invariant Natural Killer T Cells Modulate the Peritoneal Macrophage Response to Polymicrobial Sepsis

Abstract

Introduction: A dysregulated immune system is a major driver of the mortality and long-term morbidity from sepsis. With respect to macrophages, it has been shown that phenotypic changes are critical to effector function in response to acute infections, including intra-abdominal sepsis. Invariant natural killer T cells (iNKT cells) have emerged as potential central regulators of the immune response to a variety of infectious insults. Specifically, various iNKT cell:macrophage interactions have been noted across a spectrum of diseases, including acute events such as sepsis. However, the potential for iNKT cells to affect peritoneal macrophages during an abdominal septic event is as yet unknown.

Methods: Cecal ligation and puncture (CLP) was performed in both wild type (WT) and invariant natural killer T cell knockout (iNKT-/-) mice. 24 h following CLP or sham operation, peritoneal macrophages were collected for analysis. Analysis of macrophage phenotype and function was undertaken to include analysis of bactericidal activity and cytokine or superoxide production.

Results: Within iNKT^-/- mice, a greater degree of intraperitoneal macrophages in response to the sepsis was noted. Compared to WT mice, within iNKT^-/- mice, CLP did induce an increase in CD86⁺ and CD206⁺, but no difference in CD11b⁺. Unlike WT mice, intra-abdominal sepsis within iNKT-/- mice induced an increase in Ly6C-int (5.2% versus 14.9%; P < 0.05) and a decrease in Ly6C-high on peritoneal macrophages. Unlike phagocytosis, iNKT cells did not affect macrophage bactericidal activity. Although iNKT cells did not affect interleukin-6 production, iNKT cells did affect IL-10 production and both nitrite and superoxide production from peritoneal macrophages.

Conclusions: The observations indicate that iNKT cells affect specific phenotypic and functional aspects of peritoneal macrophages during polymicrobial sepsis. Given that pharmacologic agents that affect iNKT cell functioning are currently in clinical trial, these findings may have the potential for translation to critically ill surgical patients with abdominal sepsis.

Keywords: Intra-abdominal sepsis; Invariant natural killer T cells; Peritoneal macrophages; Sepsis.

Figure1:

A) Representative flow cytometry image of B) apoptosis among peritoneal macrophages from both wild type (WT) and iNKT^−/− mice. While CLP induced an increase in peritoneal macrophage apoptosis among WT mice, no effect was noted within iNKT^−/− mice. (Bars on the box plots indicate median and lower and upper edge of the box indicate lower and upper quartiles. *=p<0.05 by ANOVA. N=5-6 mice/group).

Figure 2)

Phenotype of peritoneal macrophages. 2a - Representative flow cytometry image denoting that among iNKT^−/− mice, CLP was noted to induce an increase in the Ly6C-int population among peritoneal macrophages. 2b – iNKT-cells affected phenotypic profile of peritoneal macrophages (Bars on the box plots indicate median and lower and upper edge of the box indicate lower and upper quartiles; *=p<0.05 versus all others by ANOVA. N=5-6 mice/group)

Figure 2)

Phenotype of peritoneal macrophages. 2a - Representative flow cytometry image denoting that among iNKT^−/− mice, CLP was noted to induce an increase in the Ly6C-int population among peritoneal macrophages. 2b – iNKT-cells affected phenotypic profile of peritoneal macrophages (Bars on the box plots indicate median and lower and upper edge of the box indicate lower and upper quartiles; *=p<0.05 versus all others by ANOVA. N=5-6 mice/group)

Figure 3)

Macrophage bactericidal activity assay. CLP was noted to increase bactericidal activity from both WT and iNKT−/− mice. It was noted that iNKT-cells did not appear to affect peritoneal macrophage bactericidal activity. (Bars on the box plots indicate median and lower and upper edge of the box indicate lower and upper quartiles; *=p<0.05 by ANOVA. N=5-6 mice/group).

Figure 4:

Peritoneal Macrophage cytokine production with respect to a) IL-6, b) TNF-α and c) IL-10 following sepsis from both WT and iNKT^−/− mice. (Bars on the box plots indicate median and lower and upper edge of the box indicate lower and upper quartiles; *=p<0.05 by ANOVA; †=p<0.05 by ANOVA. N=5-6 mice/group).

Figure 4:

Peritoneal Macrophage cytokine production with respect to a) IL-6, b) TNF-α and c) IL-10 following sepsis from both WT and iNKT^−/− mice. (Bars on the box plots indicate median and lower and upper edge of the box indicate lower and upper quartiles; *=p<0.05 by ANOVA; †=p<0.05 by ANOVA. N=5-6 mice/group).

Figure 4:

Peritoneal Macrophage cytokine production with respect to a) IL-6, b) TNF-α and c) IL-10 following sepsis from both WT and iNKT^−/− mice. (Bars on the box plots indicate median and lower and upper edge of the box indicate lower and upper quartiles; *=p<0.05 by ANOVA; †=p<0.05 by ANOVA. N=5-6 mice/group).

Figure 5a) –

Superoxide production declined following CLP in WT mice, whereas superoxide increased following CLP in iNKT^−/− mice. (Mean ± SEM; *=p<0.05 by ANOVA. N=5-6 mice/group).

Figure 5b) –

In WT Sepsis induced increased nitrite production from peritoneal macrophages, an effect that was enhanced with the addition of IFN-γ. This effect was not noted among macrophages from iNKT^−/− mice (Bars on the box plots indicate median and lower and upper edge of the box indicate lower and upper quartiles; *=p<0.05 by ANOVA. N=5-6 mice/group).