Article

A Liver Capsular Network

of Monocyte-DerivedMacrophages Restricts Hepatic Dissemination ofIntraperitoneal Bacteria by Neutrophil Recruitment

Graphical Abstract

Highlights

d A distinct subset of resident macrophages (LCMs) occupies

the hepatic capsule

d LCMs are replenished from blood monocytes in the

steady state

d LCMs recruit neutrophils in response to bacteria reaching the

liver capsule

d LCM depletion decreases neutrophil recruitment and

increases liver pathogen load

Sierro et al., 2017, Immunity 47, 374–388August 15, 2017 ª 2017 Elsevier Inc.http://dx.doi.org/10.1016/j.immuni.2017.07.018

Authors

Frederic Sierro, Maximilien Evrard,

Simone Rizzetto, ..., Lai Guan Ng,

David G. Bowen, Patrick Bertolino

Correspondencefrederis@ansto.gov.au (F.S.),d.bowen@centenary.org.au (D.G.B.),p.bertolino@centenary.org.au (P.B.)

In Brief

The hepatic sinusoids harbor a well-

characterized resident macrophage

population, Kupffer cells. Sierro et al.

report an additional liver-resident

macrophage population occupying the

hepatic capsule, phenotypically and

developmentally distinct from Kupffer

cells, which plays a role in

immunosurveillance by sensing

peritoneal pathogens and recruiting

neutrophils to control intrahepatic

bacterial dissemination.

Immunity

Article

A Liver Capsular Network of Monocyte-DerivedMacrophages Restricts Hepatic Dissemination ofIntraperitoneal Bacteria by Neutrophil RecruitmentFrederic Sierro,1,12,* Maximilien Evrard,2 Simone Rizzetto,3 Michelle Melino,4 Andrew J. Mitchell,5 Manuela Florido,6

Lynette Beattie,7 Shaun B. Walters,6 Szun Szun Tay,1 Bo Lu,1,8 Lauren E. Holz,7 Ben Roediger,6 Yik Chun Wong,1

Alessandra Warren,9 William Ritchie,6 Claire McGuffog,1 Wolfgang Weninger,6 David G. Le Couteur,9 Florent Ginhoux,2

Warwick J. Britton,6 William R. Heath,7 Bernadette M. Saunders,6,10 Geoffrey W. McCaughan,1 Fabio Luciani,3

Kelli P.A. MacDonald,4 Lai Guan Ng,2 David G. Bowen,1,11,* and Patrick Bertolino1,11,13,*1Centenary Institute and AW Morrow Gastroenterology and Liver Centre, University of Sydney and Royal Prince Alfred Hospital, Sydney,

NSW, Australia2Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Biopolis, Singapore, Singapore3Systems Immunology, Viral Immunology Systems Program, the Kirby Institute, UNSW, Sydney, NSW, Australia4Antigen Presentation and Immunoregulation Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia5Department of Chemical & Biomolecular Engineering, Materials Characterization and Fabrication Platform, University of Melbourne,

Melbourne, VIC, Australia6Centenary Institute and the University of Sydney, Newtown, NSW, Australia7Department of Microbiology and Immunology at Peter Doherty Institute for Infection and Immunity and the ARC Centre of Excellence in

Advanced Molecular Imaging at the University of Melbourne, Melbourne, VIC, Australia8Immunology Research Centre, St. Vincent’s Hospital, Melbourne, VIC, Australia9CERA and ANZAC Research Institute, Concord RG Hospital and University of Sydney, Sydney, NSW, Australia10School of Life Sciences, Faculty of Science, University of Technology Sydney, Sydney, NSW 2007, Australia11These authors contributed equally12Present address: Human Health, Australian Nuclear Science and Technology Organisation, Lucas Heights, NSW, Australia13Lead Contact

*Correspondence: frederis@ansto.gov.au (F.S.), d.bowen@centenary.org.au (D.G.B.), p.bertolino@centenary.org.au (P.B.)

http://dx.doi.org/10.1016/j.immuni.2017.07.018

SUMMARY

The liver is positioned at the interface between tworoutes traversed by pathogens in disseminatinginfection. Whereas blood-borne pathogens are effi-ciently cleared in hepatic sinusoids by Kupffer cells(KCs), it is unknown how the liver prevents dissemi-nation of peritoneal pathogens accessing its outermembrane. We report here that the hepatic capsuleharbors a contiguous cellular network of liver-resi-dent macrophages phenotypically distinct fromKCs. These liver capsular macrophages (LCMs)were replenished in the steady state from bloodmonocytes, unlike KCs that are embryonicallyderived and self-renewing. LCM numbers increasedafter weaning in a microbiota-dependent process.LCMs sensed peritoneal bacteria and promotedneutrophil recruitment to the capsule, and theirspecific ablation resulted in decreased neutrophilrecruitment and increased intrahepatic bacterialburden. Thus, the liver contains two separate andnon-overlapping niches occupied by distinct resi-dent macrophage populations mediating immuno-surveillance at these two pathogen entry points tothe liver.

374 Immunity 47, 374–388, August 15, 2017 ª 2017 Elsevier Inc.

INTRODUCTION

The ontogeny and function of resident myeloid populations

within solid organs has been an area of intense research over

recent years (Ginhoux and Guilliams, 2016). In the liver, Kupffer

cells (KCs) are the best characterized and represent one of the

largest subsets of tissue-resident macrophages. Like most tis-

sue-resident macrophages, they are highly phagocytic and

largely originate from the differentiation of local precursors that

seed the liver during embryogenesis (Bouwens et al., 1984).

Most KCs aremaintained by self-renewal in the adult and require

little input from bone marrow-derived precursors, although a

minor proportion of KCs can be derived from circulating blood

precursors after birth (Scott et al., 2016). Regardless of their

origin, steady-state KCs can be considered a homogeneous

liver-resident macrophage population with a specific transcrip-

tional program defined by their unique niche within the hepatic

sinusoids (Beattie et al., 2016). This location underscores the

important role of KCs in clearing blood-borne pathogens and

cellular debris (Beattie et al., 2016). However, in the setting of

inflammation, different macrophages may be recruited to the

liver via other pathways: monocyte-derived inflammatory mac-

rophages invade the liver following injury, while pro-repair

mature peritoneal macrophages can access the liver via the peri-

toneum during the recovery phase (Wang and Kubes, 2016).

Almost 30 years ago, a population of MHC class II-expressing

cells was observed in the hepatic capsule (Prickett et al., 1988).

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Figure 1. A Myeloid Cell Subset Expressing Macrophage Markers and Distinct from KCs Is Located within the Capsule of the Adult

Mouse Liver

(A) Cells (red) were stained in vivo by injecting PE-conjugated mAbs into C57BL/6 (WT) mice. Collagen fibers (blue) were detected using second harmonic

generation signals (SHG). Images represent 2PM maximal intensity projection of the hepatic capsule (top, 10 mm thickness centered on collagen fibers) and of

deeper parenchymal liver tissue containing KCs (bottom, 10 mm thickness). Bar, 50 mm.

(B) Densities of F4/80+ and CD45+ cells displaying dendrites in the liver capsules of WT mice.

(C) Flow cytometric analysis of WT liver leucocytes gated on live CD45+ MHCII+ cells.

(D) Top: 2PM images comparing the liver capsules of WT or Flt3l�/� mice stained in vivo for F4/80. F4/80+ cells densities in the liver capsule of the two lines are

quantified in the rightmost middle panel. Leftmost middle and lower panels: Flow cytometry quantitation of cDC1 and cDC2 subsets in the livers and spleens of

WT or Flt3l�/� mice.

(legend continued on next page)

Immunity 47, 374–388, August 15, 2017 375

These cells have dendritic morphology and have been initially

classified as dendritic cells (DCs) but not characterized further.

Hepatic capsular cells with dendritic processes have also been

noted in subsequent studies, in which they were variously cate-

gorized as KCs (Aoyama et al., 2010), fibroblasts or hepatic stel-

late cells (Lee et al., 2010), or myeloid DCs (Heymann et al.,

2015). More recently, a study of various hepatic myeloid cell

subsets also highlights the presence of cells with dendritic

morphology in the hepatic capsule, which the authors suggested

were dendritic cells (David et al., 2016).

In this study, we provide evidence that these MHC class II-ex-

pressing cells in the liver capsule are a macrophage population

that is developmentally and phenotypically distinct from KCs.

We also provide data supporting a role for this contiguous

cellular network of liver capsular macrophages in defending

the liver from infections traversing the peritoneal cavity.

RESULTS

Hepatic Capsular Cells with Dendritic MorphologyExpressMacrophageMarkers but Are Distinct fromKCsTo further characterize MHC class II+ cells with dendritic

morphology in the liver capsule, we used two-photonmicroscopy

(2PM) together with in vivo labeling. Tissue structures were

resolved to a depth of 50–80 mm to include the collagen fiber-

containing capsule (0 to 15 mm depth) as well as deeper paren-

chymal tissue containing KCs. In vivo-labeled CD45+ andMHCII+

cells exhibited distinct morphologies in the capsule and paren-

chyma.MHCII+ cells in the capsule displayed very long dendrites,

while those in the parenchyma were of more uniform size and

shape and did not possess extensive dendrites (Figure 1A).

MHCII+ capsular cells expressed F4/80, with staining faint at

10 min but clearly detected at 60 min after antibody injection,

suggesting that the antibody does not easily access the liver

capsule (Figure S1A). In contrast, KCs in the sinusoids stained

with anti-F4/80 as soon as 10 min after antibody injection (Fig-

ure S1A). This staining delay might explain why a recent study

concluded that capsular cells did not express F4/80 (David

et al., 2016). Similar densities of CD45+ and F4/80+ capsular cells

were detected in the capsule (Figure 1B), and in vivo labeling

confirmed co-expression of MHCII, CD45, and F4/80 by most

capsular cells (Figures S1B and S1C). Capsular cells exhibited

low expression of CD11c and expressed the key macrophage

markers CD64, CSF-1R, and CD14 (Figure 1A) but lacked

CD103 expression (Figure 1A). Most CD45+ capsular cells co-ex-

pressed CD64 (Figure S1D), suggesting that they were macro-

phages rather than DCs.We estimate that the entire adult mouse

liver capsule contains 300,000 CD45+F4/80+CD64+ cells, based

on their densities (Figure 1B) and the approximate total surface

area of the capsule.

While parenchymal KCs expressed the prototypical KC

marker Tim4 (Zigmond et al., 2014), capsular cells did not

(Figure 1A). Unlike KCs, capsular cells were CX3CR1hi (Fig-

(E) 2PM images and quantitation of hepatic capsular F4/80+ cells in liver capsule

(F) 2PM images and quantitation of hepatic capsular F4/80+ cells in liver capsule

multiple antibody doses (twice weekly for 3 weeks); bottom: single dose.

Images are representative of at least three animals, and bars represent mean ± SE

more than three individual mice (pooled from at least two independent experime

376 Immunity 47, 374–388, August 15, 2017

ure 1A). Consistent with CX3CR1 expression and confirming

findings in a recent study (David et al., 2016), the liver capsule

of Cx3cr1-GFP mice contains reporter+ cells with dendrites (Fig-

ure S1F). However, most GFP+ liver capsular cells co-expressed

F4/80 and the key macrophage marker CD64 (Figure S1F),

providing further evidence that they were not DCs but rather rep-

resented a macrophage subset distinct from KCs.

Further flow cytometric analysis of cell suspensions isolated

from the liver confirmed the presence of distinct liver macro-

phage subsets with mutually exclusive expression of Tim4 and

CX3CR1 and low expression of CD11c (Figure 1C). Cells with a

similar phenotype were not detected in the blood (Figure S1E),

suggesting that they were liver resident.

Hepatic Capsular Cells Are Dependent on CSF-1RSignaling but Not Flt3LThe density of liver capsular cells was assessed in mice deficient

for Flt3L, a growth factor critical for the differentiation of conven-

tional DCs (cDCs) from hematopoietic stem cells but dispensable

for the development of monocytes and macrophages (Liu et al.,

2009). The density of F4/80+ cells in the hepatic capsule was not

reduced in Flt3l�/�mice compared towild-type controls, whereas

cDCs (including CD103+CD11b� cDC1 and CD103�CD11b+

cDC2subsets)weredecreased inboth liverandspleen (Figure1D).

In addition, GFP+ cell density in the liver capsules of Cx3cr1-GFP

Flt3l�/� mice was not affected by Flt3L deficiency (Figure S2A).

Likewise, the b subunit of the GM-CSF receptor that promotes

the survival and homeostasis of CD103+CD11b+ DCs in non-

lymphoid tissues in the steady state without affecting most tissue

macrophage populations (Greter et al., 2012a) was dispensable

for the differentiation of hepatic capsular myeloid cells as as-

sessed by similar densities in wild-type and Csf2rb�/� mice

(Figure 1E). Although CSF-1R is expressed by several myeloid

cells including DCs, CSF-1R signaling is critical for the differentia-

tion and maintenance of macrophages in several tissues (Greter

et al., 2012b; MacDonald et al., 2010). Blockade of CSF-1R

signaling using multiple doses of the mAb M279 (Greter et al.,

2012b; MacDonald et al., 2010) resulted in the depletion of most

hepatic F4/80+ capsular cells (Figure 1F, top). A single dose of

M279 was sufficient to deplete most hepatic capsular cells, sug-

gesting that they were highly dependent on this factor (Figure 1F,

bottom). M279 treatment of Cx3cr1-GFP mice resulted also in

complete depletion of GFP+ liver capsular cells (Figure S2B).

Transcriptomic Profiling of Hepatic Capsular CellsIdentifies Them as a Macrophage SubsetIn order to purify hepatic capsular cells and to avoid inadvertent

activation by antibody binding during isolation, we sought to

identify a suitable fluorescent myeloid cell reporter that could

identify the F4/80+ population in the capsule (Figures 2A

and 2B). In most reporters assessed, capsular F4/80+ cells ex-

pressed the fluorescent protein (Figure S3A) but these lines

were unsuitable for identification of hepatic capsular cells for

s of WT or Csf2rb�/� mice.

s of WT mice treated with control Ig or anti-CSF-1R blocking Ab (M279). Top:

M numbers obtained from averaging counts from at least five random areas in

nts). See also Figures S1 and S2.

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Figure 2. Expression of Langerin in the Liver

Is Largely Restricted to Capsular Cells

(A) 2PM maximal intensity projection images of

GFP+ or YFP+ cells in the hepatic capsule (top) and

parenchymal liver tissue 10 mm deeper (bottom) of

various myeloid reporter mice. Collagen fibers are

blue. Bar, 50 mm.

(B) Left: densities of GFP+ or YFP+ cells with den-

dritic morphology in the liver capsule; Right: per-

centages of cells expressing the reporter protein

among liver capsular F4/80+ cells. Bars represent

mean ± SEM of cell numbers obtained from aver-

aging counts from at least five random areas per

animal in at least three individual mice (pooled from

at least two independent experiments).

(C)Representative cross-section imageof aCd207-

EGFPmouse liver showingGFPhi cells (green) in the

hepatic capsule (dashed line). The image is a

mosaic reconstitution of high-resolution adjacent

fields generated using the epifluorescence micro-

scope. Stitching has been applied following data

acquisition to generate the image. The variation in

background color observed in some areas of the

image corresponds to stitched areas. Bar, 50 mm.

(D) Flow cytometric analysis of liver leucocytes

from Cd207-EGFP mice.

(E) In vivo staining of hepatic capsular cells with

anti-langerin antibody. Bar, 50 mm.

(F) CyTOF analysis of liver leukocytes (represen-

tative of two independent experiments). The top

diagram on the left is a schematic representation

of the major GFP+ expressing populations (in red)

among reference populations identified based on

the expression of the indicated surface markers

(right panel). Each population has been assigned a

roman numeral: I, macrophages; II, CD103+ DCs;

III, DCs; IV, CD301b+ mononuclear phagocytes; V,

neutrophils; VI, eosinophils; VII, monocytes and

other CD11b-expressing cells. The middle and

bottom plots on the left show the location of gated

CD45+ GFP+ cells (middle) on a tSNE map of

lineage-neg liver non-parenchymal cells (bottom).

GFP expression is coded by the color palette.

See also Figure S3.

sorting because KCs and/or other myeloid cells were also

reporter positive (Figures 2A and S3B). However, in Cd207-

EGFP reporter mice, in which Langerhans cells (LCs) and

other DC subsets are GFP+, high GFP expression was suffi-

ciently restricted to hepatic capsular cells (Figures 2A and 2C).

GFPhi cells were Tim4�CD64+CD8�CD24�CD11cneg to loF4/80+

CD11bloMHCIIhiCX3CR1hiLy6Clo, further distinguishing them

from monocytes, KCs, and DCs within the liver (Figure 2D).

Expression of langerin (CD207) was also detected in some

hepatic capsular cells after in vivo labeling with an anti-langerin

antibody (Figure 2E). To determine the extent of heterogeneity

within the GFPhi population in liver capsular cells isolated from

Cd207-EGFPmice, we performed CyTOF analysis of 21 different

Im

markers. Unbiased analysis placed the

majority of GFPhi cells within a macro-

phage cluster, whereas GFPlo/int cells

clustered with CD103+ DCs (Figure 2F).

Further analysis restricted to MHCII+-

CD64+F4/80+ cells revealed that GFPhi cells were contained

within a CX3CR1+Tim4� macrophage cluster and were distinct

from CX3CR1�Tim4+ KCs (Figure S3C).

These results together with imaging analysis suggest that F4/

80+ capsular cells form a homogeneous macrophage subset

distinct from KCs but with variable langerin expression and indi-

cated that sorting GFPhi liver cells from Cd207-EGFP mice, with

CD103 as an exclusion marker, would isolate an appropriate

population of hepatic capsular cells for gene expression

profiling. Their mRNA expression profile was compared to

unaffiliated published sets of several steady-state tissue macro-

phages, DCs from tissues and lymphoid tissues, blood mono-

cytes (ImmGen Consortium database), dermal DCs and dermal

munity 47, 374–388, August 15, 2017 377

macrophages (Tamoutounour et al., 2013), and steady-state

KCs (Zigmond et al., 2014). Hepatic DCs (CD103+GFPint) and

skin LCs (CD103�GFPhi) were also isolated and sorted in the

same experiment as internal controls.

Hierarchical clustering that considered the ‘‘core DC’’ and

‘‘core macrophage’’ gene expression signatures as defined by

the Immgen Consortium placed hepatic capsular cells within a

group containing macrophages rather than DCs (Figure S4A;

Gautier and Yvan-Charvet, 2014;Miller et al., 2012). Accordingly,

principal component analysis (PCA) (Figure S4B) and hierarchi-

cal clustering analysis (Figure 3A) indicated that hepatic capsular

cells were more closely related to macrophages than to DCs.

Hepatic capsular cells did not express genes reported to be

specifically associated with KCs, DCs, or the two MHCII�F4/80hi and MHCII+F4/80lo peritoneal macrophage subsets (Fig-

ure 3B). Consistent with their phenotype determined by imaging

(Figure 1A), flow cytometry (Figure 1C), and CyTOF (Figures 2F

and S3C), hepatic capsular cells also expressed Cx3cr1, a

gene not expressed by KCs or peritoneal macrophages (Fig-

ure 3B). Cd207-EGFP+ hepatic capsular cells clustered more

closely with liver CD11b+ DCs than with other DC subsets (Fig-

ures 3A and S4A), but this clustering was likely affected by the

liver CD11b+ DC dataset being influenced by cross-contamina-

tion between CD11b+ DCs and CD11b+ macrophages as previ-

ously noted for datasets contained in the Immgen database

(Gautier et al., 2012; Miller et al., 2012; Guilliams et al., 2016).

PCA analysis considering macrophage transcriptomes re-

vealed a distinct hepatic capsular cell program that clustered

closer to the transcriptomes of dermal or gut macrophages

than to those of embryonically derived macrophages such as

KCs (Figure 3C). This relationship was further supported by hier-

archical clustering analysis restricted to gene sets specifically

enriched in prototypical embryo-derived macrophages (KCs,

lung, spleen, peritoneal cavity, microglia) and in prototypical

steady-state monocyte-derivedmacrophages (dermis, intestine)

(Figures S4C and S4D). Functional annotation clustering analysis

also identified a variety of differentially enriched processes dis-

tinguishing hepatic capsular cells and KCs (Figures 3D and S4E).

The above data identifiedCd207-EGFPhi F4/80+ capsular cells

as a distinct macrophage subset morphologically, anatomically,

and phenotypically different from KCs, leading us to name them

‘‘liver capsular macrophages’’ (LCMs).

LCMs in Adult Livers Are Bone Marrow Derived and CanBe Replenished from Circulating MonocytesTo determine whether LCMs could be replenished from adult

bone marrow (BM), C57BL/6 mice were irradiated and reconsti-

tuted with BM cells from Itgax-YFP mice, in which LCMs ex-

pressed the YFP reporter (Figures 2A, 2B, S3A, and S3B).

8 weeks post-reconstitution, LCMs of donor origin were present

in the liver capsule at densities similar to non-irradiated Itgax-

YFP mice, suggesting that LCMs were radiosensitive and could

be replenished by BM-derived cells (Figure 4A). As irradiation

induces systemic changes, LCM replenishment was examined

after transplantation in non-irradiated recipients. Livers from

Itgax-YFP mice were transplanted into B6.tdTomato recipient

mice. Recipient-derived Tomato+ LCMs but not KCs were de-

tected in the livers of transplanted animals as early as 5 days

after transplantation (Figure 4B).

378 Immunity 47, 374–388, August 15, 2017

In addition to the transplantation approach, which is associ-

ated with inflammation that might alter maintenance and/or

development of LCMs or their precursors, LCMs were also

examined by parabiosis. We analyzed CD45.1+ congenic and

CD45.2+ Cx3cr1-GFP parabionts (Figure 4C) for chimerism in

blood and lung by flow cytometry and in the liver capsule by

2PM. Consistent with previous reports (Guilliams et al., 2013),

relatively long-lived peripheral blood T cells from each animal

were evenly distributed in the blood of the parabionts, while alve-

olar macrophages that self-renew in situ independently of blood

precursors (Guilliams et al., 2013) were not exchanged (Fig-

ure 4C). Approximately 20% of Ly-6Chi and 40% of Ly-6Clo

bloodmonocyteswere exchanged betweenpartners (Figure 4C):

this lower percentage of chimerism and incomplete exchange of

blood monocytes, but not T cells, has been reported in previous

studies and attributed to a precursor half-life that is too short to

allow cells to reach the opposite parabiont (Liu et al., 2007). The

percentage of non-host chimerism for LCMs was approximately

half that observed for Ly6Chi blood monocytes (Figure 4C).

These results show that LCMs can be replenished from bone-

marrow-derived cells in the absence of irradiation or ischemia re-

perfusion-induced inflammation.

To evaluate whether LCMs could be derived from blood

monocytes, CD45.1+ WT mice were joined by parabiosis to

CD45.2+ Cx3cr1-GFP mice lacking CCR2 (Serbina and Pamer,

2006). Consistent with previous reports (Hashimoto et al.,

2013), CCR2-deficient monocytes that exhibit defects in

bone marrow exit were outcompeted by WT monocytes,

which made up >90% of circulating monocytes in both para-

bionts (Figure 4D, bottom). When compared to WT/WT para-

bionts, in the Ccr2�/�/WT setting, the density of WT LCMs

doubled in the liver capsule of the Ccr2�/� parabionts, while

Ccr2�/� LCMs could not be detected in the liver capsule of

WT parabionts (Figure 4D), suggesting that circulating mono-

cytes were LCM precursors. To demonstrate that LCMs can

derive directly from circulating monocytes, sorted GFP+ cells

from MacGreen (Csf1r-EGFP) mice that were enriched in

monocytes were adoptively transferred into syngeneic mice

pre-treated with a single dose of M279 antibody. 9 days after

adoptive transfer of sorted monocytes, GFP+ LCMs were de-

tected in the liver capsules of recipient mice (Figure 4E). In

contrast, sorted MacGreen peritoneal macrophages trans-

ferred into M279-treated recipients did not give rise to LCMs

(Figure 4E), suggesting that although these cells are recruited

to the liver after inflammation (Wang and Kubes, 2016), they

did not contribute to the LCM pool in the absence of

inflammation.

LCM Accumulation Accelerates at Weaning andCoincides with Establishment of the MicrobiotaLCM numbers were assessed during the postnatal period using

MacGreen mice. LCMs were almost absent during the first days

after birth; their accumulation accelerated after weaning at

3–5 weeks, and numbers remained relatively stable in adult ani-

mals (Figure 5A). To examine whether LCM development was

influenced by the microbiota as shown for gut-resident macro-

phages (Bain et al., 2014), we compared LCM densities

in germ- and specific-pathogen-free mice. LCMs were present

in adult germ-free mice (Figure 5B), but at lower density than in

B

pDCs Mono. LC Tissue MFs Lymphoid Tissue DCs LC

Skin LCs*

organ DCs & “CD11b+ DCs”

Dendritic cells Macrophages

C D

KC enriched functions

Hepatic capsular cell

enriched functions

A

Fcgr1 Mafb

Cx3cr1 Itgam

Siglec1 Clec4f Vsig4 Timd4 Gata6

Clec7a Tgfb2 Arg1 Fn1

Ccr7 Zbtb46

Itgax

KCs

Hepatic capsular

cells*

MHCII-F4/80hi

PC MFs

MHCII+

F4/80lo

PC MFs

Genes associated with Macrophages Genes associated with KCsGenes associated with PC MFs Genes associated with DCs

CD103+

CD207lo

Liver DCs

Gut MFs Splenic MFs Lymph node MFs Bone Marrow MFs Microglia Hepatic capsular cells*

Liver DCs* Hepatic Capsular cells*

-4 -2 0 2 4 6Fold change

Transport

Oxidation reduction process

Positive regulation of IkappaB kinase NFkappaB signaling

Metabolic pathways

Lysosome

Signal transduction

G protein coupled receptor signaling pathway

Hematopoietic cell lineage

Cytokine - cytokine receptor interaction

Figure 3. Genomic Expression Profile of Liver Capsular Cells Reveals a Distinct Macrophage Program

Hepatic capsular cells (CD103�GFPhi), liver DCs (CD103+GFPint), and epidermal LCs (CD103�GFPhi) were sorted from Cd207-EGFP mice. Gene expression of

the three cell subsets from three experimental replicates per subset was analyzed by microarray from purified mRNA and gene datasets compared to those of

plasmacytoid DCs (DCs), monocytes, LCs, various DCs,macrophages from the Immgen Consortium (GEO: GSE15907), dermal macrophages (GEO: GSE49358),

and steady-state KCs (GEO: GSE55606); asterisks after the subset name correspond to gene datasets generated in the current study.

(A) Hierarchical clustering based on transcriptome data.

(B) Heatmaps of relative expression of genes reported to be specifically expressed in KCs, peritoneal macrophages, and DCs.

(C) Principal component analysis based on macrophage transcriptomes.

(D) Functional enrichment analysis of the top gene-ontology processes that were enriched in hepatic capsular cells versus embryo-derived KCs.

See also Figure S4.

age-matched specific-pathogen-free mice (Figure 5B). Treat-

ment with antibiotics for 2 weeks from before weaning also led

to a decrease in LCM density (Figure 5C). Therefore, while not

essential, the microbiota could promote the development or

expansion of LCMs. This appeared to be independent of TLR

signaling, as adult mice lackingMyD88 or TLR4 contained similar

LCM densities as WT animals (Figure 5D).

Increased numbers of LCMs at weaning could have resulted

from increased precursor recruitment, enhanced proliferation of

local precursors, and/or increased proliferation of differentiated

Immunity 47, 374–388, August 15, 2017 379

cells. LCM proliferation was assessed both during the weaning

phase and in adults using FUCCI transgenicmice that allowdeter-

mination of cells in G1 and/or S/G2/M phases of the cell cycle

using dual color imaging. None of the cells analyzed in the hepatic

capsule (>100 cells/time point/mouse) were proliferating (Fig-

ure S5), indicating that the increased numbers of LCMs at wean-

ing were not driven by increased local proliferation of LCMs or of

their precursors, but rather resulted from increased replenishment

from blood-borne precursors, which we postulate could be

monocytes. The positive correlation between the number of

monocytes and LCM density was supported by (1) increased

numbers of circulating monocytes and LCMs after weaning

(Figure 5E), (2) decreased numbers of circulating monocytes

and LCMs in antibiotic-treated mice (Figure 5F), (3) decreased

numbers of circulating monocytes after treatment with M279

that depleted LCMs (Figure 5G), and (4) the reported decrease

in hematopoiesis, including monocytes, in germ-free mice (Khos-

ravi et al., 2014).

CSF-1 Is Enriched in the Liver Capsule and Is Critical forLCM HomeostasisCCR2 regulates recruitment ofmonocytes and their differentiation

into tissue-resident CX3CR1hi macrophages in the small intestinal

lamina propria (Bain et al., 2014) and could play a similar role in re-

cruitingmonocytes to the liver capsule. AsCcr2�/� LCMnumbers

in the liver capsule of WT parabionts (Figure 4D) may have been

influenced by lack of CCR2 regulation of monocyte exit from the

bone marrow, we tested whether circulating monocytes in

Ccr2�/� mice could be recruited to the liver capsule and give

rise to LCMs in the absence of competing WT monocytes. The

density of LCMs in liver capsules of CCR2-deficient mice was

similar to that in CCR2-sufficient mice (Figure 5D), indicating that

althoughCCR2playeda key role in regulating the number of circu-

lating monocytes, it was dispensable for their recruitment to the

liver capsule and/or their intrahepatic differentiation into LCMs.

The chemokine receptors CXCR4, CCR5, and CX3CR1 and

chemokine CCL5, also known to influence macrophage and

monocyte recruitment, were found to be dispensable for the re-

cruiting of circulating LCM precursors and/or for their intrahe-

patic differentiation as determined by LCM density in deficient

animals (Figure 5D). In addition, sorted purified CX3CR1-defi-

cient monocytes adoptively transferred into M279-treated recip-

ient mice reconstituted the liver capsule and gave rise to LCMs

(Figure 5H).

LCMs are sensitive to anti-CSF-1R antibody (Figure 1F) and

thus depend on CSF-1R signaling. IL-34, one of the two CSF-

1R ligands (Greter et al., 2012b), was not critical, as Il34�/�

mice contained the same LCM density as WT mice (Figure 5I).

CSF-1, the second CSF-1R ligand, was enriched in the hepatic

capsule (Figure 5J) while LCM density was significantly

decreased when CSF-1 was blocked (Figure 5K). Together,

these results indicate that LCMs were dependent on the

CSF-1R ligand CSF-1 rather than IL-34.

LCMs Form a Contiguous Cellular Network at theInterface between Liver Parenchyma and PeritonealCavityLCMs formed a contiguous cellular network in the hepatic

capsule (Figure 6A) reminiscent of LCs in the epidermis and in

380 Immunity 47, 374–388, August 15, 2017

the oral epithelium (Capucha et al., 2015). LCs from these tissues

express langerin and have been reported to be GFP+ in Cd207-

EGFP mice (Capucha et al., 2015). To explore the distribution of

GFP-expressing cells resembling LCMs in Cd207-EGFP mice,

we examined the superficial layers of other organs, including

the peritoneal membrane and serosa of other abdominal organs.

Although GFP+ cells arranged in networks and possessing

distinctive dendrites could easily be detected in the liver capsule

and epidermis of Cd207-EGFP mice, they were not detected in

the capsule of other organs examined (Figure 6B). GFP+ cells

were detected in the thymus and Peyer’s patches, but these

were not of dendritic morphology. These cells have previously

been described in all secondary lymphoid organs and character-

ized as blood-derived CD8a+ DCs (Douillard et al., 2005).

LCMs displayed long dendrites wrapped around collagen

fibers (Figures 6C and 6D). Their restricted anatomical location

was suggestive of a specific role at the interface between the

peritoneal cavity and the hepatic parenchyma. To assess

whether these cells could access the peritoneal cavity, recon-

structions of the hepatic capsule of Itgax-YFP or MacGreen

crossed with tdTomato mice were generated to outline how

LCMs interacted with collagen fibers, hepatocytes, and liver

sinusoids (Figures 6E and 6F). LCMs were seen extending long

dendrites into the lumen of underlying hepatic sinusoids (white

arrows in Figures 6E and 6F), while some extended dendrites

outside the capsule, toward the peritoneal cavity (Figure 6F,

white asterisks). To observe their steady-state dynamics,

LCMs were imaged for 4–5 hr using in vivo live 2PM. Although

they did not migrate within this time frame, LCMs exhibited a

behavior similar to that described in skin LCs and brainmicroglial

cells, termed ‘‘dendrite surveillance extension and retraction

cycling habitude’’ (dSEARCH) (Figure 6G; Movie S1; Ng et al.,

2008). These imaging studies suggested that LCMs extended

dendrites toward both the blood and the peritoneal cavity and

could potentially sample these compartments.

LCMs Are Able to Sense Bacteria Reaching the HepaticCapsuleThe capsule of the liver is exposed to the peritoneal cavity, a

compartment traversed by pathogens such as M. tuberculosis

(Guirat et al., 2011) and L. monocytogenes (Havell et al., 1999)

to disseminate infection. Infection of mycobacteria via the

intra-peritoneal route is a physiological model of abdominal

tuberculosis (Guirat et al., 2011), a major site of infection before

milk pasteurization was adopted. We thus sought to determine

whether LCMs could sense bacteria or bacteria products and

prevent pathogen dissemination.

Fluorescent polystyrene beads administered i.p. with or

without LPS were in close association with LCMs within 2 hr,

suggesting that LCMs were able to pick up particulate antigens

(Figure S6A). To explore whether LCMs could also take up

bacteria, we performed similar experiments using M. bovis

bacillus Calmette-Guerin (BCG) expressing mCherry. When

BCG-mCherry was injected intravenously (i.v.), most bacteria

were associated with KCs (Figure S6B), while very few were de-

tected inside YFP+ or GFP+ LCMs. In contrast, some BCG-

mCherry administered i.p. was associated with LCMs within

2 hr (Figure S6C). To directly image the dynamics of this pro-

cess, bacteria were directly applied to the capsule and then

Figure 4. LCMs Are Derived from Monocytes and Are Continuously Replenished in the Steady State(A) WT mice were irradiated and reconstituted with Itgax-YFP bone marrow. Representative images depict YFP+ cells in the hepatic capsule and skin. Graph

represents the mean ± SEM of total counts of YFP+ cells in the liver capsule before (non-irradiated age-matched controls) and after reconstitution in eight animals

analyzed in two independent experiments.

(B) Itgax-YFP livers were transplanted into tdTomato recipients. Representative images of the liver capsule and parenchyma 5 days after transplantation (n = 5

animals analyzed in two independent experiments).

(legend continued on next page)

Immunity 47, 374–388, August 15, 2017 381

immediately imaged by 2PM intravital microscopy. LCM den-

drites were seen extending toward bacteria (Figure 7A; Movie

S2), suggesting that LCMs sensed peritoneal bacteria accessing

the liver capsule.

Increased Hepatic Dissemination of PeritonealPathogens in the Absence of LCMsTo assess the potential for LCMs to inhibit dissemination of perito-

neal pathogensbreaching the liver capsule,wedepleted this pop-

ulation by administering a single dose of anti-CSF-1R (M279) anti-

body,which completely ablatedLCMsbetween2 and 5days after

treatment (Figure 1F) but did not have an effect on the numbers of

KCs or peritoneal macrophages (Figure 7B). 3 days after injection

with anti-CSF-1R or control antibody, mice were infected i.p. with

BCG or L. monocytogenes. 6 hr after bacterial injection, the livers

of M279-treated mice contained approximately 10 times more

BCG than the livers from mice treated with control antibody (Fig-

ure 7C). Similar results were obtained after infection with Listeria

(Figure 7C). M279 treatment did not result in increased numbers

of bacteria in the peritoneal cavity at the same time point (Fig-

ure S6D). These results confirm a role of LCMs in preventing peri-

toneal bacteria dissemination in the liver.

LCMs Prevent Bacterial Dissemination by RecruitingNeutrophilsTo assess whether LCMs directly eliminate peritoneal pathogens

or recruit other phagocytic cells, we investigated whether the

bacteria were mostly associated with LCMs or other cell sub-

sets. Both monocytes and neutrophils were recruited to the

capsule (Figure 7D) after intraperitoneal BCG-mCherry injection.

Although some LCMs interacted with BCG, some bacteria were

clearly associated with neutrophils but very rarely with mono-

cytes (Figure 7E). After exposure of the liver capsule to LPS or

live bacteria, circulating neutrophils were recruited from the he-

patic sinusoids and reached the liver capsule by crawling along

LCM dendrites (Figure 7E; Movies S3 and S4). Once in the

capsular region, recruited neutrophils remained localized around

LCMs (Movies S3 and S4). LCMdepletion with anti-CSF-1R anti-

body did not decrease circulating neutrophils (Figure 7F) but re-

sulted in reduced neutrophil numbers in the hepatic capsule (Fig-

ure 7G), indicating that LCMs promoted neutrophil recruitment.

Collectively, these observations indicate that LCMs, likely

through recruitment of neutrophils, play a role in preventing

dissemination of peritoneal pathogens breaching the liver

capsule.

DISCUSSION

We have identified a distinct subset of resident macrophages

that coexists with KCs in the uninjured liver. These cells are

(C and D) CD45.1+ mice were parabiotically joined to (C) congenic CD45.2+ Cx

parabiont were identified by in vivo anti-CD45.1-PE labeling (red) or GFP (green); m

Graphs show the degree of non-host chimerism in the different parabionts. The pr

calculated by flow cytometry while the frequency of KCs and LCMs was calculate

per group.

(E) WT recipient mice were treated with a single dose of M279 antibody to deplete

mice adoptively transferred 7 days later. 2PM maximal intensity projection image

See also Figure S5.

382 Immunity 47, 374–388, August 15, 2017

located in the hepatic capsule, just under a layer of mesothelial

cells where they seem to form a defensive line against pathogens

within the peritoneal cavity.

MHCIIhi capsular cells have been observed in previous studies

(Prickett et al., 1988) but were misclassified as KCs (Aoyama

et al., 2010), fibroblasts, or hepatic stellate cells (Lee et al.,

2010). A recent study (David et al., 2016) reported GFP+ cells

with dendrites in the liver capsules of Cx3cr1-GFP reporter

mice, assessing them to be F4/80 negative and classifying

them as DCs via transcriptomic analysis of sorted GFP+F4/80�

cells. While we do not exclude the presence of Cx3cr1-GFP+

DCs within the liver parenchyma, our study shows that GFP+

cells in the liver capsule of Cx3cr1-GFP mice expressed F4/80,

as well as CD64 and CSF1R, macrophage markers that were

not assessed in the study of David et al. (2016), and further

were Flt3L independent. Differences in assessment of F4/80

expression between the current study and that of David et al.

(2016) may be related to lower F4/80 expression in LCMs

compared to KCs, as suggested by CYTOF analysis, and/or

slower access of intravenously administered antibody to

the matrix-rich capsule than to the hepatic sinusoids. As F4/

80+CX3CR1-GFP+ capsular cells would have been excluded

from assessment via the sort strategy performed by David

et al. (2016), the transcriptomic analysis performed in that study

likely characterized a population of Cx3cr1-GFP+ F4/80neg

cells enriched in liver DCs and monocytes that did not contain

hepatic capsular cells. In contrast, the current phenotypical,

transcriptomic, and ontogenetic analyses consistently demon-

strate hepatic capsular cells to be macrophages.

Our results suggest that LCMs are derived from circulating

monocytes. The dependence of LCMs on CSF-1R suggests

that signaling via this pathway is critical for their recruitment

and/or maintenance. As the number of circulating monocytes

is dependent on CSF-1R signaling, it is difficult to exclude regu-

lation of LCMs by CSF-1 via control of the number of circulating

precursors. However, the concentration of CSF-1 detected in the

hepatic capsule favors a model in which local CSF-1:CSF-1R in-

teractions play a role in the recruitment of LCM precursors and/

or maintenance of LCMs in the liver capsule.

A recent study demonstrated that during liver injury, peritoneal

macrophages expressing GATA-6 can migrate via the hepatic

capsule to repair damaged liver tissue (Wang and Kubes,

2016), raising the possibility that LCMs could also be derived

fromperitoneal macrophages. However, several arguments sug-

gest that this is unlikely, at least in the steady state. First, tran-

scriptomic analysis of LCMs indicates that they do not express

Gata6 and other genes expressed by peritoneal macrophages.

Second, in contrast to monocyte transfer, transfer of peritoneal

macrophages under the same conditions did not give rise to

LCMs. While our results suggest that peritoneal macrophages

3cr1-GFP mice or (D) CD45.2+ Cx3cr1-GFP Ccr2�/� mice. LCMs from each

ultiphoton images are representative of the hepatic capsule in each parabiont.

oportion of CD45.1+ blood monocytes, T cells, and alveolar macrophages was

d by 2PM. Bar graphs represent mean ± SEM of at least three individual mice

LCMs andGFP+ sortedmonocytes or peritoneal macrophages fromMacGreen

s of the hepatic capsule were obtained 9 days after cell transfer. Bars, 20 mm.

Day 22 Day 4 Day 28 A C

apsu

le

Den

sity

of r

epor

ter p

os

LCM

s (c

ells

/mm

2 )

400

300

200

100

0 0 50 100 200 400 Time after birth (days)

Weaning

Den

sity

of F

4/80

+

LCM

s (c

ells

/mm

2 )

Germ FreeSPF housing

*400

300

200

100

0

SPF Germ free

Cap

sule

SPF housing Germ free B

Den

sity

of F

4/80

+

LCM

s (c

ells

/mm

2 )

D 400

300

200

100

0

Den

sity

of F

4/80

+

LCM

s (c

ells

/mm

2 )300

200

100

0

CSF-1 WTIl34-/-

Den

sity

of F

4/80

+

LCM

s (c

ells

/mm

2 )

300

200

100

0

*106

5 x105

0

Blo

od m

onoc

ytes

co

unts

(cel

ls/m

l)

Pre- injection (day 0)

Anti- CSF-1R (day 4)

*

Control Ig G KJIH

0

0

0

0*

300

200

100

0

Den

sity

of F

4/80

+

LCM

s (c

ells

/mm

2 )

SPF Antibiotics

Antibiotics

Cap

sule

SPF housingC

2 4 6

Blo

od m

onoc

ytes

co

unts

(cel

ls/m

l)

2 4 60

5

6

*

106

5 x105

0

Time after birth (weeks)

E F Water Antibiotics

106

5 x105

0

*

Blo

od m

onoc

ytes

co

unts

(cel

ls/m

l)

WTCcr2

-/-

Cx3cr1

-/-

Ccr5-/-

Ccl5-/-

Tlr4-/-

Myd88

-/-

Figure 5. LCMs Accumulate after Weaning and Their Recruitment and/or Maintenance Depends on CSF-1

(A) Left: representative 2PMmaximal intensity projection images of LCMs in MacGreen mice. Right: kinetics of LCM accumulation in the liver capsule after birth.

Graph represents pooled data from at least three animals (from at least two independent litters) per time point. Bar, 50 mm.

(B and C) 2PM images (left) and quantitation (right) of F4/80+ in vivo labeled LCMs from (B) 8-week-oldWTmice housed in SPF or germ-free conditions and (C) 5-

week-old untreatedWTmice housed in SPF conditions or treated with antibiotics for 2 weeks from the day of weaning. Graph represents counts from at least four

animals per group pooled from two independent litters. Bar, 50 mm.

(D) Density of LCMs in WT mice and mice deficient for CCR2, CX3CR1, CCR5, CCL5, TLR4, or MyD88.

(E–G) Bloodmonocyte counts: (E) at different ages before and after weaning; (F) at 5 weeks of age after 2 weeks of antibiotic treatment; (G) after treatment of adult

mice with a single dose of M279.

(H) WT recipient mice were treated with a single dose of M279 antibody to deplete LCMs and injected i.v. 7 days later with GFP+ sorted monocytes from Cx3cr1-

GFPgfp/gfp mice. 2PM maximal intensity projection images of the hepatic capsule were obtained 9 days after cell transfer. Bar, 20 mm.

(I) Density of LCMs in WT and IL-34-deficient mice.

(J) Immunostaining for CSF-1 (red) in the liver. Bar, 50 mm.

(K) Density of LCMs in mice treated with anti-CSF-1 or isotype control antibody (*p = 0.029, Mann Whitney test, n = 4).

In all graphs, bars represent mean ± SEM of at least three individual mice per group.

do not differentiate into LCMs in the uninflamed liver, it is

tempting to speculate that they potentially use LCMs as gate-

ways when they migrate via the hepatic capsule to repair

damaged liver tissue (Wang and Kubes, 2016).

Recent studies suggest that the origin, distribution, specific

tissue location, and functional specialization of macrophages is

imparted by micro-environmental cues that shape the epigenetic

chromatin landscape of macrophages or their precursors rather

than by an imprinted precursor program (Lavin et al., 2014;

Mass et al., 2016). LCMs occupy a separate liver niche to KCs,

and it is thus not unexpected that they have a different transcrip-

tomic signature to the embryonically derived and monocyte-

derived KCs recently described by Scott et al. (2016). Whole-tran-

scriptome analysis demonstrated that LCMs were more closely

Immunity 47, 374–388, August 15, 2017 383

Stomach Small Int. serosaBladder Diaphragm Thymus

PeritoneumKidney asores.tnIegraL.acirepv.rtraeHLarge Int. mucosa

Skin (ear) Liver Lung Spleen Peyer’s Patch serosaB

GFP (LCMs) Collagen (SHG)

H

M

col

col

D

GFP Collagen (SHG)

GFP (LCMs) Collagen(SHG)

A

C

E F

G

YFP (LCM) Collagen (SHG) mTom (Hep. + other cells)

LCM

MHCII GFP (Hepatocytes) mTom (Other cells incl. sinusoids)

llt=0 t=15 min t=30 min

Figure 6. LCMs Form a Contiguous Network within the Liver Capsule and Scan Their Environment by Extending Dendrites

(A) 1 mm2 mosaic maximal intensity projection of the liver capsule from a Cx3cr1-GFP mouse. Bar, 100 mm.

(B) 2PM maximal intensity projection images (>50 mm thickness) at the surface of various organs from Cd207-EGFP mice. Collagen is blue. Bar, 50 mm.

(C) High-resolution maximal intensity projection image of GFP+ cells in the liver capsule of a Cd207-EGFP mouse. Bar, 50 mm.

(D) Electron microscopy image of WT mouse liver capsule, depicting an LCM interacting with collagen fibers (col). Abbreviations: M, mesothelial cells; H,

hepatocytes. Bar, 5 mm.

(E) Representative cross-section of a Itgax-YFP3 tdTomato mouse liver showing a YFP+ LCM (yellow) extending a long dendrite between hepatocytes (gray) and

into the lumen of the nearest sinusoid (white arrow). Bar, 5 mm.

(F) Section of the liver capsule of an Alb-cre 3 tdTomato mouse stained with anti-MHCII antibody showing an MHCII+ LCM (yellow) extending a long dendrite

between hepatocytes (green) into the lumen of the nearest sinusoid (arrow) and projections toward the outer part of the capsule toward the peritoneal cavity (white

stars). Bar, 50 mm.

(G) Dynamics of LCMs in a Itgax-YFPmouse liver depicted by overlaying a series of 2P-IVM time lapse images at t = 0, 15min, and 30min reveals typical scanning

behavior. Bar, 50 mm.

See also Movie S1.

384 Immunity 47, 374–388, August 15, 2017

Figure 7. LCMs Sense Bacteria and Control

Hepatic Ingress of Bacteria from the Perito-

neal Cavity via Recruitment of Neutrophils

(A) Representative 2P-IVM time-lapse images of

an LCM (green) in a Itgax-YFP mouse liver capsule

exposed to mCherry-labeled BCG (purple) ex-

tending dendrites toward bacteria. Times repre-

sent exposure time to bacteria. Bars, 20 mm.

(B) Following treatment with M279 or isotype

control antibody 3 days prior, KCs were enumer-

ated by in vivo labeling using anti-F4/80-PE anti-

body and 2PM analysis at parenchymal depth.

Peritoneal cavity macrophages were enumerated

by flow cytometry. Bar graphs represent mean ±

SEM of at least three individual mice per group in

at least two independent experiments.

(C) Bacterial loads (CFU) of liver tissue harvested

after 6 hr, frommice injected i.p. with 23 105 cfu of

BCG-mCherry or Listeria. Mice were treated with

M279 or isotype control antibody 3 days prior. Bar

graphs represent mean ± SEM of at least four in-

dividual mice per group in at least two indepen-

dent experiments.

(D) Representative maximal intensity projection

mosaic of the liver capsule from a Itgax-YFPmouse

(LCM yellow) injected i.p. with LPS 6 hr prior

showing that most neutrophils were in close prox-

imity to LCMs. Neutrophils (red) were stained by

in vivo labeling using PE-conjugated anti-Ly6G

antibody. Bar, 100 mm.

(E) Top: Representative 2PM time-lapse images of

a cross-section of aMacGreenmouse liver capsule

exposed to LPS (10 mg/mL) showing three different

neutrophils extravasating at the samepoint of entry

in the liver capsule. Bottom left: Still 2PM image of a

MacGreen mouse liver capsule exposed to BCG-

mCherry. Lines represent tracks from neutrophils

that extravasated into thecapsular spacewithin the

time frame of imaging. Bottom right: Representa-

tive 2PM image of a MacBlue3MacGreen mouse

4 hr after i.p. injection with 2 3 105 cfu of BCG-

mCherry and 1 hr after i.v. injection with anti-Ly6G;

arrows indicate neutrophils containing bacteria.

Bars, 25 mm for both upper and lower panels.

(F) Blood neutrophil counts assessed by flow cy-

tometry 3 days after treatment with M279 or iso-

type control antibody.

(G) Representative images (left) and density (right)

of neutrophils in the hepatic capsule of WT

mice 3 days after treatment with M279 or isotype

control antibody and 6 hr after i.p. injection with

LPS or 2 3 105 cfu of BCG. Bar, 25 mm.

In (F) and (G), bar graphs represent mean ± SEM

of at least three individual mice per group in at

least two independent experiments. See also

Movies S2, S3, and S4 and Figure S7.

related to monocyte-derived tissue macrophages described

in the steady-state adult gut or skin. The intestine contains

mostlymonocyte-derivedmacrophages, while skin and pancreas

contain distinct niches harboring phenotypically distinct macro-

phage subsets (Calderon et al., 2015; Tamoutounour et al.,

2013). The different niches in the skin and pancreas are occupied

bymacrophages of distinct ontogeny, similar to our findings in the

liver. The skin contains prototypical embryonically derived tissue

macrophages (LCs) in the epidermis andmonocyte-derivedmac-

rophages in the dermis. The pancreas has three separate niches:

the islets of Langerhans contain a monocyte-derived subset,

while the interacinar stroma contains both monocyte-derived

and embryonically derived macrophages (Calderon et al., 2015).

It is unclear whether continuous differentiation of monocyte-

derived tissue-residentmacrophages is restricted to these organs

or whether it occurs in other tissues.

KCs have previously been considered to be the only liver-resi-

dent macrophages in the liver. They are positioned to clear

Immunity 47, 374–388, August 15, 2017 385

blood-borne pathogens in the lumen of the hepatic sinusoids.

The location of LCMs at the interface between blood and perito-

neal cavity and their ability to interact with bacteria accessing the

liver capsule raised the possibility of a similar role at the liver

surface. Pathogens such as M. tuberculosis (Guirat et al.,

2011), Listeria (Havell et al., 1999), C. albicans, and Chlamydia

traverse the peritoneal cavity to spread systemically (M€uller-

Schoop et al., 1978) from the gut or the fimbriated ends of the fal-

lopian tubes in females. Our findings that LCM dendrites could

reach both blood and peritoneal cavity and capture beads or

bacteria applied to the capsule or present in the peritoneal cavity

suggest that LCMs sense and/or sample particulate peritoneal

antigens or bacteria reaching the liver capsule. LCMs were

indeed closely associated with i.p. injected BCG and were able

to extend dendrites toward the bacteria. Consistent with a role

of LCMs in preventing the dissemination of bacteria from the

peritoneal cavity into the liver, selective LCM depletion led to

higher bacterial counts in the liver without affecting counts in

the peritoneal cavity. Our data suggest that this protective im-

mune role of LCMs is mediated by neutrophils that are recruited

to the liver capsule after LPS treatment and exposure to BCG,

extravasating in close proximity to LCMs. A role for LCMs in

the recruitment of neutrophils to the liver capsule was supported

by transcriptomic analysis demonstrating their expression of a

number of factors, including CXCL1, CXCL2, CCL2, CCL3, and

CCL4 that have potential to serve as neutrophil chemoattrac-

tants. Specific depletion of LCMs was associated with reduced

capsular neutrophil recruitment and higher hepatic bacterial

counts. These results suggest that LCMs are physiological sen-

sors of peritoneal pathogens that play a role in preventing bacte-

rial dissemination into the liver by rapidly recruiting neutrophils to

the hepatic capsule. Of note, neutrophils have been reported to

be critical in controlling liver infection after i.p. injection of Listeria

(Conlan and North, 1994) and immune-competent mice die

within a few days from this infection when neutrophils are

depleted (Rogers and Unanue, 1993).

Although infection via the peritoneum would affect other or-

gans, it is unclear whether cells similar to those described in

the liver prevent bacterial dissemination to these other tissues.

It is also unknown whether LCMs interact or cooperate with peri-

toneal macrophages to eliminate peritoneal pathogens.

In conclusion, LCMs constitute a unique monocyte-derived

macrophage subset that is ontogenetically and phenotypically

distinct from KCs, forming a capsular network in the normal adult

liver that is not detected in the capsule of other organs. This

macrophage subset appears to play a role in immunosurveillance

at the interface between the peritoneal cavity and the liver, medi-

ating neutrophil recruitment in the presence of peritoneal bacteria,

with associated control of intrahepatic bacterial loads.

STAR+METHODS

Detailed methods are provided in the online version of this paper

and include the following:

d KEY RESOURCES TABLE

d CONTACT FOR REAGENT AND RESOURCE SHARING

d EXPERIMENTAL MODEL AND SUBJECT DETAILS

386 I

B Mice

mmunity 47, 374–388, August 15, 2017

d METHOD DETAILS

B Antibiotic treatment

B Bacterial cultures

B Bacterial infections

B Liver transplantation

B Parabiosis

B BM chimeras

B CSF-1R monoclonal antibody treatment

B Purification ofmonocytes and peritoneal macrophages

for adoptive transfer

B Surgery for intravital multiphoton imaging

B Multiphoton imaging

B Isolation of liver myeloid cells and flow cytometry

B Epifluorescence Analysis of Liver Sections

B Electron microscopy

B Cell isolation and sorting for transcriptomic microarray

analysis

B Pre-processing and data analysis

B Mass cytometry sample preparation and analysis

B Mass cytometry data analysis

d QUANTIFICATION AND STATISTICAL ANALYSIS

d DATA AND SOFTWARE AVAILABILITY

SUPPLEMENTAL INFORMATION

Supplemental Information includes six figures and four movies and can be

found with this article online at http://dx.doi.org/10.1016/j.immuni.2017.

07.018.

AUTHOR CONTRIBUTIONS

Conceptualization, F.S., P.B., and D.G.B.; Methodology, F.S., K.P.A.M.,

L.G.N., B.R., and B.L.; Software, S.R., W.R., F.L., and A.J.M.; Formal Analysis,

F.S., M.E., S.R., W.R., M.M., K.P.A.M., and L.E.H.; Investigation, F.S., M.E.,

L.B., S.B.W., B.L., A.W., M.M., K.P.A.M., M.F., A.J.M., L.E.H., and S.S.T.; Re-

sources, M.E., K.P.A.M., W.W., B.M.S., L.G.N., F.G., W.J.B., andW.R.H.; Data

Curation, F.S., S.R., W.R., C.M., and M.M.; Writing – Original Draft, F.S. and

P.B.; Writing – Review & Editing, D.G.B., M.E., K.P.A.M., Y.C.W., S.S.T.,

B.R., D.G.L., B.M.S., and L.G.N.; Supervision, P.B. and D.G.B.; Project Admin-

istration, P.B.; Funding Acquisition, P.B., D.G.B., and G.W.M.

ACKNOWLEDGMENTS

We would like to thank Prof. L. Guidotti and Dr. M. Iannacone for sharing their

technical expertise in 2PM liver imaging, Prof. B. Malissen for Cd207-EGFP

mice, Dr. Matthew Sweet for Mac-Blue mice, and Dr. H. Wu for TLR4- and

MyD88-deficient mice. We would also like to thank the Centenary Institute An-

imal Facility and Advanced Cytometry Facility for technical support. This work

was supported by the NHMRC Australia (project grants APP1108565 and

APP1086451) and by SIgN, A*STAR, Singapore.

Received: August 6, 2016

Revised: March 3, 2017

Accepted: July 23, 2017

Published: August 15, 2017

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STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies

Anti-CD19 conjugate BD PharMingen 1D3; RRID:AB_395050 for flow cytometry

Anti-CD3 conjugate BD PharMingen 145-2C11; RRID:AB_312673 for flow cytometry

Anti-NK1.1 conjugate ebioscience PK136; RRID:AB_466051 for flow cytometry

Anti-CD45 conjugates BD PharMingen 30-F11; RRID: AB_394003 for flow cytometry;

RRID: AB_394611 for in vivo labeling

Anti-CD11b conjugates BioLegend M1/70; RRID: AB_2566568 for flow cytometry;

RRID: AB_312791 for in vivo labeling

Anti-Ly6G conjugate BioLegend 1A8; RRID: AB_1186104 for in vivo labeling

Anti-Ly6C conjugates BioLegend HK1.4; RRID: AB_2562353 for flow cytometry;

RRID: AB_1186132 for in vivo labeling

Anti-CD64 conjugates BioLegend X54-5/7.1.1; RRID: AB_2566561 for flow cytometry;

RRID: AB_647241 for in vivo labeling

Anti-F4/80 conjugates BioLegend BM8; RRID: AB_893478 for flow cytometry;

RRID: AB_893486 for in vivo labeling

Anti-CD11c conjugates BioLegend N418; RRID: AB_830649 for flow cytometry;

RRID: AB_313776 for in vivo labeling

Anti-CD103 conjugates BD PharMingen M290; RRID: AB_396732 for both flow cytometry

and in vivo labeling

Anti-Tim4 conjugates BD PharMingen RMT4-54; RRID: AB_2201843 for both flow cytometry

and in vivo labeling

Anti-CX3CR1 conjugates BioLegend SA011F1; RRID: AB_2565697 for flow cytometry;

RRID: AB_2564315 for in vivo labeling

Anti-Langerin conjugate ebioscience eBioRMUL.2; RRID: AB_494070 for in vivo labeling

Anti-IA/IE conjugates BioLegend M5/114.15.2; RRID: AB_493527 for flow cytometry;

RRID: AB_313323 for in vivo labeling

Anti-CD115 conjugate BioLegend AFS98; RRID: AB_1937253 for in vivo labeling

Anti-CD11c conjugated to 142 Nd In-house N418

Anti-CD64 conjugated to 143 Nd In-house X54-5/7.1

Anti-CD16/32 conjugated to 144 Nd Fluidigm 93

Anti-F4/80 conjugated to 146 Nd Fluidigm BM8

Anti-CD45 conjugated to 147 Sm In-house 30-F11

Anti-CD11b conjugated to 148 Nd In-house M1/70

Anti-CD19 conjugated to 149 Sm In-house 6D5

Anti-CD3e conjugated to 152 Sm In-house 145-2C11

Anti-CD335 conjugated to 153 Eu Fluidigm 29A1.4

Anti-NK1.1 conjugated to 158 Gd In-house PK136

Anti-B220 conjugated to 159 Tb In-house RA3-6B2

Anti-CD103-FITC Biolegend 2E7; RRID: AB_10709438

Anti-Siglec-F conjugated to 162 Dy In-house E50-2440

Anti-CD301 conjugated to 164 Dy In-house LOM14

Anti-CD326 conjugated to 165 Ho Fluidigm G8.8

Anti-TIM4 conjugated to 166 Er In-house RMT4-54

Anti-CX3CR1 conjugated to 170 Er In-house SA011F11

Anti-IA/IE conjugated to 174 Yb In-house M5/114.15.2

Anti-Ly6C conjugated to 175 Lu In-house HK1.4

Anti-Ly6G conjugated to 176 Yb In-house 1A8

(Continued on next page)

Immunity 47, 374–388.e1–e6, August 15, 2017 e1

Continued

REAGENT or RESOURCE SOURCE IDENTIFIER

Anti-FITC conjugated to 160 Gd In-house FIT-22

Anti-GFP conjugated to 169 Tm Fluidigm FM264G

GoInVivo Purified Rat IgG1, k Isotype Ctrl BioLegend RTK2071

Purified NA/LE Rat Anti-Mouse CSF-1 BD PharMingen 5A1; RRID: AB_396901

Purified Rat Anti-Mouse CSF-1R Amgen M279

Chemicals, Peptides, and Recombinant Proteins

DAPI Biolegend 422801

LPS from E.Coli Sigma L4130

Collagenase IV from Clostridium

histolyticum

Sigma C5138

DNase I Roche Cat#04716728001

Critical Commercial Assays

Ovation Pico WTA System V2 NuGEN 3302

RNeasy Mini Kit QIAGEN 74104

Deposited Data

Raw and analyzed data This paper ArrayExpress: E-MTAB-5932

Experimental Models: Organisms/Strains

C57BL/6 Australian BioResources

B6-SJL Australian BioResources Ptprca Pepcb/BoyJ

Flt3l�/� McKenna et al., 2000 C57BL/6-Flt3Ltm1Imx/ Flt3Ltm1Imx; RRID: MGI:3656072

Csf2rb�/� Robb et al., 1995 Csf2rbtm1Cgb/ Csf2rbtm1Cgb; RRID: MGI:3723079

Ccr2�/� Boring et al., 1997 B6.129S4-Ccr2tm1Ifc/J

Tlr4�/� Poltorak et al., 1998 Tlr4lps-del/ Tlr4lps-del; RRID: MGI:3588730

Myd88�/� Yamamoto et al., 2003 Myd88�/�

Ccl5�/� Makino et al., 2002 Ccl5tm1Hso/Ccl5tm1Hso; RRID: MGI:2660697

Il-34�/� Greter et al., 2012b Il34LacZ/LacZ

Cxcr5�/� Forster et al., 1996 Cxcr5tm1Lipp/ Cxcr5tm1Lipp; RRID: MGI:2658681

Itgax-EYFP Lindquist et al., 2004 Itgax (CD11c) EYFP

MacGreen Sasmono et al., 2003 B6N.Cg-Tg(Csf1r-EGFP)1Hume; RRID:

IMSR_JAX:018549

ROSAmT/mG Muzumdar et al., 2007 B6.129(Cg)Gt(ROSA)26Sortm4

(ACTB_tdTomato,_EGFP)Luo/J

Lyz2-EGFP Faust et al., 2000 B6.129(Cg)-Lyz2tm1.1Graf; RRID: MMRRC_012039-MU

Cx3cr1-EGFP Jung et al., 2000 Cx3cr1tm1Litt/ Cx3cr1tm1Litt; RRID: MGI: 4834191

Cd207-EGFP Kissenpfennig et al., 2005 B6.129S2-Cd207tm2Mal/J; RRID: IMSR_JAX:016939

Ccr2-RFP Saederup et al., 2010 B6.129(Cg)-Ccr2tm2.1Ifc/J; RRID: IMSR_JAX:017586

Fucci-G1-#639 and FucciS/G2/M-#474 Sakaue-Sawano et al., 2008 B6.Cg-Tg(FucciS/G2/M)#474BsiTg(FucciG1)#639Bsi/Rbrc

MacBlue Ovchinnikov et al., 2008 Tg(Csf1r*-GAL4/VP16,UAS-ECFP)1Hume;

RRID: IMSR_JAX:026051

Software and Algorithms

GenePattern Broad Institute N/A

GraphPad Prism 6 GraphPad Software Inc http://www.graphpad.com/scientific-software/prism/

FlowJo 2 (v9.8) FlowJo https://www.flowjo.com/solutions/flowjo

IMARIS software (v7.6) Bitplane N/A

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to Lead Contact, Patrick Bertolino (p.bertolino@

centenary.org.au).

e2 Immunity 47, 374–388.e1–e6, August 15, 2017

EXPERIMENTAL MODEL AND SUBJECT DETAILS

MiceAll experimental procedures involving mice were carried out according to protocols approved by relevant institutional Animal Ethics

Committees andwere performed according to relevant national guidelines for husbandry and experimentation on laboratory animals.

All mice except germ free animals were housed in the animal facilities of the Centenary Institute, Biological Resource Centre (BRC),

A*STAR, or QIMR Berghofer Medical Research Institute under specific pathogen-free conditions. Cd207-EGFP mice were kindly

provided by Prof B Malissen, Mac-Blue mice by Dr Matthew Sweet and both TLR4 and MyD88 deficient mice by Dr H Wu. Germ

free mice were housed in sterile isolators and provided by the WEHI (Melbourne, Australia). The mouse lines used are listed in the

KEY RESOURCES TABLE and procedures are described below.

METHOD DETAILS

Antibiotic treatmentOnweaning day, 3weeks oldmicewere treatedwith the following antibiotics ad libitum in drinking water for 2weeks: ampicillin (1g/L),

vancomycin (0.5g/L), neomycin (1g/L) and metronidazole (1g/L).

Bacterial culturesmCherry-expressingMycobacterium bovis bacillus Calmette–Guerin (BCG)-Pasteur were cultured in Middlebrook 7H9 broth (Difco,

Detroit, MI) supplemented with 0.5% glycerol, 0.05% Tween 80 and 10% albumin-dextrose-catalase enrichment (Difco, Detroit, MI)

in the presence of 50mg/ml kanamycin, at 37�C. Mid-logarithmically grown, single bacterial cell cultures were sonicated for 10 s, and

concentration was determined by measuring OD at 600 nm. L. monocytogenes (EGD strain) was grown until exponential phase in

tryptic soy broth (Difco, Detroit, MI). Bacterial stocks were frozen in 20% (v/v) glycerol and stored at �80�C.

Bacterial infectionsC57BL/6 mice pretreated 3 days prior with anti-CSF-1R (M279) mAb or isotype control antibody were injected i.p. with 2e5 CFU of

either BCG or Listeria. The numbers of viable bacteria in the liver and peritoneal cavity were determined by plating serial dilutions of

organ homogenates and peritoneal lavage on tryptic soy agar plates (Difco, Detroit, MI) for Listeria or on oleic acid-albumin-dextrose-

catalase (OADC)-enriched Middlebrook 7H10 agar (Difco, Detroit, MI) for BCG. Enumeration of bacterial colonies was performed af-

ter incubation at 37�C for 24h-48h (Listeria) or 2-3 weeks (BCG).

Liver transplantationItgax-YFP donor livers were dissected from surrounding tissues and a stent placed in the bile duct. Cholecystectomywas performed.

Livers were perfused via the portal vein with University of Wisconsin solution, then excised. Donor livers were kept at 4�C while

awaiting engraftment. Following excision of recipient TdTomato livers, Itgax-YFP donor livers were placed orthotopically and the in-

frahepatic vena cava and portal vein anastamosed using the cuff technique; the suprahepatic inferior vena cava was anastamosed.

The common bile duct was anastomosed over the stent; hepatic re-arterialization was not performed. Anhepatic times were less

than 25 min.

ParabiosisFemale mice were anesthetized using 2.5% avertin (15 mL/kg), shaved at the corresponding lateral sides of the body, and then sur-

gically joined. Briefly, for each mouse, matching skin incisions were made from the olecranon to the knee joint. The subcutaneous

fascia was then dissected to create about 1/2 cm of free skin. The matching olecranon and knee joints were attached to each other

by a single 2-0 silk suture and tie, and the dorsal and ventral skins were approximated by staples or continuous suture. After surgery,

the mice were treated with Baytril (0.05-2mg/kg s.c.) and buprenorphine (5-20 mg/kg s.c.) for 3 days and allowed to recover for

8 weeks. Mice were analyzed after 8 weeks.

BM chimerasTibias, femurs, and humeruses from Itgax-GFP BM donor mice were flushed with RPMI 1640 (GIBCO) supplemented with 10% fetal

calf serum (GIBCO BRL), 2 mmol L-glutamine (JRH Biosciences, Lenexa, KS), and 50 mmol 2-mercaptoethanol (Sigma). Bone

marrow cells were washed 3 times before being counted, and 5 3 106 cells were injected intravenously into 7–8-week-old

C57BL/6 mice that had been lethally irradiated at 950 R. BM chimeric mice were analyzed at 8 weeks post-reconstitution.

CSF-1R monoclonal antibody treatmentFor single dose treatment, mice were injected IP with 400 mg of either anti–CSF-1R Ab (M279; Amgen) or rat IgG control Ab. For

multiple dose treatment, mice were injected IP with 400 mg of either anti–CSF-1R Ab (M279; Amgen) or rat IgG control Ab twice

per week for 3 weeks.

Immunity 47, 374–388.e1–e6, August 15, 2017 e3

Purification of monocytes and peritoneal macrophages for adoptive transferCells from peripheral blood, bone marrow and spleen of MacGreen mice were pooled. Monocytes were first enriched for with anti-

CD11b conjugated MACS beads (Miltenyi), then flow sorted for GFP expression within a FSC/SSC gate appropriate for monocytes.

The sorted population contained approximately 40% monocytes and 4% macrophages. The remaining populations were CD11b

negative and contained around 9% neutrophils, 8% dendritic cells, with the remainder likely B cells. CX3CR1gfp/gfp monocytes

were enriched via the same sorting strategy, which led to GFP+ monocyte purity of > 90%.

To purify peritoneal MacGreen macrophages, cells were first enriched using anti-CD11b conjugated MACS beads (Mylteni), then

flow sorted for GFP expression by selecting leucocytes according to their size and excluding doublets. Granulocytes, B cells and

eosinophils were excluded using anti-CD19 and Ly6G antibodies. A cell mixture containing both large (LPM) and small peritoneal

macrophages (SPM) was used for transfer.

0.53 106 monocytes or peritoneal macrophages were adoptively transferred i.v. into C57BL/6 mice pre-treated, 7 days prior, with

400 mg of anti–CSF-1R Ab (M279; Amgen). Livers were harvested at 9 days after monocyte transfer to image the hepatic capsule

using 2PM.

Surgery for intravital multiphoton imagingMice were anesthetized by IP injection of ketamine/xylazine (80mg/10mg/kg). The abdomen and left flank were then shaved. The

peritoneal cavity was accessed via midline and left subcostal incisions. The falciform ligament was resected and mice placed in a

left lateral recumbent position. The left lobe was gently exteriorized onto a glass coverslip attached to a custom-made imaging stage.

The liver was covered with a small sheet of plastic wrap to limit motion artifacts. Abdominal organs and the incision were then packed

with moist gauze, and the imaging platform sealed to prevent dehydration, leaving a small window on the upper right flank for further

subcutaneous half-dose anesthetics injections as required.

In some experiments, following the surgery described above, the left liver lobe was exteriorized onto a glass coverslip with a pre-

viously deposited 20ml drop of PBS containing either an indicated concentration of LPS, 1 3 106 mCherry-BCG or Crimson-conju-

gated 200nm FluoSpheres carboxylate–modified microspheres (Invitrogen).

Multiphoton imagingIn most experiments used for multiphoton imaging analysis, cells were labeled in vivo by injecting relevant fluorochrome-conjugated

antibodies intravenously via the tail vein. Some large high-resolution multi-photon images were generated by stitching several fields

together following acquisition using the stitching function of the ImSpector software.

For live intravital imaging, animal were maintained at 35�C in a blacked-out environmental chamber with controlled heated air.

Maintenance of a core body temperature within a narrow 37 ± 1�C range was monitored using a rectal probe. Live animals prepared

as described above or tissue explants were imaged via multiphoton laser scanning microscopy, using a Nikon Ti-U inverted fluores-

cencemicroscopewith a 25x Nikonwater immersion objective (1.1 NA,WD 2mm) and a dedicated single-beam LaVision TriM Scope

I (LaVision Biotec: Germany) controlled by Imspector software (LaVision). The microscope was outfitted with one Ti:Sapphire laser

(MaiTai DeepSee: Spectra-physics) and one Synchronously Pumped Optical Parametric Oscillator (OPO) pumped by a Chameleon

Ultra II Ti:Sapphire laser (Coherent). Emission wavelengths were collected with ultrasensitive photomultiplier tubes (GaSP Hama-

matsu) for 412-472 nm (CFP, second harmonic signal), 490-520 nm (GFP), 500-550 nm (YFP), 573-633 nm (TdTomato, PE) and

650-673 nm (Alexa647). Image stacks of up to 60 optical sections with 1 to 3 mm z-spacing were acquired. The DeepSee laser

was tuned to 830 or 940 nm and OPO was tuned to 1080nm. Parfocal alignment of lasers was adjusted using fluorescent beads

before each experimental session. Each xy-plane spanned 512 pixels in each dimension with a minimal resolution of 0.86 mm per

pixel. Imaris software (Bitplane) was used for 3D rendering and analysis of image stacks.

Isolation of liver myeloid cells and flow cytometryIsolation and detection of liver immune cells together with hepatocytes and other cell types was performed via liver perfusion. Livers

were exposed and retrograde perfusion performed in situ with 1mg/ml collagenase type IV (Sigma). Livers were then excised and

minced, followed by further enzymatic digestion for 30min at 37�C in HBSS containing 1mg/ml collagenase type IV and 1 mg/ml

DNase type I (Roche), and dissociation through a 100 mm-pore cell strainer (BD). After washing homogenates, hepatocytes and

debris were removed via Percoll gradient. Recovered cells were incubated with anti-CD16/CD32 antibody (2.4G2; BD Biosciences)

prior to staining to reduce non-specific FcR mediated staining. Samples were then stained with antibodies following standard pro-

cedures, washed twice and stained with DAPI (0.1 mg/ml) before acquisition on a 5 lasers Fortessa� flow cytometer (Becton

Dickinson). Analysis was carried out using FlowJo� software. The fluorochrome-conjugated antibodies (purchased from BD,

eBioscience, BioLegend, AbD Serotec, or R&D Systems) against cell surface molecules used are listed in the KEY

RESOURCES TABLE.

Epifluorescence Analysis of Liver SectionsLivers were perfused in situwith PBS followed by 10% formalin before excision. Livers were further incubated in 10% formalin, 10%,

then 30% sucrose in PBS buffer, before freezing in O.C.T. (TissueTek) in liquid nitrogen. 8-12 mm sections were cut on a cryostat

(Leica) at �13 to �15�C, before staining. MHC-II+ and F4/80+ or CSF-1 expression was detected using rat-anti-IA/IE (M5/114.5.2)

and rat-anti F4/80 (CI:A3-1) or anti-CSF-1 (5a1) respectively, followed by AlexaFluor647-conjugated anti-rat IgG (Invitrogen). Images

e4 Immunity 47, 374–388.e1–e6, August 15, 2017

were acquired using the epifluorescence Deltavision microscope. To generate a high-resolution mosaic image of the liver tissue,

stitching has been applied following data acquisition using the Stiching function of the Softworx software.

Electron microscopyLivers were exposed and retrograde perfusion performed in situ with a 23-gauge needle at a rate of 3 to 4 mL/min with phosphate-

buffered saline and fixative [1% glutaraldehyde, 4% paraformaldehyde, 2 mmol/L CaCl2, 2%(w/v) sucrose 0.1 mol/L cacodylate

buffer pH 7.4]. The livers were then removed, cut into small pieces and fixed in the same fixative for 1 hour at 4�C. Fixed tissue

was embedded in Spurr’s and LR White resin. Ultrathin (70-90 nm) sections were cut and collected on formvar-coated nickel slot

grids (ProSciTech, Australia). Fields of interest were photographed using a Hitachi H7100FA Transmission Microscope.

Cell isolation and sorting for transcriptomic microarray analysisEpidermal LCs fromCd207-EGFPmice were obtained from ears. Ear skin obtained from ears split into dorsal and ventral halves were

separated into epidermal and dermal sheets were treated with 5 U/mL dispase (Sigma-Aldrich), then digested with 2 mg/mL colla-

genase IV (Sigma-Aldrich) followed by filtration through an 80 mm stainless steel mesh. Liver cells (obtained as described above for

flow cytometry) and epidermal immune cell suspensions fromCd207-EGFPmicewere prepared and stained for flow cytometric anal-

ysis with cell populations identified as follows: LCMs (DAPI�CD103negGFPhi), liver CD103+ dendritic cells (DAPI�CD103hiGFPint), skin

LCs (DAPI-GFPhi). Cells were sorted directly into 500mm of Trizol� reagent (a minimum of 10,000 cells for each type depending on

experiment) using an Influx� sorter (BD). RNA was extracted following Trizol� manufacturer’s guidelines. RNA quality and quantity

was assessed using a Bioanalyser 2100 instrument (Agilent). cDNA was prepared and amplified using the Ovation RNA-Seq System

V2 kit (NuGen). Samples were then hybridized to Affymetrix Gene Arrays 1.0ST.

Pre-processing and data analysisRaw microarray data were downloaded from GEO from the following studies (GSE55606, GSE15907, GSE49358, GSE75225).

Unaffiliated downloaded or in house generated files in CEL format were integrated and normalized with Robust-Multi Array (RMA)

using themodule Expression File Creator of Gene Pattern (Reich et al., 2006). Combat module of Gene Pattern was utilized to remove

the undesired variation due to the different gender between some samples. From the resulting expression matrix, probes with un-

known gene symbol were excluded as per ImmGen guidelines. Furthermore, probes with a coefficient of variation (CV) within repli-

cates above 0.5 in at least one set of replicate were removed. Differentially expressed genes were detected using Comparative

Marker Selection module of Gene Pattern.

For PCA analysis only probes with CV above the 80th percentile were considered. Distances for hierarchical clustering were calcu-

lated as the Pearson’s correlation coefficient considering genes with CV above 95th percentile.

To identify enriched gene ontologies, pathways and processes, we used the Functional Annotation Clustering tool in DAVID.

Mass cytometry sample preparation and analysisSingle cell suspensions of liver mononuclear cells were centrifuged at 350xg at 4�C for 5 min and the supernatant removed by aspi-

ration. Cells were resuspended in 1mL CyTOF staining buffer (CFB – PBS containing 0.1% BSA, 2mM EDTA and 0.05% sodium

azide) and washed in CFB a second time. Primary antibody staining was performed using the antibodies listed in the KEY

RESOURCES TABLE and diluted in CFB, for 40 min on ice. A mixture of pre-conjugated (Fluidigm) and in house conjugated (using

Maxpar X8 labeling kits – Fluidigm) was used. Following primary antibody staining, cells were washed as described previously using

1mLCFB and resuspended in 0.5mL of a 1/4000 dilution of cisplatin viability reagent (Cell-ID Cisplatin – Fluidigm, Ca) in PBS for 5min

at room temperature. Cisplatin staining was terminated by addition of 1mL of CFB and cells were washed twice by centrifugation.

Secondary antibody staining was then performed using the antibodies listed in the KEY RESOURCES TABLE for 20 min on ice,

and cells were then washed twice with 1mL CFB. Next, cells were resuspended in 400 mL of 4% paraformaldehyde in PBS (PFA)

and fixed at room temperature (RT) for 20 min, then washed twice by centrifugation with 1mL CFB. Cell pellets were resuspended

in 1mL permeabilization buffer (PB - Ebioscience), pelleted (900xg, 10 min, RT) and the supernatant aspirated. Following a second

wash in PB, cells were stained with anti-GFP antibody diluted in PB (40min, RT). Cells were then washed twice with 1mL PB. Fixation

and nuclear staining was performed in 400 mL of 4%paraformaldehyde containing 0.1 mMcell-ID� Intercalator-Ir (Fluidigm) at 4�C for

48 hours. On the day of sample acquisition, cell suspensions were washed twice by centrifugation in distilled water (1mL, 900xg,

10 min, RT) and resuspended in water containing a 1/10 dilution of EQ-beads (fluidigm) at a concentration of �1x106 cells/mL.

Data was acquired on a Helios mass cytometer (Fluidigm), and post-acquisition signal normalization was performed using Helios

control software (Fluidigm).

Mass cytometry data analysisInitial gating for Figure 2F was performed using FlowJo v10.2 (Treestar). CD45+, GFPhi cells were mapped onto Lin- (CD3-, CD19-,

B220-, NK1.1-, CD335-) liver cells using t-Distributed Stochastic Neighbor Embedding (tSNE). Dimensionality reduction was per-

formed using tSNE on all CD45+, GFPhi cells and 10% of Lin- by Cytobank. Siglec-F, CD301, TIM4, CD103, CX3CR1, Ly6C,

CD11c, CD64, CD16/32, F4/80, CD11b, IA/IE and Ly6G were used as clustering parameters, with iterations = 2000, perplexity =

100, and theta = 0.5. For unbiased identification of macrophage subsets expressing GFP (Suppl. Figure S2C), macrophages

(Lin-, Ly6G-, Siglec-F-, MHC-II+, CD11b+, Ly6Clo, F4/80int/hi, CD64hi) were manually gated using FlowJo and dimensionality reduction

Immunity 47, 374–388.e1–e6, August 15, 2017 e5

was performed by tSNE (clustering parameters: CD301, TIM4, CD103, CX3CR1, CD16/32; iterations = 1000, perplexity = 30,

theta = 0.5).

QUANTIFICATION AND STATISTICAL ANALYSIS

Results are expressed as mean ± SEM. All statistical analyses were performed in Prism 5 (GraphPad Software). Statistical analysis

was performed using a Mann-Whitney test.

DATA AND SOFTWARE AVAILABILITY

The microarray data was uploaded onto the ArrayExpress database. The accession number is ArrayExpress: E-MTAB-5932.

e6 Immunity 47, 374–388.e1–e6, August 15, 2017