Neutrophil Proteome: Lessons from Different Standpoints

Fontes

et al. 2020

IJMS

Intestinal ischemia reperfusion injury (iIRI) is a severe clinical condition presenting high morbidity and mortality worldwide. Some of the systemic consequences of IRI can be prevented by applying ischemic preconditioning (IPC), a series of short ischemia/reperfusion events preceding the major ischemia. Although neutrophils are key players in the pathophysiology of ischemic injuries, neither the dysregulation presented by these cells in iIRI nor the protective effect of iIPC have their regulation mechanisms fully understood. Protein phosphorylation, as well as the regulation of the respective phosphatases and kinases are responsible for regulating a large number of cellular functions in the inflammatory response. Moreover, in previous work we found hydrolases and transferases to be modulated in iIR and iIPC, suggesting the possible involvement of phosphatases and kinases in the process. Therefore, in the present study, we analyzed the phosphoproteome of neutrophils from rats submitted to mesenteric ischemia and reperfusion, either submitted or not to IPC, compared to quiescent controls and sham laparotomy. Proteomic analysis was performed by multi-step enrichment of phosphopeptides, isobaric labeling, and LC-MS/MS analysis. Bioinformatics was used to determine phosphosite and phosphopeptide abundance and clustering, as well as kinases and phosphatases sites and domains. We found that most of the phosphorylation-regulated proteins are involved in apoptosis and migration, and most of the regulatory kinases belong to CAMK and CMGC families. An interesting finding revealed groups of proteins that are modulated by iIR, but such modulation can be prevented by iIPC. Among the regulated proteins related to the iIPC protective effect, Vamp8 and Inpp5d/Ship are discussed as possible candidates for control of the iIR damage.

Section: Introductionmentioning

confidence: 99%

Phosphoproteomic Analysis of Rat Neutrophils Shows the Effect of Intestinal Ischemia/Reperfusion and Preconditioning on Kinases and Phosphatases

Fontes

et al. 2020

IJMS

“…The current understanding of the interconnections among the many signal transduction pathways that regulate neutrophil activation is incomplete. Proteomics research has improved the understanding of the neutrophil biology in the past, and few publications describe the effect of the inflammatory response on the PMNs proteome (Morris et al, 2008 ). There is one proteomic study on the response to intestinal ischemia and reperfusion, however limited on molecules expressed by the intestinal epithelium, and there is lack of understanding regarding the PMNs response against IR.…”

Section: Introductionmentioning

confidence: 99%

Analysis of the Effect of Intestinal Ischemia and Reperfusion on the Rat Neutrophils Proteome

Fontes

et al. 2018

Front. Mol. Biosci.

Intestinal ischemia and reperfusion injury is a model system of possible consequences of severe trauma and surgery, which might result into tissue dysfunction and organ failure. Neutrophils contribute to the injuries preceded by ischemia and reperfusion. However, the mechanisms by which intestinal ischemia and reperfusion stimulate and activate circulating neutrophils is still not clear. In this work, we used proteomics approach to explore the underlying regulated mechanisms in Wistar rat neutrophils after ischemia and reperfusion. We isolated neutrophils from three different biological groups; control, sham laparotomy, and intestinal ischemia/reperfusion. In the workflow, we included iTRAQ-labeling quantification and peptide fractionation using HILIC prior to LC-MS/MS analysis. From proteomic analysis, we identified 2,045 proteins in total that were grouped into five different clusters based on their regulation trend between the experimental groups. A total of 417 proteins were found as significantly regulated in at least one of the analyzed conditions. Interestingly, the enzyme prediction analysis revealed that ischemia/reperfusion significantly reduced the relative abundance of most of the antioxidant and pro-survival molecules to cause more tissue damage and ROS production whereas some of the significantly up regulated enzymes were involved in cytoskeletal rearrangement, adhesion and migration. Clusters based KEGG pathways analysis revealed high motility, phagocytosis, directional migration, and activation of the cytoskeletal machinery in neutrophils after ischemia and reperfusion. Increased ROS production and decreased phagocytosis were experimentally validated by microscopy assays. Taken together, our findings provide a characterization of the rat neutrophil response to intestinal ischemia and reperfusion and the possible mechanisms involved in the tissue injury by neutrophils after intestinal ischemia and reperfusion.

“…Trauma, infection, hemorrhage, the response to both elective and emergency surgical interventions, and other pathological processes are incredibly prevalent in the human population. Such conditions are often complicated by nefarious immune responses that arise from these events that are, at least partially, mediated by neutrophils [ 1 – 4 ], since these are cells known for their central role in the mechanisms of inflammation in mammals [ 5 , 6 ].…”

Section: Introductionmentioning

confidence: 99%

Reconciling the IPC and Two-Hit Models: Dissecting the Underlying Cellular and Molecular Mechanisms of Two Seemingly Opposing Frameworks

Morris

Journal of Immunology Research

et al. 2015

Inflammatory cascades and mechanisms are ubiquitous during host responses to various types of insult. Biological models and interventional strategies have been devised as an effort to better understand and modulate inflammation-driven injuries. Amongst those the two-hit model stands as a plausible and intuitive framework that explains some of the most frequent clinical outcomes seen in injuries like trauma and sepsis. This model states that a first hit serves as a priming event upon which sequential insults can build on, culminating on maladaptive inflammatory responses. On a different front, ischemic preconditioning (IPC) has risen to light as a readily applicable tool for modulating the inflammatory response to ischemia and reperfusion. The idea is that mild ischemic insults, either remote or local, can cause organs and tissues to be more resilient to further ischemic insults. This seemingly contradictory role that the two models attribute to a first inflammatory hit, as priming in the former and protective in the latter, has set these two theories on opposing corners of the literature. The present review tries to reconcile both models by showing that, rather than debunking each other, each framework offers unique insights in understanding and modulating inflammation-related injuries.