Allantoin, a metabolite generated in the purine degradation pathway, was primarily considered an intermediate for recycling of the abundant nitrogen assimilated in plant purines. More specifically, tropical legumes utilize allantoin and allantoic acid as major nodule-to-shoot nitrogen transport compounds. In other species, an increase in allantoin content was observed under different stress conditions, but the underlying molecular mechanisms remain poorly understood. In this work, Arabidopsis thaliana was used as a model system to investigate the effects of salt stress on allantoin metabolism and to know whether its accumulation results in plant protection. Plant seedlings treated with NaCl at different concentrations showed higher allantoin and lower allantoic acid contents. Treatments with NaCl favored the expression of genes involved in allantoin synthesis, but strongly repressed the unique gene encoding allantoinase (AtALN). Due to the potential regulatory role of this gene for allantoin accumulation, AtALN promoter activity was studied using a reporter system. GUS mediated coloration was found in specific plant tissues and was diminished with increasing salt concentrations. Phenotypic analysis of knockout, knockdown and stress-inducible mutants for AtALN revealed that allantoin accumulation is essential for salt stress tolerance. In addition, the possible role of allantoin transport was investigated. The Ureide Permease 5 (UPS5) is expressed in the cortex and endodermis of roots and its transcription is enhanced by salt treatment. Ups5 knockout plants under salt stress presented a susceptible phenotype and altered allantoin root-to-shoot content ratios. Possible roles of allantoin as a protectant compound in oxidative events or signaling are discussed.

A deficiency in cystic fibrosis transmembrane conductance regulator (CFTR) function in CF leads to chronic lung disease. CF is associated with abnormalities in fatty acids, ceramides, and cholesterol, their relationship with CF lung pathology is not completely understood. Therefore, we examined the impact of CFTR deficiency on lipid metabolism and pro-inflammatory signaling in airway epithelium using mass spectrometric, protein array. We observed a striking imbalance in fatty acid and ceramide metabolism, associated with chronic oxidative stress under basal conditions in CF mouse lung and well-differentiated bronchial epithelial cell cultures of CFTR knock out pig and CF patients. Cell-autonomous features of all three CF models included high ratios of ω-6- to ω-3-polyunsaturated fatty acids and of long- to very long-chain ceramide species (LCC/VLCC), reduced levels of total ceramides and ceramide precursors. In addition to the retinoic acid analog fenretinide, the anti-oxidants glutathione (GSH) and deferoxamine partially corrected the lipid profile indicating that oxidative stress may promote the lipid abnormalities. CFTR-targeted modulators reduced the lipid imbalance and oxidative stress, confirming the CFTR dependence of lipid ratios. However, despite functional correction of CF cells up to 60% of non-CF in Ussing chamber experiments, a 72-h triple compound treatment (elexacaftor/tezacaftor/ivacaftor surrogate) did not completely normalize lipid imbalance or oxidative stress.Protein array analysis revealed differential expression and shedding of cytokines and growth factors from CF epithelial cells compared to non-CF cells, consistent with sterile inflammation and tissue remodeling under basal conditions, including enhanced secretion of the neutrophil activator CXCL5, and the T-cell activator CCL17. However, treatment with antioxidants or CFTR modulators that mimic the approved combination therapies, ivacaftor/lumacaftor and ivacaftor/tezacaftor/elexacaftor, did not effectively suppress the inflammatory phenotype.We propose that CFTR deficiency causes oxidative stress in CF airway epithelium, affecting multiple bioactive lipid metabolic pathways, which likely play a role in CF lung disease progression. A combination of anti-oxidant, anti-inflammatory and CFTR targeted therapeutics may be required for full correction of the CF phenotype.

The airway mucosal microenvironment is crucial for host defense against inhaled pathogens but remains poorly understood. We report here that the airway surface normally undergoes surprisingly large excursions in pH during breathing that can reach pH 9.0 during inhalation, making it the most alkaline fluid in the body. Transient alkalinization requires luminal bicarbonate and membrane-bound carbonic anhydrase 12 (CA12) and is antimicrobial. Luminal bicarbonate concentration and CA12 expression are both reduced in cystic fibrosis (CF), and mucus accumulation both buffers the pH and obstructs airflow, further suppressing the oscillations and bacterial-killing efficacy. Defective pH oscillations may compromise airway host defense in other respiratory diseases and explain CF-like airway infections in people with CA12 mutations.