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Enhancement of Respiratory Mucosal Antiviral Defenses
by the Oxidation of Iodide
Anthony J. Fischer
1–3
, Nicholas J. Lennemann
4
, Sateesh Krishnamurthy
3
,Pe
´ter Po
´cza
5
, Lakshmi Durairaj
6
,
Janice L. Launspach
6
, Bethany A. Rhein
3
, Christine Wohlford-Lenane
3
, Daniel Lorentzen
5
, Botond Ba
´nfi
5,6
,
and Paul B. McCray, Jr.
1–4,6
1
Interdisciplinary PhD Program in Genetics,
2
Medical Scientist Training Program,
3
Department of Pediatrics,
4
Department of Microbiology,
5
Department of Anatomy and Cell Biology, and
6
Department of Internal Medicine, Carver College of Medicine, University of Iowa, Iowa City, Iowa
Recent reports postulate that the dual oxidase (DUOX) proteins
function as part of a multicomponent oxidative pathway used by the
respiratory mucosa to kill bacteria. The other components include
epithelial ion transporters, which mediate the secretion of the
oxidizable anion thiocyanate (SCN
2
) into airway surface liquid,
and lactoperoxidase (LPO), which catalyzes the H
2
O
2
-dependent
oxidation of the pseudohalide SCN
2
to yield the antimicrobial
molecule hypothiocyanite (OSCN
2
). We hypothesized that this
oxidative host defense system is also active against respiratory
viruses. We evaluated the activity of oxidized LPO substrates against
encapsidated and enveloped viruses. When tested for antiviral
properties, the LPO-dependent production of OSCN
2
did not in-
activate adenovirus or respiratory syncytial virus (RSV). However,
substituting SCN
2
with the alternative LPO substrate iodide (I
2
)
resulted in a marked reduction of both adenovirus transduction and
RSV titer. Importantly, well-differentiated primary airway epithelia
generated sufficient H
2
O
2
to inactivate adenovirus or RSV when LPO
and I
2
were supplied. The administration of a single dose of 130 mg
of oral potassium iodide to human subjects increased serum I
2
concentrations, and resulted in the accumulation of I
2
in upper
airway secretions. These results suggest that the LPO/I
2
/H
2
O
2
system can contribute to airway antiviral defenses. Furthermore,
the delivery of I
2
to the airway mucosa may augment innate antiviral
immunity.
Keywords: respiratory syncytial virus; adenovirus; lactoperoxidase;
thiocyanate; iodide
Acute viral lower respiratory tract infections represent enor-
mous disease burdens for infants, children, and adults through-
out the world. In children, acute lower respiratory infections are
the most frequent cause of mortality worldwide (1, 2). Despite
the prevalence and severity of these infections, few specific
prophylactic or therapeutic interventions are available for many
of these common diseases, including effective vaccines or anti-
viral treatments. For example, Group B and C adenoviruses are
frequent causes of bronchiolitis and pneumonia, and occasion-
ally cause postinfectious bronchiolitis obliterans (3, 4). Although
respiratory syncytial virus (RSV) is the most common cause of
acute lower respiratory infections throughout the world (5), no
effective vaccine or specific, widely used antiviral therapy is
available.
In evaluating novel approaches to antiviral defenses, we
considered the redundant layers of innate immunity at play in
the airways. We questioned whether an existing host defense
mechanism might be induced or augmented to achieve en-
hanced antiviral protection in the lung. Both nonoxidative and
oxidative host defense systems contribute to the antimicrobial
activity of airway surface liquid (ASL) (6). Although the host
defense roles of many secreted proteins and peptides are well-
characterized, recognition that the airway epithelium orches-
trates an oxidative extracellular microbicidal system is recent
(7–10). This reactive oxygen species–producing system consists
of the airway epithelial proteins dual oxidase (DUOX) 1 and 2,
lactoperoxidase (LPO; secreted by submucosal glands), and the
pseudohalide ion (thiocyanate, SCN
2
; secreted by epithelia)
(11). However, the efficacy of this LPO/halide/H
2
O
2
airway
mucosal defense system has not been investigated for viral
respiratory pathogens.
The LPO-catalyzed oxidation of SCN
2
produces hypothio-
cyanite (OSCN
2
) (12), whereas the oxidation of I
2
produces
hypoiodous acid (HOI) (13). Both OSCN
2
and HOI are short-
lived, reactive intermediates that are known to be bactericidal.
We hypothesized that this oxidative antimicrobial system might
possess functional relevance for preventing respiratory viral
infections, through the generation of OSCN
2
or HOI. Here we
show that the delivery of I
2
to respiratory epithelial cells
supports the generation of HOI in ASL. HOI, but not OSCN
2
,
readily inactivates adenovirus and RSV. Supplementation with
I
2
may serve to augment mucosal antiviral defenses.
MATERIALS AND METHODS
Chemicals and Enzymes
Sodium iodide (catalogue number S324–500; Fisher, Pittsburgh, PA)
and sodium thiocyanate (catalogue number 251410–500G; Sigma, St.
Louis, MO) were dissolved in deionized, distilled water, sterile-filtered,
and stored at 2208C. Thirty percent H
2
O
2
(catalogue number H325–
500) was purchased from Fisher. Bovine milk LPO was obtained from
Sigma (catalogue number L2005–25MG). The entire sample was
resuspended in deionized, distilled water to a final concentration of 1
CLINICAL RELEVANCE
This study shows that the lactoperoxidase/I
2
/H
2
O
2
system
can contribute to airway antiviral defenses. Furthermore,
the delivery of I
2
to airway mucosa may augment innate
antiviral immunity. The identification of a new prophylac-
tic or therapeutic approach to prevent or ameliorate
respiratory viral infections could have broad implications
for human health.
(Received in original form September 30, 2010 and in final form March 18, 2011)
This study was supported by National Institutes of Health grants P50 HL-61234
(P.B.M.), PO1 HL-091842 (P.B.M. and B.B.), N01 AI-30040 (P.B.M.), and R01 HL-
090830 (B.B.), the Roy J. Carver Charitable Trust (P.B.M.), and Cystic Fibrosis
Foundation Therapeutics Grant BANFI07A0 (B.B.). This study was also supported
by the Cell and Tissues and Cell Morphology Cores, which is partly supported by
the Center for Gene Therapy for Cystic Fibrosis (through National Institutes of
Health grant P30 DK-54759) and the Cystic Fibrosis Foundation.
Correspondence and requests for reprints should be addressed to Paul B.
McCray, Jr., M.D., Department of Pediatrics, University of Iowa, 240F EMRB,
Iowa City, IA 52242. E-mail: paul-mccray@uiowa.edu
This article has an online supplement, which is accessible from this issue’s table of
contents at www.atsjournals.org
Am J Respir Cell Mol Biol Vol 45. pp 874–881, 2011
Originally Published in Press as DOI: 10.1165/rcmb.2010-0329OC on March 25, 2011
Internet address: www.atsjournals.org
mg/ml (39 U/ml). Bovine liver catalase was obtained from Sigma
(catalogue number C1345).
Culture of Cell Lines
Vero cells (ATCC, Manassas, VA) were grown in Dulbecco’s minimum
essential medium (DMEM; Invitrogen, Carlsbad, CA), supplemented
with 10% FBS and 1% penicillin–streptomycin (PS). A549 and HEp-2
cells (ATCC) were grown in DMEM/F12 with 10% FBS and 1% PS.
Primary Culture of Airway Epithelia
Primary human or porcine airway epithelial cells grown at the air–
liquid interface were prepared at the In Vitro Models Core Facility at
the University of Iowa, using previously described methods (14). The
use of human tissue samples in this study was approved by the
Institutional Review Board of the University of Iowa. The use of
porcine tissue was approved by the Institutional Animal Care and Use
Committee of the University of Iowa. All primary cells used in this
study were well-differentiated (.2 weeks of culture), and were
maintained in DMEM/F12 medium containing 1% PS, 50 mg/ml
gentamicin sulfate, and 2% Ultroser G (BioSepra, Villeneuve, La
Garenne, France).
Adenovirus
Replication-deficient recombinant adenovirus expressing enhanced
green fluorescent protein (eGFP) (Ad5–CMV–eGFP) was produced
and tittered, using previously described methods (15). Sucrose gradi-
ent–purified virus preparations were obtained from the Gene Transfer
Vector Core at the University of Iowa. The virus was titered at 1 310
12
particles/ml (z1310
10
transducing units (TU)/ml).
Respiratory Syncytial Virus
RSV strain A2 (provided by Barney Graham at the National Institutes
of Health and Dr. Steven Varga at the University of Iowa) was grown
in HEp-2 cells and prepared as a clarified crude lysate, with a titer of
approximately 2 310
7
plaque forming units (PFU)/ml. Virus was
titered via syncytia titration on Vero cells (16). Red fluorescent protein
(RFP)–expressing virus (rrRSV), provided by Dr. Steven Varga
(University of Iowa), was previously described (17).
In Vitro Virus Inactivation Assays
See the online supplement for details.
Cell-Dependent Viral Inactivation
For experimental details, see the online supplement.
Measurement of HOI
The production of HOI was detected based on the iodination of
nicotinamide adenine dinucleotide phosphate reduced (NADPH), as
described previously (18). For additional details, see the online
supplement.
Cell Toxicity Assay
The release of lactate dehydrogenase (LDH) was measured in well-
differentiated human airway epithelia, using a commercially available kit
according to the manufacturer’s instructions (LDH-Cytotoxicity Assay
Kit, catalogue number K311–400; BioVision, Mountain View, CA).
pH Dependence of Oxidative Antiviral Activity
For experimental details, see the online supplement.
RSV Syncytia Titration
RSV was titrated by a variation of the microtiter assay originally
described by Trepanier and colleagues (16). For experimental details,
see the online supplement.
Administration of Iodide to Human Subjects, and Analysis of
Nasal Airway Surface Liquid Composition
For experimental details, see the online supplement.
RESULTS
Oxidized Forms of I
2
, but Not SCN
2
, Are Virucidal
against Adenovirus
We first asked whether the oxidized halide and pseudohalide
substrates of LPO-catalyzed oxidation were active against ade-
novirus. We selected adenovirus, a protein-encapsidated DNA
virus, because it is an important respiratory pathogen. To test
whether reactions generating OSCN
2
or HOI inhibit infection
with adenovirus, we mixed both complete and incomplete LPO
mixtures with recombinant Ad5–CMV–eGFP under cell-free
conditions, and used the expression of eGFP to monitor the
virus transduction of target cells. In Figure 1A, we show that the
addition of the replete HOI-generating reaction completely
abrogated transduction by Ad5–CMV–eGFP. By contrast,
a comparable OSCN
2
-generating reaction failed to block trans-
duction. None of the incomplete LPO reactions possessed
discernable antiviral activity. H
2
O
2
alone did not inhibit in-
fection by adenovirus.
Inactivation of Adenovirus by Hypoiodous
Acid Is pH-Dependent
The pH of airway surface liquid is slightly acidic, with pH values
ranging from 6.57–7.18 (19–21). To determine whether or not
the inactivation of adenovirus by HOI was pH-dependent, we
monitored adenovirus titers after exposure to HOI across pH
ranges from 5–8. At a pH between 6 and 7, the application of
LPO, I
2
, and H
2
O
2
was significantly more virucidal than at
physiologic pH (Figure 1B). At pH 8, the oxidation of I
2
also
significantly reduced the adenovirus titer, compared with the
PBS control.
Airway Epithelia Produce Hypoiodous Acid in an Iodide
Concentration–Dependent Manner
We previously reported that well-differentiated airway epithelia
generate H
2
O
2
apically, in a DUOX-dependent manner (8).
Here we asked whether airway epithelia produced sufficient
H
2
O
2
to support the production of HOI in the presence of the
physiological LPO concentration of airway surface liquid and
I
2
. Well-differentiated primary airway epithelia were cultured
at the air–liquid interface and stimulated by the apical addition
of ATP, which maximizes the DUOX-mediated production of
H
2
O
2
(22). The apical buffer (PBS) also contained a range of I
2
concentrations and LPO. Although a high concentration of
LPO is maintained in the airway surface fluid by the LPO-
secreting submucosal glands (23), the cultured surface airway
epithelia do not secrete LPO (data not shown) (9). The
production of HOI by airway epithelia was determined accord-
ing to the iodination of extracellular NADPH, as previously
described (18). As shown in Figure 2A, under these conditions,
well-differentiated porcine airway epithelia supported the gen-
eration of HOI at a rate similar to that of previously reported
H
2
O
2
production (8). Similar results were observed using
primary human airway epithelia (data not shown). The addition
of the H
2
O
2
scavenger catalase to the apical buffer, or the
omission of either LPO or I
2
, inhibited the generation of HOI,
confirming that the production of HOI was dependent on apical
H
2
O
2
, LPO, and I
2
.
Production of HOI by Well-Differentiated Airway Epithelia
Inhibits Adenovirus Transduction without Evidence
of Cytotoxicity
To test whether airway epithelia could generate enough reactive
product to inactivate adenovirus, Ad5–CMV–eGFP (multiplic-
ity of infection [MOI], 12.5 TU/cell) was inoculated apically
Fischer, Lennemann, Krishnamurthy, et al.: Mucosal Oxidative Virucidal Defense System 875
onto well-differentiated primary porcine airway epithelial cells.
Because the Coxsackie virus and adenovirus receptor is
expressed basolaterally (24), adenovirus requires prolonged
incubation to enter when applied to the apical surface (25).
Therefore, the virus was allowed to adsorb overnight to allow
for transduction. As shown in Figure 2B, supplementing epithe-
lia with LPO and I
2
at 5 mMor500mM significantly reduced the
number of eGFP-expressing cells compared with no I
2
controls,
indicating that viral transduction was inhibited. Quantification of
the foci of infection indicated that the inactivation of adenovirus
was I
2
dose–dependent (Figure 2C). The addition of 5 mMI
2
was sufficient to decrease the foci of infection significantly.
Figure 1. (A) Cell-free inactivation of adenovirus by hypoiodous acid.
Replication-deficient recombinant adenovirus expressing enhanced green
fluorescent protein (eGFP) (Ad5–CMV–eGFP) was mixed with combina-
tions of H
2
O
2
,NaSCN,NaI,andlactoperoxidase(LPO)(20mg/ml), as
described in the text, and incubated for 10 minutes. The virus was then
applied to monolayers of A549 cells. Cell monolayers were imaged by
fluorescence microscopy at 48 hours after transduction to monitor the
expression of GFP, and photomicrographs were captured. Representative
en face views are shown. The LPO-catalyzed oxidation of I
2
resulted in
a complete block of adenovirus transduction. Scale bars,500mm. Results
are representative of three independent experiments. (B) Dependence on
pH of adenovirus inactivation by HOI. Cell-free inactivation reactions for
Ad5–CMV–eGFP were prepared with incomplete or complete (5 mMNaI,
20 mg/ml LPO, and 100 mMH
2
O
2
) LPO reactions at different pH
conditions and incubated for 10 minutes. Samples were then diluted
and applied to A549 cells at approximately 50 multiplicity of infection
(MOI), and 48 hours later, numbers of eGFP-positive cells were counted
by fluorescence microscopy. The percentage of eGFP-positive cells was the
average counted from three separate fields. Results are representative of
three independent experiments. Values shown represent the mean 6SE.
*P,0.0001, two-way ANOVA with Tukey post hoc test.
Figure 2. (A) Rates of hypoiodous acid (HOI) production by ATP-
stimulated (100 mM) porcine airway epithelial cells in the presence of
apical LPO (6.5 mg/ml) and the indicated I
2
concentrations (open
squares, mean 6SE; n53). The production of HOI was determined by
adding nicotinamide adenine dinucleotide phosphate reduced
(NADPH) (100 mM) to the apical buffer, and measuring changes in
absorbance at 282 and 340 nm. The apical buffer of negative control
cultures lacked LPO (solid square), or was supplemented with 200 U/ml
of catalase (solid triangle). (B) Cell-dependent inactivation of adenovirus
by the generation of HOI. Porcine airway epithelia were stimulated with
ATP to generate H
2
O
2
and were supplemented with 0 mM, 5 mM, or
500 mM NaI, as indicated. LPO and Ad5–CMV–eGFP were added to the
apical surface after 30 minutes of stimulation, and were allowed to
adsorb overnight. Representative micrographs from three independent
specimens are presented from 48 hours after transduction. The
addition of 5 mM or 500 mMI
2
reduced the transduction of adenovirus,
compared with no I
2
.Scale bar 5500 mm. (C) Cell-dependent
inactivation of adenovirus by the generation of HOI (quantitation of
data presented in B). Porcine airway epithelia were stimulated with ATP
and supplemented with 0 mM, 5 mM, or 500 mM NaI. LPO and Ad5–
CMV–eGFP were added to the apical surface after 30 minutes of
stimulation, and allowed to adsorb overnight. Error bars represent
standard error for three independent porcine airway epithelial cell
cultures. *P,0.05, **P,0.005, Student ttest with Bonferroni
correction for multiple comparisons.
876 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 45 2011
We previously showed that OSCN
2
was not toxic to airway
epithelia (8). We asked whether the production of HOI by
airway epithelia was associated with evidence of cell injury by
measuring the release of LDH. The addition of a complete
mixture of LPO, NaI, and H
2
O
2
to well-differentiated epithelia
did not result in significant cell toxicity (Figure 3).
Oxidized Forms of I
2
, but Not SCN
2
, Are Virucidal
against RSV
We extended the observations with adenovirus to an enveloped
respiratory virus, RSV. We hypothesized that OSCN
2
and HOI
might exhibit antiviral activity against RSV. We incubated wild-
type RSV for up to 2 hours with 100 mMH
2
O
2
at pH 7.4. Under
these conditions, the virus titer was unchanged compared with
control PBS (Figure 4A). To test whether oxidized or unoxidized
LPO substrates are virucidal, we assembled various mixtures of
LPO, I
2
,SCN
2
,H
2
O
2
, and approximately 1 310
5
PFU/ml of
RSV (Figure 4A). We found that neither SCN
2
and I
2
alone,
nor LPO delivered with either I
-2
or SCN
2
, decreased the
recovery of RSV, compared with PBS. These controls demon-
strated that neither LPO nor its substrates were virucidal under
these conditions. When a complete reaction mixture of LPO, I
2
,
and H
2
O
2
was assembled to yield HOI, the RSV titer was rapidly
reduced to undetectable levels. Therefore, the generation of HOI
is significantly virucidal against RSV, whereas the oxidation of
SCN
2
to OSCN
2
failed to reduce the RSV titer.
Inactivation of RSV by Hypoiodous Acid Is Most Effective
at Low pH
We evaluated the pH dependence of RSV inactivation by
applying either 5 mM NaI or 500 mM NaSCN in cell-free LPO
reactions. Because the application of 5 mM NaI results in a
suboptimal reduction in RSV titer at pH 7.4 (Figure 4A), we
used this concentration to monitor the dependence of buffer pH
on virus inactivation in hypoiodous acid–generating reactions.
We found that at a pH between 6 and 6.5, the application of
LPO, I
2
, and H
2
O
2
was significantly more virucidal than at
physiologic pH, with at least a 10-fold reduction in RSV titer
compared with pH 7 (Figure 4B). Under high pH conditions
(pH 8.0), the oxidation of I
2
did not substantially reduce the
virus titer. The oxidation products of SCN
2
failed to show sig-
nificant antiviral activity across a range of pH conditions.
Airway Epithelia Produce Sufficient Hypoiodous Acid for the
Inactivation of RSV
We next asked whether airway epithelia produce enough HOI
to inactivate viruses in situ via the LPO and I
2
antimicrobial
system. Well-differentiated primary human airway epithelia,
cultured at the air–liquid interface, were stimulated to produce
H
2
O
2
by an apical addition of ATP, and I
2
was supplied to the
cells by addition to the basolateral and apical media. LPO was
added to the apical surface at a concentration of 6.5 mg/ml (8).
We added rrRSV to the apical surface in a total volume of 50 ml,
with combinations of ATP, LPO, catalase, and varying concen-
trations of I
2
. If the epithelia produced sufficient H
2
O
2
,a
significant loss of RFP expression should occur. Indeed, when
I
2
was present in the reaction, a dose-dependent decrease in
foci of infection was evident, 48 hours after infection (Figure 5).
When I
2
was present at a concentration of 5 mM, the number of
foci of infection decreased, but this trend did not reach sta-
tistical significance. Increasing the concentration of I
2
to 500 mM
resulted in a marked and significant decrease in the number of
foci of infection. The addition of the enzyme catalase rescued
RSV infection. Based on these results, we conclude that human
airway epithelia can generate sufficient H
2
O
2
to inactivate RSV
when supplemented with pharmacologic concentrations of I
2
in
the presence of physiologic concentrations of LPO.
After Bolus Intake of I
2
, the Concentration of I
2
in ASL
Exceeds That of SCN
2
in Humans
The secretion of SCN
2
by the airway epithelium is thought to be
mediated by the sodium–iodide symporter (NIS) in the basolateral
plasma membrane, and by cystic fibrosis transmembrane conduc-
tance regulator (CFTR) and pendrin in the apical plasma mem-
brane (9, 10). Because these SCN
2
-transporting proteins are also
capable of I
2
transport, we hypothesized that I
2
is absent from
ASL only because of its low serum concentration (10–100 nM). To
examine the I
2
-secreting capacity of the airway, we performed
a study with nine human subjects. After the verification of normal
thyroid function (based on blood thyroid-stimulating hormone
[TSH] and free thyroxine [T4] levels; data not shown), subjects
took a potassium iodide tablet orally (130 mg KI, Iosat; Anbex
Inc., Williamsburg, VA). Twenty-four hours before the intake of
KI, and 2, 6, and 24 hours later, nasal ASL was harvested using
microsampling probes (26), and blood was drawn from arm veins
for inorganic ion analysis by anion-exchange chromatography (27).
Before the intake of KI, the I
2
content of ASL was below the
detection limit of our assay (,0.25 mM in 50-fold diluted ASL).
However, 2 hours after the intake of KI, I
2
accumulatedinASLat
approximately 500 mM (Figures 6A and 6B). The concentration of
I
2
in ASL remained in the range of several hundred micromolar
for many hours, and exceeded the concentration of serum I
2
by
more than 50-fold at 6 hours (Figures 6B). Interestingly, the
concentration of SCN
2
was reduced in ASL after the intake of KI
(Figures 6A and 6C), whereas the serum concentration of SCN
2
remained stable. These results suggests that I
2
and SCN
2
compete
for transepithelial secretion in the respiratory tract. Our data also
show that the oral intake of a single KI dose (130 mg) leads to the
accumulation of I
2
in ASL at concentrations that (based on our
cell-culture assays) can support antiviral activities.
DISCUSSION
Viral respiratory-tract infections are significant causes of mor-
bidity and mortality throughout the world (1). Here we show that
Figure 3. Airway epithelium–dependent production of HOI is not toxic
to epithelial cells. Cytotoxicity was monitored by lactate dehydrogenase
release assay, as described in MATERIALS AND METHODS. Well-differentiated
primary human airway epithelial cells were treated overnight under the
indicated conditions. These included assay buffer alone (PBS, pH 6.6),
and assay buffer containing the indicated combinations of catalase
(750 U/ml), ATP (100 mM), LPO (20 mg/ml), and I
2
(400 mM). Media
from both apical and basolateral cell surfaces were harvested and
pooled. The positive control represents cells exposed to 1% Triton X-
100 for 30 minutes at 378C. Results shown represent mean 6SE, and
are from three independent experiments with cells from three different
human donors. *P,0.05, Student ttest.
Fischer, Lennemann, Krishnamurthy, et al.: Mucosal Oxidative Virucidal Defense System 877
the LPO/I
2
/H
2
O
2
oxidative host defense system demonstrated
robust activity against two major respiratory viral pathogens,
adenovirus and RSV. Although OSCN
2
was not effective
against either adenovirus or RSV, we discovered that the delivery
of the alternative halide substrate I
2
to the airway epithelium
converted the DUOX/LPO enzymes into a HOI-producing
antiviral system. Remarkably, HOI exhibited virucidal activity
against both encapsidated and enveloped respiratory viruses.
Importantly, the oral administration of KI in human subjects
increased serum I
2
concentrations and yielded I
2
concentrations
in the upper respiratory secretions that supported the antiviral
activity of airway epithelial cells ex vivo. Because the concentra-
tion of LPO in our cell-based study was similar to that of in vivo
airways (28), we speculate that a topical or systemic supplemen-
tation of I
2
alone might be sufficient to inactivate virus.
The LPO/halide/H
2
O
2
antimicrobial system is a host defense
mechanism previously proposed to serve as a nonspecific anti-
viral defense (12, 29), particularly in the saliva (30, 31), tears
(32), and milk (33). The discovery of DUOX gene products
revealed their distribution in human airways (34). The expres-
sion of DUOX in epithelia stimulated interest in the role of the
LPO/halide/H
2
O
2
system in airway mucosal immunity, because
it identified a mechanism for the production of epithelial H
2
O
2
(11). The two DUOX isoforms, DUOX1 and DUOX2, are
encoded by closely spaced genes on human chromosome 15,
and are transcribed in opposite directions (35, 36). DUOX uses
cytoplasmic NADPH as an electron donor to transfer two
electrons to oxygen, which is reduced to H
2
O
2
and released
extracellularly. The functions of DUOX1 and DUOX2 are also
dependent on the co-expression of the regulatory proteins
DUOXa1 and DUOXa2, respectively (37). Although they are
functionally indistinguishable, DUOX1 and DUOX2 are differ-
entially regulated. Harper and colleagues previously reported
that DUOX2 is inducible by IFN-g, as well as by rhinovirus
infection and polyinosinic:polycytidylic acid (poly[I:C]) (38).
DUOX1 is induced by IL-4 and IL-13 (10, 38), as well as by
bacterial products such as Pseudomonas aeruginosa endotoxin,
flagellum, and Type III secretion system products (39).
Three reports showed that airway epithelial cells supple-
mented with both SCN
2
and LPO can kill two types of bacteria,
Pseudomonas aeruginosa and Staphylococcus aureus (8, 9, 40).
This provides evidence that the airway LPO/halide/H
2
O
2
system
Figure 4. (A) Cell-free inactivation of respiratory syncytial
virus (RSV) by the generation of HOI. RSV (1.7 310
5
plaque forming units [PFU]/ml) was incubated for 5 min-
utes with various combinations of H
2
O
2
(100 mM), NaSCN
(500 mM), NaI (500 nM, 5 mM, and 500 mM), and LPO (20
mg/ml). Treatment categories are indicated on the x-axis,
and the resultant virus titer is presented on the y-axis. SFU,
syncytia-forming units. The limit of detection for this assay
is 10 SFU/ml. Values represent mean 6SD for one of three
independent experiments. *P,0.001, one-way ANOVA
with Tukey post hoc test. (B) Inactivation of RSV by HOI is
dependent on pH. RSV was mixed with complete or
incomplete LPO reactions (5 mMNaI,100mMH
2
O
2
)and
incubated for 10 minutes. Samples were then diluted and
applied to Vero cell monolayers for syncytia titration. At
a pH less than 7, the I
2
-dependent inactivation of virus
was significantly greater than at physiologic pH (open
diamonds). No other conditions caused dramatic shifts in
RSV titer, including buffer pH, hydrogen peroxide, or LPO
substrates alone. Values represent the mean 6SD for n53
replicate wells. Results are representative of three indepen-
dent experiments. *P,0.001, two-way ANOVA with
Tukey post hoc test.
Figure 5. Cell-dependent inactivation of RSV by the generation of HOI.
Human airway epithelia were stimulated with ATP and supplemented
with 0 mM, 5 mM, or 500 mM NaI, as indicated. LPO and red
fluorescent protein–expressing virus were added to the apical surface
after 30 minutes of stimulation, and allowed to adsorb overnight. Foci
of infection were identified by the expression of red fluorescent protein.
Data are presented as the percentage of foci formed in the absence of
I
2
. Values shown represent mean 6SE for results from three separate
human donor cultures. * P,0.05, ** P,0.005, one-way ANOVA with
Tukey post hoc test.
878 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 45 2011
is active against bacteria. The recognition that the CFTR anion
channel conducts SCN
2
raised the possibility that patients with
cystic fibrosis (CF) might manifest impaired LPO/halide/H
2
O
2
microbicidal activity. Indeed, airway epithelia from patients with
CF exhibited defects in CFTR-dependent SCN
2
transport and
defective bacterial killing (8–10). These studies suggest a possible
mechanistic link between CF lung disease and altered function of
the LPO/halide/H
2
O
2
system.
In vitro, LPO has a high affinity for both SCN
2
and I
2
.Before
this study, the I
2
concentration in ASL had not (to the best of
our knowledge) been reported, but SCN
2
is present in ASL at
approximately 450 mM (41). Therefore, SCN
2
is thought to be
the physiologic LPO substrate in the respiratory tract. What
mechanism favors the accumulation of SCN
2
over I
2
in the
airways? Animal cells cannot synthesize SCN
2
. Thus, the diet is
the only source of both SCN
2
and I
2
(8, 11, 42, 43). The average
concentration of SCN
2
in serum is approximately 20 mM(44),
whereas the average concentration of I
2
in serum is only 10–
100 nM (45). With a serum concentration of SCN
2
200–2,000-
fold higher than that of I
2
, the secretion of SCN
2
predominates.
Airway epithelial cells generate an approximately 20-fold serosal-
to-mucosal concentration gradient for the secretion of SCN
2
by
importing SCN
2
on the basolateral side through NIS (46), and
exporting it on the apical side via CFTR and perhaps the anion
transporter pendrin (SLC26A4) (9, 10). I
2
can follow the same
secretory pathways. In fact, the NIS favors I
2
threefold over
SCN
2
(47). Thus, if serum I
2
reaches micromolar concentra-
tions, I
2
can also be secreted into the respiratory tract. The data
from our investigations of human subjects (Figure 6) support
this concept. Therefore, supplementation with I
2
may allow
sufficient I
2
concentrations in ASL to support the production of
HOI. Although chronic treatment with I
2
may be associated
with toxicity, short-term daily administration to humans is safe
(48, 49). The topical delivery of I
2
may further reduce any risk
of systemic toxicity.
The mechanism by which hypohalides eliminate bacteria is
not yet known, but OSCN
2
can oxidize thiol groups in surface
proteins, thereby mediating conformational changes (13, 50).
Although we do not yet know how HOI inactivates adenovirus
and RSV, similar mechanisms are likely in play. Adenovirus
and RSV represent two different classes of viruses. Adenovirus is
a nonenveloped, protein-encapsidated double-stranded DNA
virus that enters through endocytosis, whereas RSV is an
enveloped, single-stranded RNA virus that enters the cell
through receptor-mediated membrane fusion. The results of
our cell-free virus inactivation assays are consistent with
observations by Belding and colleagues that the peroxidase-
mediated oxidation of I
2
is strongly antiviral (29). Belding and
colleagues further reported that HOI was active against polio
and vaccinia viruses, particularly at a low pH (29). Interestingly,
the LPO/I
2
/H
2
O
2
system was most virucidal against both adeno-
virus and RSV at slightly acidic pH. Because the pH of ASL is
slightly acidic (19–21), these conditions are expected to favor HOI-
dependent virucidal activity. We speculate that HOI modifies viral
surface proteins and prevents the binding or the entry of
adenovirus and RSV into the epithelium. Alternatively, HOI
may inhibit the synthesis or assembly of viral nucleic acids and
proteins, or prevent the release of virus from cells. Our results
suggest that the innate oxidative antiviral system present in the
airways is a nonspecific mechanism to inactivate invading viruses.
To test the in vivo efficacy of the antiviral activity of this
HOI-generating mechanism, large animal models of viral in-
fections are needed, because murine airways lack both LPO and
DUOX (8), and rat airways contain few LPO-secreting cells
(51). In contrast, the respiratory tracts of larger mammals such
as sheep and pigs are similar to those of humans in regard to the
expression of LPO and DUOX, anatomy, physiology, innate
and adaptive immunity, and susceptibility to viral infection (23,
52). An important question for future study involves whether
the inactivation of virus in vivo is limited by the generation of
H
2
O
2
, the concentration of I
2
, or both.
In conclusion, the airway LPO/I
2
/H
2
O
2
system exhibits ro-
bust antiviral activity against relevant human pathogens. The
identification of a new prophylactic or therapeutic approach to
Figure 6. Accumulation of I
2
in airway surface liquid (ASL) after bolus
intake of KI. (A) A representative result of nasal ASL analysis before (blue
trace) and 2 hours after (red trace) intake of KI (130 mg) by a human
subject. (Band C) Summarized data show concentrations of I
2
(B) and
oxidizable anion thiocyanate (SCN
2
)(C) in nasal ASL (triangles) and
serum (closed squares) at indicated time points before and after intake
of KI (0 hour). Results are presented as mean 6SE (n59). For ASL
SCN
2
, repeated-measures ANOVA, P50.0032; post hoc Dunnett test,
control time point at 224 hours, *P,0.05 and **P,0.01. For ASL I
2
,
repeated-measures ANOVA, P,0.0001; post hoc Dunnett test, control
time point at 224 hours, *P,0.05 and **P,0.01.
Fischer, Lennemann, Krishnamurthy, et al.: Mucosal Oxidative Virucidal Defense System 879
prevent or ameliorate respiratory viral infections could have
broad implications for human health.
Author Disclosure:None of the authors have a financial relationship with a
commercial entity that has an interest in the subject of this manuscript.
Acknowledgments: The authors thank Jennifer Bartlett and Jeydith Gutierrez for
critically reviewing and commenting on the manuscript, and Dr. Steven Varga for
providing stocks of RSV and technical assistance.
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