Absence of the cystic fibrosis transmembrane regulator (Cftr) from myeloid-derived cells slows resolution of inflammation and infection

Abstract

The absence or reduction of CFTR function causes CF and results in a pulmonary milieu characterized by bacterial colonization and unresolved inflammation. The ineffectiveness at controlling infection by species such as Pseudomonas aeruginosa suggests defects in innate immunity. Macrophages, neutrophils, and DCs have all been shown to express CFTR mRNA but at low levels, raising the question of whether CFTR has a functional role in these cells. Bone marrow transplants between CF and non-CF mice suggest that these cells are inherently different; we confirm this observation using conditional inactivation of Cftr in myeloid-derived cells. Mice lacking Cftr in myeloid cells overtly appear indistinguishable from non-CF mice until challenged with bacteria instilled into the lungs and airways, at which point, they display survival and inflammatory profiles intermediate in severity as compared with CF mice. These studies demonstrate that Cftr is involved directly in myeloid cell function and imply that these cells contribute to the pathophysiological phenotype of the CF lung.

Figure 1.. Molecular characterization of conditional, myeloid expression.

Cre-mediated recombination was first assessed in a panel of tissues from mice homozygous for the Cftr^fl10 allele with LysMCre transgene (A), with or without the LysMCre transgene (B). Tissues surveyed include thymus (T), spleen (S), lung (Lu), intestine (I), heart (H), and liver (Li). Recombination, as evidenced by the 148-bp band, was only found in Cre-containing mice. To examine the extent of recombination, DNA from bone marrow-derived macrophages was examined by PCR for the exon 10 deletion (C). Bone marrow-derived macrophages from Cftr^fl10 homozygous mice lacking Cre (Cftr^Δfl10) show only the 408-bp nonrecombined form of the allele, whereas macrophages carrying the LysMCre transgene (Cftr^fl10+LysMCre) show only the 148-bp recombined product, identical to mice with systemic absence of exon 10 (Cftr¹⁰). To examine cell-type specificity of recombination, purified populations of various myeloid and nonmyeloid immune cells were surveyed (D). Controls, in the absence of Cre (−) show only the 408-bp product, whereas systemic absence of exon 10 (+) shows only the 148-bp product. Alveolar macrophages (AM), peritoneal neutrophils (PN), splenic T cells (ST), and bone marrow-derived macrophages (BM) were compared from mice carrying Cre (LysMCre+) and mice lacking Cre (Cre−). Only myeloid-derived cells showed recombination, as T cells showed no sign of recombination.

Figure 2.. Gastrointestinal epithelial Cftr function is unaffected by myeloid-specific deletion of Cftr exon 10.

(A–D) Representative traces of short-circuit current across sections of excised duodenum from each of the mouse lines. At the time indicated by the arrow, forskolin (10 μM) and IBMX (100 μM) were added to the basolateral bathing solution to stimulate CFTR-mediated short-circuit current. (E) Summary data for forskolin/IBMX-stimulated short-circuit current in each of the four mouse lines (mean±sem; n=4–7 animals in each group; *P<0.05 using the standard paired t-test comparing C57BL/6J mice with Cftr^tm1Unc or comparing Cftr^fl10 mice with Cftr^fl10+LysMCre). One-way ANOVA analysis was used for the comparison between C57BL/6J and Cftr^fl10 and Cftr^fl10 + LysMCre.

Figure 3.. Age and weight of animals.

C57BL/6J (n=51), Cftr^fl10 (n=47), Cftr^fl10 + LysMCre (n=80), and Cftr^tm1Unc (n=31) were evaluated at a comparable age range (A) for weight differences (B). (A) Animals of each genotype group were of comparable age. (B) Animals homozygous for the floxed allele, with or without the LysMCre transgene, were indistinguishable by weight from C57BL/6J mice. Cftr^tm1Unc were considerably smaller than the other groups (*P≤0.05 using one-way ANOVA analysis).

Figure 4.. NPD is not affected by absence of Cftr from myeloid cells.

Change in NPD (ΔNPD) was measured as a response to low Cl⁻ plus forskolin. C57BL/6J mice have the greatest hyprpolarization of NPD (A), and Cftr^tm1Unc mice (C) have no detectable response, but Cftr^fl10 mice (B) and Cftr^fl10 carrying the LysMCre transgene (D) showed significant responses to stimulation and are comparable with each other. (E) Mean responses are shown (mean±sem; n=4–7 animals/group). *P ≤ 0.05 using the standard paired t-test comparing C57BL/6J mice with Cftr^tm1Unc or comparing Cftr^fl10 mice with Cftr^fl10 + LysMCre. One-way ANOVA analysis was used for the comparison between C57BL/6J and Cftr^fl10 and Cftr^fl10 + LysMCre.

Figure 5.. Survival analysis of mice infected with P. aeruginosa.

Mice homozygous for the Cftr^fl10 and C57BL/6J WT controls were combined (n=114; individual curves were almost identical) and showed better survival following infection than Cftr^fl10 mice carrying the LysMCre transgene (n=53) or Cftr^tm1Unc mice (n=47; P=0.04 and P<0.0001, respectively, using Cox regression analysis stratifying on experiment). The survival curves of Cftr^tm1Unc and Cftr^fl10 mice carrying the LysMCre transgene did not differ significantly (P=0.09; see text for detailed description of statistical applications).

Figure 6.. Clinical scores postinfection with P. aeruginosa.

Cftr^fl10 + LysMCre (n=42), Cftr^tm1Unc (n=49), and controls (n=32) were infected with P. aeruginosa-laden agarose beads and euthanized at Day 3 or Day 10. For the duration of the study, animals were evaluated for clinical scores based on the criteria outlined in Table 2. Clinical scores at Day 3 were not different among the three animal groups. At Day 10, Cftr^fl10 + LysMCre and Cftr^tm1Unc (*P≤0.05) were significantly higher than the Cftr^fl10 controls. Nonparametric Mann-Whitney tests were done to determine significance of Cftr^fl10 + LysMCre and controls and Cftr^tm1Unc and controls.

Figure 7.. Immune cell composition in the lung in the absence of infection.

BALF was obtained from Cftr^fl10 (n=12), Cftr^fl10 + LysMCre (n=14), and Cftr^tm1Unc (n=16) mice and evaluated for total cell count (A) and relative composition of neutrophils (B), macrophages (C), and lymphocytes (D). Total cell counts were elevated in Cftr^fl10 + LysMCre and Cftr^tm1Unc mice at baseline relative to controls (*P≤0.05). The shift in differential was toward neutrophils in the Cftr^tm1Unc mice (*P≤0.05) with elevated neutrophils and macrophages in the BAL of Cftr^fl10 + LysMCre (*P≤0.05). Nonparametric Mann-Whitney tests were done to determine significance.

Figure 8.. Immune response to infection with P. aeruginosa.

Cftr^fl10 + LysMCre (n=42), Cftr^tm1Unc (n=49), and controls (n=32) were infected with P. aeruginosa-laden agarose beads and euthanized at Day 3 or Day 10. BALF was obtained for total cell count (A) and the relative composition of neutrophils (B), macrophages (C), and lymphocytes (D). There was no difference in total white cell recruitment at Day 3 between the groups. Cftr^fl10 + LysMCre (·P=0.07) and Cftr^tm1Unc (*P≤0.05) had elevated white blood cells compared with controls at Day 10. The composition of the elevated cell counts was associated with elevated neutrophils (Fig. 7B; *P≤0.05) but not alveolar macrophages or lymphocytes. Nonparametric Mann-Whitney tests were done to determine the significance between Cftr^fl10 + LysMCre and controls and Cftr^tm1Unc and controls.

Figure 9.. Gross lung pathology at Day 10.

At Day 10, animals were euthanized, and lungs were evaluated for gross lung pathology as outlined in Table 2. Cftr^fl10 + LysMCre (n=14) and Cftr^tm1Unc (n=11) mice had elevated gross lung pathology scores compared with controls (n=15 from three different experiments; *P≤0.05). Nonparametric Mann-Whitney tests were done to determine significance comparing Cftr^fl10 + LysMCre and controls and Cftr^tm1Unc and controls.

Figure 10.. Bacteriology at Days 3 and 10 postinfection.

BALF and whole lung homogenate were obtained from Cftr^fl10, Cftr^fl10 + LysMCre, and Cftr^tm1Unc mice and controls and cultured for P. aeruginosa growth from three different experiments. All values of P ≤ 0.05 are indicated with a star. At Day 3, Cftr^tm1Unc mice had a higher bacterial load than Cftr^fl10 (P=0.03; n=10 mice), but Cftr^fl10 and Cftr^fl10 + LysMCre were not different (P>0.05). At Day 10, Cftr^tm1Unc (P=0.001; n=11) and Cftr^fl10 + LysMCre (P=0.07; n=15) mice had elevated P. aeruginosa growth, relative to Cftr^fl10 (n=15). Nonparametric Mann-Whitney tests were done to determine the significance Cftr^fl10 + LysMCre and controls and Cftr^tm1Unc and controls.