Abstract:

Neutrophils in type 2-diabetes are associated with elevated levels of oxidative stress. Hyperglycemia, a characteristic downstream effect of poorly controlled type 2-diabetes, generate reactive oxygen species (ROS) by activating NADPH-oxidase directly or via ligation of advanced glycation end-products (AGEs) to pattern recognition receptors expressed on the surface of neutrophils-RAGE. Reactive oxygen species thus generated serves as a second messenger in determining neutrophil cell fate by modifying various cellular pathways. While there are several reports on production of ROS, how elevated levels of ROS in those with type 2-diabetes impact neutrophil functions remains to be elucidated. Neutrophils isolated from those with type 2-diabetes are shown to spontaneously release neutrophil extracellular traps (NETs). Spontaneous NETosis in diabetes have been attributed to hyperglycemia mediated elevated levels of proinflammatory cytokines and end products of metabolic rearrangement however, the exact cellular mechanism implicating ROS in neutrophil impairment remains to be determined. In this study, the pathway initiated by elevated levels of ROS in hyperglycemic conditions are investigated, and its role in induction of NETs and phagocytosis in those with type 2-diabetes is determined. Results of this study demonstrate that hyperglycemia, directly and through increased levels of AGE and concomitant increase in expression of RAGE-cognate receptor for AGE, generates ROS by activation of cytoplasmic NADPH oxidase. ROS thus generated induces autophagy which serves as a primer for induction of NETs most likely leading to decrease in phagocytosis. The in vitro observations were further confirmed by using blood samples from those with type 2-diabetes, The results indicated the following:

(i) partial assembly of NADPH and its concomitant elevated levels of ROS in neutrophils from diabetes, 

(ii) levels of ROS were directly related to induction of NETs and reduction in phagocytosis 

(iii) ROS was also associated with elevated levels of LCIIIB which subsequently led to NETosis 

(iv) Blocking ROS and cellular pathways upstream of autophagy led to significant reduction in NETosis 

(v) NETs released by neutrophils isolated from patients with type 2-diabetes are impaired in killing S. pneumoniae. These results suggest the importance of ROS mediated induction of autophagy and its role in induction of NETs. This study is the first to demonstrate the relationship between hyperglycemia mediated ROS, induction of, autophagy, leading to increase in NETosis and decrease in phagocytosis, in the context of type 2- diabetes.

Publications:

1. Farhan A, Hassan G, Ali SH, Yousaf Z, Shafique K, Faisal A, bin Younis B, Mirza S*. Spontaneous NETosis in diabetes: A role of hyperglycemia mediated ROS and autophagy. Frontiers in Medicine. 2023; 10. 

2. Martinez PJ, Farhan A, Mustafa M, Javaid N, Darkoh C, Garrido-Sanabria E, Fisher-Hoch SP, Briles DE, Kantarci A, Mirza S*. PspA facilitates evasion of pneumococci from bactericidal activity of neutrophil extracellular traps (NETs). Microbial pathogenesis. 2019 Nov 1; 136:103653. 

3. Farhan A, and Mirza S*. Structural and functional impairments in NETs released by diabetic neutrophils in response to S. pneumoniae (Manuscript in preparation). 

4. Farhan A, bin Younis B, Mirza S*. Prevalence and risk factors associated with Hepatitis C Virus Infection in patients with type 2- diabetes mellitus; case control study in Lahore, Pakistan (Manuscript in preparation).