Lack of cystic fibrosis transmembrane conductance regulator in CD3+ lymphocytes leads to aberrant cytokine secretion and hyperinflammatory adaptive immune responses

Abstract

Cystic fibrosis (CF), the most common fatal monogenic disease in the United States, results from mutations in CF transmembrane conductance regulator (CFTR), a chloride channel. The mechanisms by which CFTR mutations cause lung disease in CF are not fully defined but may include altered ion and water transport across the airway epithelium and aberrant inflammatory and immune responses to pathogens within the airways. We have shown that Cftr(-/-) mice mount an exaggerated IgE response toward Aspergillus fumigatus, with higher levels of IL-13 and IL-4, mimicking both the T helper cell type 2-biased immune responses seen in patients with CF. Herein, we demonstrate that these aberrations are primarily due to Cftr deficiency in lymphocytes rather than in the epithelium. Adoptive transfer experiments with CF splenocytes confer a higher IgE response to Aspergillus fumigatus compared with hosts receiving wild-type splenocytes. The predilection of Cftr-deficient lymphocytes to mount T helper cell type 2 responses with high IL-13 and IL-4 was confirmed by in vitro antigen recall experiments. Conclusive data on this phenomenon were obtained with conditional Cftr knockout mice, where mice lacking Cftr in T cell lineages developed higher IgE than their wild-type control littermates. Further analysis of Cftr-deficient lymphocytes revealed an enhanced intracellular Ca(2+) flux in response to T cell receptor activation. This was accompanied by an increase in nuclear localization of the calcium-sensitive transcription factor, nuclear factor of activated T cell, which could drive the IL-13 response. In summary, our data identified that CFTR dysfunction in T cells can lead directly to aberrant immune responses. These findings implicate the lymphocyte population as a potentially important target for CF therapeutics.

Figure 1.

Total serum IgE levels in congenic C57B6–cystic fibrosis (CF) transmembrane conductance regulator (CFTR)^−/− mice and their littermates after Aspergillus fumigatus (Af) sensitization and challenge. Mice were sensitized with 200 μg of Af crude extract dissolved in 100 μl of PBS on Days 1 and 14. Blood samples were collected on Day 32, 48 hours after the third aerosol Af challenge. Total serum IgE in the Af-sensitized mice was measured by ELISA. Data are shown as group averages (±SEM).

Figure 2.

Adoptive transfer of CF splenocytes confers elevated IgE phenolype. CFTR-ΔF508 (CFTR^−/−) mice on the B6 background or matched control C57BL/6 mice (CFTR^+/+) were used as donors for splenocyte adoptive transfer into B6-Rag^−/− mice. Total serum IgE (A and C) and IgM (B and D) were measured after Af challenge. (A and B) CFTR-ΔF508 (n = 6) and C57BL/6 littermates (n = 6) were sensitized with Af–crude protein extract (Af-cpe), and 2 weeks after sensitization their splenocytes were transferred into Rag^−/− mice, where they were allowed to engraft for 8 weeks before airway challenging the mice. (C and D) Naive CFTR-ΔF508 (n = 10) and naive C57BL/6 littermates (n = 10) were used to harvest naive splenocytes for transfer into Rag^−/− mice. Splenocytes were allowed to engraft for 8 weeks, and then Rag^−/− mice were sensitized and airway challenged with Af-cpe.

Figure 3.

In vitro antigen recall with mixed cell populations. Splenocytes from congenic CFTR^−/− or CFTR^+/+ mice sensitized to ovalbumin (OVA) were separated into both CD11b⁺ and CD4⁺ cell populations with magnetic beads. Cells were mixed and “crossed” to make four groups of cell preparations: (1) CF CD11b⁺ with CF CD4⁺ cells; (2) CF CD11b⁺ with wild-type (WT) CD4⁺ cells; (3) WT CD11b⁺ with CF CD4⁺ cells; and (4) WT CD11b⁺ with WT CD4⁺ cells. Cells were then stimulated with OVA for 5 days. The cell culture supernatants were analyzed for cytokine profiles at Day 5. Data are shown as group averages (±SEM).

Figure 4.

Total serum IgE levels in naive conditional CFTR knockout mice. T cell conditional knockout mice created by crossing the floxed Cftr mouse with mice expressing the Cre recombinase expressed from the leukocyte-specific protein tyrosine kinase (Lck) promoter were compared with control animals that were Cftr floxed, but Cre recombinase negative. Basal serum IgE levels were analyzed by ELISA. Data are shown as group averages (±SEM).

Figure 5.

Total serum IgE levels in Af-cpe–sensitized conditional CFTR knockout mice. T cell Cftr knockout mice (Cftr floxed and Lck Cre⁺) along with WT controls (Cftr floxed and Cre⁻) were sensitized with 200 μg of Af crude extract dissolved in 100 μl of PBS on Days 1 and 14. Blood samples were collected on Days 21, 28, and 32. Total serum IgE in the Af-sensitized mice was measured by ELISA. Data are shown as group averages (±SEM).

Figure 6.

Intracellular calcium (iCa²⁺) flux. Changes in the concentration of intracellular free Ca²⁺ ions are measured by monitoring the change in its emission spectrum of Indo-1 dye from blue to violet upon binding to Ca²⁺. The blue emission is measured through a 530/30 BP filter, and the violet through a 405/20 BP and 405 LP filter; thus, a shift in the violet:blue ratio over time is a reflection of the increase in iCa²⁺ concentration. CD4⁺ cells were enriched (see inset, top left) from Cftr-ΔF508 and Cftr^+/+ mice and stimulated with anti–CD3/CD28 antibodies (Abs), followed by 500 ng/ml of ionomycin (as a positive control; see inset on bottom right). (A) Representative iCa²⁺ flux for Cftr-ΔF508 and Cftr^+/+ CD4⁺ T cells. (B) The area under the curve for the traces from Cftr-ΔF508 (n = 6) and Cftr^+/+ (n = 6) in response to CD3/CD28 Abs. (C) Slopes for the traces from Cftr-ΔF508 (n = 6) and Cftr^+/+ (n = 6) in response to CD3/CD28 Abs.

Figure 7.

Nuclear factor of activated T cell (NFAT) c1 nuclear translocation and cytokine secretion in Cftr^−/− and Cftr^+/+ mice after T cell receptor stimulation. (A) Nuclear translocation of NFATc1 was measured using a modified ELISA from nuclear splenocyte extracts after CD3/CD28 stimulation at 10, 30, and 90 minutes after stimulation. Parallel plated cells were assessed for cytokine expression after CD3/CD28 stimulation at 1.5, 3, 6, and 24 hours. (B) Time course expression for TNF-α. (C) Expression of IL-13 at 24 hours after stimulation. Data are shown as group averages (±SEM) for a total of n = 5.