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Regulation of monocyte subset proinflammatory responses within the lung microvasculature by the p38 MAPK/MK2 pathway
Kieran P O'Dea1, Justina O Dokpesi, Kate C Tatham
1Section of Anaesthetics, Pain Medicine and Intensive Care, Faculty of Medicine, Imperial College London, Chelsea and Westminster Hospital, London, United Kingdom.
Abstract:
Margination and activation of monocytes within the pulmonary microcirculation contribute substantially to the development of acute lung injury in mice. The enhanced LPS-induced TNF expression exhibited by Gr-1(high) compared with Gr-1(low) monocytes within the lung microvasculature suggests differential roles for these subsets. We investigated the mechanisms responsible for such heterogeneity of lung-marginated monocyte proinflammatory response using a combined in vitro and in vivo approach. The monocyte subset inflammatory response was studied in vitro in mouse peripheral blood mononuclear cell-lung endothelial cell coculture and in vivo in a two-hit model of intravenous LPS-induced monocyte margination and lung inflammation in mice, by flow cytometry-based quantification of proinflammatory genes and intracellular phospho-kinases. With LPS stimulation in vitro, TNF expression was consistently higher in Gr-1(high) than Gr-1(low) monocytes, markedly enhanced by coculture with endothelial cells, and abrogated by p38 MAPK inhibitors. Expression of IL-6, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) was only detectable under coculture conditions, was substantially higher in Gr-1(high) monocytes, and was attenuated by p38 inhibition. Consistent with these differential responses, phosphorylation of p38 and its substrate MAPK-activated protein kinase 2 (MK2) was significantly higher in the Gr-1(high) subset. In vivo, p38 inhibitor treatment significantly attenuated LPS-induced TNF expression in "lung-marginated" Gr-1(high) monocytes. LPS-induced p38/MK2 phosphorylation was higher in lung-marginated Gr-1(high) than Gr-1(low) monocytes and neutrophils, mirroring TNF expression. These results indicate that the p38/MK2 pathway is a critical determinant of elevated Gr-1(high) subset responsiveness within the lung microvasculature, producing a coordinated proinflammatory response that places Gr-1(high) monocytes as key orchestrators of pulmonary microvascular inflammation and injury.
Insights
The p38/MK2 pathway drives higher inflammatory responses in Gr-1(high) monocytes within the lung microvasculature. This pathway highlights Gr-1(high) monocytes as key orchestrators of acute lung injury and pulmonary microvascular inflammation.
Area of Science:
- Immunology
- Pulmonary Medicine
- Cell Biology
Background:
- Monocyte margination and activation in lung microcirculation contribute to acute lung injury.
- Distinct inflammatory responses between Gr-1(high) and Gr-1(low) monocyte subsets in the lung suggest specialized roles.
- Understanding the mechanisms behind this monocyte heterogeneity is crucial for addressing lung inflammation.
Purpose of the Study:
- To investigate the mechanisms underlying the differential inflammatory responses of lung-marginated monocyte subsets.
- To elucidate the role of the p38 mitogen-activated protein kinase (MAPK) pathway in monocyte activation within the pulmonary microcirculation.
- To identify key cellular players in LPS-induced pulmonary microvascular inflammation.
Main Methods:
- Utilized a combined in vitro and in vivo approach using mouse models.
- Co-cultured peripheral blood mononuclear cells with lung endothelial cells to mimic the microenvironment.
- Quantified proinflammatory gene expression and intracellular phospho-kinases via flow cytometry in response to LPS stimulation and p38 MAPK inhibitors.
Main Results:
- In vitro, LPS-induced TNF expression was higher in Gr-1(high) monocytes, enhanced by endothelial cell co-culture, and blocked by p38 MAPK inhibitors.
- Expression of IL-6, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) was significantly higher in Gr-1(high) monocytes under co-culture conditions and attenuated by p38 inhibition.
- In vivo, p38/MK2 pathway activation and LPS-induced TNF expression were significantly higher in lung-marginated Gr-1(high) monocytes compared to Gr-1(low) monocytes and neutrophils.
Conclusions:
- The p38/MK2 pathway is a critical determinant of the heightened responsiveness of Gr-1(high) monocytes in the lung microvasculature.
- Gr-1(high) monocytes, through p38/MK2 pathway activation, orchestrate a proinflammatory response contributing to pulmonary microvascular inflammation and injury.
- Targeting the p38/MK2 pathway may offer therapeutic strategies for mitigating acute lung injury.
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