Narasimhan 2019 Classical Non Classical Monocyte

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cls March 30, 2019 10:27
Annual Review of Immunology

Nonclassical Monocytes in
Health and Disease
Prakash Babu Narasimhan, Paola Marcovecchio,
Access provided by Stockholm University - Library on 04/27/19. For personal use only.
Anouk A.J. Hamers, and Catherine C. Hedrick

Annu. Rev. Immunol. 2019.37:439-456. Downloaded from www.annualreviews.org
Division of Inflammation Biology, La Jolla Institute for Allergy and Immunology, La Jolla,
California 92037, USA; email: prakash@lji.org, pmarco@lji.org, anouk@lji.org, hedrick@lji.org
Annu. Rev. Immunol. 2019. 37:439–56 Keywords

The Annual Review of Immunology is online at
monocytes, patrolling monocytes, hematopoiesis, autoimmunity,
immunol.annualreviews.org
atherosclerosis, rheumatoid arthritis, myocardial infarction
https://doi.org/10.1146/annurev-immunol-042617-
053119 Abstract
Copyright © 2019 by Annual Reviews.
Monocytes are innate blood cells that maintain vascular homeostasis and
All rights reserved
are early responders to pathogens in acute infections. There are three
well-characterized classes of monocytes: classical (CD14+ CD16− in hu-
mans and Ly6Chi in mice), intermediate (CD14+ CD16+ in humans and
Ly6C+ Treml4+ in mice), and nonclassical (CD14− CD16+ in humans and
Ly6Clo in mice). Classical monocytes are critical for the initial inflammatory
response. Classical monocytes can differentiate into macrophages in tissue
and can contribute to chronic disease. Nonclassical monocytes have been
widely viewed as anti-inflammatory, as they maintain vascular homeostasis.
They are a first line of defense in recognition and clearance of pathogens.
However, their roles in chronic disease are less clear. They have been shown
to be protective as well as positively associated with disease burden. This re-
view focuses on the state of the monocyte biology field and the functions of
monocytes, particularly nonclassical monocytes, in health and disease.
439
IY37CH18-Hedrick ARjats.cls March 30, 2019 10:27
HISTORY OF MONOCYTES
Monocytes have been reported in the literature for well over a hundred years. Exudate studies of
rabbits, rodents, and human tissue yielded evidence of phagocytic mononuclear cells capable of
taking up pathogens and debris (1, 2). Although peripheral blood monocytes were first observed
by Metchnikoff in 1905, they were simply grouped together with tissue macrophages and not
recognized as a distinct cell type until 1925 (3). The experiments published by van Furth and
Cohn in the 1960s and 1970s are often cited as the definitive papers on the mononuclear phagocyte
system (MPS) (4–6). However, this system of promonocytes in the bone marrow → monocytes
in circulation → macrophages in tissue has been rewritten over the last 50 years. In the 1970s
and early 1980s, based on their wide variation in functional activity and Fc receptor reactivity,
the possibility of functionally distinct monocyte subpopulations was first postulated (7–10). Later
work revealed that human monocytes are indeed heterogeneous and comprise at least two distinct
subsets of mononuclear phagocytes (11–15). A more detailed and accurate picture of monocyte
development has been achieved in the last two decades with the development of sophisticated
mouse models, confocal microscopy, RNA and transcriptome sequencing, and high-dimensional
analysis of cell surface and intracellular proteins.
MONOCYTE HETEROGENEITY
As early as 1989, with the use of monoclonal antibodies and two-color flow cytometry,
three distinct human monocyte subsets could be identified based on the expression of CD14
and CD16 surface antigens: the classical CD14+ CD16− , intermediate CD14+ CD16+ , and
nonclassical CD14− CD16+ subsets (16). The first evidence of monocyte subsets in mice
was based on a CX3 CR1-promoter-driven GFP transgene (17). This study identified a
F4/80+ CD11b+ CD62L+ CCR2+ CX3 CR1mid and a F4/80+ CD11b+ CD62L− CCR2− CX3 CR1hi
monocyte subset. Subsequent reports confirmed these findings and showed that these two subsets
had different phenotypic and functional properties (18, 19). Antigen expression patterns and ge-
nomic data suggest that the human equivalents of these inflammatory Ly6Chi CX3 CR1mid CCR2+
and patrolling Ly6Clo CX3 CR1hi CCR2− mouse subsets are the classical CD14+ CD16− and the
nonclassical CD14− CD16+ populations, respectively (18, 20, 21).
In mouse, gene expression profiling suggests that Treml4, a member of the triggering receptor
expressed on myeloid cells, can be used to distinguish mouse intermediate monocytes (22). This
study shows that Ly6Chi/mid Treml4+ monocytes are an intermediate stage of differentiation
between Ly6Chi Treml4− and Ly6Clo monocytes and that Treml4 expression increases along
with Nr4a1. Differential PU.1 expression has been found within the Ly6Chi CD115+ population,
leading to a spectrum of heterogeneous classical monocytes in the bone marrow and blood
compartments (23). Segregation of populations based on CD11c, MHC-II, Ly6C, CD115, Flt3,
and Sirpα distinguishes the ability of classical monocytes to differentiate into either inflamma-
tory macrophages or CD209a+ dendritic cells (DCs), depending on the contextual cues of the
microenvironment.
In healthy human donors, a CD2+ FcεR1+ classical monocyte subset was identified that can
potentially influence the generation of allergic inflammation via IgE clearance (8, 24, 25). These
monocytes occupy less than 5% of total peripheral blood monocytes, and their numbers are in-
creased in patients with severe allergies (26). Interestingly, the magnitude of FcεR1 expression on
the surface of these CD2+ monocytes can be correlated to serum IgE levels (25, 27). Considering
that CD2+ monocytes rapidly obtain DC-like features in culture (28, 29) and that peripheral DCs
also express FcεR1 (30, 31), it is conceivable that this subset differentiates to DCs in the periph-
eral tissues. Recently, Villani et al. (32) also identified this CD2+ monocyte as a specific subset by
440 Narasimhan et al.
single-cell RNA sequencing. Even though rodents do not express CD2 or any of the IgE receptors
in monocytes, some Fcγ receptors, including FcγRII, -III, and -IV, can actually bind IgE in mice,
indicating a possible role for IgG receptors in clearing IgE in mice (33, 34).
Cytometry by time-of-flight (CyTOF or mass cytometry), t-distributed stochastic neighbor
embedding (t-SNE), and clustering algorithms have proven to be extremely useful tools for in-
vestigating the heterogeneity of monocytes. CyTOF has enabled immunologists to define pop-
ulations using more than 40 markers, while t-SNE and unbiased clustering allow viewing of
the high-dimensional datasets generated by CyTOF in order to finely segregate populations of
cells based on marker expression. Using these techniques, Thomas et al. (35) demonstrated a re-
fined gating strategy to obtain more phenotypically pure populations of human monocytes by
incorporating CD36, HLA-DR, CD11c, and CCR2 to improve the CD14/CD16 subset defini-
tion. These newly defined gates for classical/intermediate/nonclassical monocytes can now be de-
fined as CD14+ CD16− HLA-DRlo CD11clo , CD14+ CD16+ CD36hi CCR2hi HLA-DRhi CD11chi ,
and CD14− CD16+ CD36lo CCR2lo , respectively.

MONOCYTE DEVELOPMENT AND DIFFERENTIATION

Transcriptional Control of Monocyte Lineage
The lineage and differentiation of monocytes is a major focus of immune research. The ability to
identify and isolate progenitor cells that give rise to unique and distinct subsets via flow cytome-
try allows for more refined immunological questions about development and disease. Monocyte
differentiation from hematopoietic stem cells takes place in the bone marrow through a step-
wise process of progressively more committed progenitors until they become one of two major
subsets: classical or nonclassical (36–38). Both of these subsets, as well as their progenitors, rely
on colony-stimulating factor 1 (Csf-1) for survival (39). The human counterpart of the previ-
ously identified mouse common monocyte progenitor (cMoP), which gives rise to Ly6Chi and
possibly Ly6Clo monocytes, was recently found using CLEC12A and CD64 markers (40). This
multipotent progenitor differentiates into both CD14+ and CD16+ monocytes. However, these
human cMoPs also express CD135, unlike mouse cMoPs. Functional assays showed that these
cells make for poor antigen-presenting cells (APCs), secrete inflammatory cytokines in response
to lipopolysaccharide and interferon stimulation, and can phagocytose dextran particles equal to
peripheral blood monocytes.
IRF8 and KLF4 transcription factor interactions are crucial for classical (Ly6C+ CCR2+ )
monocyte development from the cMoP as well as to prevent neutrophil differentiation from the
monocyte-DC progenitor (MDP) (41, 42), but they are dispensable for maintenance in the pe-
riphery (43). Further downstream, the nuclear receptor Nr4a1 has been shown to control the dif-
ferentiation of nonclassical (Ly6Clo CCR2− ) monocytes in the periphery and bone marrow (44).
These studies in mice and human tissue begin to illuminate the heterogeneity of myeloid progen-
itors and the intricate transcriptional programming necessary to generate a myriad of monocyte
subsets.
There is now substantial evidence that Ly6Clo monocytes arise from Ly6Chi monocytes in
the periphery (45–48). Recently, however, Satoh et al. (49) demonstrated that a separate Ly6Clo
monocyte subset precursor, SMP (SatM precursor), arises from granulocyte-macrophage progen-
itors in the bone marrow and differentiates into segregated-nucleus-containing atypical mono-
cytes (SatM) by the transcription factor C/EBPβ. These Ceacam1+ Msr1+ Ly6C− F4/80− Mac1+
monocytes, which were found in the lung, are prominent during the fibrotic phase of bleomycin-
treated mice and contributed directly to the initiation of fibrosis through increased levels of
www.annualreviews.org • Nonclassical Monocytes 441

TGF-β and Spp1. What is particularly interesting about this finding is that Ceacam1 is expressed
on these cells, since it is typically a marker associated with granulocytes. Mildner et al. (50) picked
up where Satoh et al. left off, by examining the peripheral blood monocyte subsets in C/EBPβ-
deficient mice. While MDP and cMoP cells in the bone marrow are relatively unaffected, more
mature monocyte subsets were drastically reduced upon ablation of C/EBPβ, with the greatest
effect seen in the Ly6Clo nonclassical subset, suggesting an interaction with Nr4a1. The authors
showed that while C/EBPβ could bind to a part of the Nr4a1 promoter region, there is still the
possibility of independent as well as dependent interactions between the two transcription factors
for control over Ly6Clo differentiation, with possible overlap by other C/EBP members. Lastly,
PU.1 transcription factor levels have been shown to dynamically regulate conversion of Ly6Chi
monocytes to highly varied myeloid effector subsets (23). With the clever use of mouse models,
researchers showed a dose-dependent effect of PU.1 on monocyte differentiation into inflamma-
tory inducible nitric oxide synthase–producing macrophages, or a more DC-like PD-L2+ 209a+
subset, upon stimulation with lipopolysaccharide or GM-CSF (23).

Gene regulation of nonclassical monocytes has expanded beyond transcription factor binding.
An enhancer region upstream of the Nr4a1 gene has been shown to be critical to induce Nr4a1
expression through KLF2 binding in order to generate Ly6Clo monocytes (51). Deletion of the E2
superenhancer region upstream of Nr4a1 proved essential for maintaining Ly6C− MHCII− sub-
sets specifically without affecting macrophage polarization or the lymphoid compartment. This
region is also found upstream of human CD16+ monocytes, showing evolutionary conservation
between mouse and human. The exact specificity of this deletion to generate a Ly6Clo monocyte
mouse model will prove useful to dissect the exact role of nonclassical monocytes in disease states.
Fate, Longevity, and Survival in the Periphery

Thus far it has been shown that monocytes exist as two major subsets in bone marrow that egress
into the periphery through the vasculature. For classical monocytes, CCR2 is the crucial receptor
responsible for their migration out of bone marrow following the binding of CCL2 (52). For non-
classical monocytes, the receptor necessary for their migration out of the bone marrow appears to
be an S1P receptor, S1PR5 (53). S1PR5−/− mice lacked Ly6Clo monocytes in the periphery but
maintained normal numbers in bone marrow. Interestingly, S1P disruption did not alter migration
of Ly6Clo monocytes, nor did it decrease Ly6Clo survival in the periphery, suggesting that S1PR5
acts through an unidentified ligand. Although both monocyte subsets express the fractalkine re-
ceptor (CX3 CR1), it appears that nonclassical monocytes particularly depend on CX3 CL1 while
in circulation for homeostasis and survival (54–56).
As mentioned before, there is a paradigm that nonclassical monocytes originate from Ly6Chi
monocytes in the circulation, or return to the bone marrow to differentiate into Ly6Clo mono-
cytes (45, 48, 57). New evidence has emerged about the survival of monocytes once they egress
out of the bone marrow. Within vascular niches of mouse bone marrow and the marginal zone
of spleen, Ly6Chi monocytes bind to endothelial cell ligand DLL1 via the Notch2 receptor and
induce conversion to Ly6Clo monocytes (58). Adoptive transfers of Notch2−/− Ly6Chi monocytes
show reduced conversion to Ly6Clo monocytes, while in vitro coculture of Ly6Chi monocytes with
DLL1 ligand shows increased conversion to Ly6Clo monocytes. However, it remains to be seen
if this Notch2-DLL1 axis can be manipulated in vivo to yield higher levels of Ly6Clo monocytes;
this would present a beneficial tool for studying effects of Ly6Clo monocyte populations in various
diseases. Additionally, conversion of classical monocytes to Ly6Clo patrolling monocytes during
bacterial infections can be induced by NOD2 receptors (59). These receptors bind to fragments
of bacterial peptidoglycans, which can then increase intermediate and nonclassical monocyte

populations for both human and mouse monocyte cultures, although the mechanism is not clear.
When thioglycolate was used to induce an inflammatory response, activation of NOD2 receptors
through binding of muramyl dipeptide (MDP) increased the nonclassical monocyte population
and decreased markers of inflammation such as IL-6 and tumor necrosis factor alpha (TNF-α).
Most interesting was the ability of MDP to elicit an increase in Ly6Clo monocytes in Nr4a1−/−
mice, suggesting either that the Ly6Clo monocyte population is genetically diverse, or that an-
other key transcription factor is involved with nonclassical monocyte differentiation. This work
demonstrates a potential in vivo mechanism for mouse and human monocyte subset regulation.
It has been shown in both human and mouse that monocytes require fractalkine (CX3 CL1)
for survival through binding of its cognate receptor CX3 CR1 on monocytes by inhibiting apop-
totic pathways (56, 60). More recently, TNF has been shown to play a crucial role in regulating
monocyte survival and function in the periphery. In an adoptive transfer with wild-type and TNF
receptor 1 or 2 (TNFR1 or TNFR2) knockout monocytes, only monocytes with TNF receptors
showed an ability to persist in the periphery by signaling through autonomous TNF production
(61). By two days after transfer, these deficient monocytes had increased levels of apoptosis as
measured by annexin V and propidium iodide staining, with little effect on monocyte progenitors.
Investigating human monocytes in vivo presents a significant challenge that often leads to ex
vivo or in vitro experiments. However, in an elegant study by Patel et al. (45), the kinetics of pe-
ripheral human monocytes in steady state and during inflammation were quantified using in vivo
deuterium glucose labeling of human subjects. All three monocyte subsets, classical (CD14+ ), in-
termediate (CD14+ CD16+ ), and nonclassical (CD16+ ), displayed similar generation kinetics to
those observed in rodent studies: bone marrow → classical monocytes → intermediate mono-
cytes → nonclassical monocytes, with a postmitotic bone marrow residence of ∼1.6 days and
circulation for 1 day (classical), 3 days (intermediate), and 7 days (nonclassical). When challenged
with endotoxemia, human monocytes were depleted from circulation and repopulated by classi-
cal monocytes released from the bone marrow, thus recapitulating mouse studies and establishing
kinetic generation of monocytes into the periphery.
In the last 18 years, it has become increasingly clear that monocytes consist of two major sub-
sets and can survey tissue during homeostasis as well as respond to infection, inflammation, and
cancer (19, 62–65). Both subsets possess unique effector functions and differentiation capacity to
replenish macrophages and DC populations in certain tissues such as lung, gut, heart, spleen, and
skin (45, 46, 66–69). Depending on the context, i.e., infection, autoimmunity, wound healing, etc.,
each subset has been reported to have inflammatory or anti-inflammatory properties, making their
programming malleable to the microenvironment and type of stimulus (21, 70–74). The discus-
sion of what monocytes do after receiving a signal depends heavily on the type of ligand:receptor
interaction, the tissue involved, and the experimental conditions used to assess the outcome. It has
become apparent that mature monocytes are a medley of poised effector cells that are tightly con-
trolled by transcriptional programming to exert their function given the appropriate signal. While
the identification of monocytic progenitors has funneled into more precise and narrow definitions
of what cell type gives rise to peripheral monocytes, the heterogeneous phenotypes and functions
of circulating monocytes have broadened well beyond simply being macrophage precursors.
MONOCYTES IN HEALTH, INFLAMMATION, AND AUTOIMMUNITY

Homeostasis
Monocytes are mononuclear phagocytes that serve a key role in regulation of inflammation (75).
Though monocytes share some common features, circulating monocytes are heterogeneous, as

mentioned above, and have different abilities to produce cytokines (75, 76). Distinct functions
are attributed to the different subsets of monocytes. During bacterial infection, classical mono-
cytes are recruited to sites of inflammation, recognize and phagocytose pathogens, secrete various
proinflammatory cytokines, and recruit other immune cells for regulation of the inflammatory
response (75–77). Nonclassical monocytes display a distinct motility and crawling pattern along
the vasculature and hence have acquired the nickname patrolling monocytes (18, 19, 21, 78). Pa-
trolling monocytes survey the luminal side of vascular endothelium in an LFA-1- and integrin-
α4 -dependent manner (19). Studies by Geissmann and colleagues have shown that nonclassical
monocytes recognize and clear dying endothelial cells in a TLR7-dependent manner to main-
tain vascular homeostasis (78, 79). Thus, during homeostasis, nonclassical monocytes appear to be
sentinels and caretakers of the vascular tissue.
Although classical monocytes have been shown to differentiate into macrophages, it is unclear
whether nonclassical monocytes become bona fide macrophages. There have been several reports
that nonclassical monocytes can differentiate into alternatively activated types of macrophages (74,
80, 81). However, mice lacking nonclassical monocytes show normal numbers of macrophages in
most tissues. We only observed a lack of a specialized thymic macrophage, whose function was to
clear apoptotic thymocytes, during T cell negative selection in Nr4a1−/− mice that lacked non-
classical monocytes (44). These data suggest that, at least at homeostasis, nonclassical monocytes
do not readily differentiate into macrophages (44).
Though nonclassical patrolling monocytes seem to be involved primarily in promoting the
resolution of inflammation, studies have shown that this monocyte subset can also contribute to
the pathogenesis of disease. The roles of monocytes in regulating autoimmune and inflammatory
diseases are discussed below. Important points are shown in Figure 1.
Rheumatoid Arthritis
Rheumatoid arthritis (RA) is a complex autoimmune disease characterized by persistent inflam-
mation of the synovium and local destruction of bone and cartilage, leading to deformation of
joints and impaired movement (82). Most therapeutic interventions center on monocytes and
macrophages, as they play a major role in the pathophysiology of RA (83, 84).
Nonclassical monocytes have been implicated in the initiation and progression of arthritis (81).
Researchers have used the serum transfer–induced arthritis (STIA) mouse, a model of human
RA (85, 86). In STIA mice treated with clodronate-loaded liposomes to deplete monocytes and
macrophages, adoptive transfer of Ly6Chi and Ly6Clo monocytes immediately after initiation of
arthritis showed that Ly6Clo monocytes increased the development of arthritis, whereas Ly6Chi
monocytes exhibited a marked delay in development (81). These investigators reported that
Ly6Clo monocytes entered the arthritic joint and developed into MHCII− synovial macrophages
(81), which proved to be necessary for the initiation and propagation phase of RA (81). However,
these same investigators reported that these synovial macrophages were resident macrophages,
suggesting that they may not have originated from Ly6Clo monocytes. In further support of a role
for Ly6Clo monocytes in tissue destruction, Puchner et al. (87) have shown that levels of nonclas-
sical monocytes in the blood of STIA mice positively correlate with markers of joint destruction.
However, in a contradictory study using the STIA model, Brunet et al. (88) demonstrated that
patrolling monocytes are not required for the initiation and progression of arthritis and instead
contribute to reducing joint inflammation. Using cytosporone (Csn-B), an agonist for Nr4a1, the
authors showed that Ly6Clo monocytes enhance regulatory T cell mobilization and activity, pos-
sibly through production of CXCL12 and TGF-β, thereby controlling excessive inflammation in
arthritic mice (88).

Atherosclerosis Multiple sclerosis

Increased patrolling during vascular Nr4a1 is important for controlling
inflammation inflammatory response by
macrophages in EAE
Sensing of pathogenic lipid
molecules by Ly6Clo monocytes CD16+ frequency positively correlates
with MS-causal or compensatory?
Minimal entry into the plaque itself
Less severe plaque formation and
endothelial cell damage correlate
with Ly6Clo patrolling Rheumatoid arthritis
Ly6Clo monocytes develop into
initiating MHCII– macrophages in the
Ischemic reperfusion injury synovium
Ly6Clo monocytes decreased renal Ly6Clo monocytes recruit Tregs and
injury by suppressing ICAM-1 help suppressive activity through
expression on kidney vascular CXCL12 and TGF-β
endothelium during IRI
CD16+ blood monocytes migrate into

Ly6Clo monocytes dominate reparative
synovial tissue (intermediate

stages of myocardial IRI monocytes?) in RA patients
Increased angiogenesis and
myofibroblast accumulation by Ly6Clo
monocytes
Development and fate Systemic lupus erythematosus
Requires S1PR5 to egress from bone Nonclassical monocytes patrol uninflamed
marrow glomeruli
Regulated by Nr4a1, C/EBPβ, and KLF2 During LN, patients have higher CD16+
binding upstream to enhancer region monocyte counts in glomeruli and
of Nr4a1 decreased frequency in peripheral blood
Both humans and mice show long SLE patients have enriched frequencies of
residence times of nonclassical CD16+ blood monocytes, which positively
monocytes in the blood correlate with Th17 differentiation and IgG
production
Conversion of Ly6Chi to Ly6Clo
monocytes mediated by Notch2 and
NOD2 binding to ligands in the
vasculature
Initiates/propagates disease
Resolves/prevents disease
Homeostasis
Figure 1
Nonclassical monocyte functions in health and disease. The development of nonclassical monocytes in bone marrow relies on the
transcription factor Nr4a1. Nonclassical monocytes have high expression of S1P5 and require S1P5 to egress from bone marrow into
circulation. In circulation, nonclassical monocytes play critical roles in maintaining vascular homeostasis. Although nonclassical
monocytes are primarily involved in promoting the resolution of inflammation, in a few cases they may actually contribute to disease
progression. An overview of the known functions of nonclassical monocytes in several important autoimmune and inflammatory
diseases is shown here. Abbreviations: EAE, experimental autoimmune encephalomyelitis; IRI, ischemia reperfusion injury; LN, lupus
nephritis; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus.
Multiple studies in humans have shown that circulating monocytes are activated in RA (89–91).
Kawanaka et al. (89) showed increased migration of CD16+ monocytes from peripheral blood to
synovial tissue in RA patients. However, the authors classified both intermediate and nonclassical
monocytes as CD16+ monocytes, which makes the results of this study difficult to interpret. More
recent studies have examined frequencies of monocyte subsets in peripheral blood of patients
with RA (92, 93) and showed increased frequencies of nonclassical monocytes. However, more
mechanistic studies are needed to determine possible functional roles of nonclassical monocytes
in human RA.

Multiple Sclerosis
Multiple sclerosis (MS) is a chronic autoimmune and demyelinating disease of the central nervous
system (CNS) characterized by inflammatory and degenerative changes in the brain and spinal
cord (94). Monocytes, macrophages, and microglia play a major role in CNS inflammation in MS.
Several studies have shown that monocytes secrete inflammatory cytokines that contribute to the
increased pathogenesis of MS (95, 96).
Experimental autoimmune encephalomyelitis (EAE) is a clinically relevant murine model of
MS, as many of the pathologies observed in animal models have strong similarities to those found
in human MS patients (97, 98). Using these models, several studies have shown that monocyte-
derived macrophages are critical for the development of MS (99–101). Studies in mice have
shown that the most potential contributor to MS lesions and CNS inflammation are inflammatory
Ly6Chi monocytes (65, 102–105). Increased expression of CCL2 (106) concomitant with recruited
CCR2+ Ly6Chi inflammatory monocytes is critical for initiation and progression of EAE (104).
Blocking CCR2-dependent Ly6Chi inflammatory monocyte entry across the blood-brain barrier
prevents EAE disease progression (99). In support of a protective role for nonclassical monocytes
in EAE, Shaked et al. (101) demonstrated much earlier onset and exacerbated disease development
of induced EAE in Nr4a1−/− mice that lacked nonclassical monocytes.
In humans with MS, Waschbisch et al. (107) reported a reduction in frequency of nonclassi-
cal monocytes and increased infiltration of intermediate CD14+ CD16+ monocytes to the cere-
brospinal fluid. Gjelstrup et al. (108) showed that in both treated and untreated MS patients,
there is a significant expansion of CD16+ nonclassical monocytes with a concomitant reduction
in CD14+ classical monocytes compared with healthy individuals. Although the mouse studies
suggest a protective role for nonclassical monocytes in MS, whether the increased frequencies
of nonclassical monocytes that are observed in humans with MS are compensatory or causal in
disease remains unclear.
Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is another chronic inflammatory autoimmune disease with
profound effects on multiple organs. One of the prominent features associated with SLE is the
dysfunction and altered homeostasis of monocytes (109). Monocytes play an important role in
alterations of immune complex recognition and clearance by expressing a receptor specific for
the FC region of IgG (FcγR) (110). Monocytes from SLE patients had reduced expression of
FcγRII and FcγRIII receptors, resulting in defective phagocytic clearance of apoptotic cell debris
in the bloodstream (111). Lupus nephritis (LN) is the major cause of morbidity in patients with
SLE. Accumulation of nonclassical monocytes to the glomerular vessels in the kidneys of lupus
patients has been documented (112). Recently, in a model of in situ immune complex–mediated
glomerulonephritis, Finsterbusch et al. (113) showed that nonclassical monocytes patrol unin-
flamed glomeruli and are involved in maintaining glomerular homeostasis. In support of this,
Barrera García et al. (114) showed that patients with severe forms of LN with a higher degree of
infiltrates of CD16+ cells in the glomerulus had lower levels of nonclassical monocytes in periph-
eral blood, suggesting that the nonclassical monocytes were recruited to renal tissues (114). These
studies enumerate that the nonclassical monocytes patrol the lumen of glomerular vessels in the
kidney and increase their infiltration of kidney tissues concomitant with disease severity.
In a recent study of 62 SLE patients, Zhu et al. (115) reported that nonclassical CD16+ mono-
cytes were enriched in the blood and positively correlated with anti-dsDNA antibody levels, which
suggests that patients with elevated frequencies of CD16+ monocytes would be at higher risk of

autoantibody development. Another study also showed the expansion of the CD16+ population
in SLE patients, by using a combination of flow cytometry and a high-dimensional automated
clustering algorithm (72). Moreover, CD16+ monocytes were shown to promote a Th17 pheno-
type in SLE patients (115). However, this latter study grouped all CD16+ monocytes together
in cocultures with T cells, which in this case would contain both intermediate and nonclassical
monocytes. Because a distinction was not made between these two subsets, it is possible some of
the functions associated with intermediate monocytes were attributed to nonclassical monocytes.
However, more mechanistic studies are needed to identify whether nonclassical monocytes are
involved in directly mediating tissue injury in SLE-mediated nephritis.
Atherosclerosis
Atherosclerosis is a chronic inflammatory and immune disease that is the underlying cause of car-
diovascular disease and cardiovascular mortality. Monocytes and macrophages are the important
and dominant innate immune cells associated with atherosclerotic plaques (65, 116, 117). Non-
classical and classical monocytes are both recruited to the atherosclerotic aorta, but via distinct
chemokine receptor pathways. Nonclassical monocytes that are Ly6Clo remain longer in circula-
tion and enter atherosclerotic plaques much less efficiently than do classical monocytes, and their
recruitment relies, at least in part, on CCR5 (118).
In animal models of atherosclerosis, several studies have shown that adhesion of monocytes to
the aortic endothelium wall is an initial, critical stage of early atherosclerosis development (116,
119). Circulating classical monocytes in mice are the major monocyte subset that migrates into
the aortic wall and is involved in the progression of atherosclerosis (120, 121). These classical
monocytes readily differentiate into macrophages within the subendothelial space of the aortic
wall and contribute to atherogenesis.
Nonclassical monocytes are also recruited to the aortic plaque but remain within the blood
vessel compartment. For the first time it was reported that, through intravital live cell imaging sys-
tem (ILTIS), CX3 CR1gfp/+ patrolling monocytes could be observed crawling in large blood vessels
with plaque formation (122). In a follow-up study, Quintar et al. (79) showed a significant increase
in numbers of patrolling monocytes crawling along the lumen of murine atherosclerotic arteries
compared to the arteries of mice that were fed a chow diet. These authors reported that Western
diet–fed Nr4a1/ApoE−/− mice that lacked nonclassical monocytes had increased apoptotic en-
dothelial damage in the vessel wall, which confirms the protective role of patrolling monocytes in
maintaining vascular homeostasis (78). This increase in patrolling by nonclassical monocytes in
mice fed a Western diet is not observed in plaque-forming vessels but was observed in large blood
vessels distal to the aortic arch early in the development of atherosclerosis (123). This increase
in patrolling seems to be mediated by uptake of OxLDL primarily through scavenger receptor
CD36.
Several clinical studies have enumerated the importance of monocytes in modulating coronary
artery disease (116). A growing body of evidence demonstrates a significant role of CD16+ mono-
cytes in the development of atherosclerosis in humans (124–128). However, most of these studies
have grouped and studied intermediate and nonclassical monocytes together as CD16+ mono-
cytes, so it is difficult to tease apart the role of nonclassical monocytes here. However, recently
Urbanski et al. (129) showed that in coronary artery disease patients, high levels of nonclassical
monocytes in blood are associated with more advanced vascular dysfunction and vascular oxidative
stress.
To understand a direct role for nonclassical monocytes in atherosclerosis, several groups have
studied Western diet–fed Nr4a1−/− mice that lacked nonclassical monocytes. Our laboratory

and that of DeVries have both shown increased atherosclerosis development in the absence of
Nr4a1 and nonclassical monocytes. This increase in atherosclerosis was associated with increased
macrophage accumulation in the plaque, increased SDF-1a, and proinflammatory cytokine pro-
duction (130, 131). This increase in atherosclerosis in Western diet–fed mice in which Nr4a1 ex-
pression was modulated was also confirmed by Wang and colleagues (132). However, some studies
have reported the opposite finding—that there is a reduction in atherosclerosis in mice lacking
nonclassical monocytes, suggesting that nonclassical monocytes are proinflammatory (133, 134).
The reasons for these conflicting reports are unclear but may involve the type of high-cholesterol
diet, the length of time the diet was fed, and the mouse model used. A second mouse model
specifically lacking nonclassical monocytes while maintaining normal macrophage phenotypes
confirmed that mice develop larger plaques when nonclassical monocytes are absent (51, 123).
Thus although this is still somewhat controversial, the bulk of these in vivo studies collectively
show that at least in mice, nonclassical monocytes are atheroprotective. Key studies in humans are
needed to confirm this.
Ischemia-Reperfusion Injury
Ischemia reperfusion injury (IRI) is characterized by initial restriction of blood and oxygen supply
to an area or organ, followed by sudden perfusion of blood and concomitant reoxygenation (135).
Several studies have shown that IRI is mediated by infiltration of inflammatory cells (136). Mono-
cyte recruitment is involved in the pathogenesis of IRI (137). Though several organ and tissues
are affected by IRI, only the role of monocytes in renal and myocardial IRI is reviewed here.
Acute kidney injury is a significant clinical problem in both native and transplanted kidneys
(138, 139) and involves infiltration of monocytes and macrophages to kidney (139, 140). Li et al.
(138) have shown that monocytes migrate from blood to kidneys and promote early tubular in-
jury. In a mouse model of acute kidney injury, depletion of CD169+ monocytes and macrophages
increases progressive renal injury by upregulating the proinflammatory cytokines in the kidney.
Adoptive transfer of Ly6Clo monocytes into CD169–diphtheria toxin receptor (DTR) mice de-
creased renal injury by suppressing ICAM-1 expression on kidney vascular endothelium, suggest-
ing that these Ly6Clo monocytes resolved inflammation in kidney IRI (141).
Myocardial infarction (MI) associated with reperfusion injury is the most common form of
acute cardiac injury and results in the ischemic death of cardiomyocytes (142). Current research
indicates that human monocyte subset dynamics following MI hold predictive value for cardio-
vascular events and patient outcome (143).
In mice, chemokine expression in the infarcted heart results in active recruitment of both
Ly6Chi and Ly6Clo monocytes via CCR2 and CX3CR1, respectively (144). Nahrendorf et al. (144)
showed that in the early inflammatory phase of myocardial ischemic injury, Ly6Chi monocytes are
recruited actively, peak on day 3, and play a key role in phagocytosis and inflammation, whereas
Ly6Clo monocytes dominate in a later reparative phase, which peaks at day 7. The Ly6Clo mono-
cytes are involved in the resolution and reparative process by promoting myofibroblast accumu-
lation and angiogenesis (144, 145). In another study, Nakano et al. (146) showed recruitment of
inflammatory Ly6Chi monocytes at 3–24 h after MI, followed by Ly6Clo monocytes at 24–48 h af-
ter IRI. Both studies report the appearance of Ly6Clo monocytes at later stages after MI, although
the timeframes studied were slightly different. Such studies clearly report the proangiogenic and
anti-inflammatory properties of nonclassical monocytes in MI (144). To further address the im-
portance of patrolling monocytes in MI, Hilgendorf and colleagues (145) demonstrated that in
MI inflammatory response is heightened and healing is compromised in Nr4a1-deficient mice
that lack nonclassical monocytes, further enumerating the role of patrolling monocytes in the

healing process. Combined, these data on both kidney and heart IRI models generally demon-
strate a beneficial effect of nonclassical monocytes in vascular repair and restoring organ function.
FUTURE PERSPECTIVES
In sum, monocytes are key early innate immune cells that play important roles in both homeostasis
and disease. Monocytes are involved in maintenance of vascular homeostasis and are one of the
earliest responder cell types to pathogens that lead to acute infections. However, as helpful as they
are, monocytes can also contribute to inflammation that is associated with chronic diseases.
The role of nonclassical monocytes has been widely viewed as protective, as they play a critical
role in maintaining vascular endothelial homeostasis. They are also a first line of defense in recog-
nition and clearance of pathogens and even cancer cells. However, their roles in chronic disease
are less clear. Although they appear to be atheroprotective and protective in IRI, in many studies
they have been shown either to be associated with or to contribute to disease burden.
In the last several years, new high-dimensional methodologies to study monocytes have shown
their heterogeneity. Nonclassical monocyte heterogeneity in particular will be important to shed
light on the sometimes conflicting Jekyll-versus-Hyde functions of this monocyte subset in disease.
Ongoing studies identifying the heterogeneity and functions of monocyte subsets in both home-
ostasis and disease will allow for new and better therapeutic approaches using selectively targeted
monocyte populations in contrast to current therapies that target all monocytes as a whole. As we
refine our understanding of how nonclassical monocytes differ in their response to disease onset,
we can potentially harness their altruistic behavior toward preventing and treating a variety of
chronic human illnesses.
DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
This work was supported by the National Institutes of Health: NIH F31 HL132538 (to P.M.M.)
and NIH R01 HL134236, HL118765, P01 HL055798, and P01 HL136275 (all to C.C.H.).
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Annual Review of Immunology

Anouk A.J. Hamers, and Catherine C. Hedrick

Annu. Rev. Immunol. 2019. 37:439–56 Keywords

and CD14− CD16+ CD36lo CCR2lo , respectively.

MONOCYTE DEVELOPMENT AND DIFFERENTIATION

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subset, upon stimulation with lipopolysaccharide or GM-CSF (23).

Fate, Longevity, and Survival in the Periphery

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MONOCYTES IN HEALTH, INFLAMMATION, AND AUTOIMMUNITY

www.annualreviews.org • Nonclassical Monocytes 443

444 Narasimhan et al.

Atherosclerosis Multiple sclerosis

CD16+ blood monocytes migrate into

synovial tissue (intermediate

www.annualreviews.org • Nonclassical Monocytes 445

Systemic Lupus Erythematosus

446 Narasimhan et al.

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needed to confirm this.

448 Narasimhan et al.

www.annualreviews.org • Nonclassical Monocytes 449

450 Narasimhan et al.

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gression and metastasis. Cancer Sci. 96(6):317–22

452 Narasimhan et al.

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couples sympathetic and inflammatory cues in CNS-recruited macrophages to limit neuroinflammation.

454 Narasimhan et al.

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with 2-year cardiovascular events. Medicine 95(18):e3466

456 Narasimhan et al.

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