Modulation of Human Neutrophil Peptides on P. aeruginosa Killing, Epithelial Cell Inflammation and Mesenchymal Stromal Cell Secretome Profiles

Figures & data

Table 1 Demographic and HNP Levels in Healthy Subjects and Patients with Sepsis

Variables	Healthy	Sepsis
Number (n)	9	62
Age (year)	46.3 ± 2.2	67.4 ± 4.5
Gender (M/F)	5/4	34/28
Lactate (mmol/L)		4.5 ± 0.5
HNP (ng/mL)	1.4 ± 0.3	4.5 ± 0.5^a
WBC (10^9/L)	5.5 ± 0.3	17.6 ± 1.1^a
Neutrophils (10^9/L)	1.5 ± 0.6	14.8 ± 7.2^a
APACHE II		26.0 ± 0.4
MODS Score		8.1 ± 3.3
Site of infection^a (n)
Bacteremia		26 (41.9%)
Lung		28 (45.2%)
Abdomen		5 (8.1%)
Urinary tract		13 (21.0%)
Soft tissue and skin		3 (4.8%)
Nervous system		2 (3.2%)
Other		1 (1.6%)

Notes: ^ap < 0.05 vs Healthy; ^aSome patients had more than one site of infection.

Abbreviations: HNP, human neutrophil peptide; WBC, white blood cell; APACHE II, acute physiology and chronic health evaluation II; MODS, multiple organ dysfunction syndrome.

Figure 1 Bacterial count and IL-8 production. (A) P. aeruginosa was incubated with HNP at indicated concentrations for indicated time points, and bacterial CFU was counted in TSB agar plates after overnight culture (n=4) (B) P. aeruginosa was treated with a solution containing HNP (50 μg/mL) and indicated doses of MRS2578 for 6 h, CFU was counted in diluted cell supernatants after overnight incubation (n=6). (C) Human small airway epithelial cells were co-cultured with and without P. aeruginosa in the medium containing indicated concentration of MRS2578 or DMSO (Vehicle), followed by 30 min later by administration of PBS or HNP at 50 μg/mL for 6 h. IL-8 levels were measured in cell supernatants by specific ELISA (n=6). *p < 0.05 vs vehicle at identical conditions at 4 h, respectively; #p < 0.05 vs vehicle at identical MRS concentration, respectively; †p < 0.05 vs identical conditions at HNP 50 μg/mL, respectively.

Abbreviations: HNP, human neutrophil peptide; CFU, colony-forming unit; TSB, tryptic soybroth. IL, interleukin; PBS, phosphate-buffered saline; HNP, human neutrophil peptide; ELISA, enzyme-linked immunosorbent assay; DMSO, dimethylsulfoxide.

Figure 2 MSC adhesion and integrin β1 expression increased upon stimulation with HNP in a dose-dependent manner. (A) MSCs were stimulated with PBS (Vehicle), 5 or 10 μg/mL HNP for 24 h. After cell detachment with trypsin-EDTA, the remaining cells were fixed and nuclei were stained with DAPI (in blue), and detached cells were counted (n=3); (B) Expression of integrin β1 in MSCs after stimulation with PBS or 10 μg/mL HNP for 24 h, and the mean value of integrin β1 expression (n=31). *p < 0.05 vs Vehicle, ^#p < 0.05 vs other groups.

Abbreviations: MSC, mesenchymal stem cell; HNP, human neutrophil peptide; EDTA, ethylene diamine tetraacetic acid; DAPI, diaminophenyl indole.

Figure 3 Changes of secretome profiles by MSCs after stimulation with PBS (Vehicle) or HNP at 10 μg/mL for 24 hrs. The cell culture supernatants were added in antibody-coated array membranes and incubated overnight at 4°C. Signal was detected by chemiluminescence after biotinylated antibody and horseradish peroxidase-conjugated streptavidin interactions. Quantification of protein expression was compared between Vehicle and HNP-treated groups (n=3). Expression of 6 out of 14 proteins detected reached statistical difference. Levels of PDGF-AA were measured by human-specific ELISA (n=17). *p < 0.05 vs Vehicle.

Abbreviations: MSC, mesenchymal stem cell; PBS, phosphate-buffered saline; HNP, human neutrophil peptide; PDGF, platelet-derived growth factor.