Expression and LPS-Induced Elevation of Nod2 and Calprotectin in the Submandibular Gland of Wild-Type and TLR4-Knockout Male Mice

The salivary gland produces a number of inflammation-related cytokines and at least some of them including S100A8 and S100A9 (calprotectin subunits) in the gland are induced by endotoxins. The signaling pathway to induce these cytokines is complex and has not been thoroughly elucidated. Thus, we examined effects of lipopolysaccharide (LPS) on the level of salivary gland S100A8/A9 and investigated whether any signaling molecule is participated in elevation of these cytokines in this tissue. Toll-like receptor-4 (TLR4)-knockout C57BL/6 (TLR4-/-) mice and their wild-type (WT), C57BL/6, counterpart (TLR4+/+) were used in the experiment. Quantitative real-time RT-PCR, RT-PCR, and Western blotting were employed for analyses of mRNA and protein levels of S100A8/A9, nucleotidebinding oligomerization domain-containing protein (Nod) 1, and Nod2. Following the injection of E. coli LPS, the mRNA levels of S100A8/A9 were strongly elevated in the submandibular gland (SMG) of WT mice and at small degree but clearly so even in the same tissue of TLR4-knockout counterparts; this suggests the existence of a signaling pathway other than TLR4 to induce S100A8/A9. In the SMG of WT mice but not TLR4-knockout counterparts, both mRNA and protein levels of Nod2 were elevated by LPS, these increases were completely blocked by cycloheximide (CHX). CHX simultaneously suppressed LPSinduced elevation of S100A8/A9 mRNAs. These results, although more experiment using Nod2 specific inhibitors is required, imply the possibility that S100A8/A9 mRNA levels were increased by LPS via both TLR4 and Nod2 signaling pathways in the mouse SMG.


Introduction
S100A8 and S100A9 belong to the S100 protein family, whose members contain 2 Ca² + binding sites of the EF-hand type (Donato, 2001).S100A8 and S100A9 in this family make a heterodimeric complex called calprotectin, which is a pleiotropic protein associated with inflammation.Namely, calprotectin binds divalent cations, such as zinc ions and shows a bacteriostatic effect (Stříž and Trebichavský, 2004) Calprotectin is produced abundantly and constitutively in the neutrophils and monocytes.This pro-inflammatory protein is expressed also in the squamous mucosal keratinocytes and innate immune cells present at the surface of mucosal tissue (Hsu et al., 2009).In the patient suffering from inflammatory diseases, the calprotectin level in the plasma, feces, dental calculus, gingival crevicular fluid,

Abstract
The salivary gland produces a number of inflammation-related cytokines and at least some of them including S100A8 and S100A9 (calprotectin subunits) in the gland are induced by endotoxins.The signaling pathway to induce these cytokines is complex and has not been thoroughly elucidated.Thus, we examined effects of lipopolysaccharide (LPS) on the level of salivary gland S100A8/A9 and investigated whether any signaling molecule is participated in elevation of these cytokines in this tissue.Toll-like receptor-4 (TLR4)-knockout C57BL/6 (TLR4-/-) mice and their wild-type (WT), C57BL/6, counterpart (TLR4+/+) were used in the experiment.Quantitative real-time RT-PCR, RT-PCR, and Western blotting were employed for analyses of mRNA and protein levels of S100A8/A9, nucleotide-binding oligomerization domain-containing protein (Nod) 1, and Nod2.Following the injection of E. coli LPS, the mRNA levels of S100A8/A9 were strongly elevated in the submandibular gland (SMG) of WT mice and at small degree but clearly so even in the same tissue of TLR4-knockout counterparts; this suggests the existence of a signaling pathway other than TLR4 to induce S100A8/A9.In the SMG of WT mice but not TLR4-knockout counterparts, both mRNA and protein levels of Nod2 were elevated by LPS, these increases were completely blocked by cycloheximide (CHX).CHX simultaneously suppressed LPS-induced elevation of S100A8/A9 mRNAs.These results, although more experiment using Nod2 specific inhibitors is required, imply the possibility that S100A8/A9 mRNA levels were increased by LPS via both TLR4 and Nod2 signaling pathways in the mouse SMG.

Preparation of Total RNA, RT-PCR, and Quantitative Real-Time RT-PCR
The mice were euthanized by cervical dislocation and the SMG and PG were carefully removed from each mouse.

Preparation of Tissue Extracts and Western Blotting
The SMG, spleen, and adipose tissue were

Effects of LPS on expression of S100A, S100A9, Nod1, and Nod2 mRNAs in the salivary glands of WT and TLR4-KO mice
The effects of the endotoxin LPS on mRNA expression levels of S100A8 and S100A9 (the subunits of calprotectin) in the SMG and PG of WT and TLR4-KO mice were first examined (Fig. 1a, b).In the SMG, LPS elevated the mRNA levels of S100A8 and S100A9 in not only the WT mice but also in the TRL4-KO ones (Fig. 1a).This mRNA elevation in the TLR4-KO mice was not limited to S100A8 and S100A9; for the Fig.

(
WT) mice in this paper.All animals were housed under standard conditions (12-h light/12-h darkness cycle at 22-25°C) with free access to food and water.They were used for experiments at the age of 8 to 9 weeks.In the RT-PCR experiment to estimate the salivary gland mRNA levels of S100A8, S100A9, IL-1β, Nod1/Nod2, TLR4, and β-actin, mice were injected i.p. with LPS dissolved in saline at a dose of 0.4 mg/kg body weight (Yao et al., 2005a); and control animals, with saline.The animals were euthanized by cervical dislocation at 3 h after the saline-or LPS-treatment.In the quantitative real-time RT-PCR experiment to examine the effects of LPS and CHX on the mRNA levels of S100A8 and S100A9, animals were divided into 4 groups and injected with CHX and LPS either singly or in combination.CHX or control saline was injected into mice i.p., 30 min prior to LPS injection; and the animals were euthanized by cervical dislocation at 3 h after the LPS injection.Dosages of CHX and LPS were 50 mg (Tait et al., 2005) and 0.4 mg/kg body weight, respectively.In the Western blotting experiment to examine the effects of CHX and LPS on the Nod1 and Nod2 protein levels, animals were divided into 4 groups, each given these 2 reagents singly or in combination similarly as described above.In all of the above experiments, each group consisted of 4-5 mice for quantitative analyses; whereas 2-3 mice were used for other assays.The protocols used in the present animal experiments were approved by the Institutional Review Board of the Animal Committee of The University of Tokushima.
homogenized in 5 mM HEPES buffer (pH 7.5, containing 50 mM mannitol, 10mM MgCl2, 1mM PMSF and 1×Protease inhibitor cocktail) and centrifuged at 14,000 rpm for 15 min at 4°C to obtain their supernatants, the protein concentrations of which were determined by the Bradford method (Bradford et al., 1976).The tissue extract prepared was denatured under reducing conditions (95°C for 5 min).Sixty micrograms of protein were separated by electrophoresis on a 10% sodium dodecyl sulfate-polyacrylamide gel.After electrophoresis, the separated proteins were transferred onto Immobilon-P membranes, which were next blocked with PVDF blocking reagent at room temperature for 3 h.The membranes were then incubated at 4°C overnight with 0.5 µg/ml of anti-Nod1 antibody and 0.1 µg/ml of anti-Nod2 antibody in "Solution 1" provided in Can Get Signal ® kit.They were subsequently washed and incubated at room temperature for 1 h with 8,000 times-diluted rabbit anti-goat IgG horse radish peroxidase-conjugate in Can Get Signal ® , "Solution 2." For the internal standard, β-actin was visualized by reacting the membrane with 50,000-times diluted mouse monoclonal anti-β-actin-peroxidase at room temperature for 1 h.All membranes were subjected to the ECL reaction and exposed to X-ray films.Statistics ANOVA and Fisher's PLSD (Protected Least Significant Difference) were applied for statistical analysis of the data obtained by quantitative real-time RT-PCR.
1a, b mRNA level of the cytokine, IL-1β was also increased by LPS in these mice.Similar results were obtained when total RNA from the PG was analyzed (Fig.1b)._________________________________________________________________________________ ______________ Purevjav Javkhlan, Guangfei Xu, Gang Chen, Chenjuan Yao, Yuka Hiroshima, Hiroshi Yoshimura, Toshihiko Nagata and Kazuo Hosoi (2015) , Journal of Research and Practice in Dentistry, DOI: 10.5171/2015.290259

Figure 1 :
Figure 1: Effects of injection of LPS on the mRNA levels of S100A8, S100A9, and IL-1β in the SMG and PG of WT and TLR4-KO mice.S100A8, S100A9, IL-1β, and β-actin mRNA levels in the SMG (a) and PG (b) were determined by RT-PCR.WT, WT mice; KO, TLR4-KO mice, -R; control experiment in the absence of RNA.Arrowheads at the right indicate the size of cDNA amplified (bp) Since Nod1 and Nod2 are cytosolic components of the innate immune system capable of binding with LPS to activate NF-κB (Inohara et al., 2001; Pauleau and Murra, 2003), we examined if these components were involved in the induction of the S100A8 and S100A9 mRNAs.RT-PCR experiments were first conducted to detect the Nod1 and Nod2 mRNAs in the SMG from WT and TLR4-KO mice (Fig. 2a).Both Nod1 and Nod2 mRNAs were expressed in this tissue; and the mRNA for Nod2 in the WT mice, but not that in the TLR4-KO mice,

Figure 2 :
Figure 2: Effects of injection of LPS on the mRNA levels of Nod1 and Nod2 in the SMG and PG of WT and TLR4-KO mice.TLR4, Nod1, Nod2, and β-actin mRNA levels were determined by RT-PCR in the SMG (a) and PG (b).WT, WT mice; KO, TLR4-KO mice; -R; control experiment in the absence of RNA.Arrowheads indicate the size of cDNA amplified (bp)

Figure 3 :
Figure 3: Effects of CHX on LPS-induced elevation of S100A8 and S100A9 in the SMG of WT and TLR4-KO mice.a, WT mice; b, TLR4-KO mice.The S100A8 and S100A9 mRNA levels were normalized by the level of β-actin mRNA, which are regarded as 1.The absolute value of β-actin mRNA was scattered within 10%.The SEM of the β-actin mRNA show the variation of the sample applied.WT, WT mice; KO, TLR4-KO mice.Relative mRNA levels ± SEM are shown.Statistical Analysis was conducted by ANOVA, followed by Fisher's Protected Least Significant Difference (PLSD).*p<0.05,significantly different.NS, not significant

Figure 4 :
Figure 4: Effects of CHX on LPS-induced changes in Nod1 and Nod2 levels in the SMG of WT and TLR4-KO mice.a, Detection of Nod1 and Nod2 proteins in the SMG and verification of the specificity of the antibodies.Ab, intact antibody; p-Ab, peptide pre-absorbed antibody; Sm, SMG; Sp, spleen; Ad, adipose tissue.The Sp and Ad were used as positive controls for detection of Nod1 and Nod2, respectively.Arrowheads, positions of Nod1 and Nod2, respectively.b, Effects of CHX and LPS on Nod1 and Nod2 levels in the SMG of WT and TLR4-KO mice.β-Actin bands in each sample are shown as an internal standard.WT, WT mice; KO, TLR4-KO mice
β-actin by RT-PCR and quantitative real-time RT-PCR Quantitative real-time RT-PCR analysis was performed to measure the expression level of S100A8, S100A9, and β-actin transcripts.The sequential reactions were performed by using a One Step SYBR Green Primescript TM RT-PCR kit according to the manufacturer's instruction, and using primers as reported previously (Javkhlan et al 2011; Table 1