Surfactant protein D alters allergic lung responses in mice and human subjects

Abstract

Background: Surfactant protein (SP) D has been proposed to be protective in allergic airway responses.

Objective: We aimed to determine the effect of SP-D deficiency on murine and human airway allergy.

Methods: Immunologic responses of SP-D gene-deficient mice (Sftpd-/-) at baseline and after 4 intranasal Aspergillus fumigatus exposures were assessed. In addition, the significance of a single nucleotide polymorphism (Met(11)Thr) in the human SP-D gene (known to decrease SP-D function) was investigated.

Results: Macrophage and neutrophil bronchoalveolar lavage fluid levels and large airway mucus production were increased in naive Sftpd-/- mice in association with increased lung CCL17 levels and CD4+ T cell numbers. T(H)2-associated antibody levels (IgG1 and IgE) were significantly lower in 4- to 5-week-old Sftpd-/- mice (P < .05). Accordingly, naive Sftpd-/- splenocytes released significantly less IL-4 and IL-13 on anti-CD3/CD28 stimulation (P < .01). After intranasal allergen exposures, a modest decrease in bronchoalveolar lavage fluid eosinophilia and IL-13 levels was observed in Sftpd-/- mice compared with values seen in wild-type mice in association with decreased airway resistance (P < .01). A single nucleotide polymorphism in the SFTPD gene, affecting SP-D levels and pathogen binding, was associated with decreased atopy in black subjects and potentially lower asthma susceptibility in white subjects.

Conclusion: Sftpd-/- mice have an impaired systemic T(H)2 response at baseline and reduced inflammation and airway responses after allergen exposure. Translational studies revealed that a polymorphism in the SFTPD gene was associated with lower atopy and possibly asthma susceptibility. Taken together, these results support the hypothesis that SP-D-dependent innate immunity influences atopy and asthma.

Figure 1. Increased cellular infiltrate in the lungs of sftpd^−/− mice

(A) BALF cells were collected in 4–6 week old wild type (WT) and Sftpd^−/− mice (KO) and total cells counted. Lungs cells were isolated as described in the method section and stained for FACS analysis. (B) After excluding dead cells and CD-45 negative cells, the percentage of CD-4 positive cells was determined. (C) Representative density plots of CD-69 positive lung cells from wild type and SFTPD^−/− mice are shown.

Figure 2. Chemokine and cytokine lung levels in wild type and sftpd^−/− mice

Lungs were collected and homogenized as described in the method section. Lung supernatants were used to measure the chemokines (A) CCL2/JE/MCP1, (B) CCL17/TARC and the Th2/Th1 cytokines (C) IL-4, (D) IL-13, (E) IFNγ and (F) IL-12p70 in 4 (open circle) and 6–7 week old (black circle) Sftpd^−/− mice and their wild type controls. Statistical significance was determined by Student’s T-test.

Figure 3. Impaired systemic Th2 profile in Sftpd^−/− mice at baseline

Total plasma (A) IgA, (B) IgE and (C) IgG1 levels were measured in 4–5 week old mice. Spleen cells from 4–5 week old wild type and Sftpd^−/− mice were stimulated with anti-CD3 and anti-CD28 as described in the method section. (D) IL-4, (E) IL-13 and (F) IFNγ levels were measured in culture supernatants at 48h. Statistical significance was determined by Mann-Whitney or Student’s T-test when appropriate.

Figure 4. Decreases BALF eosinophilia despite increased lung cytokine levels in Sftpd^−/− mice following repeated Aspergillus exposures

(A) Mice were subjected to two consecutive intranasal Aspergillus fumigatus exposures followed one week later by two subsequent exposures. BALF were collected 20h following the last intranasal challenge. Total BALF cells (B), eosinophils (C) and (D) eotaxin-2 BALF levels were assessed in 5–6 week old Sftpd^−/−− mice and their controls (17–18 mice total; 5 separate experiments). Lungs were homogenized 20h after the last challenge and the supernatant used to measure (E) IL-13 (F) IFNγ and (G) TNFα levels in Sftpd^−/− mice and their WT controls (8 mice/group; 2 separate experiments). Statistical significance was determined by Mann-Whitney or Student’s T-test when appropriate.