PBMC Adhesion Response: A Physiologic Biomarker for Immune Cell Exhaustion in Septic Shock?

Introduction: Immune response in sepsis transitions from hyper-inflammation to persistent inflammation and immunosuppression (PII). During PII, immune cells undergo immune exhaustion with inability to clear pathogen. Endotoxin-tolerance is a known marker of immune cell-exhaustion, but a rapid test it is not available. Using in vivo leukocyte adhesion in mice, we showed that the endotoxin-tolerant phase of PII occurs via sirtuin 2 (SIRT2). We hypothesized, that the ex vivo peripheral blood mononuclear cell (PBMC) adhesion is a physiological biomarker of immune cell-exhaustion in septic shock (SS) and is regulated by SIRT2. We developed PBMC adhesion assay to test our hypothesis and used human monocyte THP-1 cell line to study mechanisms.


Methods: We isolated blood PBMCs from SS patients from medical intensive care units and age/sex matched healthy controls (H). We excluded patients with COVID-19 infection, and immunosuppression. We stimulated the SS and H-PBMCs with E. coli lipopolysaccharide (LPS) to assess TNF-α, IL-6, IL-1β, and IL-10 mRNA and SIRT2 expression. In endotoxin-tolerant SS-PBMCs, we further studied adhesion (assay time: 15 minutes) and SIRT2 expression. Mechanistically, in endotoxin-tolerant THP-1 cells, we studied SIRT2 expression, adhesion, and adhesion receptor PSGL-1 expression. In endotoxin-tolerant THP-1 cells treated with SIRT2 inhibitor AK-7, we studied cytokine expression and adhesion ± LPS.


Results: SS-PBMCs showed muted TNF-α, IL-6 and IL-1β mRNA response to LPS (endotoxin-tolerance) and increased IL-10 mRNA and SIRT2 expression. In endotoxin-tolerant SS-PBMCs, we found muted adhesion with LPS vs. H-PBMCs (adhesion-tolerance). Endotoxin-tolerant THP-1 cells showed significantly increased SIRT2 expression, like SS PBMCs. AK-7-treated endotoxin-tolerant THP-1 cells showed a robust increase in TNF-α mRNA and adhesion with LPS. Together, these results suggest the adhesion assay can rapidly detect immune cell-exhaustion and it is regulated by SIRT2.


Conclusions: 1) PBMC-adhesion assay is a potential physiological marker of immune cell-exhaustion in septic shock patients 2) SIRT2 modulates immune cell exhaustion in septic shock patients. SIRT2 inhibition is a potential therapy to reverse immune cell-exhaustion in septic shock patients.