Critical Transition in Tissue Homeostasis Accompanies
Murine Lung Senescence
Carla L. Calvi1., Megan Podowski1., Franco R. D’Alessio1, Shana L. Metzger1, Kaori Misono1, Hataya
Poonyagariyagorn1, Armando Lopez-Mercado1, Therese Ku1, Thomas Lauer2, Christopher Cheadle3, C.
Conover Talbot Jr.4, Chunfa Jie4, Sharon McGrath-Morrow2, Landon S. King1, Jeremy Walston5, Enid R.
1Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America, 2Pediatric Pulmonary,
Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America, 3Lowe Family Genomics Core, Division of Allergy and Clinical Immunology,
Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America, 4JHMI Microarray Core, Johns Hopkins University School of Medicine,
Baltimore, Maryland, United States of America, 5Division of Geriatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America
Background: Respiratory dysfunction is a major contributor to morbidity and mortality in aged populations. The
susceptibility to pulmonary insults is attributed to ‘‘low pulmonary reserve’’, ostensibly reflecting a combination of age-
related musculoskeletal, immunologic and intrinsic pulmonary dysfunction.
Methods/Principal Findings: Using a murine model of the aging lung, senescent DBA/2 mice, we correlated a longitudinal
survey of airspace size and injury measures with a transcriptome from the aging lung at 2, 4, 8, 12, 16 and 20 months of age.
Morphometric analysis demonstrated a nonlinear pattern of airspace caliber enlargement with a critical transition occurring
between 8 and 12 months of age marked by an initial increase in oxidative stress, cell death and elastase activation which is
soon followed by inflammatory cell infiltration, immune complex deposition and the onset of airspace enlargement. The
temporally correlative transcriptome showed exuberant induction of immunoglobulin genes coincident with airspace
enlargement. Immunohistochemistry, ELISA analysis and flow cytometry demonstrated increased immunoglobulin
deposition in the lung associated with a contemporaneous increase in activated B-cells expressing high levels of TLR4
(toll receptor 4) and CD86 and macrophages during midlife. These midlife changes culminate in progressive airspace
enlargement during late life stages.
Conclusion/Significance: Our findings establish that a tissue-specific aging program is evident during a presenescent
interval which involves early oxidative stress, cell death and elastase activation, followed by B lymphocyte and macrophage
expansion/activation. This sequence heralds the progression to overt airspace enlargement in the aged lung. These
signature events, during middle age, indicate that early stages of the aging immune system may have important correlates
in the maintenance of tissue morphology. We further show that time-course analyses of aging models, when informed by
structural surveys, can reveal nonintuitive signatures of organ-specific aging pathology.
Citation: Calvi CL, Podowski M, D’Alessio FR, Metzger SL, Misono K, et al. (2011) Critical Transition in Tissue Homeostasis Accompanies Murine Lung
Senescence. PLoS ONE 6(6): e20712. doi:10.1371/journal.pone.0020712
Editor: Rory Edward Morty, University of Giessen Lung Center, Germany
Received December 21, 2010; Accepted May 11, 2011; Published June 21, 2011
Copyright: ? 2011 Calvi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by the National Institutes of Health (grants RO1-HL085312 to E.R.N.). This work has also been supported by the
National Institute on Aging (NIA) through the Johns Hopkins Older Americans Independence Center (OAIC), P30 AG021334 (to E.R.N. and J.D.W.). The funders had
no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
A stereotyped pattern of structural changes which occur in the
human lung as it ages, termed ‘‘senile lung’’, is characterized by
airspace enlargement that is similar but not identical to acquired
emphysema [1,2]. Although the chronicity of this process is poorly
understood with respect to time of onset or progression, the
reproducibility of the underlying pattern suggests that the lung
harbors instructions from birth that orchestrate the timing and
morphology of age-related structural changes. We hypothesized
that by studying an informative inbred strain of mice, the aging
DBA/2 strain, the molecular signatures of these age-related
changes could be identified. Furthermore, these signatures could
serve to construct a candidate genetic profile that may define those
persons at risk for lung dysfunction with aging.
A limitation of previous surveys of organ-specific aging
programs is the use of binary constructs of the aging phenotype,
focusing on ‘‘young’’ versus ‘‘old’’. Since the young organ is not
necessarily the ‘‘control’’ for the old organ, we sought to develop
an alternative approach to describe tissue aging. By performing a
genome-wide transcriptional time course survey of the aging
murine lung (over six time points), we were able to extract genes
PLoS ONE | www.plosone.org1June 2011 | Volume 6 | Issue 6 | e20712
that not only displayed more complex patterns of expression with
aging but also reflected known histologic events that could not be
replicated by simple pair-wise comparisons. In this study, we focus
on the gene cluster which corresponds to the transcriptional
transition attending the onset of airspace enlargement, e.g. 8–12
months of age.
Previous genomic surveys of murine lung aging showed that 1)
the terminal structural changes seen in the aged lung are
associated with an altered transcriptome and 2) that the aging
lung harbors both tissue-specific and aging specific molecular
signatures. Misra and colleagues found that airspace enlargement
in senescent DBA/2 mice is associated with the down-regulation of
elastin and several collagen genes despite increased collagen
content compared with the young adult controls [3,4]. However,
whether this pattern temporally approximated the onset of
structural changes in the aging lung was not established. Thus,
the senescent transcriptional program could reflect either an active
pro-aging process or terminal changes in a failing tissue. Recently,
Zahn reported tissue-specific transcriptomes, including the lung, of
aging C57Bl/6 mice over four time points . However, no
correlation with architectural changes in tissues was pursued.
These important findings augur a need for a more detailed
assessment of the molecular signatures of aging lung pathology.
In this study, we show that airspace enlargement develops
during the mid-range of the murine life-span and progresses
through the late, preterminal time points and is accompanied by
early oxidative stress, cell death and elastase activation. We also
show that several genes are transiently induced during the onset of
this structural change, and may possibly be the first signature of a
tissue-specific aging program. This period, we further demon-
strate, is punctuated by a marked, transient induction of
immunoglobulin genes, accompanied by B lymphocyte (B-cell)
activation/expansion, immunoglobulin deposition and macro-
phage infiltration. Taken together, our strategy has shown that
time course data informed by structural surveys can reveal relevant
pathways involved in tissue-specific aging that might be over-
looked with conventional young-old pairwise analyses.
Strategy of time course survey of the aging lung
In order to delineate the critical signaling events which attend
the onset of airspace enlargement during the aging of DBA/2
mice, we performed a detailed histologic and molecular analysis of
the lungs of the mice at six different time points during adult life: 2
months, 4 months, 8 months, 12 months, 16 months and 20
months. These time points loosely correspond to specific
maturation stages in humans: early adulthood (2–4 months),
middle-age (8–12 months) and old age (16–20 months) (Figure 1A).
At each time point, lungs were processed for histology, expression
profile analysis and protein immunoblotting (Figure S1).
Lung histology and morphometry in aging mice
We found a nonlinear pattern of airspace enlargement, denoted
by MLI (mean linear intercept), that commenced at 12 months of
age and progressed thereafter (Figure 1B,C). The airspace
enlargement was homogeneous without any histologic stigmata
of tissue destruction. Of note, the large vessels and microvascu-
lature showed no evidence of morphologic change with aging.
Consistent with the 8 to 12 month period representing a critical
transition with respect to organismal aging, we found that the
trend toward significant weight loss with age started at 12 months
(Figure 1D). When MLI was adjusted for weight, a quadratic (but
not linear) association was identified resulting in a p-value of 0.002
with an adjusted R2of 0.28. Since weight has a bimodal curve
morphology with age, we considered whether analysis of mice $8
months of age might show an independent association between
MLI and weight. The observed linear association between MLI
and weight in this group, however, was eliminated with adjustment
for age (p=0.68). A significant association existed between MLI
and age (R20.89) with no evidence of improved association when
weight was included in the model. Taken together, these data
show that even when corrected for weight, age remains an
independent factor contributing to MLI.
Measures of lung injury with aging
Airspace enlargement frequently accompanies various forms of
lung injury that result in oxidative stress, cell death, reduced
proliferation and/or local inflammation. Increased oxidative stress
is a signature of systemic aging and likely contributes to the higher
incidence of malignancy, fibrosis and low-grade inflammation in
elderly persons [6,7]. Immunohistochemical staining for nitrotyr-
osine, a marker of oxidative stress, revealed a progressive increase
in oxidative stress from 2 month to 20 months of age (Figure 2A,B).
The site of the staining was in the airspace epithelial compartment,
especially type II cells. Because iNOS (inducible Nitric Oxide
Synthase) activity is frequently associated with inflammation-
associated oxidative stress, we examined iNOS expression by
immunoblotting in the aging lung lysates. We saw no increase in
iNOS expression in the aging lung (data not shown). We also
measured cell death in the airspace compartment by performing
TUNEL (Terminal Transferase dUTP Nick End Labeling)
staining. We saw a different temporal pattern of staining,
compared with nitrotyrosine, with a statistically significant
enhancement in staining evident between 2 months and 12
months (Figure 2C). Using a caspase 3 bioassay, we saw no
evidence of increased caspase activity in the aging lungs until 20
months of age (Figure S2). This suggests that the early cell death
represents either caspase-independent apoptosis and/or necrosis.
Thus oxidative stress appears to precede both the development of
exuberant cell death and significant airspace enlargement in the
aging lung. Of note, the levels of oxidative stress in the aging lung
is much less than that observed in other models of airspace disease
such as the tight-skin mouse .
K-Means Clustering Profiles
Nine clustering profiles of the time course transcriptome were
generated using a K-means strategy. Of the nine cluster patterns,
one was selected for further interrogation based on the timing of
induction consistent with the onset of the airspace phenotype
(Figure 3A, B). This cluster contained 2220 genes out of the 25,000
known genes on the Illumina chip. The genes in this cluster were
termed the ‘‘airspace peak’’. The top pathways identified by Gene
Ontology included those involved in humoral immune response,
transport, negative regulation of cell cycle and response to
wounding (Table 1). The top 200 genes in the cluster are shown
in Table S1. Confirming the pathway analysis, immunoglobulin
genes were disproportionately represented in this peak (25 of top
50 genes). We selected eight genes represented in the top 100
within the airspace peak for further validation in triplicate by real
time RT-PCR (reverse transcriptase polymerase chain reaction).
Since our gene list is based on a profile over six time points, we
recognize that two-point comparison is not a true validation. We
elected to use three of these comparisons for these eight genes
spanning the 4 month to 12 month interval, 4 vs 8, 4 vs 12 and 8 vs
12. We specifically chose non-immunoglobulin genes for this
validation given the known overlapping specificity of immuno-
Impaired Tissue Homeostasis in the Aging Lung
PLoS ONE | www.plosone.org2 June 2011 | Volume 6 | Issue 6 | e20712
Figure 1. Time course analysis of aging lung phenotype. A. Schematic diagram of the lung harvest time points and the corresponding phases
of human aging. B. Representative lung histology from hematoxylin and eosin stains of 2 month, 12 month and 20 month old mice depict a
progressive increase in airspace size with age. Photomicrographs are representative of N=4–6 mice per group. Original magnification 206. C.
Morphometric analysis of airspace caliber denotes transition point to progressive airspace enlargement between 8 and 12 months of age. N=6 mice
per group. D. Body weight measurement establishes trend to weight loss initiated between 8 and 12 months of age. N=6 mice per group. MLI-Mean
linear intercept (mm). BW-Body weight. The pale versus dark shades show different phases in the evolution of the noted parameter.
Impaired Tissue Homeostasis in the Aging Lung
PLoS ONE | www.plosone.org3 June 2011 | Volume 6 | Issue 6 | e20712
globulin transcript analysis. Five out of the 8 genes surveyed in the
airspace peak were increased by RT-PCR (Table S2).
Excessive immunoglobulin synthesis and deposition in
the aging lung
Although immunosenescence involves overall dampened im-
mune responses with aging, autoimmunity is an observed
accompanying pathology . In fact, this low grade inflammation
has been termed inflama-aging and may contribute to organ-
specific disease pathology [10,11]. We explored whether the
exuberant immunoglobulin production observed in the 12 month
old lung suggested that immunoglobulin deposition might
contribute to the airspace pathology. ELISA (enzyme-linked
immunosorbent assay) analysis of serum showed that a trend
towards a significant increase in IgG (immunoglobulin G) in serum
was present in the 8 and 12 month old specimens compared with
the 2 or 18 month old samples (Figure 4A). Analysis of lung tissues,
by contrast, demonstrated a significant increase in immunoglob-
ulin deposition in the 8 and 12 month old samples (Figure 4B).
Quantitative densitometry of immunoblots of whole lung lysates
for IgG and IgM also showed an induction of 25 and 55 kDa
bands at the 8 and 12 month time points (Fig. 4C and Figure S3).
Immunohistochemical staining for mouse IgG showed that in 8
month old mice, compared with 2 month old mice, there was
increased staining for these complexes in the septal walls
(Figure 4D top panel and data not shown). By 12 months of
age, a marked increase in staining was evident in both epithelial
cells and in the septal walls (Figure 4D bottom panel).
Colocalization of IgG and complement C3 confirms immune
complex deposition in the 12 and 20 month old lungs that is
predominantly cell-associated (Figure 4E). Thus, immunohisto-
chemistry, ELISA, immunoblotting and transcriptional analysis all
support an elaboration of immunoglobulin production and
deposition in the presenescent lung parenchyma at a time point
that coincides with structural changes in the airspace morphology.
Immune cell dysregulation accompanies lung
Flow cytometry on whole lung preparations and BAL
(bronchoalveolar lavage) specimens from 2 months, 8 months,
12 month and 20 month old lungs showed an increase in B-cell
content between 8 and 12 months (Figure 5A,B). B-cell
immunophenotyping showed an increase in CD86+ and TLR4+
(toll receptor 4+) lymphocytes attending the 8 to 12 month
transition (Table 2). Since CD86 is a costimulatory molecule that
is induced in activated lymphocytes, these data are consistent with
B-cell activation accompanying the airspace simplification ob-
served in the aging lung. No change in the abundance of total T-
cells, CD4+, or FoxP3+ T regulatory subsets was seen (Figure 5A,B
and Figure S4B). Interestingly, a modest increase in the CD8+
compartment was evident between 2 months and 8 months and
maintained at 12 months (Figure S4A). Despite the lack of a
triggering insult, we examined macrophage influx and activation
in the aging lungs. We found that macrophages were increased in
the 12 month lung compared with the 8 month time point
(Figure 5C). Flow cytometric evaluation showed an increase in
MHCII (major histocompatibility complex class II) expression in
the monocyte compartment at 12 months of age, reflecting an
activated phenotype (Table 2).
Autoimmunity has been proposed as a component of immuno-
senescence, however to date no culprit immunogenic lung proteins
have been identified and the lung pathology accompanying most
rheumatologic illnesses is fibrosis rather than airspace enlarge-
ment. We examined whether selected autoimmune cytokines
(IL17, IL12/IL23heterodimer) were induced in the aging lung
[12,13]. We saw a reduction in lung levels of these cytokines from
8 months to 20 months when compared to 2 month old samples
(Figure 5D). Although these results do not eliminate an
autoimmune contribution to the immunoglobulin deposition and
airspace enlargement, they suggest that the more conventional
cytokines participating in autoimmunity are downregulated.
Taken together, these data show that an induction of B-cell
expansion/activation and immunoglobulin production in the
aging lung accompanies the earliest stage of airspace enlargement.
Figure 2. Increased oxidative stress and cell death evident in
murine mid-life. A. Representative immunohistochemical staining for
nitrotyrosine in mice at 2 months, 8 months, 12 months and 20 months
of age. Robust staining is evident by 8 months of age. Site of staining is
in alveolar epithelial cells (arrowheads). N=4–6 mice per group. Original
magnification, 206. B. Quantitative immunohistochemistry of nitrotyr-
osine staining shows enhanced staining in the 8 month old lung which
progresses through later time points. N=6 mice per group. C.
Quantitative immunohistochemistry of TUNEL staining of lungs from
aging mice shows increased cell death at 8 months of age which
persists through later time points. N=6 mice per group. Reported data
are mean values +/2 SEM from at least five mice for each group.
Impaired Tissue Homeostasis in the Aging Lung
PLoS ONE | www.plosone.org4 June 2011 | Volume 6 | Issue 6 | e20712
Further, the immunoglobulin deposition is associated with an
influx of macrophages.
Matrix deposition and turnover in the aging lung
Our finding of early oxidative stress followed by immune cell
infiltration and immunoglobulin deposition prompted us to
determine whether the enhanced oxidative stress might contribute
to matrix turnover that could trigger an immune response. Recent
reports show that both aging and non-aging related redox changes
can alter matrix homeostasis [14,15]. We first assessed elastin and
collagen content and localization in the aging lung by Movat
staining. Both matrix elements were preserved in localization and
morphology across the aging time frame of 2 months to 20 months
(Figure 6A). Of note, we did see reduced elastin deposition in the
airspace wall at 12 months when compared to 8 months of age.
Increased peribronchiolar collagen deposition in the 12 month
and 20 month old mice was consistent with previous observations
of the aging rodent lung . Although we saw no overt evidence of
elastin fragmentation or discontinuity, usual features of elastase-
associated tissue destruction, in the 12 or 20 month lungs, we
performed zymography to assess lung elastase activity. Zymogra-
phy showed an early increase in MMP9 activation in the 8month
old lung that was maintained at 12 months (Figure 6B). By
contrast, MMP12 expression decreased with age, suggesting that
MMP12, a known contributor to cigarette smoke induced
emphysema in murine models, is not the source of elastin turnover
with murine aging (Figure S5). A progressive reduction in the
number of airway alveolar attachments, a signature of alveolar
wall destruction, was observed from 8–20 months of age
(Figure 6C). These data suggest that early elastase activity is not
only destructive and precedes the onset of airspace enlargement,
but is also a plausible trigger for the immunoglobulin liberation
and macrophage influx that initiates the airspace lesion (schema-
ticized in Figure 7).
Airspace enlargement is a well-recognized pathological signa-
ture of respiratory aging [3,16]. Whether this process recapitulates
or incorporates known pathways of organismal aging is unknown.
We sought to delineate the molecular profile of age-related
airspace enlargement by performing a time course transcriptional
survey of the aging DBA/2J lung throughout adult life. We found
a distinct gene induction pattern punctuated by immunoglobulin
production and cell cycle dysregulation which attended the onset
of airspace enlargement. Whereas cell cycle changes have been
linked to organismal aging, we show a novel role of oxidant-
triggered matrix remodeling and dysregulated lymphocyte func-
tion in the aging lung.
Despite the fact that airspace enlargement is a known feature of
the aged lung, whether the lesion develops from injury or simply
reflects reduced matrix abundance in tissue is a subject of debate.
Recent observations, including those reported here, support the
former paradigm. The oxidant injury, elastase activation and cell
death preceding the onset of airspace enlargement in our studies
strongly implicate age-associated tissue stressors. Consistent with
this paradigm, Sato reported that SMP30-deficient mice, a
proposed model for the ‘‘senile lung’’, not only develop accelerated
age-associated airspace enlargement but also display increased
oxidative stress, cell death and susceptibility to cigarette smoke
induced pulmonary emphysema .
Several investigators have shown or postulated reduced immune
function in aged persons and in murine models of aging
[18,19,20]. Alterations in immune responses with aging likely
contribute to the increased susceptibility to infectious insults and
malignancy in elderly persons. Unfortunately, no unifying pattern
of changes has been reported in humans or rodent models. Defects
in humoral immunity can accompany aging, manifest in both
reduced specific antibody responses and enhanced nonspecific
antibody production [21,22]. Impaired function of hematopoietic
stem cells in the aging bone marrow seems to result in both
reduced production of naı ¨ve B-cells and marked restriction of the
Figure 3. Schematic of selection of K-means cluster profile that temporally approximates airspace enlargement. Nine profiles
generated by random selection for difference from evenly spaced profiles are depicted. The profile whose peak corresponded to the onset of airspace
enlargement is enlarged on the left. Red shows the top 200 genes. Blue shows the remainder of genes within the cluster.
Table 1. Overrepresented pathways within airspace peak.
Pathway Representation (GO)p-value
Humoral Immune Response961025
Negative regulation of cell cycle161024
Response to wounding161024
Impaired Tissue Homeostasis in the Aging Lung
PLoS ONE | www.plosone.org5 June 2011 | Volume 6 | Issue 6 | e20712
Figure 4. Immunoglobulin deposition in lungs of aging mice. A. ELISA analysis of IgG containing complexes in serum from mice at designated
ages. A trend towards a significant increase is apparent between 8 and 12 months of age. N=6 mice per group. B. ELISA analysis of lung lysates from
mice at different ages. Data are mean +/2 SEM. A significant increase in immunoglobulin deposition in the lung occurs between 8 and 12 months of
age. N=4–6 mice per time point. C. Densitometric analysis of immunoglobulin expression in lung lysates from mice at the denoted ages show that
IgM (top panel) and IgG (bottom panel) are induced in the 8 and 12 month old lung, respectively. D. Immunohistochemical staining for IgG in lungs
of representative mice at designated ages. Arrows denote immunoglobulin deposition in airspace wall. Arrowheads denote cell-associated
deposition. N=6–8 mice per group. A generalized increase in deposition in the airspace compartment occurs in the 12 month old lung. All images are
206, except for insets on right, which are 406. E. Coimmunofluorescent staining for IgG (red) and C3 complement (green) with phase overlay (L)
shows evidence of granular colocalization (white arrowheads) in alveolar epithelial cells along with sites of predominant IgG deposition (black arrow)
and C3 deposition (white arrow). Coimmunofluroescent staining for IgG and C3 complement in 12 month old lung shows strong colocalization in the
perivascular region. V-vessel lumen. 406magnification.
Impaired Tissue Homeostasis in the Aging Lung
PLoS ONE | www.plosone.org6 June 2011 | Volume 6 | Issue 6 | e20712
B-cell immune repertoire in a murine system . Elevated serum
immunoglobulin levels and increased antibody-producing cells in
the spleen and bone marrow have also been reported in aging
mice . We found that the upregulation of immunoglobulin
genes during aging-related airspace enlargement is accompanied
by B-cell expansion, B-cell activation and enhanced synthesis and
deposition of immunoglobulin complexes in the lungs of aging
mice. B-cell activation is a feature of several chronic inflammatory
conditions such as COPD (chronic obstructive pulmonary disease),
rheumatoid arthritis and multiple sclerosis. An increased number
of lymphoid follicles in the airway submucosa has been identified
not only in mice exposed to cigarette smoke, but also in patients
with advanced emphysema [25,26]. Others have invoked a
pathogenic contribution of parenchymal and airway lymphoid
collections, possibly representing an exaggerated immune response
triggered by microbial, matrix or tobacco smoke antigens . A
recent study showed an increase in autoantibodies directed against
both lung epithelial and endothelial determinants in the serum of
patients with COPD . In our aging lung model, there is no
exposure to known airspace insults like microbes or tobacco
smoke; nonetheless, oxidative stress, elastase activation and
epithelial cell death occur. Thus, the trigger for the elaboration
of immunoglobulins in the aging lung may involve oxidative stress
promoted matrix degradation, an established mechanism for
aging-associated tissue remodeling [14,15]. While no significant
matrix turnover is evident by histochemical staining at the 12
month time point, our zymography data suggests that low-level
turnover is present at 8 months and may be sufficient to drive the
initiation of the immune response (Figure 6B).
We propose three possible mechanisms connecting the immune
signature with the airspace lesion (summarized in Figure 7). First,
the enhanced elastase activity in the absence of histologic evidence
of tissue damage might generate matrix degradation products that
are immunogenic. This is a proposed but imperfectly supported
mechanism for emphysema . The critical omission in the
theory is the identification of a consistently triggering matrix-
derived antigen. A second possibility is that an alteration in
immunosurveillance at midlife could create a permissive environ-
ment for an immune response to develop to a variety of stimuli.
Since the lung is an organ that is constantly exposed to foreign
antigens, any impairment in immune function can translate into
dysregulated innate and adaptive responses to antigen and lung-
specific pathology. However, immunomodulatory mechanisms
may be sufficiently preserved that the dysregulated response is
eventually arrested. By this view, the ongoing airspace enlarge-
ment, oxidative stress and cell death manifest a tissue-specific
inability to repair/regenerate the airspace compartment and low
grade inflammation as reflected by macrophage infiltration. This
paradigm is quite similar to the progressive airspace and airway
pathology observed after smoking cessation in persons with
COPD/emphysema. A third possible mechanism is that a primary
alteration in oxidant/antioxidant balance, conferred by midlife,
results in the generation of neoantigens secondary to oxidation of
resident proteins in the lung, an organ exquisitely susceptible to
oxidant injury. Such neoantigens could trigger a local immune
response and initiate the sequelae described above. Both of these
Figure 5. Alterations in immune cell compartments in the aging
lung. A. Representative histograms depicting lymphocyte subsets
identified in lungs of mice at indicated ages. An increase in CD19+ cells
occurs at 12 months of age compared with earlier time points (2 and 8
months). N=4–6 mice per time point. B. Relative proportion of
lymphocyte subsets quantified by flow cytometry in lung mononuclear
cells isolated from mice at designated ages. N=4–6 mice per time
point. C. Quantitative immunohistochemistry of macrophage abun-
dance in lungs of aging mice. An increase in macrophage infiltration
occurs at 12 months of age compared with 8 months. Data are mean +/
2 SEM. N=3–6 mice per time point. D. IL12/23 and IL17 ELISA analyses
of lung lysates from mice at designated ages. A reduction of IL17 levels
is evident in the lungs of mice 8 months of age and older compared
with 2 month old mice.
Impaired Tissue Homeostasis in the Aging Lung
PLoS ONE | www.plosone.org7 June 2011 | Volume 6 | Issue 6 | e20712
mechanisms rely on a relatively preserved immunomodulatory
axis. Consequently, an active direction of our lab is the dissection
of these immunomodulatory pathways as they relate to lung aging.
The studies presented here demonstrate two intriguing findings.
First, we show that aging associated airspace enlargement develops
during middle age and that a contemporaneous innate and
adaptive immune signature heralds its onset. This signature
consists of exuberant immunoglobulin production, B-cell activa-
tion, local immunoglobulin deposition and macrophage infiltra-
tion. Second, we demonstrate that early aging-associated oxidative
stress and elastase activation precedes overt inflammation,
immunoglobulin deposition and airspace enlargement. We present
a novel pathogenetic scheme for aging-associated airspace
enlargement with presenescent oxidative stress triggering both
canonical and noncanonical mechanisms of emphysema. These
findings suggest that the crucial point of intervention for aging
Table 2. Flow cytometric analysis of mononculear cell subsets in aging lungs.
% B Lymphocytes 8 month 12 month p-value
% Monocytes 8 month12 month p-value
Figure 6. Matrix localization in aging lung. A. Movat’s staining of lungs from mice at designated ages. Left panel shows representative high
power (406) images of lung parenchyma. Right panel shows low power (106) images of bronchioles. Arrowheads denote sites of elastin deposition
(blue-black). Line demarcation indicates areas of enhanced collagen content (yellow). Elastin deposition in the alveolar walls is modestly reduced at
12 months but shows recovered abundance at 20 months. Peribronchiolar collagen deposition is increased in the 12 month and 20 month old lungs.
N=4–6 mice per time point. B. Zymographic analysis of lungs from mice at the designated ages. Top arrow denotes MMP9 band. Bottom arrow
denotes MMP2 band. N=4–6 mice per time point. C. Quantitation of alveolar attachments in the lungs of mice at the designated ages show a
progressive reduction in attachments with increasing age. N=4–6 mice per time point. *p,0.05 compared with 2 months.
Impaired Tissue Homeostasis in the Aging Lung
PLoS ONE | www.plosone.org8 June 2011 | Volume 6 | Issue 6 | e20712
related lung dysfunction may be well before airspace disease is
Materials and Methods
Aged male DBA/2 mice (2–20 months of age) were obtained
from the specific pathogen-free Charles River-National Institute of
Aging (NIA) facility. These mice were temporarily housed in a
Johns Hopkins Medical Institution mouse facility accredited by the
American Association of Laboratory Animal Care until time of
euthanization. The animal studies were reviewed and approved by
the institutional animal care and use committee of Johns Hopkins
School of Medicine.
RNA Extraction and Illumina Chip Hybridization
Total RNA was extracted from murine lung using the Trizol
Reagent method (Invitrogen, Carlsbad, California 92008, cat.
no. 15596-026). The six RNA samples from each time point were
pooled into two groups comprised of three murine specimens.
RNA samples were labeled and hybridized to Illumina Sentrix
MouseRef-8 Expression Beadchips (Illumina, San Diego, CA
92121-1975, cat.no. BD-26-201) according to manufacturer’s
The microarray data was normalized with Affy, a Bioconductor
package (http://www.bioconductor.org), utilizing the quantile
normalization method to reduce the variation between micro-
arrays that can develop during the processes of sample
preparation, manufacture, fluorescence labeling and hybridization
. Assuming that there is an underlying common distribution of
signal intensities across microarrays, the quantile normalization
method makes the distribution of signal intensities for each
microarray in a set of microarrays the same by forcing the values
of quantiles to be equal. An underlying assumption of the quantile
normalization method is that only a small fraction of genes is
differentially expressed between the sample conditions. When
analyzing the gene expression changes with age within individual
tissue types, normalization was separately made for the data of
each tissue type in order to avoid any ‘‘reduced’’ differences that
could be introduced by normalizing the data across tissue types
together. With the normalized signal data, principal component
analysis (PCA) was performed in R to assess sample variability.
Differentially-expressed genes were categorized using modified
Best K-Means clustering (Spotfire 9.1.1). The number of clusters
was chosen empirically based on visual inspection as differing from
evenly spaced profiles. To reduce the background and ensure the
clustering quality, only genes with detectable hybridization signals
in the arrays from all aging groups were included. Nine profiles
were generated using Spotfire cluster initialization with a data
centroid based sea. One cluster was selected for further analysis
based upon a peak transcriptional induction coincident with the
onset of airspace enlargement. Genes within this cluster were
ranked based on similarity to exemplar and examined by Gene
Ontology Definition for pathway assignment. The top 200 genes,
ranked by similarity to exemplar, are shown in Table S1.
Differentially expressed genes were classified into functional
categories based on the Gene Ontology (GO) definition using
publicly available web-based tools Onto-express and David (data
base of annotation, visualization, and integrated discovery). For
each level of annotation, the calculated p-value represents the
probability that the specific gene-function was randomly distrib-
uted between groups .
Real Time PCR
Total RNA isolated from lung tissues was treated with DNase
and reverse-transcribed using a first-strand DNA sysnthesis kit
from Invitrogen. The PCR was performed on an ABI Fast 7500
System (Applied Biosystems, Foster City, CA). TaqMan probes for
the respective genes were custom-generated by Applied Biosystems
based on the sequences in the Illumina array and used per
manufacturer’s instructions. The expression levels of target genes
were determined in triplicate from the standard curve and
normalized to Gapdh mRNA level.
Western Blot Analysis
Western blot analysis was performed using standard methods.
Primary antibodies and dilutions were as follows: IgG1 (goat
polyclonal, Abcam, 1:1000), IgA (goat polyclonal, Abcam 1:1000),
IgM (goat polyclonal, Abcam 1:1000), CTGF (Abcam 1:5000),
psmad2 (Cell Signaling 1:1000), iNOS (Abcam 1:200), beta-actin
(rabbit polyclonal, Abcam, 1:1000), MMP12 (Santa Cruz, 1:250).
Histology, Morphometry and Immunohistochemistry
Histologic, morphometric and immunohistochemical methods
were as described previously . Alveolar attachments were
quantified in a blinded fashion from 206 H&E images and
normalized to airway perimeters. Antibodies were used at the
following concentrations: Nitrotyrosine (mouse monoclonal, Ab-
cam, 1:250), MAC-3 (rat monoclonal, BD Pharmingen, 1:100),
Neutrophil (rat monoclonal, Serotec 1:50), psmad2 (rabbit
polyclonal 1:5000) and SP-D (mouse monoclonal, Santa Cruz,
1:200). Movat’s stain was performed on selected sections utilizing a
TUNEL staining was performed using the Calbiochem TdT-
FragEL DNA Fragmentation Detection Kit per standard protocol
Figure 7. Schematic depiction of the ontogeny of injury events
associated with airspace enlargement in the aging murine
Impaired Tissue Homeostasis in the Aging Lung
PLoS ONE | www.plosone.org9 June 2011 | Volume 6 | Issue 6 | e20712
as published . Caspase 3 activity was measured in whole lung
lysates using the Promega Caspase Glo 3/7 Assay kit.
Serum samples and lung lysates from mice at designated ages
were subjected to Mouse IgG ELISA analysis per Roche protocol.
IL17 and IL12/23 ELISA assays were performed per Invitrogen
and R&D protocols, respectively.
Isolation of Lung mononuclear cells
Lungs were minced and incubated at 37uC and single cell
suspensions were prepared. Lymphocytes were gated with
characteristic low forward scatter/side scatter, using a FACSAria
instrument and FACSDiva for data acquisition (Becton Dick-
inson), and Flowjo for analysis (Tree Star Inc) as previously
Lung tissue lysates were prepared in a cold room at 4C. Tissue
was homogenized in 50 mL PBS and centrifuged at 14000 RPM
for 20 min. The supernatant was removed and used as sample
lysates. Fifty mg of lung lysates were loaded on a10% Criterion
Zymography Precast Gel (Biorad) and run at 120 V. Twenty-five
mL of recombinant mouse MMP9 protein (R&D Systems,
Minneapolis, MN) was loaded as a positive control. The gel was
soaked in 16 Renaturing Buffer (Biorad) twice for 30 minutes
each at room temperature and incubated in 16 Development
Buffer (Biorad) overnight at 37C. The gels were stained with
Coomassie Brilliant Blue R-250 Staining Solution (Biorad),
followed by 16Destain Coomassie R-250 Solution (Biorad) until
a clear band appeared against a blue background.
Results are expressed as means 6 SEM unless otherwise stated.
Screening comparisons across multiple time points were per-
formed by one-way ANOVA. These were followed by pairwise-
comparisons using the two-sample t-tests or Mann-Whitney rank
sum tests. All statistical analyses were performed with Sigmastat
(version 3.5; Systat Software, Chicago, IL). A p,0.05 was
Strategy for analysis of lung phenotype at
activity in lung lysates from mice at designated ages. N=4–6 mice
per time point. *p,0.01.
Caspase activity in the aging lung. A. Caspase
lung. Western blotting for immunoglobulins in lung lysates from
mice at designated ages. Top-IgM blot, Bottom-IgG blot.
Immunoglobulin expression in the aging
Proportion of CD4+ and CD8+ cells in CD3+ lymphocyte subset
quantified by flow cytometry in lung mononuclear cells isolated
from mice at designated ages. N=4–6 mice per time point. B.
Relative proportion of FoxP3+ cells in CD4+ lymphocyte subset
from mice at designated ages. N=4–6 mice per time point.
Asterisk designates p,0.05 compared with 2 month time point.
T cell subsets in the aging lung. A. Relative
tometric analysis of MMP12 protein expression in lung lysates
from mice at designated ages normalized to actin. N=4–6 mice.
MMP12 expression in the aging lung. Densi-
Top 200 genes in airspace peak.
Real-time PCR validation of genes in airspace
We thank Rubin Tuder, Dean Sheppard, Robert Senior, Ari Zaiman and
Clarke Tankersley for thoughtful input. We thank Kathryn Carson for help
with statistical analysis.
Conceived and designed the experiments: ERN. Performed the experi-
ments: CLC MP FRD SLM KM HP AL TK TL CC. Analyzed the data:
FRD KM CC CCT CJ ERN. Contributed reagents/materials/analysis
tools: FRD CC CCT CJ LSK JW SM. Wrote the paper: ERN.
1. Richards DW (1956) The aging lung. Bull N Y Acad Med 32: 407–417.
2. Verbeken EK, Cauberghs M, Mertens I, Clement J, Lauweryns JM, et al. (1992)
The senile lung. Comparison with normal and emphysematous lungs. 1.
Structural aspects. Chest 101: 793–799.
3. Huang K, Mitzner W, Rabold R, Schofield B, Lee H, et al. (2007) Variation in
senescent-dependent lung changes in inbred mouse strains. J Appl Physiol 102:
4. Misra V, Lee H, Singh A, Huang K, Thimmulappa RK, et al. (2007) Global
expression profiles from C57BL/6J and DBA/2J mouse lungs to determine
aging-related genes. Physiol Genomics 31: 429–440.
5. Zahn JM, Poosala S, Owen AB, Ingram DK, Lustig A, et al. (2007) AGEMAP:
A Gene Expression Database for Aging in Mice. PLoS Genet 3: e201.
6. Benz CC, Yau C (2008) Ageing, oxidative stress and cancer: paradigms in
parallax. Nat Rev Cancer 8: 875–879.
7. Chung HY, Sung B, Jung KJ, Zou Y, Yu BP (2006) The molecular inflammatory
process in aging. Antioxid Redox Signal 8: 572–581.
8. Podowski M, Calvi CL, Cheadle C, Tuder RM, Biswals S, et al. (2009) Complex
integration of matrix, oxidative stress, and apoptosis in genetic emphysema.
Am J Pathol 175: 84–96.
9. Hasler P, Zouali M (2005) Immune receptor signaling, aging, and autoimmu-
nity. Cell Immunol 233: 102–108.
10. Boren E, Gershwin ME (2004) Inflamm-aging: autoimmunity, and the immune-
risk phenotype. Autoimmun Rev 3: 401–406.
11. Larbi A, Fulop T, Pawelec G (2008) Immune receptor signaling, aging and
autoimmunity. Adv Exp Med Biol 640: 312–324.
12. Langrish CL, Chen Y, Blumenschein WM, Mattson J, Basham B, et al. (2005)
IL-23 drives a pathogenic T cell population that induces autoimmune
inflammation. J Exp Med 201: 233–240.
13. Kolls JK, Linden A (2004) Interleukin-17 family members and inflammation.
Immunity 21: 467–476.
14. Fisher GJ, Quan T, Purohit T, Shao Y, Cho MK, et al. (2009) Collagen
fragmentation promotes oxidative stress and elevates matrix metalloproteinase-1
in fibroblasts in aged human skin. Am J Pathol 174: 101–114.
15. Dasgupta J, Kar S, Liu R, Joseph J, Kalyanaraman B, et al. (2010) Reactive
oxygen species control senescence-associated matrix metalloproteinase-1
through c-Jun-N-terminal kinase. J Cell Physiol 225: 52–62.
16. Huang K, Rabold R, Schofield B, Mitzner W, Tankersley CG (2007) Age-
dependent changes of airway and lung parenchyma in C57BL/6J mice. J Appl
Physiol 102: 200–206.
17. Sato T, Seyama K, Sato Y, Mori H, Souma S, et al. (2006) Senescence marker
protein-30 protects mice lungs from oxidative stress, aging, and smoking.
Am J Respir Crit Care Med 174: 530–537.
18. Dorshkind K, Montecino-Rodriguez E, Signer RA (2009) The ageing immune
system: is it ever too old to become young again? Nat Rev Immunol 9: 57–62.
19. McElhaney JE, Effros RB (2009) Immunosenescence: what does it mean to
health outcomes in older adults? Curr Opin Immunol 21: 418–424.
Impaired Tissue Homeostasis in the Aging Lung
PLoS ONE | www.plosone.org10 June 2011 | Volume 6 | Issue 6 | e20712
20. Pawelec G (1999) Immunosenescence: impact in the young as well as the old? Download full-text
Mech Ageing Dev 108: 1–7.
21. Listi F, Candore G, Modica MA, Russo M, Di Lorenzo G, et al. (2006) A study
of serum immunoglobulin levels in elderly persons that provides new insights
into B cell immunosenescence. Ann N Y Acad Sci 1089: 487–495.
22. Gibson KL, Wu YC, Barnett Y, Duggan O, Vaughan R, et al. (2009) B-cell
diversity decreases in old age and is correlated with poor health status. Aging
Cell 8: 18–25.
23. Guerrettaz LM, Johnson SA, Cambier JC (2008) Acquired hematopoietic stem
cell defects determine B-cell repertoire changes associated with aging. Proc Natl
Acad Sci U S A 105: 11898–11902.
24. Speziali E, Santiago AF, Fernandes RM, Vaz NM, Menezes JS, et al. (2009)
Specific immune responses but not basal functions of B and T cells are impaired
in aged mice. Cell Immunol 256: 1–5.
25. Hogg JC, Chu F, Utokaparch S, Woods R, Elliott WM, et al. (2004) The nature
of small-airway obstruction in chronic obstructive pulmonary disease.
N Engl J Med 350: 2645–2653.
26. van der Strate BW, Postma DS, Brandsma CA, Melgert BN, Luinge MA, et al.
(2006) Cigarette smoke-induced emphysema: A role for the B cell? Am J Respir
Crit Care Med 173: 751–758.
27. Feghali-Bostwick CA, Gadgil AS, Otterbein LE, Pilewski JM, Stoner MW, et al.
(2008) Autoantibodies in patients with chronic obstructive pulmonary disease.
Am J Respir Crit Care Med 177: 156–163.
28. Bolstad BM, Irizarry RA, Astrand M, Speed TP (2003) A comparison of
normalization methods for high density oligonucleotide array data based on
variance and bias. Bioinformatics 19: 185–193.
29. Tavazoie S, Hughes JD, Campbell MJ, Cho RJ, Church GM (1999) Systematic
determination of genetic network architecture. Nat Genet 22: 281–285.
30. D’Alessio FR, Tsushima K, Aggarwal NR, West EE, Willett MH, et al. (2009)
CD4+CD25+Foxp3+ Tregs resolve experimental lung injury in mice and are
present in humans with acute lung injury. J Clin Invest 119: 2898–2913.
Impaired Tissue Homeostasis in the Aging Lung
PLoS ONE | www.plosone.org11 June 2011 | Volume 6 | Issue 6 | e20712