Deficiency of formyl peptide receptor 1 and 2 is associated with increased inflammation and enhanced liver injury after LPS-stimulation

Abstract

Introduction: Formyl peptide-receptor 1 and 2 (FPR1 and FPR2) in mice were identified as receptors with contrary affinity for the PAMP fMLF. Formyl-methionyl-leucyl-phenylalanine is either part of the bacterial membrane and is secreted by the mitochondria of eukaryotic ceslls during apoptosis. Furthermore FPR1 and 2 are described as highly relevant factors for the chemotaxis of immune cells. Their role during the acute liver injury has not been investigated yet.

Materials and methods: Constitutive knockout mice for FPR1 (mFPR1-/-), FPR2 (mFPR2-/-) and wild type (WT) mice were challenged with LPS i.p. for 3 h and 6 h. Liver and serum were sampled for further analysis.

Results: Liver transaminases were elevated in all mice 3 h and 6 h post LPS stimulation. Gene expression analysis displayed a reduced expression of the pro-inflammatory cytokines IL-6 and CXCL1 after 3 h in the mFPR1-/- compared to wild type and mFPR2-/- mice. After 6 h, IL-6, TNF-α and CXCL1 were significantly higher in mice lacking mFPR1 or 2. Consistent to these findings the numbers of CD11b+ and Ly6G+ immune cells were altered in the livers. The analysis of TLR2 and TLR4 revealed time and genotype specific changes in theirs gene expression. Additionally, the liver in mFPR1- and mFPR2-deficient mice seem to be more susceptible to apoptosis by showing a significant higher number of TUNEL+-cells in the liver than WT-mice and displayed less Ki67-positive nuclei in the liver.

Conclusion: The results suggest a prominent role of FPRs in the regulation of the hepatic inflammatory response after LPS induced liver injury. Deletion of mFPR1 or mFPR2 leads to deregulation of the inflammatory response compared to WT mice, associated with more severe liver injury represented by higher levels of transaminases, apoptotic cells and a reduced regenerative capacity.

Figure 1. The clinical and histological analysis after LPS-stimulation includes measuring of ALT (A) and AST (B).

The histological analysis was performed using H&E staining (C). Overview pictures at 0 h were taken in 100-fold magnification, detail microphotographs at 3 h and 6 h post LPS such as infiltrating immune cells are magnified 200-fold (* = p<0.05; *** = p<0.005).

Figure 2. To identify the infiltrating immune cells after LPS stimulation according to their surface markers, CD11b was used to detect infiltrating monocytes and macrophages in the liver (A).

Pictures were taken in 200-fold magnification and the CD11b⁺-cells were counted. The results were displayed as a graph (B) indicating differences among WT, mFPR1^-/- and mFPR2^-/- (* = p<0.05; ** = p<0.01; # = p<0.0001).

Figure 3. The second subset of immune cells were analysed by Ly6G-staining.

Mainly neutrophil granulocytes were identified as Ly6G⁺ (A). Pictures were taken in 200-fold magnification and the Ly6G⁺-cells were counted. The results were displayed as a graph (B) indicating differences among WT, mFPR1^-/- and mFPR2^-/- (* = p<0.05; ** = p<0.01; # = p<0.0001).

Figure 4. To better understand mechanism underlying the infiltration of immune cells into the liver of mFPR-deficient mice the gene expression of the pro-inflammatory cytokines IL-6 (A), TNF-α (B), CXCL1 (C), TLR2 (D) and TLR4 (E) were analyzed by qPCR.

Changes in gene expression were related to GAPDH as a housekeeping gene. (* = p<0.05; ** = p<0.01; # = p<0.0001.)

Figure 5. For determination of the liver injury FFPE-section were stained for DNA-strand breaks using a TUNEL-assay.

Tunel⁺-cells were counted. Counterstaining was perfomed using Haematoxylin (A). Pictures were taken in 200 fold magnification. Results displaying an increase of TUNEL⁺-cells in regard to mFPR1 and mFPR2-deficiency are shown as a Graph (B). (* = p<0.05; ** = p<0.01.)

Figure 6. The Expression of the three mFPRs mFPR1 (A), mFPR2 (B) and mFPR3 (C) was analysed by qPCR and the gene expression was related to GAPDH.

For the analysis of inflammation in the BDL-model immune cells were stained for CD11b-positivity. Graphical score for the amount of CD11b⁺-cells in the liver displays an increase over time. The cells are displayed as numbers per view field (D).

Figure 7. To investigate liver proliferation FFPE-sections were stained with the universal cell cycle marker Ki67.

At 3⁺-nuclei were counted and analyzed as percentage of proliferative cells. Photomicrographs were taken at 200-fold and representative images are shown. Ki67⁺-nuclei are indicated by arrows (* = p<0.05; ** = p<0.01).