Mechanisms underlying induction of allergic sensitization by Pru p 3

Abstract

Background: Recently, the nature of the lipid-ligand of Pru p 3, one of the most common plant food allergens in southern Europe, has been identified as a derivative of the alkaloid camptothecin bound to phytosphingosine. However, the origin of its immunological activity is still unknown.

Objective: We sought to evaluate the role of the Pru p 3 lipid-ligand in the immunogenic activity of Pru p 3.

Methods: In vitro cultures of different cell types (monocyte-derived dendritic cells [moDCs], PBMCs [peripheral blood mononuclear cells] and epithelial and iNKT-hybridoma cell lines) have been used to determine the immunological capacity of the ligand, by measuring cell proliferation, maturation markers and cytokine production. To study the capacity of the lipid-ligand to promote sensitization to Pru p 3 in vivo, a mouse model of anaphylaxis to peach has been produced and changes in the humoral and basophil responses have been analysed.

Results: The lipid-ligand of Pru p 3 induced maturation of moDCsc and proliferation of PBMCs. Its immunological activity resided in the phytosphingosine tail of the ligand. The adjuvant activity of the ligand was also confirmed in vivo, where the complex of Pru p 3-ligand induced higher levels of IgE than Pru p 3 alone. The immunological capacity of the Pru p 3 ligand was mediated by CD1d, as maturation of moDCs was inhibited by anti-CD1d antibodies and Pru p 3-ligand co-localized with CD1d on epithelial cells. Finally, Pru p 3-ligand presented by CD1d was able to interact with iNKTs.

Conclusions and clinical relevance: The Pru p 3 lipid-ligand could act as an adjuvant to promote sensitization to Pru p 3, through its recognition by CD1d receptors. This intrinsic adjuvant activity of the accompanying lipid cargo could be a general essential feature of the mechanism underlying the phenomenon of allergenicity.

Keywords: iNKT; CD1d; Pru p 3; lipid transfer proteins; peach allergy.

Figure 1. Characterization of immunogenic activity of the lipid-ligand of Pru p 3

(a) Structural formula of the lipid-ligand of Pru p 3 (10-hydroxy-camptothecin linked to phytosphingosine) (b) Proliferation assays of human PBMCs (n=6) stained with CSFE were incubated with Pru p 3 and Complex (Pru p 3; 10 µg/ml and the lipid-ligand 1 µg/ml). After 5 days, the proliferation of PBMCs was measured by loss of CSFE staining. Stimulation index (SI) was calculated as (% CSFE^neg-PBMCs + antigen)/(% CSFE^neg-PBMCs – antigen). Means and SE (bars) are shown. *p < 0.05. (c) Cytokine produced by PMBCs by Bioplex system. The results are expressed as the fold change between the amount (pg/mL) produced in the presence of the stimulus (Pru p 3, Complex), and the amount without stimulus. Means and SE (bars) are shown. *p < 0.001. (d) Phenotype of human monocyte-derived DCs (moDC) induced by Pru p 3 and its lipid-ligand. Changes in the expression of costimulatory molecules (CD80 and CD86) in monocyte-derived DCs from healthy donors, after stimulation with Pru p 3 alone (Pru p 3; 10 µg/mL), with the lipid-ligand (Complex; 10 µg/ml), and with the lipid-ligand only (Ligand; 1 µg/mL). Maturation indices of CD80 and CD86 expression are shown (p<0.05). (e, f) NF-κB/AP-1 activation using the transfected cell line THP1-XBlue™. Cells (1×10⁶ cells/ml) were incubated with Pru p 3 (Pru p 3; 5 µg/ml), Pru p 3 with lipid-ligand (Complex; 5 µg/ml) and only lipid-ligand (Ligand; 1 µg/ml) in (e), or with the lipid-ligand (Ligand), phytosphingosine (Phytosphingosine) and campthotecin (CPT) in (f). In both, LPS was used as positive control. Activation of NF-κB/AP-1 was revealed with QUANTI-Blue™. *p < 0.05 **p < 0.01 ***p<0.001.

Figure 2. Impact of the lipid-ligand of Pru p 3 on peach allergy

(a) Mice were left naïve or topically exposed to 100 ug of Pru p 3 alone (Pru p 3 label) or Pru p 3 together with 10 ug of its lipid-ligand (Complex) once per week for six weeks. After sensitization, intraperitoneal challenge was performed with Pru p 3. (b) Antigen-specific IgE and IgG were measured prior to allergen challenge. Data are mean ± SEM. (c) Pru p 3-induced basophil activation was measured by up-regulation of CD200R. Change in MFI (median fluorescence intensity) was calculated with respect to the stimulation with media alone. Data are mean ± SEM. (d) Mice were challenged with increasing doses of Pru p 3 by intraperitoneal challenge. Body temperature prior to the challenge and 30 minutes after each ip injection are shown. n = 5 mice per group. *p < 0.05, **p < 0.01.

Figure 3

(a) Inhibitory effect in the activated phenotype (positive CD80 and/or CD86) of moDCs, after stimulation with Pru p 3 (Pru p 3; 10 µg/mL) or Pru p 3 with its lipid-ligand (Complex; 10 µg/ml) and specific antibodies against TLR2, TLR4 and CD1d. (*p < 0.05). (b–i)) Immunolocalization of Pru p 3 (Red) or Complex (Red), and CD1d surface proteins (Green) in polarized CaCo 2 monolayer after 5 minutes incubation. (b) DAPI staining (e) Phase contrast. (c, d) Co-localization was just found between CD1 protein expression and Pru p 3. Bar 20 microns.

Figure 4

(a) CD1d-based artificial antigen-presenting cells (aAPC) were incubated with a positive control (α-Galactosylceramide; 5 µgr), and with the lipid-ligand (0.5 ug) during 48h at 37 °C. After that, aAPC were co-cultured with iNKT cells at a 1:1 ratio in a 96 well flat bottom. The supernatant was collected at 24h and the production of IL-2 was measured by ELISA assays. (b) Cyc5-labeled CD1d-tetramers (red) were loaded with the lipid-ligand of Pru p 3 and incubated with healthy volunteer’s PBMCs during 2 h. iNKTs were detected with a monoclonal antibody specific for the TCR of iNKTs labelled FITC (Vα24-Jα18 combined with Vβ11). (c) Confocal microscopy image of CD1d-tetramers-lipid-ligand probe (red) and TCR iNKTs (green) and phase contrast image of two different examples.

Figure 5. Structures of saposins and Pru p 3

(a) Alignment of sequences of saposins A–D and sequence of Pru p 3. Colours labelling the residues that form α and 3₁₀ helices are also used in (b)–(d) below. Black lines connecting marked cysteines represent disulphide bridges. Red box indicates conserved N-linked glycosylation sites and green box indicates conserved tyrosines at the kink of helix α3 shown in (d) for saposin A. (b) X-ray crystal structures of saposins A–D. (c) X-ray crystal structure of Pru p 3. (d) Kink in the longest helix α1 in Pru p3 and α3 in saposin A. Dashed lines in these helices represent main chain hydrogen bonds. (e) Structural superposition of saposin A (blue) and Pru p 3 (orange) computed with FATCAT rigid.