Cellular Immunology xxx (xxxx) xxx–xxx

Contents lists available at ScienceDirect

Cellular Immunology
journal homepage: www.elsevier.com/locate/ycimm

Research paper

Alveolar Macrophages
⁎
Nikita Joshi, James M. Walter, Alexander V. Misharin
Division of Pulmonary and Critical Care Medicine, Northwestern University, Chicago, IL, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Alveolar macrophages are the most abundant innate immune cells in the distal lung parenchyma, located on the
Alveolar macrophages luminal surface of the alveolar space. They are the first to encounter incoming pathogens and pollutants and to
Macrophages help orchestrate the initiation and resolution of the immune response in the lung. Similar to other tissue-resident
macrophages, alveolar macrophages also perform non-immune, tissue-specific, homeostatic functions, most
notably clearance of surfactant. In this review we will discuss how ontogeny and local lung environment shape
the role of alveolar macrophages in health and disease.

1. Introduction
Alveolar macrophages are the most abundant innate immune cells
in the distal lung, located on the luminal surface of the alveolar space.
They are the first to encounter incoming pathogens and pollutants and
help orchestrate the initiation and resolution of the immune response in
the lung. Similar to other tissue-resident macrophages, alveolar mac-
rophages also perform non-immune, tissue-specific, homeostatic func-
tions, most notably clearance of surfactant. The immune and homeo-
static functions of alveolar macrophages have been recently reviewed
[1–4]. Considerable debate has surrounded the origin of these cells:
while some studies indicated that alveolar macrophages were capable
of proliferation, others suggested that circulating monocytes help
maintain the alveolar macrophage pool. Recent scientific and technical
advances have substantially improved our understanding of alveolar
macrophage ontogeny. Here we will discuss how ontogeny and the lung
microenvironment shape the role of alveolar macrophages in health and
disease.
2. Identification and tracking of alveolar macrophages in the
laboratory
Exposure to high partial oxygen pressure, surfactant and signals
provided by alveolar type I and II cells produce a distinct alveolar
macrophage phenotype which can be identified using multiparameter
flow cytometry. The phenotype of mouse alveolar macrophages is well-
described: similar to macrophages in other tissues they express MerTK,
CD64, CD68, CD206 and F4/80. However, because of their adaptation
to the unique lung environment, steady state mouse alveolar macro-
phages do not express fractalkine receptor CX3CR1 or integrin CD11b
[5–8]. Instead, they express high levels of integrin CD11c, and sialic
acid-binding lectin Siglec F [5–12]. These markers allow separation of
true long-living alveolar macrophages (i.e. cells that have adapted to
the local microenvironment) from transient monocyte-derived cells that
were recruited to the alveolar space during injury [7,10,13–16].
Normal human alveolar macrophages express CD206, CD169, CD11c,
CD163 and MARCO [17–21] which are also expressed by alveolar
macrophages in non-human primates [22]. Alveolar macrophages are
known to be highly autofluorescent. Although high autofluorescence
may complicate flow cytometric analysis of alveolar macrophages,
making them to appear falsely positive for a given marker, this feature
can aid in their identification [23–25].
Numerous genetic systems exist for tracking and targeting macro-
phages, including alveolar macrophages. These include: simple trans-
gene systems [26–28], Cre/lox systems (in particular Cx3cr1, LysM,
Cd11c, Runx1) [13,14,26,28,29], tetracycline-inducible Tet-on systems
[16], bone marrow chimeras including chimeras with thoracic shielding
[5,12,30–32], adoptive transfer of macrophages and their precursors
[8,33,34], and parabiosis and depletion of alveolar macrophages
[5,12]. Use of Cre/lox-based systems for targeting macrophages and
tools for depletion of macrophages, including alveolar macrophages,
has been extensively discussed [35–37]. Importantly, none of these
systems are specific for alveolar macrophages and often target other cell
types, particularly other myeloid cells (such as monocytes and dendritic
cells) and in some cases non-immune cells, for example, the CreLysM
system, targets myeloid cells in addition to alveolar type II cells [38].

⁎
Corresponding author.
E-mail address: a-misharin@northwestern.edu (A.V. Misharin).

https://doi.org/10.1016/j.cellimm.2018.01.005
Received 10 December 2017; Received in revised form 7 January 2018; Accepted 11 January 2018
0008-8749/ © 2018 Elsevier Inc. All rights reserved.

Please cite this article as: Joshi, N., Cellular Immunology (2018), https://doi.org/10.1016/j.cellimm.2018.01.005
N. Joshi et al.
3. Ontogeny and maintenance of alveolar macrophages in the
steady state
The origin of alveolar macrophages has long been debated. Studies
performed by van Furth over a century ago suggested that circulating
monocytes give rise to all tissue-resident macrophages, including al-
veolar macrophages at steady state [39,40]. This concept has been re-
vised during the past decade. Multiple studies have unambiguously
demonstrated that some prototypical tissue-resident macrophages, such
as microglia in the brain, Kupffer cells in the liver and large peritoneal
macrophages in the peritoneal cavity, originate from various progeni-
tors, populate tissues during different stages of embryogenesis and
maintain their population throughout their lifetime with minimal to no
contribution from circulating monocytes [35,41]. Although several
waves of macrophages populate the lung during embryogenesis and
participate in organogenesis, these do not give rise to the definitive
alveolar macrophage population, since the alveolar niche does not exist
until birth [8,11,28,42]. Thus, the first breath of a newborn may be
viewed as the lung’s first “wound”: it creates a niche, which gets rapidly
populated by circulating fetal monocytes.
The differentiation of recruited monocytes into alveolar macro-
phages and subsequent engraftment in the alveolar niche depends on
the production of granulocyte-macrophage colony-stimulating factor
(GM-CSF) by alveolar type II cells [8,11]. Loss of GM-CSF signaling, due
to genetic mutation or development of autoantibodies against GM-CSF
or its receptor, results in the loss of mature alveolar macrophages, ac-
cumulation of surfactant and the development of alveolar proteinosis
[43]. Rescue of GM-CSF signaling by adoptive transfer of alveolar
macrophages or their precursors with functional GM-CSF receptor or
administration of exogenous GM-CSF can reverse this condition in
mouse models [8,44–48]. In addition, there are reports of successful
resolution of alveolar proteinosis after bone marrow transplantation in
humans [49–51]. In contrast, M-CSF signaling is dispensable for sur-
vival of mature alveolar macrophages [52]. GM-CSF signaling is re-
quired for the expression of peroxisome proliferator-activated receptor
gamma (PPARG) – a key transcription factor necessary for realization of
the transcriptional program driving macrophage adaptation to the lung
environment and acquisition of a high lipid load [6,8,53–55]. PPARG
deletion in myeloid lineage prevented formation of mature alveolar
macrophages, which resulted in delayed bacterial clearance after in-
fection with S. pneumoniae and resulted in alveolar proteinosis. Other
transcriptional factors, such as Bach2 and C/EBPβ are also required for
development and maintenance of alveolar macrophages [56,57]. L-
plastin is another protein that is required for the transmigration and
retention of alveolar macrophage precursors into the alveolar niche:
mice deficient for the Lcp1 gene in myeloid cells fail to develop func-
tional alveolar macrophages and have impaired clearance of pulmonary
pathogens [42]. Recently, a crucial role of autocrine TGFβ signaling in
development and maintenance of alveolar macrophages has been also
demonstrated [58].
Using various fate-mapping techniques, several groups have de-
monstrated that in naïve, unchallenged adult mice housed in clean fa-
cilities, alveolar macrophages, microglia in the brain, Kupffer cells in
the liver and large peritoneal macrophages maintain their population
via proliferation in situ for months without contribution from circu-
lating monocytes [5,8,14,26,29,32]. Supporting these observations
made in mice, two recent clinical reports demonstrated that following
lung transplant, the population of alveolar macrophages is remarkably
stable with donor cells comprising the bulk of the alveolar macrophage
pool even 5 years after transplant [59,60]. However, it is not known
whether the initial seeding of alveolar macrophages into the lung
during the postnatal period occurs as a single wave with subsequent
expansion via local proliferation, or if additional alveolar macrophages
derived from adult bone marrow-derived monocytes contribute to the
alveolar macrophage pool during the rapid period of physiological body
growth and lung niche expansion. Since alveolar macrophages are
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Cellular Immunology xxx (xxxx) xxx–xxx
relatively sessile cells, [27,61] it is possible that during the period of
active postnatal lung growth, alveolar macrophages that populated the
lung soon after birth cannot migrate into newly formed alveoli. Hence,
recruitment of monocytes from the circulation may be necessary in
order to fully populate the niche [62]. Supporting this hypothesis,
Gomez-Pedriguero and colleagues, using a Flt3-Cre reporter system
which labels all cells derived from definitive hematopoiesis, found that
at 4 weeks of age, 10–20% of alveolar macrophages were positive for
the fluorescent reporter and that the proportion of these cells increased
up to 40% by one year of life [63]. Perhaps stereological techniques
combined with new “Microfetti” fate mapping models [64] which can
track the clonal expansion of individual macrophages, might help fur-
ther address the question of alveolar macrophage ontogeny and main-
tenance during physiological lung growth.
While it is certain that alveolar macrophages are long-lived, current
fate mapping studies do not cover the entire lifespan of the animal. It
was recently demonstrated that normal unperturbed aging is associated
with decreased number of alveolar macrophages and downregulation of
genes involved in cell cycle pathways [65]. Thus, it is possible that in
the context of aging, increased environmental stress (such as exposure
to airborne particulate matter over the long period of time) may lead to
the recruitment of monocyte-derived alveolar macrophages in adults.
This raises the question: does the origin of alveolar macrophages
matter? Several investigators have leveraged powerful genomic tools to
help find an answer. Building on work from the ImmGen consortium
[6], Lavin and colleagues demonstrated that the transcriptome and
epigenetic landscape of tissue macrophages are determined by the
tissue-specific microenvironment [66]. Using bone marrow transfer
into whole body-irradiated hosts, they found that the lung micro-
environment shapes the epigenetic landscape of bone marrow-derived
alveolar macrophages, inducing a convergence with the epigenetic
landscape of tissue-resident alveolar macrophages from naïve mice.
Moreover, upon adoptive transfer into the alveolar space, mature
peritoneal macrophages adapt to the niche, rapidly reshape their
transcriptome and become somewhat alveolar macrophage-like cells. In
another study, Gibbings and colleagues used congenic (CD45.1/
CD45.2) bone marrow chimeras with thoracic shielding to protect the
recipient’s tissue-resident alveolar macrophages (a system originally
introduced by Janssen and colleagues [32]), followed by partial de-
pletion of these cells using clodronate-loaded liposomes, to generate a
mouse that harbors alveolar macrophages of embryonic and adult
origin simultaneously [12]. Upon opening the niche, monocytes entered
the uninjured alveolar space and differentiated into cells which were
virtually indistinguishable from tissue-resident alveolar macrophages
both immunophenotypically and functionally. They further isolated
tissue-resident and monocyte-derived alveolar macrophages via FAC-
Sorting and subjected them to transcriptomic profiling via microarray.
Only 35 probes were differentially expressed between tissue-resident
and monocyte-derived cells. Similarly, van de Laar and colleagues de-
monstrated that upon adoptive transfer into a permissive niche, yolk
sac macrophages, fetal monocytes and adult bone marrow monocytes
all gave rise to cells that were functionally and transcriptionally in-
distinguishable from tissue-resident alveolar macrophages derived from
fetal monocytes [33]. Together, these studies clearly demonstrate that
in the unperturbed lung, at steady state, the origin of alveolar macro-
phages does not matter, as cells of fetal and adult origin as well as
differentiated alveolar macrophages can occupy the permissive lung
niche and give rise to fully functional, long-living, self-maintaining al-
veolar macrophages, which are virtually identical at the phenotypic,
functional, genomic and epigenomic levels.
4. Origin and function of alveolar macrophages during aging,
stress and lung injury
The aforementioned studies show that lung environment, and not
cell ontogeny (embryonic versus adult), is a major driver of alveolar
N. Joshi et al.
macrophage development. However, it should be noted, that all studies
discussed above were performed under steady state conditions in the
unperturbed lung. This raises two questions: 1) does the environment of
an injured lung alter differentiation of newly recruited monocytes into
alveolar macrophages and 2) does the environment reshape tissue-re-
sident alveolar macrophages that were present in the lung before lung
injury? Several studies have attempted to answer this question using
various models of lung injury coupled with fate mapping systems to
track tissue-resident and monocyte-derived alveolar macrophages.
Janssen and colleagues used bone marrow chimeras with thoracic
shielding and adoptive transfer of bone marrow from pan-GFP mice
[32]. They were one of the first groups to demonstrate the remarkable
longevity and stability of tissue-resident alveolar macrophages: 80% of
alveolar macrophages were found to be of host origin up to 240 days
after the generation of chimeras. Using intratracheal instillation of
bacterial lipopolysaccharide (LPS) – a popular model of lung injury –
they demonstrated that the numbers of tissue-resident alveolar mac-
rophages did not change over the course of injury. Thus, the alveolar
niche remained occupied by tissue-resident alveolar macrophages and
was not permissive for engraftment of recruited cells. They also found
that while most of the recruited macrophages underwent apoptosis
during the resolution phase, some of the recruited monocyte-derived
macrophages persisted for up to 12 days after injury. Moreover, some of
these cells obtained a surface phenotype similar to tissue-resident al-
veolar macrophages, characterized by the absence of CD11b expression
and increased expression of CD11c. Recently, this group performed an
in-depth characterization of tissue-resident alveolar macrophages and
recruited monocyte-derived macrophages using gene expression pro-
filing via RNA-seq during the course of LPS-induced lung injury [15].
They found that tissue-resident alveolar macrophages and recruited
monocyte-derived macrophages exhibited strikingly different gene ex-
pression profiles during the acute phase of lung injury and concluded
that cell origin dictates macrophage programming in this setting. It
must be noted, however, that the focus of this study was on acute lung
injury, and that while recruited macrophages were recovered from the
alveolar space, they were not bona fide alveolar macrophages (i.e. cells
adapted for long-term persistence in the niche), as evidenced by a lack
of an alveolar macrophage-specific phenotype (CD169, CD206, Siglec
F). Importantly, since LPS is a spontaneously resolving model of lung
injury, the physiological significance of the differences in expression
between the two cell types is unclear.
Long-term models of lung injury may provide more informative
platforms to investigate the differential contribution of tissue-resident
and recruited monocyte-derived alveolar macrophages. Recently, two
independent studies utilized a bleomycin model of lung injury and fi-
brosis to identify distinct contributions of tissue-resident and monocyte-
derived alveolar macrophages to fibrosis pathogenesis [13,14].
Bleomycin-induced models of lung fibrosis are characterized by an
early inflammatory phase, followed by a fibrotic response [67]. During
the early phase of injury, there is an influx of monocytes and monocyte-
derived interstitial macrophages while the fibrotic phase is character-
ized by an expansion of alveolar macrophages with low expression of
the surface marker Siglec F [7]. Using bone marrow chimeras with
thoracic shielding the same group has later demonstrated that during
the first weeks following lung injury, tissue-resident and monocyte-
derived alveolar macrophages can be distinguished based on the dif-
ferential expression of Siglec F: tissue-resident alveolar macrophages
maintained high expression of Siglec F, whereas monocyte-derived al-
veolar macrophages expressed lower levels of this molecule [14]. They
then used two overlapping Cre-drivers, namely LysMCre and CD11cCre, to
delete pulmonary macrophages by removing caspase 8 and inducing
necroptosis via the unchecked RIPK3 pathway [68]. Since it takes time
for the effects of Cre-mediated deletion to take place, circulating
monocytes or monocyte-derived interstitial macrophages were not af-
fected in this system. Using bone marrow transfer, the authors found
that only cells that escaped Cre-mediated recombination reconstituted
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Cellular Immunology xxx (xxxx) xxx–xxx
the alveolar niche and differentiated into alveolar macrophages. No-
tably, the deletion of monocyte-derived alveolar macrophages led to the
amelioration of pulmonary fibrosis, highlighting a crucial role of these
cells in disease pathogenesis. Combined deletion of Casp8 and Ripk3
prevented necroptosis of monocyte-derived alveolar macrophages and
“rescued” the fibrotic phenotype. Gene expression profiling of FAC-
Sorted monocytes, monocyte-derived interstitial macrophages, mono-
cyte-derived alveolar macrophages and tissue-resident alveolar mac-
rophages demonstrated distinct transcriptomes for all cell types despite
their prolonged coexistence in the same environment. Moreover, only
monocyte-derived alveolar macrophages were enriched for genes pre-
viously associated with development of fibrosis in various organs. Fi-
nally, since bleomycin-induced pulmonary fibrosis resolves sponta-
neously over time, the authors asked whether these recruited
monocyte-derived alveolar macrophages persist in the lung after injury
resolution. Using bone marrow chimeras with thoracic shielding to
unambiguously track tissue-resident and monocyte-derived cells in two
models of lung injury (bleomycin- and influenza A virus-mediated),
they demonstrated that monocyte-derived alveolar macrophages persist
for at least 10 months after the resolution of initial injury at which time
they are phenotypically and transcriptionally indistinguishable from
tissue-resident alveolar macrophages.
Similarly, McCubbrey and colleagues used a previously validated
tetracycline-inducible hCD68rtTA system [16] to delete c-FLIP from
monocyte-derived alveolar macrophages making them susceptible to
apoptosis [13]. They demonstrated that deletion of monocyte-derived
alveolar macrophages ameliorated the development of pulmonary fi-
brosis. Analysis of gene expression profiles of FACSorted tissue-resident
and monocyte-derived alveolar macrophages showed that these cells
exhibit markedly different gene expression profiles with only mono-
cyte-derived alveolar macrophages exhibiting a profibrotic gene sig-
nature. Together, these studies demonstrate that tissue-resident and
monocyte-derived alveolar macrophages differ in their capacity to be
programmed by their environment and that this programming has a
distinct impact on their function, resulting in clinically apparent phe-
notypes.
The recruitment of Siglec Flow monocyte-derived alveolar macro-
phages has also been observed in mouse models of asthma [69,70].
Sanfilippo and colleagues demonstrated that the recruitment and per-
sistence of monocyte-derived Siglec Flow alveolar macrophages during
allergic inflammation protects against pulmonary pneumococcal in-
fection [69]. Allergic inflammation was associated with enhanced early
clearance of S. pneumoniae from the lung, decreased bacterial invasion
from the airway into the lung tissue, a blunted inflammatory cytokine
and neutrophil response to infection, and enhanced expression of TGF-
β1. Non-selective depletion of alveolar macrophages using clodronate-
loaded liposomes abrogated this effect.
In a reverse system, Machiels and colleagues subjected mice to
gammaherpesvirus infection and then induced allergic airway in-
flammation using a house dust mite model [71]. They found that pre-
vious infection with murid herpesvirus 4 inhibits subsequent house dust
mite-induced experimental asthma. In a series of experiments, they
attributed this protective effect to alveolar macrophages. Using bone
marrow chimeras with thoracic shielding, they demonstrated that
murid herpesvirus 4 infection depletes alveolar macrophages and in-
duces monocyte recruitment. Over time these recruited monocytes
differentiated into long-lived alveolar macrophages. In agreement with
previous reports, these recruited alveolar macrophages also expressed
low levels of Siglec F. The authors also performed gene expression
profiling of the bulk alveolar macrophage population and found that
herpesvirus 4 and house dust mite challenge, alone or in combination,
produced significant changes in the gene expression profiles of alveolar
macrophages.
Collectively, these studies demonstrate that the environment of the
injured lung can reshape the transcriptome of both monocyte-derived
and tissue-resident alveolar macrophages. It is not yet known whether
N. Joshi et al.
less acutely injurious, but chronic stimuli produce a similar effect. For
example, the lung is constantly exposed to air pollutants and ambient
particular matter – a serious public health concern in both the devel-
oping and developed world [72,73]. Exposure to ambient particular
matter does not result in acute lung injury and recruitment of mono-
cyte-derived alveolar macrophages. However, in response to particulate
matter exposure, alveolar macrophages produce IL-6, which leads to
activation of the coagulation cascade in the liver and increased risk of
thrombosis [74,75]. There is evidence that exposure to particulate
matter can modify the epigenetic landscape of macrophages in vitro
[76,77]. While detailed epigenetic profiling of alveolar macrophages
after long-term exposure has not yet been reported, it is tempting to
speculate that epigenetic changes may also modulate macrophage re-
sponse, IL-6 production and subsequent risk of thrombosis.
As discussed above, both tissue-resident and monocyte-derived al-
veolar macrophages respond to changes in the local environment
during lung injury. However, quantitative and qualitative changes in
the transcriptome of monocyte-derived alveolar macrophages are more
prominent than in tissue-resident alveolar macrophages. Nevertheless,
upon resolution of lung injury, alveolar macrophages, irrespective of
their origin, become increasingly similar, as evident by convergence of
their transcriptomes. Despite these similarities, tissue-resident and
monocyte-derived macrophages may respond differently to a sub-
sequent stimulus. Recent studies have shown that monocyte to macro-
phage differentiation is associated with a global reshaping of the epi-
genetic landscape [78,79]. Moreover, previous exposure of monocytes
to stimulus can leave long-lasting epigenetic marks, which can be re-
tained upon their differentiation into macrophages. Depending on the
type of stimulus, this may result in the development of a trained im-
munity, characterized by enhanced inflammatory status, or tolerance,
characterized by a blunted inflammatory response [78]. Recently,
Schmidt and colleagues demonstrated that in vitro differentiated in-
flammatory macrophages are characterized by a unique network of
transcriptional and epigenetic regulators, which respond to activation
in a stimulus-specific manner [80]. This “inflammation specific” net-
work was characterized by globally permissive histone modifications.
While the authors did not investigate the response of tissue-resident
macrophages to stimulation directly, they obtained contrasting data
from a re-analysis of a previously published dataset [66]. Tissue-re-
sident macrophages from naïve animals, including alveolar macro-
phages, were characterized not only by the absence of expression of
inflammatory genes, but also by the presence of repressive chromatin
marks. However, it is still possible that inflammation can reshape the
epigenetic landscape of differentiated macrophages and activate latent
enhancers in differentiated cells or reshape epigenetic changes caused
by a previous stimulation [81,82]. Unfortunately, all these experiments
were performed in vitro in very simplistic and short-term models. Ulti-
mately, the validity and significance of “the second hit” hypothesis
needs to be tested in long-term animal models with measurable and
clinically relevant physiological outcome.
5. Conclusions and open questions
Our understanding of macrophage biology has improved dramati-
cally in the past decade. Irrespective of their origin, macrophages, in-
cluding alveolar macrophages, demonstrate remarkable plasticity to
their surrounding environment. The environment of the injured lung
can reshape alveolar macrophages from protective and homeostatic
cells into pathogenic cells. Better understanding of the mechanisms
through which inflammatory and environmental insults affect alveolar
macrophage function may guide development of novel therapies for
pulmonary diseases with macrophage involvement. The importance of
the environment in guiding alveolar macrophage development and
function suggest that future research should take a systems biology
approach and investigate the function of alveolar macrophages in the
context of their partnering cell types, in particular alveolar type I and
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Cellular Immunology xxx (xxxx) xxx–xxx
type II cells.
Acknowledgments
James M Walter is supported by Northwestern University’s Lung
Sciences Training Program 5T32HL076139-14 and Northwestern
University’s Dixon Young Investigator Translational Research Grant.
Alexander V Misharin is supported by NIH NHLBI 1R56HL135124-01,
BD Bioscience Immunology Research Grant and by the Office of the
Assistant Secretary of Defense for Health Affairs, through the Peer
Reviewed Medical Research Program under Award W81XWH-15-1-
0215. Opinions, interpretations, conclusions and recommendations are
those of the author and are not necessarily endorsed by the Department
of Defense.
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