Expression of toll-like receptors 2 and 4 in subjects with asthma by total serum IgE level

IgE plays a prominent role in the pathophysiology of allergic asthma. IgE binds to receptors on the surface of different types of immune effector cells causing them to release a variety of mediators that promote airway hyperresponsiveness, mucus secretion and increased vascular permeability [1]. Several strategies for decreasing IgE have been developed as a possible treatment for asthma [1]. Some studies have reported that the ligation of Toll-like receptors (TLRs) by bacterial or viral antigens can affect IgE-dependent mast cell degranulation and release of preformed mediators, as well as eicosanoid production, thus providing evidence on the involvement of innate immunity in the pathogenesis of asthma [2‐5]. Among the various agents involved in innate immunity in the pathogenesis of asthma, the TLRs may bear an important role [6‐10].

TLRs are a family of cell surface proteins involved in the recognition of pathogen-associated molecular patterns (PAMPs). Upon stimulation, TLRs can modulate subsequent adaptive immune responses. TLR2 and 4 are expressed on the cellular surface and migrate to phagosomes after activation on recognising the ligand. Just because these receptors are expressed on the cellular surface makes them easy to measure. Our laboratory has expertise in measuring them [6].

TLRs 2 and 4 function as signal receivers for gram-positive and negative bacteria (endotoxin recognition) [6‐8]. In some studies, a relationship between microbial products and regulation of the T helper (Th)1 and Th2 responses through TLRs 2 and 4 was observed in allergic diseases [11‐13]. Interestingly, TLRs 2 and 4 can modulate the Th response according to the bacterial burden, where a high lipopolysaccharide (LPS) level induces a Th1 response and a low LPS level promotes a Th2 response [9, 14, 15]. Furthemore, there is evidence that exposure to endotoxin during early life [16, 17] may be protective against the development of atopy and asthma, an argument in support of the hygiene hypothesis, although such association remains poorly understood [17].

Previous studies addressing the role of TLRs 2 and 4 in asthmatic patients had several limitations. No studies approached a differential analysis of TLRs 2 and 4 in blood versus induced sputum. Most of the studies were limited to blood cells (frequently monocytes) [18, 19], in experimental asthma models or in cell cultures [9, 13]. The few studies conducted in humans were done on diseases other than asthma, such as COPD, bronchiectasis, and cystic fibrosis [19‐23]. The induced sputum technique is a noninvasive procedure that provides direct information on cells and mediators involved in airway inflammation. Thus, we expected induced sputum analysis to be particularly suited to study the role of the innate immune response in the pathogenesis of asthma. The main objective of the present study was to assess the expression of TLRs 2 and 4 in monocytes/macrophages and neutrophils, both from peripheral blood and induced sputum, in a group of adult asthmatic patients stratified by their level of total serum IgE.

The study was approved by the Institutional Review Board and registered at ClinicalTrials.gov with identifier NCT02028637.

The main outcome of the present study was that the percentage of macrophages expressing TLR4 in induced sputum was higher in asthmatic patients with a high total serum IgE than in asthmatics with normal total serum IgE. Furthermore, we observed a correlation (but weak) between total serum IgE and the percentage of macrophages expressing TLR4 in induced sputum. This result is consistent with a possible link between innate immunity and the adaptive immune response in the pathogenesis of IgE-mediated asthma. The role of TLRs in the inflammatory process has been the subject of recent investigations. However, most of the studies have evaluated cellular responses in vitro or systemic reactions, and few studies focused on the role of TLRs in lung inflammation. In the present study, we analyzed the cellular expression of TLRs 2 and 4 on concurrent induced sputum and peripheral blood samples in adult asthmatics, and the data were stratified according to the type of asthma in terms of high versus normal level of total serum IgE. The study was limited to an observational assessment of TLR expression, yet the work was performed on a large sample of clinically well characterized patients, where all data were collected together at a single time point.

TLRs are a family of proteins responsible for the recognition of PAMPs, which include repetitive microbial molecular domains such as lipopolysaccharides, flagellin, mannose and nucleic acids from viruses and bacteria. TLRs 2 and 4 are expressed on the cell surface and translocate to phagosomes after activation, upon binding their ligands. TLR4 activation leads to a series of events including bronchoconstriction, expression of adhesion molecules by vascular endothelial cells and the release of cytokines. Such actions result in neutrophil recruitment and activation of pulmonary dendritic cells and macrophages [36], as well as a Th2 polarization of subsequent adaptive immune responses [37]. Previous studies showed that, in genetically susceptible subjects, allergen exposure along with low PAMP doses favors allergic responses [38]. In contrast, high-dose PAMP exposure, as it occurs in livestock farms, rural environments in developing countries and traditional lifestyles, leads to antigen tolerance [39]. Such protective “farm effect” has been attributed to an immunomodulatory role of the exposure to LPS and other TLR ligands during early childhood, which results in inhibiting the development of allergic immune responses [9, 40‐42]. Some recent studies [6] have led to interesting expectations by observing that in neutrophilic asthma there is an increase in the expression TLR2, TLR4, CD14 and surfactant protein-A (SP-A), and that the activation of TLR, by an allergen for example, generates a cascade of signals driven by the activation and nuclear translocation of NF-kB, that results in a cytokine-mediated inflammatory response. The discovery of TLR and their actions provides an immunological basis for the study of the hygiene hypothesis. In this study, we measure the TLR2 and 4 for several reasons: 1) because they are receptors expressed on the cell surface and they are easy to measure 2) because in our laboratory we have more experience in the measurement of receptors expressed on the cell surface and 3) because these receptors are associated in the pathogenesis of asthma, specifically with the hygiene hypothesis and the respiratory infections [16, 17]. In this study, none of the patients included had a respiratory infection within the previous month, nor suffered from bronchiectasis.

There are findings showing that bacterial and viral infections can modify the course of allergic diseases by affecting high-affinity IgE receptor (FcεRI)-dependent mast cell activation. Such studies reported that the ligation of TLRs by bacterial or viral antigens can affect IgE-dependent mast cell degranulation and preformed mediator release, as well as eicosanoid production [2]. Furthermore, it has been reported that a synergistic interaction between TLR ligands and allergens can also modify cytokine synthesis by mast cells stimulated via their high-affinity IgE receptor, FcεRI [3‐5]. Our present findings showing higher numbers of induced sputum macrophages expressing TLR4 in asthmatic subjects with high total serum IgE, along with a correlation (weak) between the percentage of TLR4⁺ macrophages and the total serum IgE level, are consistent with the idea of a relationship between respiratory exposure to PAMPs and asthma with increased IgE, and suggest an involvement of the macrophage in such relationship.

Macrophages are key cells in the pulmonary innate immune responses, since they are the most abundant leucocyte in the air spaces and one of the first cells to encounter inhaled proteins that may act as allergens. Depending on the signals received, macrophages can be pro- or anti-inflammatory. Macrophage stimulation can result from a variety of stimuli including TLR engagement in the presence of IL-10 or other cytokines [37]. Our analysis of TLR 2 and 4 expression by different inflammatory cells types in induced sputum and blood showed a higher expression of these receptors in monocytes/macrophages than in neutrophils, both in sputum and peripheral blood, which may reflect the relevance of the macrophage in the pulmonary innate immune responses. Our findings are also consistent with previous studies showing that TLR4 stimulation can induce further recruitment of macrophages to the airways [37].

The role of TLR2 in airway allergic inflammation is not fully clear. In our work we did not find any significant relationship between TLR2 expression and total serum IgE. However, TLR2 stimulation by its ligands was reported to potentiate Th2 responses and exacerbate airway hyperresponsiveness [43, 44]. The relationship between TLR2 activation and allergic responses is complex and may depend on the antigen nature and dose, the timing, and the TLR associated ligands [45]. Variability in the techniques employed, and the cell populations and specimens analyzed, may also account for limitations in the comparability among different studies.

Our study has some limitations: (i) the effect of inhaled corticosteroids on expression of TLRs in induced sputum is unknown; (ii) we only analyze receptors that are expressed on the cell surface (TLR4 and 2); (iii) it is a descriptive study. Our results were significant but, should be studied with a higher population size.

In summary, this work is the first evaluation of the expression of TLRs by blood and induced sputum leukocytes in subjects with asthma. The main finding was that asthmatic patients with a high total serum IgE have a higher percentage of macrophages expressing TLR4 in induced sputum, compared with patients with normal total serum IgE values. This outcome supports the possibility of therapeutic approaches for some forms of immune-mediated lung disease through stimulating or blocking TLRs with agonists or antagonists [46], and may be exploited in the future for therapeutic target discovery.