Article

Niche-Specific Reprogram

ming of EpigeneticLandscapes Drives Myeloid Cell Diversity inNonalcoholic Steatohepatitis

Graphical Abstract

Highlights

d Myeloid cell diversity in NASH is associated with distinct

microanatomical niches

d Reprogramming of LXR activity leads to impaired Kupffer cell

identify and survival

d ATF3 collaborates with LXRs to promote a scar-associated

macrophage phenotype

d Altered enhancer landscapes enable inference of disease

mechanisms

Seidman et al., 2020, Immunity 52, 1057–1074June 16, 2020 ª 2020 Elsevier Inc.https://doi.org/10.1016/j.immuni.2020.04.001

Authors

Jason S. Seidman, Ty D. Troutman,

Mashito Sakai, ..., Joseph L. Witztum,

Ronald N. Germain,

Christopher K. Glass

Correspondencettroutman@health.ucsd.edu (T.D.T.),ckg@ucsd.edu (C.K.G.)

In Brief

Kupffer cells and recruited myeloid cells

contribute to the pathology of

nonalcoholic steatohepatitis (NASH), but

molecular mechanisms specifying their

distinct identities and functions are not

known. Seidman and colleagues address

this problem by defining cell- and

disease-specific enhancer landscapes

that enable inference of key transcription

factors that drive myeloid cell diversity

in NASH.

ll

mailto:ttroutman@health.ucsd.�edumailto:ckg@ucsd.�eduhttps://doi.org/10.1016/j.immuni.2020.04.001http://crossmark.crossref.org/dialog/?doi=10.1016/j.immuni.2020.04.001&domain=pdf

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Article

Niche-Specific Reprogramming of EpigeneticLandscapes Drives Myeloid Cell Diversityin Nonalcoholic SteatohepatitisJason S. Seidman,1,10 Ty D. Troutman,1,2,10,* Mashito Sakai,1,10 Anita Gola,3 Nathanael J. Spann,1 Hunter Bennett,1

Cassi M. Bruni,1 Zhengyu Ouyang,1 Rick Z. Li,1 Xiaoli Sun,2 BaoChau T. Vu,1 Martina P. Pasillas,1 Kaori M. Ego,1

David Gosselin,4 Verena M. Link,1,5 Ling-Wa Chong,6 Ronald M. Evans,6,7 Bonne M. Thompson,8 Jeffrey G. McDonald,8

Mojgan Hosseini,9 Joseph L. Witztum,2 Ronald N. Germain,3 and Christopher K. Glass1,2,11,*1Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA, USA2Department of Medicine, University of California, San Diego, La Jolla, CA, USA3Lymphocyte Biology Section, Laboratory of Immune SystemBiology, National Institute of Allergy and Infectious Diseases, National Institutes

of Health, Bethesda, MD 201892, USA4Department of Molecular Medicine, Université Laval, Quebec City, QC, Canada5Faculty of Biology, Division of Evolutionary Biology, Ludwig-Maximilian University of Munich, Munich, Germany6Gene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, USA7Howard Hughes Medical Institute, The Salk Institute for Biological Studies, La Jolla, CA, USA8Center for Human Nutrition, UT Southwestern Medical Center, Dallas, TX, USA9Department of Pathology, University of California, San Diego, La Jolla, CA, USA10These authors contributed equally11Lead Contact*Correspondence: ttroutman@health.ucsd.edu (T.D.T.), ckg@ucsd.edu (C.K.G.)

https://doi.org/10.1016/j.immuni.2020.04.001

SUMMARY

Tissue-resident and recruited macrophages contribute to both host defense and pathology. Multiple macro-phage phenotypes are represented in diseased tissues, but we lack deep understanding ofmechanisms con-trolling diversification. Here, we investigate origins and epigenetic trajectories of hepatic macrophages dur-ing diet-induced non-alcoholic steatohepatitis (NASH). The NASH diet induced significant changes in Kupffercell enhancers and gene expression, resulting in partial loss of Kupffer cell identity, induction of Trem2 andCd9 expression, and cell death. Kupffer cell loss was compensated by gain of adjacent monocyte-derivedmacrophages that exhibited convergent epigenomes, transcriptomes, and functions. NASH-inducedchanges in Kupffer cell enhancers were driven by AP-1 and EGR that reprogrammed LXR functions requiredfor Kupffer cell identity and survival to instead drive a scar-associated macrophage phenotype. These find-ings reveal mechanisms by which disease-associated environmental signals instruct resident and recruitedmacrophages to acquire distinct gene expression programs and corresponding functions.

INTRODUCTION

Tissue-resident macrophages exhibit different transcriptomes in

response to environmental signals, enabling tissue-specific

functions (Gautier et al., 2012; Gosselin et al., 2014; Lavin

et al., 2014; Mass et al., 2016). Most tissues acquire resident

macrophage populations early in development from yolk sac or

fetal liver erythromyeloid progenitor (EMP) cells (Ginhoux et al.,

2010; Gomez Perdiguero et al., 2015; McGrath et al., 2015;

Schulz et al., 2012). In the brain and liver, EMP-derived microglia

and Kupffer cells (KCs) continue as exclusive, self-renewing

populations. In other tissues, EMP-derived macrophages are

partially or completely replaced over time by hematopoietic

stem cell (HSC)-derived cells (Bain et al., 2014; De Schepper

et al., 2018; Shaw et al., 2018; Yona et al., 2013).

Resident macrophages and infiltratingmonocyte-derived cells

contribute to a diverse array of human diseases (Holtman et al.,

2017; Kiss et al., 2018; Pirzgalska and Domingos, 2018). Nonal-

coholic steatohepatitis (NASH) is a form of nonalcoholic fatty

liver disease (NAFLD) and develops through combinatorial ac-

tions of steatosis and growing inflammation in response to

cellular stresses of the perturbed fatty liver environment (Haas

et al., 2016; Hardy et al., 2016; Machado and Diehl, 2016; Musso

et al., 2016; Rinella and Sanyal, 2016; Younossi et al., 2016). Dur-

ing NASH or acute liver injury, new macrophages are derived

from circulating monocytes (Karlmark et al., 2009; Seki et al.,

2009a, 2009b; Tacke and Zimmermann, 2014; Zigmond et al.,

2014). These recruited monocyte-derived cells can have both

detrimental and supportive roles, contributing to increases in pa-

thology during fibrosis onset, but hastening recovery when the

Immunity 52, 1057–1074, June 16, 2020 ª 2020 Elsevier Inc. 1057

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damage-evoking agent is removed (Ju and Tacke, 2016; Karl-

mark et al., 2009; Mitchell et al., 2009; Seki et al., 2009a,

2009b; Zigmond et al., 2014). These observations raise ques-

tions as to the mechanisms underlying the phenotypic diversity

of disease-associated macrophages.

Here, we define the identities, developmental origins, and mi-

cro-anatomic locations of the major myeloid populations in

response to a NASH-inducing diet. By sorting these cell popula-

tions and performing deep transcriptomic and epigenomic anal-

ysis, we provide evidence for combinatorial effects of diet and

anatomic location on regulatory pathways and transcription fac-

tors (TFs) that explain the emergence of disease-associated

macrophage phenotypes.

RESULTS

Single-Cell RNA-Seq Defines Myeloid Diversity DuringDietary NASHTo investigate immune cell heterogeneity during NASH, we per-

formed single-cell RNA sequencing (scRNA-seq) on liver non-

parenchymal cells from healthy mice fed a control diet or

NASH diet. As noted in previous studies (Hall et al., 2010; Pous-

sin et al., 2011), C57BL/6J mice exhibited rapid weight gain

when fed the NASH diet (Figure S1A) and developed steatosis,

inflammation, and a modest degree of fibrosis (Figure S1B).

Non-parenchymal cells from control mice and mice fed the

NASH diet for 30 weeks were purified using fluorescence-acti-

vated cell sorting (FACS) (Figure S1C). Over 6,000 scRNA-seq li-

braries that passed quality thresholds were created from these

isolated cells.

The NASH diet induced qualitative transcriptional differences

in cell clustering between cells from control livers and NASH

livers (Figure 1A left, Figure 1B; Table S1). Further, three of 17

clusters were primarily derived from cells from control diet

mice while eight clusters were primarily derived from NASH

diet mice (Figures 1A and S1D; Table S1). Interrogation of the

most abundant and most differentially expressed (DE) tran-

scripts in each cluster allowed a cell identity to be readily as-

signed to most clusters. Further studies were focused on the

five most abundant macrophage clusters. (Figure S1E;

Table S1).

The three most abundant clusters (KC-H, healthy KC;

KC-N, NASH KC; and KN-RM, named for being a recruited

macrophage occupying the KC niche, as described later)

had high expression of Adgre1, encoding the tissue macro-

phage marker F4/80, and high expression of putative KC

lineage-determining transcription factors (LDTFs) Mafb

and Maf, and KC-specific genes, as well as low expression

of Itgam, encoding CD11b (Figures 1C and S1E; Lavin et al.,

2014). KC-H consisted almost entirely of cells from controls,

while KC-N and KN-RM consisted of cells from the NASH

diet animals (Figures 1A and 1B; Table S1). The other two

major macrophage clusters were predominantly found in

NASH livers and had characteristic gene expression of

previously described monocyte-derived liver macrophages

(Figures 1C and S1E; Krenkel et al., 2018). Cluster 4 (hereafter

referred to as Ly6Chi-RM) expressed higher amounts of

transcripts typical of Ly6Chi monocytes such as Ly6c2,

Chil3, F13a1, and Fn1, while cluster 9 (hereafter referred to

1058 Immunity 52, 1057–1074, June 16, 2020

as Ly6Clo-RM) highly expressed Cd209a, Cd7, and Itgax

(Figure S1E).

Using flow cytometry, we observed both Tim4+ and Tim4�

cells in the CD11bloF4/80hi KC gate during NASH in similar

proportions to KC-N and KN-RM and collected them for bulk

RNA-seq and epigenomic profiling (Figure 1D). We also

collected CD11bhiF4/80lo-Ly6Chi-RM and Ly6Clo-RM for RNA-

seq and assay for transposase-accessible chromatin (ATAC-

seq) (Figure 1E; Buenrostro et al., 2013). RNA-seq analysis of

these populations yielded concordant expression profiles to

scRNA-seq when comparing Seurat marker genes, indicating

that we isolated the corresponding macrophage populations

defined by scRNA-seq (Figure 1F).

We next compared these mouse macrophages to human

scRNA-seq populations of non-parenchymal liver cells from in-

dividuals with either advanced cirrhosis or without evident liver

disease (Ramachandran et al., 2019). Using orthogonal genes

to cluster the combined set of human and mouse cells under

control and disease conditions resulted in eight clusters (0–7,

Figures S1F and S1G). Human livers exhibited a greater diver-

sity of myeloid cells and, in contrast to the mouse model, all cell

populations were present to some extent in individuals with or

without cirrhosis. Importantly, mouse KC-H cells overlapped

with human KCs (clusters 0 and 1). A key finding of the human

studies was the increased presence of a ‘‘scar-associated

macrophage’’ (SAM) population in cirrhotic livers (Ramachan-

dran et al., 2019). These cells were found to reside in close

proximity to areas of fibrosis and were characterized by

increased expression of TREM2 and CD9. Importantly, the

cluster containing SAMs also contained substantial fractions

of the NASH-associated KC-N and KN-RM cells (cluster 3),

but not KC-H cells, consistent with the former cell type’s

increased expression of Trem2 and Cd9 (Figure 1F). Thus,

despite relatively sparse fibrosis, a SAM phenotype emerges

in this model.

Ontogeny and Environment Contribute to Diversity ofMyeloid Cells During NASHWe hypothesized that, similar to experimental KC ablation (Bon-

nardel et al., 2019; Sakai et al., 2019; Scott et al., 2016), Tim4�

KC-like macrophages that arise during NASH (KN-RM) may be

ontogenically distinct from the embryonically derived Tim4+

KCs (KC-N). To test this hypothesis, we performed lineage-

tracing experiments using Cx3cr1CreERT2; Rosa26tdTomato/+

mice (Figure S2A; Theurl et al., 2016; Yona et al., 2013). This

model labels tissue macrophages if tamoxifen is administered

perinatally, while only certain macrophages such as microglia,

but not KCs, are labeled if mice are pulsed during adulthood

(Dick et al., 2019; Theurl et al., 2016; Yona et al., 2013). In

contrast, during adulthood, tamoxifen labels peripheral mono-

cytes and thus monocyte-derived macrophages.

Tamoxifen administration on days 1 and 2 post-parturition re-

sulted in ~50% labeling of embryonic KCs (Figures S2B–S2D).

After 20 weeks on the NASH diet, nearly all tdTomato+ cells

were Tim4+ KCs (KC-N) (Figures S2B–S2D), indicating that

Tim4� KC-like macrophages (KN-RM) are not long-lived daugh-ters of embryonic KCs. Conversely, when adult mice fed the

NASH diet for 20 weeks were given tamoxifen 4 or 1 week prior

to sacrifice, ~90% of circulating monocytes were TdTomato+

A

B

F

C

D E

Figure 1. Transcriptional Diversity of Hepatic Macrophages during NASH

(A) tSNE projections of identified graph-based cell clusters from scRNA-seq data derived from hepatic CD45+CD146� cells from control mice ormice fed a NASHdiet for 30 weeks. (Left) cells are colored based on dietary condition. (Right) cells are colored based on cell identity of the five major myeloid cells identified. Data

represent two independent donor mice per group.

(B) Macrophage proportions from (A) for control mice (left) or mice with NASH (right).

(C) Gene expression of normalized scRNA-seq data of genes supporting cluster identities. Boxes denote quartiles, and whiskers denote data range excluding

outliers.

(D) Terminal FACS gates for purification of KCs from control mice (left), or CD11bloF4/80hiTim4+ and Tim4� cells from mice with NASH (right).(E) Terminal FACS gates for purification of Ly6Chi and Ly6Clo CD11bhiF4/80loLy6G�CX3CR1+ recruited hepatic macrophages (RM). ‘‘PMN’’ stands for poly-morphonuclear leukocyte.

(F) Comparison of myeloid clusters defined by scRNA-seq (left) and bulk RNA-seq (right) for the corresponding sorted populations. Left heatmap (100 cells per

column) depicts normalized and scaled expression values for marker genes identified using a Wilcoxon rank sum test through Seurat. Right heatmap shows z-

normalized row expression of each gene for RNA-seq from bulk purified cell populations from independent biological duplicates.

Please see also Figure S1.

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2 days after the final tamoxifen injection (data not shown), and

more than 10% of Tim4� KC-like macrophages (KN-RM) wereTdTomato+ (Figures S2C and S2D). We also observed a small

degree of labeling of Tim4+ KCs (Figures S2C and S2D), which

could be due to low expression of Cx3cr1 in Tim4+ KC-N cells

or to the gradual upregulation of Tim4 by recruited cells (Scott

et al., 2016). Overall, these results indicate that embryonic KCs

remain in the liver during development of NASH and are Tim4+,

Immunity 52, 1057–1074, June 16, 2020 1059

0.01

1

100

1

10

100

1000

KC−N KN−RM Ly6Chi&lo−RMTU

NE

L ne

ares

t nei

ghbo

rdi

stan

ce (

μm)

0

25

50

75

100

0 10 20 30

Weeks on AMLN NASH diet

Per

cent

KN

-RM

KC−HKN

−RM

Ly6C

hi&lo −R

MKC

−NKN

−RM

Ly6C

hi&lo −R

M

NASHControl

E

ANASH diet

nirehdac-Eota

moTdt+

08/4F+

ot amoTdt

F4/80

KC-N

KN-RM

B

Tim4

IFM

KC-NKN-RM

F4/80

2lgM

Mgl2+(Ly6Clo-RM)

Mgl2–(Ly6Chi-RM)

D

cv

PV

PV

PV

H

NAS

H d

iet

F4/80 TUNEL Tim4

KC-N

KC-N

KN-RM

KN-RM

KN-RM

F4/80 TUNEL Tim4

I J

6.94%

17%

75.3%

F

G

K

E-cadherin Mgl2+ Mgl2–

Tota

l TU

NE

L+ c

ell c

ount

per

area

1,0

00 μ

m2

0

250

500

750

1000

KN−R

M

Ly6C

hi −RM

Ly6C

lo −RM

KC

−N

nea

rest

neig

hbor

dis

tanc

e ( μ

m)

0

200

400

600

800

Dis

tanc

e to

nea

rest

CV

or

PV

(μm

)0

25

50

75

100

125

KC−NKN

−RM

Ly6C

hi&lo −R

M

Dis

tanc

e to

nea

rest

larg

e ve

ssel

(μ m

) *** ***

******

*****

***

***

******

***

***

***

CV PV

CV PVLy6Chi−RM

CV PVLy6Clo−RM

Ly6Chi&lo-RMLy6Chi&lo-RM

E-cadherin KN-RM KC-NLy6Chi&lo-RM

PV

PV

PV

cv

CV

PV

PV

C

(legend on next page)

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1060 Immunity 52, 1057–1074, June 16, 2020

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while most recruited CD11bloF4/80hi macrophages are Tim4�.These results also indicate that recruited KN-RM cells can

survive at least 4 weeks in the NASH liver while Ly6Chi&lo-RMs

are shorter lived.

To determine the localization of myeloid cells in the liver during

NASH, immunofluorescence (IF) microscopy was performed on

livers from Cx3cr1CreERT2; Rosa26tdTomato/+ mice pulsed with

tamoxifen 7 days prior to tissue collection (Figures 2A, 2B,

S2A, S2E, and S3A). As expected, tdTomato+ monocyte-derived

cells were abundant in NASH livers but nearly absent in control

livers (Figure S2E). Using multi-parameter imaging and histo-cy-

tometry (Gerner et al., 2012), we distinguished KC-H or KC-N,

KN-RM, and Ly6Chi&lo-RM in the control and NASH livers based

on Tim4, F4/80, and tdTomato expression. As Ly6C histological

staining could not be performed, Mgl2 was used as a surrogate

marker for Ly6Chi&lo-RM, with the Ly6Chi fraction being Mgl2�

and Ly6Clo being represented byMgl2+ (Figures 2A–2C, see Fig-

ure 3G for Ly6Clo-specific expression of Mgl2). Following in situ

confirmation of these myeloid cell subsets, we assessed their

spatial distribution using the positional data preserved in histo-

cytometry and nearest-neighbor distance analyses. The two

subsets (KN-RM and Ly6Chi&lo-RM) that were increased during

NASH displayed significant differences in their spatial distribu-

tions. By comparing nearest-neighbor distances from KC-N,

KN-RM cells were found to be distributed significantly closer

to KC-N cells than Ly6Chi&lo-RM cells (Figure 2D). In addition,

the small number KN-RM cells observed in control livers were

also in close proximity to KC-H cells (Figure S3B and data

not shown).

KCs reside within the hepatic sinusoids, but the sub-anatom-

ical organization of other liver macrophages during NASH is

less certain (Ju and Tacke, 2016). High-magnification imaging

using Collagen IV to visualize the endothelial basement mem-

brane demonstrated that KC-N and KN-RM cells resided within

hepatic sinusoids in this model (Figure S3C). In contrast, Ly6-

Chi&lo-RM cells were predominantly not found within the liver

Figure 2. Expanded Macrophage Diversity during NASH Is Supported

Niches

(A) Confocal image of NASH liver showing E-cadherin (to demark the peri-portal

white circles are the central veins (CV). Demarked at the periphery of the liver lobu

Scale: 50 microns.

(B) Histo-cytometry analysis of NASH liver sample. Statistical information of se

subsequently gated on F4/80 and tdTomato expression to quantify KC-N, KN-RM

population are shown in the middle panel, and Mgl2 expression on Ly6Chi&lo-R

(Mgl2+) cells.

(C) Confocal image of NASH liver showing distribution of rendered surfaces of

regions as in (A). Scale: 50 microns.

(D) Distance and phenotype (KN-RM, Mgl2lo-RM, or Mgl2hi-RM) of closest neighb

test; p < 0.001(***).

(E) Distance to nearest portal or central vein vasculature (large diameter vessels de

cells. Kruskal-Wallis with Dunn test; p < 0.001(***).

(F) Zoom-in representative confocal image of NASH liver showing distribution

demarks the peri-portal regions, and highlighted by white circles are CV and PV

(G) Distance to nearest center of CV or PV of Ly6Chi and Ly6Clo RM cells (mm). K

(H) Immunofluorescence (IF) image assessing in situ cell death via TUNEL staini

intensity projection (MIP) of a 20-mm z stack. Scale: 5 microns for the top row an

(I) Quantification of total TUNEL+ hepatic macrophages per area (1000 mm2) in C

(J) Nearest neighbor distance of all TUNEL+ cells to KN-RM, Ly6Chi or lo-RM, and K

test; p < 0.001(***).

(K) Temporal assessment of percentage of total CD11bloF4/80+CD146� cells thaPlease see also Figure S2 and Figure S3.

sinusoids, but were highly enriched around both portal and cen-

tral vein vasculature identifiable by the large vessel diameter

(15 mm or larger) (Figures 2D and 2E). Further, Ly6Chi-RM

(Mgl2�) cells were bi-modally distributed around portal andcentral veins, while Ly6Clo-RM (Mgl2+) cells were more uniformly

distributed in closer proximity to central veins (Figures 2F and

2G). Together, these findings further supported Ly6Chi&lo-RM

cells as being positionally separated from KC-N and KN-RM

cells based on measurements that were independent of

anatomical landmarks. These results provide evidence that

KC-N and KN-RM cells reside in a similar niche within the liver

sinusoids, which is spatially distinct from the anatomic positions

occupied by Ly6Chi&lo-RMs.

Previous work found that DLL4 expression by liver sinusoidal

endothelial cells (LSECs) is graded toward higher expression

patterns in periportal zones (Halpern et al., 2018). Further,

DLL4 mediated-activation of RBPJ participates in driving differ-

entiation of monocytes to Kupffer-like cells in a repopulation

model (Sakai et al., 2019). During NASH, we confirmed the

peri-portal (E-cadherin+) enrichment of DLL4 on LSECs (Figures

S3D and S3E). Further, KC-N and KN-RM cells were significantly

closer to DLL4+ CD138+ LSECs compared to Ly6Chi&lo-RMs

(Figures S3F and S3G). These results provide support for DLL4

as a factor that may support niche specialization of distinct

macrophage populations during NASH, consistent with its

known function in instructing a KC program in recruited

monocytes.

NASH Diet Induces KC Death and ReplacementKCs are considered a self-renewing population that is normally

closed to HSC-derived cells (Perdiguero and Geissmann,

2016). However, the observation that the NASH diet resulted in

recruitment of HSC-derived KN-RM cells raised the question of

whether the NASHdiet induces loss of embryonic KCs, providing

an open niche. TUNEL staining of NASH livers showed apoptosis

of Tim4+ KC-Ns during NASH diet, but not of KC-H cells from

by Monocyte Recruitment and Occupancy of Distinct Anatomical

regions) and tdTomato F4/80+ summed channel. Highlighted in the center by

le, portal-venous or arterial vessels (PV). Data from n = 3–4 mice per condition.

gmented objects (F4/80+tdTomato+ surfaces) was imported into FlowJo and

, and Ly6Chi&lo-RM cells. Tim4mean fluorescence intensity (MFI) for each gated

M cells macrophage is shown at right to distinguish Ly6Chi (Mgl2�) or Ly6Clo

KC-N, KN-RM, and Ly6Chi&lo-RM (Rec) cells. Dashed lines denote peri-portal

or to KC-N cells in NASH livers. Data pooled from n = 4 mice. Wilcox two-sided

fined to be greater than 15 mm in diameter) of KC-N, KN-RM, and Ly6Chi&lo-RM

of rendered surfaces of Mgl2lo (Ly6Chi) and Mgl2hi (Ly6Clo)-RMs. E-cadherin

blood vessels. Scale: 30 microns.

ruskal-Wallis with Dunn test; p < 0.01(**), p < 0.001(***).

ng in addition to Tim4 and F4/80 staining. Image from NASH mice; maximum

d 10 microns for the bottom row.

trl and NASH mouse livers.

C-N cells in NASH livers, data pooled from n = 4mice. Kruskal-Wallis with Dunn

t are Tim4� KN-RM from mice fed a NASH diet as indicated.

Immunity 52, 1057–1074, June 16, 2020 1061

Clu

ster

1C

lust

er 4

Clu

ster

2C

lust

er 3

0 5 10 15

0

5

10

15

685

1442

Blood Ly6Chi Mono

Ly6C

Hi -R

M

Blood Ly6Chi

KC-H KC-N KN-RMLy6Chi

-RMLy6Clo

-RM

0 5 10 15

0

5

10

15

521

316

KC

-H

KC-N

Mean expression (log2(TPM+1))

A

0 5 10 15

0

5

10

15

40

53

KN

-RM

Mean expression (log2(TPM+1))

KC-N

F

B

C D

E

−2 −1 0 1 2z-scaled (Log2(TPM+1))

0 5 10 15

0

5

10

15

1193

1046

Ly6Chi-RM

KN

-RM

2,21

0 ge

nes;

Log

2FC

> 2

; p.a

dj <

0.0

5;T

PM

> 4

EMP

Sinusoids Large vessels

Ctrl NASH

−20 0 20 40 60

−30

−20

−10

0

10

20

30

PC1 (57%)

PC2

(19%

)

Ly6Clo-RM

Ly6Chi-RM

KC-H

KC-N

KN-RM

RLM 14d

RLM 12h

RLM 1dBloodLy6Chi

Monocytes(Ctrl and NASH)

0

500

1000

1500

Mea

n E

xpre

ssio

n (T

PM

)

0

100

200

0

200

400

600

800

KC-H

KC-N

KN-R

M

Ly6C

hi -RM

Ly6C

lo -RM

Ly6C

hi -Mo

0

25

50

75Cd9

Trem2Clec10aMgl2

Arg2

Txnrd1

C6

Cd163

KC-H

KC-N

KN-R

M

Ly6C

hi -RM

Ly6C

lo -RM

Ly6C

hi -Mo

KC-H

KC-N

KN-R

M

Ly6C

hi -RM

Ly6C

lo -RM

Ly6C

hi -Mo

KC-H

KC-N

KN-R

M

Ly6C

hi -RM

Ly6C

lo -RM

Ly6C

hi -Mo

Kupffer cell signature

Response to wounding

ROS metab. signature

Scar-assoc. mac (SAM)

G

H

DTR–DTR+

Clec4f+Tim–

KC

0

1

2

3

Clec4f+Tim+

KC

0

2

4

6

Total KC

0

5

10

Per

cent

CD

45+

J K NASH CRN Score

0

2

4

6

DT

R–

DT

R+

DTR–DTR+

I

0 5 10 15

0

5

10

15

DTR–, n = 10

DT

R+, n

= 1

2

Mean expression (log2(TPM+1))

(legend on next page)

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1062 Immunity 52, 1057–1074, June 16, 2020

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mice fed a control diet (Figures 2H and 2I). In both control and

NASH livers, TUNEL+ KN-RM and Ly6Chi&lo-RM cells were rare

(Figure 2I). By nearest-neighbor analysis of TUNEL+ cells, KN-

RM cells were significantly enriched in nearby areas as

compared to Ly6Chi&lo-RM and KC-N cells (Figure 2J). Time

course experiments indicated that KN-RM cells were detected

by 10 weeks after initiation of the NASH diet and progressively

accumulated to account for more than 50%of the KC population

at 30 weeks (Figure 2K). These results indicate that Tim4+ KCs

undergo cell death during NASH, potentially resulting in partial

opening of the niche and enabling repopulation by HSC-derived

KN-RM cells.

Effect of NASH Diet on Resident and Recruited MyeloidCell TranscriptomesThe ability to sort distinct populations of hepatic myeloid cells

defined by scRNA-seq analysis enabled deep transcriptomic

profiling of each population (Table S2). Unsupervised hierar-

chical clustering of 2,210 DE genes among KC-H, KC-N,

KN-RM, Ly6Chi-RM, and Ly6Clo-RM tightly grouped KC-N

and KN-RM and distinguished KC-H, KC-N, and KN-RM cells

from Ly6Chi&lo-RMs (Figure 3A). With inclusion of DE genes in

blood monocytes, principal component analysis (PCA) also

indicated that KC-N and KN-RM cells from NASH mice group-

ed most closely with KC-H cells from healthy mice (Figure 3B).

After experimental KC ablation, blood Ly6Chi monocytes are

recruited to the liver and rapidly differentiate into KC-like liver

macrophages (Bonnardel et al., 2019; Sakai et al., 2019; Scott

et al., 2016). At 14 days after KC ablation, these repopulating

macrophages (GEO: GSE128662) also grouped closely with

KC-H, KC-N, and KN-RM (Figure 3B). Collectively, these re-

sults suggest that monocyte-derived KN-RMs follow a similar

developmental trajectory in the context of NASH to that of re-

populating liver macrophages (RLMs) in the KC abla-

tion model.

Pairwise comparisons of KC-H and KC-N cells indicated

that the NASH diet had a large impact on resident KC gene

expression (>800 DE genes), consistent with scRNA-seq

data (Figure 3C). Notably, fewer than 100 genes were identi-

fied as significantly altered when comparing KC-N and KN-

RM KCs from mice with NASH (Figure 3D), despite their

distinct origins (EMP versus HSC). In contrast, during NASH,

Figure 3. Highly Divergent Gene Expression Patterns across Myeloid P

(A) Unsupervised hierarchical clustering of DE genes in the indicated cell types i

(B) PCA of 2,000 most variable genes in RNA-seq data frommyeloid cells in liver a

from Sakai et al. (2019), n = 2–3 per group.

(C) Scatterplot of RNA-seq data in healthy (KC-H) or NASH (KC-N) Tim4+ KCs. D

(D) Comparison of NASH Tim4+ KCs (KC-N) and Tim4� Kupffer niche recruited m(E) Comparison of NASH Tim4� Kupffer niche recruited macrophages (KN-RM) a(F) Comparison of NASH blood Ly6Chi monocytes and Ly6Chi-RM.

(G) RNA-seq expression (mean TPM ± SD) of representative genes. TPM = trans

(H) Percentage of hepatic CD45+F4/80hiCD11bloLiveSinglets fromClec4f-cre R26

with DT, allowed to rest for 4 weeks, then fed the choline-deficient, L-amino acid

(I) RNA-seq from whole liver tissue from Clec4f-cre R26-iDTR�/� (n = 10) or Clec4quartiles, and whiskers denote data range.

(J) Representative (n > 5) H&E staining of liver tissue.

(K) NASH Clinical Research Network (CRN) scoring of stained liver sections. Scor

using Kruskal-Wallis rank sum test (p = 0.71). Boxes denote quartiles, and whisk

Please see also Figure S4.

KN-RM cells exhibited more than 2,200 DE genes in compar-

ison to Ly6Chi-RM, despite both cells originating from an HSC

precursor (Figure 3E). The divergent differentiation programs

of KN-RM and Ly6Chi-RM were further reinforced by pairwise

comparisons with gene expression in Ly6Chi circulating

monocytes, indicating more than 2,000 DE genes in each

case (Figures 3F and S4A).

Prior studies defined a set of KC identity genes that distinguish

the KC from other tissue-resident macrophages (Lavin et al.,

2014). Twenty-eight of the genes downregulated during the tran-

sition of KC-H cells to KC-N cells in the NASH model are among

this set, exemplified by Cd163 and C6 (Figures 3G and S4B).

However, many KC-specific genes maintained similar expres-

sion in cells from mice on the NASH diet, including C1qa, Cd5l,

Id3, and Il18bp, indicating that only a subset of the KC-specific

gene expression program is altered during NASH. Furthermore,

KC identity genes such as Clec4f, Vsig4, and Cdh5 were highly

expressed in KN-RM, but neither Ly6Chi&lo-RM population.

Considering the differential spatial distribution of these cell pop-

ulations, such findings suggest that the KC niche is necessary to

promote induction of these genes (Figure S4B).

Gene ontology analyses of genes whose expression distin-

guishes KC-H, KC-N, and KN-RM cells from Ly6Chi&lo-RM

(clusters 1 and 2, Figure 3A) were consistent with a stronger

pro-inflammatory and wound repair phenotype of the Ly6-

Chi&lo-RMs (Figure S4C), in agreement with prior studies (Hey-

mann and Tacke, 2016). Examples of DE genes associated

with the functional categories of ‘‘ROS metabolism’’ and

‘‘response to wounding’’ are illustrated in Figure 3G. Ly6Clo-

RM preferentially expressed Mgl2 (CD301b), a gene recently re-

ported to be expressed in skin macrophages that activates a

specific myofibroblast population implicated in tissue repair

and aging (Shook et al., 2018). In addition, we observed

increased expression of Trem2 and Cd9 in both KC-N and

KN-RM cells, consistent with recent observations identifying

these genes as markers for macrophages associated with

increased lipid burden and the presence of tissue scarring

(Hill et al., 2018; Jaitin et al., 2019; Ramachandran et al.,

2019; Xiong et al., 2019). Collectively, these results indicate

that the NASH diet acts to reprogram the endogenous KC pop-

ulation and induce divergent programs of differentiation of KN-

RM cells and Ly6Chi&lo-RMs.

opulations in NASH

n control and NASH liver.

nd blood from healthy and NASH-diet-fed mice, or recruited liver macrophages

E genes identified by DESeq2 (FC > 2, p-adj < 0.05) are colored in red.

acrophages (KN-RM).

nd Ly6Chi-RM.

cripts per kilobase million).

-iDTR�/� (n = 6) or Clec4f-cre R26i-DTR+ (n = 6)mouse livers. Mice were treated-defined, high-fat diet (CDAHFD) for 4 weeks.

f-cre R26-iDTR+ mice (n = 12) as in (H). Zero DE genes (DESeq2). Boxes denote

es > 5 are indicative of steatohepatitis. No significant difference was identified

ers denote data range.

Immunity 52, 1057–1074, June 16, 2020 1063

llArticle

Functional Convergence of Embryonic KCs and KN-RMsin NASHTo clarify the functional biological roles of embryonic KCs and

monocyte origin KN-RM cells, we used the Clec4f-cre R26-

iDTR system (Sakai et al., 2019). We treated DTR+ or DTR�

mice with diphtheria toxin (DT) to ablate embryonic KCs and

then allowed the KC niche to repopulate with monocyte precur-

sors. After a 4-week recovery period, the mice were subjected to

ad libitum feeding for 4weekswith a rapid NASH-inducingmodel

(Matsumoto et al., 2013). Ablation of embryonic (KC-H) KCs

before feeding mice the NASH diet led to increased proportions

of Tim4� KN-RM cells, as expected (Figure 3H). To assess theglobal molecular consequence of replacing embryonic KCs

with monocyte-derived KN-RMs, we performed RNA-seq on to-

tal liver tissue (Figure 3I). Notably, we found no DE genes be-

tween the two groups. We also found no differences in 23 circu-

lating inflammatory cytokines (Figure S4D). Finally, quantitative

histological assessment of liver sections indicated no effect of

replacing KCs with KN-RM. These results are consistent with

the largely convergent transcriptomes observed in comparison

of KC-N and KN-RM (Figures 3J and 3K).

Niche Occupancy Reprograms the EpigeneticLandscapesTo investigate mechanisms responsible for environment-spe-

cific programs of gene expression, we identified accessible

chromatin defined by ATAC-seq in five populations of cells

from mice with NASH: KC-N and KN-RM cells, Ly6Chi&lo-RMs,

and Ly6Chi blood monocytes. Examples of ATAC-seq peaks in

these five populations of cells from mice fed the NASH diet in

the vicinity of theClec4f and Itgam genes, which are highly differ-

entially expressed in KCs compared to Ly6Chi&lo-RMs, are illus-

trated in Figure 4A. Genome-wide comparisons of ATAC-seq

peak tag counts for Ly6Clo-RM versus Ly6Chi blood monocytes

are illustrated in Figure 4B, and the same comparison for KN-RM

and Ly6Chi blood monocytes is shown in Figure 4C. Differential

regions from the five myeloid populations in NASH mice, as

well as healthy KC-H cells and 24- and 48-h RLMs after KC abla-

tion (Sakai et al., 2019), were used for PCA illustrated as in

Figure 4D. PC1, accounting for ~65% of variance, primarily

distinguished KC-H, KC-N, and KN-RM populations from Ly6Chi

blood monocytes and Ly6Chi&lo-RM. RLMs 24 and 48 h after KC

ablation became incrementally closer to KCs along PC1, reflect-

ing chromatin remodeling after arriving at the KC niche. PC2, ac-

counting for ~13% of variance, primarily separated Ly6Chi blood

monocytes from Ly6Chi&lo-RM.

Hierarchical clustering of ATAC-seq data further support the

relationships suggested by PCA, with KC-N and KN-RMcells ex-

hibiting highly similar patterns of open chromatin that are distinct

from the transitional patterns observed in Ly6Chi&lo-RM (Fig-

ure S5A). Based on these findings and the results of lineage-

tracing experiments, we considered the open chromatin regions

of KN-RM and Ly6Clo-RM populations as divergent endpoints of

chromatin remodeling events following entry of Ly6Chi blood

monocytes into the NASH liver. ATAC-seq peaks specific for

KN-RM or Ly6Clo-RM in comparison to circulating Ly6Chi mono-

cytes (red data points in Figures 4B and 4C, respectively) indi-

cated that approximately 75% of these peaks were specific to

KN-RM or Ly6Clo-RM cells (Figure 4E). Peaks specific for KN-

1064 Immunity 52, 1057–1074, June 16, 2020

RM cells were highly enriched for motifs recognized by LXR

and members of the MAF and TFE families (Figure 4E). In

contrast, ATAC-seq peaks specific for Ly6Clo-RM cells were

highly enriched for NF-kB motifs and for motifs recognized by

RUNX, ZEB, and KLF TFs. Open chromatin regions lost from

blood monocytes during acquisition of either the KC or Ly6Chi&lo

-RM niche signatures (Figures 4B and 4C, blue points) were en-

riched for KLF and C/EBP motifs (Figures S5B and S5C). The

open chromatin regions lost during acquisition of the KC niche

additionally included motifs for RUNX and CTCF (Figure S5B).

The pattern of motif enrichment in KC-N and KN-RM cells im-

plies that liver niche signals increase the expression and/or activ-

ities of TFs that bind to LXR, MAF, and TFE motifs. Consistent

with this possibility, Nr1h3 (encoding LXRa), Mafb, Tfec, and

Id3 (Lavin et al., 2014; Mass et al., 2016) are among a set of

KC LDTFs higher expressed in KC-N and KN-RM cells than in

Ly6Chi&lo-RM cells (Figure 4F; Table S2). Importantly, these

TFs are highly induced in RLMs within 12 h of entry into the

open KC niche of the liver (Sakai et al., 2019). These findings

further support a model in which KN-RM cells largely follow the

developmental program taken by RLMs following KC depletion

upon adherence to LSECs. In contrast, the lack of induction of

KC LDTFs in Ly6Chi&lo-RM cells is consistent with their location

outside of the sinusoidal space. NF-kB motifs are enriched in

KC enhancers in comparison to other tissue-resident macro-

phages (Sakai et al., 2019), but the particularly strong enrichment

for this motif in Ly6Clo-RM cells implies that the niche occupied

by these cells provides additional signals that activate NF-kB. In

addition, while RUNX factors are lower in KC-N and KN-RM cells

in comparison to blood Ly6Chi monocytes, their expression is

maintained or increased in Ly6Chi&lo-RMs (Figure S5D). In con-

cert, analysis of open chromatin provides evidence that the

divergent patterns of gene expression observed in HSC-derived

KN-RM and Ly6Chi&lo-RM cells are in part determined by

whether or not they receive niche-specific signals necessary to

adequately induce KC LDTFs.

NASH Diet Reprograms the KC Enhancer LandscapeWe next sought to understand the basis for the altered expres-

sion of the more than 900 mRNAs during the transition of KC-H

cells in the healthy liver to KC-N cells in the NASH liver (Figures

3A and 3C). Corresponding changes in the regulatory land-

scapes during this transition would potentially enable inference

of TFs and upstream signaling pathways that mediate re-

sponses to the NASH diet. In contrast to changes in open chro-

matin observed in transition of Ly6Chi blood monocytes to KN-

RM cells (Figures 4C and 4D), relatively few differences in open

chromatin were observed comparing KC-H and KC-N cells

(Figure 5A, left). The enriched motifs associated with the

increased chromatin accessibility included sites for AP1 and

EGR factor binding, whereas sites of decreased chromatin

accessibility were enriched for PU.1 and SpiC motifs (Figure 5A

right).

To investigate potential changes in the transcriptional func-

tions of these regions, we performed chromatin immunoprecip-

itation sequencing (ChIP-seq) for acetylation of histone H3 lysine

27 (H3K27ac) in KC-H cells in the healthy liver and KC-N cells in

the NASH liver. H3K27ac is deposited by histone acetyltrans-

ferases (HATs) associated with transcriptional co-activators

0 2 4 6 8 10 12 14

0

2

4

6

8

10

12

14

Ly6C

lo-R

M

8507

3509

Ly6Chi Blood

KC-N

KN-RM

5 kb

01e32e33e3

01e32e33e3

01e32e33e3

01e32e33e3

01e32e33e3

Clec4f10 kb

02e24e2

02e24e2

02e24e2

02e24e2

02e24e2

Itgam

ATAC-seq ATAC-seqRNA RNAA B C

E

F

0 2 4 6 8 10 12 14

0

2

4

6

8

10

12

14

KN

-RM

6732

2458

Gained during niche acquisition versus Ly6Chi peripheral blood monocytes

P- value

%targ/%bkgd

BestMatch

NF-κBZEBRUNX

TFE/MITFLXREMAF

ATAC-seq peaks

4,500 only up in KN-RM

6,279 only up in Ly6Clo

-RM

2,226 upin both

Motif

1e-1011e-911e-79

30/175.1/0.933/21

1e-5221e-1481e-102

34/1037/2339/27

TCAGCTGACTGAGCTACGTAGACTGATCGTACTGACGATC

CGATTGAC

ACGT

GTACTCGAATCGATCGAGCTCTGACTAG

TGACCTGAGTCATCGA

AGTC

G TACGCTAG TACCGTACTAG

CATG

AC TGCTAGTCGACGTACGATAGCTGATCG TACG TACGTACCATGCTGATACGACTGC TGACGTAC TGACG TACGTAATGCCTAGGACTACTGC TAGCG TACG TACG TACG TAACGTGCATACTGCGTAAGTCTCAG

CGATGTACCGTAACGTAGTC

C CG T ACG ACG CG ACG G T CG T A ACG ACG ACG ACG AG T ACG ACG

NF-κBNFATJUN-CRE

1e-911e-791e-75

20/6.82.1/.0231/16

TCGACTGATCAGGCAT

AG TCCG TAAGCTCTAGCGATATCGCTGAAGTCCGAT

CTAGTGCATGAC

AGTCGACTGACTCAGT

CGATCTAG

ACGTTCAGCG TAG TACAG TCAGCTTAGC

TACG

CGATGATCC TGACATGAG TCCGTARBPJ1e-22 12/7.6

GATCCATG

AGCTCTAG

CTAGTCAG

CTGACTGATACGGATC

−100 −50 0 50 100

−80

−60

−40

−20

0

20

40

PC1 (65%)

PC

2 (1

3%)

KC-N

KN-RM

Ly6ChiBlood NASH

Ly6ChiBlood Control

Ly6Clo-RM

Ly6Chi-RM

KC-H

D

RLM 24hr

RLM 48hr

Nr1h3 Mafb Id3

KC

-HK

C-N

KN

−R

MLy

6Chi-R

M

0

100

200

300

400

0

20

40

60

0

100

200

300

400

0

200

400

600

800

Mea

n E

xpre

ssio

n (T

PM

) ATAC-seq (log2(norm. tags +1))

ATAC-seq (log2(norm. tags +1))

Ly6Chi Blood

Tfec

Ly6Chi -RM

Ly6Clo -RM

Ly6Chi Blood

Ly6C

lo-R

MLy

6Chi B

lood

KC

-HK

C-N

KN

−R

MLy

6Chi-R

MLy

6Clo-R

MLy

6Chi B

lood

KC

-HK

C-N

KN

−R

MLy

6Chi-R

MLy

6Clo-R

MLy

6Chi B

lood

KC

-HK

C-N

KN

−R

MLy

6Chi-R

MLy

6Clo-R

MLy

6Chi B

lood

Figure 4. Niche-Specific Reprogramming of Epigenetic Landscapes

(A) UCSC genome browser tracks of ATAC-seq signals in the vicinities of theClec4f and Itgam genes in the indicated cell types. Bar plots to the right of each track

represent the RNA-seq gene expression (mean TPM ± SD) in each population.

(B) Genome-wide comparison of normalized ATAC-seq peak tags at enhancer-like regions (>3 kb removed from transcription start site [TSS]) comparing Ly6Chi

blood monocytes and Ly6Clo-RM during NASH. Differential regions were identified using DESeq2 (FC > 2 and p-adj < 0.05 using independent biological du-

plicates).

(C) Genome-wide comparison of normalized ATAC-seq peak tags at enhancer-like regions (>3 kb removed from TSS) comparing open chromatin in Ly6Chi blood

monocytes and KN-RM.

(D) PCA of ATAC-seq datasets (n = 2–3) for the top 10,000most variable distal (>3 kb from TSS) regions in myeloid cell populations during NASH, or recruited liver

macrophages (RLM) from Sakai et al. (2019).

(E) De novo motifs enriched in distal open chromatin regions (>3 kb from TSS) enriched in KN-RM (top), Ly6Clo-RM (bottom), or enriched in both populations

(middle) compared to Ly6Chi blood monocytes during NASH. The background for motif enrichment analysis is the distal open chromatin from Ly6Chi blood

monocytes.

(F) Expression (mean TPM ± SD) of KC LDTFs in the indicated cell types.

Please see also Figure S5.

llArticle

Immunity 52, 1057–1074, June 16, 2020 1065

F

A

C

D

0 2 4 6 8 10 12 14

0

2

4

6

8

10

12

14

KC-H

KC

-N

402

90

Differential Kupffer ATAC-seq

MotifP- value

%targ/%bkgd

Bestmatch

1e-87 69/22 AP1/ATF1e-15 19/7 EGR2

1e-24 60/13 PU.1/SpiC

Up 10%

Down8%

All enhancers

Up 3.5%Down 24%

KC signature

B

1e-137 35/19 AP1/ATF

1e-20 11/7.3 RUNX1e-25 0.7/.04 NFAT

1e-19 16/11 EGR

Differential Kupffer H3K27ac ChIP-seq

MotifP- value

%targ/%bkgd

Bestmatch

1e-19 0.3/0.0

1e-33 49/39 NR half-site1e-25 0.4/0.0 MITF

MAF1e-17 0.3/0.0 IRF

CGATGTCA

C TAG

AC TGACTGACGTAG TCCG TACG TAAC TGCGATC TAGACTGCGATAGTCCG TAG TACCGTAAC TGATCGCG TAAGTC

AGCT

G TAC

ACGTATCGACGTATGCCG TAAC TGATGCG TACACGTC TGAACGTAG TCACGTATGCAC TGCG TACG TACG TAACTGGACTG TCACG TACG TACG TAATCGATGCCG TAAG TC

ACG AC CGT ACG G T CG TCTAGTCGATTGATAGCTACAAGCT

A

ACGTACGTACGTAGTCCGATG TCAG TCAC TGAAGCTAGCTAGCTACGTG TACAGTCACGTAC TGACGTAC TGAC TGAGCTCGATCGATTGAC

CTAG

ATGC

AGTCGATCCTGA

AGTCAGTC

0 2 4 6 8 10 12 14

0

2

4

6

8

10

12

1443352 total 10452 KC sig4201365 KC sig

35832553 KC sig

CTAG

CATG

CATG

AGCTGACTCGTAGATCAGCTGTCAATGCTCGACTAGCATGACGTAGTCCTGA 1e-15 3.5/1.6 LXRE

(known)

E H3K27ac ChIP-seq(log2(norm. tags +1))

−6 −4 −2 0 2 4 6

−5

0

5

10

log2(KC-N/KC-H) H3K27ac ChIP-seq

log2

(KC

-N/K

C-H

) R

NA

-seq

R = 0.70

RNA- vs H3K27ac ChIP-seq

***

SpicIrf1

TfecMafIrf5

Nr1h3Irf7Mitf

Nr1h2Mafb

Runx1Irf8Jun

Srebf1FosAtf3

Egr2

0 2 4log2(NASH/Ctrl)RNA-seq (TPM)

***

************

**

*******

TCGA

ACGTACTGG TCAATCGACGTG TACCG TA

TCGA

ATGC

ACGTACGTAGTCAGTCCGATAGTCGCATGCATGATCCTAG

ATGC

G TAC

AGTC

GTCAGTACCTAGTGACACGT

0

50

100

RN

A-s

eq (

TP

M)

Atf3Egr1Egr2Spic

0

400

800

0 1 4 10 20 30

Weeks on AMLN diet

RN

A-s

eq (

TP

M) C6

Cd163Cd9Trem2

G

Effect of NASH on H3K27ac

5 kb

10 kb 5 kb

20 kbCd9

Trem2Cd163

C6ATAC

H3K27ac

ATAC

H3K27ac

ATAC

H3K27ac H3K27ac

ATAC

KC-HKC-N

ATAC-seq (log2(norm. tags +1))

***

KC

-N

KC-H

Figure 5. The NASH Diet Alters the Activity States of Resident KC Enhancers(A) Left: Normalized ATAC-seq signal at all distal open chromatin regions (>3 kb from TSS) in Tim4+ KCs from healthy mice (KC-H) or Tim4+ KCs from mice on

NASH diet (KC-N). Regions with significantly more chromatin accessibility during NASH are colored in red, while regions with less accessibility during NASH are

colored blue. Right: De novo motif enrichment from differentially accessible chromatin region shown at right.

(B) Left: H3K27ac ChIP-seq signal around distal (>3 kb from TSS) ATAC-seq peaks in a 2,000 bp window. Differentially acetylated regions were determined using

DESeq2 (FC > 2, p-adj < 0.05). Regions overlapping with KC signature enhancers (Figure S6A) are colored green. Enhancers with more acetylation during NASH

are colored red, or orange if also a KC signature enhancer. Enhancers with less acetylation during NASH are colored blue, or purple if also a KC signature

enhancer. Right: Representative motif enrichment from differentially acetylated chromatin regions.

(legend continued on next page

llArticle

1066 Immunity 52, 1057–1074, June 16, 2020

)

llArticle

and is highly correlated with regulatory element activity

(Creyghton et al., 2010). Out of 43,352 total distal open chro-

matin regions defined by ATAC-seq, 4,201 putative enhancers

gained H3K27ac and 3,583 putative enhancers lost H3K27ac

in response to the NASH diet (Figure 5B), affecting 18% of the

enhancer-like regions overall (Figure 5C).

A comparison of H3K27ac associated with ATAC-seq peaks in

KC-H cells versus in other macrophage populations enabled

definition of a set of 10,452 putative KC-H signature enhancers

(Figure S6A; Sakai et al., 2019). A profound overlap was

observed between KC signature enhancers and enhancers

downregulated by the NASH diet, in total identifying 2,553 KC

signature downregulated enhancers (24% of KC signature en-

hancers, 71% of all downregulated enhancers, Fisher’s exact

test p value < 0.001) (Figures 5B and 5C). Conversely, only 365

KC signature enhancers were upregulated by NASH diet (3.5%

of KC signature enhancers, 8.7% of all upregulated enhancers,

Fisher’s exact test p value = 1) (Figures 5B and 5C). Changes

in H3K27ac were highly correlated with changes in expression

of the nearest gene (Figure 5D). These findings suggest a prefer-

ential suppressive effect of the NASH diet on gene regulatory

networks governing the function of KC enhancers, in line with

the corresponding downregulation of KC identity genes

(Figure S4B).

ATAC-seq and H3K27ac ChIP-seq tracks associated with the

downregulated KC identity genes C6 and Cd163 illustrated sub-

stantial loss of H3K27ac at their respective promoters and distal

regions of open chromatin (Figure 5F). The opposite pattern was

observed at the upregulated lipid-associatedmacrophage (LAM)

and SAM-defining genes Trem2 and Cd9 (Jaitin et al., 2019;

Ramachandran et al., 2019). In addition, Trem2 and Cd9 both

provided examples of genes in which new regions of open chro-

matin are established that are associated with H3K27ac (Fig-

ure 5F), representing putative NASH-dependent enhancers.

Both pre-existing and NASH-induced regions associated with

Trem2were shifted but conserved at the TREM2 locus in human

microglia (Figure S6C). Additional examples of NASH-induced

genes in KCs and associated gene ontologies are shown in

Figure S6B.

Motif enrichment analysis of regions of open chromatin

exhibiting loss of H3K27ac in response to the NASH diet

showed enrichment for binding sites recognized by LXR,

MAF, and IRF TFs (Figure 5B right). Each of these motifs are

recognized by TFs that are established or proposed to drive

KC identity (Lavin et al., 2014; Mass et al., 2016; Sakai

et al., 2019; Scott et al., 2018). In contrast, open chromatin

regions with gained H3K27ac were enriched for de novo

(C) Summaries of percent representation of (left) differentially activated enhancer

intersected with the KC signature enhancers.

(D) Ratio-ratio plot depicting fold change in H3K27acChIP-seq signal at enhancers

fold change inmRNA expression of closest gene annotated to enhancer region in T

in blue are significantly different (FC > 2, p-adj < 0.05) for both H3K27ac ChIP-se

relationship between the highlighted differential data points.

(E) Log2FC of candidate TFs known to bind DNA elements found enriched in (B) fo

0.01, and ***p-adj < 0.001 using DESeq2.

(F) UCSC genome browser tracks of ATAC-seq or H3K27ac ChIP-seq signals in

(G) Mean TPM (LOESS fit) of the indicated genes in Tim4+ KCs from mice fed the

week: n = 3; 20 week: n = 3; 30 week: n = 4).

Please see also Figure S6.

motifs matching AP1, NFAT, RUNX, and EGR TFs (Fig-

ure 5B right).

Comparison of KC-H to KC-N cells indicated significant induc-

tion of Atf3, Fos, Jun, Egr2, and Runx1 mRNAs (Figures 5E and

5G), suggesting that increased expression of these TFs during

NASH may contribute to activation of enhancers with corre-

sponding DNA binding elements (Figure 5B). Conversely,

mRNAs encoding a subset of KC LDTFs were downregulated

in response to the NASH diet, including Spic, Irf1, and Tfec.

Notably, SpiC (encoded by Spic) binds to a motif nearly identical

to that recognized by PU.1 that is enriched at genomic regions

exhibiting loss of open chromatin in NASH. In contrast, while

LXR motifs were highly enriched at genomic regions exhibiting

loss of open chromatin, expression of Nr1h3, encoding LXRa,

was not affected by the NASH diet, and expression ofNr1h2, en-

coding LXRb, was slightly increased (Figure 5E).

To relate changes in TF expression to global changes in gene

expression, we performed RNA-seq analysis of KCs following

1, 4, 10, 20, and 30 weeks of the NASH diet. Coordinated

changes in gene expression began to occur between 4 and

10 weeks of the NASH diet, with upregulation of Atf3 and

Egr2 associated with increases in Trem2 and Cd9 expression

and downregulation of Spic associated with decreases in

Cd163 and C6 expression (Figure 5G). These findings indicate

that acquisition of features corresponding to the LAM or SAM

phenotype characterized by high expression of Trem2 and

Cd9 requires prolonged exposure to the NASH diet, which ulti-

mately results in substantial reprogramming of the KC regula-

tory landscape.

NASH Diet Selectively Impairs LXR Regulation of KCIdentity GenesThe observation that LXR binding motifs were highly enriched in

genomic regions exhibiting diet-induced loss of H3K27ac led to

a focused analysis of LXR function. Comparison of effects of

LXR deletion with effects of the NASH diet on KC gene expres-

sion indicated that a subset of LXR target genes associated

with its role as a KC LDTF were strongly downregulated by

the NASH diet. For example, Cd5l, Timd4, Cd209l, Pcolce2,

and Plac8 require LXRa for expression (Sakai et al., 2019),

and all but Cd5l exhibited significantly reduced expression in

response to the NASH diet (Figure 6A). However, many canon-

ical LXR target genes such as Abca1, Abcg1, Mylip, and Srebf1

were significantly upregulated in response to the NASH diet

(Figure 6B; Table S3), arguing against a general loss of LXR

function. Targeted lipidomic analysis of known LXR ligands

(Peet et al., 1998) in liver indicated that the concentration of

s between control and NASH KCs, or (right) differentially acetylated enhancers

(2,000 bpwindow centered on ATAC-seq peaks >3 kb from TSS) compared to

im4+ KCs in NASH dietmice (KC-N) versus healthymice (KC-H). Points colored

q signal at enhancers and closest mRNA. Pearson correlation of 0.70 denotes

r KCs from healthy mice (KC-H) and NASHmice (KC-N). *p-adj < 0.05, **p-adj <

the vicinities of the indicated genes.

AMLN NASH diet as indicated (0 week: n = 3; 1 week: n = 2; 4 week: n = 2; 10

Immunity 52, 1057–1074, June 16, 2020 1067

D

A B C

E

F

G

Figure 6. Genome-wide Occupancy of LXR Binding in KCs during NASH

(A) RNA-seq expression (mean TPM ± SD) of LXR-dependent KC signature genes. *p-adj < 0.05, **p-adj < 0.01, and ***p-adj < 0.001 using DESeq2.

(B) RNA-seq expression (mean TPM ± SD) of canonical LXR target genes.

(C) Quantification of desmosterol, 24-, 25-, and 27-OHC and 24,25-EC in livers from control or NASH mice. *** = p < 0.001.

(D) Mean LXR ChIP-seq signal at merged irreproducibility discovery rate (IDR) peaks from KC nuclei. Nuclei were sorted from Clec4f-Cre-NLS-TdTomato mice fed

either a control diet (n = 2) or the NASH diet for 20 weeks (n = 2). Peaks with significantly altered LXR binding were determined using DESeq2 (FC > 2, p-adj < 0.05).

(E) ATAC-seq, H3K27ac ChIP-seq, and LXR ChIP-seq signals in the vicinities of the indicated genes.

(F) Normalized distribution of LXR ChIP-seq density or H3K27ac ChIP-seq density at enhancers with either significantly altered LXR binding during NASH or all

LXR bound enhancers.

(G) Left: De novo motif enrichment for NASH-gained LXR peaks (right) or NASH-lost LXR peaks (right) shown in Figure 7D.

Please see also Figure S7.

llArticle

1068 Immunity 52, 1057–1074, June 16, 2020

A B

LXR gained LXR lost LXR total

G H

NASHControl

25,507

13,291

2,808

ATF3 ChIP-seqIDR peaks

01234567

Log2

(Tag

s+1)

1,000 bp

I

AP1/ATF3NR/LXREgr2PU1

0.000

0.002

0.004

0.006

0.008

−200−100 0 100 200

Mot

ifs p

er b

p pe

r pe

ak

0.000

0.001

0.002

0.003

0.004

−200−100 0 100 200

Distance from peak (bp)

0.000

0.001

0.002

0.003

−200−100 0 100 200

E

Control

NASH

ChIP-seq tag densities centered on genomic regions co-bound by LXRs and ATF3

ChI

P fr

agm

ent d

epth

per

bp p

er p

eak

ATF3 LXR P300 H3K27ac

−2000

−1000 0

1000

2000

−2000

−1000 0

1000

2000

−2000

−1000 0

1000

2000

−2000

−1000 0

1000

2000

2

4

6

8

3

6

9

12

2.5

5.0

7.5

10.0

0

5

10

15

20

Distance from enhancer (bp)

DC

P-value

Best match

1e-671 49.26/23.38 AP1/ATF3

1e-111 11.06/5.27 Egr2/Egr1

1e-69 42.57/33.94 MITF/USF1

NASH specific ATF3 vs all ATF3 peaks

Motif%targ/

%bkgd

NASH TF ocupancy atNASH gained enhancers

None 32%ATF3 10%LXR 3%

Both 54%

F

5 kb 10 kb

LXR

AT

F3

P30

0

Trem2 Cd9

Control NASH

Arhgap22

20 kb 10 kb

Kcnn4

0.0

2.5

5.0

7.5

10.0

0.0 2.5 5.0 7.5 10.0

Control − KC-H

NA

SH

− K

C-N

Mean expression(log2(TPM+1))

Gpnmb Mmp12Trem2

Cd9

ItgaxArhgap22

0.0

2.5

5.0

7.5

10.0

12.5

0.0 2.5 5.0 7.5 10.0 12.5

Clec4f-Cre- − KN-RM

Cle

c4f-

Cre

+ − K

N-R

M

Mean expression(log2(TPM+1))

43 significant

146

sign

ifica

nt

(LX

Rα/

β K

C-D

KO

)

(LXRα/β WT)

0

100

200

300

020406080

020406080

05

10152025

050

100150

0

10

20

30

Kcnn4 Tgfb3 Trem2

Arhgap22 Cd9 Itgax

Mea

n E

xpre

ssio

n (T

PM

)

Clec4f

-Cre-

Clec4f

-Cre+

Clec4f

-Cre-

Clec4f

-Cre+

Clec4f

-Cre-

Clec4f

-Cre+

**** ***

** n.s.***

Figure 7. Combinatorial Actions of LXR and ATF3 Coordinate NASH Responsive Gene Expression in KCs during NASH

(A) Motif frequency within 500 bp of LXR binding sites described in Figure 6F.

(B) Normalized ATF3 ChIP-seq signal at merged IDR peaks from KC nuclei. Nuclei were sorted from Clec4f-Cre-NLS-TdTomato mice fed either a control diet or

the NASH diet for 20 weeks (n = 2). Significant ATF3 peaks had Poisson enrichment p value < 0.0001 and FC > 4.

(legend continued on next page)

llArticle

Immunity 52, 1057–1074, June 16, 2020 1069

llArticle

desmosterol, which is by far the most abundant endogenous

LXR ligand in the liver (Sakai et al., 2019; Yang et al., 2006),

was significantly increased in the setting of the cholesterol-

rich NASH diet in comparison to the control diet (Figure 6C).

All other natural LXR agonists were present at much lower con-

centrations (data not shown). The increase in desmosterol is

consistent with the upregulation of general LXR target genes

involved in cholesterol homeostasis. A corresponding increase

in desmosterol was reported in livers of human subjects with

NASH and cirrhosis (Gorden et al., 2015).

To investigate whether changes in gene expression are asso-

ciated with corresponding changes in LXR binding, we per-

formed ChIP-seq for LXRa + LXRb in the combination of KC-

N and KN-RM cells marked by sorting nuclei prepared from

rapidly fixed control or NASH livers for nuclear Clec4f-tdTo-

mato expression (Figure S7A). These studies yielded high qual-

ity datasets that could not be obtained using conventionally

sorted cells and indicated that the NASH diet induced LXR

binding by more than 2-fold at more than 1,700 locations and

resulted in a loss of binding at more than 1,000 locations (Fig-

ure 6D). Notably, DNA binding was relatively unchanged at

most of the LXR binding sites associated with canonical LXR

activity (e.g., Abca1), which are highly LXR-dependent but not

affected at the mRNA level by the NASH diet (Figure 6E). In

contrast, LXR binding in the vicinity of Timd4, Pcolce2, and

Plac8 was markedly reduced. Overall, LXR ChIP-seq peaks

were preferentially depleted from KC signature enhancers

(405/1046, or ~39% of total downregulated LXR peaks in en-

hancers, blue points, Fisher’s exact test p value < 0.001)

compared to LXR signal gained at KC signature enhancers

(128/1795, or ~7% of total upregulated LXR peaks in en-

hancers, red points, Fisher’s exact test p value = 1) (Figures

6D and S7B). Sites at which LXR binding was gained were

associated with increased H3K27ac, whereas the opposite

pattern was observed at sites at which LXR binding was lost,

consistent with a positive regulatory function of LXR at these lo-

cations (Figure 6F).

NASH-Induced Collaborative Binding PartnersRepurpose LXRsMotif analysis of genomic regions exhibiting gain or loss of LXR

binding in response to the NASH diet indicated enrichment for

AP-1 or ATF, EGR, and MITF motifs at gained sites, and PU.1

or SPIC, GLIS3, and nuclear receptor half site motifs at lost sites

(Figure 6G). The distance relationships of thesemotifs to the cen-

ter of the LXR peak were consistent with collaborative binding in-

teractions observed for other LDTFs (Figure 7A; Heinz et al.,

2015). These findings suggested the possibility that NASH-

(C) De novo motif enrichment for NASH-specific ATF3 peaks as in (B).

(D) Intersection of ATF3 and/or LXR IDR ChIP-seq peaks from KC nuclei isolated

(from Figure 5B).

(E) Normalized distribution of ChIP-seq tag density at sites co-bound by LXRs an

(F) UCSC genome browser tracks of LXR, ATF3, and P300 ChIP-seq signals in the

both LXR and ATF3 are shaded yellow, and peaks with only gained ATF3 are sh

(G) Mean expression (TPM) for genes nearest to NASH-specific LXR peaks co-b

(H) RNA-seq (n = 2 per group) of KN-RM from control mice or Clec4fCre-LXRafl/

(I) As in (H) for selected genes. **p-adj < 0.01; ***p-adj < 0.001.

Please see also Figure S7.

1070 Immunity 52, 1057–1074, June 16, 2020

induced changes in the binding and function of LXRs were the

consequence of altered collaborative binding interactions with

AP-1 factors, EGRs, and members of the ETS TF family PU.1

and/or SpiC. To examine this possibility, we focused on ATF3,

as it was the most highly induced AP-1 factor in response to

the NASH diet (Figure 5E) and recognized the AP-1 motif en-

riched in NASH-specific enhancers (Figure 5B). Similar to

expression amounts of Atf3, we detected ~2.5 times as many

peaks in nuclei from mice with NASH (data not shown). This

observation correlated with an overall increased magnitude of

ATF3 ChIP-seq signal during NASH (Figures 7B and S7D). En-

hancers with gained ATF3 binding during NASH were enriched

for de novo motifs corresponding to AP-1, EGR, and MITF2

TFs (Figures 7C and S7C).

Intersection of the LXR and ATF3 ChIP-seq datasets indicated

~28,000 instances of overlapping peaks (Figure S7E). Of the

1,793 LXR binding sites increased by >2-fold in NASH, ~80%

overlapped with ATF3 peaks, consistent with a collaborative

binding relationship. Notably, the combination of LXR and

ATF3 was observed at 54% of NASH-induced enhancers (Fig-

ure 7D). Themajority of these sites were occupied by LXRs under

control conditions, but exhibited substantial increases in

H3K27ac in the context of NASH (Figure 7E). This result sug-

gested that diet-induced binding of ATF3 to these sites resulted

in the subsequent recruitment of HATs. We therefore performed

ChIP-seq for the HAT P300 and found its recruitment to the LXR

and ATF3 peaks in response to the NASH diet, indicating a likely

role in contribution to acetylation of H3K27 and subsequent

enhancer function (Figures 7E and S7F–S7H). Representative

examples of NASH-induced co-binding of LXRs and ATF3 or

for NASH-induced binding of ATF3 at pre-existing LXR binding

sites are illustrated for Trem2, Cd9, Arhgap22, and Kcnn4 in

Figure 7F.

Analysis of the genes nearest to enhancers bound by ATF3

and LXR in the context of the NASH diet revealed a shift in

expression distribution toward a higher mean during NASH (Fig-

ure 7G). To directly evaluate NASH-specific functions of LXRs re-

sulting from these induced binding sites, we crossed

LXRafl/flLXRbfl/fl mice to the Clec4f-cre-TdTomato mouse to

generate KC-N and KN-RM-specific LXRa�/�LXRb�/� mice.The progeny were then fed either a control diet or the NASH

diet for 20 weeks. Due to the near complete absence of KC-N

cells in theClec4f-cre-TdTomato LXRafl/flLXRbfl/fl mice, compar-

isons were made of control and Clec4f-cre-TdTomato

LXRafl/flLXRbfl/fl KN-RMs. By comparing LXR-dependent gene

expression in KN-RMs under these two conditions, we identified

a set of 146 NASH-specific LXR-dependent genes (Figures 7H

and 7I), 33 of which were part of the set defined in Figure 7G.

from NASH-diet-fed mice with enhancers with increased activity during NASH

d ATF3 indicated in (D).

vicinities of the indicated genes. Peakswith NASH-gained (HOMER) binding for

aded green.

ound by ATF3. DE genes identified using p-adj < 0.05 and FC > 2.flLXRbfl/fl mice fed the NASH diet for 20 weeks. DE genes identified as in (G).

llArticle

Notably, LXR dependency was observed both at genes

exhibiting NASH-induced binding of LXR (e.g., the SAM-associ-

ated gene Cd9) and genes exhibiting recruitment of ATF3 to pre-

existing LXR binding sites (e.g., Arhgap22 and Kcnn4). Trem2

expression was reduced in LXRa�/�LXRb�/� KN-RMs, but didnot retain statistical significance after correction for multiple

testing. Collectively, these findings provide evidence that

NASH-induced changes in collaborative TFs result in altered

binding and function of LXRs, contributing to the NASH-specific

program of KC gene expression.

DISCUSSION

Here, investigation of genomic and anatomic relationships

among phenotypically distinct macrophage populations in a

mouse model of NASH revealed a strong association of niche

occupancy with the transcriptional and phenotypic states of

different EMP- and HSC-derived myeloid cells. HSC-derived

macrophages observed in this model of NASH reside in at least

two distinct niches associated with divergent pathways of dif-

ferentiation. One pathway corresponds to the development of

Ly6Chi&lo-RMs, which reside proximal to large portal and cen-

tral vein vessels in contact with conventional (but most likely in-

flamed) vascular endothelium. An alternative pathway results in

the accumulation of KN-RM cells, which colocalized with KC-N

in contact with the LSECs and adopted a similar pattern of

gene expression. This process occurred concordantly with pro-

gressive loss of resident KC-Ns, most likely due to apoptosis.

These findings provide strong evidence for instructive roles of

the LSECs and the NASH diet in determining the molecular

phenotypes of both KN-RM and KC-N cells. Of particular inter-

est, we observed that a subset of KC-N and KN-RM cells ac-

quire similarities to the recently identified SAM that resides in

areas of fibrosis in human cirrhotic livers (Ramachandran

et al., 2019).

The transitions of open chromatin, H3K27ac and genome-

wide locations of LXRs and ATF3 provide evidence for

NASH-induced alterations in expression and activities of

ATF3, resulting in reprogramming KC-specific functions of

LXRs. In the context of relatively constant LXR expression,

increased ATF3 and likely other AP-1 TFs are suggested to

drive redistribution of LXR to NASH-specific enhancers as

well as activate poised enhancers bound by LXRs under control

conditions. The decreased expression of Spic in the context of

the NASH diet may also contribute to loss of collaborative LXR

binding at a subset of KC-specific enhancers. The delineation

of NASH-induced regulatory elements associated with the

Cd9 and Trem2 genes that are occupied by LXRs and ATF3

suggest roles of these TFs in establishing the SAM and/or

LAM phenotypes. A key remaining question is to determine

whether the altered functions of LXRs in KCs are pathogenic

or represent an adaptive response to the NASH diet.

Overall, these studies are consistent with a model in which

distinct microenvironments within the NASH liver drive diver-

gent patterns of differentiation of resident and infiltrating cells

by remodeling the open chromatin landscapes of recruited

monocytes and altering the activities of pre-existing enhancers

of the resident KC population. The inference of TFs and up-

stream signaling pathways associated with these distinct cell

populations provides a basis for understanding how disease-

promoting environmental signals instruct resident and recruited

macrophages to acquire distinct pathogenic programs of gene

expression. Application of the methods utilized in this study

should prove valuable in gaining a better understanding of

how distinct myeloid phenotypes are established in other dis-

ease contexts.

STAR+METHODS

Detailed methods are provided in the online version of this paper

and include the following:

d KEY RESOURCES TABLE

d RESOURCE AVAILABILITY

B Lead Contact

B Materials Availability

B Data and Code Availability

d EXPERIMENTAL MODEL AND SUBJECT DETAILS

B Mice

B NASH-Model Diets

B Diphtheria toxin (DT)-mediated depletion of

Kupffer cells

d METHOD DETAILS

B Microscopy and Histo-Cytometry

B Nuclei Sorting

B Cell Sorting and Flow Cytometry

B Single-Cell RNA-Seq

B ATAC-Seq

B Poly A RNA-Seq

B Chromatin Immunoprecipitation

B ChIP-Seq Library Preparation

B Lipid Measurements

d QUANTIFICATION AND STATISTICAL ANALYSIS

B Sequencing Data Analysis

SUPPLEMENTAL INFORMATION

Supplemental Information can be found online at https://doi.org/10.1016/j.

immuni.2020.04.001.

ACKNOWLEDGMENTS

These studies were supported by NIH grants DK091183, HL088083,

DK063491, and GM085764 and Fondation Leducq grant 16CVD01. J.S.S.

was supported by American Heart Association Fellowship 16PRE30980030

and NIH Predoctoral Training grant 5T32DK007541. T.D.T. was supported

by P30 DK063491, T32DK007044, and NRSA T32CA009523. M.S. was sup-

ported by the Manpei Suzuki Diabetes Foundation of Tokyo, Japan and the

Osamu Hayaishi Memorial Scholarship for Study Abroad, Japan. X.S. was

supported by the American Heart Association grant 18POST34060088.

This work was also supported in part by the Intramural Research program

of NIAID, NIH.

AUTHOR CONTRIBUTIONS

Conceptualization, J.S.S., T.D.T., M.S., A.G., R.N.G, and C.K.G.; Methodol-

ogy, J.S.S., T.D.T., M.S., A.G. and N.J.S.; Formal Analysis, J.S.S, T.D.T.,

A.G., Z.O., R.Z.L., and M.H.; Investigation, J.S.S., T.D.T., M.S., A.G.,

C.M.B., N.J.S., H.B., B.T.V., M.P.P., K.M.E., D.G., and L.-W.C.; Data Curation,

J.S.S., T.D.T, and Z.O; Writing – Original Draft, J.S.S., T.D.T, A.G., R.N.G, and

C.K.G.; Writing – Review & Editing, J.S.S, T.D.T., M.S., A.G., V.M.L., R.N.G.,

Immunity 52, 1057–1074, June 16, 2020 1071

https://doi.org/10.1016/j.immuni.2020.04.001https://doi.org/10.1016/j.immuni.2020.04.001

llArticle

and C.K.G.; Funding Acquisition, J.S.S., T.D.T., R.N.G, and C.K.G.; Visualiza-

tion, J.S.S., T.D.T., A.G., and Z.O.

DECLARATION OF INTERESTS

J.L.W. and X.S. are named inventors on patent applications or patents

related to the use of oxidation-specific antibodies that are held by UCSD.

J.L.W. is a consultant to Ionis Pharm and is a scientific founder of Oxi-

tope, Inc.

Received: February 3, 2020

Revised: April 1, 2020

Accepted: April 8, 2020

Published: May 1, 2020

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