A mechanism for matrikine regulation in acute inflammatory lung injury

Abstract

Proline-glycine-proline (PGP) and its acetylated form (Ac-PGP) are neutrophil chemoattractants generated by collagen degradation, and they have been shown to play a role in chronic inflammatory disease. However, the mechanism for matrikine regulation in acute inflammation has not been well established. Here, we show that these peptides are actively transported from the lung by the oligopeptide transporter, PEPT2. Following intratracheal instillation of Ac-PGP in a mouse model, there was a rapid decline in concentration of the labeled peptide in the bronchoalveolar lavage (BAL) over time and redistribution to extrapulmonary sites. In vitro knockdown of the PEPT2 transporter in airway epithelia or use of a competitive inhibitor of PEPT2, cefadroxil, significantly reduced uptake of Ac-PGP. Animals that received intratracheal Ac-PGP plus cefadroxil had higher levels of Ac-PGP in BAL and lung tissue. Utilizing an acute LPS-induced lung injury model, we demonstrate that PEPT2 blockade enhanced pulmonary Ac-PGP levels and lung inflammation. We further validated this effect using clinical samples from patients with acute lung injury in coculture with airway epithelia. This is the first study to our knowledge to determine the in vitro and in vivo significance of active matrikine transport as a mechanism of modulating acute inflammation and to demonstrate that it may serve as a potential therapeutic target.

Keywords: COVID-19; Extracellular matrix; Inflammation; Neutrophils; Peptides.

Figure 1. The measurement of labeled Ac-PGP distribution and labeled Ac-PGP–induced airway neutrophilic inflammatory response.

Labeled Ac-PGP peptide (250 μg in 50 μl) was intratracheally administered to 6- to 8-week-old C57BL/6J mice (n = 5 animals per time point). The bronchoalveolar lavage (BAL) (A) and lung tissue (B) was collected at multiple time points after treatment for the measurement of labeled Ac-PGP by electrospray ionization–liquid chromatography–tandem mass spectrometry (ESI-LC-MS/MS). The neutrophil cell counts (C) and MMP-9 level (D) were determined in BAL samples. Data are presented as the mean ± SD and were analyzed by 1-way ANOVA and Tukey’s multiple comparisons post test.

Figure 2. The transport of Ac-PGP in human distal lung epithelial cell line NCl-H441 was inhibited by blocking PEPT2.

The expression of PEPT2 protein (green fluorescence) in polarized NCl-H441 (H441) cells (nuclei, blue fluorescence) was examined by immunohistological staining. Scale bar: 50 μm. Z-stacking images are shown below. Scale bar: 20 μm (A). The transepithelial transport of labeled Ac-PGP (50 ng/ml) in the apical-to-basal directions across PEPT2-knockdown H441 cell monolayers was measured by ESI-LC-MS/MS (B). The transepithelial transport of labeled Ac-PGP (Ac-PGP*, 50 ng/ml) in the apical-to-basal directions across H441 cell monolayers was measured in the presence of PEPT2 inhibitor cefadroxil (0.2 mM, 2.0 mM and 5.0 mM; Cef.0.2, Cef.2.0, and Cef.5.0, respectively) by ESI-LC-MS/MS (C). Data (mean ± SD) are expressed with 3–9 wells per group and were analyzed by Mann-Whitney test or 1-way ANOVA with Tukey’s multiple comparisons post test. crRNA, CRISPR RNA.

Figure 3. Ac-PGP uptake in the lung was inhibited by blocking PEPT2.

Labeled Ac-PGP peptide (Ac-PGP*, 250 μg in 50 μl saline) was intratracheally administered to C57BL/6J female mice 30 minutes after intratracheal pretreatment with PEPT2 inhibitor cefadroxil (Cef, 250 μg/mouse). The concentration of labeled Ac-PGP in BAL (A), lung tissue (B), and pulmonary window tissue (C) was measured by ESI-LC/MS/MS. Data (mean ± SD) are expressed with 4–7 mice per group and were analyzed by 1-way ANOVA and Tukey’s multiple comparisons post test.

Figure 4. The application of PEPT2 inhibition increased LPS-induced acute lung inflammation and blocked Ac-PGP transport in patients with ARDS.

C56BL/6J mice were intratracheally (i.t.) treated with cefadroxil (Cef) and 100 μg LPS (P. aeruginosa). BAL was collected after treatment for total cell counts (A), MPO (B), and MMP-9 (C) levels. The level of Ac-PGP peptides in the BAL was analyzed by ESI-LC-MS/MS (D). Statistical analysis was performed using 1-way ANOVA and Tukey’s multiple comparison post test, n = 3–9 mice/group. All values represent mean ± SD. The transepithelial transport of Ac-PGP peptides in BAL of patients with ARDS in the apical-to-basal directions across polarized H441 cell monolayers was measured with or without cefadroxil by ESI-LC-MS/MS (E). Data (mean ± SD) are expressed with 4–5 wells per group and were analyzed by Mann-Whitney test.