Lysozyme association with circulating RNA, extracellular vesicles, and chronic stress

Sarah K Abey, Yuana Yuana, Paule V Joseph, Natnael D Kenea, Nicolaas H Fourie, LeeAnne B Sherwin, Gregory E Gonye, Paul A Smyser, Erin S Stempinski, Christina M Boulineaux, Kristen R Weaver, Christopher K E Bleck, Wendy A Henderson, Sarah K Abey, Yuana Yuana, Paule V Joseph, Natnael D Kenea, Nicolaas H Fourie, LeeAnne B Sherwin, Gregory E Gonye, Paul A Smyser, Erin S Stempinski, Christina M Boulineaux, Kristen R Weaver, Christopher K E Bleck, Wendy A Henderson

Abstract

Background: Stress has demonstrated effects on inflammation though underlying cell-cell communication mechanisms remain unclear. We hypothesize that circulating RNAs and extracellular vesicles (EVs) in patients with chronic stress contain signals with functional roles in cell repair.

Methods: Blood transcriptome from patients with Irritable Bowel Syndrome versus controls were compared to identify signaling pathways and effectors. Plasma EVs were isolated (size-exclusion chromatography) and characterized for effectors' presence (immunogold labelling-electron microscopy). Based on transcriptome pathways and EV-labelling, lysozyme's effects on cell migration were tested in human colon epithelial CRL-1790 cells and compared to the effects of CXCL12, a migration inducer (wound assay). The effect of lysozyme on immune-linked mRNA and protein levels in cells which survived following serum starvation and scratch wound were investigated (NanoString).

Results: Blood transcriptomes revealed pyridoxal 5'phosphate salvage, pyrimidine ribonucleotides salvage pathways, atherosclerosis, and cell movement signaling with membrane CD9 and extracellular lysozyme as effectors. Plasma EVs showed labelling with CD9, mucins, and lysozyme. This is the first identification of lysozyme on plasma EVs. In CRL-1790 cells, lysozyme induced migration and repaired scratch wound as well as CXCL12. Immune mRNA and protein expressions were altered in cells which survived following serum starvation and scratch wound, with or without lysozyme in serum-free media post-wounding: CD9, IL8, IL6 mRNAs and CD9, NT5E, PD-L1 proteins.

Conclusions: Repair and inflammatory signals are identified in plasma EVs and circulating RNAs in chronic stress. Registered clinicaltrials.gov #NCT00824941.

General significance: This study highlights the role of circulating RNAs and EVs in stress.

Keywords: CD9; CD9, cluster of differentiation 9; CXCL12; CXCL12, Chemokine (C-X-C motif) ligand 12; EVs, Extracellular vesicles; IL, interleukin; NT5E, 5′-Nucleotidase Ecto; PD-L1, programmed death ligand 1; inflammation; lysozyme; stress; wound.

Figures

Fig. 1
Fig. 1
A network generated from gene expression profiling of whole blood from patients with chronic stress-induced GI dysfunction versus controls.
Fig. 2
Fig. 2
(A) EVs isolated using size exclusion chromatography and detection by uranyl acetate negative staining followed by transmission electron microscopy visualization. Example of EV diameter size quantification by transmission electron microscopy. (B) A histogram of EV size distribution from a representative pool of 914 EVs from patients and controls. (C) Plasma EVs from patients (Pt) and controls (Ct) showed immunogold-labelling with CD9, lysozyme (LYZ), mucin 1 (MUC1), and mucin 2 (MUC2) by transmission electron microscopy. All scale bars are 100 nm.
Fig. 2
Fig. 2
(A) EVs isolated using size exclusion chromatography and detection by uranyl acetate negative staining followed by transmission electron microscopy visualization. Example of EV diameter size quantification by transmission electron microscopy. (B) A histogram of EV size distribution from a representative pool of 914 EVs from patients and controls. (C) Plasma EVs from patients (Pt) and controls (Ct) showed immunogold-labelling with CD9, lysozyme (LYZ), mucin 1 (MUC1), and mucin 2 (MUC2) by transmission electron microscopy. All scale bars are 100 nm.
Fig. 3
Fig. 3
(A) Human fetal colon epithelial cells CRL-1790 migrated towards lysozyme-containing media. Negative control was serum-free media (“Serum-Free”). Positive control was CXCL12-containing media. Fluorescent index values are means ± standard deviations from three biological replicates. (B) Scratch wound assay: Following an overnight incubation in serum-free media (serum starvation), CRL-1790 cells were scratch-wounded at time 0 h followed by incubation in serum-free media containing lysozyme, CXCL12, or 10% fetal bovine serum. Negative control was serum-free media. Images were taken and wound gaps were measured at 0 h and 8 h post-wounding.
Fig. 4
Fig. 4
Results of the nCounter RNA:Protein Immune Panel analyses of cellular alterations in response to serum starvation and scratch wound, in the presence or absence of lysozyme, are plotted. In (A), a volcano plot shows differential mRNA levels in CRL-1790 cells surviving at 8 h post-wounding in the presence of lysozyme (+ Lyz) versus in cells harvested at 0 h post-wounding (Baseline). In (B), a volcano plot shows differential mRNA levels in cells surviving at 8 h post-wounding in the absence of lysozyme (− Lyz) versus baseline. In (C), all genes whose mRNA levels were altered in the presence of lysozyme (+ Lyz) versus baseline were plotted against all genes whose mRNA levels were altered in the absence of lysozyme (− Lyz) versus baseline were plotted, regardless of their significance ranking (p-values). (D) Immune-related proteins whose levels were altered 8 h post-wounding in the presence (+ Lyz) or absence (− Lyz) of lysozyme, both compared to Baseline, are shown. (E) Lysozyme-dependent alterations of three highest expressed immune-linked proteins and their corresponding mRNAs levels in CRL-1790 cells which survived at 8 h post-wounding are shown.
Fig. 4
Fig. 4
Results of the nCounter RNA:Protein Immune Panel analyses of cellular alterations in response to serum starvation and scratch wound, in the presence or absence of lysozyme, are plotted. In (A), a volcano plot shows differential mRNA levels in CRL-1790 cells surviving at 8 h post-wounding in the presence of lysozyme (+ Lyz) versus in cells harvested at 0 h post-wounding (Baseline). In (B), a volcano plot shows differential mRNA levels in cells surviving at 8 h post-wounding in the absence of lysozyme (− Lyz) versus baseline. In (C), all genes whose mRNA levels were altered in the presence of lysozyme (+ Lyz) versus baseline were plotted against all genes whose mRNA levels were altered in the absence of lysozyme (− Lyz) versus baseline were plotted, regardless of their significance ranking (p-values). (D) Immune-related proteins whose levels were altered 8 h post-wounding in the presence (+ Lyz) or absence (− Lyz) of lysozyme, both compared to Baseline, are shown. (E) Lysozyme-dependent alterations of three highest expressed immune-linked proteins and their corresponding mRNAs levels in CRL-1790 cells which survived at 8 h post-wounding are shown.
Fig. 4
Fig. 4
Results of the nCounter RNA:Protein Immune Panel analyses of cellular alterations in response to serum starvation and scratch wound, in the presence or absence of lysozyme, are plotted. In (A), a volcano plot shows differential mRNA levels in CRL-1790 cells surviving at 8 h post-wounding in the presence of lysozyme (+ Lyz) versus in cells harvested at 0 h post-wounding (Baseline). In (B), a volcano plot shows differential mRNA levels in cells surviving at 8 h post-wounding in the absence of lysozyme (− Lyz) versus baseline. In (C), all genes whose mRNA levels were altered in the presence of lysozyme (+ Lyz) versus baseline were plotted against all genes whose mRNA levels were altered in the absence of lysozyme (− Lyz) versus baseline were plotted, regardless of their significance ranking (p-values). (D) Immune-related proteins whose levels were altered 8 h post-wounding in the presence (+ Lyz) or absence (− Lyz) of lysozyme, both compared to Baseline, are shown. (E) Lysozyme-dependent alterations of three highest expressed immune-linked proteins and their corresponding mRNAs levels in CRL-1790 cells which survived at 8 h post-wounding are shown.

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