Can birch pollen directly influence the IL-4/IL-4R interaction to modulate Th2 responses?

Abstract

Birch pollen (BP) is a clinically relevant aeroallergen source affecting up to 16% of the European population. In predisposed individuals, BP exposure triggers Th2 immune responses that orchestrate allergic sensitization.1 The key cytokine for Th2 differentiation, interleukin-4 (IL-4), binds with high affinity to IL-4R alpha (IL-4Rα) to form the active type-I IL-4 receptor with the common gamma (γC) chain. Downstream, induced GATA3 mainly regulates the expression of type 2 cytokines. Although the initiation of Th2 differentiation by pollen sources is still unclear, the reported adjuvant function of specific BP-derived compounds other than the major allergen2, 3 suggests that Th2-inducing signals originate from the BP source itself. We, therefore, aimed to study the role of the IL-4/IL-4R pathway in BP-induced Th2 responses by (i) investigating the ability of BP extracts (BPE) compared with other pollen species to bind IL-4Rɑ via ELISA, (ii) screening for latent IL-4Rɑ ligands by fractionating BPE via size exclusion chromatography (SEC), and (iii) assessing associated Th2 responses in vivo using IL-4 reporter mice. Finally, (iv) the functional downstream signaling elicited by the interaction with the IL-4R was examined in pilot in vitro assays. Commercial (c)BPE showed a dose-dependent binding activity to IL-4Rα (Figure 1A), which was not attributable to the major allergen, Bet v 1 (Figure 1B). The activity of the natural ligand IL-4 was higher than for cBPE. By contrast, neither timothy grass (cGPE) nor short ragweed (cRPE) pollen extracts exhibited this IL-4Rα-binding activity (Figure 1A; Figure S1-S3A), indicating a BP-specific effect. Proteinase K-mediated degradation of cBPE (PK-BPE) abolished the binding signal (Figure 1C; Figure S1-S3B), inferring a protein ligand. In inhibition and competition approaches, cBPE was able to block the binding of IL-4Rα to IL-4 dose-dependently and, conversely, IL-4 hindered IL-4Rα to interact with cBPE (Figure 1D,E; Figure S1-S3C), implying similar binding interaction. Interestingly, BPE from self-collected pollen (sBPE) displayed a stronger binding activity than cBPE (Figure 2A). Via interpolation from an IL-4 standard curve, an IL-4Rα-binding activity of 37% for sBPE versus 20% for cBPE was measured. SEC-generated fractions of sBPE (sF1-7) and cBPE (cF1-5) both showed an increased binding activity from low (LMW) to high (HMW) molecular weight fractions. The augmented binding activity observed for sF4-sF1 (86, 65, 55, 39%) and cF1 (47%) suggests the presence of a HMW ligand (Figure 2A; Figure S1-S3). Consistently, compared with original cBPE, only the HMW fraction significantly induced a Th2 response in vivo, measured by the induction of IL-4/eGFP expression in CD4-positive (+) lymphocytes (Figure 2B). Immunization with sBPE similarly induced a significant increase in 13% IL-4/eGFP+CD4+ cells (Figure 2C). Blocking of IL-4Rα on sBPE-stimulated human naïve CD4+T cells increased anti-inflammatory IL-10 secretion, and in HEK reporter cells cBPE stimulation interfered with IL-4-induced STAT6 phosphorylation (Figure S1-S3A,B), preliminarily suggesting the activation of a non-canonical pathway downstream of IL-4R. BPE was demonstrated earlier to act on T cells favoring IL-5 and IL-13 expression compared with Th1-associated TNFα and IL-2.3 IL-4R signaling was also shown to regulate IL-10 secretion to establish Th2 dominance4 and IL-4Rα-deficiency in FoxP3+Tregs exacerbated airway inflammation in a house dust mite sensitization model.5 By exploring the existence of intrinsic Th2 inducers within BPE, our results revealed the peculiar feature of BP-derived compound(s) contained in the HMW fraction and affected by protein degradation to interact with IL-4Rα, associated with the induction of Th2 response in vivo. Several microbial as well as plant-derived cytokine mimics with immunomodulating functions have already been described,6 yet this is the first study to present a natural IL-4Rα ligand other than IL-4. Considering IL-4R expression on various immune cells, concrete downstream effects mediated by BP via the IL-4R pathway to prime Th2 response in vivo remains to be investigated. The identification of BP-intrinsic immunostimulators is relevant to better understand the mechanisms initiating BP allergy and will provide novel therapeutic targets. The authors thank Univ. Prof. Dr. Fatima Ferreira of the Department of Biosciences and Medical Biology for her great support endorsed by productive discussions and providing lab equipment. The research was supported by Austrian/German Joint Science Funds (FWF/DFG Project I5312), by the University of Salzburg priority program Allergy-Cancer-BioNano Research Centre, by the doctoral program Immunity in Cancer and Allergy—ICA funded by the Austrian Science Fund (FWF W1213) and by the Doctoral School Program Biomolecules of the University of Salzburg. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Appendix S1. Figures S1-S3. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.