous studies because of their unusual
SSC-A<sup>high</sup> phenotype.

Dynamics and origin of dermal melanophages

To assess the dynamics of dermal melanophages replenishment
after DT-induced depletion, CD64<sup>dtr</sup> mice were treated with DT
twice and 24 h apart. Melanophages were all ablated within 24 h,
and their numbers fully reconstituted ~20 d after the last DT
injection (Fig. 5, A and B). The granularity of SSC-A<sup>high</sup> cells, a
proxy for melanin content, increased with time. The upper sec-
tion (>190 K) of the SSC-A<sup>high</sup> gate was only filled between days
20 and 90 after DT treatment, and a melanin content distribution
identical to pre-DT-treatment levels was regained 90 d after DT
treatment (Fig. 5, A and B). Although the dermis of CCR2-defi-
cient mice is only populated by macrophages of prenatal origin
that self-maintain without any input from adult monopoiesis
(Tamoutounour et al., 2013), it showed a melanophage to macro-
phage ratio and melanin content distribution identical to that of
WT dermis (Fig. 6 A). Therefore, the macrophages found in the
ear dermis have the same propensity to capture melanin regard-
less of their prenatal (Fig. 6 A) or adult (Fig. 5) origin.

To demonstrate without relying on DT ablation that replen-
ishment of the pool of adult dermal macrophages and mela-
nophages relied on continuous input from BM-derived circu-
lating precursors, parabiosis was established between adult
mice expressing CD45.1 and CD45.2 (Fig. 6 B). Blood monocytes
were allowed to exchange between parabions for 8 wk before

assessing the presence of donor-derived macrophages and
melanophages in the ear skin of each parabiont. Dermal MHC
II⁺ macrophages contained ~15% of cells originating from the
parabiotic partner (Fig. 6 B), a distribution comparable to that of
Ly-6Chigh blood monocytes and demonstrating that they contin-
uously develop from BM monocytes (Tamoutounour et al., 2013;
Sheng et al., 2015). Consistent with the view that a fraction of
dermal MHCII⁻ macrophages is of embryonic origin and self-re-
news in situ (Sheng et al., 2015), MHCII⁻ macrophages showed
a twofold reduced rate of exchange with blood-borne precur-
sors compared with dermal MHCII⁺ macrophages. In parabi-
osis experiments involving WT and CCR2-deficient mice, the
percentages of donor-derived cells found in the CCR2-deficient
parabions reached 80% and 40% of MHCII⁺ and MHCII⁻ mac-
rophages, respectively, compared with 80% for Ly-6Chigh blood
monocytes (Fig. 6 B). As expected for cells linked by a precur-
sor-product relationship, in both types of parabiosis MHCII⁺ and
MHCII⁻ melanophages achieved a degree of chimerism identical
to that of MHCII⁺ and MHCII⁻ macrophages (Fig. 6 B). Altogether,
these data indicate that dermal macrophages and melanophages
are related through a precursor-product relationship and that
dying dermal macrophages and melanophages are continuously
replenished with new ones via CCR2-dependent recruitment of
circulating Ly-6Chigh blood precursors.

Comparative transcriptomic analysis of dermal macrophages
and melanophages

To assess whether melanophages show a unique transcriptomic
signature compared with dermal SSC-A<sup>low</sup> macrophages that
have not ingested melanin, MHCII<sup>+</sup> and MHCII<sup>-</sup> melanophages
and macrophages from the ear dermis were sorted in triplicates
and subjected to gene-expression profiling. Principal compo-
nent analysis (PCA) on all the genes found to be differentially
expressed between at least two of the four subsets showed that
the MHCII<sup>+</sup> and MHCII<sup>-</sup> status accounted for most of the over-
all variability observed in the dataset (47%) and that differences
between melanophages and macrophages was only the second
component of variability (17%; Fig. 7 A). Insights into the biolog-
ical pathways underlying these transcriptomic differences were
obtained through ingenuity pathway analysis (IPA) of eight
lists of genes corresponding to those differentially expressed
(1) in one out of the four subsets, (2) between MHCII<sup>+</sup> versus
MHCII<sup>-</sup> cells, and (3) between melanophages and macrophages
(Fig. 7 B). When compared with their MHCII<sup>-</sup> counterparts,
MHCII<sup>+</sup> macrophages and melanophages were significantly

DAPI and analyzed by microscopy for the presence of melanin. Bars: (top) 100 µm; (bottom) 20 µm. (D) Morphological characteristics of CD64⁺CCR2⁻SSC-A<sup>low</sup> macrophages and CD64⁺SSC-A<sup>high</sup> melanophages sorted from ear skin and analyzed by hematoxylin and eosin staining after cytospin onto glass slides. Two representative cells are shown for each population. Bar, 10 µm. (E) Morphological characteristics of CD64⁺SSC-A<sup>high</sup> melanophages sorted from ear skin and analyzed by electron microscopy. Three representative cells are shown. Melanophages display an irregular nucleus surrounded by a prominent cytoplasm crowded with elongated melanin granules. Bars, 1 µm. Higher magnification shows pigment granules with a homogeneous and extremely dense content, attesting for its mature stage. Melanosomes are grouped together forming melanosome complexes (asterisks), but they are also singly dispersed in the cytoplasm, all of them are surrounded by a membrane. Bars, 0.5 µm. (F) En face section of ear dermis from CX3CR1<sup>Cre/+</sup> Rosa<sup>sl/-tdt/+</sup> mice analyzed by confocal microscopy for the colocalization of melanin with CD64⁺ CX3CR1-fate-mapped macrophages (tdtomato<sup>+</sup>). Bars: (top) 50 µm; (bottom) 10 µm. Data are representative of two to three experiments. (G) CD11b⁺CD64⁺CCR2⁻ cells from the ear skin and trunk skin of B6 (telogen and anagen phases) of B6 mice were analyzed for the percentages of SSC-A<sup>low</sup> and SSC-A<sup>high</sup> cells. Data are representative of at least two experiments involving three animals per group.
<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /><figcaption>Figure 4. SSC-A<sup>high</sup>CD64<sup>+</sup> dermal cells correspond to melanophages. (A) CD45<sup>+</sup>Lin<sup>-</sup> cells from the ear skin of B6, B6-albino, and BALB/c mice, the tail skin of B6 mice, and the lamina propria of the large intestine of B6 mice were analyzed for the percentages of SSC-A<sup>low</sup> and SSC-A<sup>high</sup> cells. As expected on the basis of the anatomical distribution of mouse melanocytes (Aoki et al., 2009), the lamina propria of B6 mice lacked melanophages. (B) Pie charts and corresponding frequencies of the indicated cells among CD11b<sup>+</sup> noncDC2 cells found in the specified anatomical location and mice strain. Data corresponding to each pie chart were averaged from six mice and the corresponding percentages (mean values ± SD) are shown below the charts. Data are representative of at least three experiments involving three to six animals per group.</figcaption></figure>

enriched in annotations related to the adaptive immune response such as antigen presentation pathway, dendritic cell maturation, and cytokine signaling (IL-4, IL-6, IL-10, and IL-17). MHCII expression was also associated with a pro-inflammatory signature with higher expression of *Ccr7*, *Il1b*, *Il1r2*, and *Il6* and to immune cell chemotaxis with higher expression of *Ccl17*, *Ccl22*, *Ccl5*, and *Cxcl13* (Fig. 7 C). When compared with SSC-A<sup>low</sup> macrophages, melanophages were significantly enriched in annotations related to aryl hydrocarbon receptor signaling and glutathione-mediated detoxification (Fig. 7 B) because of their expression of two genes of the glutathione S transferase family (Gstm1 and 3; Table S1). High and selective expression by melanophages of genes coding for a lysosomal ATPase and a cystein protease (*Atp6v0d2* and *Ctsk*, respectively) led to their enrichment for the annotation phagosome maturation (Fig. 7 B and Table S1), a feature consistent with the fact that they are laden with melanosomes.

## Melanophages are found in melanocytic melanoma

Tumors depend on the supportive functions of macrophages. Clusters of melanophages have been observed in the periphery

of the nevi found in the dermis of mice expressing oncogenic mutated form (BrafV600E) of the serine-threonine-specific protein kinase BRAF (Dhomen et al., 2009). To determine whether such melanoma-associated melanophages correspond to CD64<sup>+</sup>SSC-A<sup>high</sup> cells, B6-albino mice, which lack melanophages, were injected with melanocytic (B16) or amelanoctytic (Braf<sup>V600E</sup>) melanoma cells. Analysis of CD45<sup>+</sup>Lin<sup>-</sup>Siglec-F<sup>-</sup> cells isolated from tumor masses 14 to 18 d after inoculation showed that SSC-A<sup>high</sup> cells were present only in the melanocytic melanoma (Fig. 8 A). Their morphology was similar to that of the melanophages identified in the B6 dermis (Fig. 8 B) and they had a CD11b<sup>+</sup>CD64<sup>+</sup>F4/80<sup>+</sup> phenotype (Fig. 8 C).

The melanophages that are identifiable in melanocytic melanoma constitute a unique thread permitting revisiting the phenotype of tumoral macrophages. For instance, the presence of CD64 and absence of Ly-6C permit readily distinguishing macrophages in steady-state tissues (Tamoutounour et al., 2013), and it has been assumed that such a feature holds true within the tumor microenvironment (Salmon et al., 2016). Our analysis of the melanophages found in melanocytic tumors showed, however,
<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /><figcaption>Figure 5. SSC-A<sup>high</sup>CD64<sup>+</sup> melanophages showed a protracted kinetics of replenishment after DT treatment compared with SSC-A<sup>low</sup> macrophages. (A) CD45<sup>+</sup>Lin<sup>-</sup> cells from the ear skin of CD64<sup>dtr</sup> mice were analyzed for the percentages of SSC-A<sup>low</sup> and SSC-A<sup>high</sup> cells before (-DT) or at the specified time points after DT treatment. (B) Quantification of the results in A are shown for the MHCII<sup>+</sup> and MHCII<sup>-</sup> cells found among the SSC-A<sup>high</sup> melanophages. Each symbol corresponds to a mouse and the mean (horizontal bar) is indicated. Data are representative of two to three independent experiments involving three to six animals per group. n.s., P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; unpaired Student's t test.</figcaption></figure>

that most of them expressed Ly-6C (Fig. 8 C). Therefore, analysis of the melanophages found in melanoma constitute a unique decision support tool permitting to determine whether markers defined as macrophage-specific under steady-state conditions keep or lose their discriminatory potential within the tumor microenvironment.

## Skin macrophages capture and retain tattoo pigment particles

Using the CD64<sup>dtr</sup> mouse model and benefiting of our knowledge on melanophages, we determined next the identity, origin, and dynamics of the skin myeloid cells that are capable of capturing and retaining tattoo pigment particles. The frequency of melanophages in the tail skin of B6 mice is fourfold lower than that of melanophages in ear dermis and reflects its scarcity in

melanocytes. Moreover, because of their reduced melanin content tail skin melanophages barely merged in the SSC-A<sup>high</sup> gate, the lower limit of which that was defined by using B6 albino ear (Fig. 4, A and B). Accordingly, we used tail skin of B6 mice to monitor the appearance in the almost-empty SSC-A<sup>high</sup> gate of those cells that have captured the bright green pigment particles that are contained in the tattooing ink paste used in the present study. Analysis of the percentages of SSC-A<sup>low</sup> and SSC-A<sup>high</sup> cells found among the CD45<sup>+</sup>Lin<sup>-</sup> cells isolated from the skin of tails that were tattooed 3 wk before analysis showed that they contained a unique population of SSC-A<sup>high</sup> cells compared with untreated mice (Fig. 9 A). Further characterization of those SSC-A<sup>high</sup> cells showed that they had a CD24<sup>low</sup>CD11b<sup>+</sup>CD64<sup>+</sup>CCR2<sup>-</sup> to low phenotype (Fig. 9 A), which was identical to that observed for SSC-A<sup>high</sup>
<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /><figcaption>Figure 6. Prenatal and adult origins of SSC-A<sup>high</sup>CD64<sup>+</sup> melanophages. (A) CD45<sup>+</sup>Lin<sup>-</sup> cells from the ear skin of B6 (WT) and Ccr2<sup>2/-</sup> mice were analyzed for the percentages of SSC-A<sup>low</sup> and SSC-A<sup>high</sup> cells. (B) Percentages (mean values ± SD) of donor chimerism in MHCII<sup>+</sup> and MHCII<sup>-</sup> dermal macrophages and MHCII<sup>+</sup> and MHCII<sup>-</sup> melanophages found in the skin dermis of parabiotic mice involving either CD45.1 and CD45.2 WT parabionts or CD45.1 WT and CD45.2 Ccr2<sup>2/-</sup> parabionts. No macrophages or melanophages originating from CCR2-deficient mice were found in the dermis of WT partners. Data are representative of two to three independent experiments involving two to four animals per group.</figcaption></figure>

melanophages and SSC-A<sup>low</sup> macrophages (Fig. 3 B). Cytospin analysis of SSC-A<sup>high</sup> cells sorted from tattooed tail skin showed that they were large cells containing prominent cytoplasmic vacuoles loaded with green material, whereas SSC-A<sup>low</sup> dermal macrophages and cDCs contained no green tattoo pigment (Fig. 9 B). None of the CD45<sup>-</sup> skin cells, which mostly comprise keratinocytes, contained green material (not depicted). Therefore, 3 wk after tail tattooing, the green pigment particles present at the site of tattooing were primarily found within dermal macrophages.

## Cycles of pigment capture-release-recapture account for long-term tattoo persistence

We used CD64<sup>dtr</sup> mice to ablate dermal CD64<sup>+</sup> macrophages laden with green pigment particles (“green” macrophages in short) and to determine whether their death results in tattoo vanishing. Accordingly, CD64<sup>dtr</sup> mice that had the skin of their tail tattooed with green tattoo ink paste 3 wk before were treated with

DT twice and 24 h apart. Analysis of tattooed skin of those mice for the presence of SSC-A<sup>high</sup> green macrophages showed that they were as expected all ablated within 2 d after DT treatment and that, akin to melanophages, their pool was slowly reconstituted after DT injection (Fig. 9 C). Their granularity, reflecting green-particle content per cell, increased with time and 90 d after DT injection reached levels identical to those present just before DT treatment (Fig. 9, A and C). The pool of SSC-A<sup>high</sup> cells that was replenished 90 d after DT treatment was exclusively composed of green macrophages with a morphology and CD24<sup>low</sup>CD11b<sup>+</sup>CD64<sup>+</sup>CCR2<sup>-</sup> to <sup>low</sup> phenotype resembling those of green macrophages observed pre-DT treatment (Fig. 9 D and not depicted).

Considering that no green tattoo ink paste was reapplied after DT treatment, it is likely that most of the green pigment particles released after DT-induced green macrophage death remained in an extracellular form at the site of tattooing where they were progressively recaptured by incoming dermal macrophages originating from circulating Ly-6C<sup>high</sup> blood monocytes (see model A in Fig. 10). Consistent with that model, cytospin preparation of the total cell suspension from tail skin showed that before DT treatment green pigment particles were all associated with cells that had typical features of tissue macrophages, whereas 2 d after the last DT injection no green macrophages were left (Fig. 9 C) and the green pigment particles that were spun down with the cells were all extracellular (Fig. 9 E). Importantly, macroscopic examination of tattooed tails showed that even at time points when green macrophages were absent and green pigment particles were extracellular (for instance, day 2 after DT), the tattoo kept the appearance it had just before DT treatment (Fig. 9 F). Therefore, despite the fact that the green tattoo pigments went, over 90 d, through a full cycle of capture-release-recapture, the tattoo kept its macroscopic appearance.

To further support our pigment capture-release-recapture model without relying on DT ablation, CD45.1<sup>+</sup> B6 mice were tattooed on the tail. The tattooed tail skin was explanted and grafted on the skin of the back of CD45.2<sup>+</sup> B6 albino mice. Analysis of the myeloid cells present in the graft 6 wk after transplantation showed that most of the SSC-A<sup>high</sup> cells found among CD11b<sup>+</sup>CD64<sup>+</sup>CCR2<sup>-</sup> cells were of host origin, suggesting that green tattoo pigments were released from the CD45.1<sup>+</sup>CD11b<sup>+</sup>CD64<sup>+</sup>CCR2<sup>-</sup> SSC-A<sup>high</sup> cells originally present in the donor graft and captured by incoming CD45.2<sup>+</sup>CD11b<sup>+</sup>CD64<sup>+</sup>CCR2<sup>-</sup> macrophages of host origin (Fig. 9 G). As discussed below, those findings provide a cellular basis for long-term tattoo persistence and have implications for improving strategies aimed at removing tattoos.

## Discussion

We used a novel model of DT-induced depletion of macrophages to generate niche availability without triggering inflammation and showed that the pool of adult dermal macrophages can be fully reconstituted from circulating Ly-6C<sup>high</sup> monocytes. By using a model of DT-mediated depletion of liver Kupffer cells, circulating Ly-6C<sup>high</sup> monocytes were also found capable of colonizing the emptied liver niches and of adopting a Kupffer cell identity (Scott et al., 2016). Likewise, transferred BM-derived monocytes were able to colonize the empty alveolar macrophage
<figure><img src="image_26.png" /></figure>

A

<figure><img src="image_27.png" /></figure>

C

<figure><img src="image_28.png" /></figure>

B

<figure><img src="image_29.png" /><figcaption>**Figure 7.** Gene expression profiling of MHCII⁺ and MHCII⁻ melanophages and macrophages from steady-state dermis. MHCII⁺ and MHCII⁻ CD64⁺CCR2⁻ SSC-Alow macrophages and MHCII⁺ and MHCII⁻ CD64⁺SSC-Ahigh melanophages were sorted in triplicates from ear skin and subjected to gene-expression profiling (Affymetrix 1.0ST). (A) PCA of gene expression by MHCII⁺ and MHCII⁻ dermal macrophages and MHCII⁺ and MHCII⁻ melanophages, based on all the 1,054 unique genes differentially expressed between at least one pair of experimental conditions. The percentage of overall variability of the dataset is indicated along each PC axis. (B) Heat map representation of selected functions and pathways found enriched by IPA for the lists of genes differentially expressed in one of the three cell types, between MHCII⁺ versus MHCII⁻ cells, or between melanophages and macrophages. (C) Heat map showing the expression pattern across</figcaption></figure>
<figure><img src="image_30.png" /></figure>

niche of neonatal CSF2RB-deficient mice and generate alveolar macrophages (van de Laar et al., 2016). Therefore, our data showed that the capacity for Ly-6<sup>high</sup> blood monocytes to replenish macrophages after DT-induced ablation also extends to the skin dermis. Moreover, we characterized the melanophages that are present in large numbers in the normal ear dermis of mice with a black coat, demonstrating that they correspond to dermal macrophages that have ingested melanosomes originating from neighboring melanocytes.

Limited information has been provided on the identity of the cells that are found in tattooed skin biopsies and capable of retaining tattoo pigment particles. By capitalizing on the knowledge gained on melanophages and on the functionalities offered by CD64<sup>dtr</sup> mice, we conducted the first systematic analysis of the identity, origin, and dynamics of the skin myeloid cells that are capable of capturing and retaining tattoo pigment particles. The tattooing process consists of inserting water-insoluble pigment of the desired color into the dermis layer of the skin. Tattoo pigments are primarily metal salts in the submicrometer range. Immediately after tattooing, a fraction of pigments is shed with the epidermis or transported away from the dermis via lymphatic vessels. We demonstrated that the pigment particles that remain at the site of injection and cause the long-term tattoo color were exclusively found within dermal macrophages. Zaba et al. (2007) previously identified a population of cells located in the human dermis that was positive for factor XIIIA and the macrophage scavenger receptor CD163 that lacked CD11c and BDCA-1 and weakly activated allogenic T cells. Those cells have structural and ultrastructural features of macrophages and in biopsies of tattoos they were selectively laden with granular pigments. The fate of tattoo pigment injected into dermal tissues has been studied in the past, and fibroblasts were considered the primary long-term reservoir of the pigment granules (Ferguson et al., 1997; Elsaie et al., 2009). However, the morphological study of Zaba et al. (2007) and our present data combining multiparameter cytometry and the use of CD64<sup>dtr</sup> "depleter" mice demonstrate that dermal macrophages constitute the primary store of dermal tattoo pigment in both mice and humans.

Although the dermis lacked macrophages for approximately 1 wk after DT treatment and it took several more weeks for the incoming macrophages to scavenge the released cell-free tattoo pigments, no macroscopic modification of the tattoo occurred. Before our study, the longevity of the undefined tattoo-pigment-laden cells found in the dermis was thought to be in the same order of that of a human adult, thereby accounting for long-term tattoo persistence (Fig. 10 B). Based on the present results, an alternative model for long-term tattoo persistence can be proposed. It takes into consideration the facts that the macrophages found in the dermis of adult mice are continuously replenished from circulating monocytes to compensate

for the loss of dying macrophages and that most of the tattoo particles that are released from macrophages after DT treatment of CD64<sup>dtr</sup> mice remain in situ and are recaptured by incoming macrophages. According to the pigment capture-release-recapture model (Fig. 10 A), in physiological conditions, when tattoo-pigment-laden macrophages die during the course of adult life, neighboring macrophages recapture the released pigments and ensure in a dynamic manner the macroscopic stability and long-term persistence of tattoos. Note that, in contrast to dermal cDCs, dermal macrophages do not have the capacity to migrate to draining LNs under normal and inflammatory conditions (Tamoutounour et al., 2013), an essential property for insuring tattoo persistence. Whether the pigment capture-release-recapture model of tattoo persistence applies to humans remains to be determined. In the case of the melanophages found in the human dermis, it has been suggested that they persist for years through longevity rather than continual renewal (Bigley et al., 2011).

Professional tattoos last for years or decades, and when no longer desired, they can be removed through the use of quality-switched lasers. Laser pulses lead to lysis of the tattoo-pigment-laden cells, fragmentation of the tattoo pigments, and their lymphatic transportation. Several cycles of laser treatment are required to achieve tattoo removal, and some tattoos remain immune to full removal. These difficulties are generally accounted for by the fact that a fraction of the fragmented pigment particles remains at the tattooing site and are recaptured by neighboring cells, a possibility formally demonstrated here by using the CD64<sup>dtr</sup> model. Considering the long duration needed for the reconstitution of the pool of dermal macrophages and the recapture of the whole released pigments, tattoo removal can be likely improved by combining laser surgery with the transient ablation of the macrophages present in the tattoo area. As a result, the fragmented pigment particles generated by using laser pulses will not have the possibility to be immediately recaptured, a condition increasing the probability of having them drained away via the lymphatic vessels.

In conclusion, we have validated a new model of DT-mediated depletion of tissue-resident macrophages, monocytes, and monocyte-derived cells in which DCs are spared. By permitting the emptying of the niche occupied in the skin dermis and intestinal lamina propria by CD64⁺ myeloid cells, the CD64<sup>dtr</sup> mouse model showed that all the mo-DC and macrophage subsets that were present in adult skin and intestine before DT treatment can be readily reconstituted from circulating Ly-6<sup>high</sup> monocytes. Furthermore, we showed that the myeloid cells of the ear skin dermis are dominated by melanophages, and we determined their origin and dynamics. Benefiting by our knowledge on melanophages, we determined the identity, origin, and dynamics of the skin myeloid cells that are capable of capturing tattoo-pigment particles and showed that

<footnote>skin monocytes (P1), mo-DCs (P2, P3), macrophages (P4, P5, Mac), melanophages (Mel), and cDC2s of selected genes contributing to IPA enrichments for the SSC-A<sup>low</sup> macrophage, SSC-A<sup>hi</sup> melanophage, and MHCII<sup>-</sup> versus MHCII<sup>+</sup> transcriptomic signatures. The cell samples which names are in black font on the top of the heat map originate from Tamoutounour et al. (2013), where the P4 and P5 cells correspond to the MHCII<sup>-</sup> and MHCII<sup>+</sup> macrophages from the present study. The exhaustive lists of genes responsible for the IPA enrichments shown in B are provided in Table S1.</footnote>
<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /><figcaption>Figure 8. SSC-A<sup>high</sup>CD64<sup>+</sup> melanophages can be found in melanocytic melanoma. (A) B6-albino mice were injected subcutaneously with 10<sup>5</sup> amelanocytic (Braf<sup>V600E</sup>) or melanocytic (B16) melanoma cells. CD45<sup>+</sup>Lin<sup>−</sup>Siglec-F<sup>−</sup> cells were isolated from tumor masses 14–18 d after inoculation and analyzed for the percentages of SSC-A<sup>low</sup> and SSC-A<sup>high</sup> cells. Note that Siglec-F staining permits elimination of eosinophils. (B) Morphological characteristics of representative SSC-A<sup>high</sup> melanophages and SSC-A<sup>low</sup> macrophages sorted from B16 melanoma tumor masses after cytospin onto glass slides. Four representative cells are shown for each population. Panels are made of two to four concatenated images. Bars, 10 µm. (C) Analysis of CD45<sup>+</sup>Lin<sup>−</sup>Siglec-F<sup>−</sup> SSC-A<sup>high</sup> cells isolated from B16 melanoma tumor masses 14 d after inoculation for CD11b, CD64, F4/80, MHCII, Ly6C, and CD11c expression. Appropriate isotype controls are shown for each of the analyzed markers. Data are representative of at least three experiments involving three animals per group.</figcaption></figure>

they correspond exclusively to dermal macrophages. Based on
those results, we proposed a dynamic model that accounts for
long-term tattoo persistence and suggest that tattoo removal

can be improved by combining laser surgery with the transient
and specific ablation of the macrophages present in the tattooed
area. Finally, we showed that melanocytic melanoma host read-
ily identifiable melanophages that permit determining whether
markers defined as macrophage-specific under steady-state
conditions keep or lose their discriminatory potential within
the tumor microenvironment.

**Materials and methods**

Mice

Ccr2<sup>-/-</sup> mice have been described (Jung et al., 2000). Cx3cr1<sup>cre/+</sup> (MW126Gsat) mice were generated by N. Heintz (The Rockefeller University, GENSAT, New York) and purchased from MMRRC. Rosa<sup>sl1-tdt/+</sup> (Ai14[RCL-tdT]-D) have been described (Madisen et al., 2010) and purchased from The Jackson Laboratory. Generation of *Fcgr1-IRES-EGFP-hDTR* gene-targeted mice is described below. C57BL/6 (B6) and BALB/c mice were purchased from January and B6 albino (B6[Cg]-Tyr<sup>c-2j</sup>l) and CD45.1 C57BL/6 mice from Charles River. All mice, except BALB/c, were maintained on a B6 background. Mice were housed under specific pathogen-free conditions, and in vivo procedures were performed following protocols approved by the Ethics Committee of Marseille in accordance with institutional, national, and European directives for animal care (approvals APAFIS 779-2015 0605 10534083 and APAFIS 781-2015 0605 14006182).

**Construction *Fcgr1-IRES-EGFP-hDTR* mice**

A 6.2-kb genomic fragment encompassing exons 4 to 6 of the *Fcgr1* gene (ENSMUSG00000015947) was isolated from a BAC clone of B6 origin (clone RP23-427P23; http://www.lifesciences.sourceforge.com). By using ET recombination, an IRES-EGFP-2A-hDTR-loxP-Cre-neoR-loxP cassette was introduced in the 3' untranslated region of the *Fcgr1* gene, 33 bp downstream of the stop codon. The targeting construct was abutted to a cassette coding for the DT fragment A expression cassette and linearized with Pme1. JM8.F6 C57BL/6N embryonic stem (ES) cells (Pettitt et al., 2009) were electroporated with the targeting vector. After selection in G418, ES cell clones were screened for proper homologous recombination by Southern blot. When tested on Sca1-digested genomic DNA, the 3' single-copy probe used to identify proper recombination events hybridized to a 7.0-kb WT fragment and to a 10.3-kb recombinant fragment. When tested on HindIII-digested genomic DNA, the 5' single-copy probe used to identify proper recombination events hybridized to a 10.7-kb WT fragment and to a 6.3-kb recombinant fragment. A neomycin-specific probe was used to ensure that adventitious nonhomologous recombination events had not occurred in the selected ES clones. Properly recombined ES cells were injected into FVB blastocysts. Germline transmission led to the self-excision of the loxP-Cre-NeoR-loxP cassette in male germinal cells. *Fcgr1-IRES-EGFP-hDTR* mice were identified by PCR of tail DNA. The pair of primers, sense 5'-CCCTTCCTCCCAGTGACAGTACTG-3' and antisense 5'-TGAACCCATCCACCCTGTGAG-3', amplifies a 402-bp band in case of the WT *Fcgr1* allele, whereas the pairs of primers, sense 5'-CCCTTCCTCCCAGTGACAGTACTG-3' and antisense 5'-CCTGCAAAGGGTCGCTACAGA-3', amplified a 349-bp
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**Figure 9.** Dermal macrophages are responsible for uptake and storage of tattoo pigment particles. (A) CD45⁺Lin⁻ cells from the tail skin of B6 mice that were left untreated or tattooed with green tattoo ink paste 3 wk before isolation were analyzed for the percentages of SSC-A<sup>low</sup> and SSC-A<sup>high</sup> cells. SSC-A<sup>high</sup> cells were further analyzed by using a gating strategy resolving cDC2s, monocytes, mo-DCs, and macrophages (Fig. S1 A). (B) Morphological characteristics of representative SSC-A<sup>high</sup> cells (green macrophages), SSC-A<sup>low</sup> cells (macrophages), and cDC2s sorted from tail skin of B6 mice tattooed with green tattoo paste 3 wk before isolation and analyzed by hematoxylin and eosin staining after cytospin onto glass slides. Bar, 10 µm. (C) Tails of CD64<sup>dtr</sup> mice were tattooed with green tattoo paste. 3 wk after, mice were treated twice and 24 h apart with 1 µg DT, and tail skin was analyzed 2 or 90 d after the last DT injection for the percentages of SSC-A<sup>low</sup> and SSC-A<sup>high</sup> cells found among CD45⁺Lin⁻ cells. (D) SSC-A<sup>high</sup> cells were sorted from the tail skin of CD64<sup>dtr</sup> mice that were tattooed with green tattoo paste and either left untreated (-DT) or treated with DT and analyzed 90 d (DT + d90) after DT treatment. Sorted cells were analyzed by hematoxylin and eosin staining after cytospin onto glass slides. One representative cell is shown for each population. Bar, 10 µm. (E) Tail skin of CD64<sup>dtr</sup> mice that were tattooed with green tattoo paste and either left untreated (-DT) or treated with DT and analyzed 2 d (DT + d2) after DT treatment was digested, and the resulting cell suspension was directly analyzed by hematoxylin and eosin staining after cytospin onto glass slides. Insets show magnification of selected areas. Bar, 10 µm. (F) Macroscopic view of the tails of CD64<sup>dtr</sup> mice that were tattooed with green tattoo ink paste and either left untreated (-DT) or treated
<figure><img src="image_41.png" /></figure>

band in the case of the *Fcgr1-IRES-EGFP-hDTR* allele. Because of its low level of expression, the EGFP reporter was not used in the present study.

## Isolation of monocytes, macrophages, and DCs from tissues and tumors

To isolate myeloid cells from ear skin, ears were processed as described (Bouladoux et al., 2017). In brief, ears were split into ventral and dorsal parts and incubated for 105 min at 37°C in RPMI containing 0.25 mg/ml Liberase TL (Roche Diagnostic Corp.) and 0.5 mg/ml DNase I (Sigma Aldrich). Digested tissue was homogenized by using Medicons and Medimachine (Becton Dickinson) to obtain homogenous cell suspensions. To isolate myeloid cells from tail skin, the skin was separated from the muscle and the vertebra and cut into small pieces and then digested in RPMI with Liberase TL and DNase as described for ear skin. The digested tissue was homogenized by using C Tubes and GentleMACS Dissociator (Miltenyi Biotec). To isolate myeloid cells from the lamina propria of the large intestine, the large intestine was opened longitudinally, cut into pieces, and washed in HBSS. The epithelial layer was eliminated by two incubations of 20 and 30 min in HBSS containing 2 mM EDTA. Intestinal pieces were washed twice in HBSS and then digested in RPMI medium containing 10% FCS, 1 mg/ml Collagenase 8 (Sigma Aldrich), and 30 ng/ml DNase I. Tissue was homogenized in C Tubes and GentleMACS Dissociator. Light-density cells were then purified by centrifugation on a 70-40% Percoll gradient (GE Healthcare). To isolate myeloid cells from tumor mass, tumors were cut into small pieces and then digested in RPMI medium containing 5% FCS, 0.8 mg/ml Collagenase 4 (Worthington), and 0.2 mg/ml DNase I for 40 min at 37°C. The digested tissue was homogenized by using C Tubes and GentleMACS Dissociator (Miltenyi Biotec).

## Analysis of blood cells

Blood was collected in EDTA-containing tubes and then transferred to Trucount Absolute Counting Tubes (Becton Dickinson). Red blood cells were lysed with BD FACS Lysing Solution (Becton Dickinson). The absolute number of cells present in a given volume was determined by using the bead-to-cell ratio.

## Antibodies

APC-Vio770-conjugated anti-NK1.1 (PK136), anti-CD3 (17A2), anti-Ly-6G (1A8), and anti-CD19 (6D5) were from Miltenyi Biotec. Biotin-conjugated anti-mouse-MERTK (BAF591) and PE-conjugated anti-CCR2 (475301) were from R&D. BV711-conjugated anti-CD11b (M1/70); BV605-conjugated anti-CD11c (N418); FITC-conjugated anti-CD43 (S7); Alexa Fluor 700-conjugated anti-CD45.1 (A20); Alexa Fluor 700-conjugated rat IgG2b κ isotype control (R35-38);

FITC-conjugated anti-CD45.2 (104); APC-conjugated anti-CD64 (X54-5/7.1); FITC-, PE-CF594-, and BV421-conjugated anti-Ly-6C (AL-21); BV650-conjugated anti-NK1.1 (PK136); BV421-conjugated anti-Siglec-F (E50-2440); biotin-conjugated anti-Ly-6C (AL-21); and biotin-conjugated anti-CD103 (M290) were from BD PharMingen. PE-Cy5-conjugated anti-CD24 (M1/69), BV650-conjugated anti-CD45.1 (A20), PE-conjugated anti-Ly-6G (1A8), Pe-Dazzle-conjugated anti-CD45.2 (104), APC-conjugated anti-F4/80 (BM8), APC-conjugated rat IgG2a κ isotype control (RTK2758), APC-conjugated anti-CD11c (N418), APC-conjugated Armenian hamster IgG isotype control (HTK888), APC-conjugated anti-CD5 (53-7.3), APC-conjugated anti-CD19 (6D5), APC-Cy7-conjugated anti-CD11b (M1/70), FITC-conjugated anti-CD11b (M1/70), FITC-conjugated rat IgG2b κ isotype control (MRG25-8T), BV711-conjugated anti-CD64 (X54-5/7.1), BV711-conjugated mouse IgG1 κ isotype control (MOPC-21), and BV510- and Alexa-700-conjugated anti-MHC Class II (IA/I-E; M5/114.15.2) were from Biolegend. PE-Cy5.5-conjugated anti-CD45 (30-F11) and Pe-Cy7-conjugated anti-CD115 (AFS98) were from eBioscience. Biotin-conjugated antibodies were detected by using streptavidin conjugated with PE-CF594 or Pe-Cy7 (BD PharMingen).

## Flow cytometry

Cells were stained and acquired on a FACS LSRII system and analyzed with FlowJo software (Tree Star). Neutrophils and B, T, and NK cells were systematically gated out by using a Lin⁺ gate (Fig. S1 and Fig. 2).

## Cell sorting

The different cell types were sorted on a FACS Aria II according to the marker combination specified on Fig. S1 A and Fig. 3. For microarrays (Affymetrix 1.0ST), cells were sorted directly in lysis buffer provided from the Qiagen RNAeasy PLUS extraction kit.

## Cell morphology

Sorted cells were spun onto Shandon Cytoslides with a Thermo Shandon Cytospin 4 cytofuge (4 min at 400 g), fixed with methanol and stained with hematoxylin and eosin.

## Electron microscopy

Sorted cells were incubated at 37°C on poly-Lysine-coated cover slides. After 30 min, attached cells were fixed by using 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4 for 1 h, postfixed for 1 h with 2% buffered osmium tetroxide, dehydrated in a graded series of ethanol solution, and then embedded in epoxy resin. Images were acquired with a digital camera Quemesa (SIS) mounted on a Tecnai Spirit transmission electron microscope (FEI Company) operated at 80 kV.

with DT and analyzed 2 (DT + d2) and 90 d (DT + d90) after DT treatment. Data are representative of at least three experiments. (C) CD11b⁺CD64⁺CCR2⁻ cells from the back of untreated CD45.2 albino mice or from the back of CD45.2 albino mice previously grafted with green-tattooed tail skin from CD45.1 B6 animals were analyzed for the percentages of SSC-Alow and SSC-Ahigh cells. The SSC-Ahigh CD64⁺ cells found in the back of the grafted CD45.2 albino mice were further analyzed for the percentages of CD45.1 (donor) and CD45.2 (host) expression. Data are representative of two to three independent experiments involving two to four animals per group.
<figure><img src="image_42.png" /></figure>

<figure><img src="image_43.png" /><figcaption>**Figure 10.** Models accounting for tattoo persistence. (A) The pigment capture-release-recapture model. Analysis of parabiotic mice that share a blood supply showed that the macrophage pool found in the noninflammatory dermis of adult mice is continuously replenished from circulating monocytes, thereby permitting replacement of dying macrophages. Upon tattooing, pigment particles (green) are captured by dermal macrophages. With time, macrophages laden with tattoo pigment particles die and release the tattoo pigment particles. Because of their size, those particles remain in an extracellular form at the site of tattooing where they are recaptured by neighboring or incoming macrophages. During the adult life, several cycles of pigment capture-release-recapture can occur, accounting for long-term tattoo persistence. (B) The “longevity” model. Although data on dermal macrophage dynamics (Tamoutounour et al., 2013; McGovern et al., 2014; and this paper) speak in favor of a pigment capture-release-recapture model, the possibility that a few dermal macrophages including those laden with green tattoo pigments had a longevity of the same order of adult mice cannot be excluded.</figcaption></figure>

## Confocal microscopy

Trunk and ear skin pieces were fixed in Antigen Fix (Microm Microtech) for 2 h, washed in 0.1 M phosphate buffer, and dehydrated overnight in 30% sucrose in 0.1M phosphate buffer. Tissues were snap frozen, and 25-µm sections were stained with DAPI. To obtain an en face section of ear dermis, the dorsal part was split from the ventral one 2 h after intradermal ear injection of CD64-APC (2 µg), fixed in Antigen Fix for 2 h, washed in phosphate buffer, stained with DAPI, and clarified with Rapiclear 1.47 (SunJin Lab). Immunofluorescence confocal microscopy was performed by using a Zeiss LSM 880 confocal microscope. Separate

images were collected for each fluorochrome and merged to obtain a multicolor image. Final image processing was performed with Imaris software (Bitplane) and Adobe Photoshop.

## RNA isolation and microarray analysis

Transcriptomic and bioinformatics analyses were performed as previously described (Tamoutounour et al., 2013). To perform a PCA focused on all variability across the four biological samples examined (Fig. 7 A), we selected all ProbeSets showing a fold change ≥1.2 between at least one pair of conditions, taking into account only those ProbeSets nonambiguously annotated
<figure><img src="image_44.png" /></figure>

to one gene only and further keeping only the highest ProbeSet for each gene. This led to selection of 1,054 unique genes. To select the genes showing a significant differential expression in one type of condition compared with the other three, between MHCII⁺ versus MHCII⁻ cells, or between melanophages and macrophages (Fig. 7 B), we used the GeneSign module of the BubbleGUM software using the metrics Minimal pairwise (Mean(test)/Mean(ref)) with a linear fold change threshold of 1.5 and a P value threshold of 0.05. The microarray data have been deposited in the GEO database under accession number GSE103339.

### Generation of parabiotic mice

Parabiotic mice were generated by using 8-9-wk-old male mice as described previously (Tamoutounour et al., 2013). For WT-WT parabiosis, CD45.1 B6 mice were sutured together with CD45.2 B6 mice, whereas for WT-Ccr2⁻/⁻ parabiosis, WT CD45.1 B6 mice were sutured with Ccr2⁻/⁻ CD45.2 B6 mice and subsequently treated with Bactrim for 8 wk before analysis.

### In vivo depletion of CD64⁺ cells

CD64<sup>dtr</sup> mice were injected intraperitoneally twice and 24 h apart with 1 µg DT (Calbiochem, EMD Millipore) in PBS. CD64<sup>dtr</sup> mice injected with PBS showed no ablation of monocyte-derived cells and macrophages.

### Tumor model

B6-albino mice were injected subcutaneously with 10⁵ amelanocytic (Braf<sup>V600E</sup>) or melanocytic (B16) melanoma cells (Brown et al., 2001; Zelenay et al., 2015).

### Tattooing

Tattooing was performed on the tail skin of mice under general anesthesia. Tail skin was tattooed by using the Animal Tattoo Ink Paste (Ketchum Mfg. Co, Inc.) and a 25-gauge needle. The ink-coated needle permits ink delivery in a topical manner. Before analysis, mice were kept for at least 3 wk until complete healing of the treated skin.

### Skin grafting

CD45.1 B6 mice were tattooed as described above. After 5 wk, the tattooed tail skin was explanted and grafted on the back of CD45.2 albino mice under general anesthesia. The back skin of the host was analyzed 6 wk after the graft.

### Statistical analyses

The unpaired Student t test was used for statistical analyses with GraphPad Prism software.

### Online supplemental material

Fig. S1 shows the gating strategy used to identify CD11b⁺ cell subsets within the skin of WT and CD64<sup>dtr</sup> mice. Fig. S2 shows the ablation of CD64⁺ cells present in the lamina propria of the large intestine upon DT treatment of CD64<sup>dtr</sup> mice. Fig. S3 shows the replenishment kinetics of the monocyte precursors and monocytes found in the BM after DT treatment of CD64<sup>dtr</sup> mice. Fig. S4 shows the lack of major impact of DT treatment on cDCs found in the skin of CD64<sup>dtr</sup> mice. Table S1, a separate Excel file, shows

the exhaustive lists of genes responsible for the IPA enrichments shown in Fig. 7 B.

### Acknowledgments

We thank M.M. Haniffa, C. Wohn, M. Guilliams, and N. Malissen for discussions; A. Adda-Benatia and F. Danjan for construction of the mice; C. Reis e Sousa for the Braf<sup>V600E</sup> melanoma; F. Devaux, L. Chasson, and the CIML flow cytometry core facility for technical help. Microarray experiments were performed at the Plateforme GenomEast (Strasbourg, France; http://www.igbmc.fr/technologies/5/team/54/).

This work was supported by Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Medicale, PHENOMIN, European Research Council (FP7/2007-2013 grant 322465 to B. Malissen), Marie Skłodowska-Curie Actions (PIOF-GA-2013-625328-MeTaPATH to S. Henri), ERA-NET Infect-ERA (ABIR: ANR-13-IFEC-0005-03 to B. Malissen), the DCBIOL Labex (ANR-11-LABEX-0043, grant ANR-10-IDEX-0001-02 PSL), and the A*MIDEX project (ANR-11-IDEX-0001-02). A. Baranska is supported by a fellowship from the ABIR project. We acknowledge PICT-IBiSA, a member of the France-BioImaging national research infrastructure, supported by the CelTisPhyBio LabEx (ANR-10-LBX-0038) as part of the IDEX PSL (ANR-10-IDEX-0001-02 PSL).

The authors declare no competing financial interests.

Author contributions: B. Malissen, S. Henri, and A. Baranska conceived the project. B. Malissen and F. Fiore designed the *Fcgr1-IRES-EGFP-hDTR* mice. A. Baranska and S. Henri did the experiments with the technical assistance of C. Malosse and O. Voluzan. M. Malissen contributed data in Fig. 2. A. Shawket, T.-P. Vu Manh, and M. Dalod performed electron microscopy. M. Baratin and M. Bajénoff performed the fluorescent microscopy analysis.

Submitted: 31 August 2017

Revised: 14 December 2017

Accepted: 6 February 2018

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