Post‐septic Down‐regulation of Sulfatase‐1 Suppresses Pulmonary Endothelial ICAM‐1 Expression

Rationale Post‐septic compensatory anti‐inflammatory syndrome (CARS) is a state of immunoparalysis that leaves patients susceptible to secondary infections. Interestingly, CARS coincides with post‐septic reconstitution of the pulmonary endothelial glycocalyx, a heparan sulfate (HS)‐enriched layer that regulates endothelial‐neutrophil adhesion and is degraded during early sepsis. CARS is generally described as a consequence of impaired leukocyte function, with little understanding of the role of the post‐septic endothelium. We hypothesized that the post‐septic pulmonary endothelial glycocalyx is characterized by changes in HS sulfation (via changes in sulfatase expression), leading to impaired endothelial inflammatory responses. Methods We induced sepsis in male C57BL/6J mice with cecal ligation and puncture (CLP). To recapitulate CARS, post‐septic mice (3 days after CLP) animals were treated with intratracheal lipopolysaccharide (LPS), and the degree of inflammation was assessed thereafter by bronchoalveolar lavage (BAL) cell count and protein concentration. To determine changes in post‐septic pulmonary endothelial gene expression during CARS, we flow‐sorted pulmonary endothelial cells and performed RNA microarray and confirmatory qRT‐PCR. To determine post‐septic changes in HS sulfation, we performed mass spectrometry of HS isolated from either the pulmonary endothelial glycocalyx or the plasma of post‐CLP or post‐sham mice. We determined the impact of altered endothelial HS sulfation on CARS by either (a) degrading HS from the endothelial glycocalyx by intravenous heparinase I or (b) administering exogenous sulfated HS intravenously prior to intratracheal LPS. We specifically measured the effect of sulfatase‐1 (sulf‐1, an extracellular enzyme that specifically cleaves 6‐O sulfate of HS) on endothelial cell ICAM‐1 expression using siRNA. Results In comparison to sham mice, post‐septic mice showed decreased cellular and protein infiltration in BAL fluid 48 hrs after intratracheal LPS (n=5–6, P < 0.05). Microarray and qRT‐PCR analyses of flow‐sorted endothelial cells (harvested 48 hrs after CLP or sham) detected downregulation of sulf‐1 in CLP mice (n=3–4, P < 0.05). While these changes correlated with increased 6‐O sulfation in the endothelial glycocalyx and circulating HS, experimental modulation of these HS compartments did not influence CARS. However, in vitro, transfection of siRNA targeted to sulf‐1 resulted in 90% reduction of sulf‐1 mRNA and down‐regulation of ICAM‐1 by 57–70% (n=2). These in vitro changes surprisingly occurred independently of HS 6‐O sulfation. Conclusions Concurrently with CARS, post‐septic pulmonary endothelial cells undergo downregulation of sulf‐1, causing loss of endothelial ICAM‐1 expression. Surprisingly, this impaired ICAM‐1 expression occurred in the absence of changes in HS 6‐O sulfation. Support or Funding Information Department of Defense PR150655 (to EPS) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .