Quantitative phosphoproteomics reveals diverse stimuli activate distinct signaling pathways during neutrophil activation

Abstract

Neutrophils display functional heterogeneity upon responding diversely to physiological and pathological stimulations. During type 2 diabetes (T2D), hyperglycemia constitutively activates neutrophils, leading to reduced response to infections and on the other hand, elevated metabolic intermediates such as homocysteine induce bidirectional activation of platelets and neutrophils leading to thrombosis. Hence, in the context of T2D-associated complications, we examined the influence of high glucose, homocysteine, and LPS representing effector molecules of hyperglycemia, thrombosis, and infection, respectively, on human neutrophil activation to identify distinct signaling pathways by quantitative phosphoproteomics approach. High glucose activated C-Jun-N-Terminal Kinase, NTRK1, SYK, and PRKACA kinases associated with Rho GTPase signaling and phagocytosis, whereas LPS induced AKT1, SRPK2, CSNK2A1, and TTN kinases involved in cytokine signaling and inflammatory response. Homocysteine treatment led to activatation of LRRK2, FGR, MAPK3, and PRKCD kinases which are associated with neutrophil degranulation and cytoskeletal remodeling. Diverse inducers differentially modulated phosphorylation of proteins associated with neutrophil functions such as oxidative burst, degranulation, extracellular traps, and phagocytosis. Further validation of phosphoproteomics data on selected kinases revealed neutrophils pre-cultured under high glucose showed impeded response to LPS to phosphorylate p-ERK1/2Thr202/Tyr204, p-AKTSer473, and C-Jun-N-Terminal KinaseSer63 kinases. Our study provides novel phosphoproteome signatures that may be explored to understand neutrophil biology in T2D-associated complications.

Immunology of chronic low-grade inflammation: relationship with metabolic function

“…This observation is supported by various animal studies indicating that obesity predisposes neutrophils to spontaneously release weak NETs without stimulation (Cichon et al 2021, Burczyk et al 2022. This process is thought to involve C-Jun-N-terminal kinase (JNK) induced Rho GTPase kinase activity which is known to play an important role in NADPH oxidase activation as well as regulation of actin cytoskeletal rearrangement, both of which are required for NETs formation (Lacy & Eitzen 2008, Gavillet et al 2018, Thimmappa et al 2022. The externalized components of NETs which include chromatin, citrullinated histones, cell-free DNA, nucleosomes, and NE persist in vasculature and have been linked to the pathogenesis of disease progression (Kaplan & Radic 2012, Papayannopoulos 2018).…”

“…Defects in intracellular glucose cycling can therefore negatively impact key neutrophil functions, whereas a disproportionate increase in glycolysis due to the excessive availability of extracellular glucose fuels persistent neutrophil activity (Sadiku et al 2021). Thimmappa et al (2022) elucidated specific neutrophil responses following various activating signals by assessing the differential phosphorylation of proteins using an unbiased quantitative phosphoproteomics approach. The authors indicated that the ex vivo exposure of neutrophils to hyperglycaemia for a period of 3 h induced a 6-fold increase in NETs formation compared to only a 3-fold increase upon stimulation with either lipopolysaccharide (LPS) (bacterial infection) or the metabolic intermediate homocysteine for the same period.…”

“…It is furthermore unclear if blocking persistent NADPH oxidase activity will restore the normal functional responses of neutrophils. Despite the high glycolytic potential of neutrophils and their ability to oxidize fatty acids, their continuous activation under high glucose conditions limits the responsiveness of these leukocytes to subsequent stimulation with LPS and impairs chemotaxis (Joshi et al 2020, Roy et al 2022, Thimmappa et al 2022. A delay in neutrophil trafficking and inability to mount a sufficient phagocytic response together with limited production of additional stronger NETs due to this 'exhausted phenotype' make diabetic patients vulnerable to infection.…”

Immunoporosis: Role of immune system in the pathophysiology of different types of osteoporosis

“…Previous research found that hyperglycemia increases the number of circulating neutrophils ( 103 ). However, the later studies found that the function of neutrophils is impaired in high glucose conditions, and their phagocytic and killing functions are inhibited to a certain extent ( 104 , 105 ). These results reflected that hyperglycemia induces massively impaired neutrophils in peripheral blood, resulting in chronic inflammation.…”

Neutrophil sub-types in maintaining immune homeostasis during steady state, infections and sterile inflammation

“…Accordingly, cell surface markers such as olfactomedin, CD177, CXCR4 + CD62L − and many others have also been basis of defining neutrophil subtypes which showed functional alterations in diseases [ 29 – 31 ]. Moreover, studies from many laboratories, including ours, have shown activation of stimulus-specific signalling pathways in neutrophils [ 32 , 33 ]. For example, high glucose, LPS and homocysteine representing stimuli for diabetes, infections and thrombosis, respectively, induced distinct set of kinases which were associated with specific functions of neutrophils [ 33 ].…”