Resolvin D2 induces anti-microbial mechanisms in a model of infectious peritonitis and secondary lung infection

Abstract

In severe bacterial infections, there is a pro-inflammatory response to promote bacterial clearance but this response can cause tissue injury. Later, the immune system becomes dysregulated and the host is unable to clear a secondary or a pre-existing infection. Specialized Pro-resolving Mediators (SPMs) such as resolvin D2 (RvD2) have been shown to be beneficial for inflammation/infection resolution in animal models of sepsis but in vivo mechanisms by which RvD2 may promote bacterial clearance and/or attenuate deleterious effects of a secondary infection have not been fully established. In this study, we used the 2-hit model of cecal ligation and puncture (CLP) induced infectious peritonitis and secondary lung infection with Pseudomonas aeruginosa to find possible antimicrobial and immunomodulatory mechanisms of RvD2. We show that RvD2 given as late as 48h after CLP surgery reduced blood bacterial load without altering plasma cytokines compared to mice given saline vehicle. RvD2 increased splenic neutrophil accumulation as well as average reactive oxygen species (ROS) production. There was also an increase in an immature leukocyte population the myeloid derived suppressor cells (MDSCs) in the spleen of RvD2 treated mice. RvD2 reduced lung lavage bacterial load 24h after P. aeruginosa administration and significantly decreased lung lavage levels of IL-23, a cytokine essential in the Th-17 inflammatory response. In addition, we show that RvD2 increased the number of non-inflammatory alveolar macrophages after P. aeruginosa administration compared to saline treated mice. The study uncovered an antimicrobial mechanism of RvD2 where RvD2 increases mature neutrophil and MDSC accumulation into the spleen to promote blood bacterial clearance. The study showed that in this 2-hit model, RvD2 promotes lung bacterial clearance, increased non-inflammatory alveolar macrophage number and inhibits an adaptive immune pathway providing evidence of its resolution mechanism in secondary pulmonary infection.

Keywords: Pseudomonas aeruginosa; alveolar macrophages; cytokines; inflammation; innate immunity; myeloid-derived suppressor cells; neutrophils; specialized pro-resolving mediators.

Figure 1

Schematic representation of our model of infectious peritonitis and secondary lung infection.

Figure 2

CLP mice were given vehicle saline or RvD2 48h after surgery. Mice were sacrificed 24h later and blood sample were taken. Samples were serially diluted, plated on TSA plates and colony forming units (CFUs) were counted 24 hours after plating. RvD2 reduced the blood bacteria load. * P < 0.05, for n = 5 - 6 mice in each group.

Figure 3

Sham or CLP surgery was performed on mice. CLP mice were given vehicle saline or RvD2 48h after surgery. Mice were sacrificed 24h later and blood samples were taken. Using flow cytometry, cytokines including TNF-α, IL-6, IFN-γ, IFN-β, IL-1β, IL-12, IL-17, and MCP-1 were quantified using bead-based immunoassay. CLP increased plasma levels of TNFα, IL-6. IFN-γ, IFN-β, IL-1β and MCP-1 but RvD2 did not change levels further. * P < 0.05, ** P < 0.01 for n = 6 – 14 in all groups.

Figure 4

(A) CLP mice were given vehicle saline or RvD2 48h after surgery. Mice were sacrificed 24h later and spleen samples were taken. Ly6G⁺ splenic neutrophils were gated and counted based on side scatter and Ly6G expression. RvD2 increased the number of Ly6G⁺ splenic neutrophils. Data are mean ± S.E.M. for n = 8 in each group, **P<0.01. (B) ROS production (DHR⁺ cells) in CD11b⁺ Ly6G⁺ splenic neutrophils was measured in unstimulated (basal) and fMLP stimulated cells. Cell density plots (Y-axis: count; X-axis: DHR; oxidative burst) shows oxidative burst in cells taken from CLP and CLP + RvD2 mice. (C) The percentage change in the number of the fMLP stimulated neutrophils (CD11b⁺ Ly6G⁺ DHR⁺) was measured. RvD2 decreased the number of fMLP stimulated neutrophils. (D) The percentage change in median fluorescence intensity (MFI) in CD11b⁺ Ly6G⁺ DHR⁺ cells (basal and stimulated) between cells taken from CLP+RvD2 treated mice and cells from CLP mice was measured. RvD2 increased the MFI of ROS producing splenic neutrophils. All data are mean ± S.E.M. *P<0.05 for n = 3 in all groups.

Figure 5

(A) CLP mice were given vehicle saline or RvD2 48h after surgery. Mice were sacrificed 24h later and spleen samples were taken. Myeloid-derived suppressor cells (MDSCs: CD11b⁺ Ly6C⁺ Ly6G⁺) were identified as shown. (B) Cells from Sham, CLP, and CLP + RvD2 groups were quantified. Splenic MDSC numbers were increased after CLP and RvD2 administration increased the numbers even further. All data are mean ± S.E.M. *P<0.05, *** P < 0.001 for n = 7-11 in all groups. (C) In separate experiments, the percentage change in the number of fMLP stimulated ROS producing MDSCs (CD11b⁺ Ly6G⁺ Ly6C⁺ DHR⁺) was counted for cells from CLP and CLP+RvD2 mice. RvD2 had no effect on the number of MDSC stimulated cells. (D) The percentage change in median fluorescence intensity (MFI) in ROS producing CD11b⁺ Ly6G⁺ Ly6C⁺ DHR⁺ cells (basal and stimulated) between CLP + RvD2 treated mice cells and cells from CLP mice was measured. RvD2 did not affect the MFI of ROS producing MDSCs.

Figure 6

CLP surgery was performed on mice. Mice were given vehicle saline or RvD2 48h after surgery. 24h after injections, mice were given P. aeruginosa intranasally. 24h after the 2^nd hit with P. aeruginosa, mice were sacrificed and lungs lavaged. Lavage fluid was serially diluted and plated on TSA plates. RvD2 administration reduced lung bacteria load. Data are mean ± S.E.M. *P<0.05 for n = 9 in all groups.

Figure 7

CLP surgery was performed on mice. Mice were given vehicle saline or RvD2 48h after surgery. 24h after injections, mice were given P. aeruginosa intranasally. 24h after the 2^nd hit of p. aeruginosa, mice were sacrificed and lungs lavaged. Using flow cytometry, cytokines in lungs including TNF-α, IL-6, IL-10, IFN-γ, IFN-β, IL-1β, IL-17, IL-23, and MCP-1 were quantified by bead-based immunoassay. RvD2 significantly reduced lung lavage fluid IL-23 levels. Data are mean ± S.E.M. ** P < 0.01 for n = 10 – 12 in all groups.

Figure 8

CLP surgery was performed on mice. Mice were given vehicle saline or RvD2 48h after surgery. 24h after injections, mice were given P. aeruginosa intranasally. 24h after the 2^nd hit of P. aeruginosa, mice were sacrificed and lungs were lavaged. Using flow cytometry, non-inflammatory alveolar macrophages (CD11b^- Siglec F⁺) were counted in lung lavage. There was an increase in non-inflammatory alveolar macrophages in lavage fluid of RvD2 treated mice. Data are mean ± S.E.M. * P < 0.05 for n = 5 for both groups.