Synthesized Heparan Sulfate Competitors Attenuate Pseudomonas aeruginosa Lung Infection

Abstract

Several chronic respiratory diseases are characterized by recurrent and/or persistent infections, chronic inflammatory responses and tissue remodeling, including increased levels of glycosaminoglycans which are known structural components of the airways. Among glycosaminoglycans, heparan sulfate (HS) has been suggested to contribute to excessive inflammatory responses. Here, we aim at (i) investigating whether long-term infection by Pseudomonas aeruginosa, one of the most worrisome threat in chronic respiratory diseases, may impact HS levels, and (ii) exploring HS competitors as potential anti-inflammatory drugs during P. aeruginosa pneumonia. P. aeruginosa clinical strains and ad-hoc synthesized HS competitors were used in vitro and in murine models of lung infection. During long-term chronic P. aeruginosa colonization, infected mice showed higher heparin/HS levels, evaluated by high performance liquid chromatography-mass spectrometry after selective enzymatic digestion, compared to uninfected mice. Among HS competitors, an N-acetyl heparin and a glycol-split heparin dampened leukocyte recruitment and cytokine/chemokine production induced by acute and chronic P. aeruginosa pneumonia in mice. Furthermore, treatment with HS competitors reduced bacterial burden during chronic murine lung infection. In vitro, P. aeruginosa biofilm formation decreased upon treatment with HS competitors. Overall, these findings support further evaluation of HS competitors as a novel therapy to counteract inflammation and infection during P. aeruginosa pneumonia.

Keywords: Pseudomonas aeruginosa infections; anti-inflammatory drugs; chronic respiratory diseases; glycosaminoglycans; mouse models.

Figure 1

Disaccharide products of the digestion of heparin/HS from murine lungs after chronic P. aeruginosa infection. C57Bl/6NcrlBR mice were intratracheally injected with 1–2 × 10⁶ colony forming units (CFUs) of P. aeruginosa isolate AA43 embedded in agar-beads (Infected) or with sterile agar-beads (Ctrl). After 28 days, lungs were perfused, recovered, homogenized and separated into pellets and supernatants. After removal of proteins, lipids and DNA, the presence of GAGs was verified by NMR. Samples were digested with a cocktail of heparin lyases to selectively degrade heparin/HS and recovered digestion products were desalted and finally analyzed by HPLC-MS. (a) Base Peak Chromatogram of digestion products from the pellet of one Ctrl uninfected (upper panel) and one infected (lower panel) lung homogenate from C57Bl/6NcrlBR mice. The unsaturated bond of the terminal uronic acid is indicated by Δ, and the number of monomers, the number of sulfates and the number of acetyls are reported; (b) The graph shows the amount of each disaccharide species detected in the whole lung of an infected and an uninfected Ctrl mouse; 100% is considered the sum of peak areas of one whole lung from infected mouse lungs containing the highest amount of disaccharides. The data are pooled from at least two independent experiments (n = 6–15). Data are the mean ± standard error of the mean (SEM) of at least three samples per type which have been processed independently. Statistical significance is indicated: *** p < 0.001.

Figure 2

Structures and characteristics of synthesized HS competitors. The repeating disaccharide unit of compounds (R₁ and R₂ = H/SO₃⁻, R₃ = H/SO₃⁻/COCH₃) is shown. The uronic acid is predominantly in the form l-iduronic acid (l-IdoA and l-IdoA-2-O-sulfate; ~80%) with d-glucuronic acid (d-GlcA; ~20%) making up the remainder. (a) Structure of the canonical disaccharide building block of heparin; (b) The glycol-split uronic acid residue present in compounds C3_gs20, MMW C3_gs90 and MMW C3_gs45.

Figure 3

Modulation of the host response by synthesized HS competitors in a mouse model of acute P. aeruginosa lung infection. C57Bl/6NcrlBR mice were intratracheally injected with 5 × 10⁶ CFUs of the highly virulent P. aeruginosa isolate AA2. Mice were subcutaneously treated with HS competitors (30 mg/kg) or their vehicle two hours before and two hours after the challenge and sacrificed 6 h post-infection. BALF and lung were recovered. (a) Total CFUs in the lungs were evaluated; (b) Total cell and (c) neutrophil recruitment was analyzed in BALF. The data are pooled from at least two independent experiments (n = 7–8). CFUs in individual mice are represented as dots and horizontal lines represent median values. Cells are represented as mean ± SEM. Statistical significance is indicated: * p < 0.05.

Figure 3

Modulation of the host response by synthesized HS competitors in a mouse model of acute P. aeruginosa lung infection. C57Bl/6NcrlBR mice were intratracheally injected with 5 × 10⁶ CFUs of the highly virulent P. aeruginosa isolate AA2. Mice were subcutaneously treated with HS competitors (30 mg/kg) or their vehicle two hours before and two hours after the challenge and sacrificed 6 h post-infection. BALF and lung were recovered. (a) Total CFUs in the lungs were evaluated; (b) Total cell and (c) neutrophil recruitment was analyzed in BALF. The data are pooled from at least two independent experiments (n = 7–8). CFUs in individual mice are represented as dots and horizontal lines represent median values. Cells are represented as mean ± SEM. Statistical significance is indicated: * p < 0.05.

Figure 4

Modulation of the host response by synthesized HS competitors in a mouse model of chronic P. aeruginosa lung infection (14 days). C57Bl/6NcrlBR mice were intratracheally injected with 1–2 × 10⁶ CFUs of the P. aeruginosa isolate AA43 embedded in agar-beads. Mice were treated subcutaneously with HS competitors (30 mg/kg) or vehicle every day starting from the day of infection for 14 days. At the sacrifice, BALF and lung were recovered. (a) Bacterial clearance (white) and incidence of colonization (green) were determined; (b) CFUs were evaluated in total lung of mice still infected at the sacrifice. Total cell (c) and neutrophil (d) recruitment was analyzed in BALF. The data are pooled from two independent experiments (n = 14–15). CFUs in individual mice are represented as dots and horizontal lines represent median values. Cells are represented as mean ± SEM. Statistical significance is indicated: * p < 0.05, ** p < 0.01.

Figure 5

Modulation of the host response by synthesized HS competitors in a mouse model of long-term chronic P. aeruginosa lung infection (28 days). C57Bl/6NcrlBR mice were intratracheally injected with 1–2 × 10⁶ CFUs of the P. aeruginosa isolate AA43 embedded in agar-beads. Mice were treated subcutaneously with HS competitors (30 mg/kg) or vehicle every day starting from ten days post-infection. At the sacrifice, BALF and lung were recovered. (a) Changes from initial body weight were calculated for each group of mice at regular intervals. (b) Total CFUs in the lungs were evaluated. (c) Total cell and (d) neutrophil recruitment was analyzed in BALF. The data are pooled from at least two independent experiments (n = 20–26). CFUs in individual mice are represented as dots and horizontal lines represent median values. The other parameters are represented as mean ± SEM. Statistical significance is indicated: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 6

Effect of synthesized HS competitors on P. aeruginosa biofilm formation. P. aeruginosa strain AA43 was grown for 24 h at 37 °C either in the absence or presence of different concentrations of C3_gs20 (a) and C23 (b). Biofilm biomass was quantified by staining with crystal violet and absorbance measurements at OD₆₀₀. Absorbance of planktonic bacteria in the culture medium was measured at OD₆₀₀. Results are expressed as the ratio between biofilm absorbance and planktonic bacteria absorbance normalized on the value obtained for AA43 treated with isotonic saline (vehicle). The data derive from three independent experiments in triplicate. Values represent the mean ± SEM. Statistical significance is indicated: * p < 0.05.