Limited replication of influenza A virus in human mast cells

Candy W. Marcet • Chris D. St. Laurent •

Tae Chul Moon • Nav Singh • A. Dean Befus

Published online: 30 September 2012

� Springer Science+Business Media New York 2012

Abstract Mast cells are important in innate immunity

and protective against certain bacterial infections. How-

ever, there is limited evidence that mast cells respond to

viruses. As mast cells are abundant in mucosal tissues of

the lung, they are in a prime location to detect and respond

to influenza virus. In this study, we characterized for the

first time the replication cycle of influenza A virus in

human mast cells by measuring influenza A virus tran-

scription, RNA replication, protein synthesis, and forma-

tion of infectious virus as compared to the replication cycle

in epithelial cells. We detected the presence of influenza A

viral genomic RNA transcription, replication, and protein

synthesis in human mast cells and epithelial cells. How-

ever, there was no significant release of infectious influenza

A virus from mast cells, whereas epithelial cells produce

*100-fold virus compared with the inoculating dose. We

confirmed that influenza A virus infects human mast cells,

begins to replicate, but the production of new virus is

aborted. Thus, mast cells may lack critical factors essential

for productive infection or there are intrinsic or inducible

anti-influenza A mechanisms in mast cells.

Keywords Mast cell � Influenza A � Myxovirus �Innate immunity � Antiviral � Hemagglutinin

Introduction

Influenza virus causes a febrile respiratory disease in

humans that ranges from self-limiting infection to primary

viral pneumonia that has the potential to be fatal [1]. Of the

three influenza virus types A, B, and C, influenza A virus

(FluA) causes the majority of human disease [2]. Although

FluA can be associated with pandemics, yearly seasonal

influenza results in significant morbidity and mortality,

with an estimated half a million deaths worldwide every

year [2].

An improved understanding of how the immune system

defends against FluA is critical in the development of novel

preventative and therapeutic strategies for influenza

infection. Research on innate immunity against FluA has

focused on the inflammatory and antiviral responses of

epithelial cells, the primary target of FluA replication

[2, 3]. However, the complexity of host defences against

the pathogens reveals the interplay of many cell types. By

distinguishing the roles of different cells in the response

against FluA, we will gain a better understanding of the

dynamic interactions involved and elucidate the critical

players involved in viral immunity.

Mast cells are emerging as an important cell type in

innate immunity and host defenses [4]. Mast cells are

widely distributed and abundant at mucosal surfaces and

thus are in prime locations to encounter microbes and alert

the immune system [5]. Evidence was initially developed

for mast cell responses to bacterial and fungal infections

and more recently to viruses such as human immunodefi-

ciency virus (HIV), vesicular stomatitis virus, dengue,

influenza, and many others [6–11]. For example, dengue

virus is capable of infecting mast cells and can induce their

degranulation and release of cytokines IL-1b, TNF, type

I interferons, and IL-6, as well as several chemokines

C. W. Marcet � C. D. St. Laurent � T. C. Moon � N. Singh

Pulmonary Research Group, Department of Medicine, Room

567, HMRC, University of Alberta, Edmonton, AB T6G 2S2,

Canada

A. D. Befus (&)

AstraZeneca Canada Chair in Asthma Research, Pulmonary

Research Group, Department of Medicine, Room 550A, HMRC,

University of Alberta, Edmonton,

AB T6G 2S2, Canada

e-mail: dean.befus@ualberta.ca

123

Immunol Res (2013) 56:32–43

DOI 10.1007/s12026-012-8377-4

[10, 12–14]. Moreover, dengue virus induces other antivi-

ral responses in mast cells, including upregulation of

nucleic acid sensors such as melanoma differentiation-

associated gene 5 (MDA5), retinoic acid inducible gene 1

(Rig-I), and protein kinase R (PKR) [10, 14].

Currently, there is little evidence that human mast cells

respond to FluA. However, FluA has been associated with

increased histamine levels in the lungs of patients, imply-

ing potential interactions between FluA and mast cells [8].

Also, human mast cells respond to UV-inactivated FluA by

producing interferon-a [15]. To study human mast cell

responses to FluA, we investigated whether or not FluA

infects human mast cells and how FluA infection proceeds.

We defined infection of mast cells as when there was

evidence that FluA was using host machinery, that is,

mRNA transcription, since this is the first process we

studied that requires host components. Our results show

that FluA infects mast cells and that viral RNA replication,

transcription and protein synthesis occur, but little infec-

tious FluA is released compared to epithelial cells. The

paucity of release of FluA progeny from mast cells sug-

gests that mast cells do not support certain components of

viral replication and budding, perhaps as a result

of intrinsic deficiencies of permissive factors or presence of

non-permissive factors [16] and/or induced antiviral

mechanisms. Since mast cells are closely associated with

epithelial cells in the lungs, mast cells may have modula-

tory effects on FluA infection of epithelial cells that

influence the course of infection in the host.

Materials and methods

Cell culture

The human mast cell line, LAD2 (laboratory of allergic

diseases 2), developed from human bone marrow mononu-

clear cells (generously provided by Dr. D. D. Metcalfe and A

Kirshenbaum, National Institutes of Health, Bethesda, MD)

was cultured as previously described [17, 18]. Human

peripheral blood-derived primary cultured mast cells

(HCMC) were developed from CD34? progenitors [19].

Briefly, 100 ml of blood was drawn from healthy human

donors in 10-ml heparinized VacutainerTM tubes (BD Can-

ada, Oakville, ON, Canada) and layered on Histopaque 1077

(Sigma-Aldrich Canada Ltd., Oakville, ON, Canada). The

mononuclear cell fraction was obtained after centrifugation

at 4509g for 30 min. After washing the mononuclear cell

fraction twice with 10 mM phosphate buffer containing

150 mM NaCl [phosphate-buffered saline (PBS)], CD34?

progenitors were isolated using the EasySep� human CD34

positive selection kit (StemCell Technologies, Vancouver,

BC, Canada). CD34? cells were cultured at 5 9 104 cells/ml

in StemSpan� SFEM (StemCell Technologies) supple-

mented with 100 ng/ml rhSCF and 100 ng/ml rhIL-6 for

8 weeks, with 30 ng/ml rhIL-3 for the first week only. The

volume of StemSpan� SFEM was hemidepleted every

3.5 days. At 4 weeks, the entire volume of old media was

replaced once by fresh media and then hemidepleted every

3.5 days until 8 weeks. Primary mast cell cultures were used

after 8 weeks and confirmed as [99 % mast cells by tryp-

tase/chymase staining before use [20, 21].

The human lung adenocarcinoma epithelial cell line

Calu-3 (ATCC� number: HTB-55TM) was obtained from

American Type Culture Collection (ATCC, Manassas, VA)

and cultured as previously described [22] with MEM ?

Earle’s salts (Invitrogen, Grand Island, NY) and L-gluta-

mine supplemented with 10 % fetal bovine serum, 0.1 mM

non-essential amino acids, 1 mM sodium pyruvate, 100 U/

ml penicillin, and 100 lg/ml streptomycin.

Influenza virus and ultraviolet (UV) inactivation

Influenza A virus (FluA) of the A/PR/8/34 strain (H1N1)

grown in allantoic fluid of duck eggs was obtained from

Dr. K. P. Kane (University of Alberta). UV inactivation of

virus was performed using a UV lamp (ENF-280C,

Spectroline, Westbury, NY) at 254 nm, placed 20 cm from

the sample for 20 min. Effective inactivation was confirmed

by positive hemagglutination and negative hemadsorption.

Virus exposure

LAD2 and HCMC were seeded at 1 9 106 cells/ml in 1 ml

of media in 12-well plates, and Calu-3 was plated at

1.5 9 105 cells/ml in 5 ml of media in 6-well tissue culture

plates. Mast cells were rested for 1 h at 37 �C prior to FluA

treatment. Calu-3 was incubated at 37 �C overnight prior to

FluA treatment to allow monolayer formation; 1 ml of

FluA was added to the cells at various concentrations for

1 h to allow for virus adsorption onto the cell surface.

Then, cells were washed 3 times and no FluA was detected

in the 3rd wash as determined by lack of hemagglutination

in assays described below.

Cell viability

Cell number was counted using a BRIGHT-LINETM

hemacytometer (Hausser Scientific, Horsham, PA). Cell

viability was calculated as percentage of live cells by try-

pan-blue (0.4 % in PBS) exclusion.

Hemagglutination assay

Hemagglutination assay was performed to quantify total

FluA in a sample by the ability to bind red blood cells

Immunol Res (2013) 56:32–43 33

123

(RBC) as previously described [23]; 50 ll supernatant

samples were placed in a round-bottom 96-well microtitre

plate (BD Biosciences, Mississauga, ON, Canada) and

serially diluted using PBS/0.1 % BSA. An equal volume of

0.5 % human RBC was mixed with the diluted samples and

incubated for 2–3 h at room temperature. Positive hem-

agglutination was verified with the presence of lattice

formation, while negative hemagglutination was verified

by a dot formation of RBC at the bottom of the well. The

lowest dilution at which hemagglutination was present was

determined as the hemagglutination unit (HAU) of that

sample. Viral titers were expressed in hemagglutination

unit (HAU)/ml.

Hemadsorption assays

We performed the hemadsorption assay [24] with modifi-

cations. Calu-3 monolayers in 48-well plates at [80 %

confluency were treated with 125 ll samples for 1 h to

allow for the virus adsorption, then washed three times

with PBS and fresh media was added. After 5 days, we

removed the media, added 2 ml of 0.5 % RBC and placed

the plates at 4 �C for 30 min. After washing 2 times with

PBS, cells were observed with an inverted microscope to

detect positive hemadsorption as evidenced by rosette or

plaque formations.

Determination of 50 % tissue culture infectious dose

(TCID50)/ml

TCID50/ml was determined by the dilution at which 50 %

of FluA-infected wells shows hemadsorption. Calu-3

monolayers at[80 % confluency were treated with 125 ll

of serial 10-fold dilutions of each sample in eight identical

wells. Plates were incubated for 5 days at 37 �C and

assayed for hemadsorption. TCID50/ml was calculated

using the Reed-Muench method [25].

Reverse transcription-polymerase chain reaction

(RT-PCR) for specific species of FluA RNA

Total RNA was extracted with the RNAqueous�-4PCR kit

(Life Technologies Inc., Burlington, ON, Canada) accord-

ing to the manufacturer’s instructions and quantified by

measuring optical density at 260 nm, and then cDNA

synthesis was completed using the SuperScriptTM III first-

strand synthesis kit (Invitrogen) according to the manu-

facturer’s recommendations. Oligo(dT)20 was used to

reverse transcribe all messenger RNA (mRNA). Forward

(50-GGA GAA GGA GGG CTC ATA CC-30) and reverse

(50-AAA CAA GGG TGT TTT TCC TCA-30) primers

specific for the hemagglutinin (HA) gene were used to

reverse transcribe negative-sense viral genomic RNA

(vRNA) and complementary RNA (cRNA), respectively.

PCR was performed for the vRNA, cRNA, and mRNA of

HA, as well as the mRNA of polymerase B1 (PB1) using

JumpStartTM RED Taq DNA polymerase (Sigma-Aldrich

Canada Ltd.) with specific primer sets: HA (469 bp), for-

ward 50- GGA GAA GGA GGG CTC ATA CC-30, reverse

50-CCT GAC CGT ATT TTG GGC ACT-30; PB1 (427 bp),

forward 50-AAC GAT GGA GGT TGT TCA GC-30,reverse 50-AAA CCC CCT TAT TTG CAT CC-30. b-actin

(326 bp) was used as internal control (forward 50-GGC

ATC CTC ACC CTG AAG TA-30, reverse 50-AGG GCA

TAC CCC TCG TAG AT-30). The conditions for PCR

amplification were denaturing at 94 �C for 45 s, annealing

at 58 �C for 45 s, and extension at 72 �C for 1 min. We

optimized the cycle number for PB1, HA, and b-actin to be

32, 28, and 28 cycles, respectively, to be within the

exponential phase of amplification. PCR products were

visualized under UV light after separation of a 1 % agarose

gel containing ethidium bromide. Specificity of HA cRNA

and mRNA detection were confirmed using another

HA-specific primer set (forward 50-CAG GGA AAA GGT

AGA TGG AGT G-30, reverse 50-AAA CAA GGG TGT

TTT TCC TCA-30). PCR product was detected when the

reverse primer was used for reverse transcription, but not

detected when oligo(dT)20 was used (data not shown). The

PCR products for mRNA, vRNA and cRNA of HA, and

mRNA of PB1 and b-actin were confirmed by sequencing

(DNA Core Services Lab, University of Alberta).

Western blot

Proteins were precipitated using the ReadyPrepTM 2-D

Cleanup Kit (Bio-Rad Laboratories, Mississauga, ON,

Canada) and reconstituted to 1 lg/ml. Protein samples

were separated using 10 % SDS–polyacrylamide gel elec-

trophoresis and transferred onto PVDF membranes using

semi-dry transfer (Bio-Rad Laboratories). FluA proteins

were detected using rabbit antiserum to FluA (A/PR/8/34)

(generous gift from Dr. Earl G. Brown, University of

Ottawa) [26]. Isotype controls were performed using nor-

mal rabbit serum (also from Dr. Earl G. Brown). b-actin

was detected using a mouse monoclonal antibody (AC-15)

against b-actin (Santa Cruz Biotechnology, Santa Cruz,

CA) and used as a loading control. IRDye�680-conjugated

goat anti-mouse IgG and IRDye�800CW-conjugated anti-

rabbit IgG were used with the Odyssey� Infrared Imaging

System for immunodetection of proteins (Li-cor Biosci-

ences, Lincoln, NE). Membranes containing FluA-treated

samples were scanned together with membranes containing

UV inactivated and mock control-treated samples, and

subtraction of background was performed simultaneously

on all membranes using the image curve adjustment tool in

the odyssey software to optimize consistency.

34 Immunol Res (2013) 56:32–43

123

Data and statistical analysis

Densitometry was performed using ImageJ (NIH Image,

Bethesda, MD). Statistical analysis was performed using

GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla,

CA). The one-way analysis of variance (ANOVA) with

Bonferroni post-test was used when comparing greater than

two sets of values. Two-way ANOVA was used when

comparing data sets with responses based on more than two

variables. A p value of \0.05 was considered significant.

Data are expressed as mean ± SEM.

Results

FluA treatment affects viability of the human mast cell

line, LAD2

To analyze the response of mast cells to FluA treatment,

we exposed mast cells to FluA in vitro. We investigated

whether FluA treatment affects the viability and cell

number of LAD2 and HCMC, as well as the epithelial cell

line Calu-3 (Fig. 1). The cell number and viability of

LAD2 followed similar trends in response to 0–100 HAU/

ml of FluA at 2 days post-treatment (Fig. 1a). FluA at a

dose of 10 HAU/ml (78.9 ± 6.6 %) and 100 HAU/ml

(58.1 ± 2.5 %) significantly decreased the viability of

LAD2 compared with mock infection (90.0 ± 2.3 %), and

also cell number (65.4 ± 9.7 %) compared to mock

(Fig. 1a). Viability was determined for LAD2, HCMC, and

Calu-3 from 0 to 8 days post-mock or FluA treatment using

20 HAU/ml (Fig. 1b–d). Viability between mock and FluA

treatments in Calu-3 was not significantly different

(Fig. 1b). Over the course of the 8 days cultures, LAD2

exposed to FluA showed significantly lower viability than

mock-exposed cells (Fig. 1c). In HCMC, there was

decreased viability with both FluA and mock treatments as

the number of days post-treatment increased (Fig. 1d).

Transcription and RNA replication of FluA takes place

in human mast cells

In the FluA life cycle, after the virus releases its genomic

contents into the cytoplasm, mRNA transcription from the

negative-sense viral RNA (vRNA) takes place followed by

replication of the vRNA into full-length complementary

RNA (cRNA). The cRNA is then used to generate more

copies of vRNA to be packaged into newly formed virus.

We investigated whether transcription and replication of

the different FluA RNA species occur in the mast cell line

LAD2. To evaluate, mast cells were exposed to 0.01–

100 HAU/ml of FluA for 2 days. As shown in Fig. 2, we

detected the expression of vRNA, mRNA, and cRNA for

the hemagglutinin (HA) gene of FluA in mast cells after

2 days of treatment with various doses of FluA. RT-PCR of

stock FluA showed genomic vRNA, but not mRNA and

cRNA (data not shown), confirming that the detected

mRNA and cRNA in infected cells were newly synthe-

sized. Study of the different RNA types in the mock

treatments did not generate a signal above background

(Fig. 2a).

Next, we performed a time course analysis of FluA

vRNA, mRNA, and cRNA expression to compare the

amount and duration of viral RNA in epithelial cells and

mast cells. In both Calu-3 and mast cells, vRNA, mRNA,

and cRNA of HA were detected (Fig. 3). mRNA tran-

scription for both HA (Fig. 3) and PB1 (Fig. 4) was

detected as early as 0.25 days (6 h) post-FluA treatment,

but there was no detectable signal for FluA in the mock

treatments for all cell types (data not shown). Of note, the

0-h time point indicates the beginning of the post-FluA

treatment period, after 1 h of FluA adsorption and sub-

sequent washes. Thus, the presence of FluA transcripts at

0 h in Figs. 3 and 4 is not unexpected. In Calu-3, the HA

(Fig. 3a, c) and PB1 (Fig. 4a, b) mRNA signal is signifi-

cantly increased up to 4 days post-FluA treatment com-

pared to 0 day. However, in both LAD2 and HCMC, HA

(Fig. 3e, g) and PB1 (Fig. 4c–f) mRNA was significantly

increased up to 2 days of post-FluA treatment and then

decreased. These results indicate that human mast cells

begin transcription of FluA mRNA at a similar time as

Calu-3 epithelial cells, but mRNA transcription in mast

cells is not sustained compared to Calu-3.

FluA protein synthesis takes place in human mast cells

After confirmation that FluA gene transcription and repli-

cation occur in mast cells, we investigated LAD2 and

HCMC for FluA protein expression in comparison with

Calu-3. The FluA proteins recognized by an anti-FluA

serum are as follows: hemagglutinin (HA); nucleoprotein

(NP); matrix protein 1 (M1); and non-structural protein 1

(NS1) (confirmed by correspondence with Dr. E. Brown)

[26].

All FluA proteins recognized by the anti-FluA serum

were detected in at least one experiment in Calu-3, LAD2,

and HCMC (Fig. 5). HA (70 kDa), NP (58 kDa), and M1/

NS1 (26–27 kDa with similar gel mobilities) were detected

by 6 h in all 3 cell types, after infection with 20 and

100 HAU/mL FluA (Fig. 5), although there is variability in

detection of HA in Calu-3 (4/8 experiments), HA in HCMC

(4/5 experiments), and M1/NS1 in HCMC (3/6 experi-

ments). None of these proteins were detected in cells from

mock or UV-inactivated FluA treatments, with the excep-

tion of a faint band near the molecular weight of NP in a

single experiment of HCMC treated with UV-inactivated

Immunol Res (2013) 56:32–43 35

123

FluA (Fig. 5c). Non-specific bands at approximately 37–

40 kDa were detected in all treatments including mock and

UV inactivated, as well as several other faint non-specific

bands that do not correspond to viral proteins.

Detection of FluA released from human mast cells

compared with Calu-3

After we determined that the production of FluA in Calu-3

is comparable in production in MDCK, a standard canine

cell line used for FluA (data not shown), we investigated

whether FluA undergoes productive infection in human

mast cells by measuring the amount of FluA released from

mast cells using the hemagglutination assay. Release of

FluA from Calu-3, LAD2, and HCMC was measured from 0

to 6 days post-FluA treatment at 20 HAU/ml. At 20 HAU/

ml of FluA treatment, Calu-3 release of FluA was detected

at 1 day (416 ± 298 HAU/ml) with increased release at

2 days (971 ± 411 HAU/ml), 3 days (1410 ± 410 HAU/

ml), and 4 days (1710 ± 453 HAU/ml) (Fig. 6a). LAD2

and HCMC did not release significant amounts of FluA

using 20 HAU/ml of initial FluA-infecting dose (Fig. 6a),

and this was not changed by increasing initial FluA-

infecting dose to 100 HAU/ml (data not shown). No release

of FluA was detected by hemagglutination assay at 0 day

treatments at both 20 and 100 HAU/ml with any of the cell

types (Fig. 6a), and there was no detectable virus release in

experiments with the cell types treated with 100 HAU/ml of

UV-inactivated FluA (data not shown). This data indicate

that little FluA is released from mast cells after infection.

Assessment of infectivity of FluA released from human

mast cells compared with Calu-3

As the hemagglutination assay detects HA of FluA whether

the virus is inactivated or infectious, we tested whether

human mast cells release infectious FluA by measuring the

infectivity of supernatants from FluA-treated HCMC and

LAD2 in comparison with Calu-3. After we determined the

sensitivity of Calu-3 as a host cell in the hemadsorption assay

was comparable with MDCK (data not shown), FluA-treated

supernatants after 2 days from HCMC, LAD2, and Calu-3

Fig. 1 Cell number and viability of human mast cells and lung

epithelial cells treated with FluA. Mock treatment (0 HAU/ml)

consisted of media only. Cell number was calculated as percentage of

mock number where mock was set at 100 %. Viability was

determined as percentage of live cells (% viability of total cell

number). Note that SCF was added in fresh media at 0 days post-FluA

treatment. a Cell number and viability of the human mast cell line

LAD2. Cells were treated with FluA from 0 to 100 HAU/ml

for 2 days. Repeated measures one-way ANOVA with Bonferroni

post-test was performed and statistical significance for cell number is

represented as ##p \ 0.01 and for viability as *p \ 0.05,

***p \0.001, when compared to mock treatment (n = 4). b, c, dViability of the human lung epithelial cell line Calu-3, LAD2, and

primary human cultured mast cells (HCMC), respectively, was

determined at 0–8 days post-treatment with mock or FluA at 20 HAU/

ml. Two-way ANOVA was performed, and statistical significance for

viability is represented as ***p \ 0.001, when compared to mock

treatments in panels b–d (b n = 1–5; c n = 2–6; d n = 1–2)

36 Immunol Res (2013) 56:32–43

123

were tested for infectious virus by detecting positive

hemadsorption on Calu-3 monolayers. Dose response

experiments using varying initial infecting doses of FluA

were conducted for Calu-3 and LAD2. Calu-3 released

infectious FluA with 0.002 HAU/ml in 2 of 3 experiments

and in 3 of 3 experiments at higher doses tested up to

20 HAU/ml (Table 1, top panel). In contrast, LAD2 only

showed detectable release of infectious FluA when treated

with 100 HAU/ml of FluA in 6 of 6 experiments and

20 HAU/ml in 1 of 6 experiments (Table 1, bottom panel).

These results show that release of infectious FluA from mast

cells was only detectable at [4 log higher doses of initial

FluA infection compared with the FluA dose used in Calu-3

cells.

Next, we compared the time course of infectious FluA

release from human mast cells with that of Calu-3. Using

20 HAU/ml of FluA treatment by 6 h, Calu-3 supernatants

contained infectious FluA in 4 of 6 experiments (Fig. 6b).

There was positive FluA infectivity in Calu-3 supernatants

at 1–4 days post-FluA treatment in all experiments

(Fig. 6b). At the same 20 HAU/ml FluA dose, LAD2

supernatants contained infectious FluA at both 0 and 6 h in 2

of 3 experiments, but at subsequent time points did not

demonstrate any release of infectious virus (with the

exception of 1 experiment at 2 days) (Fig. 6b). No detectable

infectious FluA was released by HCMC at any time points

when treated with 20 HAU/ml of FluA (Fig. 6b). However,

by increasing the dose to 100 HAU/ml, we were able to

detect the release of infectious FluA from LAD2 and HCMC

at 0 h to 2 days, but none at the later time points of 3 and

4 days (data not shown). Given 100 HAU/ml, Calu-3

released infectious FluA in all experiments from 0 h to

4 days (data not shown). The observation that there is

infectious FluA in supernatants of human mast cells at earlier

time points but not at later time points, especially with the

higher initial infecting dose of FluA, suggests that the

detected virus is a detached virus that was adsorbed to the cell

rather than actual new viral progeny being produced from

inside the mast cells. We investigated this issue by using mild

acid treatment of the mast cells after 1 h of FluA adsorption

to remove any virus that were adhered on the cell surface, but

still allowing internalized virus to replicate. However, we

were unable to complete these experiments because in con-

trast to epithelial cells, mast cells were extremely sensitive to

Fig. 2 Transcription and RNA replication of FluA in mast cells. PCR

analysis was conducted for the viral RNA species: viral genomic RNA

(vRNA, n = 4), messenger RNA (mRNA, n = 3), and complemen-

tary RNA (cRNA, n = 3) of the FluA hemagglutinin (HA) gene. FluA

doses from 0.01 to 100 HAU/ml were used, whereas mock treat-

ment consisted of media only. All samples were taken at 2 days

post-treatment. a Dose response of FluA in the human mast cell line

LAD2. b–d Densitometric data of a was plotted by calculating the

signal as a percentage of the 100 HAU/ml treatment. Repeated

measures one-way ANOVA with Bonferroni post-test was performed

and statistical significance is represented as *p \ 0.05, **p \ 0.01,

and ***p \ 0.001 when compared to the 100 HAU/ml FluA

treatments

Immunol Res (2013) 56:32–43 37

123

acid treatment and high cell death occurred even at low

concentrations of acid treatments (data not shown).

We also determined the titer of infectious FluA using

TCID50 experiments. If the FluA titers decrease with time

from mast cells, it would suggest that new virus is not

being produced, but instead reflect the residual inoculating

FluA. We determined the log TCID50/ml values of Calu-3,

LAD2, and HCMC supernatants from 0 to 6 days post-

Fig. 3 vRNA, cRNA, and

mRNA level of the FluA

hemagglutinin gene after FluA

infection in human mast cells

and lung epithelial cells. Time

course analysis of the FluA

hemagglutinin (HA) gene in

a–d the human lung epithelial

cell line Calu-3 and e–h the

human mast cell line LAD2 by

PCR. Cells were treated with

20 HAU/ml FluA for 1 h to

allow for adsorption of virus,

media was removed then the

cells were washed 3 times, and

fresh media added. Cells were

harvested at 0 h, 6 h, 1 days,

2 days, 3 days, and 4 days post-

infection, and PCR was

performed. The 0-h time point

indicates the time at which fresh

media was added back to the

cells after virus treatment and

washes. Gels representing time

course expression of FluA

vRNA, mRNA, and cRNA from

0 h to 4 days post-FluA

treatment in a the human lung

epithelial cell line Calu-3 and

e the human mast cell lines

LAD2. Densitometric analysis

of HA of vRNA, mRNA, and

cRNA levels was performed for

b–d Calu-3 and f–h LAD2.

Densitometric data for panels

c and g were plotted by

calculating the FluA mRNA-to-

b-actin (b-act) ratios expressed

in arbitrary units (AU).

Repeated measures one-way

ANOVA with Bonferroni post-

test was performed, and

statistical significance is

represented as *p \ 0.05,

**p \ 0.01, and ***p \ 0.001

when compared to 0 days post-

treatments (n = 3–4)

38 Immunol Res (2013) 56:32–43

123

treatment with initial FluA-infecting dose of 100 HAU/ml.

The sensitivity of this method was 0.5 log TCID50/ml. The

infectious titer of Calu-3 supernatants was detectable at 2–

6 days of FluA treatment, ranging from 3.5 log TCID50/ml

at 2 days to 2.9 log TCID50/ml at 6 days but that of both

LAD2 and HCMC supernatants were below the detectable

limit of this assay (data not shown).

Discussion

Although respiratory epithelial cells are the primary target of

FluA infection, the spectrum of cell types that are infected

in vivo is not well known. Recent studies with GFP-reporter

FluA identified several other GFP-positive cells, including

dendritic cells, monocytes, neutrophils, macrophages, NK

Fig. 4 FluA polymerase B1 mRNA level after FluA infection in

human mast cells and lung epithelial cells. FluA polymerase B1 (PB1)

mRNA expression was analyzed by PCR. Cells were treated with 20

HAU/ml FluA for 1 h to allow for adsorption of virus, media was

removed then the cells were washed 3 times, and fresh media added.

Cells were harvested at 0 h, 6 h, 1 days, 2 days, 3 days, and 4 days

post-infection, and PCR was performed. The 0-h time point indicates

the time at which fresh media was added back to the cells, that is,

after the 1 h adsorption and washes. UVI indicates treatment with

UV-inactivated FluA for 2 days. Gels representing time course

expression of FluA PB1 mRNA from 0 h to 4 days post-FluA

treatment in a the human lung epithelial cell line Calu-3, c the human

mast cell line, LAD2, and e primary human cultured mast cells

(HCMC). Densitometric analysis of PB1 expression was performed

for b Calu-3, d LAD2, and f HCMC. Densitometric data were plotted

by calculating the FluA PB1 mRNA-to-b-actin (b-act) ratios

expressed in arbitrary units (AU). Repeated measures one-way

ANOVA with Bonferroni post-test was performed, and statistical

significance is represented as *p \ 0.05, **p \ 0.01 when compared

to 0 day treatments (n = 3)

Immunol Res (2013) 56:32–43 39

123

and T and B cells [27]. We have established that human mast

cells can also be infected with FluA, but as has been reported

for neutrophils [28], in some studies with macrophages [29],

and recently with mouse bone marrow-derived dendritic

cells [30], there is a paucity of infectious virus released from

mast cells.

Using the human mast cell line, LAD2, as well as pri-

mary human mast cells cultured from peripheral blood

progenitors, FluA mRNA transcription was evident as early

as 6 h, similar to in epithelial cells, but the levels of mRNA

for HA and PB1 were not sustained in mast cells as long as

in Calu-3 (Figs. 3, 4). Although FluA proteins were

detectable by 6 h post-FluA treatment in both mast cells

and Calu-3, their abundance varied among experiments

(Fig. 5). While epithelial cells generated greater numbers

of progeny than the initial infecting dose of FluA, infected

mast cells did not produce significant amounts of virus

(Fig. 6). Moreover, while release of infectious FluA from

Calu-3 was detected with an initial exposure of

0.002 HAU/ml, release of infectious FluA from mast cells

was rarely detected at 20 HAU/ml, a 4 log higher dose

(Table 1). These observations indicate that mast cells can

be infected with FluA, but the FluA life cycle is impeded in

mast cells. Given that viral RNA is produced at an early

time after FluA infection and viral protein levels are sus-

tained in mast cells for up to 6 days, there are either

deficiency in essential factors to support productive infec-

tions, intrinsic antiviral mechanisms [16] in mast cells, or

inducible mechanisms that markedly limit the production

of infectious virus. The mechanism could involve inhibi-

tion of FluA gene transcription or translation (e.g., M2,

NS2) and/or malfunction or mal-localization of FluA pro-

teins in mast cells. Decreased FluA mRNA expression in

mast cells compared to epithelial cells (Figs. 3, 4) suggests

that mast cells do not support FluA transcription as well as

epithelial cells do. Because M1 protein of FluA regulates

viral transcription in the nucleus through interactions with

vRNPs [31–33], decreased FluA mRNA levels after

infection could involve a defect of M1 function or trans-

port. Our preliminary evidence suggests that mast cells also

produce inducible antiviral proteins after FluA infection

and these may inhibit the production of new virus (data not

shown). Recently, St John et al. [10] and Brown et al. [14]

identified that dengue virus infection induced both viral

Fig. 5 Time course comparison

of FluA proteins in human mast

cells and lung epithelial cells.

Western blot analysis of

translated FluA proteins from

0 h to 6 days post-FluA

treatment using 20 or 100 HAU/

ml FluA virus in a Calu-3,

b LAD2, and c HCMC. Gels are

representative of independent

experiments (n = 3–8). UV-

inactivated FluA (UV FluA)

dose used was 100 HAU/ml.

Mock treatment consisted of

media only. FluA proteins

appear in the green channel and

b-actin appears in the redchannel. MM indicates the

molecular weight markers. FluA

proteins detected were HA

(hemagglutinin) at 70 kDa, NP(nucleoprotein) at 58 kDa, M1

(matrix protein 1) at 27 kDa,

and NS1 (non-structural protein

1) at 26 kDa. M1 and NS1

cannot be distinguished because

of their similar molecular

weights

40 Immunol Res (2013) 56:32–43

123

sensors and antiviral pathways in mast cells. We are

investigating these potential antiviral mechanisms in mast

cells infected with FluA.

Our study is the first to show that human mast cells are

susceptible to infection by FluA. We measured the log

TCID50/ml of our stock FluA and determined that 100 HAU/

ml, the dose at which mast cells produce small amounts of

infectious FluA, is equivalent to 4.5 log TCID50/ml. This

FluA dose is comparable to peak median FluA titers found in

nasopharyngeal washes of influenza patients (4.8 log

TCID50/ml) [34] and in human experimental influenza

(4.2 log TCID50/ml) [35], and thus, the infection of mast

cells with FluA in vivo is possible. We are also the first to

show that FluA transcription and protein synthesis takes

place in mast cells. Our direct comparison of FluA infection

in mast cells and epithelial cells identifies the varied

responses of different cell types to FluA.

Although others have not investigated in detail the life

cycle of viruses in mast cells and how viral replication

differs in mast cells compared to other cell types, our

Fig. 6 Time course of FluA

release from human mast cells

and lung epithelial cells.

a Hemagglutination assay of

supernatants from epithelial

cells and mast cells from 0 to

4 days post-FluA treatment.

Release of FluA from Calu-3,

LAD2, and HCMC using

20 HAU/ml of FluA treatment.

Statistical significance was

determined using two-way

ANOVA with Bonferroni post-

test and is represented as

*p \ 0.05, **p \ 0.01,

***p \ 0.001 when compared

to HCMC, and ##p \ 0.01,###p \ 0.001 when compared to

LAD2 (n = 4–8).

b Hemadsorption assay of Calu-

3 monolayers after addition of

supernatants from FluA-treated

Calu-3 and mast cells (LAD2

and HCMC). Samples were

taken from 0 h to 4 days of

post-FluA treatment. Percentage

of positive results was plotted

and number of positive

hemadsorption experiment/total

independent experiment is

indicated inside the bar. UVIindicates UV-inactivated FluA

at 2 days post-treatment

Table 1 Release of infectious FluA from human epithelial cells and mast cellsa

FluA (HAU/ml) 20 2 0.2 0.02 0.002 0.0002 Mockb

Calu-3 (?)3 (?)3 (?)3 (?)3 (?)2 (-)1 (-)3 (-)3

FluA (HAU/ml) 100 20 10 1 0.1 0.01 Mockb

LAD2 (?)6 (?)1 (-)5 (-)5 (-)5 (-)5 (-)5 (-)5

a Hemadsorption assay on Calu-3 monolayers after treatment with supernatants from FluA-infected Calu-3 and LAD2. Samples were taken at

2 days post-treatment with supernatants from infected Calu-3 and LAD2. Positive hemadsorption is indicated by (?); lack of hemadsorption is

indicated by (-). Superscript numbers indicate n-values for each outcome (from a total of 3 to 6 experiments)b Mock was media only

Immunol Res (2013) 56:32–43 41

123

results complement previous studies of virus infection of

mast cells. King et al. [12] demonstrated that dengue virus

is capable of infecting a human mast cell/basophil line to

produce infectious virus and that virus production can be

enhanced using a dengue-specific antibody. Patients with

acquired immunodeficiency syndrome (AIDS) have HIV-

infected mast cells, and mast cells cultured from uninfected

subjects can be infected with HIV [36]. Interestingly, mast

cells infected with reovirus can mediate chemotaxis of NK

cells, providing evidence that mast cells may contribute to

the host response against viral stimuli [37], a postulate

supported by recent studies of FluA in mice [11]. Inter-

estingly, respiratory syncytial virus (RSV) does not infect

mast cells, but instead activates mast cell degranulation

through virus-specific IgE [38]. Our initial studies have

identified that FluA itself does not induce degranulation of

cultured human mast cells, but as shown for other viruses

such as dengue, mast cells produce type I interferon and

several cytokines and chemokines in response to FluA

(data not shown).

Although until recently [11] mast cells have been largely

overlooked in FluA infection, our results suggest that

additional investigations are warranted. It will be important

to establish whether mast cells in the respiratory tract are

infected in murine models (e.g., [27]) or in influenza

patients. Secondly, there is limited information about the

production of type I interferons by mast cells following

exposure to viruses [9, 10, 14, 15], and the role of intrinsic

and inducible antiviral mechanisms by mast cells has

received limited attention. Mast cells respond to viral

stimuli by releasing various cytokines and chemokines

[14, 39], and we have also detected cytokine and chemo-

kine release from mast cells after FluA infection.

Also, Shirato et al. [40] showed that mast cell degran-

ulation is induced when cocultured with RSV-infected

epithelial cells, suggesting an important role for epithelial–

mast cell interactions in the pathogenesis of viral infec-

tions. As mentioned before, mast cells and epithelial cells

exist in close proximity in the lungs and communicate; for

example, coculture models show that mast cells increase

epithelial cell proliferation, while epithelial cells promote

mast cell survival [41, 42]. As mast cells are normally

found in the subepithelial region and between epithelial

cells in the lung, it is tempting to postulate that a pathogen

that targets the epithelium would have direct or indirect

effects on adjacent mast cells. By studying epithelial–mast

cell interactions in the context of FluA infection, we may

discover that mast cells are protective against FluA infec-

tion of epithelial cells. Alternatively, FluA-infected epi-

thelial cells may send signals to activate mast cell defense

mechanisms against viral invasion of the host.

In conclusion, our results show for the first time that

human mast cells are infected by FluA in vitro and undergo

FluA mRNA transcription and protein synthesis. Infectious

virus can be detected in FluA-infected mast cell superna-

tants, albeit at a much lower level than from epithelial

cells. The mechanisms by which mast cells are less per-

missive to a complete FluA replication cycle need to be

clarified as one component of the involvement of mast cells

in innate immunity against viruses.

Acknowledgments This work was supported by the Canadian

Institutes of Health Research (CIHR). Candy W. Marcet is a recipient

of the Walter and Jessie Boyd and Charles Scriver MD/PhD Stu-

dentship from CIHR. We thank Drs. Kevin P. Kane and Earl G.

Brown for FluA virus (A/PR/8/34 strain) and rabbit antiserum to

FluA, respectively, and Dr Yokananth Sekar for repeating Western

blots shown in Fig. 5.

References

1. Rothberg MB, Haessler SD, Brown RB. Complications of viral

influenza. Am J Med. 2008;121(4):258–64.

2. Julkunen I, Melen K, Nyqvist M, Pirhonen J, Sareneva T, Mati-

kainen S. Inflammatory responses in influenza A virus infection.

Vaccine. 2000;19(Suppl 1):S32–7.

3. Wang JP, Kurt-Jones EA, Finberg RW. Innate immunity to

respiratory viruses. Cell Microbiol. 2007;9(7):1641–6.

4. Boyce JA. Mast cells: beyond IgE. J Allergy Clin Immunol.

2003;111(1):24–32; quiz 3.

5. Galli SJ, Maurer M, Lantz CS. Mast cells as sentinels of innate

immunity. Curr Opin Immunol. 1999;11(1):53–9.

6. Marshall JS. Mast-cell responses to pathogens. Nat Rev Immunol.

2004;4(10):787–99.

7. Sundstrom JB, Ellis JE, Hair GA, Kirshenbaum AS, Metcalfe DD,

Yi H, et al. Human tissue mast cells are an inducible reservoir of

persistent HIV infection. Blood. 2007;109(12):5293–300.

8. Clementsen P, Bisgaard H, Pedersen M, Permin H, Struve-

Christensen E, Milman N, et al. Staphylococcus aureus and

influenza A virus stimulate human bronchoalveolar cells to release

histamine and leukotrienes. Agents Actions. 1989;27(1–2):107–9.

9. Dietrich N, Rohde M, Geffers R, Kroger A, Hauser H, Weiss S,

et al. Mast cells elicit proinflammatory but not type I interferon

responses upon activation of TLRs by bacteria. Proc Natl Acad

Sci USA. 2010;107(19):8748–53.

10. St. John AL, Rathore AP, Yap H, Ng ML, Metcalfe DD,

Vasudevan SG et al. Immune surveillance by mast cells during

dengue infection promotes natural killer (NK) and NKT-cell

recruitment and viral clearance. Proc Natl Acad Sci USA.

2011;108(22):9190–5.

11. Hu Y, Jin Y, Han D, Zhang G, Cao S, Xie J, et al. Mast cell-

induced lung injury in mice infected with H5N1 influenza virus.

J Virol. 2012;86(6):3347–56.

12. King CA, Marshall JS, Alshurafa H, Anderson R. Release of

vasoactive cytokines by antibody-enhanced dengue virus infection

of a human mast cell/basophil line. J Virol. 2000;74(15):7146–50.

13. King CA, Anderson R, Marshall JS. Dengue virus selectively

induces human mast cell chemokine production. J Virol.

2002;76(16):8408–19.

14. Brown MG, McAlpine SM, Huang YY, Haidl ID, Al-Afif A,

Marshall JS, et al. RNA sensors enable human mast cell anti-viral

chemokine production and IFN-mediated protection in response

to antibody-enhanced dengue virus infection. PLoS ONE. 2012;

7(3):e34055.

42 Immunol Res (2013) 56:32–43

123

15. Kulka M, Alexopoulou L, Flavell RA, Metcalfe DD. Activation

of mast cells by double-stranded RNA: evidence for activation

through Toll-like receptor 3. J Allergy Clin Immunol. 2004;

114(1):174–82.

16. Yan N, Chen ZJ. Intrinsic antiviral immunity. Nat Immunol.

2012;13(3):214–22.

17. Kirshenbaum AS, Akin C, Wu Y, Rottem M, Goff JP, Beaven

MA, et al. Characterization of novel stem cell factor responsive

human mast cell lines LAD 1 and 2 established from a patient with

mast cell sarcoma/leukemia; activation following aggregation of

FcepsilonRI or FcgammaRI. Leuk Res. 2003;27(8):677–82.

18. Sekar Y, Moon TC, Slupsky CM, Befus AD. Protein tyrosine

nitration of aldolase in mast cells: a plausible pathway in nitric

oxide-mediated regulation of mast cell function. J Immunol.

2010;185(1):578–87.

19. Kirshenbaum AS, Goff JP, Semere T, Foster B, Scott LM, Met-

calfe DD. Demonstration that human mast cells arise from a

progenitor cell population that is CD34(?), c-kit(?), and

expresses aminopeptidase N (CD13). Blood. 1999;94(7):2333–42.

20. Moon TC, Lee E, Baek SH, Murakami M, Kudo I, Kim NS, et al.

Degranulation and cytokine expression in human cord blood-

derived mast cells cultured in serum-free medium with recom-

binant human stem cell factor. Mol Cells. 2003;16(2):154–60.

21. Yoshimura T, Moon TC, St Laurent CD, Puttagunta L, Chung K,

Wright E et al. Expression of nitric oxide synthases in leukocytes in

nasal polyps. Ann Allergy Asthma Immunol. 2012;108(3):172–7.

22. Duta V, Duta F, Puttagunta L, Befus AD, Duszyk M. Regulation

of basolateral Cl(-) channels in airway epithelial cells: the role of

nitric oxide. J Membr Biol. 2006;213(3):165–74.

23. Cottey R, Rowe CA, Bender BS. Influenza virus. Curr Protoc

Immunol. 2001;Chapter 19:Unit 19 1.

24. Gaush CR, Smith TF. Replication and plaque assay of influenza

virus in an established line of canine kidney cells. Appl Micro-

biol. 1968;16(4):588–94.

25. Reed LJ, Muench H. A simple method of estimating fifty percent

endpoints. Am J Hyg. 1938;27:493–7.

26. Brown EG, Bailly JE. Genetic analysis of mouse-adapted influ-

enza A virus identifies roles for the NA, PB1, and PB2 genes in

virulence. Virus Res. 1999;61(1):63–76.

27. Manicassamy B, Manicassamy S, Belicha-Villanueva A, Pisanelli

G, Pulendran B, Garcia-Sastre A. Analysis of in vivo dynamics of

influenza virus infection in mice using a GFP reporter virus. Proc

Natl Acad Sci USA. 2010;107(25):11531–6.

28. Cassidy LF, Lyles DS, Abramson JS. Synthesis of viral proteins

in polymorphonuclear leukocytes infected with influenza A virus.

J Clin Microbiol. 1988;26(7):1267–70.

29. Lee SM, Dutry I, Peiris JS. Editorial: macrophage heterogeneity

and responses to influenza virus infection. J Leukoc Biol.

2012;92(1):1–4.

30. Ioannidis LJ, Verity EE, Crawford S, Rockman SP, Brown LE.

Abortive replication of influenza virus in mouse dendritic cells.

J Virol. 2012;86(10):5922–5.

31. Ye ZP, Pal R, Fox JW, Wagner RR. Functional and antigenic

domains of the matrix (M1) protein of influenza A virus. J Virol.

1987;61(2):239–46.

32. Ye ZP, Baylor NW, Wagner RR. Transcription-inhibition and

RNA-binding domains of influenza A virus matrix protein map-

ped with anti-idiotypic antibodies and synthetic peptides. J Virol.

1989;63(9):3586–94.

33. Baudin F, Petit I, Weissenhorn W, Ruigrok RW. In vitro dis-

section of the membrane and RNP binding activities of influenza

virus M1 protein. Virology. 2001;281(1):102–8.

34. Kaiser L, Fritz RS, Straus SE, Gubareva L, Hayden FG. Symptom

pathogenesis during acute influenza: interleukin-6 and other

cytokine responses. J Med Virol. 2001;64(3):262–8.

35. Hayden FG, Treanor JJ, Fritz RS, Lobo M, Betts RF, Miller M,

et al. Use of the oral neuraminidase inhibitor oseltamivir in

experimental human influenza: randomized controlled trials for

prevention and treatment. JAMA. 1999;282(13):1240–6.

36. Li Y, Li L, Wadley R, Reddel SW, Qi JC, Archis C, et al. Mast

cells/basophils in the peripheral blood of allergic individuals who

are HIV-1 susceptible due to their surface expression of CD4 and

the chemokine receptors CCR3, CCR5, and CXCR4. Blood.

2001;97(11):3484–90.

37. Burke SM, Issekutz TB, Mohan K, Lee PW, Shmulevitz M,

Marshall JS. Human mast cell activation with virus-associated

stimuli leads to the selective chemotaxis of natural killer cells by

a CXCL8-dependent mechanism. Blood. 2008;111(12):5467–76.

38. Dakhama A, Lee YM, Ohnishi H, Jing X, Balhorn A, Takeda K

et al. Virus-specific IgE enhances airway responsiveness on

reinfection with respiratory syncytial virus in newborn mice.

J Allergy Clin Immunol. 2009;123(1):138–45.

39. Marshall JS, King CA, McCurdy JD. Mast cell cytokine and

chemokine responses to bacterial and viral infection. Curr Pharm

Des. 2003;9(1):11–24.

40. Shirato K, Taguchi F. Mast cell degranulation is induced by A549

airway epithelial cell infected with respiratory syncytial virus.

Virology. 2009;386(1):88–93.

41. Artuc M, Steckelings UM, Grutzkau A, Smorodchenko A, Henz

BM. A long-term coculture model for the study of mast cell-

keratinocyte interactions. J Invest Dermatol. 2002;119(2):411–5.

42. Hsieh FH, Sharma P, Gibbons A, Goggans T, Erzurum SC,

Haque SJ. Human airway epithelial cell determinants of survival

and functional phenotype for primary human mast cells. Proc

Natl Acad Sci USA. 2005;102(40):14380–5.

Immunol Res (2013) 56:32–43 43

123