Am J Physiol Lung Cell Mol Physiol 308: L1189–L1201, 2015.
First published April 17, 2015; doi:10.1152/ajplung.00028.2015.
Review
Respiratory protease/antiprotease balance determines susceptibility to viral
infection and can be modified by nutritional antioxidants
Megan Meyer1 and Ilona Jaspers1,2,3,4
1
Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina;
Center for Environmental Medicine, Asthma and Lung Biology, University of North Carolina at Chapel Hill, Chapel Hill,
North Carolina; 3Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; and
4
Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
2
Submitted 26 January 2015; accepted in final form 13 April 2015
protease/antiprotease; viral infection; nutritional antioxidants
of cells have been identified in the
respiratory tract, including neutrophils, dendritic cells, macrophages, natural killer (NK) cells, B cells, and T cells (125).
Because of this diverse population of cells, a central “orchestrator” is necessary for coordinating an appropriate immune
response to inhaled stimuli. The respiratory epithelium is one
of the first sites within the respiratory tract to be exposed to
inhaled stimuli and functions as a focal orchestrator to organize
and initiate appropriate responses in three distinct ways (Fig. 1).
First, the respiratory epithelium forms a physical barrier
through tightly regulated cell-cell interactions. These interactions promote basic biological functions, protect against invading pathogens, and establish mechanical strength. Second,
epithelial cells express receptors and ligands that coordinate
cellular responses and activate immune cells. These processes
are important for detecting foreign stimuli, communicating
among different cells, and guiding immune responses. Third,
respiratory epithelial cells secrete soluble factors such as cytokines, chemokines, and host defense mediators such as proteases and antiproteases. These secreted, soluble factors are
crucial for the overall host defense response. In the context of
antimicrobial host defense responses, epithelial function can be
modulated by intrinsic factors, such as age, and extrinsic
NEARLY 40 DIFFERENT TYPES
Address for reprint requests and other correspondence: I. Jaspers, Dept. of
Pediatrics, Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 275997310 (e-mail: [email protected]).
http://www.ajplung.org
factors, such as microbial infection, exposure to inhaled toxicants, and supplementation with nutritional antioxidants. As
such, this review will examine the role of epithelial cellderived proteases and antiproteases, in the context of lung
disease and viral infection and offer a potential strategy to
modulate this balance with nutritional antioxidants.
Respiratory Viruses Infect Respiratory Epithelial Cells and
Cause Exacerbations of Lung Disease
Respiratory epithelial cells are targets for respiratory viruses and differ in protease and/or antiprotease sensitivity.
Respiratory epithelial cells are the primary targets for respiratory viral infection based on extracellular ligands and residues
that are found on these cells. More than 200 antigenically
different viruses can infect respiratory epithelial cells (110).
These viruses include coronaviruses, rhinoviruses, metapneumoviruses, enteroviruses, adenoviruses, respiratory syncytial
viruses, measles virus, parainfluenza viruses, and influenza
viruses. These respiratory viruses differ in genome composition, viral structure, specific target cell type, and sensitivity to
components of the respiratory protease/antiprotease balance
(Table 1).
Upon viral infection, respiratory epithelial cells replicate
viral genomes and propagate infectious virions for productive
viral infection. Additionally, respiratory epithelial cells mount
intracellular immune responses to warn neighboring cells as
well as orchestrate recruitment of immune cells to sites of
1040-0605/15 Copyright © 2015 the American Physiological Society
L1189
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Meyer M, Jaspers I. Respiratory protease/antiprotease balance determines
susceptibility to viral infection and can be modified by nutritional antioxidants. Am
J Physiol Lung Cell Mol Physiol 308: L1189 –L1201, 2015. First published April
17, 2015; doi:10.1152/ajplung.00028.2015.—The respiratory epithelium functions
as a central orchestrator to initiate and organize responses to inhaled stimuli.
Proteases and antiproteases are secreted from the respiratory epithelium and are
involved in respiratory homeostasis. Modifications to the protease/antiprotease
balance can lead to the development of lung diseases such as emphysema or chronic
obstructive pulmonary disease. Furthermore, altered protease/antiprotease balance,
in favor for increased protease activity, is associated with increased susceptibility
to respiratory viral infections such as influenza virus. However, nutritional antioxidants induce antiprotease expression/secretion and decrease protease expression/
activity, to protect against viral infection. As such, this review will elucidate the
impact of this balance in the context of respiratory viral infection and lung disease,
to further highlight the role epithelial cell-derived proteases and antiproteases
contribute to respiratory immune function. Furthermore, this review will offer the
use of nutritional antioxidants as possible therapeutics to boost respiratory mucosal
responses and/or protect against infection.
Review
L1190
PROTEASE/ANTIPROTEASE BALANCE AND VIRAL INFECTION
1. Barrier/mechanical function
2. Receptor-mediated interactions
3. Soluble factor production
Surfactant protein,
Lactoferrin
Pathogen
receptor-mediated
interactions
Defensins/
Cathelicidins
Proteases/
Antiproteases
Cellular
receptor-mediated
interactions
Cytokines
Chemokines
infection (152). The respiratory epithelium also secretes soluble mediators such as type II transmembrane serine proteases
(TTSPs) and antiproteases, such as secretory leukocyte protease inhibitor (SLPI), which are implicated in viral infection.
Many respiratory viruses, such as coronaviruses, metapneumoviruses, parainfluenza viruses, and influenza viruses, are sensitive to TTSP-dependent cleavage (Table 1). However,
whereas some respiratory viruses, such as coronaviruses, induce antiprotease expression, only parainfluenza and influenza
are sensitive to SLPI activity (Table 1). Thus investigating the
role of the protease/antiprotease balance in the context of a
parainfluenza or influenza infection could elucidate mechanisms and strategies to prevent viral infections.
Understanding the role of the protease/antiprotease balance
in the context of an influenza infection is of public health
importance, since influenza infects 2–5 million people worldwide each year and can cause worldwide pandemics, as seen
with the recent 2009 H1N1 strain (162). Moreover, in the US
alone, over 20,000 people die because of influenza infection
and related complications (29a). Influenza remains a significant
public health burden due to yearly virus mutations and the
ability of the virus to infect a wide range of hosts. Despite
large-scale vaccination programs and the development of antiviral
therapeutics, influenza-associated morbidity and mortality rates
have not changed in recent years (99). This is of particular
importance in years when the strains used in the influenza vaccine
Table 1. Genome and structure, target cell type, and sensitivity to protease/antiprotease balance of respiratory viruses
Respiratory Virus
Genome; Structure
Adenovirus
dsDNA; Nonenveloped
Rhinoviruses
⫹ssRNA; Nonenveloped
Enteroviruses
⫹ssRNA; Nonenveloped
Coronaviruses
⫹ssRNA; Enveloped
Measles viruses
-ssRNA; Enveloped
Metapneumoviruses
-ssRNA; Enveloped
Respiratory syncytial
viruses
Parainfluenza viruses
-ssRNA; Enveloped
Influenza viruses
-ssRNA; Enveloped
-ssRNA; Enveloped
Target Cell
Epithelial cells using clathrin-dependent
entry mechanisms (119)
Epithelial cells of the upper airway
expressing ICAM-1 (69)
Epithelial cells expressing ICAM-1 or
DAF (129)
Epithelial cells expressing ACE2 or
DPP4 (107, 139)
Epithelial cells expressing nectin 4
(132, 133)
Upper and lower airway epithelial cells
expressing ␣V␤1 integrin (39)
Epithelial cells expressing sulfated
glycosamino-glycans (70)
Epithelial cells expressing 2,3 or 2,6
linked sialic acid residues (55)
Epithelial cells expressing 2,3 or 2,6
linked sialic acid residues (36)
Require Proteolytic Cleavage by
Secreted Respiratory Proteases?
Sensitive to Secreted
Respiratory Antiproteases?
No
No
No
No
No
No
Yes- cleave S protein (17,
19, 20, 64, 77, 157, 194)
No
Yes (96, 142)
Yes- cleave F protein (158)
No
No
No
Yes- cleave F protein (3,
94)
Yes- cleave HA protein (10,
11, 16, 19, 22, 23, 56,
75, 148, 178, 194)
Yes (52, 93, 94)
AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00028.2015 • www.ajplung.org
No
Yes (14, 91, 93, 95)
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Fig. 1. Respiratory epithelial cells function as an “orchestrator” to initiate, coordinate, and respond to inhaled stimuli. 1: Respiratory epithelial cells form a
physical and mechanical barrier to protect against respiratory stimuli. 2: Respiratory epithelial cells express receptors and ligands that interact with neighboring
epithelial cells, respiratory immune cells, and inhaled stimuli such as respiratory pathogens. 3: Respiratory epithelial cells secrete soluble factors such as
cytokines, chemokines, and antimicrobial peptides to contribute to respiratory host defense.
Review
L1191
PROTEASE/ANTIPROTEASE BALANCE AND VIRAL INFECTION
Proteases and antiproteases of the Lung: a Delicate
Enzymatic Balance
Epithelial cell-derived proteases. Proteases are key components of respiratory host defense. In the healthy lung, proteases
maintain tissue homeostasis and their activities are regulated
by antiproteases. Elevated net protease activity is associated
with lung destruction and the development of chronic lung
diseases such as emphysema and chronic obstructive pulmonary disease (COPD) (1, 90). Lung destruction is induced by
proteolytic degradation of extracellular matrix (ECM) components, including collagens, laminins, and elastin, with type I
collagen serving as the major target (164). Cysteine proteases,
matrix metalloproteinases (MMPs), and serine proteases are
prevalent proteases found in the lung, with serine proteases
serving as the most predominant class. Moreover, each family
has unique target substrates, cellular sources, and active sites
(Table 2).
Cathepsins. Cysteine proteases, or cathepsins, are synthesized as proenzymes, require cleavage for activation, and
contain conserved cysteine and histidine residues, with the
cysteine serving as a nucleophile for target motifs. Cathepsin
B, K, L, and S are highly abundant in the lung; cathepsin K,
mainly produced in bronchial epithelial cells, is one of the most
potent matrix-degrading proteases identified to date (26).
Cathepsins cleave elastin, laminin, and collagen as well as
antiproteases such as SLPI and are important for major histocompatibility complex II formation and antigen presentation
(60, 144, 174). Cathepsins have been linked to respiratory virus
infection since cathepsin L cleaves and activates the spike
protein of severe acute respiratory syndrome coronavirus
(SARS-CoV), and cathepsin B is induced during IAV infection
in mice (20, 27).
MMPs. MMPs are a family of metal endopeptidases that
target ECM components. Secreted by immune cells and epithelial cells, MMPs possess a common prodomain and catalytic
domain. Because MMPs are synthesized as inactive zymogens,
the signal domain must be cleaved for MMPs activity. The
catalytic domain contains a conserved zinc binding site that
confers protease activity. MMP1, 2, 3, 7, 8, 9, 12, and 13 are
the main MMPs found in the respiratory tract and are induced
by growth factors, reactive oxygen species, and proinflammatory cytokines (42, 68, 100). MMP1, 8, and 13 cleave collagen,
whereas MMP2 and 9 act as gelatinases and also degrade
elastin and type IV collagen (46, 84).
Additionally, increased MMP activity is associated with
inflammation. Baseline MMP levels such as MMP1, 2, 3, 8,
and 9 are elevated in smokers and patients with COPD (40).
Moreover, MMP9 cleaves proinflammatory cytokines such as
interleukin-8 (IL-8), resulting in the release of a modified,
more potent neutrophil chemotactic mediator (134). MMP12
degrades elastin but also inactivates key antiproteases in the
lung such as SLPI (140). Although MMP activity has not been
implicated in viral activation, MMPs are involved in virusinduced inflammation. In epithelial cell lines and mouse models, MMP2 and 9 are induced during IAV infection and
contribute to the IAV-associated lung destruction (105, 185,
190). Tacon et al. (171) demonstrated that MMP levels are
Table 2. Target substrates, sources, and activity of respiratory proteases
Target Substrates
Sources in the Lung
Activity
Cathepsin B, K, L, and S
Proteases in the Respiratory Tract
Elastin, laminin, antiproteases, MHC II,
respiratory viruses (20, 60, 144, 174)
Bronchial epithelial cells, macrophages,
dendritic cells (26, 58)
MMP1, 2, 3, 7, 8, 9, 12, and 13
Collagen, elastin, inflammatory
cytokines such as IL-1␤ and IL-8,
defensins, antiproteases (46, 84, 188)
Elastin, collagen, fibronectin, laminin,
proteoglycans, immunoglobulins,
complement components, T cell
receptors. antiproteases (47, 65, 116,
121, 167, 170, 180, 186)
Fibrinogen, urokinase, respiratory
viruses (17, 23, 104, 158, 176)
Epithelial cells, alveolar macrophages,
monocytes, neutrophils, mast cells,
eosinophils (41, 51, 147)
Mature neutrophils (80)
Cysteine proteases with cysteine
serving as a nucleophile for
target motifs
Metal endopeptidases with
conserved zinc binding site
Neutrophil-derived proteases (NE,
cathepsin G, proteinase 3)
TTSPs
Epithelial cells (25)
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Serine proteases with conserved
catalytic triad of serine,
aspartic acid, and histidine
Serine proteases with conserved
catalytic triad of serine,
aspartic acid, and histidine
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do not antigenically match the circulating influenza virus strains,
as is the case in the current 2014 –2015 influenza epidemic (54).
Therefore, examining strategies such as modulating the protease/
antiprotease balance may serve as alternative targets to prevent
influenza infection.
HA must be proteolytically cleaved for productive IAV
infection. For influenza A virus (IAV) to infect cells, the viral
membrane protein hemagglutinin (HA) must be proteolytically
cleaved and activated by respiratory serine proteases. HA
resides on the virion as a fusion-inactive precursor (HA0), and
upon proteolytic cleavage a fusion-active trimer with disulfidelinked HA1 and HA2 subunits is generated (16, 18, 22, 23).
After entry into the cell, the virion enters endosomal compartments, and upon acidification of the endosome viral and host
membranes fuse together. The membrane fusion allows for
viral entry into the cell, so viral replication can initiate (98).
Most HA0 will be cleaved at either a single arginine or
lysine residue to generate active HA1 and HA2. For lowpathogenic IAV strains, respiratory trypsin-like serine proteases cleave at monobasic arginine or lysine residues (21,
102). However, highly pathogenic avian influenza viruses possess multibasic cleavage sites that can be cleaved by ubiquitous
intracellular furin-like serine proteases (166, 182). This difference in HA cleavage motifs increases susceptibility for systemic, widespread infection to sites such as the central nervous
system rather than localized, restricted infection to sites within
the respiratory tract. Consequently, investigating strategies to
block protease-induced HA activation and increase antiprotease activity may serve as a potential approach to protect
against IAV infection.
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PROTEASE/ANTIPROTEASE BALANCE AND VIRAL INFECTION
either during attachment and entry into host cells or during
budding and shedding of virions from an infected cell (21).
TMPRSS2 possesses a wide range of functions specific to
epithelial cell biology. Donaldson et al. (45) demonstrated that
TMPRSS2 reduced epithelial sodium channel activity, indicating a role for TMPRSS2 in ion transport. TMPRSS2 can be
expressed as a full-length 70-kDa protein or a variety of
smaller, truncated forms, depending on tissue site and localization (3, 30). Additionally, TMPRSS2 is implicated in the
activation of respiratory viruses. Cells expressing TMPRSS2
show enhanced replication of human metapneumovirus as well
as increased activation and replication of SARS-CoV (19,
158). Bottcher et al. (22, 23) reported that cells stably expressing TMPRSS2 resulted in the monobasic cleavage of HA,
which elevated IAV replication. In recent reports, TMPRSS2
has been shown to preferentially cleave H1N1, and to a lesser
degree H3N2. Hatesuer et al. (75) revealed that viral titers in
H1N1-infected TMPRSS2-deficient mice were significantly
reduced compared with wild-type animals. Furthermore, this
group reported that although H3N2 replicated at similar levels
in both knockout and wild-type mice, the knockout mice had
increased survival and body weight post-H3N2 infection (75).
Recent reports have further expanded on the HA specificity
of TMPRSS2. One group demonstrated that TMPRSS2 knockout mice were protected from lethal H1N1 but not lethal H3N2
infection, whereas another group showed that TMPRSS2
knockout mice were protected against both H1N1 and H3N2
infection (148, 178). These opposing findings suggest that HA
activation not only depends on HA structure but can vary
among different viral strains bearing the same HA subtype.
Despite these differences, there is substantial evidence implicating TTSPs in IAV pathogenesis.
Epithelial cell-derived antiproteases. Antiproteases are a
broad class of proteins that inhibit proteases and modulate
immune responses in the lung. Respiratory antiproteases are
comprised of four families: tissue inhibitors of metalloproteinases (TIMPs), serine protease inhibitors (serpins), trappin-2/
elafin, and SLPI. Each antiprotease family has unique target
substrates, cellular sources, and antiprotease function (Table 3).
Although the primary function of antiproteases is to inhibit
protease function, new and emerging studies indicate that
antiproteases inhibit overt inflammatory cascades, protect
against the development of chronic respiratory diseases, and
block microbial infection. As such, antiproteases are critical
components of respiratory host defense responses.
TIMPs. TIMP1-4 are produced by alveolar epithelial cells
and alveolar macrophages (97). TIMPs inhibit MMP activity
through the formation of an NH2-terminal reactive ridge domain that inserts into the active site of the target MMP (24,
103). The TIMP family share about 40% homology and have
overlapping anti-MMP activity. TIMPs induce cell growth and
proliferation. TIMP1 and 2 activate growth of keratinocytes,
fibroblasts, and erythroid cells (15, 76).
Furthermore, TIMPs have been shown to be associated with
chronic lung diseases such as asthma and COPD. Circulating
granulocytes from patients with asthma have an impaired
ability to release TIMP1, the main inhibitor of MMP9, after
stimulation with phorbol 12-myristate 13-acetate (28). Additionally, although it has been shown that sputum samples from
stable COPD patients contain elevated TIMP1 levels during a
COPD exacerbation, others have reported that TIMP1 levels
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elevated in vitro and in vivo during human rhinovirus (HRV)
infection, which was dependent on NF-␬B, further illustrating
the role of MMPs in virus-induced respiratory inflammation.
TTSPs. Serine proteases make up the largest portion of
respiratory proteases (66). Whereas neutrophil elastase (NE),
proteinase 3, and cathepsins G are neutrophil-derived proteases, other serine proteases such as TTSPs are produced and
secreted by the respiratory epithelium. TTSPs are a family of
proteases that possess an NH2-terminal transmembrane domain, a stem region, and a COOH-terminal serine protease
domain that contains the conserved catalytic triad composed of
histidine, aspartic acid, and serine (25). Protease activity is
attained through the nucleophilic serine, which attacks the
substrate’s carbonyl functional group. This activity generates
an acyl intermediate, transfers a proton from the positively
charged histidine to the substrate, and allows for the hydrolysis
and cleavage of the substrate (5). TTSPs are synthesized as a
single-chain, inactive proenzyme and require cleavage following a basic arginine or lysine (82). The catalytic domains are
located on the terminal extracellular region to permit direct
exposure to the extracellular environment.
TTSPs are comprised of four subfamilies: the human airway
trypsin-like protease (HAT)/differentially expressed in squamous cell carcinoma (DESC) subfamily that include HAT,
DESC1, and HAT-like 1–5; the hepsin/transmembrane protease
serine (TMPRSS) subfamily contains hepsin, TMPRSS2-5 and 13,
mosaic serine protease large-form, and enteropeptidase; the
corin protease; and the matriptase subfamily that includes
matriptase 1–3 and polyserase-1 (5). Although TTSPs are
transmembrane proteases, release of the extracellular domains
has been reported for several of the TTSPs such as matriptase,
HAT, and TMPRSS2 (3, 189). Moreover, matriptase, HAT,
and TMPRSS2 are widely expressed in epithelial tissues,
including the respiratory tract.
Matriptase cleaves and activates protease-activated receptor
2 (PAR2), hepatocyte growth factor, and urokinase, indicating
that matriptase plays a role not only in ECM degradation but
also in epithelial cell differentiation and remodeling (104, 176).
In the respiratory epithelium, matriptase is expressed on the
basolateral side of the epithelial cells. Upon activation, matriptase migrates to the apical side of the epithelium, where it can
be secreted into the lumen of the respiratory tract. Recent
studies have revealed that intracellular matriptase localizes to
the plasma membrane and endosomes, allowing for interaction
and activation of the IAV virion (11, 73). Moreover, secreted
matriptase cleaves HA protein on the IAV virion, including
H1, but not H2 or H3, which activates the virion and allows for
enhanced rates of replication (11, 73).
HAT cleaves fibrinogen, activates PAR2, and urokinase
(11). In sputum samples from patients with chronic airway
disorders such as bronchitis or asthma, secreted HAT levels are
elevated (189). Moreover, Chokki et al. (34) demonstrated that
HAT enhanced MUC5AC gene expression, resulting in increased mucus production. HAT is produced and secreted by
ciliated epithelial cells and is not found in basal or goblet cells
of the respiratory tract (175). HAT activates the SARS-CoV
viral spike protein necessary for host cell entry (17). Moreover,
HAT cleaves the monobasic site of the HA protein required for
HA activation, enhancing IAV replication (21–23). Cleavage
of HA by HAT has been shown to occur at the cell surface
Review
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PROTEASE/ANTIPROTEASE BALANCE AND VIRAL INFECTION
Table 3. Target substrates, sources, and activity of respiratory antiproteases
Antiproteases in the
Respiratory Tract
Target Substrates
TIMPs
MMPs (24)
Serpin
Serine and cysteine proteases,
caspases, bacteria, viruses (9, 57,
138, 141, 154)
NE and proteinase-3, enveloped
viruses (193)
NE, cathepsin G, chymase, bacteria,
and viruses (62, 184)
Trappin/Elafin
SLPI
Sources in the Lung
Alveolar epithelial cells, alveolar
macrophages, granulocytes (28, 97)
Epithelial cells, alveolar macrophages
(35, 115, 159)
Tracheal epithelial cells, club cells, type
II cells, alveolar macrophages (123)
Epithelial cells, neutrophils, macrophages
(48, 121)
Activity
Insert reactive ridge NH2-terminal domain into MMP
active site, inhibit apoptosis (103, 120)
Insert RCL into the active site of the targeted
protease, inhibit LPS-induced inflammation, block
microbial infection (101, 130)
Contain WAP domain for antiprotease activity,
inhibit viral infection (59, 150, 156, 187)
Contain WAP domain for antiprotease activity,
antimicrobial effects due to NH2-terminal domain
and high cationicity, block inflammatory cascades,
inhibit viral infection (67, 91, 93, 150, 156, 172,
173, 187)
are decreased (40, 120). Moreover, Mercer et al. (120) demonstrated that during a COPD exacerbation there was a shift in
the protease/antiprotease molar balance, with an increase in
MMP9 and a decrease in TIMP1. In the context of viral
infection, overexpressing TIMP-1 blocks respiratory syncytial
virus syncytia formation (191). However, since MMP activity
is directly implicated in viral activation and viral entry/fusion,
there are limited studies that investigate the direct antiviral
effects of TIMPs.
Serpins. Serpins are the largest and most broadly distributed
superfamily of protease inhibitors. The majority of serpins
inactivate serine proteases, but a small portion of serpins also
inhibits caspase and cysteine proteases (141, 154). Serpins
exert their antiprotease activity by undergoing irreversible
conformational changes that allow for the interaction and
inactivation of serine proteases. To inhibit protease activity,
serpins form long, flexible reactive center loops (RCL) that
insert into the active site of the targeted protease. These RCL
structures form above the serpin framework and allow for the
NH2-terminal portion of the RCL to insert into the protease
active site. At this time, the protease and serpin remain covalently linked, the protease structure is conformationally
changed, and the protease activity is significantly reduced
(101). This action leads to the permanent deactivation of both
serpin and protease, often described as a “suicide” event.
SerpinA1 encodes the most abundant antiprotease in the
lung, alpha-1 antitrypsin (␣-1AT). Point mutations of ␣-1AT
generates a misfolded version of this protein, resulting in an
␣-1AT deficiency (109). ␣-1AT-deficient individuals are at a
high risk for developing emphysema and COPD (168, 169).
Respiratory epithelial cells secrete ␣-1AT in both the apical
and basolateral compartments of the cell (159). Additionally,
serpins have been shown to block viral entry of enveloped
viruses such as human immunodeficiency virus (HIV) and
herpes simplex virus (9, 138). Smee et al. (161) showed that in
vitro serpin antithrombin III treatment inhibited IAV infection
nearly 100-fold more than ribavirin, a nucleoside inhibitor used
to halt IAV replication. The authors found that this effect was
dependent on viral HA with decreasing efficacy in order of
H1N1⬎H3N2⬎H5N1 (161), suggesting that serpin activity
may parallel the TTSP activity in the context of IAV infections, which is dependent on amino acid sequence, protein
structure, and/or HA substrate accessibility. A recent study
demonstrated that plasminogen activator inhibitor 1 (PAI-1),
encoded by SerpinE1, inhibited HA cleavage and reduced
infectivity of progeny virions (44). Specifically, these studies
demonstrated that antiproteases, such as PAI-1, regulate the
spread of IAV infection by blocking proteolytic maturation of
newly formed virus particles and propagation of the infection.
Thus protease/antiprotease balances may be important mucosal
regulators of IAV spreading.
Trappin/elafin. Trappin-2 is a cationic 9.9-kDa serine antiprotease secreted into the lung as a nonglycosylated preprotein.
Upon proteolytic cleavage, trappin-2 is converted to elafin, a
6-kDa protein. Structurally, trappin-2 and elafin contain a
cysteine-rich region, joined by four disulfide bonds, termed the
whey acidic protein domain (WAP) (153). The structure of
elafin is divided into two regions, the COOH-terminal region,
which comprises the antiprotease active site, and NH2-terminal
region, which allows for interactions with ECM proteins (149).
Elafin specifically inhibits NE and proteinase-3 and is secreted
from tracheal epithelial cells, club cells, type II cells, and
alveolar macrophages (123, 187).
Trappin-2 and elafin function as alarm antiproteases, as both
are produced in response to inflammatory stimuli (136). In
respiratory epithelial cells, elevated elafin levels are protective
against recombinant NE treatment and products derived from
activated human neutrophils in vitro (160). In addition to
functioning as an alarm antiprotease, elafin possesses antimicrobial effects. Ghosh et al. (59) revealed that recombinant
elafin was protective during HIV infection only when elafin
was preincubated with HIV, suggesting that elafin directly
interacts with HIV virion and prevents viral infection. Interestingly, this group found that secreted elafin levels were
elevated in the cervicovaginal lavages of HIV-negative women
compared with HIV-positive women, albeit not statistically
significant, suggesting that elafin secretions may be decreased
after HIV infection. Similarly, after HRV infection, elafin is
downregulated in subjects with COPD (113). These findings
suggest that, despite elafin functioning as an alarm mediator,
elafin secretions may be decreased after viral infection, thus
enhancing susceptibility to viral infection.
SLPI. SLPI is an additional alarm antiprotease that is highly
expressed by epithelial cells, neutrophils, and macrophages.
SLPI can be detected in respiratory secretions, saliva, seminal
fluid, cervical mucus, tears, and cerebral spinal fluid (78, 151).
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MMPs, matrix metalloproteinases; NE, neutrophil elastase; RCL, reactive center loops; SLPI, secretory leukocyte protease inhibitor; TIMPs, tissue inhibitors
of metalloproteinases; WAP, whey acidic protein domain.
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PROTEASE/ANTIPROTEASE BALANCE AND VIRAL INFECTION
released from infected cells were secreted as noninfectious
HA0 precursor virions (95). Additionally, we have shown that
recombinant human SLPI inhibits IAV replication in vitro (91).
Although SLPI contributes to respiratory host defense by
decreasing inflammation and infection, SLPI can also induce
proinflammatory responses. Mulligan et al. (126) demonstrated
that posttranslational modifications of SLPI increased IL-8
secretion, neutrophil recruitment, and vascular permeability.
Extracellular SLPI can be posttranslationally modified by respiratory proteases such as cathepsin L, MMP12, chymase, and
NE, resulting in cleavage of SLPI (12, 116, 140, 174). Cleaved
SLPI levels are elevated in COPD patients during respiratory
infection, which renders SLPI nonfunctional (1, 113, 135).
Moreover, we have shown that in nasal lavage fluid from
smokers, SLPI is posttranslationally processed and cleaved
(121). Reactive oxygen intermediates generated in the context
of environmental exposures to oxidant pollutants, such as
cigarette smoke, modify the active site and decrease the antiprotease properties of SLPI (29, 183). Whereas SLPI exerts
powerful protective effects in the respiratory tract, posttranslational alterations and/or oxidative modifications can drastically shift the activity of SLPI toward potentially damaging
effects and may reduce its antiviral properties.
The protease/antiprotease balance is a delicate interaction of
enzymes and proteins that are involved in respiratory function.
In the healthy lung, proteases are critical for cellular regeneration, cellular repair, and tissue homeostasis. Furthermore,
antiproteases are important for protease inhibition and contribute to host defense. In the healthy lung, although there are
many types of proteases and antiproteases expressed, a balance
between proteases and antiproteases is established to ensure
respiratory homeostasis. Balance will depend on the overall
presence, timing of protease and antiprotease production/release, and activity. A shift in the balance toward increased
protease expression and activity can lead to overt inflammation
and the development of chronic lung disorders such as COPD
and emphysema (1, 90). Moreover, respiratory serine proteases, such as the TTSP family, activate a variety of respiratory viruses for enhanced rates of viral infection. Additionally,
antiproteases, such as SLPI, inhibit the function of serine
proteases and block viral entry into the target cell. Hence the
protease/antiprotease balance not only is a critical component
for respiratory homeostasis but also is a powerful determinant
of respiratory viral pathogenesis.
Nrf2 Decreases Oxidative Stress, Modifies Components of
the Protease/antiprotease Balance, and Protects Against the
Development of Lung Disease and Viral Infection
The respiratory epithelium is constantly being exposed to
inhaled insults, which results in the generation of reactive
oxygen species, free radicals, and peroxides (13). Oxidative
stress has been associated with cancer, cardiovascular diseases,
neurodegenerative diseases, and chronic inflammation (49, 89).
Cells are equipped with various mechanisms, such as antioxidant responses, to block these damaging effects (71, 72). One
mechanism involves nuclear factor (erythroid-derived 2)-like 2
(Nrf2), a key transcription factor that regulates that transcription of many antioxidant and detoxifying enzymes (32, 63).
Under baseline conditions, Nrf2 is sequestered in the cytoplasm by kelch-like ECH-associated protein 1 (Keap1), and
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Comprised of two closely related domains, the tertiary structure of SLPI resembles a boomerang (48). Similar to the
trappin family, SLPI contains a cysteine-rich WAP COOHterminal domain that is responsible for protease inhibition as
well as the NH2-terminal domain that stabilizes the protease/
antiprotease complex and exerts antimicrobial activity (192).
The COOH-terminal active site, which confers antiprotease
activity, is comprised of leucine and methionine residues (50,
181).
The main function of SLPI is to inhibit serine proteases such
as NE (its main target), cathepsin G, elastase, and chymase (62,
184). SLPI further contributes to respiratory host defense by
modulating inflammation and inducing wound healing. SLPI
exerts cellular anti-inflammatory effects by preventing the
degradation of regulatory components of the NF-␬B pathway
and by directly competing for NF-␬B binding sites in the
promoter regions of proinflammatory cytokines such as IL-8
and tumor necrosis factor-␣ (TNF-␣) (67, 172, 173). SLPI is
also implicated in wound healing as reported by Ashcroft et al.
(8), who investigated the role of SLPI in a mouse model of
dermal wounds. Using this model, the authors found that SLPI
knockout mice had impaired wound healing due to increased
NE activity.
SLPI possesses broad antimicrobial effects. The NH2-terminal domain of SLPI possesses extracellular antibacterial activity by disrupting Staphylococcus aureus and Escherichia coli
membranes (79). Additionally, SLPI binds to Opa protein of
Neisseria gonorrhoeae, which leads to the lysis of bacterial
membranes (37). Fahey et al. (53) reported that increased
extracellular SLPI secretions were correlated with elevated
bactericidal activity and treatment with an anti-SLPI antibody
significantly diminished bactericidal activity. SLPI has been
shown to exert intracellular antibacterial effects by disrupting
translation of E. coli during bacterial replication (124). Furthermore, SLPI exerts fungicidal and fungistatic activity toward Aspergillus fumigatus and Candida albicans that is also
dependent on the NH2-terminal domain of SLPI (179).
In the context of viral infections, SLPI inhibits HIV, human
papillomavirus (HPV), and respiratory viruses. McNeely et al.
(117) first detailed the anti-HIV activity of SLPI in human
saliva. The authors demonstrated that SLPI was highly expressed in the saliva and inhibited HIV infection in monocytes
and T cells in dose-dependent fashion. Further studies revealed
that SLPI exerts anti-HIV properties not through direct interaction with the virus, but rather through interacting with the
target host cell (86). However, this group found that, despite
decreasing viral load, SLPI did not alter the infectivity of
progeny virions, suggesting that SLPI inhibits HIV at the early
stages of viral infection (118).
SLPI is also associated with protection from human papillomavirus, with two recent studies demonstrating that high
levels of SLPI, possibly induced by cigarette smoking, correlate with protection against HPV infection (81, 137). Additionally, SLPI is induced and protective during respiratory virus
infection. Kido et al. (93) first demonstrated that SLPI decreased infectivity of IAV and Sendai viruses. Moreover,
reports have revealed that intranasal administration with recombinant SLPI (rSLPI) decreased progeny virus nearly 3,000fold compared with vehicle-treated mice infected with mouseadapted IAV (95). Furthermore, the authors also reported that
in mice administered rSLPI nearly all the progeny viruses
Review
L1195
PROTEASE/ANTIPROTEASE BALANCE AND VIRAL INFECTION
and decreased influenza replication (91). Furthermore, we
previously reported that EGCG blocked influenza entry into
target epithelial cells (92). Together, these results suggest that
antioxidant supplementation with flavan-3-ols may serve as an
attractive therapy to protect against lung disease and infection.
SFN alters SLPI and TMPRSS2 levels, enhances lung host
defense responses, and protects against infection. Another
potent antioxidant is sulforaphane (SFN), an isothiocyanate
that induces respiratory host defense by decreasing oxidative
stress and inflammation (111). SFN also exerts broad antimicrobial effects. SFN supplementation of alveolar macrophages
from COPD patients increases clearance of Pseudomonas
aeruginosa (74). Furthermore, SFN elevates bacterial detection
and induces phagocytosis in alveolar macrophages from COPD
patients, which is dependent on Nrf2 activation (74).
SFN is derived from the hydrolysis of glucosinolates found
in cruciferous vegetables such as broccoli, brussels sprouts,
and cabbage. Using in vitro models of respiratory epithelial
cells, SFN supplementation enhances Nrf2 activity, induces
cellular antioxidants such as HO-1 and NQO1, and inhibits
proinflammatory cytokine release (106, 145, 165, 177). Furthermore, Riedl et al. (143) reported that in vivo SFN suppleO
S
C
S
H3C
N
Sulforaphane
Decrease inflammation
Reduce oxidative stress and
cellular toxicity
**Modify protease/antiprotease balance
Nrf2
Keap1
Nrf2
ARE
TMPRSS2
Nrf2
SLPI
Cytoprotective genes,
Antioxidants
Fig. 2. Sulforaphane (SFN) dampens inflammation, alleviates oxidative
stress, and alters the protease/antiprotease balance. SFN enters the cell and
induces Nrf2 release from its cytoplasmic repressor, Keap1. Nrf2 translocates into the nucleus and binds antioxidant response element (ARE)
regions on various cytoprotective promoters. Secretory leukocyte protease
inhibitor (SLPI) contains ARE sites on its promoter and SFN induces
Nrf2-dependent activation of SLPI. Additionally, SFN decreases type II
transmembrane serine proteases (TTSP) expression such as hepsin/transmembrane protease serine 2 (TMPRSS2).
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upon stimulation Nrf2 is released from Keap1 and Nrf2 translocates into the nucleus and binds to antioxidant response
elements (ARE) (85). ARE binding sites are located on the
promoter regions of phase II enzymes such as NADPH quinone
oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) (33).
The Nrf2-dependent phase II response has been implicated in
the development of chronic lung diseases since antioxidant
capacity and Nrf2 protein levels are decreased in the lungs of
COPD patients (112). Iizuka et al. (83) reported that Nrf2deficient mice exposed to cigarette smoke developed emphysema-like qualities and were unable to induce antiproteases
such as SLPI. SLPI contains ARE regions on its promoter and
we have previously demonstrated that Nrf2 agonists enhance
SLPI expression in airway epithelial cells in an Nrf2-dependent
manner (122), suggesting that oxidative stress responses modulate SLPI regulation.
In addition to its role in maintaining oxidant/antioxidant
homeostasis, we and others have previously shown that Nrf2 is
critical for respiratory epithelial responses to respiratory viral
infection (31, 92). Nrf2 has been shown to regulate and
decrease TMPRSS2 expression in prostate cancer cell lines,
suggesting that in addition to inhibiting respiratory oxidative
stress, Nrf2 alters components of the protease/antiprotease
balance and protects against respiratory infection (155). Thus
strategies activating Nrf2 may be a potential mechanism to not
only reduce oxidative stress, but also protect against viral
infection.
Antioxidants are an attractive strategy to modulate the
protease/antiprotease balance and protect against chronic
lung disease and viral infection. Antioxidants have garnered
much attention due to the inhibition of respiratory oxidative
stress yet may also serve as therapies to modify the protease/
antiprotease balance and inhibit viral infection (88). Antioxidants are a broad class of molecules that can be found in a
variety of foods and have been shown to exert potent host
defense effects. One potent class of antioxidants includes
flavonoids, which are nonessential, polyphenolic phytonutrients that occur naturally in many plant-based foods and possess
profound effects on human health (38). Over 8,000 flavonoid
compounds have been identified, and they can be broken down
into six groups: flavonols, flavones, isoflavones, flavanones,
anthocyanidins, and flavan-3-ols (43). Flavan-3-ols (or commonly referred to as flavanols or catechins) are the most
common flavonoid consumed in the American diet and can be
found in red wine, tea, apple, grapes, and chocolate (7).
Flavan-3-ols found in green tea, such as epigallcatechin-3gallate (EGCG), have been studied extensively in the context
of a viral infection. EGCG has been shown to bind to CD4 T
cell receptors and prevent interaction with gp120, a HIV
envelope protein (87). Nance et al. (128) showed that EGCG
was able to inhibit HIV infection of human T cells and
macrophages at physiological doses. Because of these effects,
use of EGCG as an anti-HIV therapeutic has entered preclinical
investigation. Furthermore, EGCG possesses potent anti-influenza properties. EGCG agglutinates influenza viruses and inhibits viral replication, which was dependent on influenza
subtype (127, 163). Ling et al. (108) corroborated these findings, showing that oral administration of EGCG in mice
infected with influenza had nearly a 50% decrease in viral titers
and a 50% increase in survival rates. We have shown that
EGCG induced SLPI secretion, reduced TMPRSS2 secretion,
Review
L1196
PROTEASE/ANTIPROTEASE BALANCE AND VIRAL INFECTION
2.
Serine
protease
reveal that SFN increases SLPI secretion and decreases TMPRSS2 expression in a variety of models, as summarized in
Fig. 2.
Indeed, our studies indicate that SFN is protective against
respiratory viruses such as IAV. We have shown that SFN
decreases IAV replication by reducing IAV entry into respiratory epithelial cells (92). Furthermore, we and others have
shown that SFN decreases TMPRSS2 expression, which we
speculate is mediating the SFN-dependent protection against
influenza (unpublished data) (61, 155). Furthermore, our group
recently showed that sulforaphane-containing broccoli sprouts
significantly decreased IL-6 and markers of viral replication in
nasal lavage fluid from smokers in subjects inoculated with the
live attenuated influenza virus vaccine (131). These results
indicate that SFN may be a safe, low-cost intervention for
decreasing influenza infection in susceptible populations such
as smokers. On the basis of the data summarized above, we
hypothesize that the anti-IAV effects of SFN may be mediated
3.
Strain dependence?
SLPI
IAV
O
S
IAV
C
S
H3C
N
IAV
Sulforaphane
1.
IAV
N
N
HA0
HA1
Serine
protease
IAV
AV
IAV
TM
C
S–S
N HA2 TM
TMPRSS2
C
SLPI
C
Egress
IAV
Uncoating
Translation/Assembly
Replication
SLPI
vRNA synthesis
TMPRSS2
Fig. 3. The protease/antiprotease balance regulates influenza A virus (IAV) infection and can be modulated by SFN supplementation. 1: For entry into target host
cells, the hemagglutinin (HA) protein on the IAV virion must be proteolytically cleaved from HA0 (purple) into fusoactive HA1 and HA2 proteins (blue). 2:
Respiratory serine proteases such as TMPRSS2 (TM), the human airway trypsin-like protease (HAT), and matriptase have been implicated in HA activation and
IAV infection, which are likely to strain specific. Although antiproteases such as SLPI inhibit serine protease activity and protect against IAV infection, it remains
unknown what HA-activating protease is inhibited by SLPI and how SLPI mediates these effects. 3: Lastly, SFN supplementation can be used to increase SLPI
secretion, reduce TMPRSS2 secretion, and decrease viral entry and downstream IAV infection.
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mentation enhanced respiratory phase II enzyme expression,
indicating that nutritional SFN supplementation induces respiratory antioxidant responses.
Beyond its antioxidant function, SFN may have important
functions in modulating components of the protease/antiprotease balance. We have shown that SFN supplementation
transcriptionally enhances SLPI expression, resulting in increased SLPI secretion in the nasal mucosa in an Nrf2-dependent fashion (122). SFN also inhibits MMP expression and
inflammation (4, 114, 146). SFN decreases TTSPs, such as
TMPRSS2, by downregulating androgen receptor (AR) signaling, a receptor involved regulation of TMPRSS2 expression
(61). Recent reports from Schultz et al. (155) have confirmed
and expanded these findings showing that Nrf2 negatively
regulates AR transactivation of androgen response genes such
as TMPRSS2. The authors found that nuclear factor (erythroidderived 2)-like 1, a cytoplasmic transactivator of the AR, is
sequestered in the nucleus when Nrf2 is induced. These studies
Review
PROTEASE/ANTIPROTEASE BALANCE AND VIRAL INFECTION
in a two-pronged approach: 1) by inducing SLPI into the nasal
mucosa, which protects against influenza entry into respiratory
epithelial cells, and 2) by decreasing TMPRSS2 secretion,
which decreases HA activation and IAV replication (Fig. 3).
11.
Summary
GRANTS
12.
13.
14.
15.
16.
17.
18.
This research was funded by National Institutes of Health Grants
R01HL095163 (I. Jaspers) and R01ES020897.
19.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
20.
M.M. and I.J. prepared figures; M.M. and I.J. drafted manuscript; M.M. and
I.J. edited and revised manuscript; M.M. and I.J. approved final version of
manuscript.
21.
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