Distinct transcriptome profiles differentiate nonsteroidal anti-inflammatory drug-dependent from nonsteroidal anti-inflammatory drug-independent food-induced anaphylaxis

Abstract

Background: Lipid transfer protein (LTP), an abundant protein in fruits, vegetables, and nuts, is a common food allergen in Mediterranean areas causing diverse allergic reactions. Approximately 40% of food-related anaphylaxis induced by LTPs requires nonsteroidal anti-inflammatory drugs (NSAIDs) as a triggering cofactor.

Objective: We sought to better understand the determinants of NSAID-dependent and NSAID-independent LTP-induced anaphylaxis (LTP-A).

Methods: Selection of patients was based on a proved clinical history of NSAID-dependent or NSAID-independent anaphylaxis to LTPs, positive skin prick test response to LTPs, and serum LTP IgE. Whole-transcriptome (RNA sequencing) analysis of blood cells from 14 patients with NSAID-related LTP-A (NSAID-LTP-A), 7 patients with LTP-A, and 13 healthy control subjects was performed to identify distinct gene expression signatures.

Results: Expression of genes regulating gastrointestinal epithelial renewal was altered in both patient sets, particularly in those with LTP-A, who also presented with gene expression profiles characteristic of an inflammatory syndrome. These included altered B-cell pathways, increased neutrophil activation markers, and increased reactive oxygen species levels. Increased expression of the IgG receptor (CD64) in patients with LTP-A was mirrored by the presence of LTP-specific IgG1 and IgG3. Conversely, patients with NSAID-LTP-A were characterized by reduced expression of IFN-γ-regulated genes and IFN-γ levels, as well as upregulated expression of adenosine receptor 3 (ADORA3) and genes related to adenosine metabolism.

Conclusions: Gene ontology analysis suggests disturbances in gut epithelial homeostasis in both groups with LTP-A, with potential integrity breaches in patients with LTP-A that might explain their distinct inflammatory signatures. Differential regulation in patients with LTP-A and those with NSAID-LTP-A of the IFN-γ pathway, IgG receptors, and ADORA3 might provide the pathogenic basis of their distinct responses.

Keywords: Anaphylaxis; food allergy; lipid transfer protein syndrome; nonsteroidal anti-inflammatory drugs; transcriptome analysis.

Fig 1. Increased reactive oxygen species and Top scored gene network in the comparison NSAID-LTP-A vs. LTP-A

A) Serum levels of free radicals (reactive oxygen species, ROS, and reactive nitrogen species, RNS) in the indicated groups. Data are expressed as mean±SEM. **p<.01, ***p<.001 compared to HV. B) Functional relationships of differentially expressed genes in NSAID-LTP-A vs. LTP-A in the top scored network “Cell death and Survival, Inflammatory response, Cancer”. The intensity of the node color indicates the degree of up-regulation (red) or down-regulation (green). Genes in uncolored nodes were of relevance for this network and integrated into the computationally generated networks on the basis of the evidence stored in the IPA knowledge memory, but they were not identified as differentially expressed. Lines indicate molecular interactions.

Fig 2. LTP-specific IgG1/IgG3 in LTP-A serums and repressed IFN-γ and IFN-γ regulated genes in NSAID-LTP-A

A) Representation of IFNG as an upstream regulator of differentially regulated genes in NSAID-LTP-A compared to LTP-A, as identified by IPA. The arrows connect genes whose expression is altered and may be regulated by IFNG. The style of the lines defines the direction of change. The color of the node indicates the direction of the gene regulation. Grey: down-regulated; blank: up-regulated. B) Serum levels of IFN-γ and C) IgG1 and IgG3 anti-Pru p 3 in the indicated groups. Data are expressed as mean±SEM. *p<.05, **p<.01, ***p<.001 compared to HV unless otherwise indicated.

Fig 3

Differentially expressed genes in NSAID-related LTP-induced anaphylaxis (NSAID-LTP-A) patients compared to LTP-anaphylaxis (LTP-A) patients. Genes in grey boxes indicate their association with Th2-type of immunity while genes in blue boxes indicate association with Th1-type of immunity. Differences between groups were statistically significant (p<0.05) unless otherwise indicated. NS: not significant. Columns 3-5 were color-coded in accordance with the fold change