Nga mouse macrophages

Immunology Letters 106 (2006) 91–95

Short communication

Defect of toll-like receptor 9-mediated activation in NC/Nga mouse macrophages Tohru Sakai a,b,∗ , Mari Kogiso a , Kaori Mitsuya a , Tatsushi Komatsu b , Sigeru Yamamoto a a

Department of International Nutrition, Institution of Health Bioscience, The University of Tokushima Graduate School, Tokushima 770-8503, Japan b Department of Clinical Nutrition, Osaka Prefecture University, Osaka 583-8555, Japan Received 2 February 2006; received in revised form 21 March 2006; accepted 26 March 2006 Available online 18 April 2006

Abstract Toll-like receptors (TLRs) control activation of adaptive immune responses by antigen-presenting cells (APCs). In this study, we examined TLR9-mediated activation in NC/Nga mice, an animal model for human atopic dermatitis. NC/Nga mouse macrophages produced significantly less TNF-␣ than did BALB/c mouse macrophages in response to CpG oligonucleotide (ODN). In addition to defective TLR9-mediated TNF-␣ production, phosphorylation of ERK1,2 and p38 was rapidly diminished after 60 min of CpG ODN stimulation, whereas phosphorylation of these molecules was sustained until 60 min in BALB/c mice. Furthermore, phosphorylation of c-Jun N-terminal kinase (JNK) was not observed in NC/Nga mouse macrophages. In contrast, B cells and dendritic cells (DCs) from NC/Nga mice showed normal responses to CpG ODN stimulation. The expression level of TLR9 in NC/Nga mouse macrophages was significantly lower than that in BALB/c mouse macrophages, whereas levels of TLR9 expression in B cells and DCs in NC/Nga mice were the same as those in BALB/c mice. These results suggest that defective TLR9-mediated activation in NC/Nga mouse macrophages contributes to the reduction of TLR9 expression levels. © 2006 Elsevier B.V. All rights reserved. Keywords: NC/Nga mice; Toll-like receptor 9; Macrophages

1. Introduction The first line of defense in the body against invading pathogens is accomplished through evolutionarily conserved sets of molecules, namely, toll-like receptors (TLRs), that recognize conserved molecular patterns associated with pathogens. Microbes, microbial products and pharmaceutical products that are ligands for TLRs have been identified. Ligands for TLR1 and 2 and those for 2 and 6 are Gram-positive bacteria and yeast cell wall components, while the predominant Gram-negative bacterial product, LPS, is a ligand for TLR4. It has also been shown that dsRNA (Poly I:C), bacterial flagellin, immiquimod and CpG oligodeoxynucleotides (ODN) are ligands for TLR3, 5, 7 and 9, respectively [1]. The interaction between a TLR and its ligand results in the secretion of anti-bacterial peptides, defensins and

proinflammatory cytokines such as TNF-␣ and IL-6, which initiate an inflammatory response to clear the invading organism. The inflammatory response results in the recruitment of cells of adaptive immunity to initiate clearance of the pathogens by generating a specific immune response [2]. Atopic dermatitis (AD) is a chronic and relapsing inflammatory skin disease with immunological disturbance, and its incidence is increasing in infants and children [3]. The NC/Nga mouse has been shown to be a model animal for human atopiclike dermatitis. When NC/Nga mice were kept in conventional conditions, they spontaneously suffered from AD-like skin lesions with marked elevation in plasma levels of total IgE [4]. In this study, we examined the function of TLRs in NC/Nga mice since TLRs are crucial for acquired immunity [2] and might contribute to the pathogenesis of allergic diseases [5,6]. 2. Materials and methods

∗

Corresponding author at: Department of International Nutrition, Institution of Health Bioscience, The University of Tokushima Graduate School, Kuramoto 3-18-15, Tokushima 770-8503, Japan. Tel.: +81 88 633 7450; fax: +81 88 633 9427. E-mail address: [email protected] (T. Sakai). 0165-2478/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2006.03.006

2.1. Mice Specific pathogen-free male NC/Nga and BALB/c mice were purchased from SLC (Hamamatsu, Japan).

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This study conformed to the guidelines for the care and use of laboratory animals of The University of Tokushima Graduate School Institution of Health Bioscience. 2.2. Measurement of TNF-α production from macrophages Resident peritoneal macrophages (2 × 106 cells/ml) were cultured with various concentrations of phosphorothioatestabilized CpG oligonucleotide (ODN) (TCC-ATG-ACG-TTCCTG-ATG-CT) for 24 h. Concentrations of TNF-␣ in the culture supernatants were measured by using a Mouse TNF-␣ ELISA kit (eBioscience, CA) according to the manufacturer’s instructions.

mAb and FITC-conjugated anti-CD11c mAb, respectively, for 30 min on ice. After washing, the cells were suspended in PBS, and an equal volume of 4% paraformaldehyde was added for fixation. After fixing for 10 min at room temperature, cells were washed with PBS twice. For intracellular staining, all reagents and washes contained 1% BSA and 0.5% saponin (Sigma, St. Louis, MO) and all incubations were performed at room temperature. After washing and a 10-min incubation with PBS/BSA/saponin, cells were stained with biotin-conjugated anti-mouse TLR9 (HyCult biotechnology b.v., The Netherlands) for 30 min and then with Cy5-conjugated streptavidin for 30 min. Samples were analyzed on a FACScalibur with CellQest software (Becton Dickinson).

2.3. Western blot analysis 3. Results and discussion Peritoneal macrophages that had been treated with 1 ␮M CpG ODN in vitro were lysed in lysis buffer containing 1% NoidetP40, 150 mM NaCl, 10 mM Tris–Cl (pH 7.5) and 1 mM EDTA. The cell lysates were dissolved by SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was blotted with an Ab to phosphorylated ERK1,2, p38, JNK or ␤-actin (Santa Cruz, CA) and visualized using an enhanced chemiluminescence system (Amersham Biosciences, UK). 2.4. Proliferation assay B cells were purified using a B cell isolation kit (Milteny Biotec, Inc., CA) according to the manufacturer’s instructions. Purified B cells (2 × 105 cells/well) were cultured with various concentrations of CpG ODN for 48 h in a 96-well plate. One microcurie of [3 H]thymidine deoxyribose (TdR) was added to the wells in last 12 h of culture, and the amount of incorporated [3 H]TdR was measured by a Matrix beta counter (Packard Instrument B.V. Chemical Operations, The Netherlands). 2.5. Expression of MHC class II/CD80 and production of TNF-α in dendritic cells (DCs) Bone marrow (BM) cells were cultured with 10 ng/ml GMCSF in a 250 cm2 flask for 6 days and further cultured with CpG ODN (1 ␮M) for 2 days. After culture, DCs were collected and stained with phycoerythrin (PE)-conjugated anti-mouse I-Ad monoclonal Ab (mAb) and fluorescein isothiocyanate (FITC)conjugated anti-mouse CD80 mAb for 30 min on ice. Stained cells were analyzed by a FACScan and Consort 30 software (Becton Dickinson, CA). Percentages of CD11c+ cells used in this study were more than 90% in cells from both BALB/c and NC/Nga mice. BM-derived dendritic cells (2 × 106 cells/ml) were cultured with 1 ␮M CpG ODN for 24 h. Concentrations of TNF-␣ in the culture supernatants were measured by using a Mouse TNF-␣ ELISA kit (eBioscience). 2.6. Flow cytometric analysis for TLR9 expression Peritoneal macrophages, spleen cells and DCs were stained with FITC-conjugated anti-CD11b, FITC-conjugated anti-B220

NC/Nga and BALB/c mouse macrophages were stimulated with PGN, Poly I:C, LPS and CpG ODN, which are ligands for TLR2, 3, 4 and 9, respectively. These stimulators activated BALB/c mouse macrophages and induced production of TNF-␣. Macrophages from NC/Nga mice showed two-fold less TNF-␣ production in response to Poly I:C and LPS than did macrophages from BALB/c mice but produced a comparable level of TNF-␣ in response to PGN (data not shown). Notably, TNF-␣ production was hardly observed in NC/Nga mouse macrophages upon stimulation with CpG ODN (Fig. 1A). Signaling through TLR9 occurs through sequential recruitment of the adaptor molecule MyD88 and serine/threonine kinase and subsequently activates mitogen-activated protein (MAP) kinases and the nuclear factor NF-␬B [7]. We analyzed the activation of MAP kinase family members, including c-Jun N-terminal kinase (JNK), ERK1,2 and p38, in NC/Nga mouse macrophages by Western blotting. As shown in Fig. 1B, p38 and ERK1,2 were phosphorylated at 20 min after stimulation with CpG ODN, and the phosphorylation was continued for 60 min in macrophages from BALB/c mice. In NC/Nga mouse macrophages, phosphorylation of p38 and ERK1,2 occurred until 40 min after stimulation with CpG ODN, but the phosphorylation had stopped at 60 min after the stimulation. Furthermore, phosphorylation of JNK was not observed in NC/Nga mouse macrophages in response to CpG ODN. These results suggest that impaired activation of the MAP kinase cascade contributes to the defect in TLR9-mediated activation in NC/Nga mouse macrophages. We next analyzed responsiveness of NC/Nga mouse B cells to CpG ODN. B cells were cultured in the presence of various concentrations of CpG ODN. B cells from BALB/c mice showed an increased proliferative response to CpG ODN in a dose-dependent manner. Similarly, B cells from NC/Nga mice responded to CpG ODN normally (Fig. 2A). We further examined TLR9-mediated activation in DCs. Immature DCs were obtained from BM cells by culturing with GM-CSF and then adding CpG to the culture to induce production of mature DCs. In this maturation process, expression of MHC class II and CD80 molecules in cells from BALB/c mice was up-regulated when the cells were stimulated with CpG ODN. The extent of this induction in cells from NC/Nga mice was similar to that

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Fig. 1. Defect of TLR9-mediated activation in NC/Nga mouse macrophages. (A) Macrophages from BALB/c and NC/Nga mice were cultured with indicated concentrations of CpG ODN for 24 h. The concentrations of TNF-␣ in the supernatants were quantified by ELISA. The data are means ± S.D. for triplicate observations. (B) BALB/c and NC/Nga mouse macrophages were stimulated for the indicated periods with 1 ␮M CpG ODN. Total lysates were used for immunoblotting. ␤-Actins were detected as a loading control.

observed in cells from BALB/c mice stimulated with in CpG ODN (Fig. 2B). In addition, the level of TNF-␣ production in DCs from NC/Nga mice was comparable to that in DCs from BALB/c mice (Fig. 2C). These results suggest that B cells and DCs from NC/Nga mice respond to CpG ODN stimulation normally. Finally, we examined the expression of intracellular TLR9 by flow cytometric analysis. Levels of TLR9 expression in B cells and DCs from NC/Nga mice were similar to those in B cells and DCs from BALB/c mice. In contrast, the level of TLR9 expression in NC/Nga mouse macrophages was significantly lower than that in BALB/c macrophages (Fig. 3). In the murine TLR9 gene, transcription is regulated via two cis-acting elements, a distal regulatory region (DRR) and a proximal promoter region (PPR). It has been shown that IFN-stimulated response element (ISRE)/IFN-regulatory factor (IRF)-1, ISRE/IRF-2 and AP-1

sites are crucial for constitutive TLR9 expression and that the transcription factors IRF-2 and AP-1 bind to these sites [8]. The difference between the expression level of TLR9 in NC/Nga mouse macrophages and the levels in B cells and DCs might result from different distributions and amounts of IRF-2 and AP-1 in these cells. We also examined the expression of TLR9 and its responsiveness to CpG ODN in thioglycollate-elicited macrophages and found that its expression and responsiveness are similar to those in macrophages from BALB/c mice (data not shown). This fact indicates that expression of TLR9 in resident macrophages of NC/Nga mice is not regulated by intrinsic polymorphism in the TLR9 promoter region. A critical role of TLR9 in systemic autoimmune disease has been reported. TLR9 contributes to the pathogenesis by inducing generation of anti-dsDNA and anti-chromatin autoantibodies [9] or by inducing production of IFN-␣ from plasmacytoid dendritic

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Fig. 2. Normal responses of NC/Nga mouse B cells and DCs to CpG ODN stimulation. (A) Splenocytes from BALB/c and NC/Nga mice were cultured with the indicated concentrations of CpG ODN for 48 h and plused with [3 H]TdR for the last 12 h. [3 H]TdR incorporation was measured by a Matrix beta counter. Data are means ± S.D. of quadruplicate cultures of one representative experiment. (B) DCs were stimulated with CpG ODN for 48 h and analyzed for cell-surface expression of the indicated molecules by flow cytometry. Dashed and open histograms represent expression of MHC class II and CD80 molecules in DCs cultured with CpG ODN and without CpG ODN, respectively. (C) DCs were stimulated with 1 ␮M CpG ODN for 24 h. The concentrations of TNF-␣ in the supernatants were quantified by ELISA. The data are means ± S.D. for triplicate observations.

cells [10]. In human allergic diseases, a possible association of TLR9 polymorphism C-1237T with asthma has been reported, but the mechanism has not been elucidated [11]. There are few reports on TLR deficiency other than that in gene-knockout mice. Alexopoulou et al. found that most human subjects who received vaccination with the Borrelia burgdorferi outer-surface lipoprotein (OspA) developed substantial specific Ab responses but that some recipients did not. Macrophages from the low responder group produced less TNF-␣ and IL-6 upon OspA stimulation and had lower surface expression levels of TLR1

[12]. It is not clear how the defect in TLR9-mediated activation in NC/Nga mice contributes to the development of dermatitis or IgE production. It has been shown that development of the dermatitis is controlled by an autosomal recessive gene in NC/Nga mice [13]. Macrophages from NC/Nga mice kept in conventional conditions remained unresponsiveness to CpG ODN and showed a low level of TLR9 expression. Although we examined the relationship between TLR9-mediated TNF␣ production and the level of serum IgE or the development of dermatitis in (BALB/c × NC/Nga)F1 × NC/Nga backcross mice

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Fig. 3. Expressions of TLR9 on macrophages, B cells and DCs in NC/Nga mice. Expressions of TLR9 on macrophages, B cells and DCs were estimated by gating CD11b+ , B220+ and CD11c+ cells, respectively, as described in Section 2. Shaded and open histograms represent cells stained with isotype-matched mAb and anti-TLR9 mAb, respectively.

housed in conventional conditions, we could not find a significant relationship between these factors (T. Sakai, M. Kogiso, K. Mitsuya, S. Yamamoto, unpublished data). Further investigation of this issue is needed. In summary, in the course of investigating causes of allergy responses in NC/Nga mice, we found a low level of TLR9 expression and a defect in TLR9-mediated activation in NC/Nga mouse macrophages. Acknowledgment This research was supported by a grant from Uehara Memorial Foundation. References [1] Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nature Immunol 2001;2:675–80. [2] Takeda K, Akira S. Toll-like receptors in innate immunity. Int Immunol 2005;17:1–17. [3] Cooper KD. Atopic dermatitis: recent trends in pathogenesis and therapy. J Invest Dermatol 1994;102:128–37. [4] Matsuda H, Watanabe N, Geba G, Sperl J, Tsudzuki M, Hiroi J, et al. Development of atopic dermatitis-like skin lesion with IgE hyperproduction in NC/Nga mice. Int Immunol 1997;9:461–6. [5] Cook DN, Pisetsky DS, Schwartz DA. Toll-like receptors in the pathogenesis of human disease. Nat Immunol 2004;10:975–9.

[6] Vandenbulcke L, Bachert C, Van Cauwenberge P, Claeys S. The innate immune system and its role in allergic disorders. Int Arch Allergy Immunol 2005;139:159–65. [7] Gobda J, Matsumura T, Inoue J-I. TNFR-associated factor (TRAF) 6 is essential for MyD88-dependent pathway but not Toll/Il-1 receptor domain-containing adaptor-inducing IFN-␤ (TRIF)-dependent pathway in TLR signaling. J Immunol 2004;173:2913–7. [8] Gou Z, Garg S, Hill K, Jayashankar L, Mooney MR, Hoelscher M, et al. A distal regulatory region is required for constitutive and IFN-␤induced expression of murine TLR) gene. J Immunol 2005;175:7407– 18. [9] Christensen SR, Kashgarian M, Alexopoulou L, Flavell RA, Akira S, Shlomchik MJ. Toll-like receptor 9 controls anti-DNA autoantibody production in murine lupus. J Exp Med 2005;202:321–31. [10] Lian Z-X, Kikuchi K, Yang G-X, Ansari AA, Ikehara S, Gershwin ME. Expansion of bone marrow IFN-␣-producing dendritic cells in New Zealand Black (NZB) mice: high level expression of TLR9 and secretion of IFN-␣ in NZB bone marrow. J Immunol 2004;173:5283–9. [11] Lazarus R, Walter T, Klimecki WT, Raby BA, Vercelli D, Palmer LJ, et al. Single-nucleotide polymorphisms in the Toll-like receptor 9 gene (TLR9): frequencies, pairwise linkage disequilibrium, and haplotypes in three U.S. ethnic groups and exploratory case–control disease association studies. Genomics 2003;81:85–91. [12] Alexopoulou L, Thomas V, Schnare M, Lobet Y, Anguita J, Schoen RT, et al. Hyporesponsiveness to vaccination with Borrelia burgdorferi OspA in humans and in TLR1- and TLR2-deficient mice. Nat Med 2002;8:878–84. [13] Tsudzuki M, Watanabe N, Wada A, Nakane Y, Hiroi J, Mastuda H. Genetic analyses for dermatitis and IgE hyperproduction in the NC/Nga mouse. Immunogenetics 1997;47:88–90.