Choline Triggers Exacerbations of Chronic Obstructive Pulmonary Disease in Patients Infected with Pseudomonas aeruginosa

Abstract

Background: Although exacerbations of chronic obstructive pulmonary disease produced by Pseudomonas aeruginosa infections are a major cause of death, the molecular mechanism that produces them is not well known. Here we focused on the energetic basis of dyspnoea, hypercapnia and acidosis symptoms.

Methods and findings: We used an in vivo exacerbation model exposing mice to cigarette smoke and LPS, to mimic emphysema and infections, and choline challenges to trigger exacerbations, that showed 31% increased in the airway resistance for naïve mice and 250% for smoke/LPS treatment. Tissue resistance was increased 32%, in naïve mice, and 169% for smoke/LPS treatment. A decreased tissue elastance, was confirmed by decreased collagen content and increased alveoli chord length. Consequently, the O₂ demanded was 260% greater for smoke/LPS treated mice, to provide the energy required to pump the same volume of air then for naïve mice. The extra CO₂ produced per ml of air pumped caused hypercapnia and acidosis by 4% decrease in pH.In addition, the bacteria grown with choline had a decrease of 67% in phosphate, 23% ATP and 85% phospholipids with an increase of 57% in polyphosphates, 50% carbohydrates, 100% LPS, consuming 45% less energy relative to the bacteria grown with succinate.

Conclusion: choline, released by P. aeruginosa, triggers exacerbation symptoms by increasing lung resistance, O₂ consumption and producing more pCO₂ in blood with dyspnea, hypercapnia and acidosis. The energetic shift of decreased O₂ bacterial demand and increased lung demand benefits the infection, thus restoring the energetic balance on the host will favor P. aeruginosa eradication.

Keywords: Bacterial infections; COPD exacerbations; LPS; animal model; emphysema; respiratory infections.

Fig. (1). Exacerbations in mice

(A) Representative scheme of the host-pathogen interaction in mice lung during exacerbations of COPD. As an extracellular pathogen P. aeruginosa releases to the medium phospholipase C (PLC) (6) and phosphoryl-choline phosphatase (PChP) (7) within vesicles (5) that degrades the membranes of lung epithelial cells from phosphatidyl choline to phosphoryl-choline and Diacylglycerol (DAG) [40], that causes Ca²⁺ mediated vaso-constriction (10). Choline and Pi released by PChP produces airway constriction in the lung tissue, and LPS and PolyP accumulation in P. aeruginosa. (B) Western blot of MMP12 from supernatant of peritoneal macrophages stimulated with or without LPS (200 ng) and incremental doses of INFγ of 5, 50 or 500 ng. (C) Representative experiment of inflammatory cells present in BAL of naïve mice (n=5), mice treated with of LPS (n= 4), smoke exposed (n=8) and smoke plus 100 ng/weekly of LPS (n= 3) from P. aeruginosa. *P= 0.01 relative to naïve mice ⁑P= 0.04 relative to naïve mice, P= 0.01 relative to smoke exposed, §P=0.01 relative to naïve mice, †P=0.05 relative to smoke exposed, ∫P=0.05 relative to naïve mice, ‡P=0.01 relative to smoke exposed. (D) Plots show the pressure offered by the lung of mice smoke naïve (black circles, n=7), LPS (black squares, n=4), smokes exposed (open circles, n=4) and smoke plus LPS exposed (open squares, n=5) due to airway resistance (top plot) tissue resistance (middle plot) and dynamic elasticity (bottom plot). Data is expressed as mean+s.e.m, *P≤0.0001 determined by t-student test.

Fig. (2). Impact of inflammation on the lung mechanics

(A) Plot showing the energy consumed by the extra work of breathing (WOB) done by the lung to exhale the same volume of air (1 ml) versus the alveoli chord length (CL) determined by morphometry, quantified in mice treated with cigarette smoke (n=8) and smoke plus LPS (n=3) compared to naïve control (n=8) or the value for LPS obtained from [44], (B) Oxygen consumed to exhale the same volume of air; dotted line separates the O₂ due to obstruction produced by inflammation from smoke exposure, LPS or combination of both after choline induced resistance, dots represent average±SD.

Fig. (3). Morphology of P. aeruginosa

Electron microscopy from P. aeruginosa grown with choline (A) or succinate (B) showing vesicles (black arrow) and zones of high refringency (white arrows). The inclusions of high electronic density are due to polyphosphate accumulation [41], Vesicle formation [5] 52,000×.