Impact of surfactant protein D, interleukin-5, and eosinophilia on Cryptococcosis

Abstract

Cryptococcus neoformans is an opportunistic fungal pathogen that initiates infection following inhalation. As a result, the pulmonary immune response provides a first line of defense against C. neoformans. Surfactant protein D (SP-D) is an important regulator of pulmonary immune responses and is typically host protective against bacterial and viral respiratory infections. However, SP-D is not protective against C. neoformans. This is evidenced by previous work from our laboratory demonstrating that SP-D-deficient mice infected with C. neoformans have a lower fungal burden and live longer than wild-type (WT) control animals. We hypothesized that SP-D alters susceptibility to C. neoformans by dysregulating the innate pulmonary immune response following infection. Thus, inflammatory cells and cytokines were compared in the bronchoalveolar lavage fluid from WT and SP-D(-/-) mice after C. neoformans infection. Postinfection, mice lacking SP-D have reduced eosinophil infiltration and interleukin-5 (IL-5) in lung lavage fluid. To further explore the interplay of SP-D, eosinophils, and IL-5, mice expressing altered levels of eosinophils and/or IL-5 were infected with C. neoformans to assess the role of these innate immune mediators. IL-5-overexpressing mice have increased pulmonary eosinophilia and are more susceptible to C. neoformans infection than WT mice. Furthermore, susceptibility of SP-D(-/-) mice to C. neoformans infection could be restored to the level of WT mice by increasing IL-5 and eosinophils by crossing the IL-5-overexpressing mice with SP-D(-/-) mice. Together, these studies support the conclusion that SP-D increases susceptibility to C. neoformans infection by promoting C. neoformans-driven pulmonary IL-5 and eosinophil infiltration.

FIG 1

SP-D^−/− mice are more resistant than WT mice to aerosol exposure of C. neoformans. (A) Mice were exposed to aerosolized H99 Stud in full-body Madison chambers, and fungal burden was assessed in the lung up to 21 days postinfection. Each time point was repeated at least twice for a total of 7 to 8 mice/group. Representative images from histological analyses of paraffin-embedded and H&E-stained lung tissue from WT mice (B) and SP-D^−/− mice (C) after aerosol exposure are shown. *, P < 0.05; **, P < 0.01.

FIG 2

Total CD45⁺ cells are decreased in SP-D^−/− mice compared to WT mice after C. neoformans infection. Flow cytometric analysis of total CD45⁺ cells in WT and SP-D^−/− mice after C. neoformans infection in lung interstitium (day 3, n = 5 to 9 mice/group; day 7, n = 14 to 21 mice/group) (A) and lung BAL fluid (day 3, n = 1 to 2 samples/group; day 7, n = 5 to 8 samples/group; samples were pooled and normalized to per mouse cell counts) (B). Shown in panels A and B are absolute numbers of cells per mouse calculated from total cellularity obtained and the percentage of CD45⁺ cells. Frequency of PMNs in lung interstitium (C) and BAL fluid (D), of CD11c⁺ MHC-II^int cells in lung interstitium (E) and BAL fluid (F), of CD11c⁺ MHC-II^hi cells in lung interstitium (G) and BAL fluid (H), and of lymphocytes/other CD45⁺ cells in lung interstitium (I) and BAL fluid (J) are shown. In panels C to J, data are shown as percentages of CD45⁺ cells. *, P < 0.05 for WT infected versus SP-D^−/− infected mice.

FIG 3

IL-5 production and eosinophil infiltration are reduced in infected SP-D^−/− mice compared to levels in WT mice. WT and SP-D^−/− mice were infected intranasally with 5 × 10⁵ H99 Stud cells. (A) Eosinophils in BAL fluid were assessed at 7 days postinfection via cytospin and H&E staining (n = 3 to 11 mice/group). (B) IL-5 protein levels were measured at 3 days postinfection in BAL fluid by ELISA (n = 8 to 10 mice/group). (C) IL-5 mRNA steady-state level was measured on days 1 to 3 postinfection in postlavage lung tissue (n = 4 to 10 mice/group). (D) Mice were administered IL-5 neutralizing antibodies at the time of infection. Eosinophils were assessed in BAL fluid at 7 days postinfection via cytospin and H&E staining. For the IL-5 blockade, data are shown as frequency of the respective isotype-treated control (n = 12 to 14 mice/group). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Photomicrographs show representative cytospins used for analysis of saline-treated WT mice (E), saline-treated SP-D^−/− mice (F), C. neoformans-infected WT mice (G), and C. neoformans-infected SP-D^−/− mice (H). Arrowheads indicate macrophages, and arrows indicate eosinophils.

FIG 4

IL-5-overexpressing mice are more susceptible to C. neoformans than WT mice. (A) Pulmonary fungal burden at 7 days post-C. neoformans infection in WT and IL-5Tg mice. ***, P < 0.001. (B) Survival analysis of WT and IL-5Tg mice (two independent experiments for a total of at least 18 mice/group; P < 0.0001).

FIG 5

IL-5 overexpression increases the susceptibility of SP-D^−/− mice to C. neoformans. (A) Pulmonary fungal burden at 14 days post-C. neoformans infection in WT, IL-5Tg, SP-D^−/−, and IL-5TgxSP-D^−/− mice. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (B) Survival analysis of WT, IL-5Tg, SP-D^−/−, and IL-5TgxSP-D^−/− mice (two independent experiments for a total of at least 3 mice/group; for the WT versus SP-D^−/−, P <0.001; for the WT versus IL-5TgxSP-D^−/−, not significant; for IL-5TgxSP-D^−/− versus SP-D^−/−, P < 0.05; and for IL-5TgxSP-D^−/− versus IL-5Tg, P < 0.001).

FIG 6

Representative photomicrographs from H&E-stained lung sections 7 days after C. neoformans infection: WT sham-infected mice (A), WT C. neoformans-infected mice (B), SP-D^−/− sham-infected mice (C), SP-D^−/− C. neoformans-infected mice (D), IL-5Tg sham-infected mice (E), IL-5Tg C. neoformans-infected mice (F), IL-5TgxSP-D^−/− sham-infected mice (G), and IL-5TgxSP-D^−/− C. neoformans-infected mice (H). Arrows indicate C. neoformans cells; the cell wall of C. neoformans is stained by hematoxylin.