Human Myeloid-derived Suppressor Cells are Associated With Chronic Immune Suppression After Severe Sepsis/Septic Shock

Abstract

Objective: We hypothesized that after sepsis in humans, MDSCs will be persistently increased, functionally immunosuppressive, and associated with adverse clinical outcomes.

Background: Cancer and sepsis have surprisingly similar immunologic responses and equally dismal long term consequences. In cancer, increased myeloid-derived suppressor cells (MDSCs) induce detrimental immunosuppression, but little is known about the role of MDSCs after sepsis.

Methods: Blood was obtained from 74 patients within 12 hours of severe sepsis/septic shock (SS/SS), and at set intervals out to 28 days, and also in 18 healthy controls. MDSCs were phenotyped for cell surface receptor expression and enriched by cell sorting. Functional and genome-wide expression analyses were performed. Multiple logistic regression analysis was conducted to determine if increased MDSC appearance was associated with in-hospital and long-term outcomes.

Results: After SS/SS, CD33CD11bHLA-DR MDSCs were dramatically increased out to 28 days (P < 0.05). When co-cultured with MDSCs from SS/SS patients, antigen-driven T-cell proliferation and TH1/TH2 cytokine production were suppressed (P < 0.05). Additionally, septic MDSCs had suppressed HLA gene expression and up-regulated ARG1 expression (P < 0.05). Finally, SS/SS patients with persistent increased percentages of blood MDSCs had increased nosocomial infections, prolonged intensive care unit stays, and poor functional status at discharge (P < 0.05).

Conclusions: After SS/SS in humans, circulating MDSCs are persistently increased, functionally immunosuppressive, and associated with adverse outcomes. This novel observation warrants further studies. As observed in cancer immunotherapy, MDSCs could be a novel component in multimodality immunotherapy targeting detrimental inflammation and immunosuppression after SS/SS to improve currently observed dismal long-term outcomes.

Figure 1. Percentage MDSCs in Patient with Severe Sepsis/Septic Shock over 28 Days

A. MDSC percentages are significantly elevated in severe sepsis/septic shock (SS/SS) patients when compared to healthy controls (HC) at all time-points. SS/SS patients have an immediate elevation in MDSCs (CD33⁺ CD11b⁺ HLA-DR^−/low) within hours of clinical symptoms that persists for at least 28 days. B. Elevated levels of MDSCs in SS/SS patients are predominantly granulocytic (CD33⁺ CD11b⁺ HLA-DR^−/low CD14⁻ CD15⁺). C. SS/SS patients have a significant decrease in monocytic-MDSCs (CD33⁺ CD11b⁺ HLA-DR^−/lowCD15⁺ CD14⁺) compared to HC subjects. D. Total number of patients per time point. The HC subjects had a single blood draw (n=18). The study cohort decreased as patients were discharged or expired.

Figure 2. MDSC Effect on T-cell Proliferation

A. A single healthy control (HC) subject’s T-cell suppression data, representative of our data set, is shown here as a histogram. HC CD3⁺ T-cells were stimulated with IL-2 and anti-CD3/CD28 in the absence and presence of MDSCs. Unstimulated HC T-cells do not actively replicate (black peak); however, stimulated HC T-cells replicate and fluorescence intensity is divided between daughter cells during each replication cycle; this results in decreased fluorescence and multiple peak shifts towards the left (green line). The height of this peak signals frequency of T-cells undergoing replication. However, HC T-cells incubated with MDSCs from severe sepsis/septic shock (SS/SS) patients are significantly suppressed in a dose-dependent fashion and remain non-replicating. B. MDSCs from either HC subjects (n=4) or SS/SS patients (n=4) were incubated with HC T-cells in increasing ratios. SS/SS MDSCs are not significantly more suppressive than HC MDSCs, suggesting that HC MDSCs also have immunosuppressive capacity. C. MDSCs suppress T-cell cytokine production. HC T-cells were incubated with SS/SS MDSCs or HC MDSC (2 MDSCs to 1 T-cell) and stimulated with IL-2 and anti-CD3/CD28 beads. SS/SS MDSCs significantly suppressed the ability of HC T-cells to produce INFγ, IL -10, and IL-4.

Figure 3. Severe Sepsis/Septic Shock Patient and Healthy Control Subject Gene Expression

Using a false discovery adjusted probability of <0.001 and a two-fold difference in expression, the temporal pattern of the expression of the sepsis responsive genes differed between MDSCs from the healthy control (HC) subjects and severe sepsis/septic shock (SS/SS) patients. A. There was a significant difference in the expression of 257 genes between MDSCs isolated from SS/SS patients and HC subjects (p <0.001). B. Principle Component Analysis (PCA) of the gene expression from the 257 genes demonstrates the overall differences in the patterns of gene expression. Gene expression patterns from HC subjects (red) are tightly grouped to the right and the SS/SS patients (blue) are tightly grouped to the left. The tight grouping and lack of overlap of data points reflects the significant difference between the two groups.

Figure 4. Increased Circulating MDSCs in Severe Sepsis/Septic Shock Patients with Subacute Mortality

A. Patients who succumb to early mortality (<14 days) have significantly elevated percentages of MDSCs when compared to patients that survive greater than 14 days at both the 12 and 24 hour time points from onset of clinical sepsis. MDSC levels then sharply decline until death. Patients that survive longer than 14 days have similar levels of MDSCs; however, MDSCs are slightly elevated at all time-points in those patients that remain in the ICU longer than 14 days. At 14 days, patients that will continue to remain in the ICU have significantly elevated percentages of MDSCs compared to those patients who are transferred out of the ICU. B. Total number of patients per time point.

Figure 5. Percentage of MDSCs is more Clinically Prognostic than Absolute Number of MDSCs

A. Patients that succumb to early mortality (<14 days) have a relative leukopenia during the first 24 hours when compared to those who survive greater than 14 days. Patients with ICU LOS greater than or equal to 14 days have a persistently higher leukocytosis when compared to those patients with ICU LOS less than 14 days. B. Patients with early mortality have similar absolute numbers of MDSCs as survivors because the relative leukopenia is offset by a significantly greater percentage of MDSCs (Figure 4). Patients with shorter ICU LOS (<14 days) have lower absolute numbers of MDSCs, this is statistically significant at days 4, 7 and 14 and correlates with the data reported on percentage of MDSCs. C & D. Total number of patients per time point.