Acute-Phase Deaths from Murine Polymicrobial Sepsis Are Characterized by Innate Immune Suppression Rather Than Exhaustion

Abstract

Sepsis, a leading cause of death in the United States, has poorly understood mechanisms of mortality. To address this, our model of cecal ligation and puncture (CLP) induced sepsis stratifies mice as predicted to Live (Live-P) or Die (Die-P) based on plasma IL-6. Six hours post-CLP, both Live-P and Die-P groups have equivalent peritoneal bacterial colony forming units and recruitment of phagocytes. By 24 h, however, Die-P mice have increased bacterial burden, despite increased neutrophil recruitment, suggesting Die-P phagocytes have impaired bacterial killing. Peritoneal cells were used to study multiple bactericidal processes: bacterial killing, reactive oxygen species (ROS) generation, and phagocytosis. Total phagocytosis and intraphagosomal processes were determined with triple-labeled Escherichia coli, covalently labeled with ROS- and pH-sensitive probes, and an ROS/pH-insensitive probe for normalization. Although similar proportions of Live-P and Die-P phagocytes responded to exogenous stimuli, Die-P phagocytes showed marked deficits in all parameters measured, thus suggesting immunosuppression rather than exhaustion. This contradicts the prevailing sepsis paradigm that acute-phase sepsis deaths (<5 d) result from excessive inflammation, whereas chronic-phase deaths (>5 d) are characterized by insufficient inflammation and immunosuppression. These data suggest that suppression of cellular innate immunity in sepsis occurs within the first 6 h.

Figure 1

Phagocyte Dysfunction Precedes Death from CLP-induced Sepsis. CLP was performed to induce ~50% mortality in ICR CD1-outbred mice. Plasma was collected at 24 hours post CLP and animals were monitored for survival. The mice who died in the first 5 days had significantly higher plasma levels of IL-6 (A). ROC curve analysis was used to generate IL-6 values predictive for mortality. None of the mice with IL-6 >3.13 ng/ml survived (B). Peritoneal bacterial loads and cellular recruitment between Live-predicted/Die-predicted mice were compared following euthanasia at 6 or 24 hours post-CLP. Panel C total peritoneal cells, Panel D bacterial CFUs. *=p<0.05, ***= p<0.0001 between the indicated groups.

Figure 2

Die-P Mice Exhibit Impaired Bacterial Killing of E. coli at 6 and 24hr post-CLP. E.coli were incubated with or without peritoneal cells for 1hr and then plated overnight for CFU counts. Data from at least 3 independent experiments. N=7-13/group at 6hr, 7-8/group at 24hr, where N represents an individual mouse. ***= p<0.0001 comparing the indicated groups.

Figure 3

Die-P Peritoneal Phagocytes have Decreased ROS Burst. (A) Live-P and Die-P peritoneal cells loaded with Dihydrorhodamine-1,2,3 were incubated with OpH-E.coli or PMA to delineate physiological-response ROS or Cellular ROS Capacity via flow cytometry. Histograms show representative staining of PMN incubated with (red histogram) or without OpH-E.coli (blue). (B-C) Bar graphs show results from 3 independent experiments (N=4-7 mice per group. (D-E) Representative ROS Kinetics for unstimulated (basal) (D) or Opsonized-E.coli (E) stimulated cells for Live-P and Die-P mice, as measured by Luminol based chemiluminescence (RLU). (F) Bar graphs show the Area Under the Curve (AUC) calculated for samples in unstimulated and stimulated conditions. (N=3 mice / group). (D). *=p<0.05, **=p<0.01,***=p<0.001 between live-P and die-P. Black bars = Die-P mice, Open bars = Live-P mice.

Figure 4

Die-P Phagocytes Are Suppressed in Phagocytosis. 3X-Labeled E.coli were incubated with peritoneal cells for 30min. Gating for Phagocytosis⁺ events: (A) Ice control left, 37°C incubation right. Trypan blue was used to quench extracellular fluorescence. The percentage of Peritoneal cells from Live-P and Die-P mice that phagocytize bacteria at 6hr (B) and 24hr post-CLP (C), and the overall amount of bacteria internalized at 6hr (D) and 24hr (E). Data collected from 3-4 independent experiments. N=4/group for 6hr, N=5-7/group for 24hr. *p<0.05, ***p<0.0001 Live-P vs Die-P.

Figure 5

Gating for Neutrophils and Macrophages based on Phagocytic Activity. 3X-Labeled E.coli were incubated with peritoneal cells for 30min and Trypan blue added to quench extracellular fluorescence. (A) Contour Plots for Phagocytosis⁺ events. (B) DCF and pHrodo fluorescence of phagocytosis⁺ events, (C) Normalization of DCF and pHrodo fluorescence to Alexa-350 fluorescence. (D) Cytospin preparations of sorted high ROS and low ROS events reveals them to be neutrophils and macrophages, respectively (D). (E) The overall amount of internalized bacteria for neutrophils and macrophages was calculated via its Alexa-350 fluorescence (E). *p<0.05,**p<0.01 ***p<0.001, Live-P vs Die-P. N=4 at 6hr and 5-7 at 24hr from 3 independent experiments.

Figure 6

Die-P Peritoneal Phagocytes are Suppressed in Phagosomal Maturation. 3X-Labeled E.coli were incubated with peritoneal cells for 30min and Trypan blue added to quench extracellular fluorescence. PMN phagosomal ROS was calculated by calibrating DCF (ROS sensitive) fluorescence to Alexa-350 fluorescence (ROS/pH insensitive) at 6hr (A) and 24hr post-CLP (B). PMN phagosomal acidification was calculated by normalizing pHrodo fluorescence to Alexa-350 at 6hr (C) and 24hr (D). Macrophage phagosome acidification at 6hr (E) and 24hr (F). *p<0.05,**p<0.01 ***p<0.001 Live-P vs Die-P. N=5-7 from 3 independent experiments.