Articles in PresS. Am J Physiol Lung Cell Mol Physiol (December 18, 2015). doi:10.1152/ajplung.00398.2015
1
Re-submitted to the AJP Lung call for papers on ‘sex differences in the respiratory system’
2
on: 11 December 2015 (MS#: L-00398-2015)
3
4
5
6
Estrogenic compounds reduce influenza A virus replication in primary human nasal
7
epithelial cells derived from female, but not male, donors
8
Jackye Peretz1, Andrew Pekosz1, 2, Andrew P. Lane4, and Sabra L. Klein1, 3
9
10
11
12
13
14
15
16
1
W. Harry Feinstone Department of Molecular Microbiology and Immunology, 2Department of
Environmental Health Sciences, and 3Department of Biochemistry and Molecular Biology,
The Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205,
4
Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University School of
Medicine, Baltimore, Maryland 21287
17
18
Running Head: estrogenic signaling and influenza
19
20
Keywords: antiviral defenses, estradiol, respiratory disease, SERMs, zinc finger proteins
21
22
Corresponding author:
23
Sabra L. Klein
24
25
26
Email: [email protected]
Phone: (410) 955-8898
Fax: (410) 955-0105
Copyright © 2015 by the American Physiological Society.
Peretz et al.
2
27
Abstract
28
Influenza causes an acute infection characterized by virus replication in respiratory epithelial
29
cells. The severity of influenza and other respiratory diseases changes over the life course and
30
during pregnancy in women, suggesting that sex steroid hormones, such as estrogens, may be
31
involved. Using primary, differentiated human nasal epithelial cell (hNEC) cultures from adult
32
male and female donors, we exposed cultures to the endogenous 17β-estradiol (E2) or select
33
estrogen receptor modulators (SERMs), then infected cultures with a seasonal influenza A virus
34
(IAV) to determine whether estrogenic signaling could affect the outcome of IAV infection and
35
whether these effects where sex-dependent. Estradiol, raloxifene, and bisphenol A decreased
36
IAV titers in hNECs from female, but not male, donors. The estrogenic decrease in viral titer was
37
dependent on the genomic estrogen receptor- 2 (ESR2) as neither genomic ESR1 nor non-
38
genomic GPR30 were expressed in hNEC cultures and addition of the genomic ER antagonist
39
ICI 182,780 reversed the antiviral effects of E2. Treatment of hNECs with E2 had no effect on
40
interferon or chemokine secretion, but significantly downregulated cell metabolic processes,
41
including genes that encode for zinc finger proteins, many of which contain estrogen response
42
elements in their promoters. These data provide novel insights into the cellular and molecular
43
mechanisms of how natural and synthetic estrogens impact IAV infection in respiratory epithelial
44
cells derived from humans.
Peretz et al.
45
3
Introduction
46
Biological differences exist between males and females, yet these differences are
47
often overlooked in experimental studies in immunology and infectious diseases (2). The
48
recent policy announcement by the National Institutes of Health (NIH) that applicants
49
consider balancing the sexes in preclinical, cell culture and animal studies speaks to the
50
importance of sex as a biological variable that can help predict the outcome of diseases (4,
51
19). Clinical observations reveal that respiratory diseases, such as asthma, chronic
52
obstructive pulmonary disorder, and influenza are often more severe in women than men,
53
being highly dependent on both age and hormonal status (3, 18). Influenza, in particular, is a
54
major recurring cause of morbidity and mortality worldwide, affecting 10% of the population
55
annually through epidemics, pandemics, and seasonal outbreaks. When age and sex are
56
included in analyses of influenza pathogenesis, young adult women experience more severe
57
outcomes compared to age-matched men (42). Young adult women were reportedly 2-6
58
times more likely to die from H5N1 or H7N9 avian influenza infection and during the initial
59
wave of the 2009 H1N1 influenza pandemic (14, 51, 64). Influenza is considered an immune-
60
mediated disease, in which the disease severity is largely determined by the strength of the
61
host inflammatory response to infection (56). Murine models have been instrumental in
62
characterizing sex differences in the outcome of influenza A virus (IAV) infection (26, 46, 47).
63
These studies collectively illustrate that young adult female mice suffer a worse outcome
64
from infection with IAV than their male counterparts. Sex differences in the outcome of IAV in
65
mice are not caused by differences in virus replication in the lower respiratory tract (i.e.,
66
lungs), but rather are associated with an exacerbated proinflammatory cytokine and
67
chemokine response in the lungs (26, 47). Regulating the balance between immune
68
responses causing protection or pathology is integral for repair of pulmonary tissue and
69
recovery from infection.
Peretz et al.
4
70
Murine models further reveal that sex differences in the outcome of respiratory
71
diseases, including IAV, are mitigated in the absence of sex steroid hormones (46, 47),
72
suggesting that the hormonal milieu plays a fundamental role in determining sex-specific
73
differences in the outcome of infection. Endogenous sex steroid hormones, such as 17β-
74
estradiol (E2), are immunomodulatory and regulate cellular immune responses to infection
75
(54, 63). In mouse models of asthma, allergy, and IAV, females suffer a worse outcome from
76
pulmonary diseases compared to males (30, 47). Among female rodents, endogenous
77
changes in circulating E2 concentrations alter lung function and exogenous E2 treatment
78
reduces pulmonary inflammation and disease symptoms (22, 30, 45, 65). Continuous E2
79
exposure, but not cyclical or ablated E2, in adult female mice prolongs survival following IAV
80
infection (47). In women, sustained concentrations of E2 are associated with improved
81
outcome of respiratory diseases, such as asthma. For example, administration of oral
82
contraceptives containing E2 reduces allergic inflammation in the respiratory tract and
83
asthma exacerbations associated with premenstrual asthma (22, 59).
84
Estrogens, primarily E2, regulate cellular function in diverse cell types, via binding to
85
intracellular, genomic estrogen receptors (ERs), such ERα (ESR1) and ERβ (ESR2), or to
86
membrane-associated, non-genomic ERs, such as the G protein-coupled estrogen receptor
87
(GPER). These receptors are cell type-specific and can activate gene expression directly by
88
serving as ligand-dependent transcriptional factors (ESR1 and ESR2) or indirectly by altering
89
signaling pathways, including ERK/MAPK, NF-κB, and STAT activity (10, 54). Both immune
90
cells and non-immune cells (e.g., respiratory epithelial cells), have intracellular ERs that
91
regulate cellular functions, including inflammatory responses (10). Targeting ER activity might
92
be therapeutic for diseases that are caused by excessive inflammation and that
93
disproportionately affect females. Selective estrogen receptor modulators (SERMs) bind to ERs
94
to cause conformational changes that alter engagement with coactivator and corepressor
Peretz et al.
5
95
proteins to initiate either induction or suppression of target gene transcription (21, 38). A recent
96
screen revealed antiviral properties of two SERMs against Ebola virus infection: clomiphene,
97
which is used to treat infertility, and raloxifene, approved for treatment of osteoporosis and to
98
decrease the risk of breast cancer in postmenopausal women (17). Environmental estrogens,
99
including xenoestrogens, such as bisphenol A (BPA), also can interact with ERs to alter
100
transcriptional activity (57), but the effects on cellular responses to viruses have not been
101
evaluated. In this study, we used primary differentiated human nasal epithelial cell (hNEC)
102
cultures isolated from healthy tissue of male and female donors (8, 20, 44) to study the
103
effects of estrogenic compounds on the human cellular response to IAV infection. Nasal
104
epithelial cells are the primary cell type infected with IAV and these cultures allowed us to
105
investigate IAV infection and pathogenesis based on the sex and hormonal milieu of the donor
106
cultures.
107
108
Materials and Methods
109
MDCK cells
110
Madin-Darby canine kidney (MDCK) cells were propagated in Dulbecco's modified Eagle's low-
111
phenol, high glucose medium (DMEM; Invitrogen) containing 10% fetal bovine serum (FBS; Life
112
Technologies), 100 U/ml penicillin (Life Technologies), 100 μg/ml streptomycin (Life
113
Technologies), and 2mM L-Glutamine (Life Technologies). The cells were maintained at 37oC in
114
a humidified environment with 5% CO2.
115
116
Virus
117
The recombinant influenza A/Udorn/307/72 H3N2 virus (IAV) used in this study was described
118
previously (41). The hemagglutinin (HA) protein contains amino acid changes (L226Q/S228G in
119
the H3 numbering system) that allows for preferential binding to α2,3-linked sialic acid. Viral
120
working stocks of were generated by infecting MDCK cells at a multiplicity of infection (MOI) of
Peretz et al.
6
121
approximately 0.01 infectious units per cell in low-phenol, high glucose DMEM containing 0.25%
122
bovine serum albumin (BSA; Life Technologies), acetylated trypsin from bovine pancreas
123
(5μg/mL; N-acetyl trypsin; Sigma), 100 U/ml penicillin, 100 μg/ml streptomycin, 6mM L-
124
Glutamine, and 1mM Sodium Pyruvate (Life Technologies), which we refer to as infection
125
media. The infected cells were incubated at 37°C for 72 hours, infected cell supernatant was
126
harvested, clarified by centrifugation, aliquoted, and stored at -80oC. Infectious virus titers were
127
determined by 50% tissue culture infectious dose (TCID50) as described previously (33).
128
129
Chemicals
130
Stock solutions of 17β-estradiol (E2; 1mM; Sigma) were made in ethanol, while raloxifene
131
(100mM; Sigma), ospemifene (100mM; Sigma), clomiphene citrate (100mM; Sigma), ICI
132
182,780 (ICI; 2mM; Tocris-Cookson), and bisphenol A (BPA; 44mM; Sigma; provided by Dr.
133
Jodi A. Flaws, University of Illinois at Urbana-Champaign) were made in dimethyl sulfoxide
134
(DMSO; Sigma). All stock solutions were stored at -20oC.
135
136
Human nasal epithelial cell (hNEC) cultures
137
Primary human nasal epithelial cells were collected from non-diseased nasal mucosa of patients
138
undergoing
139
dacryocystorhinostomy, approach to anterior skull base pathology, or removal of benign nasal
140
masses. The cells were collected from male (n = 10) and female (n = 42) donors (age range 18-
141
45). The research protocol was approved through the Johns Hopkins Institutional Review Board,
142
and all subjects gave signed informed consent. The cells were differentiated at an air-liquid
143
interface (ALI) in 24-well Falcon filter inserts (0.4-μM pore; 0.33cm2; Becton Dickinson) coated
144
with human type IV placental collagen (Sigma-Aldrich) as described previously (8, 20, 44).
endoscopic
sinonasal
145
146
Infection and treatment of hNEC cultures
surgery
for
non-sinusitis
indications,
including
Peretz et al.
7
147
Prior to infection, the apical surface of hNEC cultures was washed with Dulbecco's PBS with
148
calcium and magnesium (DPBS; Life Technologies). The cultures were infected via the apical
149
chamber with a MOI of 0.1 TCID50/cell or mock infected at 32°C in 200 μl of infection media.
150
After 2 hours, the inoculums were aspirated, the apical surfaces washed twice with DPBS, and
151
cells were incubated at 32°C. At the indicated hours post infection (hpi), 200 μl of infection
152
media was added to the apical surface, incubated at 32°C for five minutes, and then collected.
153
All samples were stored at -80°C. At the indicated times pre- or post-infection, the basolateral
154
media was replaced by media containing doses of vehicle control (EtOH or DMSO as
155
appropriate), E2 (0.1, 1.0, or 10nM), BPA (44 μM), raloxifene (50 μM), ospemifene (50 μM),
156
clomiphene citrate (50 μM), or ICI (100nM). Basolateral media was collected and replaced with
157
media containing fresh vehicle or compound at 48 and 96 hpi.
158
159
Tissue Culture Infectious Dose (TCID50) assay
160
MDCK cells were plated in a 96-well plate and cultured for 72 hours in growth DMEM until 90-
161
100% confluent. The cells were washed twice with DPBS, then covered with 180μl of infection
162
media. Serial dilutions (10-1 to 10-8) of hNEC apical samples at each time-point were made in
163
separate, 96-well plates by adding 20μl of each apical sample to 180μl of infection DMEM, then
164
serially diluted 10 fold. Twenty microliters of each dilution were then added to the MDCK plates
165
in replicates of six, resulting in final dilutions of each sample ranging from 10-2 to 10-9. The
166
infection proceeded for 7 days at 32°C, and then the cells were fixed with 4% formaldehyde and
167
stained with napthol blue-black solution. The cytopathic effect was scored visually and the Reed
168
and Muench calculation was used to determine the titer of infectious virus at each time-point.
169
170
Multiplex Chemokine Assay Analysis
Peretz et al.
8
171
The Meso Scale Discovery (MSD) multiplex assay system was used to measure chemokines
172
collected from apical samples of hNECs after infection. In each well, the Chemokine Panel 1 kit
173
quantitatively determined the concentration of eight C-C ligand motif (CCL2, CCL3, CCL4,
174
CCL11, CCL13, CCL17, CCL22, CCL26) and two C-X-C ligand motif (CXCL8, CXCL10)
175
chemokines, according to the manufacturer’s protocol. All samples were run in duplicate. MSD
176
plates were read on the MSD SECTOR Imager 2400 at the Becton Dickinson Core Facility at
177
the Johns Hopkins Bloomberg School of Public Health and analyzed on the accompanying
178
software (MSD Discovery Workbench version 4).
179
180
Enzyme-linked Immunosorbent Assay (ELISA)
181
The PBL Assay Science DIY Human IFN Lambda 1/2/3 (IL-29/28A/28B) ELISA was used to
182
measure levels of interferon lambda (IFNλ) collected from apical samples of hNECs after
183
infection. All samples were run in duplicate and read on the FilterMax F3 Multi-Mode Microplate
184
Reader (Molecular Devices). The data were analyzed with the accompanying software (SoftMax
185
Pro Data Acquisition and Analysis Software).
186
187
Real-time RT-PCR
188
At the indicated times post infection, hNEC cultures were treated with TRIzol Reagent (Life
189
Technologies) and total RNA was extracted using the PureLink™ RNA Mini Kit (Ambion by Life
190
Technologies) according to the manufacturer’s protocol. Reverse transcriptase generation of
191
complementary DNA (cDNA) was performed with 0.5μg of total RNA, primed with a blend of
192
random hexamer and oligo (dT)s against the RNA library, using an iScript RT Kit (Bio-Rad
193
Laboratories, Inc.). Real-time PCR (qPCR) using Ssofast qPCR Supermix™ with EvaGreen
194
(Bio-Rad) was conducted using the StepOnePlus™ Real-Time PCR System (Life Technologies)
195
and accompanying software (StepOne™ Software) according to the manufacturer’s instructions.
196
A dilution curve was generated from a compilation of the samples and qPCR analysis was
Peretz et al.
9
197
performed using forward and reverse primers for estrogen receptor alpha (ESR1), estrogen
198
receptor beta (ESR2), G-protein-coupled estrogen receptor 1 (GPER), and the zinc finger
199
protein genes ZNF91, ZMYM6, and ZFAND4.
200
followed by 94oC for 10s, annealing at 60oC for 10s, and extension at 72oC for 10s, for 40
201
cycles, followed by a final extension at 72oC for 10min. A melting curve was generated at 55-
202
90oC to monitor the generation of a single product. 18s RNA (RNA18S) was used as a
203
reference gene for each sample. Final values of relative gene expression were calculated and
204
expressed as the ratio normalized to RNA18S. All analyses were performed in duplicate or
205
triplicate.
An initial incubation of 95oC for 10min was
206
207
Western Blot Analysis
208
At the indicated times post infection, cells were lysed with 1% sodium dodecyl sulfate (SDS;
209
Fisher Scientific) in PBS and total protein was measured using the Pierce™ BCA Protein Assay
210
Kit (Life Technologies) according to the manufacturer’s instructions. BSA dilutions (Pierce™
211
BCA Protein Assay Kit) were used as the standards. For western blot analysis, 7-30μg of total
212
protein from each sample was separated on 4-15% Mini-PROTEAN® TGX™ Precast Gels (Bio-
213
Rad) and transferred to a polyvinylidene fluoride Immunobilon-FL membrane (PVDF; Millipore).
214
The membranes were blocked with PBS containing 5% dry milk powder and 0.05% Tween-20
215
(Sigma). Wash buffer contained PBS with 0.05% Tween-20. Membranes were blocked for 30
216
minutes at room temperature (RT), incubated for 1.5 hours at RT with primary antibody, washed
217
three times each for 5 minutes, incubated for 1 hour at RT with secondary antibody, and then
218
washed three times each for 5 minutes. Primary and secondary antibodies were diluted in
219
blocking buffer. The primary antibodies used were mouse anti-βactin (AC-15 MAb; 1:5000
220
dilution; Abcam-ab6276), rabbit anti-ESR2 (PAb; 1:100 dilution; Thermo- PA1-310B), and rabbit
221
anti-ESR1 (1:500 dilution; Genetex-GTX62423). The Alexa Fluor 647-conjugated secondary
222
antibodies used were a goat anti-mouse immunoglobulin G (IgG) and a donkey anti-rabbit IgG
Peretz et al.
10
223
(both at a 1:1000 dilution; Invitrogen). For visualization, membranes were imaged using a
224
FluorChemQ phosphorimager (ProteinSimple) and signal intensities quantified by Image J
225
analyses (NIH).
226
227
Identification of Putative Estrogen Response Elements (EREs)
228
The Dragon ERE Finder version 3.0 (http:// http://datam.i2r.a-star.edu.sg/ereV3/) was used to
229
identify putative EREs in the promoters of select innate immune factor genes (1). The NCBI
230
BLAST program was used search for the annotated start sites of genes of interest against
231
~11,000 human promoters. These sequences were then used to pinpoint the location of the
232
putative ERE pattern by expanding them to cover an extended region of [-5000, +3000] relative
233
to genes of interest start site. We used the recommended search sensitivity of 83%.
234
235
Microarray Analysis
236
Trizol reagent and the PureLink RNA Mini kit (Ambion/Life Technologies) were used for
237
extraction and purification of RNA. Cells were processed according to the manufacturer’s
238
protocol. Following elution of purified RNA from the PureLink columns with Nuclease-free water,
239
quantitation was performed using a NanoDrop spectrophotometer and quality assessment
240
determined by RNA Nano LabChip analysis on an Agilent BioAnalyzer 2100 or RNA Screen
241
Tape on an Agilent TapeStation 2200. One hundred ng of total RNA was processed for
242
hybridization to Affymetrix Human Gene ST 2.0 microarrays using the Affymetrix GeneChip WT
243
PLUS Reagent Kit according to the manufacturer’s recommended protocol. The signal
244
amplification protocol for washing and staining of eukaryotic targets was performed in an
245
automated fluidics station (Affymetrix FS450) using Affymetrix protocol FS450_0002.
246
arrays were scanned in the GCS3000 laser scanner with autoloader and 3G upgrade
247
(Affymetrix). Quality assessment of hybridizations and scans was performed with Expression
248
Console software (Affymetrix).
The
Peretz et al.
11
249
250
Statistical Analyses:
251
For analysis of virus titers, cytokine/chemokine levels, and real-time PCR data, comparisons
252
among treatment groups were performed using multivariate analysis of variance (MANOVA) (for
253
repeat measures) followed by planned comparisons or one-way analysis of variance (ANOVA)
254
with appropriate post hoc tests using SigmaPlot 12 (Systat Software). For microarray analyses,
255
Partek Genomics Suite version 6.6 was used, with the RMA (Robust Multi-chip Average)
256
Algorithm used for background correction, normalization, and summarization of probes.
257
ANOVAs with linear contrasts were performed to generate relative fold change, p-values, and
258
gene lists. Statistical significance was assigned at p≤ 0.05.
259
260
Results
261
Pre-treatment with E2 reduces viral titers in hNECs from female, but not male, donors
262
To test the hypothesis that E2 has antiviral effects on IAV infection, hNEC cultures from
263
female and male donors were pre-treated in the basolateral media with E2 (0.1, 1, or 10nM) or
264
vehicle for 24h prior to infection with IAV. During peak virus replication (i.e., 72-96hpi), E2 pre-
265
treatment of hNEC cultures from female (Fig 1A) but not male (Fig 1B) donors significantly
266
reduced virus titers as compared with vehicle treated hNEC cultures (p<0.05). We interpret
267
these data to suggest that E2 has a sex-specific antiviral activity in hNEC cultures.
268
To determine whether the duration of E2 exposure could shift the timing of the antiviral
269
effects of E2, hNEC cultures from female donors were exposed to E2 72 hours before or 24
270
hours after infection with IAV. Pre-treatment of hNEC cultures with E2 for 72 hours resulted in a
271
significant reduction in virus titers 48 and 72 hpi as compared with vehicle treated cultures
272
(p<0.05; Fig 2A), while treatment of hNEC cultures 24hpi significantly reduced virus titers at
273
72hpi (p<0.05; Fig 2B). Taken together, these data suggest that the antiviral activity of E2 in
274
hNEC cultures from female donors can be induced with treatments over an extended timeframe.
Peretz et al.
12
275
276
E2 does not affect production of cytokines in response to IAV infection in female donors
277
Respiratory epithelial cells are significant producers of cytokines, chemokines, and type
278
III interferons in response to IAV infection (36, 49). To test the hypothesis that E2 reduces IAV
279
titers by altering host immune responses to infection, hNEC cultures from female donors were
280
pre-treated with E2 (0.1, 1, and 10nM) or vehicle for 24h prior to infection, and the apical
281
supernatants were analyzed at 72hpi (i.e., during peak virus replication) for production of a
282
panel of chemokines and type III interferons. Concentrations of IFNλ and all chemokines
283
measured, with the exception of CCL13 and CCL26, were significantly increased in IAV-infected
284
hNEC cultures as compared with mock infected cultures derived from females (p<0.05 in each
285
case; Table 1). In contrast to the immunomodulatory effects of E2 reported in reproductive
286
epithelial cells (11, 50, 52), E2 did not significantly alter secretion of any chemokine or IFNλ in
287
either the mock- or IAV-infected hNECs compared to the vehicle-treated hNECs. Therefore, the
288
antiviral effects of E2 on IAV infection do not involve altered apical production of IFNλ or
289
chemokines.
290
291
The antiviral effects of E2 require intracellular ESR2 signaling in hNECs from female
292
donors
293
The ER family is comprised of many ER and ER-like proteins, including the intracellular
294
genomic E2 receptors, ESR1 and ESR2, and the nongenomic G-protein coupled receptors
295
(e.g., GPER). To test the hypothesis that the antiviral effects of E2 were mediated by signaling
296
through genomic rather than nongenomic receptors, we pre-treated hNEC cultures from females
297
with E2 (10nM), the genomic ER blocker ICI 182, 780 (100nM), E2+ICI, or vehicle for 24h prior
298
to inoculation with IAV. Pretreatment of hNEC cultures with E2 significantly reduced virus titers
299
at 72 and 96 hpi as compared with vehicle-treated cells (p < 0.05; Fig 3), but the addition of ICI
300
with E2 reversed the antiviral effects of E2 resulting in virus titers that were indistinguishable
Peretz et al.
13
301
from vehicle-treated cells (Fig 3). These data suggest that the antiviral effects of E2 are
302
mediated by genomic signaling through intracellular ERs.
303
To determine whether hNEC cultures expressed genomic and nongenomic ERs, we
304
measured transcripts of ESR1, ESR2, and GPER in hNECs derived from male or female donors
305
treated with either vehicle or E2 prior to mock- or IAV infection. At 24hpi, transcripts of GPER
306
were not detected in hNEC cultures from male or female donors (data not shown). Although
307
ESR1 was expressed in hNECs derived from both male and female donors, the expression level
308
was low and was not altered in the presence of E2 (Fig 4A). In contrast, ESR2 was expressed
309
to higher levels in hNECs derived from female than male donors following exposure to E2 (p<
310
0.05; Fig 4B). To evaluate the kinetics of genomic ER expression, transcripts of ESR1 and
311
ESR2 were measured 24 and 48hpi in hNEC cultures from female donors. Detection of ESR1
312
mRNA remained low at both 24 and 48hpi and was not altered by treatment with E2 (Fig 4C). In
313
contrast, treatment of hNECs from females with E2 significantly increased the expression of
314
ESR2 at 24hpi, but not 48hpi (p<0.05; Fig 4D).
315
To confirm that hNEC cultures derived from female donors expressed genomic ER
316
proteins, cell lysates at 24hpi were subjected to Western Blot analysis. At 24hpi, ESR2, but not
317
ESR1, was detected in hNECs derived from females (Fig 5A and B). Both proteins were
318
detected in MCF-7 cells, indicating the lack of ESR1 expression in hNEC cultures was not due
319
to an inability of the antibodies to detect the protein. Taken together, these data suggest that the
320
activation of ESR2 by E2 in female, but not male, hNECs mediate the sex-specific antiviral
321
effects of E2. .
322
323
E2 reduces metabolic processes in hNEC cultures from females following IAV infection
324
To uncover potential pathways affected by E2-ESR2 signaling in hNEC cultures derived
325
from females, we conducted microarray analyses on RNA isolated at 24 and 48hpi. To
326
determine the selective effects of E2 on gene expression during IAV infection, samples from
Peretz et al.
14
327
IAV-infected cultures were normalized to mock-infected cultures from the same hormone
328
treatment condition. Overall, significantly more genes were altered by E2-treatment at 24
329
compared with 48hpi. Using a fold change of 1.3 and p<0.05, we determined that 666
330
transcripts were differentially expressed in hNECs treated with E2 as compared with vehicle at
331
24hpi and only 234 transcripts were differentially expressed at 48hpi (Fig 6A). There were only 7
332
overlapping genes that were differentially expressed in hNECs treated with E2 as compared
333
with vehicle at both 24 and 48hpi (Fig 6A). Gene Ontology Enrichment revealed that the
334
biological functions with the greatest number of differentially expressed genes in hNECs treated
335
with E2 as compared with vehicle at 24hpi included organelle organization and cellular
336
metabolic processes, both of which received significant enrichment scores (i.e., > a score of 3;
337
Fig 6B). Further analyses revealed that a family of zinc finger protein (ZFP) genes was the
338
largest cluster of genes differentially expressed at 24hpi in response to E2 treatment of hNEC
339
cultures (Fig 6C).
340
To determine whether E2 could transcriptionally regulate the activity of these zinc finger
341
genes, we used Dragon ERE Finder version 3.0 (http:// http://datam.i2r.a-star.edu.sg/ereV3/) to
342
identify putative EREs in the promoter regions of ZFP genes. Of the 38 ZFP genes that were
343
downregulated during IAV infection by E2, 21 had at least one putative or known estrogen
344
response elements (EREs) present in their promoters (Fig 7A). Of the 21 ZFP genes that had
345
putative EREs in their promoters, 20 of these genes had two or more putative EREs, 11 of
346
which have been experimentally confirmed to bind to ERs (1). Zinc finger proteins play diverse
347
roles in regulating cellular function and responses to damage; their role in IAV infection,
348
however, is largely unknown. Of the 38 ZFP genes that were downregulated in the presence of
349
E2 during IAV infection, ZNF91, ZMYM6, and ZFAND4 were of particular interest because of
350
the large number of EREs present in their promoters (i.e., ZNF91 and ZMYM6) or because it
351
belongs to a family of ZFPs that can have antiviral activity (i.e., ZFAND4) (28). The effects of E2
352
on the expression of ZNF91, ZMYM6, and ZFAND4 transcripts were validated using real time
Peretz et al.
15
353
RT-PCR, which confirmed that E2 significantly downregulated ZNF91 and ZMYM6 at 24hpi but
354
not 48hpi (p<0.05; Fig 7B). While ZFAND4 expression was reduced at 24hpi, this did not
355
achieve statistical significance (p=0.057; Fig 7B). Taken together, these data suggest that E2
356
has the potential to directly regulate transcription of ZFP genes, which may provide a novel
357
mechanism for reducing late stage virus replication.
358
359
Estrogenic chemicals that selectively bind to ESR2 reduce IAV titers in hNECs from
360
females
361
Selective estrogen receptor modulators are a class of FDA-approved therapeutic
362
estrogenic chemicals, while BPA is a xenoestrogen forming the backbone of various plastic and
363
consumer products. These estrogenic chemicals have differential affinity for ERs in a variety of
364
tissues (21). To determine if compounds that bind genomic ERs could decrease IAV titers,
365
similar to E2, hNEC cultures from females were pre-treated with raloxifene, clomiphene citrate,
366
ospemifene, BPA, or vehicle 24h prior to infection with IAV. Raloxifene, but not clomiphene
367
citrate or ospemifene, significantly reduced viral titers at 72 and 96hpi as compared with vehicle-
368
treated cultures (p< 0.05; Fig 8A). Treatment of hNECs with BPA also significantly reduced viral
369
titer at 48 and 72hpi compared to vehicle-treated hNECs (p<0.05; Fig 8B). Both raloxifene and
370
BPA have stronger binding affinities for ESR2 than ESR1 (24), suggesting that chemicals that
371
selectively bind ESR2 have antiviral activity against IAV infection of respiratory epithelial cells
372
from females.
373
374
Discussion
375
In this study, E2 and select estrogenic chemicals had antiviral effects against IAV
376
infection. These estrogenic compounds reduced peak viral titer, which did not involve changes
377
in the secretion of chemokines or IFNλ, but rather was associated with reduced metabolic
378
processes. The effects of estrogens were specific for hNEC cultures from female but not male
Peretz et al.
16
379
donors. The antiviral effects were most likely mediated by signaling through ESR2 as ESR2, but
380
not ESR1 or GPER, was expressed in hNEC cultures and the protective effects of E2 were
381
eliminated in the presence of the genomic ER antagonist, ICI 182,780.
382
Antiviral effects of E2 and estrogenic chemicals have been reported previously against
383
viruses including HIV, Hepatitis C virus (HCV), Ebola virus, and human cytomegalovirus
384
(HCMV). Estradiol can prevent HIV transcription by blocking HIV promoter activity in peripheral
385
blood mononuclear cells (PBMCs) and preventing HIV entry in CD4+ T cell and macrophages
386
(48, 55). Estradiol also prevents the production of infectious HCV particles (12). Certain FDA-
387
approved SERMs inhibit Ebola and other Marburg viruses in vivo and in vitro (17). Further,
388
SERMs, including clomiphene and raloxifene, inhibit HCV infection at multiple stages of the
389
virus life cycle and, raloxifene, in particular, acts as an adjuvant to improve antiviral HCV
390
therapies in postmenopausal women (9, 37). While E2 has antiviral activities against a wide
391
range of viruses, it is still not clear whether the molecular mechanisms involved are conserved
392
across virus families or result from changes in different cellular pathways resulting from the
393
broad effects of E2 on cellular gene expression. Implications of these findings are far reaching
394
as a significantly greater proportion of the world population is exposed to IAV and suffer from
395
influenza annually than Ebola, HCV, HCMV, or HIV (61).
396
The inhibition of IAV infectious virus production in E2 or SERMs treated hNECs from
397
female donors only occurred at late times post-low MOI infection, with the magnitude of the
398
antiviral effects being greater when cells were treated prior to rather than after the initiation of
399
infection. Because the inhibition of virus titer occurs at a time when the infection in the hNECs is
400
no longer synchronized among the cells in the cultures, the E2-induced reduction in titer could
401
be resulting from E2 effects at multiple stages of the virus life cycle (i.e., entry, replication, re-
402
infection). Therefore, we hypothesized that there may be some combination of host response
403
and virus infection occurring synergistically to impair virus replication at later time-points of
404
infection.
Peretz et al.
17
405
Our microarray data indicate that E2 significantly alters the expression of genes involved
406
in metabolic processes. Within the differentially regulated genes comprising metabolic
407
processes, the largest family of E2-downregulated genes in IAV-infected hNECs from females
408
was those that encode for ZFPs. Very little is known about the function of many of these ZFPs,
409
let alone their potential roles in the IAV life cycle. In general, ZFPs are transcriptional regulators
410
that can induce or repress host gene transcription (25). Because these ZFPs are all similarly
411
downregulated in response to E2 treatment and IAV infection and expressed EREs in their
412
promoters, this may point to a common transcription factor or activation pathway responsible for
413
the antiviral effects of E2. There is a growing appreciation of the importance of various
414
metabolic, cell cycle, and lipid metabolism pathways for productive IAV infection (60) and
415
downregulation of these ZFPs may be exerting an antiviral activity by altering those pathways.
416
Both ESR1 and ESR2 are present in the upper and lower respiratory tract and are
417
required for the development and function of the lung, but exhibit distinct cell-type specific
418
expression patterns (5, 15, 29). ESR2 has been found in bronchial epithelial cells and alveoli,
419
whereas ESR1 is mostly limited to alveolar macrophages and endothelial blood vessel cells,
420
with modest expression in bronchial epithelial cells (15, 29, 40, 58). In primary human bronchial
421
epithelial cells (HBECs) and HBEC cell lines from males and females, the amount of ESR2 is
422
almost doubled compared to ESR1 (15). In the nasal mucosa, ESR2 is also the dominant ER
423
present and, in some studies, the only ER detectable (34, 43, 53), which is consistent with our
424
data. Similar to our results, in lung adenocarcinoma cells, only the ERs in cells from females
425
respond to E2 and other ER ligands (7). Although no studies have investigated the ESR1:ESR2
426
relationship in the respiratory tract, in mammary, uterine, and prostate tissue, where both
427
subtypes of ER are expressed, ESR2 is antagonistic to ESR1, inhibiting ESR1-mediated gene
428
expression (13, 31). The expression of ESR2 has also been shown to alter the recruitment of
429
ESR1 transcriptional co-factors and increase the degradation of ESR1, suggesting that ESR2
Peretz et al.
18
430
can mediate or dominate ESR1 signaling in the same cell (31, 32), which may explain why low
431
levels of ESR1 mRNA, but not ESR1 protein, is detectable in hNECs.
432
ESR1 and ESR2 have similar DNA binding homology but only share 57% homology in
433
their ligand binding domains, contributing to differences in transcriptional activities and tissue-
434
specific cellular responses triggered through ligand-dependent ER activation (27). For example,
435
E2, through ESR1, the dominant receptor in the female reproductive tract, decreases the
436
production of human beta-defensin 2 (HBD2) and elafin in the vaginal epithelium, but increases
437
HBD2 and elafin in the uterine epithelium (62). ESR2 is required for overall lung function,
438
whereas ESR1 is required to control inflammation in the lung (35, 58). In mice, signaling through
439
ESR1, but not ESR2, reduces pulmonary concentrations of proinflammatory cytokines and
440
chemokines in the lungs and improves the outcome of influenza in female mice (47). In addition
441
to the tissue type, the biological effects of E2 are contingent on the concentration of E2.
442
Physiological levels of E2, signaling through ESR1, promote proinflammatory type 1 IFN
443
production via TLR stimulation and induction type 1 IFN genes (23). Conversely, high E2 levels
444
consistent with those found prior to ovulation and during pregnancy are protective against
445
excessive inflammatory responses (54). In our study, physiologically relevant levels of E2 had
446
no effect on the induction of chemokines or IFNλ in cells from the upper respiratory tract. This
447
may reflect a fundamental difference between signaling through ESR2 in upper respiratory tract
448
epithelial cells and signaling through ESR1 in immune cells in the lower respiratory tract and
449
reproductive tract. Future studies will need to compare ER signaling in mouse and human
450
epithelial and immune cells in the upper and lower respiratory tract to definitively resolve the
451
differences in how E2 protects against IAV in both hNECs and mice. Collectively our data
452
illustrate that cell type-specific differences in ER expression result in differential modes of
453
protection.
454
In the current study, of the SERMs tested, only raloxifene and BPA significantly
455
decreased IAV titer in a manner similar to E2. Raloxifene can bind to both ESR1 and ESR2, but
Peretz et al.
19
456
has a higher affinity for ESR2 (16). Further, raloxifene binding to ESR2 results in an increase in
457
ESR2 reporter gene expression, but minimal expression after binding to ESR1 (39). BPA has
458
can have varying affinity for ERs depending on the cell or tissue in which it was measured (24).
459
Ospemifene can bind both ERs with similar affinity, and, while the affinities of clomiphene citrate
460
for ESR1 and ESR2 are unknown, clomiphene citrate is a well-documented ESR2 antagonist,
461
inhibiting ESR2-mediated cellular events. We hypothesize that ospemifene and clomiphene
462
citrate did not inhibit virus replication because they do not signal primarily through ESR2 (21).
463
The data from the present study implicate signaling through ESR2 as being critical for the
464
antiviral effects of E2 in hNEC cultures from female donors.
465
After the Women’s Health Initiative raised concerns about estrogen replacement
466
therapy, use of SERMs as safer alternatives has greatly increased in the United States (21).
467
Repurposing SERMs as anti-virals could be a beneficial and a novel therapeutic for the many
468
women currently prescribed SERMs for other diseases. The expression of ERs in the
469
respiratory tract implies that E2 and SERMs can affect the function of tissues and organs
470
outside of the reproductive tract. Understanding these effects will help shed light on additional
471
novel functions of E2 in pulmonary cellular physiology.
472
473
Acknowledgements
474
We thank the Klein and Pekosz laboratory members for insightful discussions about these data.
475
We thank Anne Jedlicka and Amanda Dziedzic of the JHSPH Genomic Analysis and
476
Sequencing Core for assistance with microarray analyses.
477
Grants
478
This work was funded by grants from the Center for Alternatives to Animal Testing (2013-09;
479
SLK) and NIH (R01AI097417 [AP], HHSN272201400007C [AP], R01AI72502 [APL], and T32
480
AI007417 [JP]).
481
Author contributions
Peretz et al.
20
482
SLK, JP, and AP designed the experiments, interpreted results, and wrote the manuscript; JP
483
conducted the experiments and drafted the figures and tables; APL provided cultures, discussed
484
data, and provided critical feedback on the manuscript.
485
Disclosures
486
The authors have no disclosures.
487
488
Peretz et al.
489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
21
References
1.
Bajic VB, Tan SL, Chong A, Tang S, Strom A, Gustafsson JA, Lin CY, and Liu ET. Dragon ERE
Finder version 2: A tool for accurate detection and analysis of estrogen response elements in vertebrate
genomes. Nucleic Acids Res 31: 3605-3607, 2003.
2.
Beery AK, and Zucker I. Sex bias in neuroscience and biomedical research. Neurosci Biobehav
Rev 35: 565-572, 2011.
3.
Casimir GJ, Lefevre N, Corazza F, and Duchateau J. Sex and inflammation in respiratory
diseases: a clinical viewpoint. Biol Sex Differ 4: 16, 2013.
4.
Clayton JA, and Collins FS. Policy: NIH to balance sex in cell and animal studies. Nature 509: 282283, 2014.
5.
Couse JF, Lindzey J, Grandien K, Gustafsson JA, and Korach KS. Tissue distribution and
quantitative analysis of estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta)
messenger ribonucleic acid in the wild-type and ERalpha-knockout mouse. Endocrinology 138: 46134621, 1997.
6.
Crooks GE, Hon G, Chandonia JM, and Brenner SE. WebLogo: a sequence logo generator.
Genome Res 14: 1188-1190, 2004.
7.
Dougherty SM, Mazhawidza W, Bohn AR, Robinson KA, Mattingly KA, Blankenship KA, Huff
MO, McGregor WG, and Klinge CM. Gender difference in the activity but not expression of estrogen
receptors alpha and beta in human lung adenocarcinoma cells. Endocr Relat Cancer 13: 113-134, 2006.
8.
Fischer WA, 2nd, King LS, Lane AP, and Pekosz A. Restricted replication of the live attenuated
influenza A virus vaccine during infection of primary differentiated human nasal epithelial cells. Vaccine
33: 4495-4504, 2015.
9.
Furusyo N, Ogawa E, Sudoh M, Murata M, Ihara T, Hayashi T, Ikezaki H, Hiramine S, Mukae H,
Toyoda K, Taniai H, Okada K, Kainuma M, Kajiwara E, and Hayashi J. Raloxifene hydrochloride is an
adjuvant antiviral treatment of postmenopausal women with chronic hepatitis C: a randomized trial. J
Hepatol 57: 1186-1192, 2012.
10.
Gilliver SC. Sex steroids as inflammatory regulators. J Steroid Biochem Mol Biol 120: 105-115,
2010.
11.
Haddad SN, and Wira CR. Estradiol regulation of constitutive and keratinocyte growth factorinduced CCL20 and CXCL1 secretion by mouse uterine epithelial cells. Am J Reprod Immunol 72: 34-44,
2014.
12.
Hayashida K, Shoji I, Deng L, Jiang DP, Ide YH, and Hotta H. 17beta-estradiol inhibits the
production of infectious particles of hepatitis C virus. Microbiol Immunol 54: 684-690, 2010.
13.
Heldring N, Pike A, Andersson S, Matthews J, Cheng G, Hartman J, Tujague M, Strom A,
Treuter E, Warner M, and Gustafsson JA. Estrogen receptors: how do they signal and what are their
targets. Physiological reviews 87: 905-931, 2007.
14.
Hoffmann J, Otte A, Thiele S, Lotter H, Shu Y, and Gabriel G. Sex differences in H7N9 influenza
A virus pathogenesis. Vaccine 2015.
15.
Ivanova MM, Mazhawidza W, Dougherty SM, Minna JD, and Klinge CM. Activity and
intracellular location of estrogen receptors alpha and beta in human bronchial epithelial cells. Mol Cell
Endocrinol 305: 12-21, 2009.
16.
Jisa E, Dornstauder E, Ogawa S, Inoue S, Muramatsu M, and Jungbauer A. Transcriptional
activities of estrogen receptor alpha and beta in yeast properties of raloxifene. Biochem Pharmacol 62:
953-961, 2001.
17.
Johansen LM, Brannan JM, Delos SE, Shoemaker CJ, Stossel A, Lear C, Hoffstrom BG, Dewald
LE, Schornberg KL, Scully C, Lehar J, Hensley LE, White JM, and Olinger GG. FDA-approved selective
estrogen receptor modulators inhibit Ebola virus infection. Sci Transl Med 5: 190ra179, 2013.
18.
Klein SL, Hodgson A, and Robinson DP. Mechanisms of sex disparities in influenza pathogenesis.
Peretz et al.
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
22
J Leukoc Biol 92: 67-73, 2012.
19.
Klein SL, Schiebinger L, Stefanick ML, Cahill L, Danska J, de Vries GJ, Kibbe MR, McCarthy MM,
Mogil JS, Woodruff TK, and Zucker I. Opinion: Sex inclusion in basic research drives discovery. Proc Natl
Acad Sci U S A 112: 5257-5258, 2015.
20.
Kohanski MA, and Lane AP. Sinonasal epithelial cell response to Staphylococcus aureus burden
in chronic rhinosinusitis. JAMA otolaryngology-- head & neck surgery 141: 341-349, 2015.
21.
Komm BS, and Mirkin S. An overview of current and emerging SERMs. J Steroid Biochem Mol
Biol 143C: 207-222, 2014.
22.
Kos-Kudla B, Ostrowska Z, Marek B, Ciesielska-Kopacz N, Sieminska L, Kajdaniuk D, Nowak M,
and Kudla M. Hormone replacement therapy in postmenopausal asthmatic women. J Clin Pharm Ther
25: 461-466, 2000.
23.
Kovats S. Estrogen receptors regulate innate immune cells and signaling pathways. Cell Immunol
294: 63-69, 2015.
24.
Kurosawa T, Hiroi H, Tsutsumi O, Ishikawa T, Osuga Y, Fujiwara T, Inoue S, Muramatsu M,
Momoeda M, and Taketani Y. The activity of bisphenol A depends on both the estrogen receptor
subtype and the cell type. Endocr J 49: 465-471, 2002.
25.
Laity JH, Lee BM, and Wright PE. Zinc finger proteins: new insights into structural and functional
diversity. Current opinion in structural biology 11: 39-46, 2001.
26.
Larcombe AN, Foong RE, Bozanich EM, Berry LJ, Garratt LW, Gualano RC, Jones JE, Dousha LF,
Zosky GR, and Sly PD. Sexual dimorphism in lung function responses to acute influenza A infection.
Influenza Other Respir Viruses 5: 334-342, 2011.
27.
Lee HR, Kim TH, and Choi KC. Functions and physiological roles of two types of estrogen
receptors, ERalpha and ERbeta, identified by estrogen receptor knockout mouse. Laboratory animal
research 28: 71-76, 2012.
28.
Lin W, Zhu C, Hong J, Zhao L, Jilg N, Fusco DN, Schaefer EA, Brisac C, Liu X, Peng LF, Xu Q, and
Chung RT. The spliceosome factor SART1 exerts its anti-HCV action through mRNA splicing. J Hepatol 62:
1024-1032, 2015.
29.
Massaro D, and Massaro GD. Estrogen regulates pulmonary alveolar formation, loss, and
regeneration in mice. Am J Physiol Lung Cell Mol Physiol 287: L1154-1159, 2004.
30.
Matsubara S, Swasey CH, Loader JE, Dakhama A, Joetham A, Ohnishi H, Balhorn A, Miyahara
N, Takeda K, and Gelfand EW. Estrogen determines sex differences in airway responsiveness after
allergen exposure. Am J Respir Cell Mol Biol 38: 501-508, 2008.
31.
Matthews J, and Gustafsson JA. Estrogen signaling: a subtle balance between ER alpha and ER
beta. Mol Interv 3: 281-292, 2003.
32.
Matthews J, Wihlen B, Tujague M, Wan J, Strom A, and Gustafsson JA. Estrogen receptor (ER)
beta modulates ERalpha-mediated transcriptional activation by altering the recruitment of c-Fos and cJun to estrogen-responsive promoters. Mol Endocrinol 20: 534-543, 2006.
33.
McCown MF, and Pekosz A. The influenza A virus M2 cytoplasmic tail is required for infectious
virus production and efficient genome packaging. J Virol 79: 3595-3605, 2005.
34.
Millas I, Liquidato BM, de Sousa Buck H, Barros MD, Paes RAP, and Dolci JEL. Evaluation of
estrogenic receptors in the nasal mucosa of women taking oral contraceptives. Contraception 83: 571577, 2011.
35.
Morani A, Barros RP, Imamov O, Hultenby K, Arner A, Warner M, and Gustafsson JA. Lung
dysfunction causes systemic hypoxia in estrogen receptor beta knockout (ERbeta-/-) mice. Proc Natl
Acad Sci U S A 103: 7165-7169, 2006.
36.
Muller L, and Jaspers I. Epithelial cells, the "switchboard" of respiratory immune defense
responses: effects of air pollutants. Swiss Med Wkly 142: w13653, 2012.
37.
Murakami Y, Fukasawa M, Kaneko Y, Suzuki T, Wakita T, and Fukazawa H. Selective estrogen
Peretz et al.
585
586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
23
receptor modulators inhibit hepatitis C virus infection at multiple steps of the virus life cycle. Microbes
Infect 15: 45-55, 2013.
38.
Osborne CK, Zhao H, and Fuqua SA. Selective estrogen receptor modulators: structure,
function, and clinical use. Journal of clinical oncology : official journal of the American Society of Clinical
Oncology 18: 3172-3186, 2000.
39.
Paech K, Webb P, Kuiper GG, Nilsson S, Gustafsson J, Kushner PJ, and Scanlan TS. Differential
ligand activation of estrogen receptors ERalpha and ERbeta at AP1 sites. Science 277: 1508-1510, 1997.
40.
Patrone C, Cassel TN, Pettersson K, Piao YS, Cheng G, Ciana P, Maggi A, Warner M, Gustafsson
JA, and Nord M. Regulation of postnatal lung development and homeostasis by estrogen receptor beta.
Mol Cell Biol 23: 8542-8552, 2003.
41.
Pekosz A, Newby C, Bose PS, and Lutz A. Sialic acid recognition is a key determinant of influenza
A virus tropism in murine trachea epithelial cell cultures. Virology 386: 61-67, 2009.
42.
Peretz J, Hall OJ, and Klein SL. Sex Differences in Influenza Virus infection, Vaccination, and
Therapies. In: Sex and Gender Differences in Infection and Treatments for Infectious Diseases, edited by
Klein SL, and Roberts CW. Switzerland: Springer International Publishing, 2015, p. 183-210.
43.
Philpott CM, Wild DC, Wolstensholme CR, and Murty GE. The presence of ovarian hormone
receptors in the nasal mucosa and their relationship to nasal symptoms. Rhinology 46: 221-225, 2008.
44.
Ramanathan M, Jr., and Lane AP. A comparison of experimental methods in molecular chronic
rhinosinusitis research. Am J Rhinol 21: 373-377, 2007.
45.
Robinson DP, Hall OJ, Nilles TL, Bream JH, and Klein SL. 17beta-estradiol protects females
against influenza by recruiting neutrophils and increasing virus-specific CD8 T cell responses in the lungs.
J Virol 88: 4711-4720, 2014.
46.
Robinson DP, Huber SA, Moussawi M, Roberts B, Teuscher C, Watkins R, Arnold AP, and Klein
SL. Sex chromosome complement contributes to sex differences in coxsackievirus B3 but not influenza A
virus pathogenesis. Biology of sex differences 2: 8, 2011.
47.
Robinson DP, Lorenzo ME, Jian W, and Klein SL. Elevated 17beta-estradiol protects females
from influenza a virus pathogenesis by suppressing inflammatory responses. PLoS Pathog 7: e1002149,
2011.
48.
Rodriguez-Garcia M, Biswas N, Patel MV, Barr FD, Crist SG, Ochsenbauer C, Fahey JV, and Wira
CR. Estradiol reduces susceptibility of CD4+ T cells and macrophages to HIV-infection. PLoS One 8:
e62069, 2013.
49.
Sanders CJ, Doherty PC, and Thomas PG. Respiratory epithelial cells in innate immunity to
influenza virus infection. Cell Tissue Res 343: 13-21, 2011.
50.
Schaefer TM, Wright JA, Pioli PA, and Wira CR. IL-1beta-mediated proinflammatory responses
are inhibited by estradiol via down-regulation of IL-1 receptor type I in uterine epithelial cells. J Immunol
175: 6509-6516, 2005.
51.
Sedyaningsih ER, Isfandari S, Setiawaty V, Rifati L, Harun S, Purba W, Imari S, Giriputra S, Blair
PJ, Putnam SD, Uyeki TM, and Soendoro T. Epidemiology of cases of H5N1 virus infection in Indonesia,
July 2005-June 2006. J Infect Dis 196: 522-527, 2007.
52.
Sentman CL, Meadows SK, Wira CR, and Eriksson M. Recruitment of uterine NK cells: induction
of CXC chemokine ligands 10 and 11 in human endometrium by estradiol and progesterone. J Immunol
173: 6760-6766, 2004.
53.
Shirasaki H, Watanabe K, Kanaizumi E, Konno N, Sato J, Narita S-I, and Himi T. Expression and
localization of steroid receptors in human nasal mucosa. Acta Oto-Laryngologica 124: 958-963, 2004.
54.
Straub RH. The complex role of estrogens in inflammation. Endocr Rev 28: 521-574, 2007.
55.
Szotek EL, Narasipura SD, and Al-Harthi L. 17β-estradiol inhibits HIV-1 by inducing a complex
formation between β-catenin and estrogen receptor on the HIV promoter to suppress HIV transcription.
Virology 443: 375-383, 2013.
Peretz et al.
633
634
635
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
24
56.
Teijaro JR, Walsh KB, Rice S, Rosen H, and Oldstone MB. Mapping the innate signaling cascade
essential for cytokine storm during influenza virus infection. Proc Natl Acad Sci U S A 111: 3799-3804,
2014.
57.
Vandenberg LN, Colborn T, Hayes TB, Heindel JJ, Jacobs DR, Jr., Lee DH, Shioda T, Soto AM,
Vom Saal FS, Welshons WV, Zoeller RT, and Myers JP. Hormones and endocrine-disrupting chemicals:
low-dose effects and nonmonotonic dose responses. Endocr Rev 33: 378-455, 2012.
58.
Vegeto E, Cuzzocrea S, Crisafulli C, Mazzon E, Sala A, Krust A, and Maggi A. Estrogen Receptorα as a Drug Target Candidate for Preventing Lung Inflammation. Endocrinology 151: 174-184, 2010.
59.
Velez-Ortega AC, Temprano J, Reneer MC, Ellis GI, McCool A, Gardner T, Khosravi M, and Marti
F. Enhanced generation of suppressor T cells in patients with asthma taking oral contraceptives. J
Asthma 50: 223-230, 2012.
60.
Watanabe T, Watanabe S, and Kawaoka Y. Cellular networks involved in the influenza virus life
cycle. Cell host & microbe 7: 427-439, 2010.
61.
WHO. Disease and injury regional mortality estimates, 2000–2012; global summary estimates
http://www.who.int/healthinfo/global_burden_disease/estimates/en/index1.html. [November 10,
2015].
62.
Wira CR, Fahey JV, Rodriguez-Garcia M, Shen Z, and Patel MV. Regulation of mucosal immunity
in the female reproductive tract: the role of sex hormones in immune protection against sexually
transmitted pathogens. Am J Reprod Immunol 72: 236-258, 2014.
63.
Wira CR, Rodriguez-Garcia M, and Patel MV. The role of sex hormones in immune protection of
the female reproductive tract. Nat Rev Immunol 15: 217-230, 2015.
64.
Zarychanski R, Stuart TL, Kumar A, Doucette S, Elliott L, Kettner J, and Plummer F. Correlates of
severe disease in patients with 2009 pandemic influenza (H1N1) virus infection. CMAJ 182: 257-264,
2010.
65.
Zimmerman JL, Woodruff PG, Clark S, and Camargo CA. Relation between phase of menstrual
cycle and emergency department visits for acute asthma. Am J Respir Crit Care Med 162: 512-515, 2000.
Peretz et al.
25
660
Figure Legends
661
Figure 1. 17β-estradiol (E2) inhibits infectious virus production in hNECs from female,
662
but not male, donors. hNEC cultures from (A) female or (B) male donors were pre-treated with
663
E2 (0.1, 1, and 10nM) or vehicle for 24 hours in the basolateral media, then infected with IAV
664
(MOI = 0.1 TCID50/cell) via the apical membrane. Supernatants were collected every 24 hours
665
post infection (hpi) and virus titers were analyzed by TCID50 assay. The limit of detection is
666
indicated by a dotted line. The data represent means ± SEMs. Asterisks (*) indicate that E2-
667
treated cultures were significantly different from sex-matched vehicle controls based on a
668
MANOVA followed by planned comparisons, with n= 15 hNEC wells per treatment group,
669
p<0.05.
670
671
Figure 2. Extended E2 pre-treatment and E2 treatment post-infection inhibits infectious
672
virus production in hNECs from female donors. hNEC cultures from females were (A) pre-
673
treated with E2 (10nM) for 72 hours prior to IAV infection or (B) treated with E2 (10nM) 24 hours
674
after infection with IAV (MOI = 0.1 TCID50/cell). Supernatants were collected every 24 hours
675
post infection (hpi) and virus titers were analyzed by TCID50 assay. The limit of detection is
676
indicated by a dotted line. The data represent means ± SEMs. Asterisks (*) indicate that E2-
677
treated cultures were significantly different from vehicle controls based on a MANOVA followed
678
by planned comparisons, with n= 12 hNEC wells per treatment group, p<0.05.
679
680
Figure 3. The estrogen receptor antagonist, ICI 182,780, reverses the effects of E2 on
681
virus titers in hNECs from females. hNEC cultures from female donors were pre-treated with
682
E2 (10nM), ICI (100nM), E2+ ICI, or vehicle for 24 hours in the basolateral media, then infected
683
with IAV (MOI = 0.1 TCID50/cell) via the apical membrane. Supernatants were collected every
684
24 hours post infection (hpi) and virus titers were analyzed by TCID50 assay. The limit of
685
detection is indicated by a dotted line. The data represent means ± SEMs. Asterisks (*) indicate
Peretz et al.
26
686
that E2-treated cultures were significantly different from vehicle controls based on a MANOVA
687
followed by planned comparisons, with n= 12 hNEC wells per treatment group, p<0.05.
688
689
Figure 4. E2 stimulates the expression of ESR2, but not ESR1, mRNA in hNECs from
690
female donors only. hNEC cultures from female and male donors were pre-treated with E2
691
(10nM) or vehicle for 24 hours in the basolateral media, infected with IAV (MOI = 0.1
692
TCID50/cell) via the apical membrane, and after 24 hours, the cells were collected for analysis of
693
ESR1 (A) and ESR2 (B) expression. In hNECs from females only, the kinetics of mRNA
694
expression of ESR1 (C) and ESR2 (D) was analyzed in cultures collected at 24 and 48 hours
695
post infection (hpi). The fold change of ESR1 and ESR2 mRNA was determined by the ΔΔCt
696
method using 18sRNA as the reference gene. The data represent means ± SEMs with the
697
stippled line indicating the normalized expression level of genes in vehicle-treated, mock-
698
infected cultures. An asterisk (*) indicates a significant difference from the mock vehicle control.
699
One-way ANOVA and Tukey post-hoc tests, n= 3-5 hNEC wells per treatment group per sex,
700
p<0.05.
701
702
Figure 5. hNEC cultures derived from female donors express ESR2, but not ESR1,
703
protein. hNEC cultures from female donors were pre-treated with E2 (10nM) or vehicle for 24
704
hours in the basolateral media, then infected with IAV (MOI = 0.1 TCID50/cell) via the apical
705
membrane. After 24 hours, the cells were lysed and subjected to Western Blot analysis for
706
ESR1, ESR2, and ACTB measurements (A). Protein isolated from MCF-7 cells was used as a
707
positive control for estrogen receptor expression. Quantification of ESR1 and ESR2 relative to
708
ACTB was quantified by ImageJ analyses (B).
709
710
Figure 6. E2 treatment reduces metabolic processes in IAV-infected hNECs from females.
711
hNEC cultures from female donors were pre-treated with E2 (10nM) or vehicle for 24 hours in
Peretz et al.
27
712
the basolateral media, then infected with IAV (MOI = 0.1 TCID50/cell) via the apical membrane.
713
At 24 and 48 hours post infection (hpi), cells were harvested, RNA was isolated, and spotted on
714
Affymetrix Human GeneST2.0 arrays. Data from infected hNECs were normalized to uninfected
715
hNECs within the same treatment group and ANOVAs in Partek Genomics Suite version 6.0
716
were used to determine significant differences between E2- and vehicle-treated, IAV-infected
717
hNECs. The Venn diagram (A) illustrates the number of differentially expressed genes at 24 and
718
48hpi. Gene ontology (B) was used to group genes differentially expressed between E2- and
719
vehicle-treated, IAV-infected hNECs at 24hpi into functional hierarchies based on enrichment
720
score, with scores >3 indicating significant differences. (C) A heat map was created using
721
unbiased hierarchical clustering to represent the largest family of differentially expressed genes
722
(i.e., genes that encode zinc finger proteins) between E2- and vehicle-treated, IAV-infected
723
hNEC cultures at 24hpi.
724
725
Figure 7. Identification of putative estrogen response elements in zinc finger protein
726
gene promoters. (A) The Dragon ERE Finder version 3.0 (http:// http://datam.i2r.a-
727
star.edu.sg/ereV3/) was used to identify putative estrogen response elements (EREs) in the
728
promoters of zinc finger protein (ZFP) genes. The WebLogo sequence generator was used to
729
develop a graphical representation of the ERE nucleic acid sequence conservation (GGTCA-
730
nnn-TGACC) among the identified ZFP genes (6). (B) Cells were collected for analysis by real
731
time RT-PCR to validate the ZNF91, ZMYM6, and ZFAND4 mRNA expression levels. Fold
732
change of the target genes was determined by the ΔΔCt method using 18sRNA as the
733
reference gene. The data represent means ± SEMs with an asterisk (*) indicating significant
734
differences compared to vehicle based on student’s t-tests at each time-point, n= 4-5 hNEC
735
wells per treatment group, p<0.05.
736
Peretz et al.
28
737
Figure 8. Select estrogenic compounds with affinity for ESR2 reduce virus titers in
738
hNECs from female donors. hNEC cultures from female donors were pre-treated with vehicle
739
and either (A) selective estrogen receptor modulators, including clomiphene citrate, ospemifene,
740
and raloxifene or (B) bisphenol (BPA) for 24 hours in the basolateral media and infected with
741
IAV (MOI = 0.1 TCID50/cell) via the apical membrane. Supernatants were collected every 24
742
hours post infection (hpi) and virus titers were analyzed by TCID50. The limit of detection is
743
indicated by a dotted line. The data represent means ± SEMs. An asterisk (*) indicates a
744
significant difference between vehicle-treated hNECs and raloxifene-treated (A) or BPA-treated
745
(B) based on 1-way ANOVAs and Tukey post-hoc tests, n= 9 hNEC wells per treatment group,
746
p<0.05.
747
748
Table 1. Effects of E2 on cytokine production in hNECs from female donors. hNEC
749
cultures from female donors were pre-treated with E2 (0.1, 1, and 10nM) or vehicle for 24 hours
750
in the basolateral media, then infected with IAV (MOI = 0.1 TCID50/cell) via the apical
751
membrane. Supernatants collected at 72 hours post infection (hpi) were analyzed by MSD
752
assay for human chemokines or ELISA for IFNλ. Data represent means ± SEMs. N.D. indicates
753
not detectable. E2 had no effect on chemokine or interferon production in hNECs from female
754
donors. An asterisk (*) indicates significant differences between the IAV-infected and mock-
755
infected hNECs within a treatment group, based on a 2-way ANOVA followed by Tukey post-
756
hoc tests, n= 3-9 hNEC wells per treatment group, p<0.05.
Table 1. Effects of E2 on chemokine and interferon production in hNECs from females
Mock
Protein†
(pg/mL)
CCL11
CCL26
CXCL8
CXCL10
CCL2
CCL13
CCL22
CCL3
CCL4
CCL17
IFNλ
IAV
E2 (nM)
E2 (nM)
Vehicle
13.1
±2.64
5.71
±0.51
1340
±302
2.50
±0.56
0.32
±0.09
14.7
±3.38
17.5
±2.59
21.1
±3.09
6.73
±0.70
4.31
±0.30
0.1
19.7
±1.83
6.56
±0.92
715
±78.0
1.88
±0.26
0.38
±0.08
12.4
±1.89
15.7
±4.79
28.4
±2.56
6.07
±0.81
3.93
±0.32
1
15.9
±2.58
6.31
±0.68
1100
±326
9.46
±3.65
0.17
±0.05
13.3
±1.39
17.8
±2.14
27.8
±2.27
7.04
±0.78
4.00
±0.25
10
15.6
±1.39
5.82
±0.72
824
±105
2.50
±0.42
0.32
±0.12
11.7
±2.78
16.9
±2.76
21.3
±2.07
5.16
±0.57
4.36
±0.25
N.D.
N.D.
N.D.
N.D.
Vehicle
*103
±10.5
2.27
±0.51
*2920
±386
*14100
±2600
*4.82
±0.54
18.5
±3.42
*115
±18.2
*76.4
±6.09
*42.4
±4.69
*23.1
±2.41
*20100
±7540
0.1
*116
±17.5
1.87
±0.62
*2940
±414
*14900
±3080
*5.16
±0.63
13.2
±1.47
*164
±23.8
*137
±51.3
*48.2
±6.36
*32.1
±5.61
*14200
±4060
1
*129
±16.9
3.51
±0.85
*2840
±479
*18400
±3870
*4.22
±0.53
14.3
±2.88
*164
±26.7
*86.4
±7.66
*44.2
±5.06
*25.1
±3.69
*14800
±4960
† data presented as mean ± SEM
* IAV > Mock within treatment group based on 2-way ANOVA;, p ≤ 0.001
10
*102
±9.10
2.43
±0.84
*3070
±466
*15100
±3010
*5.32
±0.61
16.0
±1.82
*110
±16.5
*83.5
±6.80
*45.2
±4.52
*23.1
±2.10
*16800
±6850