Unraveling IFN-I response dynamics and TNF crosstalk in the pathophysiology of systemic lupus erythematosus.

Introduction: The innate immune system serves the crucial first line of defense against a wide variety of potential threats, during which the production of pro-inflammatory cytokines IFN-I and TNFα are key. This astonishing power to fight invaders, however, comes at the cost of risking IFN-I-related pathologies, such as observed during autoimmune diseases, during which IFN-I and TNFα response dynamics are dysregulated. Therefore, these response dynamics must be tightly regulated, and precisely matched with the potential threat. This regulation is currently far from understood. Methods: Using droplet-based microfluidics and ODE modeling, we studied the fundamentals of single-cell decision-making upon TLR signaling in human primary immune cells (n = 23). Next, using biologicals used for treating autoimmune diseases [i.e., anti-TNFα, and JAK inhibitors], we unraveled the crosstalk between IFN-I and TNFα signaling dynamics. Finally, we studied primary immune cells isolated from SLE patients (n = 8) to provide insights into SLE pathophysiology. Results: single-cell IFN-I and TNFα response dynamics display remarkable differences, yet both being highly heterogeneous. Blocking TNFα signaling increases the percentage of IFN-I-producing cells, while blocking IFN-I signaling decreases the percentage of TNFα-producing cells. Single-cell decision-making in SLE patients is dysregulated, pointing towards a dysregulated crosstalk between IFN-I and TNFα response dynamics. Discussion: We provide a solid droplet-based microfluidic platform to study inherent immune secretory behaviors, substantiated by ODE modeling, which can challenge the conceptualization within and between different immune signaling systems. These insights will build towards an improved fundamental understanding on single-cell decision-making in health and disease.

Assessment of allelic diversity in intron-containing Mal d 1 genes and their association to apple allergenicity.

BACKGROUND: Mal d 1 is a major apple allergen causing food allergic symptoms of the oral allergy syndrome (OAS) in birch-pollen sensitised patients. The Mal d 1 gene family is known to have at least 7 intron-containing and 11 intronless members that have been mapped in clusters on three linkage groups. In this study, the allelic diversity of the seven intron-containing Mal d 1 genes was assessed among a set of apple cultivars by sequencing or indirectly through pedigree genotyping. Protein variant constitutions were subsequently compared with Skin Prick Test (SPT) responses to study the association of deduced protein variants with allergenicity in a set of 14 cultivars. RESULTS: From the seven intron-containing Mal d 1 genes investigated, Mal d 1.01 and Mal d 1.02 were highly conserved, as nine out of ten cultivars coded for the same protein variant, while only one cultivar coded for a second variant. Mal d 1.04, Mal d 1.05 and Mal d 1.06 A, B and C were more variable, coding for three to six different protein variants. Comparison of Mal d 1 allelic composition between the high-allergenic cultivar Golden Delicious and the low-allergenic cultivars Santana and Priscilla, which are linked in pedigree, showed an association between the protein variants coded by the Mal d 1.04 and -1.06A genes (both located on linkage group 16) with allergenicity. This association was confirmed in 10 other cultivars. In addition, Mal d 1.06A allele dosage effects associated with the degree of allergenicity based on prick to prick testing. Conversely, no associations were observed for the protein variants coded by the Mal d 1.01 (on linkage group 13), -1.02, -1.06B, -1.06C genes (all on linkage group 16), nor by the Mal d 1.05 gene (on linkage group 6). CONCLUSION: Protein variant compositions of Mal d 1.04 and -1.06A and, in case of Mal d 1.06A, allele doses are associated with the differences in allergenicity among fourteen apple cultivars. This information indicates the involvement of qualitative as well as quantitative factors in allergenicity and warrants further research in the relative importance of quantitative and qualitative aspects of Mal d 1 gene expression on allergenicity. Results from this study have implications for medical diagnostics, immunotherapy, clinical research and breeding schemes for new hypo-allergenic cultivars.

Silencing the major apple allergen Mal d 1 by using the RNA interference approach.

BACKGROUND: Apple allergy is dominated by IgE antibodies against Mal d 1 in areas where birch pollen is endemic. Apples with significantly decreased levels of Mal d 1 would allow most patients in these areas to eat apples without allergic reactions. OBJECTIVE: The aim of this study was to inhibit the expression of Mal d 1 in apple plants by RNA interference. METHODS: In vitro -grown apple plantlets were transformed with a construct coding for an intron-spliced hairpin RNA containing a Mal d 1-specific inverted repeat sequence separated by a Mal d 1-specific intron sequence. The presence of the construct in transformants was checked by PCR. Expression of Mal d 1 in leaves was monitored by prick-to-prick skin testing in 3 patients allergic to apples and by immunoblotting with a Mal d 1-reactive mAb and with IgE antibodies against Mal d 1. RESULTS: After transformation, plantlets were selected on the basis of having a normal phenotype and growth rate. With PCR, in 6 of 9 selected plantlets, the presence of the gene-silencing construct was demonstrated. By skin prick test it was shown that a wild-type plantlet had significantly ( P < .05) higher allergenicity than 5 of the transformants. Reduction of expression of Mal d 1 was confirmed by immunoblotting. In wild-type and unsuccessful transformants, a strong band was detected with Mal d 1-reactive mAb 5H8 at the expected apparent M r of 17 kDa. This band was virtually absent in the transformants that carried the gene-silencing construct. With human IgE antibodies, the same observations were made. CONCLUSIONS: Mal d 1 expression was successfully reduced by RNA interference. This translated into significantly reduced in vivo allergenicity. These observations support the feasibility of the production by gene silencing of apples hypoallergenic for Mal d 1.