Serum inflammatory profiles in cystic fibrosis mice with and without Bordetella pseudohinzii infection.

Cystic fibrosis (CF) is an autosomal recessive disease caused by dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) protein, and is marked by an accumulation of mucus in affected airways resulting in persistent infection and chronic inflammation. Quantitative differences in inflammatory markers have been observed in CF patient serum, tracheal cells, and bronchoalveolar lavage fluid, in the absence of detectable infection, implying that absent CFTR function alone may result in dysregulated immune responses. To examine the relationship between absent CFTR and systemic inflammation, 22 analytes were measured in CF mice (F508del/F508del) sera using the MSD multiplex platform. Pro-inflammatory cytokines IL-2, TNF-α, IL-17α, IFN-γ, IL-1ß, and MIP-3α are significantly elevated in infection-naïve CF mice (p < 0.050). Anti-inflammatory cytokines IL-10 and IL-4 are also significantly increased (p = 0.00003, p = 0.004). Additionally, six general markers of inflammation are significantly different from non-CF controls (p < 0.050). To elucidate the effects of chronic infection on the CF inflammatory profile, we examined CF mice exposed to spontaneous Bordetella pseudohinzii infections. There are no statistical differences in nearly all inflammatory markers when compared to their infection-naïve CF counterparts, except in the Th2-derived IL-4 and IL-5 which demonstrate significant decreases following exposure (p = 0.046, p = 0.045). Lastly, following acute infection, CF mice demonstrate elevations in nearly all inflammatory markers, but exhibit a shortened return to uninfected levels over time, and suppression of Th1-derived IL-2 and IL-5 (p = 0.043, p = 0.011). These results imply that CF mice have a persistent inflammatory profile often indistinguishable from chronic infection, and a dysregulated humoral response during and following active infection.

Genetic Variation Near chrXq22-q23 Is Linked to Emotional Functioning in Cystic Fibrosis.

INTRODUCTION: Cystic fibrosis (CF) is an autosomal recessive disease that affects many organ systems, most notably the pulmonary and gastrointestinal systems. Through genome-wide association studies, multiple genetic regions modifying CF-related pulmonary and gastrointestinal symptoms have been identified, but translation of these findings to clinical benefit remains elusive. Symptom variation in CF patients has been associated with changes in health-related quality of life (HRQOL), but the relationship between CF symptom-modifying genetic loci and HRQOL has not been explored. The purpose of this study was to determine whether two previously identified genetic modifiers of CF-related pathology also modify the subscales of HRQOL. METHODS: HRQOL and genotype data were obtained and analyzed. Linear regressions were used to examine the amount of variance in HRQOL subscales that could be explained by genotype for each modifier locus. RESULTS: A significant regression equation was found between genotype for rs5952223, a variant near chrXq22-q23, and emotional functioning in a sample of 129 CF patients. DISCUSSION: These data suggest that genotype for this single-nucleotide polymorphism is associated with emotional functioning in CF patients and highlight this genetic region as a potential therapeutic target, irrespective of CF transmembrane conductance regulator genotype.

Genetic Correlates of Fatigue in Individuals with End Stage Renal Disease.

Fatigue is a prevalent symptom associated with decreased quality of life and increased mortality in individuals with end stage renal disease (ESRD), yet causes of fatigue in individuals with ESRD remain poorly understood. We examined gene expression of Neuronal PAS Domain Protein 2 (NPAS2) in relation to patient-reported fatigue in 122 individuals with ESRD. Independent samples t-tests were used to examine NPAS2 gene expression profiles of: non-fatigue versus fatigue. Multivariable regression analyses were used to examine the relationship between fatigue and numerous variables including depression. Participants were approximately 58 years old (+/- 13.2 years), 78% African American (n = 95), and 72% male (n = 88). The phenotype of fatigue was not significantly associated with gene expression of NPAS2 but was significantly associated with depression (p< .001). This study suggests that further research should examine the causal mechanism between depression and fatigue in order to identify genetic factors that could explain the high comorbidity of depression and fatigue.

Relationship of HIV Status and Fatigue, Cardiorespiratory Fitness, Myokines, and Physical Activity.

People living with HIV (PLWH) experience high rates of fatigue, which can be improved with physical activity. We examined relationships between HIV infection, fatigue, cardiorespiratory fitness, physical activity, and myokines. Twenty PLWH and 20 HIV-uninfected adults completed a fatigue assessment, a maximal cardiometabolic exercise test, serum measures of myokines, and wore an accelerometer for 7 days. Measures were completed at baseline, 3 months, and 6 months. At baseline, PLWH had more fatigue (4.7 ± 2.6 vs. 2.8 ± 2.5, p = .01) and higher peak ventilatory efficiency (VE/VCO2; 33 ± 5.5 vs. 30.2 ± 2.5; p = .06). Half of PLWH engaged in at least one 10-minute bout of physical activity in the previous week, compared with control subjects (65%). Over time, HIV infection and fibroblast growth factor 21 were associated with fatigue (p < .05). People living with HIV have more fatigue and a higher ventilatory efficiency; expression of fibroblast growth factor 21 may underpin this relationship.

Dysregulation of Circadian Rhythm Gene Expression in Cystic Fibrosis Mice.

Cystic fibrosis (CF) is autosomal recessive disease that affects multiple body systems. CF patients often experience sleep disturbances, altered sleep patterns, and sleep apnea. Sleep in mammals is controlled in part by circadian clock genes, including Clock, Bmal1, Period1, Period2, Cryptochrome1, and Cryptochrome2. The purpose of this study was to gain a better understanding of the biological underpinnings of disordered sleep experienced in CF. To accomplish this, we evaluated circadian clock gene expression profiles in CF and wildtype mice, divided into two subgroups each based on sleep condition. One subgroup of each genotype was permitted to maintain their sleep-wake cycle while the other was deprived of sleep for six hours prior to sacrifice. Brain, skeletal muscle, jejunum, colon, lung and adipose tissues were collected from each mouse. Quantitative polymerase chain reaction (PCR) was used to quantify expression of Clock, Bmal1, Period1, Period2, Cryptochrome1 and Cryptochrome2, and expression levels were compared between study groups. Our comparisons showed distinct differences between the CF groups and the wildtype groups under both sleep conditions. Additionally, we found the CF mice that had been sleep deprived had severely dysregulated expression of all measured genes in the lung apart from Cry1. Our findings suggest that (1) disordered sleep in CF may be caused by circadian system dysregulation and (2) the loss of the cystic fibrosis transmembrane conductance regulator (CFTR) is a causative factor in the dysregulated circadian clock gene expression profiles of CF mice.

AGTR2 absence or antagonism prevents cystic fibrosis pulmonary manifestations.

BACKGROUND: Pulmonary disease remains the primary cause of morbidity and mortality for individuals with cystic fibrosis (CF). Variants at a locus on the X-chromosome containing the type 2 angiotensin II receptor gene (AGTR2) were identified by a large GWAS as significantly associating with lung function in CF patients. We hypothesized that manipulating the angiotensin-signaling pathway may yield clinical benefit in CF. METHODS: Genetic subset analysis was conducted on a local CF cohort to extend the GWAS findings. Next, we evaluated pulmonary function in CF mice with a deleted AGTR2 gene, and in those who were given subcutaneous injections of PD123,319, a selective AGTR2 antagonist for 12 weeks beginning at weaning. RESULTS: The genetic subset analysis replicated the initial GWAS identified association, and confirmed the association of this locus with additional lung function parameters. Studies in genetically modified mice established that absence of the AGTR2 gene normalized pulmonary function indices in two independent CF mouse models. Further, we determined that pharmacologic antagonism of AGTR2 improved overall pulmonary function in CF mice to near wild-type levels. CONCLUSIONS: These results identify that reduced AGTR2 signaling is beneficial to CF lung function, and suggest the potential of manipulating the angiotensin-signaling pathway for treatment and/or prevention of CF pulmonary disease. Importantly, the beneficial effects were not CF gene mutation dependent, and were able to be reproduced with pharmacologic antagonism. As there are clinically approved drugs available to target the renin-angiotensin signaling system, these findings may be quickly translated to human clinical trials.

Early pulmonary disease manifestations in cystic fibrosis mice.

BACKGROUND: Altered pulmonary function is present early in the course of cystic fibrosis (CF), independent of documented infections or onset of pulmonary symptoms. New initiatives in clinical care are focusing on detection and characterization of preclinical disease. Thus, animal models are needed which recapitulate the pulmonary phenotype characteristic of early stage CF. METHODS: We investigated young CF mice to determine if they exhibit pulmonary pathophysiology consistent with the early CF lung phenotype. Lung histology and pulmonary mechanics were examined in 12- to 16-week-old congenic C57bl/6 F508del and R117H CF mice using a forced oscillation technique (flexiVent). RESULTS: There were no significant differences in the resistance of the large airways. However, in both CF mouse models, prominent differences in the mechanical properties of the peripheral lung compartment were identified including decreased static lung compliance, increased elastance and increased tissue damping. CF mice also had distal airspace enlargement with significantly increased mean linear intercept distances. CONCLUSIONS: An impaired ability to stretch and expand the peripheral lung compartment, as well as increased distances between gas exchange surfaces, were present in young CF mice carrying two independent Cftr mutations. This altered pulmonary histopathophysiology in the peripheral lung compartment, which develops in the absence of infection, is similar to the early lung phenotype of CF patients.

RACK1 interacts with filamin-A to regulate plasma membrane levels of the cystic fibrosis transmembrane conductance regulator.

Mutations in cystic fibrosis transmembrane regulator (CFTR), a chloride channel in the apical membranes of secretory epithelial cells, underlie the fatal genetic disorder cystic fibrosis. Certain CFTR mutations, including the common mutation ΔF508-CFTR, result in greatly decreased levels of active CFTR at the apical membrane. Direct interactions between CFTR and the cytoskeletal adaptors filamin-A (FlnA) and Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) stabilize the expression and localization of CFTR at the plasma membrane. The scaffold protein receptor for activated C kinase 1 (RACK1) also stabilizes CFTR surface expression; however, RACK1 does not interact directly with CFTR and its mechanism of action is unknown. In the present study, we report that RACK1 interacts directly with FlnA in vitro and in a Calu-3 airway epithelial cell line. We mapped the interaction between RACK1 and FlnA to the WD4 and WD6 repeats of RACK1 and to a segment of the large rod domain of FlnA, consisting of immunoglobulin-like repeats 8-15. Disruption of the RACK1-FlnA interaction causes a reduction in CFTR surface levels. Our results suggest that a novel RACK1-FlnA interaction is an important regulator of CFTR surface localization.

COMMD1 interacts with the COOH terminus of NKCC1 in Calu-3 airway epithelial cells to modulate NKCC1 ubiquitination.

Mice deficient in Na-K-2Cl cotransporter (NKCC1) have been generated by targeted disruption of the gene encoding NKCC1 involving the carboxy terminus (CT-NKCC1) but not the amino terminus. We hypothesize that the resulting physiological defects are due to loss of proteins interacting with CT-NKCC1. Using a yeast two-hybrid approach, adaptor protein COMMD1 was found to bind to CT-NKCC1 (aa 1,040-1,212). Binding was verified in a yeast-independent system using GST-COMMD1 and myc-CT-NKCC1. Truncated COMMD1 and CT-NKCC1 peptides were used in binding assays to identify the site of interaction. The results demonstrate concentration-dependent binding of COMMD1 (aa 1-47) to CT-NKCC1 (aa 1,040-1,134). Endogenous COMMD1 was detected in pull downs using recombinant FLAG-CT-NKCC1; this co-pull down was blocked by COMMD1 (aa 1-47). CT-NKCC1 (aa 1,040-1,137) decreased basolateral membrane expression of NKCC1, and COMMD1 (aa 1-47) increased NKCC1 membrane expression. Downregulation of COMMD1 using silencing (si)RNA led to a transient loss of endogenous COMMD1 but did not affect activation of NKCC1 by hyperosmotic sucrose. Hyperosmolarity caused a transient increase in NKCC1 membrane expression, indicating regulated trafficking of NKCC1; downregulation of COMMD1 using siRNA reduced baseline (unstimulated) NKCC1 expression and blunted a transient elevation in NKCC1 membrane expression caused by hyperosmolarity. Constitutive downregulation of COMMD1 in HT29 engineered cells exhibited loss of COMMD1 and decreased NKCC1 membrane expression with no effect on activation of NKCC1. Loss of COMMD1 in Calu-3 cells and in HT29 cells led to reduced ubiquitinated NKCC1. The results indicate a role for COMMD1 in the regulation of NKCC1 membrane expression and ubiquitination.

Biochemical basis of the interaction between cystic fibrosis transmembrane conductance regulator and immunoglobulin-like repeats of filamin.

Mutations in the chloride channel cystic fibrosis transmembrane regulator (CFTR) cause cystic fibrosis, a genetic disorder characterized by defects in CFTR biosynthesis, localization to the cell surface, or activation by regulatory factors. It was discovered recently that surface localization of CFTR is stabilized by an interaction between the CFTR N terminus and the multidomain cytoskeletal protein filamin. The details of the CFTR-filamin interaction, however, are unclear. Using x-ray crystallography, we show how the CFTR N terminus binds to immunoglobulin-like repeat 21 of filamin A (FlnA-Ig21). CFTR binds to beta-strands C and D of FlnA-Ig21 using backbone-backbone hydrogen bonds, a linchpin serine residue, and hydrophobic side-chain packing. We use NMR to determine that the CFTR N terminus also binds to several other immunoglobulin-like repeats from filamin A in vitro. Our structural data explain why the cystic fibrosis-causing S13F mutation disrupts CFTR-filamin interaction. We show that FlnA-Ig repeats transfected into cultured Calu-3 cells disrupt CFTR-filamin interaction and reduce surface levels of CFTR. Our findings suggest that filamin A stabilizes surface CFTR by anchoring it to the actin cytoskeleton through interactions with multiple filamin Ig repeats. Such an interaction mode may allow filamins to cluster multiple CFTR molecules and to promote colocalization of CFTR and other filamin-binding proteins in the apical plasma membrane of epithelial cells.