Hyperglycemia impedes lung bacterial clearance in a murine model of cystic fibrosis-related diabetes

Abstract

Cystic fibrosis-related diabetes (CFRD) is the most common comorbidity associated with cystic fibrosis (CF), impacting more than half of patients over age 30. CFRD is clinically significant, portending accelerated decline in lung function, more frequent pulmonary exacerbations, and increased mortality. Despite the profound morbidity associated with CFRD, little is known about the underlying CFRD-related pulmonary pathology. Our aim was to develop a murine model of CFRD to explore the hypothesis that elevated glucose in CFRD is associated with reduced lung bacterial clearance. A diabetic phenotype was induced in gut-corrected CF transmembrane conductance regulator (CFTR) knockout mice (CFKO) and their CFTR-expressing wild-type littermates (WT) utilizing streptozotocin. Mice were subsequently challenged with an intratracheal inoculation of Pseudomonas aeruginosa (PAO1) (75 μl of 1-5 × 10(6) cfu/ml) for 18 h. Bronchoalveolar lavage fluid was collected for glucose concentration and cell counts. A portion of the lung was homogenized and cultured as a measure of the remaining viable PAO1 inoculum. Diabetic mice had increased airway glucose compared with nondiabetic mice. The ability to clear bacteria from the lung was significantly reduced in diabetic WT mice and control CFKO mice. Critically, bacterial clearance by diabetic CFKO mice was significantly more diminished compared with nondiabetic CFKO mice, despite an even more robust recruitment of neutrophils to the airways. This finding that CFRD mice boast an exaggerated, but less effective, inflammatory cell response to intratracheal PAO1 challenge presents a novel and useful murine model to help identify therapeutic strategies that promote bacterial clearance in CFRD.

Keywords: Pseudomonas; cystic fibrosis; cystic fibrosis-related diabetes; murine model; pneumonia.

Fig. 1.

Diabetic phenotypes in wild-type (WT) and cystic fibrosis (CF) transmembrane conductance regulator (CFTR) knockout (CFKO) mice. A: Increase in blood glucose levels in streptozotocin (STZ)-treated mice (n = 11–19). Under basal conditions, no significant differences between WT and CFKO mice were observed. 30 days after STZ treatments, blood glucose levels were significantly elevated in both WT and CFKO mice but were not significantly different between genotypes. B: changes in mouse weight 30 days after STZ treatment. STZ-treated animals showed a greater loss of body weight than untreated controls regardless of genotype (*P < 0.05; n = 22–31). No differences between non-STZ-treated WT and CFKO mice were observed. C: intraperitoneal glucose tolerance test. STZ-treated animals showed an exaggerated increase in blood glucose levels following intraperitoneal glucose administration where differences from baseline to peak glucose concentrations were greater (*P < 0.05; n = 3–5). Based on area-under-the-curve analyses, CFKO and WT mice treated with STZ were not statistically different, as both STZ-treated CFKO and WT mice produced similar glycemic profiles. No differences between non-STZ-treated WT and CFKO mice were observed.

Fig. 2.

Glucose concentrations in the bronchoalveolar lavage fluid (BALF). Uninfected WT and CFKO mice treated with STZ displayed significantly higher levels of glucose in BALF compared with nondiabetic, genotype-matched controls (*P < 0.05; n = 4–6). No differences between STZ-treated wild-type and CFKO mice were observed.

Fig. 3.

Pulmonary leukocytic infiltrate levels following Pseudomonas aeruginosa (PAO1) instillation. Data are presented as the means ± SE of positively identified leukocytes in 10 high-powered fields following differential staining. A: relative leukocytic infiltrates in BALF from sham and infected mice. PAO1 infection caused a significant increase in leukocyte infiltration compared with basal levels of leukocytes in sham-operated controls of both genotypes (*P < 0.05; n = 8–12). PAO1-infected diabetic CFKO mice showed statistically higher levels of leukocytic infiltration compared with infected WT, diabetic WT, and CFKO mice (*P < 0.05). B: pulmonary macrophage levels in the BALF. BALF macrophage levels were increased by ∼2-fold following infection with PAO1 in WT mice (*P < 0.05) but not in diabetic WT mice. Macrophages in CFKO mice were not higher than in WT mice but were higher in diabetic CFKO mice (*P < 0.05). C: polymorphonuclear cells (PMNs, neutrophils) in the BALF. PMNs increased in each group with infection (*P < 0.05). The relative increase in total leukocytic infiltration is primarily a consequence of an increase of PMNs in the BALF, constituting upwards of 90% of the total infiltrate. *Statistical difference (P < 0.05) between sham and postinfection mice within each group. ‡Statistical difference (P < 0.05) between diabetic CFKO and other groups.

Fig. 4.

Lung histology following PAO1 infection. Left lobes were processed and stained with hematoxylin and eosin for morphological analyses. Findings support relative levels of leukocytes in the BALF determined through differential staining, where PAO1-infected diabetic CFKO mice displayed higher levels of leukocytic infiltration.

Fig. 5.

Pulmonary bacterial clearance after 18 h of PAO1 infection. Data are presented as a scatterplot of lung homogenate colony-forming units (CFUs) from individual mice (⧫; n = 8–16 per group) 18 h after PAO1 infection (△ = average CFUs/lung ± SE). The dashed line represents an approximate clearance threshold, based on 3 times the observed standard deviation above the mean value in nondiabetic WT mice for this dose of bacteria. Lung homogenates that displayed less than 25,000 CFUs/lung were considered to be cleared of the instilled PAO1 bacteria. Most WT mice were cleared 18 h postinfection. Counts in nondiabetic WT mice were significantly lower than in nondiabetic CFKO mice. Cultures from diabetic WT mice had substantially increased CFUs/lung compared with nondiabetic WT mice. CFUs/lung from diabetic CFKO mice were also substantially increased compared with nondiabetic CFKO mice. Interestingly, lungs from diabetic CFKO mice had significantly increased numbers of PAO1 CFUs compared with lungs from diabetic WT mice, suggesting that diabetic CFKO mice have a diminished ability to properly clear PAO1 infections. *P < 0.05, **P < 0.01.