Polarization of monocyte to macrophages: NADPH expression and protein modification

Deepak RATHI

Doctoral thesis

Abstract:

In the present study, we investigated the polarization of monocytes to macrophages, ROS generation during the transition, and mediated protein modifications under the influence of different differentiation inducers. Morphological changes in monocytes in the presence of a differentiation inducer were studied using Confocal Laser Scanning Microscopy (CLSM), and NADPH oxidase expression was studied using Immunoblotting. The regulation of the enzyme complex was studied in both monocytes and macrophages, and the role of EVs secreted from monocytes and their effect on differentiating monocytes as messengers or antioxidant cargo was also studied. Our results showed that monocytes turn into macrophages when they encounter inflammation or differentiation and produce ROS. Polyunsaturated fatty acids (PUFA) are easily damaged by these reactive molecules and can lead to reactive intermediates or products such as aldehydes. Malondialdehyde (MDA), which is one of the final products of polyunsaturated fatty acids peroxidation led to protein modification. Furthermore, the involvement of EVs derived from monocytes and their co-culture with non-differentiating/differentiating cells showed that EVs through their cargo of regulatory molecules, potentially NOX isotypes contributed to the modulation of polarization of monocytes to distinct macrophage phenotypes. We studied the effect of selected exogenous antioxidant/bioactive compounds isolated from industrial waste to understand their efficacy in protecting the cells against oxidative damage. The intricate equilibrium and transition between the pro-oxidant and antioxidant effects of antioxidants were also investigated.

Abstract:

In the present study, we investigated the polarization of monocytes to macrophages, ROS generation during the transition, and mediated protein modifications under the influence of different differentiation inducers. Morphological changes in monocytes in the presence of a differentiation inducer were studied using Confocal Laser Scanning Microscopy (CLSM), and NADPH oxidase expression was studied using Immunoblotting. The regulation of the enzyme complex was studied in both monocytes and macrophages, and the role of EVs secreted from monocytes and their effect on differentiating monocytes as messengers or antioxidant cargo was also studied. Our results showed that monocytes turn into macrophages when they encounter inflammation or differentiation and produce ROS. Polyunsaturated fatty acids (PUFA) are easily damaged by these reactive molecules and can lead to reactive intermediates or products such as aldehydes. Malondialdehyde (MDA), which is one of the final products of polyunsaturated fatty acids peroxidation led to protein modification. Furthermore, the involvement of EVs derived from monocytes and their co-culture with non-differentiating/differentiating cells showed that EVs through their cargo of regulatory molecules, potentially NOX isotypes contributed to the modulation of polarization of monocytes to distinct macrophage phenotypes. We studied the effect of selected exogenous antioxidant/bioactive compounds isolated from industrial waste to understand their efficacy in protecting the cells against oxidative damage. The intricate equilibrium and transition between the pro-oxidant and antioxidant effects of antioxidants were also investigated.

Keywords

monocyte, macrophages, NADPH expression, protein modification, NOX, bioactive compounds, free radicals, extracellular vesicles

Language used: English

Date on which the thesis was submitted / produced: 10. 5. 2024

Thesis defence

• Supervisor: Mgr. Ankush Prasad, Ph.D.

Palacký University Olomouc

Faculty of Science

Doctoral programme / field:
Biophysics / Biophysics