P2Y₁₂ Null Mice Are Protected Against Sepsis-Induced Lung Injury

Abstract 615

The P2Y₁₂ receptor is expressed on the platelet surface and mediates ADP-induced aggregation, but it has recently been shown to be expressed also in other cells of the immune system. Furthermore, the P2Y₁₂ receptor gene variants correlate with pulmonary inflammation and asthma, and P2Y₁₂ receptor deficiency or platelet depletion abrogated dust mite-induced airway inflammation, suggesting an important role for the P2Y₁₂ receptor in pulmonary inflammation. Excessive neutrophil infiltration into the lungs is a hallmark of acute lung injury. The mechanisms that trigger this infiltration are poorly understood but may be the result of the development of a pro-inflammatory phenotype in the lung. Platelets are increasingly recognized for their ability to modulate immune responses through their interaction with neutrophils, dendritic cells, and T lymphocytes. These studies suggest an important regulatory role for the P2Y₁₂ receptor and activated platelets in regulating neutrophil influx into the lung.

Using a mouse model of intra-abdominal sepsis and acute lung injury (cecal ligation and double puncture (CLP)), we examined the role of the P2Y₁₂ receptor in neutrophil migration and lung inflammation using P2Y₁₂ null mice. In this study wild type (WT) and P2Y₁₂ null (KO) mice underwent Sham surgery or CLP surgery (5 animals/group). At 24 hours post surgery, blood and lung tissue samples were collected.

Our results show that the sepsis-induced increase in circulating white blood cells (WBC) was attenuated in CLP KO mice, compared with CLP WT mice (circulating WBC in CLP WT: 1.62 ± 0.41×10³cell/ml* compared with CLP KO: 0.8 ± 0.4×10³cell/ml, *p<0.05 WT CLP versus KO CLP mice). There were no significant differences in the WBC count in WT Sham surgery animals as compared to the KO sham surgery animals (Sham WT: 0.8 ± 0.2×10³cell/ml vs. Sham KO: 0.8 ± 0.1×10³cell/ml). Interestingly, no significant changes in the platelet count between the groups were observed. Next we investigated the role of P2Y₁₂ in regulating sepsis-induced elevations in plasma levels of cytokines (TNF-a, IL-10, IL-6 and MIP-1b) (Figure 1). The concentration of each cytokine was elevated during sepsis in both WT and KO animals as compared to sham surgery animals, but, the sepsis-induced increase was significantly lower in P2Y₁₂ null mice (**p < 0.01 KO CLP model versus WT- CLP).

Figure 1:

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Sepsis-induced increases in plasma cytokines are attenuated in P2Y₁₂ KO mice following CLP. Plasma levels of TNF-a, IL-10, IL-6 and MIP-1b in wild type (black) and KO mice (white). Values are expressed as pg/ml, mean ± SEM (*p < 0.05; **p < 0.01; KO CLP model versus WT CLP, n=5).

Figure 1:

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Sepsis-induced increases in plasma cytokines are attenuated in P2Y₁₂ KO mice following CLP. Plasma levels of TNF-a, IL-10, IL-6 and MIP-1b in wild type (black) and KO mice (white). Values are expressed as pg/ml, mean ± SEM (*p < 0.05; **p < 0.01; KO CLP model versus WT CLP, n=5).

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Finally we studied neutrophil infiltration in lung tissues using hematoxylin and eosin (H & E) staining and myeloperoxidase (MPO) activity in lung tissue homogenates. Analysis of H & E staining demonstrated that the increase in inflammatory cell infiltration detected in WT was diminished in KO mice, suggesting a decrease in inflammation in the lungs of P2Y₁₂ null mice. Furthermore, the MPO concentration was significantly decreased in KO CLP lung tissue as compared to WT CLP (Figure 2), (*p < 0.05; KO CLP model versus wild type).

Figure 2:

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Sepsis-induced elevations in lung MPO levels are reduced in P2Y₁₂ null mice. MPO analysis was performed in lung samples of Sham and CLP in wild type (black) and KO mice (white). Values are expressed as rfu/min/mg, mean ± SEM (*p < 0.05; KO CLP model versus WT, n=5).

Figure 2:

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Sepsis-induced elevations in lung MPO levels are reduced in P2Y₁₂ null mice. MPO analysis was performed in lung samples of Sham and CLP in wild type (black) and KO mice (white). Values are expressed as rfu/min/mg, mean ± SEM (*p < 0.05; KO CLP model versus WT, n=5).

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In conclusion, our data shows a decrease in plasma cytokine levels and in circulating WBC, as well as a diminished neutrophil infiltration in the lungs indicating that P2Y₁₂ null mice are protected against sepsis-induced lung injury. This suggests a regulatory role for P2Y₁₂ receptor during inflammation.