Search

BackgroundInsect bite hypersensitivity (IBH) is the most frequent skin allergy of horses and is highly debilitating, especially in the chronic phase. IBH is caused by IgE-mediated hypersensitivity reactions to culicoides midge bites and an imbalanced immune response that reduces the welfare of affected horses.ObjectiveIn the present study, we investigated the pathological mechanisms of IBH, aiming to understand the immune cell modulation in acute allergic skin lesions of IBH horses with the goal of finding possible biomarkers for a diagnostic approach to monitor treatment success.MethodsBy qPCR, we quantified the gene expression of cytokines, chemokines, and immune receptors in skin punch biopsies of IBH with different severity levels and healthy horses simultaneously in tandem with the analysis of immune cell counts in the blood.ResultsOur data show an increase in blood eosinophils, monocytes, and basophils with a concomitant, significant increase in associated cytokine, chemokine, and immune cell receptor mRNA expression levels in the lesional skin of IBH horses. Moreover, IL-5Ra, CCR5, IFN-γ, and IL-31Ra were strongly associated with IBH severity, while IL-31 and IL-33 were rather associated with a milder form of IBH. In addition, our data show a strong correlation of basophil cell count in blood with IL-31Ra, IL-5, IL-5Ra, IFN-γ, HRH2, HRH4, CCR3, CCR5, IL-12b, IL-10, IL-1β, and CCL26 mRNA expression in skin punch biopsies of IBH horses.ConclusionIn summary, several cytokines and chemokines have been found to be associated with disease severity, hence contributing to IBH pathology. These molecules can be used as potential biomarkers to monitor the onset and progression of the disease or even to evaluate and monitor the efficacy of new therapeutic treatments for IBH skin allergy. To our knowledge, this is the first study that investigated immune cells together with a large set of genes related to their biological function, including correlation to disease severity, in a large cohort of healthy and IBH horses.

Eosinophils play a key role in allergic diseases such as insect bite hypersensitivity (IBH). Together with Th2 cells, they shape the course of inflammation in associated type I/IVb allergies. Therefore, a virus-like particle (VLP)-based vaccine targeting equine interleukin-5 (eIL-5), eIL-5-CuMV-TT, was developed to interfere with the IL-5 dependency of eosinophils by inducing the production of anti-self-IL-5 antibodies and alleviating clinical signs in IBH-affected horses. A previous study highlighted the presence of two eosinophil subsets, steady-state resident eosinophils (rEos) and inflammatory eosinophils (iEos), circulating in the blood of healthy and IBH-affected horses, distinguishable by the expression of integrin CD49f. Furthermore, eIL-5-CuMV-TT 1st year vaccination showed a significant decrease of total eosinophils and, in particular, iEos. Nevertheless, the very few remaining eosinophils still shared an iEos phenotype, reflected by bigger size and higher granularity. The aim of this study was to follow up on the phenotype of eosinophils in the 2nd year of vaccination of IBH-affected horses with eIL-5-CuMV-TT. Using flow cytometry analysis of the blood of healthy, IBH, IBH-placebo, and IBH-vaccinated horses, the percentage and count of cells were compared between groups with a focus on pair analysis of eosinophils in 1st and 2nd year vaccinated horses. Our data showed comparably low levels of iEos and a significant increase of rEos in 2nd year compared to 1st year vaccinated horses, suggesting a phenotypic shift toward a resident-like eosinophil population, primarily associated with the phenotype of healthy horses. The reduction of size, granularity, and expression of integrin CD49f in the 2nd year suggests a benefit of long-term treatment with the eIL-5-CuMV-TT vaccine.

Urticaria, independent of or associated with allergies, is commonly seen in horses and often shows a high reoccurrence rate. Managing these horses is discouraging, and efficient treatment options are lacking. Due to an incidental finding in a study on horses affected by insect bite hypersensitivity using the eosinophil-targeting eIL-5-CuMV-TT vaccine, we observed the prevention of reoccurring seasonal urticaria in four subsequent years with re-vaccination. In an exploratory case series of horses affected with non-seasonal urticaria, we aimed to investigate the role of eosinophils in urticaria. Skin punch biopsies for histology and qPCR of eosinophil associated genes were performed. Further, two severe, non-seasonal, recurrent urticaria-affected horses were vaccinated using eIL-5-CuMV-TT, and urticaria flare-up was followed up with re-vaccination for several years. Eotaxin-2, eotaxin-3, IL-5, CCR5, and CXCL10 showed high sensitivity and specificity for urticarial lesions, while eosinophils were present in 50% of histological tissue sections. The eIL-5-CuMV-TT vaccine reduced eosinophil counts in blood, cleared clinical signs of urticaria, and even prevented new episodes of urticaria in horses with non-seasonal recurrent urticaria. This indicates that eosinophils play a leading role in urticaria in horses, and targeting eosinophils offers an attractive new treatment option, replacing the use of corticosteroids.

Simple Summary: Insect bite hypersensitivity (IBH) is a common skin allergic condition in horses caused by insect bites, mainly of Culiocides species. Affected horses develop severe itchy lesions, up to traumatic skin injuries. Eosinophils are known to play a crucial role in the pathogenesis of IBH. Recently, we described two subsets of eosinophils: inflammatory eosinophils (iEos) dominant in blood of IBH-affected horses, and resident eosinophils (rEos) present in blood of healthy horses. iEos and rEos were distinguishable by size, granularity, and proteins present on their cell surface. Interleukin (IL)-5, the main activator and regulator of eosinophils, is our vaccine target. Vaccinated horses showed a significant reduction of total eosinophils, in particular iEos, where the very few remaining eosinophils still showed iEos phenotype. In the present study, we followed the phenotype of eosinophil subsets in the 2nd year of vaccination in IBH-affected horses. Our results showed comparably lower levels of iEos and a significant increase of rEos in 2nd compared to 1st year vaccinated and unvaccinated horses. This suggests a shift from iEos to the rEos phenotype, the dominant eosinophil type of healthy horses. The change in size, granularity and migration properties suggests a benefit of long term vaccination of IBH-affected horses. Eosinophils play a key role in allergic diseases such as insect bite hypersensitivity (IBH). Together with Th2 cells, they shape the course of inflammation in associated type I/IVb allergies. Therefore, a virus-like particle (VLP)-based vaccine targeting equine interleukin-5 (eIL-5), eIL-5-CuMV-TT, was developed to interfere with the IL-5 dependency of eosinophils by inducing the production of anti-self-IL-5 antibodies and alleviating clinical signs in IBH-affected horses. A previous study highlighted the presence of two eosinophil subsets, steady-state resident eosinophils (rEos) and inflammatory eosinophils (iEos), circulating in the blood of healthy and IBH-affected horses, distinguishable by the expression of integrin CD49f. Furthermore, eIL-5-CuMV-TT 1st year vaccination showed a significant decrease of total eosinophils and, in particular, iEos. Nevertheless, the very few remaining eosinophils still shared an iEos phenotype, reflected by bigger size and higher granularity. The aim of this study was to follow up on the phenotype of eosinophils in the 2nd year of vaccination of IBH-affected horses with eIL-5-CuMV-TT. Using flow cytometry analysis of the blood of healthy, IBH, IBH-placebo, and IBH-vaccinated horses, the percentage and count of cells were compared between groups with a focus on pair analysis of eosinophils in 1st and 2nd year vaccinated horses. Our data showed comparably low levels of iEos and a significant increase of rEos in 2nd year compared to 1st year vaccinated horses, suggesting a phenotypic shift toward a resident-like eosinophil population, primarily associated with the phenotype of healthy horses. The reduction of size, granularity, and expression of integrin CD49f in the 2nd year suggests a benefit of long-term treatment with the eIL-5-CuMV-TT vaccine. [ABSTRACT FROM AUTHOR]

Background: Insect bite hypersensitivity (IBH) is the most frequent skin allergy of horses and is highly debilitating, especially in the chronic phase. IBH is caused by IgE-mediated hypersensitivity reactions to culicoides midge bites and an imbalanced immune response that reduces the welfare of affected horses. Objective: In the present study, we investigated the pathological mechanisms of IBH, aiming to understand the immune cell modulation in acute allergic skin lesions of IBH horses with the goal of finding possible biomarkers for a diagnostic approach to monitor treatment success. Methods: By qPCR, we quantified the gene expression of cytokines, chemokines, and immune receptors in skin punch biopsies of IBH with different severity levels and healthy horses simultaneously in tandem with the analysis of immune cell counts in the blood. Results: Our data show an increase in blood eosinophils, monocytes, and basophils with a concomitant, significant increase in associated cytokine, chemokine, and immune cell receptor mRNA expression levels in the lesional skin of IBH horses. Moreover, IL-5Ra, CCR5, IFN-γ, and IL-31Ra were strongly associated with IBH severity, while IL-31 and IL-33 were rather associated with a milder form of IBH. In addition, our data show a strong correlation of basophil cell count in blood with IL-31Ra, IL-5, IL-5Ra, IFN-γ, HRH2, HRH4, CCR3, CCR5, IL-12b, IL-10, IL-1β, and CCL26 mRNA expression in skin punch biopsies of IBH horses. Conclusion: In summary, several cytokines and chemokines have been found to be associated with disease severity, hence contributing to IBH pathology. These molecules can be used as potential biomarkers to monitor the onset and progression of the disease or even to evaluate and monitor the efficacy of new therapeutic treatments for IBH skin allergy. To our knowledge, this is the first study that investigated immune cells together with a large set of genes related to their biological function, including correlation to disease severity, in a large cohort of healthy and IBH horses. [ABSTRACT FROM AUTHOR]

The di‐copper center CuA is an essential metal cofactor in cytochrome oxidase (Cox) of mitochondria and many prokaryotes, mediating one‐electron transfer from cytochrome c to the site for oxygen reduction. CuA is located in subunit II (CoxB) of Cox and protrudes into the periplasm of Gram‐negative bacteria or the mitochondrial intermembrane space. How the two copper ions are brought together to build CoxB·CuA is the subject of this review. It had been known that the reductase TlpA and the metallochaperones ScoI and PcuC are required for CuA formation in bacteria, but the mechanism of copper transfer has emerged only recently for the Bradyrhizobium diazoefficiens system. It consists of the following steps: (a) TlpA keeps the active site cysteine pair of CoxB in its dithiol state as a prerequisite for metal insertion; (b) ScoI·Cu2+ rapidly forms a transient complex with apo‐CoxB; (c) PcuC, loaded with Cu1+ and Cu2+, dissociates this complex to CoxB·Cu2+, and a second PcuC·Cu1+·Cu2+ transfers Cu1+ to CoxB·Cu2+, yielding mature CoxB·CuA. Variants of this pathway might exist in other bacteria or mitochondria. [ABSTRACT FROM AUTHOR]

The CuA cofactor in cytochrome oxidase of mitochondria and many aerobic prokaryotes is a di-copper metal center that catalyzes one-electron transfer from cytochrome c to the oxygen-reducing active site. How the two copper ions are brought together for assembly of CuA is reported here. A thioredoxin-like disulfide reductase and two dedicated metal-lochaperones, one for Cu2+ and one for Cu1+, cooperate in this process. Copper insertion into cytochrome oxidase proceeds by a previously unknown mechanism. [ABSTRACT FROM AUTHOR]