Veterinary Immunology and Immunopathology 155 (2013) 270–275

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Veterinary Immunology and Immunopathology

journal homepage: www.elsevier.com/locate/vetimm

Short communication

Induction of Toll-like receptor 4 signaling in avian

macrophages inhibits infectious laryngotracheitis virus
replication in a nitric oxide dependent way夽
Siamak Haddadi, Dae-Sun Kim, Hui Jasmine, Frank van der Meer, Markus Czub,
Mohamed Faizal Abdul-Careem ∗
Department of Ecosystem and Public Health, Faculty of Veterinary Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, AB
T2N 2Z6, Canada

a r t i c l e i n f o a b s t r a c t

Article history: LPS is one of the pathogen associated molecular patterns that activates Toll-like receptor 4
Received 20 June 2013 (TLR4) signaling pathway eliciting antiviral host responses in mammals although informa-
Received in revised form 15 August 2013
tion on such responses in avian species is scarce. Our objectives were to characterize the
Accepted 19 August 2013
LPS induced innate responses particularly the expression of LPS receptors (TLR4, CD14) in
avian macrophages and observe whether TLR4 mediated induction of NO can elicit antivi-
Keywords:
ral response against infectious laryngotracheitis virus (ILTV) replication. We found that
Avian
Infectious laryngotracheitis virus LPS was capable of inducing the expression of TLR4, CD14 and NO production but not the
Lipopolysaccharide type 1 interferons in an avian macrophage cell line, MQ-NCSU. We also showed that TLR4
Macrophage mediated NO production can lead to antiviral response against ILTV replication when MQ-
Nitric oxide NCSU cells were treated with LPS and the resultant supernatant was then transferred to
Toll-like receptor 4 ILTV replicating cells to assess antiviral activity. Antiviral activity of NO was blocked by a
selective inhibitor, S-methylisothiourea sulfhate that inhibits inducible NO synthase. This
observation confirms that the antiviral activity is positively correlated with NO production.
The data show that LPS can be a potential innate immune stimulant that can be used against
ILTV infection in chickens that require further evaluation in vivo.
© 2013 The Authors. Published by Elsevier B.V. All rights reserved.

1. Introduction ligands that binds with TLR4 is LPS derived from gram-
negative bacteria such as Escherichia coli (EC-LPS) and
TLRs and their downstream signaling components are Salmonella sub type Enteritidis (SE-LPS). LPS recognition
mostly conserved in chickens (Lillehoj and Li, 2004; Lynn by TLR4 requires the activation of co-receptors such as
et al., 2003; Philbin et al., 2005), except for TLR4 (Keestra CD14 and MD2. Chicken TLR4 and co-receptors, in response
and van Putten, 2008). In mammals, TLR4 is expressed in to LPS induce pro-inflammatory mediators such as inter-
a variety of immune and non-immune cells (Arpaia et al., leukin (IL)-1␤, IL-6 and IL-18 and the gaseous free radical
2011; Tang et al., 2008). One of the well-characterized nitric oxide (NO) through nitric oxide synthase (iNOS) acti-
vation (Dil and Qureshi, 2002; Farnell et al., 2003; He et al.,
2006). NO has already been shown to mediate anti-viral
response for avian viruses such as reovirus, herpes virus
夽 This is an open-access article distributed under the terms of the Cre-
of turkey (HVT) and Marek’s disease virus (MDV) (Pertile
ative Commons Attribution-NonCommercial-ShareAlike License, which
et al., 1996; Xing and Schat, 2000).
permits non-commercial use, distribution, and reproduction in any
medium, provided the original author and source are credited. Here, we first investigated whether EC-LPS and SE-LPS
∗ Corresponding author. Tel.: +1 403 220 4462; fax: +1 403 210 9740. can increase the expression of LPS receptors, TLR4 and
E-mail address: faizal.abdulcareem@ucalgary.ca (M.F. Abdul-Careem). CD14, in avian macrophages in response to LPS. Secondly,

0165-2427/$ – see front matter © 2013 The Authors. Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.vetimm.2013.08.005
 S. Haddadi et al. / Veterinary Immunology and Immunopathology 155 (2013) 270–275 271

we evaluated whether NO in culture supernatants elic- post-treatment culture supernatants were collected and
its antiviral effects against another avian viral infection transferred to naive LMH cells. Then, LMH cells were
other than reovirus, HVT and MDV. We found that avian infected with 100 PFU/well of ILTV. Five days following the
macrophages, MQ-NCSU cells could be stimulated with LPS infection, the number of plaques was counted. The experi-
to increase in the expression of LPS receptors but not type ment was done in triplicate.
1 interferons and to elicit antiviral response against infec-
tious laryngotracheitis virus (ILTV) in a NO dependent way.
2.3.4. Stimulation of macrophages for quantification of
mRNA expression of type 1 interferons
2. Materials and methods
In chickens, since it has been shown that LPS signaling
does not lead to interferon (IFN)␤ expression in HD11 avian
2.1. Cells and virus
macrophages (Keestra and van Putten, 2008), we evaluated
whether LPS signaling in MQ-NCSU avian macrophages
Avian macrophage cell line, MQ-NCSU was a gift from
lead to similar outcome to rule out the potential involve-
Dr. Shayan Sharif (University of Guelph, Canada). Leghorn
ment of type 1 interferons as antiviral molecules against
male hepatoma line (LMH) and ILTV were purchased from
ILTV following LPS stimulation. At this end we quanti-
American Type Culture Collection (ATCC).
fied the expressions of IFN␣ and IFN␤ mRNA following
stimulation with SE-LPS (0.1 ␮g/ml) on 0.5, 1, 3, 6, 12
2.2. Cell culture
and 24 h post-treatment using real-time PCR as has been
described previously (Esnault et al., 2011; Villanueva et al.,
LMH cells were cultured on 0.1% gelatin (Sigma-Aldrich,
2011).
St. Louis, MO, USA) coated tissue culture plates. The growth
medium for LMH cells consists of complete Waymouth’s
MB 752/1 medium (Invitrogen, Burlington, ON, Canada). 2.4. Assay for endotoxin contamination
The MQ-NCSU cells were cultured in complete LM HAHN
medium. The cells were maintained under 5% carbon diox- The LPS concentrations in the used reagents and
ide (CO2 ) and 37 ◦ C (LMH) or 40 ◦ C (MQ-NCSU). all the growth media were determined using Limulus
Amebocyte Lysate LPS detection assay (E-TOXATETM kit;
2.3. Experimental design Sigma-Aldrich, St. Louis, MO, USA) according to the manu-
facturer’s protocol.
2.3.1. Stimulation of macrophages for quantification of
TLR4 and CD14 expression 2.5. Assay for NO production
The MQ-NCSU cells responded to LPS treatment produc-
ing NO in a dose dependent manner (data not shown). The Cell-free culture supernatants were assayed for nitrite,
lowest concentration of LPS (0.1 ␮g/ml) that resulted high a stable metabolite of NO, as a measure of NO produc-
amount of NO in our preliminary studies was chosen for tion, using a Griess reagent system (Promega, Madison, WI,
subsequent experiments. MQ-NSU cells were treated with Canada) according to the manufacturer’s recommendation.
either EC-LPS or SE-LPS with un-stimulated controls. Three, The absorbance (OD) of the final colorimetric product was
6 and 12 h post-treatment, 2 × 106 cells from each treat- read at 548 nm. The concentration of nitrite was quantified
ment or control stained for TLR4 and CD14 expressions. using sodium nitrite as a standard.
The experiment was done in triplicate.

2.6. Flow cytometry

2.3.2. Antiviral assay for culture supernatants produced
following MQ-NCSU stimulation with LPS in vitro
Standard flow cytometry procedures were used. Briefly,
MQ-NCSU cells were cultured in 6 well plates
the isolated MQ-NCSU cells were Fc blocked using 1:100
(2 × 106 cells/well) 24 h before treatments with 0.1 ␮g/ml
chicken serum (Invitrogen, Burlington, ON, Canada). Stain-
EC-LPS, SE-LPS or PBS. 18 h post-treatment culture super-
ing was done at final concentration of 0.02 ug/␮l mouse
natants (500 ␮l/well) were collected and transferred to
anti-Human CD284 (TLR4) phycoerythrin (PE) mAb (Clone
naive LMH cells. Then, LMH cells were infected with
HTA125, eBioscience, San Diego, CA, USA), or mouse anti-
10-fold serial dilution of ILTV (500 ␮l/well) with a
Human CD14 FITC mAb (Clone MSE2, eBioscience, San
titer of 1.27 × 106 plaque forming unit (PFU)/ml. Five
Diego, CA, USA), with respective isotype controls or 1% BSA
days following the infection, the number of plaques
(unstained controls) and incubated on ice for 30 min. The
was counted. The experiment was done in tripli-
samples were analyzed with a BD LSR II flow cytometer (BD
cate.
Biosciences, Mississauga, ON, Canada).
2.3.3. Determining the antiviral effect of LPS mediated
NO production in vitro 2.7. Data analysis
MQ-NCSU cells were cultured in 6 well plates (2 × 106
cells/well) 24 h before treatments with 0.1 ␮g/ml EC-LPS Data were subjected to ANOVA test followed by
or SE-LPS with or without inducible nitric oxide syn- Tukey’s test (Minitab Inc., State College, Pennsylvania,
thase (iNOS) inhibitor, S-methylisothiourea sulphate (SMT; USA). Before being tested, each set of data was analyzed
Sigma-Aldrich, St. Louis, MO, USA) or PBS. Six and 18 h using the Grubbs’ test (GraphPad Software Inc., CA, USA) to
272 S. Haddadi et al. / Veterinary Immunology and Immunopathology 155 (2013) 270–275

Fig. 1. MQ-NCSU cells increase the expression of TLR4 and CD14 following treatment with LPS. The experiment was done in triplicate; (a) a representative
FACS plot of isotype control for PE- mouse anti-human TLR4 Ab; (b) a representative FACS plot of isotype control for FITC-mouse anti-human CD14 Ab;
(c) a representative FACS plot showing control TLR4+ CD14+ macrophages; (d)–(e) representative FACS plots showing TLR4+ CD14+ macrophages 12 h
post-treatment with EC-LPS and SE-LPS respectively; (f)–(g) illustrates percentage TLR4+ CD14+ macrophages 3, 6 and 12 h post-treatment with EC-LPS
and SE-LPS, respectively.

Fig. 2. Culture supernatants of LPS-treated MQ-NCSU cells inhibit ILTV replication in vitro; (a) illustrates representative ILTV titration plates from EC-LPS
and SE-LPS treated groups compared to the control; (b) illustrates ILTV titers in EC-LPS and SE-LPS treated groups compared to the control.
 S. Haddadi et al. / Veterinary Immunology and Immunopathology 155 (2013) 270–275 273

Fig. 3. NO produced by MQ-NCSU cells following LPS treatment inhibits ILTV replication in vitro; (a) represents the concentration of NO in cell culture
supernatants 6 h post-treatment; (b) represents the concentration of NO in cell culture supernatants 18 h post-treatment; (c) shows the ILTV titers as a
percentage following transfer of 6 h conditioned media from MQ-NCSU cells; (d) shows the ILTV titers as a percentage following transfer of 18 h conditioned
media from MQ-NCSU cells; (e) illustrates representative ILTV titration plates of treatment and control groups. Data represent mean values ± SEM.

identify outliers. Comparisons were considered significant supernatants. Finally, we confirmed that SE-LPS stimula-
at P ≤ 0.05. tion of MQ-NCSU cells does not lead to type 1 interferon
mRNA expression (P > 0.05, Fig. 4) and unlikely to have con-
3. Results and discussion tributed to the antiviral effect elicited by MQ-NCSU cells
following LPS stimulation against ILTV.
First, LPS-TLR4 induced signaling pathway activates It has been shown that the expression of avian CD14
MQ-NCSU chicken macrophages by increasing TLR4 and and TLR4 can be quantified using anti-human mAbs against
CD14 cell surface expression and elicits an antivi- these two receptors as they are components of highly
ral response against ILTV. Secondly, the inhibition of conserved innate immune system (Dil and Qureshi, 2002;
ILTV replication was dependant on NO production in Janeway and Medzhitov, 2002). In agreement with these
274 S. Haddadi et al. / Veterinary Immunology and Immunopathology 155 (2013) 270–275

culture supernatants of MQ-NCSU cells treated with EC-LPS

alone (P = 0.031; Fig. 3c and e). Culture supernatants pro-
duced by MQ-NCSU cells 18 h post-treatment with EC-LPS
or SE-LPS with an iNOS inhibitor abrogated the inhibitory
effect of ILTV replication when compared to MQ-NCSU cells
treated with EC-LPS and SE-LPS alone (P = 0.000; Fig. 3d and
e).
The viability of the cells in our experiments was more
than 95% according to trypan blue exclusion. Endotoxin
concentrations of all the tested reagents were below
0.06 EU/ml.
The method we used for NO quantification, Griess assay
detects nitrite and not NO. For this reason we could not
relate our antiviral effect findings directly to NO. Since NO
is unstable, it is possible that by the time of treatment of
LMH cells, NO may have converted to nitrite. Unlike NO,
nitrite is stable and unlikely to possess antiviral activity. It
has also been shown that under certain conditions nitrite
can convert back to NO in biological systems (Lundberg
et al., 2008). It is a potential reason for the observed antivi-
ral effect in our experiments, however, this needs further
investigation.
In conclusion, we have shown that EC-LPS and SE-LPS
TLR4 ligands are capable of activating MQ-NCSU avian
macrophages as evidenced by increase of LPS receptors
and elicit antiviral response against ILTV in a NO depend-
ent manner. The potential role of type 1 interferons in LPS
mediated antiviral response against ILTV is not clear since
we did not observe up regulation of expressions of IFN␣ and
IFN␤ following SE-LPS treatment. It is of interest to investi-
Fig. 4. MQ-NCSU cells do not up regulate the mRNA expression of IFN␣
gate the interaction between E. coli and ILTV in respiratory
and IFN␤ following treatment with SE-LPS; (a) and (b) illustrate the IFN␣
and IFN␤ mRNA expressions, respectively. Real-time PCR was done to mucosa.
quantify the mRNA expression and fold changes are presented. Data rep-
resent mean values ± SEM.
Conflict of interest statement

observations, we observed first that MQNCSU cells express

The Authors declare that there is no conflict of interest.
TLR4 and CD14 constitutively, but in a low number of cells.
Following treatment with EC-LPS the expression of TLR4
and CD14 have been increased significantly in MQ-NCSU Acknowledgments
cells when compared to the controls at 6 h (P = 0.002) and
12 h (P = 0.002) post-treatments (Fig. 1) whereas with SE- This study was funded by University of Calgary Faculty
LPS, the MQ-NCSU cells expressing both TLR4 and CD14 of Veterinary Medicine, Natural Sciences and Engineering
increased significantly at 12 h post stimulation (P = 0.002). Research Council of Canada, Canadian Poultry Research
We also observed that culture supernatants collected fol- Council and Alberta Livestock and Meat Agency, Canada.
lowing LPS treatment of MQ-NCSU could reduce ILTV
replication in susceptible LMH cells (P = 0.00; Fig. 2). References
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