Blood Transcriptomics Identifies Multiple Gene Expression Pathways Associated with the Clinical Efficacy of Hymenoptera Venom Immunotherapy

Abstract

Allergen-specific venom immunotherapy (VIT) is a well-established therapy for Hymenoptera venom allergy (HVA). However, the precise mechanism underlying its clinical effect remains uncertain. Our study aimed to identify the molecular mechanisms associated with VIT efficiency. We prospectively included 19 patients with HVA undergoing VIT (sampled before the beginning of VIT, after reaching the maintenance dose, one year after finishing VIT, and after a sting challenge) and 9 healthy controls. RNA sequencing of whole blood was performed on an Illumina sequencing platform. Longitudinal transcriptomic profiling revealed the importance of the inhibition of the NFκB pathway and the downregulation of DUX4 transcripts for the early protection and induction of tolerance after finishing VIT. Furthermore, successful treatment was associated with inhibiting Th2, Th17, and macrophage alternative signalling pathways in synergy with the inhibition of the PPAR pathway and further silencing of the Th2 response. The immune system became activated when reaching the maintenance dose and was suppressed after finishing VIT. Finally, successful VIT restores the immune system's balance to a state similar to that of healthy individuals. Our results underline the important role of the inhibition of four pathways in the clinical effect of VIT: Th2, Th17, NFκB, and macrophage signalling. Two biomarkers specific for successful VIT, regardless of the time of sampling, were C4BPA and RPS10-NUDT3 and should be further tested as potential biomarkers.

Keywords: Hymenoptera venom immunotherapy; longitudinal transcriptomic profiling; successful venom immunotherapy; tolerance induction.

Figure 1

Follow-up analysis and overlapping of differentially expressed genes at three time points (before, after reaching maintenance dose, and after) for successful venom immunotherapy (blue) and at two time points (before and after) for treatment failure (red). (Cut-off: fold change > 5, p-value < 0.05.) VIT: venom immunotherapy; DEGs: differentially expressed genes.

Figure 2

A volcano plot of differentially expressed genes between successful venom immunotherapy and treatment failure groups. Highlighted and with red arrows marked is the transcript of interest, IKBKGP1. No difference between the two groups before the initiation of venom immunotherapy is observed (a), whereas a significant difference between the two groups after venom immunotherapy is pointed out (b). The IKBKGP1 transcript is significantly downregulated after venom immunotherapy in the group with successful treatment. The kinetics of the IKBKGP1 transcripts of individual samples from successful venom immunotherapy are shown, with significant downregulation after venom immunotherapy (p-value = 0.0156) (c). RPKM: reads per kilobase per million mapped reads; *: statistical significance.

Figure 3

Comparison of differentially expressed genes between the three groups—successful VIT, treatment failure, and healthy controls. Overlapping genes specific for successful VIT with the two transcripts characteristic of successful VIT, regardless of the time point of sampling (From: Gene Cards^®. The Human Gene Database n.d. Available online: https://www.genecards.org/, accessed on 23 January 2024) (a). Overlapping genes specific for treatment failure (b). Number of DEGs after VIT in the analysis of successful VIT and healthy controls compared to treatment failure and healthy controls (c). (Cut-off: fold change > 5, p-value < 0.05.) VIT: venom immunotherapy; DEGs: differentially expressed genes.

Figure 3

Comparison of differentially expressed genes between the three groups—successful VIT, treatment failure, and healthy controls. Overlapping genes specific for successful VIT with the two transcripts characteristic of successful VIT, regardless of the time point of sampling (From: Gene Cards^®. The Human Gene Database n.d. Available online: https://www.genecards.org/, accessed on 23 January 2024) (a). Overlapping genes specific for treatment failure (b). Number of DEGs after VIT in the analysis of successful VIT and healthy controls compared to treatment failure and healthy controls (c). (Cut-off: fold change > 5, p-value < 0.05.) VIT: venom immunotherapy; DEGs: differentially expressed genes.

Figure 4

A heat map of normalised expression values of differentially expressed genes after the sting challenge in the groups with successful venom immunotherapy (dark blue) and treatment failure (dark red). Each column represents a patient’s sample; each row represents an individual gene. For each gene, bright blue represents under-expression, and bright red over-expression. (Cut-off: fold change > 5, p-value < 0.05.) VIT: venom immunotherapy.

Figure 5

The follow-up study design. Whole-blood samples were collected before the beginning of VIT, after reaching the maintenance dose, one year after finishing VIT, and after a controlled sting challenge. VIT: venom immunotherapy.