The mannose receptor negatively modulates the Toll-like receptor 4-aryl hydrocarbon receptor-indoleamine 2,3-dioxygenase axis in dendritic cells affecting T helper cell polarization.

BACKGROUND: Dendritic cells (DCs) are key players in the induction and re-elicitation of TH2 responses to allergens. We have previously shown that different C-type lectin receptors on DCs play a major role in allergen recognition and uptake. In particular, mannose receptor (MR), through modulation of Toll-like receptor (TLR) 4 signaling, can regulate indoleamine 2,3-dioxygenase (IDO) activity, favoring TH2 responses. Interestingly, the aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor with an emerging role in immune modulation, has been implicated in IDO activation in response to TLR stimulation. OBJECTIVE: Here we investigated how allergens and lectins modulate the TLR4-AhR-IDO axis in human monocyte-derived DCs. METHODS: Using a combination of genomics, proteomics, and immunologic studies, we investigated the role of MR and AhR in IDO regulation and its effect on T helper cell differentiation. RESULTS: We have demonstrated that LPS induces both IDO isoforms (IDO1 and IDO2) in DCs, with partial involvement of AhR. Additionally, we found that, like mannan, different airborne allergens can effectively downregulate TLR4-induced IDO1 and IDO2 expression, most likely through binding to the MR. Mannose-based ligands were also able to downregulate IL-12p70 production by DCs, affecting T helper cell polarization. Interestingly, AhR and some components of the noncanonical nuclear factor κB pathway were shown to be downregulated after MR engagement, which could explain the regulatory effects of MR on IDO expression. CONCLUSION: Our work demonstrates a key role for MR in the modulation of the TLR4-AhR-IDO axis, which has a significant effect on DC behavior and the development of immune responses against allergens.

The role of lectins in allergic sensitization and allergic disease.

Allergic diseases are a global public health issue affecting millions of persons around the world. However, full understanding of the molecular basis of this group of chronic inflammatory disorders remains rather elusive. Recently, the role of carbohydrates on allergens and their counterstructures on antigen-presenting cells (lectins) have been highlighted as crucial factors in allergen sensitization, which culminates in TH2 cell differentiation and the production of deleterious specific IgE antibodies. Here we review recent progress on the role of different lectins in patients with type I hypersensitivity or allergy, their interplay with other determinants of allergenicity, and ways of developing therapeutic modalities against newly identified targets.

Retagging identifies dendritic cell-specific intercellular adhesion molecule-3 (ICAM3)-grabbing non-integrin (DC-SIGN) protein as a novel receptor for a major allergen from house dust mite.

Dendritic cells (DCs) have been shown to play a key role in the initiation and maintenance of immune responses to microbial pathogens as well as to allergens, but the exact mechanisms of their involvement in allergic responses and Th2 cell differentiation have remained elusive. Using retagging, we identified DC-SIGN as a novel receptor involved in the initial recognition and uptake of the major house dust mite and dog allergens Der p 1 and Can f 1, respectively. To confirm this, we used gene silencing to specifically inhibit DC-SIGN expression by DCs followed by allergen uptake studies. Binding and uptake of Der p 1 and Can f 1 allergens was assessed by ELISA and flow cytometry. Intriguingly, our data showed that silencing DC-SIGN on DCs promotes a Th2 phenotype in DC/T cell co-cultures. These findings should lead to better understanding of the molecular basis of allergen-induced Th2 cell polarization and in doing so paves the way for the rational design of novel intervention strategies by targeting allergen receptors on innate immune cells or their carbohydrate counterstructures on allergens.

An investigation into IgE-facilitated allergen recognition and presentation by human dendritic cells.

BACKGROUND: Allergen recognition by dendritic cells (DCs) is a key event in the allergic cascade leading to production of IgE antibodies. C-type lectins, such as the mannose receptor and DC-SIGN, were recently shown to play an important role in the uptake of the house dust mite glycoallergen Der p 1 by DCs. In addition to mannose receptor (MR) and DC-SIGN the high and low affinity IgE receptors, namely FcεRI and FcεRII (CD23), respectively, have been shown to be involved in allergen uptake and presentation by DCs. OBJECTIVES: This study aims at understanding the extent to which IgE- and IgG-facilitated Der p 1 uptake by DCs influence T cell polarisation and in particular potential bias in favour of Th2. We have addressed this issue by using two chimaeric monoclonal antibodies produced in our laboratory and directed against a previously defined epitope on Der p 1, namely human IgE 2C7 and IgG1 2C7. RESULTS: Flow cytometry was used to establish the expression patterns of IgE (FcεRI and FcεRII) and IgG (FcγRI) receptors in relation to MR on DCs. The impact of FcεRI, FcεRII, FcγRI and mannose receptor mediated allergen uptake on Th1/Th2 cell differentiation was investigated using DC/T cell co-culture experiments. Myeloid DCs showed high levels of FcεRI and FcγRI expression, but low levels of CD23 and MR, and this has therefore enabled us to assess the role of IgE and IgG-facilitated allergen presentation in T cell polarisation with minimal interference by CD23 and MR. Our data demonstrate that DCs that have taken up Der p 1 via surface IgE support a Th2 response. However, no such effect was demonstrable via surface IgG. CONCLUSIONS: IgE bound to its high affinity receptor plays an important role in Der p 1 uptake and processing by peripheral blood DCs and in Th2 polarisation of T cells.

Recognition of the major cat allergen Fel d 1 through the cysteine-rich domain of the mannose receptor determines its allergenicity.

Dendritic cells are professional antigen-presenting cells that are specialized in antigen uptake and presentation. Allergy to cat has increased substantially in recent years and has been shown to be positively associated with asthma. We have recently shown that the mannose receptor (MR), a C-type lectin expressed by dendritic cells, recognizes various glycoallergens from diverse sources and is involved in promoting allergic responses to a major house dust mite allergen in vitro. Here we investigated the potential role of MR in allergic responses to Fel d 1, a major cat allergen. Fel d 1 binding to MR was confirmed by ELISA. Using blocking, gene silencing (siRNA) experiments, and MR knock-out (MR(-/-)) cells, we have demonstrated that MR plays a major role in internalization of Fel d 1 by human and mouse antigen-presenting cells. Intriguingly, unlike other glycoallergens, recognition of Fel d 1 by MR is mediated by the cysteine-rich domain, which correlates with the presence of sulfated carbohydrates in natural Fel d 1. WT and MR(-/-) mice were used to study the role of MR in allergic sensitization to Fel d 1 in vivo. MR(-/-) mice sensitized with cat dander extract and Fel d 1 produced significantly lower levels of total IgE, Fel d 1-specific-IgE and IgG1, the hallmarks of allergic response, compared with WT mice. Our data show for the first time that Fel d 1 is a novel ligand of the cysteine-rich domain of MR and that MR is likely to play a pivotal role in allergic sensitization to airborne allergens in vivo.

The mannose receptor mediates the uptake of diverse native allergens by dendritic cells and determines allergen-induced T cell polarization through modulation of IDO activity.

The mannose receptor (MR) is a C-type lectin expressed by dendritic cells (DCs). We have investigated the ability of MR to recognize glycosylated allergens. Using a gene silencing strategy, we have specifically inhibited the expression of MR on human monocyte-derived DCs. We show that MR mediates internalization of diverse allergens from mite (Der p 1 and Der p 2), dog (Can f 1), cockroach (Bla g 2), and peanut (Ara h 1) through their carbohydrate moieties. All of these allergens bind to the C-type lectin-like carbohydrate recognition domains 4-7 of MR. We have also assessed the contribution of MR to T cell polarization after allergen exposure. We show that silencing MR expression on monocyte-derived DCs reverses the Th2 cell polarization bias, driven by Der p 1 allergen exposure, through upregulation of IDO activity. In conclusion, our work demonstrates a major role for MR in glycoallergen recognition and in the development of Th2 responses.

The detection of ADAM8 protein on cells of the human immune system and the demonstration of its expression on peripheral blood B cells, dendritic cells and monocyte subsets.

A disintegrin and metalloprotease (ADAM) proteins have wide ranging functions, including proteolytic cleavage of cell surface molecules, cell fusion, cell adhesion and intracellular signalling. Recent evidence suggests the involvement of ADAM8 in allergic responses. For instance, ADAM8 is amongst a number of genes up-regulated in experimentally induced asthma in animals. In order to further define the involvement of ADAM8 in allergic responses, we sought in the first instance to examine its distribution on human peripheral blood B cells, resting and activated T cells, monocyte subsets and monocyte derived dendritic cells. Here we demonstrate for the first time ADAM8 protein expression on B cells and dendritic cells, and its higher expression on CD14(2+)CD16(-) monocytes compared to CD14(+)CD16(+) cells. Immature dendritic cells expressed low levels of ADAM8 when treated with a combination of GM-CSF and IL-4, but stimulation with LPS resulted in a higher level of expression, which was TLR-4 independent. Up-regulation of ADAM8 expression on dendritic cells was also observed after stimulation with TNF-alpha, but not after stimulation with anti-CD40. The demonstration of ADAM8 expression on these cells provides an opportunity for addressing the potential role of inhaled protease allergens, such as Der p 1, in modulating ADAM8 functions, particularly with regards to innate immune responses by dendritic cells and IgE synthesis by B cells.

The role of indoleamine 2,3-dioxygenase-aryl hydrocarbon receptor pathway in the TLR4-induced tolerogenic phenotype in human DCs.

A controlled inflammatory response is required for protection against infection, but persistent inflammation causes tissue damage. Dendritic cells (DCs) have a unique capacity to promote both inflammatory and anti-inflammatory processes. One key mechanism involved in DC-mediated immunosuppression is the expression of tryptophan-metabolizing enzyme indoleamine 2,3-dioxygenase (IDO). IDO has been implicated in diverse processes in health and disease but its role in endotoxin tolerance in human DCs is still controversial. Here we investigated the role of IDO in shaping DCs phenotype and function under endotoxin tolerance conditions. Our data show that TLR4 ligation in LPS-primed DCs, induced higher levels of both IDO isoforms together with the transcription factor aryl-hydrocarbon receptor (AhR), compared to unprimed controls. Additionally, LPS conditioning induced an anti-inflammatory phenotype in DCs - with an increase in IL-10 and higher expression of programmed death ligand (PD-L)1 and PD-L2 - which were partially dependent on IDO. Furthermore, we demonstrated that the AhR-IDO pathway was responsible for the preferential activation of non-canonical NF-κB pathway in LPS-conditioned DCs. These data provide new insight into the mechanisms of the TLR4-induced tolerogenic phenotype in human DCs, which can help the better understanding of processes involved in induction and resolution of chronic inflammation and tolerance.

Activity profile of dust mite allergen extract using substrate libraries and functional proteomic microarrays.

Enzymatic activity in the fecal droppings from the house dust mite has been postulated to contribute to the elicited allergic response. Screening dust mite extracts through 137,180 tetrapeptide fluorogenic substrates allowed for the characterization of proteolytic substrate specificity from the potential cysteine and serine proteases in the extract. The extract was further screened against a 4000 member peptide nucleic acid (PNA) encoded inhibitor library designed to target cysteine proteases using microarray detection. Affinity chromatography coupled with mass spectrometry identified Der p 1 as one of the proteases targeted by the PNA inhibitors in the dust mite lysate. A phenotypic readout of Der p 1 function in allergy progression was demonstrated by the inhibition of CD25 cleavage from T cells by dust mite extract that had been treated with the Der p 1 inhibitor identified from the PNA-encoded inhibitor library.

The glycosylation pattern of common allergens: the recognition and uptake of Der p 1 by epithelial and dendritic cells is carbohydrate dependent.

Allergens are initiators of both innate and adaptive immune responses. They are recognised at the site of entry by epithelial and dendritic cells (DCs), both of which activate innate inflammatory circuits that can collectively induce Th2 immune responses. In an attempt to have a better understanding of the role of carbohydrates in the recognition and uptake of allergens by the innate immune system, we defined common glycosylation patterns in major allergens. This was done using labelled lectins and showed that allergens like Der p 1 (Dermatophagoides pteronyssinus group 1), Fel d 1 (Felis domisticus), Ara h 1 (Arachis hypogaea), Der p 2 (Dermatophagoides pteronyssinus group 2), Bla g 2 (Blattella germanica) and Can f 1 (Canis familiaris) are glycosylated and that the main dominant sugars on these allergens are 1-2, 1-3 and 1-6 mannose. These observations are in line with recent reports implicating the mannose receptor (MR) in allergen recognition and uptake by DCs and suggesting a major link between glycosylation and allergen recognition. We then looked at TSLP (Thymic Stromal Lymphopoietin) cytokine secretion by lung epithelia upon encountering natural Der p 1 allergen. TSLP is suggested to drive DC maturation in support of allergic hypersensitivity reactions. Our data showed an increase in TSLP secretion by lung epithelia upon stimulation with natural Der p 1 which was carbohydrate dependent. The deglycosylated preparation of Der p 1 exhibited minimal uptake by DCs compared to the natural and hyperglycosylated recombinant counterparts, with the latter being taken up more readily than the other preparations. Collectively, our data indicate that carbohydrate moieties on allergens play a vital role in their recognition by innate immune cells, implicating them in downstream deleterious Th2 cell activation and IgE production.

Laminin and fibronectin treatment leads to generation of dendritic cells with superior endocytic capacity.

BACKGROUND: Sampling the microenvironment at sites of microbial exposure by dendritic cells (DC) and their subsequent interaction with T cells in the paracortical area of lymph nodes are key events for initiating immune responses. Most of our knowledge of such events in human is based on in vitro studies performed in the absence of extracellular matrix (ECM) proteins. ECM in basement membranes and interstitial spaces of different tissues, including lymphoid organs, plays an important role in controlling specific cellular functions such as migration, intracellular signalling and differentiation. The aim of this study was, therefore, to investigate the impact of two abundant ECM components, fibronectin and laminin, on the phenotypical and functional properties of DC and how that might influence DC induced T-cell differentiation. METHODOLOGY/PRINCIPAL FINDINGS: Human monocyte derived DC were treated with laminin and fibronectin for up to 48 hours and their morphology and phenotype was analyzed using scanning electron microscopy, flow cytometry and real time PCR. The endocytic ability of DC was determined using flow cytometry. Furthermore, co-culture of DC and T cells were established and T cell proliferation and cytokine profile was measured using H(3)-thymidine incorporation and ELISA respectively. Finally, we assessed formation of DC-T cell conjugates using different cell trackers and flow cytometry. Our data show that in the presence of ECM, DC maintain a 'more immature' phenotype and express higher levels of key endocytic receptors, and as a result become significantly better endocytic cells, but still fully able to mature in response to stimulation as evidenced by their superior ability to induce antigen-specific T cell differentiation. CONCLUSION: These studies underline the importance of including ECM components in in vitro studies investigating DC biology and DC-T cell interaction. Within the context of antigen specific DC induced T cell proliferation, inclusion of ECM proteins could lead to development of more sensitive assays.

Allergen-driven suppression of thiol production by human dendritic cells and the effect of thiols on T cell function.

Dendritic cells are a major source of extracellular thiols needed for T cell activation, a process in which CD40-mediated stimulation plays a pivotal role. The Dermatophagoides pteronyssinus group 1 mite allergen (Der p 1) has previously been shown to cleave CD40 from the surface of human dendritic cells, thereby suggesting that Der p 1 might compromise the ability of these cells to sustain thiol production during T cell activation. This has therefore prompted us to examine the effect of the mite protease allergen Der p 1 on thiol production by human dendritic cells. Monocyte-derived dendritic cells were treated with either proteolytically active or inactive Der p 1 and then stimulated through CD40 for extracellular thiol detection. The effect of thiol (N-acetyl-l-cysteine) and thiol inhibitors on naïve T cell responses, including CD25 and FOXP3 expressions, cell proliferation and cytokine production, was determined. Here, we show that Der p 1-mediated cleavage of CD40 from the surface of dendritic cells suppresses the ability of these cells to produce extracellular thiols, and that reducing thiols are needed for the generation of the T helper type 1 (Th1), T cytotoxic type 1 (Tc1) and T regulatory (Treg) cell phenotypes. We conclude that Der p 1-driven suppression of thiol production by dendritic cells may disrupt Th1/Tc1 and Treg cell development, and in doing so could lead to Th2/Tc2 cell responses and allergy.

The molecular basis of allergenicity.

Allergens are mostly innocuous antigens that elicit powerful T helper cell type 2 (Th2) responses leading to hyper-immunoglobulin E (IgE) production and allergy. Research carried out over several years has highlighted the possible role of the inherent protease activity, surface features and glycosylation patterns of allergens in the engagement of a Th2 signalling pathway. It is thought that allergens possess common features and patterns that enable them to be recognized by innate immune defences as Th2-inducing antigens. These events are further amplified by proteolytically active allergens through digestion of cell surface molecules involved in regulating innate and adaptive immune functions, favouring Th2 responses. A greater understanding of the molecular features that make proteins allergenic will help define new therapeutic targets aimed at blocking allergen recognition and protease activity.

Major house dust mite allergens Dermatophagoides pteronyssinus 1 and Dermatophagoides farinae 1 degrade and inactivate lung surfactant proteins A and D.

Lung surfactant proteins (SP) A and D are calcium-dependent carbohydrate-binding proteins. In addition to playing multiple roles in innate immune defense such as bacterial aggregation and modulation of leukocyte function, SP-A and SP-D have also been implicated in the allergic response. They interact with a wide range of inhaled allergens, competing with their binding to cell-sequestered IgE resulting in inhibition of mast cell degranulation, and exogenous administration of SP-A and SP-D diminishes allergic hypersensitivity in vivo. House dust mite allergens are a major cause of allergic asthma in the western world, and here we confirm the interaction of SP-A and SP-D with two major mite allergens, Dermatophagoides pteronyssinus 1 and Dermatophagoides farinae 1, and show that the cysteine protease activity of these allergens results in the degradation of SP-A and SP-D under physiological conditions, with multiple sites of cleavage. A recombinant fragment of SP-D that is effective in diminishing allergic hypersensitivity in mouse models of dust mite allergy was more susceptible to degradation than the native full-length protein. Degradation was enhanced in the absence of calcium, with different sites of cleavage, indicating that the calcium associated with SP-A and SP-D influences accessibility to the allergens. Degradation of SP-A and SP-D was associated with diminished binding to carbohydrates and to D. pteronyssinus 1 itself and diminished capacity to agglutinate bacteria. Thus, the degradation and consequent inactivation of SP-A and SP-D may be a novel mechanism to account for the potent allergenicity of these common dust mite allergens.

Analysis of proteomic profiles and functional properties of human peripheral blood myeloid dendritic cells, monocyte-derived dendritic cells and the dendritic cell-like KG-1 cells reveals distinct characteristics.

BACKGROUND: Dendritic cells (DCs) are specialized antigen presenting cells that play a pivotal role in bridging innate and adaptive immune responses. Given the scarcity of peripheral blood myeloid dendritic cells (mDCs) investigators have used different model systems for studying DC biology. Monocyte-derived dendritic cells (moDCs) and KG-1 cells are routinely used as mDC models, but a thorough comparison of these cells has not yet been carried out, particularly in relation to their proteomes. We therefore sought to run a comparative study of the proteomes and functional properties of these cells. RESULTS: Despite general similarities between mDCs and the model systems, moDCs and KG-1 cells, our findings identified some significant differences in the proteomes of these cells, and the findings were confirmed by ELISA detection of a selection of proteins. This was particularly noticeable with proteins involved in cell growth and maintenance (for example, fibrinogen gamma chain (FGG) and ubiquinol cytochrome c) and cell-cell interaction and integrity (for example, fascin and actin). We then examined the surface phenotype, cytokine profile, endocytic and T-cell-activation ability of these cells in support of the proteomic data, and obtained confirmatory evidence for differences in the maturation status and functional attributes between mDCs and the two DC models. CONCLUSION: We have identified important proteomic and functional differences between mDCs and two DC model systems. These differences could have major functional implications, particularly in relation to DC-T cell interactions, the so-called immunological synapse, and, therefore, need to be considered when interpreting data obtained from model DC systems.

An attempt to define allergen-specific molecular surface features: a bioinformatic approach.

Allergens are proteins that elicit T helper lymphocyte type 2 (Th2) responses culminating in IgE antibody production and allergic disease. However, we have no answer to the fundamental question of why certain proteins are allergens, while others are not. We hypothesized that analysis of the surface of diverse allergens may reveal common structural features which might enable them to be recognized as Th2-inducing antigens by cells of the innate immune system. We have therefore used the ConSurf server to search for allergen-specific motifs. This has enabled us to identify residue conservation patterns in the homologues of Ara t 8 (plant profilin), Act c 1 (actinidin), Bet v 1 (plant pathogenesis-related protein) and Ves v 5 (venom allergen). The results demonstrate the presence of allergen-specific patches consisting of an unusually high proportion of surface-exposed hydrophobic residues. The patches that have been identified may represent molecular patterns recognizable by cells of the innate immune system.