A vexing problem in cystic fibrosis (CF) pathogenesis has been to explain the high prevalence of Pseudomonas aeruginosa biofilms in CF airways. We speculated that airway surface liquid (ASL) hyperabsorption generates a concentrated airway mucus that interacts with P. aeruginosa to promote biofilms. To model CF vs. normal airway infections, normal (2.5% solids) and CF-like concentrated (8% solids) mucus were prepared, placed in flat chambers, and infected with an Ϸ5 ؋ 10 3 strain PAO1 P. aeruginosa. Although bacteria grew to 10 10 cfu/ml in both mucus concentrations, macrocolony formation was detected only in the CF-like (8% solids) mucus. Biophysical and functional measurements revealed that concentrated mucus exhibited properties that restrict bacterial motility and small molecule diffusion, resulting in high local bacterial densities with high autoinducer concentrations. These properties also rendered secondary forms of antimicrobial defense, e.g., lactoferrin, ineffective in preventing biofilm formation in a CF-like mucus environment. These data link airway surface liquid hyperabsorption to the high incidence of P. aeruginosa biofilms in CF via changes in the hydration-dependent physical-chemical properties of mucus and suggest that the thickened mucus gel model will be useful to develop therapies of P. aeruginosa biofilms in CF airways.mucus ͉ rheology C ystic fibrosis (CF) lung disease reflects the chronic bacterial infection of intrapulmonary airways with Pseudomonas aeruginosa biofilms (1, 2). P. aeruginosa biofilms have mostly been studied in flow chambers (3-7) that are good models for the biofilms that form on venous or urethral catheters under conditions of high flow rates and relatively high oxygen tensions. However, biofilms in CF airways form in thickened (concentrated) mucus gels that are relatively hypoxic and adherent to airway surfaces (see Fig. 1A) (8, 9). Recently, Sriramulu et al. (10) investigated the role autoregulators and amino acids play in the tightness of biofilm formation in a mucus stimulant of constant hydration. For the present study, we designed a culture system to investigate whether dehydration of the CF mucus environment may predispose to P. aeruginosa biofilm formation.A key aspect of the model was the selection of mucus concentrations that mimic normal and CF mucus. Although measurements of mucus concentration, i.e., the percentage of solids content, from CF subjects before infection have not been reported, estimates from cell cultures (11) and sputum (12-18) suggest that CF mucus is at least three or four times more concentrated than normal. Therefore, mucus was obtained from well differentiated human airway cultures and was isotonically concentrated (1) to produce mucus of normal (2.5% solids wt/wt) and CF-like (8%) concentrations (Fig. 1B). Aliquots of each were deposited in (i) an open chamber to model a mucus plaque adherent to CF airway surfaces with a residual lumen allowing airflow and (ii) a closed chamber to mimic a mucus plug occluding a CF airway (Fig. 1 A). P....

Studies of primary ciliary dyskinesia (PCD) have been hampered by the lack of a suitable animal model because disruption of essential ciliary genes in mice results in a high incidence of lethal hydrocephalus. To develop a viable mouse model for long-term studies of PCD, we have generated a transgenic mouse line in which two conserved exons of the mouse intermediate dynein chain gene, Dnaic1, are flanked by loxP sites (Dnaic1(flox/flox)). Dnaic1 is the murine homolog of human DNAI1, which is mutated in approximately 10% of human PCD cases. These mice have been crossed with mice expressing a tamoxifen-inducible Cre recombinase (CreER). Treatment of adult Dnaic1(flox/flox)/CreER(+/-) mice with tamoxifen results in an almost complete deletion of Dnaic1 with no evidence of hydrocephalus. Treated animals have reduced levels of full-length Dnaic1 mRNA, and electron micrographs of cilia demonstrate a loss of outer dynein arm structures. In treated Dnaic1(flox/flox)/CreER(+/-) animals, mucociliary clearance (MCC) was reduced over time. After approximately 3 months, no MCC was observed in the nasopharynx, whereas in the trachea, MCC was observed for up to 6 months, likely reflecting a difference in the turnover of ciliated cells in these tissues. All treated animals developed severe rhinosinusitis, demonstrating the importance of MCC to the health of the upper airways. However, no evidence of lung disease was observed up to 11 months after Dnaic1 deletion, suggesting that other mechanisms are able to compensate for the lack of MCC in the lower airways of mice. This model will be useful for the study of the pathogenesis and treatment of PCD.

A balance sheet describing the integrated homeostasis of secretion, absorption, and surface movement of liquids on pulmonary surfaces has remained elusive. It remains unclear whether the alveolus exhibits an intra-alveolar ion/liquid transport physiology or whether it secretes ions/liquid that may communicate with airway surfaces. Studies employing isolated human alveolar type II (AT2) cells were utilized to investigate this question. Human AT2 cells exhibited both epithelial Na

Primary ciliary dyskinesia (PCD) is a genetic disorder in which impaired ciliary function leads to chronic airway disease. Exome sequencing of a PCD subject identified an apparent homozygous frameshift variant, c.887_890delTAAG (p.Val296Glyfs*13), in exon 5; this frameshift introduces a stop codon in amino acid 308 of the growth arrest-specific protein 2-like 2 (GAS2L2). Further genetic screening of unrelated PCD subjects identified a second proband with a compound heterozygous variant carrying the identical frameshift variant and a large deletion (c.867_*343þ1207del; p.?) starting in exon 5. Both individuals had clinical features of PCD but normal ciliary axoneme structure. In this research, using human nasal cells, mouse models, and X.laevis embryos, we show that GAS2L2 is abundant at the apical surface of ciliated cells, where it localizes with basal bodies, basal feet, rootlets, and actin filaments. Cultured GAS2L2deficient nasal epithelial cells from one of the affected individuals showed defects in ciliary orientation and had an asynchronous and hyperkinetic (GAS2L2-deficient ¼ 19.8 Hz versus control ¼ 15.8 Hz) ciliary-beat pattern. These results were recapitulated in Gas2l2 À/À mouse tracheal epithelial cell (mTEC) cultures and in X. laevis embryos treated with Gas2l2 morpholinos. In mice, the absence of Gas2l2 caused neonatal death, and the conditional deletion of Gas2l2 impaired mucociliary clearance (MCC) and led to mucus accumulation. These results show that a pathogenic variant in GAS2L2 causes a genetic defect in ciliary orientation and impairs MCC and results in PCD.

The Western Diet – characterized by high protein, sugar, fat and low fiber intake – is widely believed to contribute to the incidence and pathogenesis of inflammatory bowel diseases (IBD). However, high sodium chloride salt content, a defining feature of processed foods, has not been considered as a possible environmental factor that might drive IBD. We set out to bridge this gap. We examined murine models of colitis on either a high salt diet (HSD) or a low salt diet (LSD). We demonstrate that a HSD exacerbates inflammatory pathology in the IL-10-deficient murine model of colitis relative to mice fed a LSD. This was correlated with enhanced expression of numerous pro-inflammatory cytokines. Surprisingly, sodium accumulated in the colons of mice on a HSD, suggesting a direct effect of salt within the colon. Similar to the IL-10-deficient model, a HSD also enhanced cytokine expression during infection by Salmonella typhimurium. This occurred in the first three day of infection, suggesting that a HSD potentiates innate immune response. Indeed, in cultured dendritic cells we found that high salt media potentiates cytokine expression downstream of TLR4 activation via p38 MAPK and SGK1. A third common colitis model, administration of dextran sodium sulfate (DSS), was hopelessly confounded by the high sodium content of the DSS. Our results raise the possibility that high dietary salt is an environmental factor that drives increased inflammation in IBD.