Abstract
In this spotlight, we review technical issues that compromise single-cell analysis of tissue macrophages, including limited and unrepresentative yields, fragmentation and generation of remnants, and activation during tissue disaggregation. These issues may lead to a misleading definition of subpopulations of macrophages and the expression of macrophage-specific transcripts by unrelated cells. Recognition of the technical limitations of single-cell approaches is required in order to map the full spectrum of tissue-resident macrophage heterogeneity and assess its biological significance.
References
1.
Hume
DA
, Caruso
M
, Ferrari-Cestari
M
, Summers
KM
, Pridans
C
, Irvine
KM
. Phenotypic impacts of CSF1R deficiencies in humans and model organisms
. J Leukoc Biol
. 2020
;107
(2
):205
-219
.2.
Grabert
K
, Sehgal
A
, Irvine
KM
, et al. A transgenic line that reports CSF1R protein expression provides a definitive marker for the mouse mononuclear phagocyte system
. J Immunol
. 2020
;205
(11
):3154
-3166
.3.
Summers
KM
, Bush
SJ
, Hume
DA
. Network analysis of transcriptomic diversity amongst resident tissue macrophages and dendritic cells in the mouse mononuclear phagocyte system
. PLoS Biol
. 2020
;18
(10
):e3000859
.4.
Mass
E
, Nimmerjahn
F
, Kierdorf
K
, Schlitzer
A
. Tissue-specific macrophages: how they develop and choreograph tissue biology
. Nat Rev Immunol
. 2023
:1
-17
.5.
Hume
DA
. Differentiation and heterogeneity in the mononuclear phagocyte system
. Mucosal Immunol
. 2008
;1
(6
):432
-441
.6.
Hume
DA
, Irvine
KM
, Pridans
C
. The mononuclear phagocyte system: the relationship between monocytes and macrophages
. Trends Immunol
. 2019
;40
(2
):98
-112
.7.
Sehgal
A
, Carter-Cusack
D
, Keshvari
S
, et al. Fate-mapping studies in inbred mice: a model for understanding macrophage development and homeostasis?
. Eur J Immunol
. 2023
;53
(8
):e2250312
.8.
Ng
LG
, Liu
Z
, Kwok
I
, Ginhoux
F
. Origin and heterogeneity of tissue myeloid cells: a focus on GMP-derived monocytes and neutrophils
. Annu Rev Immunol
. 2023
;41
:375
-404
.9.
Guilliams
M
, Thierry
GR
, Bonnardel
J
, Bajenoff
M
. Establishment and maintenance of the macrophage niche
. Immunity
. 2020
;52
(3
):434
-451
.10.
Ginhoux
F
, Yalin
A
, Dutertre
CA
, Amit
I
. Single-cell immunology: past, present, and future
. Immunity
. 2022
;55
(3
):393
-404
.11.
Sanin
DE
, Ge
Y
, Marinkovic
E
, et al. A common framework of monocyte-derived macrophage activation
. Sci Immunol
. 2022
;7
(70
):eabl7482
.12.
Dick
SA
, Wong
A
, Hamidzada
H
, et al. Three tissue resident macrophage subsets coexist across organs with conserved origins and life cycles
. Sci Immunol
. 2022
;7
(67
):eabf7777
.13.
Bassler
K
, Schulte-Schrepping
J
, Warnat-Herresthal
S
, Aschenbrenner
AC
, Schultze
JL
. The myeloid cell compartment-cell by cell
. Annu Rev Immunol
. 2019
;37
:269
-293
.14.
Keshvari
S
, Caruso
M
, Teakle
N
, et al. CSF1R-dependent macrophages control postnatal somatic growth and organ maturation
. PLoS Genet
. 2021
;17
(6
):e1009605
.15.
Rojo
R
, Raper
A
, Ozdemir
DD
, et al. Deletion of a Csf1r enhancer selectively impacts CSF1R expression and development of tissue macrophage populations
. Nat Commun
. 2019
;10
(1
):3215
.16.
Haimon
Z
, Volaski
A
, Orthgiess
J
, et al. Re-evaluating microglia expression profiles using RiboTag and cell isolation strategies
. Nat Immunol
. 2018
;19
(6
):636
-644
.17.
Millard
SM
, Heng
O
, Opperman
KS
, et al. Fragmentation of tissue-resident macrophages during isolation confounds analysis of single-cell preparations from mouse hematopoietic tissues
. Cell Rep
. 2021
;37
(8
):110058
.18.
Mondor
I
, Baratin
M
, Lagueyrie
M
, et al. Lymphatic endothelial cells are essential components of the subcapsular sinus macrophage niche
. Immunity
. 2019
;50
(6
):1453
-1466.e4
.19.
Batoon
L
, Millard
SM
, Wullschleger
ME
, et al. CD169(+) macrophages are critical for osteoblast maintenance and promote intramembranous and endochondral ossification during bone repair
. Biomaterials
. 2019
;196
:51
-66
.20.
Kaur
S
, Raggatt
LJ
, Batoon
L
, Hume
DA
, Levesque
JP
, Pettit
AR
. Role of bone marrow macrophages in controlling homeostasis and repair in bone and bone marrow niches
. Semin Cell Dev Biol
. 2017
;61
:12
-21
.21.
Tabula Muris Consortium
. Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris
. Nature
. 2018
;562
(7727
):367
-372
.22.
Han
X
, Wang
R
, Zhou
Y
, et al. Mapping the mouse cell atlas by Microwell-seq
. Cell
. 2018
;172
(5
):1091
-1107.e1017
.23.
Han
X
, Zhou
Z
, Fei
L
, et al. Construction of a human cell landscape at single-cell level
. Nature
. 2020
;581
(7808
):303
-309
.24.
Gray
EE
, Friend
S
, Suzuki
K
, Phan
TG
, Cyster
JG
. Subcapsular sinus macrophage fragmentation and CD169+ bleb acquisition by closely associated IL-17-committed innate-like lymphocytes
. PLoS One
. 2012
;7
(6
):e38258
.25.
Baccin
C
, Al-Sabah
J
, Velten
L
, et al. Combined single-cell and spatial transcriptomics reveal the molecular, cellular and spatial bone marrow niche organization
. Nat Cell Biol
. 2020
;22
(1
):38
-48
.26.
Li
W
, Wang
Y
, Zhao
H
, et al. Identification and transcriptome analysis of erythroblastic island macrophages
. Blood
. 2019
;134
(5
):480
-491
.27.
Romano
L
, Seu
KG
, Papoin
J
, et al. Erythroblastic islands foster granulopoiesis in parallel to terminal erythropoiesis
. Blood
. 2022
;140
(14
):1621
-1634
.28.
Tay
J
, Bisht
K
, McGirr
C
, et al. Imaging flow cytometry reveals that granulocyte colony-stimulating factor treatment causes loss of erythroblastic islands in the mouse bone marrow
. Exp Hematol
. 2020
;82
:33
-42
.29.
Popescu
DM
, Botting
RA
, Stephenson
E
, et al. Decoding human fetal liver haematopoiesis
. Nature
. 2019
;574
(7778
):365
-371
.30.
Pinho
S
, Wei
Q
, Maryanovich
M
, et al. VCAM1 confers innate immune tolerance on haematopoietic and leukaemic stem cells
. Nat Cell Biol
. 2022
;24
(3
):290
-298
.31.
Pinho
S
, Marchand
T
, Yang
E
, Wei
Q
, Nerlov
C
, Frenette
PS
. Lineage-biased hematopoietic stem cells are regulated by distinct niches
. Dev Cell
. 2018
;44
(5
):634
-641.e4
.32.
Tikhonova
AN
, Dolgalev
I
, Hu
H
, et al. The bone marrow microenvironment at single-cell resolution
. Nature
. 2019
;569
(7755
):222
-228
.33.
Nestorowa
S
, Hamey
FK
, Pijuan Sala
B
, et al. A single-cell resolution map of mouse hematopoietic stem and progenitor cell differentiation
. Blood
. 2016
;128
(8
):e20
-31
.34.
Hernandez-Malmierca
P
, Vonficht
D
, Schnell
A
, et al. Antigen presentation safeguards the integrity of the hematopoietic stem cell pool
. Cell Stem Cell
. 2022
;29
(5
):760
-775.e10
.35.
Lynch
RW
, Hawley
CA
, Pellicoro
A
, Bain
CC
, Iredale
JP
, Jenkins
SJ
. An efficient method to isolate Kupffer cells eliminating endothelial cell contamination and selective bias
. J Leukoc Biol
. 2018
;104
(3
):579
-586
.36.
Bleriot
C
, Barreby
E
, Dunsmore
G
, et al. A subset of Kupffer cells regulates metabolism through the expression of CD36
. Immunity
. 2021
;54
(9
):2101
-2116.e6
.37.
De Simone
G
, Andreata
F
, Bleriot
C
, et al. Identification of a Kupffer cell subset capable of reverting the T cell dysfunction induced by hepatocellular priming
. Immunity
. 2021
;54
(9
):2089
-2100.e8
.38.
Liang
Y
, Kaneko
K
, Xin
B
, et al. Temporal analyses of postnatal liver development and maturation by single-cell transcriptomics
. Dev Cell
. 2022
;57
(3
):398
-414.e5
.39.
Hume
DA
, Offermanns
S
, Bonnavion
R
. Contamination of isolated mouse Kupffer cells with liver sinusoidal endothelial cells
. Immunity
. 2022
;55
(7
):1139
-1140
.40.
Iannacone
M
, Bleriot
C
, Andreata
F
, et al. Response to contamination of isolated mouse Kupffer cells with liver sinusoidal endothelial cells
. Immunity
. 2022
;55
(7
):1141
-1142
.41.
Guilliams
M
, Bonnardel
J
, Haest
B
, et al. Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches
. Cell
. 2022
;185
(2
):379
-396.e38
.42.
Bonnardel
J
, T'Jonck
W
, Gaublomme
D
, et al. Stellate cells, hepatocytes, and endothelial cells imprint the Kupffer cell identity on monocytes colonizing the liver macrophage niche
. Immunity
. 2019
;51
(4
):638
-654.e9
.43.
Sakai
M
, Troutman
TD
, Seidman
JS
, et al. Liver-derived signals sequentially reprogram myeloid enhancers to initiate and maintain Kupffer cell identity
. Immunity
. 2019
;51
(4
):655
-670.e8
.44.
Terkelsen
MK
, Bendixen
SM
, Hansen
D
, et al. Transcriptional dynamics of hepatic sinusoid-associated cells after liver injury
. Hepatology
. 2020
;72
(6
):2119
-2133
.45.
Kolodziejczyk
AA
, Federici
S
, Zmora
N
, et al. Acute liver failure is regulated by MYC- and microbiome-dependent programs
. Nat Med
. 2020
;26
(12
):1899
-1911
.46.
Zhou
Y
, Adewale
F
, Kim
S
, et al. Five-in-one: simultaneous isolation of multiple major liver cell types from livers of normal and NASH mice
. J Cell Mol Med
. 2021
;25
(20
):9878
-9883
.47.
Zong
C
, Meng
Y
, Ye
F
, et al. AIF1(+) CSF1R(+) MSCs, induced by TNF-alpha, act to generate an inflammatory microenvironment and promote hepatocarcinogenesis
. Hepatology
. 2023
;78
(2
):434
-451
.48.
Kumar
V
, Donthireddy
L
, Marvel
D
, et al. Cancer-associated fibroblasts neutralize the anti-tumor effect of CSF1 receptor blockade by inducing PMN-MDSC infiltration of tumors
. Cancer Cell
. 2017
;32
(5
):654
-668.e5
.49.
Tang
PC
, Chung
JY
, Liao
J
, et al. Single-cell RNA sequencing uncovers a neuron-like macrophage subset associated with cancer pain
. Sci Adv
. 2022
;8
(40
):eabn5535
.50.
Bain
CC
, MacDonald
AS
. The impact of the lung environment on macrophage development, activation and function: diversity in the face of adversity
. Mucosal Immunol
. 2022
;15
(2
):223
-234
.51.
Aegerter
H
, Lambrecht
BN
, Jakubzick
CV
. Biology of lung macrophages in health and disease
. Immunity
. 2022
;55
(9
):1564
-1580
.52.
Tan
SY
, Krasnow
MA
. Developmental origin of lung macrophage diversity
. Development
. 2016
;143
(8
):1318
-1327
.53.
Irvine
KM
, Caruso
M
, Cestari
MF
, et al. Analysis of the impact of CSF-1 administration in adult rats using a novel Csf1r-mApple reporter gene
. J Leukoc Biol
. 2020
;107
(2
):221
-235
.54.
Sikkema
L
, Ramirez-Suastegui
C
, Strobl
DC
, et al. An integrated cell atlas of the lung in health and disease
. Nat Med
. 2023
;29
(6
):1563
-1577
.55.
Hume
PS
, Gibbings
SL
, Jakubzick
CV
, et al. Localization of macrophages in the human lung via design-based stereology
. Am J Respir Crit Care Med
. 2020
;201
(10
):1209
-1217
.56.
Baillie
JK
, Arner
E
, Daub
C
, et al. Analysis of the human monocyte-derived macrophage transcriptome and response to lipopolysaccharide provides new insights into genetic aetiology of inflammatory bowel disease
. PLoS Genet
. 2017
;13
(3
):e1006641
.57.
Chakarov
S
, Lim
HY
, Tan
L
, et al. Two distinct interstitial macrophage populations coexist across tissues in specific subtissular niches
. Science
. 2019
;363
(6432
):eaau0964
.58.
Schyns
J
, Bai
Q
, Ruscitti
C
, et al. Non-classical tissue monocytes and two functionally distinct populations of interstitial macrophages populate the mouse lung
. Nat Commun
. 2019
;10
(1
):3964
.59.
Leach
SM
, Gibbings
SL
, Tewari
AD
, et al. Human and mouse transcriptome profiling identifies cross-species homology in pulmonary and lymph node mononuclear phagocytes
. Cell Rep
. 2020
;33
(5
):108337
.60.
Vanneste
D
, Bai
Q
, Hasan
S
, et al. MafB-restricted local monocyte proliferation precedes lung interstitial macrophage differentiation
. Nat Immunol
. 2023
;24
(5
):827
-840
.61.
Ural
BB
, Yeung
ST
, Damani-Yokota
P
, et al. Identification of a nerve-associated, lung-resident interstitial macrophage subset with distinct localization and immunoregulatory properties
. Sci Immunol
. 2020
;5
(45
):eaax8756
.62.
Fantom Consortium
. A promoter-level mammalian expression atlas
. Nature
. 2014
;507
(7493
):462
-470
.63.
Cheung
MD
, Erman
EN
, Moore
KH
, et al. Resident macrophage subpopulations occupy distinct microenvironments in the kidney
. JCI Insight
. 2022
;7
(20
):e161078
.64.
Sarkar
A
, Stephens
M
. Separating measurement and expression models clarifies confusion in single-cell RNA sequencing analysis
. Nat Genet
. 2021
;53
(6
):770
-777
.65.
Abadie
K
, Pease
NA
, Wither
MJ
, Kueh
HY
. Order by chance: origins and benefits of stochasticity in immune cell fate control
. Curr Opin Syst Biol
. 2019
;18
:95
-103
.66.
Hume
DA
. Probability in transcriptional regulation and its implications for leukocyte differentiation and inducible gene expression
. Blood
. 2000
;96
(7
):2323
-2328
.67.
Raj
A
, van Oudenaarden
A
. Nature, nurture, or chance: stochastic gene expression and its consequences
. Cell
. 2008
;135
(2
):216
-226
.68.
Helft
J
, Bottcher
J
, Chakravarty
P
, et al. GM-CSF mouse bone marrow cultures comprise a heterogeneous population of CD11c(+)MHCII(+) macrophages and dendritic cells
. Immunity
. 2015
;42
(6
):1197
-1211
.69.
Shalek
AK
, Satija
R
, Adiconis
X
, et al. Single-cell transcriptomics reveals bimodality in expression and splicing in immune cells
. Nature
. 2013
;498
(7453
):236
-240
.70.
Shalek
AK
, Satija
R
, Shuga
J
, et al. Single-cell RNA-seq reveals dynamic paracrine control of cellular variation
. Nature
. 2014
;510
(7505
):363
-369
.71.
Ravasi
T
, Wells
C
, Forest
A
, et al. Generation of diversity in the innate immune system: macrophage heterogeneity arises from gene-autonomous transcriptional probability of individual inducible genes
. J Immunol
. 2002
;168
(1
):44
-50
.72.
Ding
J
, Sharon
N
, Bar-Joseph
Z
. Temporal modelling using single-cell transcriptomics
. Nat Rev Genet
. 2022
;23
(6
):355
-368
.73.
Jenkins
SJ
, Allen
JE
. The expanding world of tissue-resident macrophages
. Eur J Immunol
. 2021
;51
(8
):1882
-1896
.74.
Ke
M
, Elshenawy
B
, Sheldon
H
, Arora
A
, Buffa
FM
. Single cell RNA-sequencing: a powerful yet still challenging technology to study cellular heterogeneity
. Bioessays
. 2022
;44
(11
):e2200084
.75.
Grabski
IN
, Street
K
, Irizarry
RA
. Significance analysis for clustering with single-cell RNA-sequencing data
. Nat Methods
. 2023
;20
(8
):1196
-1202
.76.
Xi
NM
, Li
JJ
. Benchmarking computational doublet-detection methods for single-cell RNA sequencing data
. Cell Syst
. 2021
;12
(2
):176
-194.e6
.77.
Freeman
TC
, Horsewell
S
, Patir
A
, et al. Graphia: a platform for the graph-based visualisation and analysis of high dimensional data
. PLoS Comput Biol
. 2022
;18
(7
):e1010310
.78.
Reshef
YA
, Rumker
L
, Kang
JB
, et al. Co-varying neighborhood analysis identifies cell populations associated with phenotypes of interest from single-cell transcriptomics
. Nat Biotechnol
. 2022
;40
(3
):355
-363
.79.
Van Hove
H
, Martens
L
, Scheyltjens
I
, et al. A single-cell atlas of mouse brain macrophages reveals unique transcriptional identities shaped by ontogeny and tissue environment
. Nat Neurosci
. 2019
;22
(6
):1021
-1035
.80.
Wu
YE
, Pan
L
, Zuo
Y
, Li
X
, Hong
W
. Detecting activated cell populations using single-cell RNA-seq
. Neuron
. 2017
;96
(2
):313
-329.e6
.81.
Koenitzer
JR
, Wu
H
, Atkinson
JJ
, Brody
SL
, Humphreys
BD
. Single-nucleus RNA-sequencing profiling of mouse lung. Reduced dissociation bias and improved rare cell-type detection compared with single-cell RNA sequencing
. Am J Respir Cell Mol Biol
. 2020
;63
(6
):739
-747
.82.
Stewart
TA
, Hughes
K
, Hume
DA
, Davis
FM
. Developmental stage-specific distribution of macrophages in mouse mammary gland
. Front Cell Dev Biol
. 2019
;7
:250
.83.
Sehgal
A
, Donaldson
DS
, Pridans
C
, Sauter
KA
, Hume
DA
, Mabbott
NA
. The role of CSF1R-dependent macrophages in control of the intestinal stem-cell niche
. Nat Commun
. 2018
;9
(1
):1272
.© 2023 by The American Society of Hematology
2023
You do not currently have access to this content.
This feature is available to Subscribers Only
Sign In or Create an Account Close Modal