As the incidence of cystic fibrosis (CF) bone disease is increasing, we analyzed CF transmembrane conductance regulator (CFTR) deficient mice (CF mice) to gain pathogenic insights. In these studies comparing adult (14 wk) CF and C57BL/6J mice, both bone length and total area were decreased in CF mice. Metaphyseal trabecular and cortical density were also decreased, as well as diaphyseal cortical and total density. Trabecular bone volume was diminished in CF mice. Female CF mice revealed decreased trabecular width and number compared with C57BL/6J, whereas males demonstrated no difference in trabecular number. Female CF mice had reduced mineralizing surface and bone formation rates. Conversely, male CF mice had increased mineralizing surface, mineral apposition, and bone formation rates compared with C57BL/6J males. Bone formation rate was greater in males compared with female CF mice. Smaller bones with decreased density in CF, despite absent differences in osteoblast and osteoclast surfaces, suggest CF transmembrane conductance regulator influences bone cell activity rather than number. Differences in bone formation rate in CF mice are suggestive of inadequate bone formation in females but increased bone formation in males. This proanabolic observation in male CF mice is consistent with other clinical sex differences in CF.

Increased life expectancy in cystic fibrosis (CF) is accompanied by an increasing incidence of CF related diabetes (CFRD). Altered immune reactivity occurs in CF, which we hypothesize, is exacerbated by hyperglycemia. Cystic fibrosis transmembrane conductance regulator deficient (CFTR-/-) mice were rendered hyperglycemic by streptozotocin (STZ) to test this hypothesis. CFTR-/-, C57BL/6J, and FVB/NJ mice received either STZ or lactated ringers (LR) (n=5-10). Four weeks later, splenocytes were harvested, mitogen stimulated, and analyzed for cytokine production (IL-2, IL-4, and IL-10) along with stimulation indices (SI). SI of STZ-treated CFTR-/- were elevated compared to LR-treated mice, although both were greater than C57BL/6J and FVB/NJ (p<0.05). Fasting glucose levels of STZ-treated CFTR-/- mice correlated with SI (p<0.003). Stimulated IL-10 concentrations were elevated in STZ-treated CFTR-/- compared to LR-treated animals and controls (p<0.05). IL-2 levels were greater in CFTR-/- mice compared to controls (p<0.05), but unrelated to STZ. Reinforcing generalized cytokine up-regulation in CFTR-/-, IL-4 levels were greater in CFTR-/- mice compared to C57BL/6J, but FVB/NJ mice demonstrated greatest concentrations following STZ. These results suggest that, hyperglycemia may exacerbate the clinical course in CF by impacting immune reactivity. There is clear need to maximize metabolic management in CFRD.

Interleukin-10 (IL-10) is a pleiotropic cytokine that plays a pivotal role in the regulation of immune responses. Hence, we evaluated the effects of a recombinant adeno-associated viral vector 1 (rAAV1) encoding rat IL-10 (rAAV1-IL-10) in a rat model of kidney allograft rejection. Dark Agouti rat kidneys were transplanted into Wistar-Furth (WF) rats 8 weeks following a single intramuscular administration of either rAAV1-IL-10 or rAAV1-green fluorescence protein (GFP). Isografts (WF-WF) served as an additional experimental control. Both allograft and isograft recipients received daily cyclosporine (10 mg/kg) for 14 days after transplantation. Serum IL-10 levels increased at 8, 12 and 16 weeks following vector administration in rAAV1-IL-10-treated animals, but not in rAAV1-GFP and isograft groups. rAAV1-IL-10 treatment resulted in lower BUN and creatinine levels (p<0.001), as well as increased allograft survival rates from 22% to 90%. Allograft histological abnormalities were significantly attenuated in the rAAV1-IL-10-treated rats compared with those of rAAV1-GFP controls. Serum levels of proinflammatory cytokines such as growth-related oncogene were also significantly higher in the rAAV1-GFP group than in the rAAV1-IL-10 group. These data suggest delivery of IL-10 using a rAAV1 vector improves renal function and prolongs graft survival in a rat model of kidney transplant rejection.

The cause of cystic fibrosis-related diabetes (CFRD) remains unknown, but cystic fibrosis transmembrane conductance regulator (CFTR) mutations contribute directly to multiple aspects of the cystic fibrosis phenotype. We hypothesized that susceptibility to islet dysfunction in cystic fibrosis is determined by the lack of functional CFTR. To address this, glycemia was assessed in CFTR null (CFTR(-/-)), C57BL/6J, and FVB/NJ mice after streptozotocin (STZ)-induced beta-cell injury. Fasting blood glucose levels were similar among age-matched non-STZ-administered animals, but they were significantly higher in CFTR(-/-) mice 4 weeks after STZ administration (288.4 +/- 97.4, 168.4 +/- 35.9, and 188.0 +/- 42.3 mg/dl for CFTR(-/-), C57BL/6J, and FVB/NJ, respectively; P < 0.05). After intraperitoneal glucose administration, elevated blood glucose levels were also observed in STZ-administered CFTR(-/-) mice. STZ reduced islets among all strains; however, only CFTR(-/-) mice demonstrated a negative correlation between islet number and fasting blood glucose (P = 0.02). To determine whether a second alteration associated with cystic fibrosis (i.e., airway inflammation) could impact glucose control, animals were challenged with Aspergillus fumigatus. The A. fumigatus-sensitized CFTR(-/-) mice demonstrated similar fasting and stimulated glucose responses in comparison to nonsensitized animals. These studies suggest metabolic derangements in CFRD originate from an islet dysfunction inherent to the CFTR(-/-) state.

To gain insight into aberrant cytokine regulation in cystic fibrosis (CF), we compared the phenotypic manifestations of allergen challenge in gut-corrected CFTR-deficient mice with background-matched C57Bl6 (B6) mice. Aspergillus fumigatus (Af) antigen was used to mimic allergic bronchopulmonary aspergillosis, a peculiar hyper-IgE syndrome with a high prevalence in CF patients. CFTR-/-, C57BL/6 and FVB/NJ mice were sensitized with Af antigen by serial intraperitoneal injections. Control mice were mock sensitized with PBS. Challenges were performed by inhalation of Af antigen aerosol. After Af antigen challenge, histologic analysis showed goblet cell hyperplasia and lymphocytic infiltration in both strains. However, total serum IgE levels were markedly elevated in CF mice. Sensitized CF mice showed a five-fold greater IgE response to sensitization as compared with B6- and FVB-sensitized controls. Additional littermate controls to fully normalize for B6-FVB admixture in the strain background confirmed the role of CFTR mutation in the hyper-IgE syndrome. Cytokine mRNA levels of IL-5 and GM-CSF in the bronchoalveolar lavage (BAL) fluid, and BAL cell differentials indicated that CFTR mutation caused a shift from an IL-5-predominant to an IL-4-predominant cytokine profile. This system models a very specific type of airway inflammation in CF and could provide insights into pathogenesis and treatment of the disease.