106 - Neutrophil elastase targets select proteins on human blood monocyte derived macrophage cell surfaces

Publication Number: 106.5628

Judith Voynow, Children's Hospital of Richmond at VCU, Richmond, VA, United States; Nadia Tasnim Ahmed, Virginia Commonwealth University, Saint Louis, MO, United States; Apparao B.. Kummarapurugu, Virginia Commonwealth University, Richmond, VA, VA, United States; Shuo Zheng, Virginia Commonwealth University School of Medicine, Richmond, VA, United States; Gamze B. Bulut, VCU, Glen Allen, VA, United States; Le Kang, Virginia Commonwealth University School of Public Health, Richmond, VA, United States; Aashish Batheja, Virginia Commonwealth University School of Medicine, Richmond, VA, United States; Adam M. Hawkridge, Virginia Commonwealth University, Richmond, VA, United States

Background: Neutrophil elastase (NE) causes macrophage phagocytic failure and activates the release of macrophage extracellular traps increasing inflammatory mediators into the airway . However, NE treatment of macrophages fails to induce inflammation.

Objective: We sought to examine the effect of NE exposure on the human macrophage proteome and evaluate its impact on pro-inflammatory signaling.

Design/Methods: Human blood monocytes from healthy deidentified volunteers (n=3) were differentiated in culture into macrophages (hBMDM) and exposed to either NE (500 nM) or control vehicle (Ctr), 2 h. The cells were harvested, lysed, digested with trypsin, and analyzed by nLC 1200 UHPLC system coupled with ThermoFisher Q-Exactive HF-X tandem mass spectrometry. LC-MS/MS data were grouped by donor and searched in Proteome Discoverer 3.0 using CHIMERYS. The ratios for label-free quantitation (LFQ) analysis comparing the ratio of Ctr/NE treatment were log2 transformed, then compared by t-test against the entire values for each patient. False discovery rates were calculated for each patient data set (Benjamini-Hochberg). Enrichment analysis was performed in g:Profiler for Homo sapiens: g:SCS threshold < 0.05, highlighting the Gene Ontology driver terms. To confirm proteomic results, western analyses of Ctr or NE-treated hBMDM protein lysates (n=3) were evaluated for proteoglycans, Neuropilin1 (NRP1), Syndecan 2 (SDC2) and Glypican 4 (GPC4) and compared using one-way nonparametric ANOVA (Kruskal-Wallis test) followed by Tukey's multiple comparison test. Our study was approved by the VCU IRB, HM20018160.

Results: A total of 5262 unique proteins were identified across all 3 donor samples. The number of proteins that were significantly different (Ctr/NE) comprised a much smaller set of 41 proteins detected in all 3 donors. Of the 26 proteins that were down-regulated, 21 included cell surface proteins and 4 were proteoglycans including NRP1, SDC2, GPC4 and CD99L2. Proteins that regulate either M1 or M2 polarization or that regulated leukocyte activation and inflammatory responses were decreased. Western analyses confirmed the proteomic results of NE-degradation of NRP1, SDC2 and GPC4.

Conclusion(s): NE exposure decreased hBMDM cell surface proteoglycans and receptors which are important for macrophage regulation of polarization and inflammation.