S100A9, a pro-inflammatory alarmin, is up-regulated in inflamed tissues. However, the role of S100A9 in regulating neutrophil activation, inflammation and lung damage in sepsis is not known. Herein, we hypothesized that blocking S100A9 function may attenuate neutrophil recruitment in septic lung injury. Male C57BL/6 mice were pretreated with the S100A9 inhibitor ABR-238901 (10 mg/kg), prior to cercal ligation and puncture (CLP). Bronchoalveolar lavage fluid (BALF) and lung tissue were harvested for analysis of neutrophil infiltration as well as edema and CXC chemokine production. Blood was collected for analysis of membrane-activated complex-1 (Mac-1) expression on neutrophils as well as CXC chemokines and IL-6 in plasma. Induction of CLP markedly increased plasma levels of S100A9. ABR-238901 decreased CLP-induced neutrophil infiltration and edema formation in the lung. In addition, inhibition of S100A9 decreased the CLP-induced up-regulation of Mac-1 on neutrophils. Administration of ABR-238901 also inhibited the CLP-induced increase of CXCL-1, CXCL-2 and IL-6 in plasma and lungs. Our results suggest that S100A9 promotes neutrophil activation and pulmonary accumulation in sepsis. Targeting S100A9 function decreased formation of CXC chemokines in circulation and lungs and attenuated sepsis-induced lung damage. These novel findings suggest that S100A9 plays an important pro-inflammatory role in sepsis and could be a useful target to protect against the excessive inflammation and lung damage associated with the disease.

Septic lung damage is associated with endothelial cell and neutrophil activation. This study examines the role of the E3 ubiquitin ligase midline 1 (Mid1) in abdominal sepsis. Mid1 expression was increased in endothelial cells derived from post-capillary venules in septic mice and TNF-α challenge increased Mid1 levels in endothelial cells in vitro. The siRNA-mediated knockdown of Mid1 decreased TNF-α-induced upregulation of ICAM-1 and neutrophil adhesion to endothelial cells. Moreover, Mid1 silencing reduced leukocyte adhesion in post-capillary venules in septic lungs in vivo. The silencing of Mid1 not only decreased Mid1 expression but also attenuated expression of ICAM-1 in lungs from septic mice. Lastly, TNF-α stimulation decreased PP2Ac levels in endothelial cells in vitro, which was reversed in endothelial cells pretreated with siRNA directed against Mid1. Thus, our novel data show that Mid1 is an important regulator of ICAM-1 expression and neutrophil adhesion in vitro and septic lung injury in vivo. A possible target of Mid1 is PP2Ac in endothelial cells. Targeting the Mid1-PP2Ac axis may be a useful way to reduce pathological lung inflammation in abdominal sepsis.

Lung endothelial cell dysfunction plays a central role in septic-induced lung injury. We hypothesized that endothelial cell subsets, capillary endothelial cells (capEC) and post capillary venules (PCV), might play different roles in regulating important pathophysiology in sepsis. In order to reveal global transcriptomic changes in endothelial cell subsets during sepsis, we induced sepsis in C57BL/6 mice by cecal ligation and puncture (CLP). We confirmed that CLP induced systemic and lung inflammation in our model. Endothelial cells (ECs) from lung capillary and PCV were isolated by cell sorting and transcriptomic changes were analyzed by bioinformatic tools. Our analysis revealed that lung capEC are transcriptionally different than PCV. Comparison of top differentially expressed genes (DEGs) of capEC and PCV revealed that capEC responses are different than PCV during sepsis. It was found that capEC are more enriched with genes related to regulation of coagulation, vascular permeability, wound healing and lipid metabolic processes after sepsis. In contrast, PCV are more enriched with genes related to chemotaxis, cell–cell adhesion by integrins, chemokine biosynthesis, regulation of actin filament process and neutrophil homeostasis after sepsis. In addition, we predicted some transcription factor targets that regulate a significant number of DEGs in sepsis. We proposed that targeting certain DEGs or transcriptional factors would be useful in protecting against sepsis-induced lung damage.