Role of invariant NKT cells in lipopolysaccharide-induced lethal shock during encephalomyocarditis virus infection

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Role of invariant NKT cells in lipopolysaccharide-induced lethal shock during encephalomyocarditis virus infection Tatsuya Ando a , Hiroyasu Ito a,∗ , Hirofumi Ohtaki b , Ayumu Kanbe a , Akihiro Hirata c , Akira Hara d , Mitsuru Seishima a a

Department of Informative Clinical Medicine, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194, Japan Department of Medical Technology, Kansai University of Health Sciences, 2-11-1 Wakaba, Kumatori, Osaka 590-0482, Japan c Division of Animal Experiment, Life Science Research Center, Gifu University, 1-1 Yanagido, Gifu 501-1194, Japan d Department of Tumor Pathology, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194, Japan b

a r t i c l e

i n f o

Article history: Received 2 June 2016 Received in revised form 12 August 2016 Accepted 17 September 2016 Available online xxx Keywords: Endotoxin shock Natural killer T cell Encephalomyocarditis virus Toll-like receptor 4 Inducible nitric oxide synthase

a b s t r a c t Viral infections can give rise to secondary bacterial infections. In the present study, we examined the role of invariant natural killer T (iNKT) cells in lipopolysaccharide (LPS)-induced lethal shock during encephalomyocarditis virus (EMCV) infection. Wild-type (WT) mice and J␣18 gene knockout (J␣18 KO) mice were inoculated with EMCV, 5 days prior to challenging with LPS. The survival rate of J␣18 KO mice subjected to EMCV and LPS was significantly higher than that of WT mice. TNF-␣ and nitric oxide (NO) production were increased in WT mice, than that in J␣18 KO mice, after the administration of EMCV and LPS. EMCV infection increased the number of iNKT cells and IFN-␥ production by iNKT cells in WT mice. Moreover, EMCV infection enhanced the expression of Toll-like receptor 4 (TLR4) in the lung and spleen. IFN-␥ also increased the expression of TLR4 in splenocytes. These findings indicated that EMCV infection activated iNKT cells, and IFN-␥ secreted from the iNKT cells up-regulated the expression of TLR4 in various tissues. As a result, EMCV-infected mice were susceptible to LPS and easily developed the lethal shock. In conclusion, iNKT cells were involved in the development of LPS-induced lethal shock during EMCV infection. © 2016 Elsevier GmbH. All rights reserved.

1. Introduction Secondary bacterial infections can be induced by pre-existing microbial infections (caused by viruses, bacteria, fungi, and parasites) (Bakaletz, 2004; Kash and Taubenberger, 2015). These microbes act synergistically to mediate complex disease processes. In particular, bacterial infections during an acute viral infection such as influenza virus is well known as an aggravating factor in infectious disease (Kash and Taubenberger, 2015; Brundage, 2006). Lipopolysaccharide (LPS) is a component of the outer membrane of gram-negative bacteria, can cause systemic inflammatory response syndrome, endotoxin shock, disseminated intravascular coagulation, and even lead to multi-organ failure (Cohen, 2002). Several reports have shown that LPS augments the aggravation of condition in a host especially during adenovirus and lymphocytic choriomeningitis virus infection (Fejer et al., 2005; Nansen and Randrup

∗ Corresponding author. E-mail address: [email protected] (H. Ito).

Thomsen, 2001). In previous study, we established a mouse secondary bacterial infection model using encephalomyocarditis virus (EMCV) and LPS (Ohtaki et al., 2012). An injection of LPS at a low dose could induce lethal shock in the mice infected by EMCV. However, the detailed mechanism underlying the development of lethal shock was unknown. Invariant natural killer T (iNKT) cells, a novel lymphoid lineage that is, distinct from mainstream T cells, B cells, and NK cells has been identified. These cells are characterized by the coexpression of NK cell receptors and semi-invariant T cell receptors encoded by V␣14 and J␣18 gene segments (Bendelac et al., 1997; Makino et al., 1995). iNKT cells are activated by glycolipids such as alpha-galactosylceramide (GalCer), and secrete a large amount of cytokines, including Th1 and Th2 cytokines (Ito et al., 2014, 2010; Rossjohn et al., 2012). Several infections also induce the activation of iNKT cells directly and/or indirectly (Holzapfel et al., 2014; Selvanantham et al., 2013). In some viral infection models, iNKT cells were activated during viral infections and were involved in the elimination of these pathogens (Godfrey et al., 2012; Reilly et al., 2012; Raftery et al., 2014). Previously, our studies estab-

http://dx.doi.org/10.1016/j.imbio.2016.09.005 0171-2985/© 2016 Elsevier GmbH. All rights reserved.

Please cite this article in press as: Ando, T., et al., Role of invariant NKT cells in lipopolysaccharide-induced lethal shock during encephalomyocarditis virus infection. Immunobiology (2016), http://dx.doi.org/10.1016/j.imbio.2016.09.005

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lished the endotoxin-induced lethal shock model using GalCer (Ito et al., 2006, 2005). The administration of LPS at a low dose induced lethal shock in the mice pre-treated with GalCer. Further, acute lung and liver injury were induced in this endotoxin shock (Ito et al., 2006) (Tumurkhuu et al., 2008) (Yokochi, 2012). Lethal shock by low-dose LPS is easily induced under the conditions in which iNKT cells are activated. Therefore, iNKT cells might be involved in the development of lethal shock during secondary bacterial infections. In the present study, we examined the role of iNKT cells in the murine secondary endotoxin shock model using EMCV and lowdose LPS. We found that EMCV infection initiated the lethal shock induced by low-dose LPS injection through the activation of iNKT cells. These results indicated that iNKT cells are strongly involved in the development of LPS-induced lethal shock during viral infection. 2. Materials and methods 2.1. Mice Mouse experiments were performed according to the guidelines of the Animal Ethics Committee of Gifu University Graduate School of Medicine. B10D2 mice, approximately 10–12 weeks old, were obtained from Japan SLC (Hamamatsu, Japan) and used as wild-type (WT) mice. J␣18 gene knockout (J␣18 KO) mice with a B10D2 background were produced by gene targeting as described previously (Cui et al., 1997). 2.2. Virus inoculation and LPS treatment A myocarditic variant of EMCV was generously provided by Dr. Seto (Keio University, Tokyo, Japan). The virus stock was stored at −80 ◦ C in Hanks’ balanced salt solution (HBSS) with 0.1% BSA. The mice were inoculated intraperitoneally with 200 plaque forming units (pfu) of EMCV in 0.2 mL saline and were treated intravenously with 0.8 mg/kg LPS (Escherichia coli O111:B4; Sigma-Aldrich, St Louis, MO, USA) in 0.2 mL saline at 0 (non-infected), and 5 days after the viral inoculation unless otherwise noted. Mice were intraperitoneally injected with 250 ␮g anti-IFN-␥ antibody (clone XMG 1.2; BioXcell, West Lebanon, NH, USA), on days 0 and days 4 after EMCV infection. The lethality was determined at each day after LPS treatment. The experiments were performed according to the institutional guidelines of Gifu University for microbiologic study. 2.3. Measurement of serum TNF-˛ and nitric oxide (NO) concentration The concentration of TNF-␣ in the serum was determined using a mouse TNF-␣ Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s recommendations. The concentration of NO in plasma was determined using the Griess reaction (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, 2.5% phosphoric acid). The plasma (100 ␮L) was mixed with 100 ␮L of Griess reagent, incubated at room temperature for 10 min, and then the absorbance was measured at 570 nm using a microplate reader.

PCR that was conducted using pre-designed primer/probe sets for TNF-␣, iNOS, IFN-␥, IL-4, TLR4, and 18S rRNA (Applied Biosystems), according to the manufacturer’s recommendations. EMCV RNA was detected using KAPA SYBR Green I (Roche Diagnostic Systems, Indianapolis IN, USA) and the following oligonucleotide primer pairs: EMCV sense, 5 -GTCGTGAAGGAAGCAGTTCC-3 , and antisense, 5 -CACGTGGCTTTTGGCCGCAGAGGC-3 , with 18S rRNA used as an internal control. Real-time PCR was carried out using a Light-Cycler 480 system (Roche Diagnostic Systems). 2.5. Flow cytometric analysis Intrahepatic lymphocytes (IHL) were obtained from the liver 5 days after EMCV infection by centrifugation of single cells with Ficoll-Conray (IBL, Gunma, Japan). Flow cytometry was used to determine the expression levels of CD3 and DX5 in IHL and splenocytes at 5 days of EMCV infection. The expression of CD11b, CD11c, and TLR4 were detected at 5 days after EMCV infection in BALF. The cells were stained with allophycocyanin (APC)conjugated anti-mouse CD3 antibody (clone 17A2; eBioscience, San Diego, CA, USA), phycoerythrin (PE)-Cy7 conjugated anti-mouse CD11b (clone M1/70; eBioscience), PE-conjugated anti-mouse TLR4 (clone MTS510; eBioscience), PE-conjugated anti-mouse CD49b (clone DX5; Biolegend, San Diego, CA, USA), fluorescein isothiocyanate (FITC)-conjugated anti-mouse IFN-␥ (clone XMG1.2; BD Biosciences, Franklin Lakes, NJ, USA), VioBlue- conjugated antimouse CD11c (clone N418; Miltenyi Biotec, Bergisch Gladbach, Germany). The stained cells were analyzed using a FACSCanto II instrument (BD Biosciences). 2.6. Intracellular cytokine staining Splenocytes (2 × 106 cells/ml) and hepatic IHL (1 × 106 cells/ml) were incubated after infectionwith EMCV. Protein Transport Inhibitor (GolgiPlug; BD Biosciences) was added to each well 4 h before harvesting the cells for fixation and permeabilization with cytofix/cytoperm (BD Biosciences). 2.7. Histopathology Histopathological examination of the heart was performed on day 5 of EMCV infection with or without LPS injection. The tissues were fixed in 10% formalin for 24 h and embedded in paraffin. Tissue sections were deparaffinized, stained with hematoxylin and eosin, and examined by light microscopy. The infiltrating mononuclear cells of hematoxylin and eosin stained heart tissue was determined at 400× magnification and nuclei were counted in 4 randomly chosen fields. 2.8. Statistical analysis In each experiment, the results were expressed as the mean ± standard deviation (SD). The statistical significance of the difference in mean values was determined by Student’s t-test or one-way analysis of variance followed by Scheffe’s test. P values of less than 0.05 were considered significant.

2.4. Real-time RT-PCR analysis 3. Results Real-time RT-PCR was used to quantify the mRNA levels of TNF-␣, iNOS, IFN-␥, IL-4, TLR4, and EMCV RNA. Total RNA in the brain, heart, lung, and spleen was isolated using Isogen (Nippon Gene, Tokyo, Japan). Total RNA in the bronchoalveolar lavage fluid (BALF) was isolated using an RNeasy Mini Kit (QIAGEN, Hilden, Germany) and transcribed to cDNA by using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Purified cDNA was used as the template for real-time

3.1. iNKT cell deficiency improved the survival rates of EMCV-infected mice after the injection of low-dose LPS To examine the effect of iNKT cells on secondly endotoxin shock, WT and J␣18 KO mice were intraperitoneally inoculated with 200 pfu of EMCV and intravenously injected with 0.8 mg/kg of LPS 5 days after the EMCV inoculation. On day 2 after LPS injection,

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Fig. 1. Survival rate in EMCV-infected wild-type (WT) mice and J␣18 knockout (KO) mice after LPS infection. (A) Survival rate of WT and J␣18 KO mice inoculated with or without EMCV followed by a low-dose of LPS. The survival curves of WT mice and J␣18 KO mice differed significantly (p < 0.01) by Kaplan Meyer’s test. (B) WT mice and J␣18 KO mice were inoculated with EMCV, and the amount of EMCV RNA in the heart and brain from each group of mice was determined using real-time RT-PCR. (C) Histopathological examination in the heart at 0, and 5 days after EMCV infection was performed. Tissue sections were deparaffinized, stained with hematoxylin-eosin, and examined under light microscopy. (D) The number of infiltrating mononuclear cells per field was counted in 4 fields from each heart tissue. (E) IL-1␤ and TNF-␣ mRNA expression in heart and brain at 5 days after EMCV infection was analyzed by real-time RT-PCR and was determined on the basis of 18S rRNA expression. The data were calculated in reference to mRNA levels of the respective tissues in control mice (Day 0). The data are represented as means ± SD of the results of 4 mice in each group. Statistically significant differences between the groups were determined using a Student’s t test; *p < 0.05.

the survival rate of J␣18 KO mice was 89%, whereas the survival rate of WT mice was 13%. On the other hand, the injection of LPS alone or EMCV alone did not induce lethal endotoxin shock in WT and J␣18 KO mice (Fig. 1A). We next measured the viral load in the brain and heart 5 days after EMCV infection by real-time RTPCR. There was no significant difference in viral loads in the brain and heart between WT and J␣18 KO mice (Fig. 1B). Histological examination of the heart was performed 5 days after inoculation with EMCV (Fig. 1C), and we measured the number of infiltrating mononuclear cells in heart at day 5 after EMCV infection (Fig. 1D). Although the numbers of infiltrating cells in heart increased after EMCV infection, there was no significant difference between WT and J␣18KO mice. Next, we examined the expression of IL-1␤ and TNF-␣ mRNA at 5 days after EMCV infection in heart and brain

(Fig. 1E). The expression of IL-1␤ and TNF-␣ mRNA were not significantly different between WT and J␣18 KO mice.

3.2. EMCV infection induced the activation of NKT cells Several previous reports demonstrated that NKT cells are involved in various pathogenic infections (Tumurkhuu et al., 2008; Emoto et al., 2002). We evaluated the effect of EMCV infection on the induction of NKT cell activation in WT mice. The expression of IFN-␥ mRNA in brain, liver, heart, and spleen from WT mice increased at 2 days after EMCV infection (Fig. 2A). Moreover, it is well-known that activated NKT cells also produce a large amount of IL-4. In the present study, the expression of IL-4 mRNA in heart, brain, and liver from WT mice also increased after EMCV infection (Supplemental Fig. S1). The frequency of the NKT cell (CD3+/DX5+)

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Fig. 2. Effect of EMCV infection on NKT cell activation. (A) The expression of IFN-␥ mRNA in heart, spleen, lung and liver at 2 days after EMCV infection was analyzed by real-time RT-PCR and was determined on the basis of 18S rRNA expression. The data were calculated in reference to mRNA levels of the respective tissues in control mice (Day 0). (B) Gated percentages of CD3+ DX5+ cells in the liver mononuclear cells and splenocytes of WT mice at 5 days after EMCV inoculation. (C) Flowcytometric analysis for intracellular IFN-␥ produced by CD3+ DX5+ cells in the liver mononuclear cells and splenocytes from WT mice at 0 and 5 days after EMCV inoculation, and cultured with GolgPlug in vitro. Statistically significant differences between the groups were determined using a Student’s t-test; *p < 0.05.

population in the liver remarkably increased 5 days after EMCV infection (Fig. 2B). Next, we measured by intracellular cytokine staining IFN-␥ production in NKT cells from the liver and spleen after infection with EMCV. As shown in Fig. 2C, the intracellular staining indicated that EMCV infection enhanced IFN-␥ production in NKT cells from both the liver and spleen.

from the EMCV-infected mice treated with LPS (Fig. 3A and B). The mRNA expressions of IFN-␥, TNF-␣, and iNOS mRNA in the lung (Fig. 3C) and spleen (Fig. 3D) after LPS treatment were determined using real-time RT-PCR. IFN-␥ and iNOS mRNA expression in both the lung and spleen of WT mice was significantly increased compared to that of J␣18 KO. TNF-␣ mRNA expression in the lung of WT mice was markedly up-regulated compared to J␣18 KO mice.

3.3. Effect of EMCV infection on LPS-induced TNF-˛, nitric oxide production, and mRNA expression of cytokines 3.4. Effect of EMCV infection on TLR4 mRNA expression in the lung The concentration of serum TNF-␣ and NO was measured at 2 h and 8 h after LPS injection in EMCV-infected mice. The serum levels of TNF-␣ in EMCV-infected WT mice after LPS injection were significantly higher than those in J␣18 KO mice (Fig. 3A). Similarly, the NO concentration in serum from WT mice at 8 h after LPS injection was significantly increased compared to that from J␣18 KO mice (Fig. 3B). On the other hand, TNF-␣ concentration and NO levels in serum from the mice treated with LPS alone were lower than those

To determine the mechanisms by which low-dose LPS promotes lethal endotoxin shock, we measured the expression of TLR4, which is an LPS-specific receptor. TLR4 mRNA expression in BALF cells isolated from WT mice significantly increased after EMCV infection. In contrast, TLR4 mRNA expression in J␣18 KO mice was not affected by EMCV infection (Fig. 4A). Furthermore, we determined the protein level of TLR4 in BALF cells 5 days after EMCV infection using

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Fig. 3. Effect of EMCV infection on LPS-induced nitric oxide (NO) expression and cytokine production. (A) EMCV-infected or non-infected WT and J␣18 KO mice were inoculated with LPS (0.8 mg/kg) and the serum levels of TNF-␣ were subsequently quantified using an ELISA. (B) EMCV-infected or non-infected WT and J␣18 KO mice were inoculated with LPS (0.8 mg/kg) and the plasma levels of nitrite were subsequently quantified by a Griess reaction. (C and D) IFN-␥, TNF-␣, and iNOS mRNA expressions in the lung (C) and spleen (D) from EMCV-infected mice at 1 h after LPS treatment were determined on the basis of 18S mRNA expression using real-time RT-PCR. The data were calculated in reference to mRNA levels of the respective tissues in control mice. The data are expressed as means ± SD of 5 mice per time point per group. Statistically significant differences between the groups were determined using a Student’s t-test; *p < 0.05.

flow cytometric analysis. Flow cytometry also indicated that TLR4 expression increased after EMCV infection (Fig. 4B and C). 3.5. The expression of TLR4 was augmented by IFN- after EMCV infection In the present study, EMCV infection enhanced IFN-␥ production in WT mice. We next examined the effect of IFN-␥ on the expression of TLR4. The expression of TLR4 mRNA in splenocytes, IHL, and BALF cells from both WT and J␣18 KO mice was significantly increased by the administration of recombinant IFN-␥ in vitro (Fig. 5A). Furthermore, we determined whether IFN-␥ aggravate the secondary lethal endotoxin shock during EMCV infection via the enhancement of TLR4 expression. As shown in Fig. 5B, the administration of anti-IFN-␥ antibody improved survival rate in the secondary lethal endotoxin shock. These data indicated the enhancement of IFN-␥ production was required for the development of lethal endotoxin shock in secondary bacterial infection. Thus, the enhancement of TLR4 expression was induced by the increase of IFN-␥ after EMCV infection. 4. Discussion iNKT cells, a novel lymphoid lineage that is, distinct from typical T cells, B cells and NK cells has been identified (Godfrey et al., 2004). These cells are found in relative abundance in tissues such as spleen, bone marrow, thymus, and liver tissue, and are characterized by the co-expression of NK cell receptors and semi-invariant T cell receptors encoded by V␣14 and J␣18 gene segments. It is well known that activated iNKT cells vigorously produce Th1 and Th2 cytokines. NKT cells are involved in anti-tumor immunity and combating bacterial and virus infections (Ito et al., 2003, 2008). Gal-

Cer is well known as one of the specific ligands of iNKT cells. iNKT cells are activated by glycolipid like GalCer presenting on CD1d molecules. Moreover, a previous study indicated that viral infection induced the activation of iNKT cells through the up regulation of IL-12 (Tyznik et al., 2014). In the present study, the expression of IFN-␥ and IL-4 mRNA in WT mice was augmented after EMCV infection compared to that in J␣18 KO mice (Fig. 2A and Supplemental Fig. S1). EMCV infection increased the frequency of iNKT cells in liver, and enhanced the production of IFN-␥ from hepatic and splenic iNKT cells (Fig. 2B and C). On the other hand, the frequency of splenic iNKT cells decreased after EMCV infection, but these cells produced IFN-␥. Previous study demonstrated that splenic iNKT cells do not expand after virus HSV infection, but these cells activate and produce IFN-␥ after virus infection. The up-regulation of chemokine receptor expression on iNKT cells after virus infection induces the chemotactic migration into peripheral blood (Raftery et al., 2014). The frequency of splenic iNKT cells after EMCV infection may decrease through the increase of chemotactic migration peripheral blood. These results indicated that EMCV infection also induced the activation of iNKT cells. Several reports have shown that the administration of LPS at a low dose can promote lethal shock during the activation of iNKT cells (Ito et al., 2006; Tumurkhuu et al., 2008; Dagvadorj et al., 2010). The administration of LPS induced an extreme immune response in mice or cells pre-treated with GalCer in vivo and in vitro (Ito et al., 2005; Ohtaki et al., 2009). Our present study showed that the survival rate of J␣18 KO mice infected with EMCV and administered low-dose LPS was significantly higher than that of WT mice (Fig. 1A). These results indicated that the activation of iNKT cells was involved in the development of virus-induced secondary endotoxin shock. Several reports have shown that iNKT cells are critical in the elimination of virus, but there was no difference in the load

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Fig. 4. Effect of EMCV infection on TLR4 expression. (A) The expression of TLR4 mRNA in bronchoalveolar lavage fluid (BALF) cells 5 days after EMCV infection was analyzed by real-time RT-PCR and was determined on the basis of 18S rRNA expression. The data were calculated in reference to mRNA levels of the respective tissues in control mice (Day 0). (B) The flow cytometric analysis of BALF cells 5 days after EMCV infection. BALF cells were stained with anti-CD4, anti-CD8, anti-CD11b, and anti-CD11c. (C) TLR4 protein levels in CD11c positive cells were measured 5 days after EMCV infection using flow cytometric analysis. The data are represented as means ± SD from 4 mice of each group. Statistically significant differences between the groups were determined using a Student’s t-test; *p < 0.05.

of EMCV between WT and J␣18 KO in brain and heart (Fig. 1B). EMCV infection induces acute myocarditis and encephalitis in various animal species (Topham et al., 1991; Kishimoto et al., 1985). In previous report, we demonstrated that EMCV loads in the brain and heart markedly increased at 5 days after the inoculation of virus (Ohtaki et al., 2012). Histological examination indicated that iNKT cell deficiency did not aggravate the pathological condition caused by EMCV infection in our model. Therefore, the survival rate of the secondary lethal endotoxin shock did not depend on the severity of EMCV infection, but rather the activation of iNKT cells (Fig. 1C–E). Previous reports have demonstrated that lethal shock by endotoxin is characterized by hypotension, decreased systemic vascular resistance, and impaired vascular reactivity. TNF-␣, NO, and IFN-␥ has been implicated as principal mediators in the pathogenesis of septic shock (Cauwels and Brouckaert, 2007). Both TNF-␣ production and NO levels in serum from J␣18 KO mice were lower than those of WT mice in the present endotoxin shock model (Fig. 3A and B). Moreover, real-time RT-PCR analysis also indicated that the mRNA expressions of TNF-␣, IFN-␥, and iNOS in the lungs of J␣18 KO mice were significantly down-regulated compared to those of WT mice (Fig. 3C and D). Previous study demonstrated that the production of IFN-␥ and TNF-␣ in J␣18 KO mice administered with low-dose LPS was lower than those in WT mice (Nagarajan and Kronenberg, 2007). Furthermore, J␣18 KO mice were resistant to the generalized Shwartzman reaction. Namely, iNKT cells may be involved in the development of lethal endotoxin shock. In the present study, the secondary lethal endotoxin shock in

EMCV-infected WT mice were induced after low-dose LPS injection. However, the development of secondary lethal endotoxin shock was impaired in J␣18 KO mice. Thus, the activation of iNKT cells is deeply involved in the lethal shock induced by low-dose LPS injection. Anti-TNF-␣ or anti-IFN-␥ antibodies increased the survival rate in the mouse endotoxin shock model using GalCer and LPS (Ito et al., 2006). Similarly, secondary lethal endotoxin shock in EMCV-infected mice could be rescued by the administration of antiIFN-␥ antibody (Fig. 5B). NO promotes endotoxin shock through the induction of vessel dilatation. These results also indicated that the activation of iNKT cells was required to develop secondary endotoxin shock during viral infection. TLR4 recognizes LPS from gram-negative bacteria, and its recognition is essential for the activation of the innate immune response. Furthermore, TLR4 and NF-␬B pathways contribute to LPS-induced lethal shock (Roger et al., 2009; Guo et al., 2012). The expression of TLRs is enhanced by various agonists in bacterial and viral infections (Tissari et al., 2005; Ghosh and Bishayi, 2015a,b; Park and Mun, 2014; Zannetti et al., 2014). In this study, the mRNA expression of TLR4 in BALF cells increased after EMCV infection in WT mice (Fig. 4A and B). On the other hand, TLR4 expression did not increase in J␣18 KO mice infected with EMCV. These results indicated that EMCV infection enhanced the TLR4 expression in BALF cells via iNKT cell activation. The activation of NKT cells enhances the adaptive immune response through the production of Th1 and Th2 cytokines (Diana and Lehuen, 2009; Kronenberg and Gapin, 2002; Taniguchi et al., 2003). In particular, activated iNKT cells

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Fig. 5. The expression of TLR4 was augmented by IFN-␥ after EMCV infection. (A) Splenocytes, intrahepatic lymphocytes, and BALF cells from WT and J␣18 KO mice were incubated with recombinant IFN-␥ (10 ␮g/ml) in vitro. Untreated cells served as a control. mRNA was isolated 24 h after treatment and the expression of TLR4 mRNA was measured using real-time RT-PCR. The data were calculated in reference mRNA levels of the respective untreated cells. The data are represented as means ± SD from 4 mice of each group. Statistically significant differences between the groups were determined using a Student’s t-test; *p < 0.05. (B) Effect of intraperitoneal administration of anti-IFN-␥ antibody on survival of EMCV-infected WT mice injected with 20 ␮g of LPS. The treatment of anti-IFN-␥ antibody (250 ␮g/mouse) injected at 0 and 4 days after EMCV infection. The survival curves of WT mice and J␣18 KO mice differed significantly (p < 0.01) by Kaplan Meyer’s test.

secrete abundant IFN-␥ (Dagvadorj et al., 2010). This study demonstrated that EMCV infection activated NKT cells and enhanced the production of IFN-␥ from iNKT cells (Fig. 2). The administration of recombinant IFN-␥ significantly increased the expression of TLR4 mRNA in splenoctyes, intra-hepatic mononuclear cells, and BALF cells of both WT and J␣18 KO mice (Fig. 5A). In a previous report, IFN-␤ was shown to up-regulate TLR7 expression (Derkow et al., 2013). Furthermore, IFN-␥ can induce the expression of TLR2 and TLR4 mRNA (Faure et al., 2001). Several TLR agonists that induce the production of IFN also up-regulate the expression of TLR (Ghosh and Bishayi, 2015a,b„2014; Iwasa et al., 2015). IFN-␥ has the ability to promote TLR expression, and the activation of NKT cells leads to enhanced TLR expression. In conclusion, we demonstrated that iNKT cells were critical for the development of EMCV-related secondary LPS-induced lethal shock. iNKT cells were activated by EMCV infection and secreted a large amount of IFN-␥. The enhancement of IFN-␥ secretion upregulated the expression of TLR4 and increased the susceptibility of mice to LPS. Therefore, there is a possibility that the regulation of iNKT cell activation could be a new strategy for the elimination of lethal shock caused by secondary bacterial infection.

Conflict of interest The authors declare that there are no conflicts of interest.

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Please cite this article in press as: Ando, T., et al., Role of invariant NKT cells in lipopolysaccharide-induced lethal shock during encephalomyocarditis virus infection. Immunobiology (2016), http://dx.doi.org/10.1016/j.imbio.2016.09.005