Rubicon protein as a marker of severity of atopic bronchial asthma.

Author: Ibragimov Bulat Rafisovich¹

Co-authors: ¹Skibo Yulia Valerievna, ^1,2Reshetnikova Irina Dmitrievna, ¹Abramova Zinaida Ivanovna.

¹Kazan (Volga Region) Federal University,

²«Kazan Research Institute of Epidemiology and Microbiology» of Rospotrebnadzor.

Russia, Kazan

Canonical autophagy is the process of degradation of proteins and organelles in conditions of nutrient deficiency. Recently, a new form of autophagy has been discovered — LC3-associated phagocytosis (LAP) is a form of non-canonical autophagy, one example of which is efferocytosis (the process by which apoptotic cells are removed by phagocytic cells, which contributes to the resolution of inflammation) [1]. It was found that this process in severe forms of pulmonary diseases, for example, atopic bronchial asthma, proceeds with disturbances. However, the relationship between the course of the mechanism and the severity of the disease has not yet been studied in detail [2].

Monocytes were isolated by centrifugation in a ficoll density gradient (ρ=1.077) (PanEco), followed by centrifugation in a 46% Percoll osmotic density gradient (Percoll plus 1l, GE Healthcare) [3]. Organelles of canonical and non-canonical autophagy were detected by electron microscopy, and the analysis of key proteins of autophagy and LC3-associated phagocytosis regulators was performed by Western blot.

Healthy monocytes are characterized by an increase in cell size in healthy samples, the nuclei of monocytes from asthma patients are usually fragmented. The number of lysosomes and mitochondria increases. During autophagy, the phagophore encapsulates cytoplasmic material such as dysfunctional or damaged organelles, proteins, and double-membrane autophagosomes (APs) are formed.

LC3-associated phagocytosis involves the conjugation of the LC3 protein to single organelles (laposomes). Whereas in canonical autophagy, the LC3 protein is conjugated to double membrane structures [4].

In patients with atopic bronchial asthma, compared with control samples, a decrease in the apoptotic protein caspase-3 and an increase in the anti-apoptotic protein Bcl-2 were noted.

The LC-1/LC3-II ratio of the autophagy progression marker shows that the level of activity is higher in the monocytes of asthma patients. In the samples of patients with a severe form of atopic bronchial asthma, a significant decrease in Rubicon (a marker of LC3-associated phagocytosis) is observed, which indicates a LAP defect in this form of the course of the disease.

In monocytes of patients with severe atopic asthma, the process of apoptosis is suppressed and components characteristic of autophagy are activated. In patients with a severe form of atopic bronchial asthma, a reduced LC3-phagocytic response is expressed, in contrast to healthy donors. The Rubicon protein, which is characteristic of LC3-associated phagocytosis [5], can be used as a marker of the severity of atopic bronchial asthma.

The work was carried out at the expense of a subsidy allocated as part of the state support of the Kazan (Volga Region) Federal University in order to increase its competitiveness among the world’s leading scientific and educational centers (Priority-2030).

Bibliography.

1. Deev, R.V. Modern ideas about cell death / R.V. Deev, A.I. Bilyalov, T.M. Zhampeisov // Genes and Cells. ‒ 2018. ‒ V. 13, № 1. ‒ P. 6–19.

2. Liang, Z. Impaired macrophage phagocytosis of bacteria in severe asthma / Z. Liang, Q. Zhang, C. Thomas, K. K. Chana, D. Gibeon, P. J. Barnes, K. F. Chung, P. K. Bhavsar, L. E. Donnelly // Respir Res. ‒ 2014. ‒ V. 15, № 72.

3. Menck, K. Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags / K. Menck, D. Behme, M. Pantke, N. Reiling, C. Binder, T. Pukrop, F. Klemm // J Vis Exp. ‒ 2014. ‒ V. 91. ‒ P. 51554.

4. Skendros, P. Autophagy in Neutrophils: From Granulopoiesis to Neutrophil Extracellular Traps /P. Skendros, I. Mitroulis, K. Ritis // Front Cell Dev Biol. ‒ 2018. ‒ V. 6. ‒ P. 109.

5. Wong, S.W. Rubicon: LC3-associated phagocytosis and beyond / S. W. Wong, P. Sil, J. Martinez // The FEBS journal. ‒ 2018. ‒ V. 285, № 8. ‒ P. 1379-1388.