- Top of page
- Materials and methods
Background: In many patients with atopic dermatitis (AD), the disease is complicated by their enhanced susceptibility to bacterial skin infections, especially with Staphylococcus aureus. The pattern recognition receptor toll-like receptor (TLR)-2 recognizes components of S. aureus, for example, lipoteichoic acid (LTA) and peptidoglycan (PGN) and, therefore, might be crucial in the pathogenesis and flare-ups of AD.
Objective: To investigate TLR-2 expression and cytokine secretion in macrophages from patients with AD compared to healthy controls upon TLR-2 stimulation with PGN, LTA and Pam3Cys.
Methods: Macrophages were cultivated from highly purified peripheral blood monocytes of AD patients and nonatopic healthy controls and stimulated with PGN, LTA and Pam3Cys in a time and dose–dependent manner. Afterwards, TLR-2 expression and cytokine secretion were measured on protein and mRNA level. TLR-1 and TLR-6 expression were investigated on the mRNA level. Immunohistochemical stainings from punch biopsies were performed to investigate TLR-2 expression in skin macrophages.
Results: We could clearly show that macrophages from patients with AD expressed significantly less TLR-2, whereas the expression pattern of TLR-1 and TLR-6 were not altered. Macrophages had a reduced capacity to produce pro-inflammatory cytokines such as IL-6, IL-8 and IL-1β after stimulation with TLR-2 ligands.
Conclusion: Our findings clearly show an impaired TLR-2 expression and functional differences of TLR-2-mediated effects on macrophages of AD patients compared to healthy controls which might contribute to the enhanced susceptibility to skin infections with S. aureus in AD.
Atopic dermatitis (AD) is one of the most frequent chronic inflammatory skin diseases with an increasing prevalence affecting 10–20% children and 1–3% adults in industrial countries (1, 2).
One hallmark of AD is the striking susceptibility to colonization with Staphylococcus aureus: 80–100% of patients with AD are colonized with S. Aureus (3, 4). In contrast, S. aureus can be isolated from the skin of only 5–30% of healthy individuals, mainly from intertriginous areas (4). Moreover, a relationship between disease severity, extent and S. aureus colonization of lesional and nonlesional skin has been noted (3) which can be due to IgE-mediated hypersensitivity (5, 6) or production of exotoxins with superantigenic properties (7, 8). It was shown that disease severity can be improved with antistaphylococcal treatment (9).
Both the innate and the adaptive immune system participate in recognition and defence of microbial pathogens. More recently, the role of the innate immune system has gained large attention in the pathogenesis of AD (10, 11).
Toll-like receptors act as PRRs comprising a family of (currently) 13 receptors in humans with distinct recognition profiles. In this context, TLR-2 has emerged as a principle receptor in combating Gram-positive bacteria, especially S. aureus (12). TLR-2 forms heterodimers with TLR-1 and TLR-6 to interact with a rather broad spectrum of ligands. Studies using knockout mice identified TLR-1 as the coreceptor required for the recognition of triacylated lipoproteins and lipopeptides such as Pam3Cys (13), while diacylated components such as lipoteichoic acid (LTA), which is a component of the cell wall of S. aureus, interact with TLR-2/TLR-6 heterodimers (13, 14). Peptidoglycan (PGN) is a major constituent of the cell wall of Gram-positive bacteria which induces signal transduction via TLR-2, NOD 1 (Card4) and NOD 2 (Card15) receptors, respectively. NOD molecules including NOD1 and NOD2, are a family of intracellular pattern recognition proteins involved in bacterial detection (15, 16).
A recent study by Hasannejad et al. showed a general impairment of TLR-2-mediated proinflammatory cytokine production by monocytes from AD. In this study the authors investigated the effect of the synthetic TLR-1/TLR-2 ligand Pam3Cys on the production of TNFα and IL-1β (17).
In this study, we chose macrophages for our investigations as they are the relevant cells in the dermis which differentiate from blood-derived monocytes in situ. In AD, macrophages are known to accumulate in acutely and chronically inflamed skin (18). Besides phagocytosis and secretion of lytic enzymes, macrophages are known to be an important source of proinflammatory cytokines such as IL-1β, TNF-α, IL-6, IL-8 and IL-12 and express TLR-2. Yet it still remains unclear, whether TLR-2 has any direct functional effect on the pathogenesis and maintenance of AD. Therefore, we investigated TLR-2 expression and effects of TLR-2 stimulation with the staphylococcal cell wall components PGN and LTA and with the synthetic TLR-2 agonist Pam3Cys on cytokine secretion (IL-6, IL-8, IL-12, IL-1β and TNF-α) in patients with AD compared to healthy controls. Our data confirm a functional role of TLR-2 on macrophage function. We found a significantly impaired TLR-2 expression and TLR-2-mediated cytokine secretion (IL-6, IL-8 and IL-1β) in macrophages from patients with AD compared to healthy controls.
Thus our data indicate that a dysregulation of TLR-2 expression and in TLR-2-mediated effects in macrophages might be an explanation for the enhanced susceptibility to skin infections with S. aureus and might be essential in the linkage between innate and adaptive immunity in the pathogenesis of AD.
- Top of page
- Materials and methods
AD is a chronic inflammatory skin disease, which is frequently complicated by an enhanced susceptibility to bacterial skin infections, especially with S. aureus. This may be due to an impaired innate immune defence against these bacteria (10, 11).
In this study, we chose macrophages for our investigations as they are a well-established model for differentiated monocytes that immigrate into the skin. Besides phagocytosis and secretion of lytic enzymes, macrophages are known to be an important source of pro-inflammatory cytokines such as IL-1β, TNF-α, IL-6, IL-8 and IL-12 and express TLR-2. Yet it still remains unclear whether TLR-2 stimulation in macrophages has any direct functional effect on the pathogenesis and maintenance of AD.
In our study, macrophages from AD patients expressed significantly less TLR-2 compared to healthy controls on protein as well as on mRNA level. Since TLR-2 forms heterodimers with TLR-1 and TLR-6, we investigated the expression pattern of these receptors as well. We did not find significant differences in TLR-1 and TLR-6 expression in macrophages from AD patients compared to healthy controls. Moreover, we show for the first time a decreased TLR-2 expression in skin macrophages from the inflammatory dermal infiltrate of AD patients compared to healthy controls as detected by immunohistochemistry. Hassanejad et al. could not find any differences in TLR-2 expression between AD patients and healthy controls in monocytes (17). Monocyte differentiation into macrophages might influence TLR-2 expression. Consecutively, an impaired TLR-2 expression in skin macrophages of AD patients compared to healthy controls might be crucial for the enhanced susceptibility to bacterial skin infection with S. aureus in these patients. TLR-2 expression was stable and could not be influenced by stimulation with TLR-2 ligands such as PGN, LTA and Pam3Cys. In this context, Mrabet-Dahbi et al. (22) could show recently a decrease of TLR-2 expression in human activated T cells from patients with AD and healthy controls upon LTA stimulation.
GM-CSF is known to ‘maturate’ macrophages from monocytes. Therefore, we added this cytokine to our cultures. CM-CSF did not influence the membrane expression of TLR-2 on monocytes (data not shown). Since we used the cytokine in cultures with cells both from AD patients and from healthy controls, GM-SCF cannot be the cause of the differences in TLR-2 expression in our study.
GM-CSF has effects on other cells such as T-lymphocytes, basophils, eosinophils and dendritic cells and is present in the skin. Interestingly, keratinocytes from AD patients are known to produce more GM-CSF and increased GM-CSF levels have been documented in peripheral blood mononuclear cells of AD patients (23).
In functional experiments we investigated TLR-2-mediated effects on macrophages and found a general impairment of TLR-2 induced effects from patients with AD compared to healthy controls. Since there is no selective TLR-2 ligand known so far and significant differences in TLR-1 and TLR-6 expression in macrophages from AD patients and healthy controls had been excluded before, we chose to stimulate our cells with two natural staphylococcal components, namely the TLR-2/TLR-6 ligand LTA and the TLR-2/NOD ligand PGN in parallel with a synthetic TLR-2/TLR-1 ligand (Pam3Cys). Macrophages from AD patients produced significantly less IL-6, IL-8 and IL-1β compared to macrophages from healthy controls in a ligand dependent manner in response to natural TLR-2 ligands. This is partly in line with Hassanejad et al. (17) who found a selective impairment in IL-1β secretion in monocytes from patients with AD upon stimulation with Pam3Cys, a synthetic TLR-1/TLR-2 agonist. However, they did not compare the effects of the TLR-1/TLR-2 ligand to effects of the ‘natural’ ligands PGN and LTA both acting via TLR-2 and other co-receptors (NOD and TLR-6, respectively). In our experiments differences in IL-1β secretion could be induced significantly by PGN and in tendency by LTA. Here, no effect was observed upon Pam3Cys stimulation indicating a different pathway and co-receptors for macrophages compared to monocytes upon TLR-2 stimulation. However, we could not reproduce an impaired TNF-α secretion which might be explained again by differences in function between monocytes and macrophages.
Recently, an enhanced TLR-2 expression and induction of TNF-α and IL-8 secretion was observed in macrophages from the joints of patients with rheumatoid arthritis (RA) which supported the notion of a potential role of TLR-2 for the inflammation and joint destruction in RA (24). In another study, IL-6, TNF-α and IL-1β were induced upon stimulation with LTA and PGN from S. epidermidis in the murine macrophage-like cell line J774.2 (25). We could confirm these findings since TLR-2 stimulation in general led to an enhanced production of TNF-α, IL-6, IL-8 and IL-1β. However, secretion of IL-6, IL-8 and IL-1β was impaired in macrophages from AD patients compared to healthy controls.
Here we also investigated IL-6 which has previously been shown to be up-regulated in human monocytes upon stimulation with PGN and LTA (26) and has been shown to play an important role as pro-inflammatory cytokine in AD which is up-regulated in the skin of humans and in animal models of AD (27). An impaired IL-6 secretion in macrophages from AD patients upon stimulation with TLR-2 ligands in our AD patients might point to an impaired S. aureus processing and consecutively explain the enhanced susceptibility of bacterial skin infections with this germ.
In a murine model with bone marrow derived macrophages stimulation with Pam3Cys yielded to an enhanced IL-6, IL-8 and TNF-α secretion (28). However, the authors did not compare Pam3Cys effects with other TLR-2 ligands such as PGN and LTA which led to an augmentation of these cytokines in our patients regardless of the diagnosis AD.
IL-12 is an important pro-inflammatory and immunoregulatory cytokine mainly produced by monocytes/macrophages which polarizes and stimulates Th1 cells (29, 30). In a mouse model TLR-2 induced IL-12 has been shown to play a significant role in host defence (31). In AD IL-12 is considered to be a ‘key player’ during the chronification of eczema (32).
Here, we failed to detect an up-regulation of IL-12 upon TLR-2 stimulation as well as differences in IL-12 secretion in macrophages from AD patients and healthy controls indicating an important role for TLR-2 in the acute more than in the chronic phase of inflammation.
In conclusion, we show for the first time an impaired TLR-2 expression and functional differences of TLR-2-mediated effects on macrophages of AD patients compared to healthy controls. Therefore, macrophages from patients with AD have a reduced capacity to produce pro-inflammatory cytokines which might be an explanation for the enhanced susceptibility to skin infections with S. aureus. Our data support the emerging concept that AD patients have a dysbalance in innate as well as acquired immunity. Further investigations are necessary to elucidate the function of TLR-2 in patients with AD to explain the high susceptibility to staphylococcal infections and provide new therapeutic options for altering the dysbalance in innate as well as acquired immunity.