In this study, peptidoglycan (PEG) from Staphylococcus aureus-stimulated, but not muramyldipeptide (MDP)-stimulated, Langerhans cells (LCs) induced a dose-dependent Th2-prone immune response. However, when LCs were stimulated with PEG in combination with MDP, the strength of Th2 immune responses was synergistically augmented by MDP. Furthermore, it was found that production of IL-10, but not of IL-12 p40, by PEG-stimulated LCs was also enhanced in the presence of MDP. These results suggest that MDP enhances Th2 cell development through up-regulation of IL-10 production from PEG-stimulated LCs, increase the importance of S. aureus colonization in patients with atopic dermatitis.
chemokine (C-C motif) ligand
nuclear factor of κ light polypeptide gene enhancer in B cells
nucleotide-binding oligomerization domain
peripheral blood mononuclear cell
phorbol 12-myristate 13-acetate
Atopic dermatitis is a chronic inflammatory skin disease with immunopathologic features that vary depending on the duration of the lesions. Most AD patients have increased numbers of Th2 cells in their peripheral blood and acute skin lesions, and also superficial skin colonization by Staphylococcus aureus . S. aureus can be isolated from 96 to 100% of skin lesions of AD patients, whereas the skins of only 0–10% of healthy individuals are colonized by this organism [2, 3]. Previously, we reported detecting S. aureus more frequently in lesioned skin of AD patients than in non-lesioned skin . Furthermore, the bacterial cell count of S. aureus was significantly higher in lesioned skin of AD patients than in non-lesioned skin. Although many attempts have been made to characterize the role of S. aureus in the skin of AD patients, most studies have focused on the role of staphylococcal exotoxins . However, there is no significant difference between lesioned and non-lesioned skin in the rate of detection of S. aureus producing superantigenic exotoxin. Because half the S. aureus strains isolated from AD patients are not capable of producing superantigens [3, 5, 6]; the roles of these strains in the skin lesions of AD patients are not fully understood. Gram-positive bacterial cell walls are composed of highly cross-linked PEG decorated to a variable extent with teichoic acid polymers. The latter are also linked to plasma membrane phospholipids; this LTA is another major cell wall component [7, 8]. Gram-positive bacteria contain no LPS; LTA and/or PEG are thought to be the major inflammatory products in their cell walls. Therefore, we postulated that LTA and/or PEG from S. aureus may play a more important pathogenic role than superantigenic exotoxins in AD patients. In previous studies, we found that LTA and PEG can induce IL-5 production by PBMCs from patients with AD , and that intradermal injection of LTA induces localized AD-like inflammation associated with significantly more numerous Th2-type cells in the dermis of allergen-sensitized mice . Furthermore, we demonstrated that percutaneous invasion of PEG from the cell walls of gram-positive bacteria induces Th2 cell infiltration of the dermis . Our recent study demonstrated that PEG from S. aureus, known to be a ligand of TLR2 , can induce a systemic Th2-dominant immune response like that seen in AD patients . These findings suggest that development of Th2 cells is an important cause of AD and that S. aureus strains not producing toxins such as superantigen would also be capable of causing Th2-type inflammation in AD lesions. However, the role of MDP (N-acetylmuramyl-L-alanyl-D-isoglutamine), which has the minimal bioactive structure of PEG, in the Th2 immune response is not sufficiently clear. Therefore, in this study we investigated the influence of MDP on the PEG-induced Th2 immune response and the involvement of IL-10 and IL-12 secretion by LCs.
Peptidoglycan derived from S. aureus and MDP were obtained from Fluka (Buchs SG, Switzerland) and EMD Biosciences (La Jolla, CA, USA), respectively, and reconstituted in PBS, pH 7.4, at a concentration of 1 mg/mL. Specific-pathogen-free BALB/c (wild type) mice and DO 11.10 TCR Tg mice (OVA323–339-specific I-Ad-restricted TCR-transgenic mice) were obtained from Japan SLC (Hamamatsu, Japan) and the Jackson Laboratory (Bar Harbor, ME, USA), respectively, and used at the age of 6–8 weeks. They were housed in plastic cages with sterilized paper bedding in a clean, air-conditioned room at 24°C and allowed free access to a standard laboratory diet and water. All procedures performed on the mice were in accordance with the Guidelines of the Animal Care and Use Committee of Meiji Pharmaceutical University, Tokyo. Th2 adjuvant activity of PEG and/or MDP was detected according to the method of Whelan et al. , with modification. LCs were separated from the epidermis of BALB/c mice as previously described , adjusted to 1 × 105 cells/mL in RPMI 1640 medium with L-glutamine (Sigma, St. Louis, MO, USA) containing 10% FBS (Sigma), 25 mM HEPES (Sigma), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco RBL, Grand Island, NY, USA) (RPMI 10) and stimulated with 0.1–10 μg/mL PEG and/or MDP for 18 hr at 37°C in a humidified atmosphere with 5% CO2, and then washed two times with RPMI 10 before coculturing with naïve Th cells. Next, Th cells were separated from DO 11.10 TCR Tg mouse spleen cells using an EasySep Negative Selection Mouse CD4+ T Cell Enrichment Kit (StemCell Technologies, Vancouver, BC, Canada) and then treated with mouse anti-CD62L monoclonal antibody (clone lam1–116, IgG2a) (1 μg per 1 × 106 cells; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in RPMI 10 for 1 hr on ice. The Th cells that had been reacted with anti-CD62L antibody were then purified using a CELLection Pan Mouse IgG Kit (Invitrogen, Dynal AS, Oslo, Norway), and used as naïve Th cells. The naïve Th cells were cultured (5 × 105 cells/mL) with the above PEG- and/or MDP-stimulated LCs in the presence of 30 nM OVA peptide (323-ISQAVHAAHAEINEAGR-339; obtained from Operon Biotechnologies, Tokyo, Japan) for 5 days at 37°C. The cells were then stimulated with 50 ng/mL PMA (Sigma) and 500 ng/mL ionomycin (Sigma) for 24 hr at 37°C. The cell supernatants were finally removed and tested for production of IFN-γ and IL-4 using ELISA kits (R&D Systems, Minneapolis, MN, USA).
To confirm the involvement of IL-12 p40, IL-12 p70 and IL-10 production from LCs in the induction of Th2-dominant immune responses, LCs were adjusted to 5 × 105 cells/mL in RPMI 10 and incubated in the presence or absence of 0.1–10 μg/mL PEG and/or MDP at 37°C in a humidified atmosphere with 5% CO2. Furthermore, in some experiments, LCs were incubated in the presence of 5 μg/mL rat anti-mouse IL-10 neutralizing monoclonal antibody (clone JES5–16E3, IgG2a) (BioLegend, San Diego, CA, USA). Rat anti-keyhole limpet hemocyanin monoclonal antibody (clone B39–4, IgG2a) (BD Pharmingen, San Jose, CA, USA) was also used as an isotype-matched control antibody. The culture supernatants were collected after incubation for 48 hr, and IL-12 p40, IL-12 p70 and IL-10 concentrations were measured using ELISA kits for quantification of each murine cytokine (R&D Systems). The data were expressed as means (±SD), and differences between means were analyzed using Student's t-test with a two-tailed test of significance. Differences at P < 0.05 were considered statistically significant.
Langerhans cells were stimulated with PEG and/or MDP for 18 hr, and then incubated with naïve Th cells for 5 days in the presence of OVA peptide to induce Th1/Th2 development. Furthermore, the growth of Th cells was expanded by stimulation with PMA and ionomycin for 24 hr; production of both IFN-γ and IL-4 was confirmed by ELISA. The data in Figure 1 indicate that LCs stimulated with PEG induce dose-dependent Th2-prone immune responses, as shown by enhanced production of IL-4. However, LCs stimulated with MDP did not induce Th2-prone immune responses. These findings indicate that LCs stimulated with PEG, but not with MDP, induce development of Th2 type cells.
To examine whether Th2 development induced by PEG-stimulated LCs is influenced by the presence of MDP, LCs were simultaneously stimulated with 0.1–10 μg/mL PEG and MDP. As shown in Figure 2, PEG-stimulated LCs dose-dependently enhanced Th2-prone immune responses in combination with MDP, as represented by enhanced production of IL-4, but not of IFN-γ, by activated CD4+ lymphocytes. There is evidence that maturation of Th cell precursors into biased Th1 or Th2 populations is strongly influenced by cytokines in the environment . In particular, IL-12 appears to be the dominant cytokine driving the differentiation of Th1 lymphocytes in vitro and in vivo [16, 17]. Furthermore, some investigators have reported that IL-10 appears to be the dominant cytokine driving the differentiation of Th2 lymphocytes in vitro and in vivo [18, 19]. However, because the concentrations of IL-17 in the culture supernatants (none; 617 ± 93 pg/mL) were not influenced by stimulation with PEG and/or MDP, respectively (data not shown), Th2-prone immune response would not involve Th17 cells.
We previously demonstrated that the ability of PEG-stimulation of LCs to induce Th2-prone immune responses is mainly associated with strong IL-12 p40 production and weak IL-12 p70 production by LCs, and that IL-12 p70 production is down-regulated through IL-10 production from LCs via an autocrine mechanism [13, 20]. Therefore, LCs were simultaneously stimulated with 0.1–10 μg/mL PEG and MDP, and concentrations of IL-12 p40, IL-12 p70 and IL-10 in the culture supernatants were assayed. As shown in Figure 3, although IL-12 p40 and IL-12 p70 production from PEG-stimulated LCs was not influenced by the presence of MDP, their IL-10 production was significantly and dose-dependently enhanced in the presence of MDP. However, LCs stimulated with MDP alone produced insignificant amounts of IL-10 (data not shown). Furthermore, although IL-12 p70 production by PEG-stimulated LCs was augmented by neutralization of IL-10 activity with anti-IL-10 antibody, it was not influenced by stimulation with MDP.
Our previous studies showed that weak IL-12 p70 production by PEG-stimulated LCs plays a critical role in Th2 development, as does excessive production of IL-12 p40 homodimer protein, the latter working as an antagonist to IL-12 p70 to induce Th1 responses, and that insufficient IL-12 p70 production from LCs is associated with IL-10 production [13, 20]. Given that neutralization of IL-10 activity enhances production of IL-12 p70 by PEG-stimulated LCs and inhibits Th2 development in mice, it has been suggested that down-regulation of IL-12 p70 production through IL-10 production is involved in induction of Th2 development by PEG-stimulated LCs. These facts indicate that IL-10 production from LCs promotes the development of Th2 immune responses to antigens. In the present study, although PEG-stimulated LCs induced Th2 development, MDP-stimulated LCs did not. Given that Werts et al. have reported that MDP-stimulated murine macrophages induce CCL5  and that we have also confirmed that MDP induces CCL5 from LCs (data not shown), the MDP used in the present study is biologically active. Therefore, these results seem attributable to inability of MDP to induce IL-10 production by LCs. However, simultaneous stimulation of LCs with PEG and MDP induced stronger Th2 development than did stimulation with PEG alone. Stimulation with MDP did not influence the extent of IL-12 p40 and IL-12 p70 production induced by PEG-stimulation of LCs. However, IL-10 production from LCs was augmented by simultaneous stimulation with both PEG and MDP. Therefore, the synergistic increase in Th2 development induced by PEG and MDP is likely attributable to further production of IL-10 from LCs. It is well known that MDP is a NOD2 agonist ; therefore, TLR2 stimulation by PEG and NOD2 stimulation by MDP would exert a synergistic inductive effect on IL-10 production from LCs. However, because further IL-10 production from PEG-stimulated LCs in the presence of MDP is not associated with further down-regulation of IL-12 p70 and restoration of IL-12 p70 production from PEG-stimulated LCs by neutralization of IL-10 activity is also not influenced by stimulation with MDP, IL-12 p70-independent Th2 development must exist. The mechanisms behind these phenomena remain largely elusive.
On the other hand, although MDP binding to NOD2 reportedly activates the NF-κB pathway , IL-10 production is associated with the Janus Kinase pathway  but not with the NF-κB pathway . Therefore, MDP itself would not induce IL-10 production by LCs and subsequent Th2 development. Given that the density of S. aureus in skin lesions of AD patients exceeds 1 × 107 organisms/cm2, which is equivalent to 1.5–8 μg PEG [2, 25, 26], it seems that 1–10 μg/mL PEG and MDP for in vitro stimulation would be close to concentrations occurring in vivo. Therefore, sustained S. aureus colonization in AD patients could cause epidermal LCs to induce Th2-prone immune responses in the presence of PEG and MDP, which are common cell components of superantigenic exotoxin-producing and -non-producing S. aureus strains. Because the skin of most AD patients shows superficial S. aureus colonization and barrier disruption due to reduced levels of filaggrin , PEG and MDP would be expected to penetrate continuously into the skin. Therefore, anti-microbial treatment of a subgroup of AD patients, irrespective of whether they show clinical signs of superinfection, may be a useful new therapeutic strategy for AD.
The authors declare that they have no conflicts of interest.