Antigen identification and characterization of adaptive immune responses in severe Equine Asthma

• Applicant: Dr. Christiane Schnabel
• Subject Area: Veterinary Medical Science
• Term: since 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 431342499

Severe equine asthma (sEA) is similar to human adult-onset asthma. Both respiratory diseases are immune-mediated and lack causative treatment options. In susceptible horses hay dust elicits recurrent airway obstruction. However, pathogenesis of sEA is controversial. Allergy to hay dust components, e.g. molds, has been assumed. Adaptive immune responses involved in allergy have been reported, such as mold-specific immunoglobulin (Ig)E, and cytokines associated with T helper (Th)2 activation. In contrast, Th1 or Th17 profiles together with typical neutrophilic inflammation in sEA have been observed which point to non-allergic mechanisms. To clarify the pathogenesis of sEA, a better understanding of critical B and T cell immunity is required and causative antigens (Ag) need to be identified on a molecular basis. I hypothesize that allergic and non-allergic pathogenesis subtypes of sEA exist. Specific Ig and reactive T cells indicate disease-relevant Ag. Polarizations of the Th responses suggest pathogenetic mechanisms and are associated with characteristic Ig isotypes. To identify causative Ag in hay dust, I will take a bottom-up approach using immune proteomics. Ig isotype binding patterns on two-dimensional immunoblots will be compared between samples (serum, bronchoalveolar lavage fluid, BALF) from horses with sEA and healthy controls. Exclusive binding of asthmatics’ Ig will point to disease-relevant Ag. These will be identified by mass spectrometry, produced as recombinant proteins and confirmed by immunoblots. Subsequently, I will establish Ag-specific Ig isotype quantification in a bead-based assay to define the B cell response. Moreover, re-stimulation in a novel assay will determine T cell Ag-reactivity and polarization by cytokine induction. To characterize sEA pathogenesis, immune responses of horses with sEA will be compared to those of healthy horses. Systemic (blood) and local (BALF) B and T cell responses to the Ags identified will be assessed, and Ag sensitization will be examined by intradermal tests (IDT) to confirm relevant Ags in vivo and define allergic vs. non-allergic pathogenesis subtypes. The allergic subtype is expected to be associated with Th2, IgE, IgG5, and immediate IDT reactions, while non-allergic pathogenesis would be accompanied by Th1/Th17, IgG4/other IgG isotypes, and delayed/negative IDT. To distinguish pathological mechanisms during the initiation and chronic phases of symptomatic sEA, immune responses will be assessed over time. Healthy and asthmatic horses’ adaptive responses will be compared during sEA remission (dust-free housing) and exacerbation (during and after controlled hay dust exposure). These comparisons will indicate stage-dependent pathologic immune responses to specific Ags in sEA, and immune regulation of healthy horses. The proposed Ag identification and in-depth characterization of adaptive immune responses will elucidate sEA pathogenesis and enable future specific immunotherapy.

• DFG Programme: Independent Junior Research Groups