Induction of Toll-like receptor 4 signaling in avian macrophages inhibits infectious laryngotracheitis virus replication in a nitric oxide dependent way

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LPS is one of the pathogen associated molecular patterns that activates Toll-like receptor 4 (TLR4) signaling pathway eliciting antiviral host responses in mammals although information on such responses in avian species is scarce. Our objectives were to characterize the 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 antiviral response against infectious laryngotracheitis virus (ILTV) replication. We found that LPS was capable of inducing the expression of TLR4, CD14 and NO production but not the type 1 interferons in an avian macrophage cell line, MQ-NCSU. We also showed that TLR4 mediated NO production can lead to antiviral response against ILTV replication when MQNCSU cells were treated with LPS and the resultant supernatant was then transferred to 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.


1. Introduction

TLRs and their downstream signaling components are mostly conserved in chickens (Lillehoj and Li, 2004; Lynnet al., 2003; Philbin et al., 2005), except for TLR4 (Keestraand van Putten, 2008). In mammals, TLR4 is expressed in a variety of immune and non-immune cells (Arpaia et al.,2011; Tang et al., 2008). One of the well-characterized ligands that binds with TLR4 is LPS derived from gram-negative bacteria such as Escherichia coli (EC-LPS) andSalmonella subtype Enteritidis (SE-LPS). LPS recognition by TLR4 requires the activation of co-receptors such asCD14 and MD2. Chicken TLR4 and co-receptors, in response to LPS induce pro-inflammatory mediators such as interleukin (IL)-1 _, IL-6 and IL-18 and the gaseous free radical 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 of turkey (HVT) and Marek’s disease virus (MDV) (Pertileet al., 1996; Xing and Schat, 2000).


Here, we first investigated whether EC-LPS and SE-LPScan increase the expression of LPS receptors, TLR4 andCD14, in avian macrophages in response to LPS. Secondly,  we evaluated whether NO in culture supernatants elicits antiviral effects against another avian viral infection other than reovirus, HVT and MDV. We found that avian macrophages, MQ-NCSU cells could be stimulated with LPSto increase in the expression of LPS receptors but not type1 interferons and to elicit an antiviral response against infectious laryngotracheitis virus (ILTV) in a NO dependent way.


2. Materials and methods

2.1. Cells and virus

Avian macrophage cell line, MQ-NCSU was a gift fromDr. Shayan Sharif (University of Guelph, Canada). Leghorn male hepatoma line (LMH) and ILTV were purchased fromAmerican Type Culture Collection (ATCC).


2.2. Cell culture

LMH cells were cultured on 0.1% gelatin (Sigma-Aldrich, St. Louis, MO, USA) coated tissue culture plates. The growth medium for LMH cells consists of complete Waymouth’sMB 752/1 medium (Invitrogen, Burlington, ON, Canada).The MQ-NCSU cells were cultured in complete LM HAHNmedium. The cells were maintained under 5% carbon dioxide (CO2) and 37?C (LMH) or 40?C (MQ-NCSU).


2.3. Experimental design

2.3.1. Stimulation of macrophages for quantification ofTLR4 and CD14 expression 

The MQ-NCSU cells responded to LPS treatment producing NO in a dose dependent manner (data not shown). The lowest concentration of LPS (0.1  _g/ml) that resulted high amount of NO in our preliminary studies was chosen for subsequent experiments. MQ-NSU cells were treated with either EC-LPS or SE-LPS with unstimulated controls. Three,6 and 12 h post-treatment, 2 × 106 cells from each treatment or control stained for TLR4 and CD14 expressions.The experiment was done in triplicate.


2.3.2. Antiviral assay for culture supernatants producedfollowing MQ-NCSU stimulation with LPS in vitro

MQ-NCSU cells were cultured in 6 well plates(2 × 106cells/well) 24 h before treatments with 0.1  _g/mlEC-LPS, SE-LPS or PBS. 18 h post-treatment culture supernatants (500  _l/well) were collected and transferred to naive LMH cells. Then, LMH cells were infected with10-fold serial dilution of ILTV (500  _l/well) with a titer of 1.27 × 106plaque forming unit (PFU)/ml. Five days following the infection, the number of plaques was counted. The experiment was done in triplicate.


2.3.3. Determining the antiviral effect of LPS mediatedNO production in vitro

MQ-NCSU cells were cultured in 6 well plates (2 × 106cells/well) 24 h before treatments with 0.1  _g/ml EC-LPSor SE-LPS with or without inducible nitric oxide synthase (iNOS) inhibitor, S-methylisothiourea sulphate (SMT; Sigma-Aldrich, St. Louis, MO, USA) or PBS. Six and 18 h post-treatment culture supernatants were collected and transferred to naive LMH cells. Then, LMH cells were infected with 100 PFU/well of ILTV. Five days following the infection, the number of plaques was counted. The experiment was done in triplicate.


2.3.4. Stimulation of macrophages for quantification ofmRNA expression of type 1 interferons

In chickens, since it has been shown that LPS signaling does not lead to interferon (IFN) _ expression in HD11 avian macrophages (Keestra and van Putten, 2008), we evaluated whether LPS signaling in MQ-NCSU avian macrophages lead to similar outcome to rule out the potential involvement of type 1 interferons as antiviral molecules againstILTV following LPS stimulation. At this end, we quantified the expressions of IFN _ and IFN _ mRNA following stimulation with SE-LPS (0.1  _g/ml) on 0.5, 1, 3, 6, 12and 24 h post-treatment using real-time PCR as has been described previously (Esnault et al., 2011; Villanueva et al.,2011). 


2.4. Assay for endotoxin contamination

The LPS concentrations in the used reagents and all the growth media were determined using LimulusAmebocyte Lysate LPS detection assay (E-TOXATETMkit; Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s protocol.


2.5. Assay for NO production

Cell-free culture supernatants were assayed for nitrite, a stable metabolite of NO, as a measure of NO production, using a Griess reagent system (Promega, Madison, WI, Canada) according to the manufacturer’s recommendation.The absorbance (OD) of the final colorimetric product was read at 548 nm. The concentration of nitrite was quantified using sodium nitrite as a standard.


2.6. Flow cytometry

Standard flow cytometry procedures were used. Briefly, the isolated MQ-NCSU cells were Fc blocked using 1:100chicken serum (Invitrogen, Burlington, ON, Canada). Staining was done at final concentration of 0.02 ug/ _l mouseanti-Human CD284 (TLR4) phycoerythrin (PE) mAb (CloneHTA125, eBioscience, San Diego, CA, USA), or mouse anti-Human CD14 FITC mAb (Clone MSE2, eBioscience, SanDiego, CA, USA), with respective isotype controls or 1% BSA(unstained controls) and incubated on ice for 30 min. The samples were analyzed with a BD LSR II flow cytometer (BDBiosciences, Mississauga, ON, Canada).


2.7. Data analysis

Data were subjected to ANOVA test followed by Tukey’s test (Minitab Inc., State College, Pennsylvania, USA). Before being tested, each set of data was analyzed using the Grubbs’ test (GraphPad Software Inc., CA, USA) to identify outliers. Comparisons were considered significant at P ≤ 0.05.3


Fig. 1. MQ-NCSU cells increase the expression of TLR4 and CD14 following treatment with LPS. The experiment was done in triplicate; (a) a representativeFACS 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 hpost-treatment with EC-LPS and SE-LPS respectively; (f)–(g) illustrates percentage TLR4+CD14+macrophages 3, 6 and 12 h post-treatment with EC-LPSand 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-LPSand SE-LPS treated groups compared to the control; (b) illustrates ILTV titers in EC-LPS and SE-LPS treated groups compared to the control.


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.


3. Results and discussion

First, LPS-TLR4 induced signaling pathway activatesMQ-NCSU chicken macrophages by increasing TLR4 andCD14 cell surface expression and elicits an antiviral response against ILTV. Secondly, the inhibition ofILTV replication was dependent on NO production in supernatants. Finally, we confirmed that SE-LPS stimulation of MQ-NCSU cells does not lead to type 1 interferonmRNA expression (P > 0.05, Fig. 4) and unlikely to have contributed to the antiviral effect elicited by MQ-NCSU cells following LPS stimulation against ILTV.


It has been shown that the expression of avian CD14and TLR4 can be quantified using anti-human mAbs against these two receptors as they are components of highly conserved innate immune system (Dil and Qureshi, 2002; Janeway and Medzhitov, 2002). In agreement with these observations, we observed first that MQNCSU cells expressTLR4 and CD14 constitutively, but in a low number of cells.Following treatment with EC-LPS the expression of TLR4and CD14 have been increased significantly in MQ-NCSUcells when compared to the controls at 6 h (P = 0.002) and12 h (P = 0.002) post-treatments (Fig. 1) whereas with SE-LPS, the MQ-NCSU cells expressing both TLR4 and CD14increased significantly at 12 h post stimulation (P = 0.002).We also observed that culture supernatants collected following LPS treatment of MQ-NCSU could reduce ILTVreplication in susceptible LMH cells (P = 0.00; Fig. 2).


Fig. 4. MQ-NCSU cells do not up regulate the mRNA expression of IFN _and IFN _ following treatment with SE-LPS; (a) and (b) illustrate the IFN _and IFN _ mRNA expressions, respectively. Real-time PCR was done to quantify the mRNA expression and fold changes are presented. Data represent mean values ± SEM.


Xing and Schat (2000) have shown that the inhibition of MDV and HVT replication in CEF stimulated by LPSfollowing recombinant ChIFN- _ is mediated by NO and not any other bioactive agents such as reactive oxygen species, IL-1 _ and IL-6 like molecules produced follow-ing the treatment (Xing and Schat, 2000). Pertile et al.(1996) also have shown that NO-mediated antiviral effect against avian reovirus in HD11 cells treated with spleen cell conditioned-medium or LPS (Pertile et al., 1996). We observed that iNOS inhibitor significantly reduced the production of NO induced by EC-LPS and SE-LPS TLR4 ligands in MQ-NCSU cells (P = 0.00; Fig. 3a and b). Culture supernatants produced by MQ-NCSU cells following treatment with EC-LPS and iNOS inhibitor (6 h post-treatment) abrogated the inhibitory effect of ILTV replication shown by culture supernatants of MQ-NCSU cells treated with EC-LPSalone (P = 0.031; Fig. 3c and e). Culture supernatants produced by MQ-NCSU cells 18 h post-treatment with EC-LPSor 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 ande).


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 below0.06 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 NOis unstable, it is possible that by the time of treatment ofLMH 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 antiviral effect in our experiments, however, this needs further investigation.


In conclusion, we have shown that EC-LPS and SE-LPSTLR4 ligands are capable of activating MQ-NCSU avian macrophages as evidenced by increase of LPS receptors and elicit an antiviral response against ILTV in a NO dependent manner. The potential role of type 1 interferons in LPSmediated antiviral response against ILTV is not clear since we did not observe up regulation of expressions of IFN _ andIFN _ following SE-LPS treatment. It is of interest to investigate the interaction between E. coli and ILTV in respiratory mucosa.


Conflict of interest statement

The Authors declare that there is no conflict of interest.



This study was funded by University of Calgary Faculty of Veterinary Medicine, Natural Sciences and Engineering Research Council of Canada, Canadian Poultry Research Council and Alberta Livestock and Meat Agency, Canada.



This article was originally published in Veterinary Immunology and Immunopathology 155 (2013) 270–275. DOI: 10.1016/j.vetimm.2013.08.005. This is an Open Access article distributed under the terms of the Creative Commons Attribution License: Attribution-NonCommercial-ShareAlike 3.0 Unported (CC BY-NC-SA 3.0).




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Dr. Careem completed his BVSc (Hons) degree at the Peradeniya University, Sri Lanka in 1991 and MVM degree from the University of Glasgow Veterinary School, UK in 1995. He obtained his PhD degree, in the field of host-viral interactions, at the Ontario Veterinary College, University of Guelph in 2008. Following the PhD degree, he was awarded a Canadian Institutes of Health Research Fellowship to conduct post-doctoral research on innate immune responses generated against mucosal viral infections at the Center for Gene Therapeutics of the McMaster University, Hamilton. Dr. Careem is a diplomate
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