Overexpression of PD-1-related molecules is associated with lymphocyte anergy, mortality, and development of nosocomial infections in septic shock patients

Introduction

Septic syndromes are a culmination of multiple partially understood dynamic processes. However, it is now established that, after a transient exacerbated proinflammatory response, a counter-regulatory phase develops, rapidly inducing immune alterations that are thought to play a major role in patients' mortality and susceptibility to nosocomial infections. Programmed Death-1 (PD-1) receptor and its ligands PD-L1 and PD-L2 constitute a newly described pathway that negatively controls immune responses. Recently, improved bacterial clearance and decreased mortality were observed in PD-1 knockout mice [1]. The objective of the present study was to investigate PD-1-related molecule expressions in septic shock patients.

Methods

PD-1-related molecule expressions were measured by flow cytometry on circulating leukocytes from 64 septic shock patients and 49 healthy individuals. Severity scores (SAPS II, SOFA), clinical events (28-day mortality, occurrence of nosocomial infections) and the usual biomarkers of sepsis-induced immunosuppression (monocyte HLA-DR expression, lymphocyte phenotyping including Treg, plasmatic IL-10 concentration) were assessed. Ex vivo functional assays such as lymphocyte proliferation ([³H] thymidine incorporation) in response to phytohemagglutinin, and cytokine release (TNFα and IL-10 assessed by Bio-Plex technique) after overnight LPS incubation, were performed in the presence of blocking antibodies against PD-1-related molecules.

Results

Patients presented with typical features of sepsis-induced immuno-suppression (decreased mHLA-DR expression, increased Treg percentage, decreased LPS-induced TNFα release). At days 1 to 2 and days 3 to 5 after the onset of shock, patients displayed increased PD-1 and PD-L1 expressions on CD4⁺ T lymphocytes and enhanced PD-1, PD-L1 and PD-L2 expressions on monocytes. See Figure 1 overleaf. Nonsurvivors presented with increased monocyte PD-L1 expression while enhanced monocyte PD-1 or PD-L2 expressions were associated with the occurrence of secondary nosocomial infections. In addition, decreased mitogen-induced lymphocyte proliferation was negatively correlated with increased lymphocyte PD-1 and PD-L1 expressions whereas monocyte PD-1-related molecule expressions were highly correlated with increased circulating IL-10 concentration. No beneficial effects of anti-PD-1-related molecule antibodies were observed.

Figure 1

PD-1, PD-L1 and PD-L2 measurements on circulating monocytes and CD4⁺ lymphocytes in septic shock patients and healthy volunteers. PD-1-related molecule expressions were measured on (a) circulating monocytes and (b) CD4⁺ lymphocytes in whole blood from healthy volunteers (n =40) and septic shock patients at D1 to D2 (n = 37) and at D3 to D5 (n =56) after the onset of shock. Results presented as percentages of positive cells among total population of monocytes or CD4⁺ lymphocytes and as boxplots and individual values. *P < 0.020, **P ≤ 0.002 (Mann-Whitney test). P < 0.025 was considered statistically significant (with correction by the number of tests).

Conclusions

We describe here for the first time the overexpression of PD-1-related molecules on circulating leukocytes in septic shock patients. Importantly, these increased expressions were significantly associated with the occurrence of immune dysfunctions, secondary nosocomial infection, and death after septic shock. Taken together, our results suggest that PD-1-related molecules may constitute an additional regulatory system involved in sepsis-induced immune alterations. This may offer innovative therapeutic perspectives for the treatment of this hitherto deadly disease.