Microbial contamination of grass pollens could affect sensitization, subsequent allergic response, and efficacy of allergen-specific immunotherapy. We investigated whether bacterial immunomodulatory substances can direct PBMC responses of allergic and nonatopic subjects against ryegrass pollen (RGP) toward Th1, Th2, or regulatory T (Treg) cells. Aqueous extracts of RGP with high or low LPS were fractionated into large and small molecular weight (MW) components by diafiltration. CFSE-labeled PBMCs from allergic and nonatopic subjects were stimulated with RGP extracts (RGPEs) and analyzed for cytokine secretion and T-cell responses. High LPS RGPE increased IFN-γ+ Th1 and IL-4+ Th2 effector cell induction and consistently decreased CD4+Foxp3hi Treg-cell induction. IL-10-producing T-cell frequency was unaltered, but IL-10 secretion was increased by high LPS RGPE. RGPE-stimulation of TLR-transfected cell lines revealed that high LPS pollen also contained a TLR2-ligand, and both batches a TLR9-ligand. Beta-1,3-glucans were detected in large and small MW fractions and were also T-cell stimulatory. In conclusion, coexposure to allergen and proinflammatory microbial stimuli does not convert an established Th2- into a Th1-response. Instead, proinflammatory responses are exacerbated and Foxp3hi Treg-cell induction is decreased. These findings show that adjuvants for specific immunotherapy should enhance Treg cells rather than target immune deviation from Th2 to Th1.
A combination of genetic predisposition and environmental factors determines the development of immune-mediated inflammatory diseases including allergy and asthma [1-3]. An important environmental factor is exposure to microbial compounds such as lipopolysaccharide (LPS), which can modulate both innate and adaptive immune responses. This is mediated by pattern recognition receptors (PRRs) on antigen-presenting cells (APCs) such as dendritic cells (DCs) that specifically recognize pathogen- or endogenous danger-associated molecular patterns (PAMPs or DAMPs). In the T helper (Th) cell response to antigen, APCs not only present antigen-derived peptides but also, via PRR detected signals, direct the type of T-cell response to be inflammatory (Th1, Th2, Th17) or tolerogenic (Tr1, anergy) (reviewed in [4, 5]). Several allergens have been shown to carry PAMPs or directly stimulate PRRs. For example, house dust mite faeces contain not only allergenic proteins but also bacterial DNA, endotoxin and chitin [6-8]. The peanut allergen Ara h 1 is a ligand for the carbohydrate receptor DC-SIGN that facilitates Ara h 1 uptake by DCs . Birch pollen-derived substances, such as phytoprostanes and adenosine, can bias immune responses toward Th2 via modulation of DCs [10, 11].
The role of exposure to microbes in allergic sensitization has been studied extensively in the context of the hygiene hypothesis, which states that high exposure to particular microbes protects against development of Th2-based disease. In murine studies, low levels of LPS enhanced Th2-type responses to inhaled antigens and high LPS doses favored a Th1 response . The incidence of allergic sensitization is inversely correlated with level of exposure to LPS in children with a particular single nucleotide polymorphism in the CD14 promoter region . Conversely, LPS found on pollen grains due to Gram-negative bacterial contamination has been suggested to facilitate sensitization to pollen allergens as well as aggravate allergic responses in already sensitized individuals .
Microbial contaminants in allergen extracts are also likely to affect efficacy of allergen-specific immunotherapy (SIT), potentially in different ways. SIT is the only curative treatment for allergy and involves subcutaneous injection of increasing doses of allergen extract (SCIT) or, more recently, sublingual administration of allergen extracts (SLIT). SIT is most successful in the treatment of bee venom and pollen allergies, but is also useful in the treatment of house dust mite and domestic pet allergies. Since allergy is characterized by a Th2-biased immune response, therapeutic approaches include the use of adjuvants to promote immune deviation to a Th1-polarized response . The mechanisms of SIT, however, are still not fully understood. Trivedi et al.  showed that pollen extracts used in SIT have highly variable LPS content and suggested that the influence of LPS contamination of allergen extracts on SIT efficacy and adverse reactions needs to be examined. Environmental LPS exposure of individuals with allergic airway diseases, including asthma, is known to exacerbate the disease . Therefore, such “accidental” coadministration of LPS with pollen allergen extracts during SIT may not be beneficial. Clarifying the role of LPS and other microbial contaminants in SIT efficacy is hampered by the high batch-to-batch variation in allergen content as well as contaminating substances .
Previous studies on the role of microbial stimuli in SIT have focused largely on shifting the Th2 bias to Th1. A deficiency in immune regulation, however, is now recognized as a major underlying mechanism of allergic responses and therapeutic approaches for allergy are now targeting induction of tolerance, in particular via antigen-specific regulatory T (Treg) cells . We recently demonstrated that allergen-mediated induction of Treg cells was impaired in allergic compared with nonatopic subjects , and in a clinical study of SLIT in patients with house dust mite allergy, we showed that in the active group, improved symptoms were associated with increased numbers of Treg cells .
In the present study, we performed a detailed investigation of the immunomodulatory effects of microbial contamination of allergen extracts on human PBMC responses to ryegrass pollen (RGP) allergens, separating small and large molecular weight (MW) components of the pollen extracts. The presence of beta-1,3-glucans and particular TLR ligands in the pollen fractions was determined and effector and Treg-cell responses of allergic and nonatopic individuals to aqueous extracts from RGP with high or low LPS content were compared.
RGP extracts have highly variable LPS content, which is not removed by diafiltration
Three different RGP batches were extracted (1:20 w/v) and extracts tested for endotoxin content by the manufacturer Greer Laboratories with results ranging from 50 to 5000 EU/mL (Supporting Information Fig. 1A). We chose the high and low LPS batches (A and C) for our further studies and prepared aqueous extracts. We then fractionated these extracts by diafiltration using a 10 kDa cut-off that separated proteins and LPS from small molecule components (Supporting Information Fig. 1B) similar to dialysis. Diafiltration is faster than dialysis and thus decreases the risk of allergen protein degradation. Furthermore, small MW components remained concentrated in the filtrate, which allowed their further analysis in comparison with the large MW fractions.
Protein and allergen content of RGPE fractions
Protein content of RGPEs was analyzed by two different protein assays, Bradford and BCA. While these assays gave similar results for the diafiltrated large MW fractions, there was a large discrepancy between the two methods when testing the small MW fractions and unfractionated total RGPE (Supporting Information Fig. 1C). The BCA protein assay detected a relatively high signal in the small MW fraction despite minimal amounts of protein detected by silver staining after SDS-PAGE (Supporting Information Fig. 2A) and a very low result in the Bradford assay. The BCA assay also gave a higher signal in the unfractionated extract than the Bradford assay. This indicates that nonprotein small MW components cause a signal in the BCA, but not the Bradford assay. Therefore, the Bradford assay was used to quantify proteins in the total unfractionated extract and the large MW fraction, while the BCA assay “protein concentration” readout was used to standardize the small MW fractions.
Allergens were identified using monoclonal antibodies against the major RGP allergens Lol p 1 and Lol p 5 as well as serum from RGP allergic subjects. Similar protein and allergen profiles were observed for high and low LPS RGPEs (Supporting Information Fig. 2). The majority of proteins were retained in the large MW fractions (Supporting Information Fig. 2A). Nonatopic donor sera had negligible IgE binding to small MW components and the allergen-specific monoclonal antibodies detected no Lol p 1 and Lol p 5 (Supporting Information Fig. 2C). Sera from RGP-allergic donors had IgE-reactivity to small MW fractions of both low (Supporting Information Fig. 2C) and high LPS RGPE (data not shown).
LPS content affects T-cell proliferative activity, but not mitogenicity of RGPEs
The protein concentration of RGPE for suboptimal stimulation of T-cell proliferation was determined by 3H-thymidine incorporation assay (Supporting Information Fig. 3A). Extracts from high and low LPS RGP differed in their capacity to stimulate proliferation of PBMCs from allergic donors. At high protein concentrations, both the total extract and the large MW fraction of the high LPS RGPE had a decreased capacity to stimulate T-cell proliferation, compared with the low LPS extracts at higher concentrations, suggesting a component that is toxic or reduces proliferation. Despite the very low actual protein concentration, the small MW extract fractions were able to induce PBMC proliferation, albeit at low levels.
Extracts were further characterized for toxic or inhibitory properties and mitogenicity. Proliferation of a latex allergen Hev b 5-specific T-cell line in response to IL-2 was measured in the presence or absence of RGPE. Decreased proliferation was observed in the presence of the high LPS RGPE, and to a lesser extent with the low LPS extracts (Supporting Information Fig. 3B). The effect was greater for both small MW fractions compared with the large MW fractions, confirming that dialysis or diafiltration is an important step for removing potentially toxic or immune modulatory components in preparation of pollen extracts for SIT (Supporting Information Fig. 3B). None of the extracts induced nonspecific T-cell proliferation in a latex allergen Hev b 5-specific T-cell line (Supporting Information Fig. 3C) showing that the RGPEs do not contain mitogens.
LPS enhances proinflammatory cytokine production as well as IL-10 secretion
Cytokine secretion was determined by a 12-cytokine multiplex assay of cell culture supernatants after 48 h based on a time-course experiment (Fig. 1A). High LPS RGPE increased proinflammatory and effector Th1 and Th2 cytokine levels in PBMCs from allergic (IFN-γ (p = 0.0039), TNF-α (p = 0.0039), IL-5 (p = 0.0156), IL-13 (p = 0.0156)) and nonatopic (IFN-γ (p = 0.0156), TNF-α (0.0039), IL-13 (p = 0.0313)) donors as well as IL-10 (allergic: p = 0.0039, nonallergic: p = 0.0156) when compared with low LPS RGPE (Fig. 1C). IL-12p70 was only above the detection limit in five subjects if they were stimulated with high LPS RGPE or low LPS RGPE with added LPS, but not in the absence of LPS. Only very low levels of IL-4 were observed and no RGPE fraction induced IL-17. LPS alone induced IFN-γ and TNF-α as well as IL-10 (Fig. 1B and D). Addition of LPS to low LPS RGPE fractions increased secretion of these cytokines in allergic (IFN-γ, p = 0.0039, TNF-α, p = 0.0039, IL-10, p = 0.0078) and nonatopic (IFN-γ, p = 0.0223, TNF-α, p = 0.0156, IL-10, p = 0.0313) donors, indicating they were induced by LPS itself. Consistently, blocking LPS with an antagonist decreased secretion of IFN-γ, TNF-α, and IL-10 (allergic: IFN-γ, p = 0.0039, TNF-α, p = 0.0039, IL-10, p = 0.0039, and nonatopic: IFN-γ, p = 0.0156, TNF-α, p = 0.0156, IL-10, p = 0.0156) in response to high LPS RGPE (Fig. 1D).
Both high and low LPS RGPEs induced higher Th2 cytokine secretion in allergic subjects compared with that of nonatopic controls (IL-5, p = 0.0120, IL-13, p = 0.0166 for combined high and low LPS RGPEs) (Fig. 1C). High LPS RGPE induced more IL-13 secretion than low LPS RGPE. Addition of LPS increased IL-13 secretion above the detection limit in PBMCs of two nonatopic subjects that did not secrete IL-13 in response to low LPS RGPE large MW fraction alone (Fig. 1D). Blocking LPS in the high LPS RGPE large MW fraction had no effect on IL-13 secretion indicating that it is induced by components other than LPS.
The small MW fractions of both low and high LPS RGPEs induced similar cytokine levels. Interestingly, the low LPS RGPE small MW fraction induced higher IL-10 secretion than the large MW fraction (p = 0.0010) (Fig. 1C). This indicates that dialysis may remove small MW components that could have a tolerogenic effect, in particular when contamination with Gram-negative bacteria is low.
LPS increases induction of both IFN-γ and IL-4 producing effector T cells
Consistent with the increased cytokine secretion, induction of Th1 and Th2 effector cells (dividing CD4+IFN-γ+ and IL-4+ T cells) was increased in response to high LPS RGPE (Fig. 2A–C). There was a trend for increased IL-4+CD4+ Th2 effector cell induction in allergic subjects with both RGPEs compared with nonatopic subjects (Fig. 2B). This indicates that the Th2-biased response in allergic subjects could not be overcome by natural adjuvants present in the high LPS RGPE. Interestingly, the induction of IFN-γ+ CD4+ T cells in response to high LPS RGPE was significantly higher in nonatopic than in allergic subjects (Fig. 2B). The ratio of IL-4+/IFN-γ+ dividing CD4+ T cells was not significantly different between high and low LPS extracts (Fig. 2C).
Differential induction of IL-10 in Foxp3hi and Foxp3int dividing CD4+ T cells by LPS
A subset of Treg cells characterized by IL-10 secretion (Tr1) has been described . Tr1 have been shown to regulate the adverse Th2 response in allergic disease . Induction of dividing IL-10+CD4+ T cells was not significantly different between high and low LPS RGPE (Fig. 2D and E). When CD4+ T cells, however, were subdivided based on Foxp3 expression, opposite effects of LPS on Foxp3hi and Foxp3int CD4+ T cells became apparent. There was a trend for fewer Foxp3hi but more Foxp3int cells to secrete IL-10 in response to high compared with low LPS RGPE (Fig. 2F) in both groups. These differences were statistically significant (Foxp3hi cells: p = 0.006; Foxp3int cells: p = 0.05) when all subjects (allergic and nonatopic) were compared.
Induction of CD4+Foxp3hi Treg cells
Recently, we described a subset of CD4+ Treg cells induced in response to RGPE stimulation. These cells are proliferating, but in contrast to T-helper cells they express very low levels of inflammatory cytokine and suppressed CD4+ T-cell proliferation and IFN-γ production. Instead, these cells are characterized by higher and more stable levels of Foxp3 expression than observed on activated Th cells . In the present study, the frequency of these induced dividing CD4+Foxp3hi Treg cells was decreased for allergic compared with nonatopic subjects (Fig. 3B) while CD4+ T-cell proliferation in response to RGPE was generally higher for allergic than nonatopic subjects (Fig. 3B). This is consistent with our previous finding that RGPE (low LPS) stimulation induces both effector T cells and Treg cells, with a reduced Treg-cell frequency in allergic compared to nonatopic subjects . Fewer Treg cells were induced with high LPS RGPE compared with low LPS in both allergic and nonatopic subjects (Fig. 3C). It is important to note, that the difference in Treg-cell induction with large MW RGPE fractions between the two subject groups (allergic and nonatopic) was only significant with low LPS RGPE, but lost when using the high LPS RGPE. Blocking LPS with its antagonist increased Treg-cell induction in some donors while adding LPS to the low LPS RGPE decreased Treg-cell induction in most donors (Fig. 3D). LPS blocking or addition had limited effects in mimicking low and high LPS RGPEs indicating that RGPE components other than LPS contribute to the differences in Treg-cell induction.
Effects of non-LPS pollen components
Overall, high LPS RGPE increased proinflammatory and effector Th1 and Th2 responses as well as IL-10 and reduced induction of Foxp3hi Treg cells. Low LPS RGPE with added LPS did resemble some high LPS RGPE effects such as high TNF-α and IL-10 secretion and low Foxp3hi Treg-cell induction. Addition of high dose LPS or antagonising LPS, however, had no effect on IL-13 secretion and no clear effect on CD4+ T-cell proliferation, indicating a role for other pollen extract components causing the differences associated with varying levels of microbial contamination. Further testing revealed the presence of beta-1,3-glucans in the extracts (Fig. 4A) that can be of plant or fungal origin. Levels of beta-1,3-glucans were higher in the high LPS extract and in the large rather than the small MW fractions.
All fractions could activate the innate immune system to some extent as assessed by activation of the NF-κB-dependent ELAM promoter linked to luciferase reporter in mouse RAW264.7 macrophages (Fig. 4B). The low LPS RGPEs only activated weakly at the highest doses, while the high LPS RGPE fractions potently induced NF-κB activation. Using TLR-transfected cell lines, the presence of a TLR4-ligand in the high LPS RGPE was confirmed (Fig. 4C). The high LPS RGPE fractions also activated TLR2- and to a lesser extent TLR9-transfected cells, consistent with contamination with other bacterial components such as bacterial lipopeptides and DNA (Fig. 4D). Low LPS RGPEs only activated TLR9 at low levels (Fig. 4E). None of the RGPE preparations were recognized by TLR3-transfected cells, indicating a lack of viral nucleic acid contamination (Fig. 4D and E).
After the initial discovery of the association of the allergic diathesis with a Th2-biased T-cell response, therapeutic approaches focused on shifting the T-cell response from Th2- to Th1-polarized using Th1-inducing adjuvants. Bacterial extracts were used by themselves in the absence of the allergen in the 1970s and 1980s with little success . Later, the less toxic LPS derivative, monophosphoryl lipid A (MPL) was tested as an adjuvant in SCIT and SLIT in combination with chemically modified rather than native allergen extract. Treatment was observed to improve rhinoconjunctivitis and asthma symptom scores, decrease allergen-specific IgE levels and increase allergen-specific IgG compared with untreated controls . However, it is not clear whether this treatment has any advantage over traditional SIT.
Grass and tree pollens have been found to be contaminated with bacteria and fungi, which introduce various known and unknown adjuvants including LPS. It is, however, not clear how this might affect the use of allergen extracts from pollen in allergen-specific immunotherapy. Although small MW components are removed from SIT reagents by dialysis, this fails to remove LPS and other large MW microbial products. In this study, we compared RGPE with low and high LPS content and found that bacterial products such as LPS augment existing T-cell responses, both Th1 and Th2. Therefore, this bacterial contamination is unlikely to be responsible for immune deviation of Th2 responses of allergic subjects to Th1 during immunotherapy. Moreover, the induction of Foxp3hi Treg cells that could suppress the unwanted Th2 response is not only decreased in allergic subjects , but also further suppressed by high LPS. These CD4+Foxp3hi Treg cells are distinct from activated CD4 T cells, that only transiently upregulate Foxp3 .
We detected no IL-17 secretion in response to RGPE fractions and have previously found that the frequency of IL-17+ Th cells induced by RGP is low compared with IL-4+ and IFN-γ+ Th cells . Th17 cells have been shown to drive chronic inflammatory disease states such as severe asthma, while our experiments are focusing on the initial antigen-specific responses [25, 26].
In our study, LPS increased the frequency of dividing IL-10+ Foxp3int CD4+ T cells consistent with Tr1 Treg cells. However, a population of IL-10 secreting activated IFN-γ+ Th1 effector cells has been described and shown to be important in defence against infection with the malaria parasite Toxoplasmodium gondi . Costaining of IL-10 and IFN-γ would be required to clarify the presence of this T-cell subset. Both dividing and nondividing IL-10+ CD4 T cells were very rare in our RGPE-stimulated PBMC cultures. Therefore, secreted IL-10, as detected by multiplex assay in this study, is likely to be mostly derived from non-T cells. Nevertheless, IL-10+ CD4 T cells could have important effects locally on other T cells.
LPS has been shown to increase allergen-specific T-cell proliferation when present in relatively small concentrations of up to 500 EU/mL [25, 26]. The high LPS RGPE used in this study contained about 10 times more LPS and the decreased proliferation that it induced may be due to toxic effects of such high dose LPS or the presence of other toxic components. The reduced proliferation was not due to increased Treg-cell induction. To the contrary, frequency of CD4+Foxp3hi Treg cells in dividing CD4+ T cells was reduced by high LPS RGPE fractions. Therefore, an important mechanism that normally controls both Th1- and Th2-type inflammation is impaired. Similarly, LPS was found to increase IL-4 as well as IFN-γ production and block Treg-cell development in vivo in mice . We also detected TLR2 ligands in the high LPS RGPE and TLR2 stimulation has been shown to favor Th2 responses by reducing IFN-γ and IL-12p70 production by dendritic cells . Interestingly, the receptor for beta-1,3-glucans (which we detected in our extracts), dectin-1, has been shown to synergize with TLR-2 and TLR-4 pathways for TNF-α production by human monocytes and macrophages . Thus bacterial contamination may contribute to the exacerbation of proinflammatory and effector Th1 and Th2 responses consistent with our study, which further emphasizes that bacterial contamination of allergen preparations for SIT is not beneficial.
Both low and high LPS RGPEs stimulated TLR9-transfected cells pointing to the presence of CpG motifs from bacterial DNA. In the case of the low LPS RGPE, the CpG DNA may be derived from contaminating Gram-positive bacteria. Both Gram-negative and Gram-positive bacteria, as well as fungi have been identified on pollen . Genetic variations of TLRs and TLR pathway genes associated with asthma and atopy have been identified, in particular for TLR2, for which we detected a ligand in the high LPS RGPE, and also for TLR9 . CpG immunostimulatory sequences (CpG ISS) are another adjuvant that has been utilized for promoting Th1 responses. When CpG ISS was packaged into virus-like particles and administered together with house dust mite allergen extract, asthma, and rhinitis symptom scores improved and allergen-specific IgG increased . There was also a transient increase in allergen-specific IgE consistent with our in vitro findings that LPS, also a Th1 adjuvant, exacerbated both Th1 and Th2 responses in allergic subjects.
The small MW fractions of RGPEs also induced T-cell proliferation and cytokine secretion, particularly in allergic subjects. This indicates that they contain T-cell activating factors, probably allergen-derived peptides comprising T-cell epitopes as well as immune modulating substances other than LPS. In addition to bacterial and fungal contaminants, pollen-derived substances, such as phytoprostanes and adenosine, can bias immune responses toward Th2 via modulation of dendritic cells [31, 32]. While small MW substances are removed from pollen extracts for immunotherapy by dialysis, they may be encountered during natural exposure to pollen by inhalation and could affect the type of immune response toward the pollen. The small MW fractions did not contain LPS, but would be expected to contain other smaller microbial substances. Their induced T-cell proliferation was generally low, consistent with the decreased amount of antigen and hence there was only a small difference between the two batches of pollen (high and low LPS) in terms of cytokine secretion and Th differentiation. We also observed a nonspecific reduction of T-cell proliferation when using high concentrations of both small MW fractions. Whether this was due to inhibition of proliferation or to cell death could be delineated in future studies.
In conclusion, coexposure to allergen and proinflammatory microbial stimuli does not further increase the Th1-bias in nonatopic or convert an established Th2- into a Th1-response in allergic individuals. Instead, both Th1- and Th2-biased proinflammatory responses are exacerbated in association with decreased induction of Foxp3hi Treg cells. These data provide important insight into the adjuvant properties of pollen microbial contaminants and will aid development of suitable adjuvants for SIT that enhance Treg cells rather than promote immune deviation from Th2 to Th1.
Materials and methods
Twenty-six RGP-allergic donors (mean age 31.1 ± 8.9 years, 17/26 male) and 12 nonatopic controls (mean age 42.1 ± 15.9 years, 6/12 male) were recruited at the Alfred Hospital Allergy Clinic. A diagnosis of allergy to RGP was based on a clinical history of seasonal rhinitis and evidence of specific sensitization by positive skin prick test to RGP and/or RGP-specific IgE CAP-FEIA score (Phadia, Uppsala, Sweden) greater than 0.70 kU/L (Class 2). Blood samples were taken outside the RGP season that in South East Australia runs from late September to early January. No donor was suffering from symptoms of allergic rhinitis or asthma at the time of blood sampling. All donors gave written informed consent with study approval by The Alfred Hospital and Monash University Ethics Committees.
RGP extracts, diafiltration
Batches of RGP were purchased from Greer Laboratories Inc. (Lenoir, NC) as dry, nondefatted pollen. For RGP extraction, 4 g of pollen were suspended in 40 mL PBS for 30 min at 37°C under constant rotation, followed by centrifugation at 5000 × g at room temperature. Supernatants were sterile filtered through a 0.2-μm pore size filter (Sartorius, Goettingen, Germany). RGP extracts (RGPEs) were separated into large and small MW fractions by diafiltration using Vivaspin 20 (Sartorius) with a 10 kDa cut-off membrane following the manufacturer's instructions. Extracts were stored at –20°C as allergens were stable under these conditions as determined by IgE-immunoblotting, while IgE reactivity was decreased after storage at 4°C for 3 weeks (data not shown).
Endotoxin and (1,3)-beta-D-glucan assay
The endotoxin content of the RGPEs was determined using the Pyrochrome Chromogenic 120 Test Kit (Associates of Cape Cod, E. Falmouth, MA) and (1,3)-beta-D-glucan was measured using the Glucatell kit (Associates of Cape Cod) as per the manufacturer's instructions.
Protein content of RGPE fractions was determined by both the Bradford assay using the Coomassie Plus kit (Pierce, Rockford, IL) and the Bicinchoninic acid (BCA) assay using the BCA protein assay kit (Pierce). Assays were performed as directed by the manufacturer's instructions.
SDS-PAGE, silver staining, and immunoblotting
RGPEs were incubated with reducing sample buffer for 5 min at 100°C and proteins separated by SDS-PAGE as described previously . Proteins were stained in the gel using the LC6100 SilverXpress Silver Staining Kit (Invitrogen, Carlsbad, CA) as per the manufacturer's instructions. Sera were tested for RGP-specific IgE by immunoblotting as described previously . In brief, proteins were transferred from the gels onto nitrocellulose membrane and the membrane blocked with 0.3% Tween20/PBS followed by overnight incubation with serum (1:10 dilution). Antigen-bound IgE was detected by incubation with a rabbit-anti-human IgE monoclonal antibody (1:500 dilution; Dako, Glostrup, Denmark), followed by an HRP-conjugated anti-rabbit IgG antibody (1:1000 dilution; Promega, Madison, WI) and 4-chloro-1-naphthol substrate (Sigma, St Louis, MO). Lol p 1 and Lol p 5 were detected by overnight incubation with specific monoclonal antibodies (A1 and A7, 1:10 dilution), and incubation with sheep-anti-mouse Ig-HRP (1:1000) followed by 4-chloro-1-naphthol substrate.
PBMCs were isolated from heparinized blood using Ficoll Paque (Pharmacia, Uppsala, Sweden) density gradient centrifugation. Cells from allergic and nonatopic donors used to compare induction of Foxp3hi cells were cryopreserved and thawed as described by Maecker et al. . The cells were frozen in 10% DMSO in fetal calf serum at 107 cells/mL and stored in liquid nitrogen for subsequent parallel testing. The viable cell recovery on thawing was 70 to 80%.
CFSE labeling of PBMCs was performed at a concentration of 1 μM CFSE and 107 cells/mL in PBS. Cells were stimulated with RGPEs at 5 μg/mL for total RGPE, 20 μg/mL for large MW RGPE fractions and 100 μg/mL for the small MW RGPE fractions unless indicated otherwise. Cells were cultured in complete medium (RPMI supplemented with 2 mM L-glutamine, 100 IU/mL penicillin-streptomycin (Invitrogen, Carlsbad, CA) and 5% screened, heat-inactivated human AB serum (Sigma)) in 24-well tissue culture plates (Greiner Labortechnik, Frickenhausen, Germany) with 2.5 × 106 PBMCs per well in 2 mL volume at 37°C in 5% CO2 in air in a Heraeus incubator (Heraeus, S. Plainfield, NJ) for 7 days. In some experiments, LPS (Sigma) or a LPS antagonist (Invivogen, San Diego, CA) was added as indicated.
Levels of IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17, IFN-γ, TNF-α, and GM-CSF in cell culture supernatants sampled at 48 h were determined simultaneously by Bioplex assay (BioRad, Hercules, CA) following the manufacturer's instructions. Samples for a standard curve were measured following the manufacturer's instructions in parallel with the test samples. Only values within the detection limits as given by the manufacturer were used for data analysis.
For labeling of surface markers, cells were incubated with specific fluorochrome conjugated antibodies to CD4, CD25, and CD127 (BD Biosciences, San Diego, CA) for 20 min at 4°C. For Foxp3 staining, surface marker stained cells were fixed, permeabilized, and stained for intranuclear Foxp3 using the Foxp3 staining kit (eBioscience, San Diego, CA) according to the manufacturer's instructions. For intracellular cytokine staining, 7-day cultured PBMCs were restimulated with phorbol myristate acetate (PMA) and ionomycin (Sigma) or with immobilized anti-CD3 monoclonal antibody (OKT-3, in-house, purified from cell culture supernatant, coated at 4 μg/mL) and 2 μg/mL soluble anti-CD28 (BD Biosciences) for 5 h (IL-4, IFN-γ) in the presence of 10 μg/mL Brefeldin A (Sigma) for the last 4 h. Staining with antibodies specific for IL-4 and IFN-γ (BD Biosciences) was performed simultaneously with staining for Foxp3. Staining for IL-10 secreting cells was performed after 16 h restimulation with anti-CD3/anti-CD28 using the cytokine secretion assay (CSA) kit (Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the manufacturer's instructions. Labeled cells were analyzed by flow cytometry on a FACS Calibur (BD Biosciences) or FACS Aria (BD Biosciences).
RAW264.7 mouse macrophage and TLR-transfected cell lines and stimulation assays
RAW264.7 mouse macrophages stably expressing the κB-dependent ELAM-luciferase reporter were generated as described  and maintained in 10% FCS/RPMI and 5% CO2. RAW-ELAM cells were seeded at 4 × 104 cells/well in 96-well format 24 h prior to stimulation with indicated ligands for a further 6 h. Cells were lysed with Passive Lysis Buffer (Promega) and assessed for luminescence using a FLUOstar Optima plate reader (BMG, Ortenberg, Germany). Results are represented as fold stimulation as compared with nonstimulated control.
HEK293 cells stably expressing individual human TLRs as indicated (kind gift of Eicke Latz and Douglas Golenbock, UMASS) were maintained in 10% FCS/DMEM in 5% CO2. Luciferase reporter assays were performed as previously described [35, 36] using the 5× κB-luciferase reporter construct (Stratagene, La Jolla, CA) concomitantly with Renilla luciferase-thymidine kinase encoding plasmid (pRL-TK) (Promega) to normalize for transfection efficiency. Transfected cells were lysed using Passive Lysis Buffer (Promega) and assayed for luciferase and Renilla activity using luciferase assay reagent (Promega) and coleonterazine substrate, respectively, and assessed using a FLUOstar Optima plate reader. Luminescence readings were corrected for Renilla activity and expressed as fold increase over nonstimulated control values.
Statistical analysis was performed using GraphPad Prism software v5 (San Diego, CA). Statistical significance was assessed by either Mann–Whitney test (nonparametric) for independent values, or two-tailed paired Students t-test for paired values. A p value <0.05 was considered significant.
We thank Lorraine Baxter for collection of blood samples, Geza Paukovics for flow cytometry advice, Kathryn Hjerrild and Pin Wang for technical assistance with the luciferase reporter assays, and Arnd Petersen for helpful discussion. We gratefully acknowledge funding for this research from the National Health and Medical Research Council, the Co-operative Research Centre for Asthma and Airways, the Victorian Government Infrastructure Support Program and the Alfred Research Trust, Australia.
Conflict of interest
The authors declare no financial or commercial conflict of interest.