Monitoring pro-resolving leukocyte responses in peripheral blood predicts clinical severity during sepsis

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Abstract Sepsis is characterized by immune cell dysfunction, which is linked to the pathophysiology of the disease with consequent organ injury and increased associated mortality. In experimental sepsis in mice, regulating leukocyte function decreases organ injury and lessens the severity of the infection. Assessment of peripheral blood leukocyte activation and function may serve as prognostic clinical biomarkers and inform new therapeutic strategies for the management of human sepsis and inflammatory diseases. Resolution of inflammation is an active process with anti-inflammatory and pro-resolving mechanisms. There are several families of resolution mediators, termed “specialized pro-resolving mediators (SPMs)”, that serve as agonists at cognate receptors to transduce cell-type specific responses on leukocytes and in experimental model systems these SPMs decrease the severity and duration of sepsis. Recently, in microliter quantities of peripheral blood, we determined that functional characterization of leukocyte responses was more informative than leukocyte counting for clinical assessment of the trajectory of critical illness. In response to the NIGMS NOT-GM-19-054 “Exploring the Scientific Value of Existing or New Sepsis Human Biospecimen Collections”, the proposed proof of concept and scale-up studies are designed to determine the scientific value of our existing and ongoing Registry of Critical Illness biorepository of human sepsis biospecimens with associated patient health record data. These biospecimens are collected at presentation, hospital day 3 and hospital 7 to align with the clinical trajectory of the patient, linked to clinical datasets and represent transformative potential for sepsis endotyping/stratification. Here, we propose the use of isodielectric separation as a contemporary cutting-edge technology in the analysis of these biospecimens to uncover leukocyte activation during the upslope and resolution of patient’s immune responses that will test the hypothesis: Sepsis hyper- inflammatory responses result from defective endogenous resolution mechanisms and that these defects are evident in peripheral blood leukocyte activation and functional responses to SPMs. To address this hypothesis, we plan four specific aims. In the R21 phase, (1) demonstrate reproducible isolation and functional analysis of neutrophils from capillary blood, and (2) demonstrate ability to obtain deep proteomic and phosphoproteomic profiles from small numbers of neutrophils. In the R33 phase, (3) longitudinal analysis of SPM pathway activation during sepsis resolution, and (4) inference and model generation and testing with ex vivo assays. The goals of this application are to develop an improved understanding of the resolution defects that unleash unrestrained inflammation of sepsis with a longer term goal for new therapeutic targets and prognostic indicators of disease progression. Important products of this application include provision of guidance on the best practices for collecting, utilizing, and analyzing human biospecimens to maximize their value for the entire sepsis research community.