New methodical approach for diagnosis of autoinflammatory diseases: flow cytometry-based investigation of intracellular proinflammatory cytokine production
Abstract
This work aims at the introduction and optimization of methods for innate immune parameters analysis with a detailed focus on proinflammatory cytokines. Autoinflammatory diseases like Familial Mediterranean Fever (FMF), Schnitzler syndrome and Hyper-IgD syndrome (HIDS), as well as autoimmune diseases that involve the inflammatory component, are usually characterized by a non-specific clinical manifestation.
Therefore, obtaining a proper diagnosis of these disorders remains complicated. Genetic examination serves as an accurate diagnostic method for the esta-blishment of monogenic autoinflammatory disease diagnosis.
However, genetic profiling cannot be provided to all patients. Moreover, some of the mutations responsible for the development of the disease haven't been defined yet.
Therapy of inflammatory diseases is currently based on molecules that are capable of blocking the function of inflammatory cytokines. In inflammatory diseases, the proper understanding of disease pathogenesis is still far from satisfactory and therefore, selecting the right therapeutic treatment modality keeps causing difficulties.
For this reason, we focused on the introduction and optimization of a novel method which has a potential to detect activation of inflammatory cytokines and may, therefore, contribute to the correct diagnosis and direct therapeutic strategy. Production of main proinflammatory cytokines (IL-1β, IL-6, TNFα), which are produced by monocytes after lipopolysaccharide stimulation, was tested with two different methods - flow cytometry, detecting the intracellular production of cytokines, and ALBIA (Addressable Laser Bead Immuno Assay) - Luminex method, which can detect extracellular cytokine release into supernatant.
Using flow cytometry we optimized the setting by detecting the optimal time of stimulation, concentration of LPS and the optimal time to stop cytokine exocytosis after adding Brefeldin A.