Joachim Riethmüller

Ceramide accumulation mediates inflammation, cell death and infection susceptibility in cystic fibrosis

Microbial lung infections are the major cause of morbidity and mortality in the hereditary metabolic disorder cystic fibrosis, yet the molecular mechanisms leading from the mutation of cystic fibrosis transmembrane conductance regulator (CFTR) to lung infection are still unclear. Here, we show that ceramide age-dependently accumulates in the respiratory tract of uninfected Cftr-deficient mice owing to an alkalinization of intracellular vesicles in Cftr-deficient cells. This change in pH results in an imbalance between acid sphingomyelinase (Asm) cleavage of sphingomyelin to ceramide and acid ceramidase consumption of ceramide, resulting in the higher levels of ceramide. The accumulation of ceramide causes Cftr-deficient mice to suffer from constitutive age-dependent pulmonary inflammation, death of respiratory epithelial cells, deposits of DNA in bronchi and high susceptibility to severe Pseudomonas aeruginosa infections. Partial genetic deficiency of Asm in Cftr−/−Smpd1+/− mice or pharmacological treatment of Cftr-deficient mice with the Asm blocker amitriptyline normalizes pulmonary ceramide and prevents all pathological findings, including susceptibility to infection. These data suggest inhibition of Asm as a new treatment strategy for cystic fibrosis.

Colistin-Tobramycin Combinations Are Superior to Monotherapy Concerning the Killing of Biofilm Pseudomonas aeruginosa

BACKGROUND Antibiotic combination therapy might be more efficient than single antibiotics to combat Pseudomonas aeruginosa biofilms in the airways of patients with cystic fibrosis. We tested the ability of colistin sulphate-tobramycin combinations and single antibiotics to kill P. aeruginosa biofilms. METHODS P. aeruginosa biofilms were generated in vitro and in rat lungs. In a pilot study, 5 patients with cystic fibrosis inhaled colistin and then tobramycin for 4 weeks. The changes in P. aeruginosa counts and lung function were assessed before and after therapy. RESULTS Antibiotic combination therapy significantly reduced the number of P. aeruginosa cells in P. aeruginosa biofilm models in vitro. When rats were challenged with 1 x 10(7) cfu of P. aeruginosa, which was embedded in alginate beads, mortality rates, lung pathologic findings, and bacterial colony-forming unit counts were significantly lower after 7 days in animals receiving antibiotic combination than in animals receiving single antibiotics. In patients with cystic fibrosis, inhaled colistin-tobramycin was well tolerated and resulted in a mean decrease of 2.52 + /- 2.5 log(10) cfu of P. aeruginosa per milliliter of sputum (P = .027). Measurements of forced expiratory volume in 1 s, obtained both before and after the study, did not differ significantly. CONCLUSION Colistin-tobramycin combinations are more efficient than respective single antibiotics for killing P. aeruginosa in biofilms in vitro, and they significantly reduced P. aeruginosa cell counts in a rat lung infection model and in patients with cystic fibrosis.

Acid Sphingomyelinase Inhibitors Normalize Pulmonary Ceramide and Inflammation in Cystic Fibrosis

Employing genetic mouse models we have recently shown that ceramide accumulation is critically involved in the pathogenesis of cystic fibrosis (CF) lung disease. Genetic or systemic inhibition of the acid sphingomyelinase (Asm) is not feasible for treatment of patients or might cause adverse effects. Thus, a manipulation of ceramide specifically in lungs of CF mice must be developed. We tested whether inhalation of different acid sphingomyelinase inhibitors does reduce Asm activity and ceramide accumulation in lungs of CF mice. The efficacy and specificity of the drugs was determined. Ceramide was determined by mass spectrometry, DAG-kinase assays, and fluorescence microscopy. We determined pulmonary and systemic Asm activity, neutral sphingomyelinase (Nsm), ceramide, cytokines, and infection susceptibility. Mass spectroscopy, DAG-kinase assays, and semiquantitative immune fluorescence microscopy revealed that a standard diet did not influence ceramide in bronchial respiratory epithelial cells, while a diet with Peptamen severely affected the concentration of sphingolipids in CF lungs. Inhalation of the Asm inhibitors amitriptyline, trimipramine, desipramine, chlorprothixene, fluoxetine, amlodipine, or sertraline restored normal ceramide concentrations in murine bronchial epithelial cells, reduced inflammation in the lung of CF mice and prevented infection with Pseudomonas aeruginosa. All drugs showed very similar efficacy. Inhalation of the drugs was without systemic effects and did not inhibit Nsm. These findings employing several structurally different Asm inhibitors identify Asm as primary target in the lung to reduce ceramide concentrations. Inhaling an Asm inhibitor may be a beneficial treatment for CF, with minimal adverse systemic effects.

Flagellin Induces Myeloid-Derived Suppressor Cells: Implications for Pseudomonas aeruginosa Infection in Cystic Fibrosis Lung Disease

Pseudomonas aeruginosa persists in patients with cystic fibrosis (CF) and drives CF lung disease progression. P. aeruginosa potently activates the innate immune system, mainly mediated through pathogen-associated molecular patterns, such as flagellin. However, the host is unable to eradicate this flagellated bacterium efficiently. The underlying immunological mechanisms are incompletely understood. Myeloid-derived suppressor cells (MDSCs) are innate immune cells generated in cancer and proinflammatory microenvironments and are capable of suppressing T cell responses. We hypothesized that P. aeruginosa induces MDSCs to escape T cell immunity. In this article, we demonstrate that granulocytic MDSCs accumulate in CF patients chronically infected with P. aeruginosa and correlate with CF lung disease activity. Flagellated P. aeruginosa culture supernatants induced the generation of MDSCs, an effect that was 1) dose-dependently mimicked by purified flagellin protein, 2) significantly reduced using flagellin-deficient P. aeruginosa bacteria, and 3) corresponded to TLR5 expression on MDSCs in vitro and in vivo. Both purified flagellin and flagellated P. aeruginosa induced an MDSC phenotype distinct from that of the previously described MDSC-inducing cytokine GM-CSF, characterized by an upregulation of the chemokine receptor CXCR4 on the surface of MDSCs. Functionally, P. aeruginosa–infected CF patient ex vivo–isolated as well as flagellin or P. aeruginosa in vitro–generated MDSCs efficiently suppressed polyclonal T cell proliferation in a dose-dependent manner and modulated Th17 responses. These studies demonstrate that flagellin induces the generation of MDSCs and suggest that P. aeruginosa uses this mechanism to undermine T cell–mediated host defense in CF and other P. aeruginosa–associated chronic lung diseases.

Invasion of human epithelial cells by Pseudomonas aeruginosa involves src-like tyrosine kinases p60Src and p59Fyn.

ABSTRACT Pseudomonas aeruginosa plays a major role in respiratory tract infections or sepsis in patients with cystic fibrosis or upon suppression of the immune system. Several P. aeruginosa strains have been shown to be internalized by human epithelial cells; however, the molecular mechanisms of the invasion process are poorly characterized. Here, we show that the internalization of P. aeruginosa into human epithelial cells results in and requires activation of the Src-like tyrosine kinases p59Fyn and p60Src and the consequent tyrosine phosphorylation of several eukaryotic proteins. The significance of Src-like tyrosine kinase activation is shown by an almost complete blockade of P. aeruginosa internalization, but not adhesion, upon inhibition of Src-like tyrosine kinases. Likewise, inhibition of P. aeruginosa binding to CFTR, which has been shown to blockP. aeruginosa internalization, prevents Src and Fyn activation, supporting a pivotal role of Src-like tyrosine kinases for invasion by P. aeruginosa.

Therapeutic Efficacy and Safety of Amitriptyline in Patients with Cystic Fibrosis

Amitriptyline, a blocker of acid sphingomyelinase and acid ceramidase, significantly reduces Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) mice with concurrent increase of survival. Our aim was to establish whether amitriptyline is safe and effective in the treatment of CF patients. In a randomised, double-blinded, placebo-controlled, cross-over pilot study, 4 adult CF patients received 37.5 mg of amitriptyline or placebo twice daily for 14 days. Subsequently in a phase II study 19 adult CF patients were randomly allocated to three treatment groups receiving amitriptyline once daily for 28 days at doses of 25 mg (n=7), 50 mg (n=8), or 75 mg (n=8) or placebo (n=13). The primary outcome was the difference of forced expiratory volume in 1 sec (FEV1) at day 14 between amitriptyline and placebo. Primary endpoint measures improved significantly in three of four patients in the pilot study after amitriptyline treatment vs placebo (relative FEV1: 14.7±5%; p = 0.006) and in the 25 mg treatment group of the phase II study (relative FEV1: 4.0±7%; p = 0.048). Amitriptyline was well tolerated in both studies and 96% of the patients completed the studies. Amitriptyline as a novel therapeutic option in patients with CF is safe and seems to be efficacious.

Relative contribution of Prevotella intermedia and Pseudomonas aeruginosa to lung pathology in airways of patients with cystic fibrosis

Background Patients with cystic fibrosis (CF) with Pseudomonas aeruginosa lung infections produce endobronchial mucus plugs allowing growth of obligate anaerobes including Prevotella spp. Whether obligate anaerobes contribute to the pathophysiology of CF lung disease is unknown. Methods The virulence of Prevotella intermedia and Ps aeruginosa was investigated in vitro and in mice, antibodies against P intermedia in CF sera were assessed and a culture-independent detection method for P intermedia/P nigrescens in CF sputum was tested. Results P intermedia reached cell numbers of >105–>107 colony-forming units (CFU)/ml sputum. The majority of patients with CF (16/17; 94.1%) produced antibodies against two immunoreactive antigens of P intermedia. Culture supernatant fluids, collected from 109 P intermedia cells, were more cytotoxic to respiratory epithelial cells in vitro and inflammatory in mouse lungs than respective fluids from anaerobically grown Ps aeruginosa, while fluids from aerobically grown Ps aeruginosa had the highest cytotoxicity and inflammation. Both pathological effects were largely reduced when culture supernatant fluids from 107 cells of either species were used. P intermedia cells (∼106CFU/lung) did not induce mortality in the agar beads lung infection mouse model, while Ps aeruginosa cells caused death in 30% of mice due to rapid multiplication. A P intermedia/P nigrescens-specific PNA probe was significantly more sensitive than culture-dependent diagnostic assays to detect these strict anaerobes. Conclusion Ps aeruginosa and P intermedia become significantly virulent in vitro and in vivo when cell numbers exceed 108 CFU/lung.

Therapy of CF-Patients with Amitriptyline and Placebo - a Randomised, Double-Blind, Placebo-Controlled Phase IIb Multicenter, Cohort-Study

Background/Aims: Several recent studies revealed an accumulation of ceramide in bronchial, tracheal and intestinal epithelial cells of mice and patients with cystic fibrosis (CF). Normalization of ceramide concentrations in lungs of CF mice employing the functional acid sphingomyelinase inhibitor amitriptyline also normalized mucociliary clearance, chronic inflammation and infection susceptibility to pulmonary P. aeruginosa in these mice. Methods: To test for a beneficial effect of amitriptyline in vivo, we performed a phase IIb randomised, double-blind, placebo-controlled study. Twenty-one CF patients were treated with 25 mg/d amitriptyline twice daily for 28 days. The placebo consisted of 19 patients and was also treated twice per day. The primary endpoint was the change in lung function in the intention-to-treat (ITT) population. Secondary endpoints were ceramide levels in epithelial cells and safety. Results: After treatment, forced expiratory volume in 1 sec predicted (FEV1) increased 6.3±11.5% (p=0.08) in the ITT population (36 of 40 CF patients) and 8.5±10% (p=0.013) in the per protocol (PP) population (29 of 40 patients). Ceramide levels decreased in nasal epithelial cells after amitriptyline treatment. Amitriptyline had no severe and only mild and mostly transient adverse effects, i.e. xerostomia and tiredness. Conclusion: Amitriptyline is safe in CF-patients, increases FEV1 and reduces ceramide in lung cells of CF patients.

Sinonasal inhalation of tobramycin vibrating aerosol in cystic fibrosis patients with upper airway Pseudomonas aeruginosa colonization: results of a randomized, double-blind, placebo-controlled pilot study

Rationale In cystic fibrosis (CF), the paranasal sinuses are sites of first and persistent colonization by pathogens such as Pseudomonas aeruginosa. Pathogens subsequently descend to the lower airways, with P. aeruginosa remaining the primary cause of premature death in patients with the inherited disease. Unlike conventional aerosols, vibrating aerosols applied with the PARI Sinus™ nebulizer deposit drugs into the paranasal sinuses. This trial assessed the effects of vibrating sinonasal inhalation of the antibiotic tobramycin in CF patients positive for P. aeruginosa in nasal lavage. Objectives To evaluate the effects of sinonasal inhalation of tobramycin on P. aeruginosa quantification in nasal lavage; and on patient quality of life, measured with the Sino-Nasal Outcome Test (SNOT-20), and otologic and renal safety and tolerability. Methods Patients were randomized to inhalation of tobramycin (80 mg/2 mL) or placebo (2 mL isotonic saline) once daily (4 minutes/nostril) with the PARI Sinus™ nebulizer over 28 days, with all patients eligible for a subsequent course of open-label inhalation of tobramycin for 28 days. Nasal lavage was obtained before starting and 2 days after the end of each treatment period by rinsing each nostril with 10 mL of isotonic saline. Results Nine patients participated, six initially receiving tobramycin and three placebo. Sinonasal inhalation was well tolerated, with serum tobramycin <0.5 mg/L and stable creatinine. P. aeruginosa quantity decreased in four of six (67%) patients given tobramycin, compared with zero of three given placebo (non-significant). SNOT-20 scores were significantly lower in the tobramycin than in the placebo group (P=0.033). Conclusion Sinonasal inhalation of vibrating antibiotic aerosols appears promising for reducing pathogen colonization of paranasal sinuses and for control of symptoms in patients with CF.

The Role of Sphingolipids and Ceramide in Pulmonary Inflammation in Cystic Fibrosis

Sphingolipids and in particular ceramide have been shown to be critically involved in the response to many receptor-mediated, but also receptor-independent, mainly stress stimuli. Recent studies demonstrate that ceramide plays an important role in the pathogenesis of cystic fibrosis, a hereditary metabolic disorder caused by mutations of the Cystic Fibrosis Transmembrane Conductance Regulator. Patients with cystic fibrosis suffer from chronic pulmonary inflammation and microbial lung infections, in particular with Pseudomonas aeruginosa. Chronic pulmonary inflammation in these patients seems to be the initial pathophysiological event. Inflammation may finally result in the high infection susceptibility of these patients, fibrosis and loss of lung function. Recent studies demonstrated that ceramide accumulates in lungs of cystic fibrosis mice and causes age-dependent pulmonary inflammation as indicated by accumulation of neutrophils and macrophages in the lung and increased pulmonary concentrations of Interleukins 1 and 8, death of bronchial epithelial cells, deposition of DNA in bronchi and high susceptibility to Pseudomonas aeruginosa infections. Genetic or pharmacological inhibition of the acid sphingomyelinase blocks excessive ceramide production in lungs of cystic fibrosis mice and corrects pathological lung findings. First clinical studies confirm that inhibition of the acid sphingomyelinase with small molecules might be a novel strategy to treat patients with cystic fibrosis.