Generation of high affinity ICAM-1-specific nanobodies and evaluation of their suitability for allergy treatment.

The nasal cavity is an important site of allergen entry. Hence, it represents an organ where trans-epithelial allergen penetration and subsequent IgE-mediated allergic inflammation can potentially be inhibited. Intercellular adhesion molecule 1 (ICAM-1) is highly expressed on the surface of respiratory epithelial cells in allergic patients. It was identified as a promising target to immobilize antibody conjugates bispecific for ICAM-1 and allergens and thereby block allergen entry. We have previously characterized a nanobody specific for the major birch pollen allergen Bet v 1 and here we report the generation and characterization of ICAM-1-specific nanobodies. Nanobodies were obtained from a camel immunized with ICAM-1 and a high affinity binder was selected after phage display (Nb44). Nb44 was expressed as recombinant protein containing HA- and His-tags in Escherichia coli (E.coli) and purified via affinity chromatography. SDS-PAGE and Western blot revealed a single band at approximately 20 kDa. Nb44 bound to recombinant ICAM-1 in ELISA, and to ICAM-1 expressed on the human bronchial epithelial cell line 16HBE14o- as determined by flow cytometry. Experiments conducted at 4°C and at 37°C, to mimic physiological conditions, yielded similar percentages (97.2 ± 1.2% and 96.7 ± 1.5% out of total live cells). To confirm and visualize binding, we performed immunofluorescence microscopy. While Texas Red Dextran was rapidly internalized Nb44 remained localized on the cell surface. Additionally, we determined the strength of Nb44 and ICAM-1 interaction using surface plasmon resonance (SPR). Nb44 bound ICAM-1 with high affinity (10-10 M) and had slow off-rates (10-4 s-1). In conclusion, our results showed that the selected ICAM-1-specific nanobody bound ICAM-1 with high affinity and was not internalized. Thus, it could be further used to engineer heterodimers with allergen-specific nanobodies in order to develop topical treatments of pollen allergy.

Immunomodulatory Properties of Blackberry Anthocyanins in THP-1 Derived Macrophages.

An anthocyanin-rich diet is considered to protect against chronic inflammatory processes although the bioavailability of anthocyanins is regarded as rather low. Moreover, the immunomodulatory role of anthocyanins is not fully understood yet. In the present study, fractions of blackberry (Rubus fruticosus) juice were investigated in plasma-relevant concentrations with respect to their immunomodulatory properties in lipopolysaccharide (LPS)-challenged THP-1-derived macrophages. The complex blackberry extract acted ineffective as well as potential degradation products. Cyanidin-3O-glucoside (Cy3glc), the main constituent of blackberry anthocyanins, diminished TNF-α levels at a concentration of 0.02 µg/mL, indicating protective effects as measured with quantitative RT-PCR and multiplex cytokine assays. LPS-boosted activity of transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) of differentiated THP-1 reporter gene cells was marginally inhibited by Cy3glc. LPS-induced microRNA-155 was further increased, supporting the evidence of protection. Of note, fractions obtained from blackberry juice, in particular cyanidin-3O-(6″-dioxalylglucoside), were displaying potential pro-inflammatory properties as these elevated IL-6 and TNF-α levels. In conclusion, highly purified anthocyanin fractions of blackberry juice display both anti- and pro-inflammatory properties at plasma-relevant concentrations depending on their structure and substitution pattern.

The mycotoxin alternariol suppresses lipopolysaccharide-induced inflammation in THP-1 derived macrophages targeting the NF-κB signalling pathway.

Alternariol (AOH) is a secondary metabolite formed by black mold of the genus Alternaria alternata. Due to limited hazard and occurrence data, AOH is still considered as an "emerging mycotoxin" and, as such, not monitored and regulated yet. Recent studies indicate immunosuppressive effects in vitro by altering the expression of CD molecules and proinflammatory cytokines, which are indispensable in mounting an innate immune response. However, the mode of action by which AOH exerts its immunosuppressive effects has not been unraveled yet. The present study aimed to characterise the impact of AOH on the nuclear factor kappa B (NF-κB) pathway, the expression of NF-κB target cytokines and involved regulatory microRNAs (miRNAs). In THP-1 derived macrophages, AOH (1-20 µM) was found to suppress lipopolysaccharide (LPS)-induced NF-κB pathway activation, decrease secretion of the proinflammatory cytokines IL-8, IL-6, TNF-α and to induce secretion of the anti-inflammatory IL-10. Thereby, a distinct pattern of cytokine mRNA levels was monitored, varying between short- and long-term exposure. Concomitantly, AOH (2-20 µM) affected the transcription levels of miR-146a and miR-155 in LPS-stimulated THP-1 derived macrophages dose-dependently by down- and upregulation, respectively. In contrast, transcription of miR-16 and miR-125b, two other immune-related miRNAs, was not modulated. In the absence of a LPS stimulus, AOH (20 µM) did not affect basal NF-κB activity, but increased IL-10 transcription. Collectively, our results indicate, that AOH itself does not induce a proinflammatory immune response in human macrophages; however, in an inflamed environment it possesses the ability to repress inflammation by targeting the NF-κB signalling pathway and regulatory miRNAs.