Murine models of Aspergillosis: Role of collectins in host defense.

Aspergillus fumigatus, a ubiquitous fungus, causes a wide spectrum of clinical conditions ranging from allergic to invasive aspergillosis depending upon the hosts' immune status. Several animal models have been generated to mimic the human clinical conditions in allergic and invasive aspergillosis. The onset, duration and severity of the disease developed in models varied depending on the animal strain/fungal isolate, quantity and mode of administration of fungal antigens/spores, duration of the treatment, and type of immunosuppressive agent used. These models provide insight into host and pathogen factors and prove to be useful for evaluation of diagnostic markers and effective therapies. A series of studies established the protective role of collectins in murine models of Allergic Bronchopulmonary Aspergillosis and Invasive Pulmonary Aspergillosis. Collectins, namely surfactant protein A (SP-A), surfactant protein D (SP-D) and mannan binding lectin (MBL), are pattern recognition molecules regulating both innate and adaptive immune response against pathogens. In the present review, we discussed various murine models of allergic and invasive aspergillosis and the role of collectins in host defense against aspergillosis.

Surfactant protein SP-D modulates activity of immune cells: proteomic profiling of its interaction with eosinophilic cells.

Surfactant protein D (SP-D), a C-type lectin, is known to protect against lung infection, allergy and inflammation. Its recombinant truncated form comprising homotrimeric neck and CRD region (rhSP-D) has been shown to bring down specific IgE levels, eosinophilia and restore Th2-Th1 homeostasis in murine models of lung hypersensitivity. SP-D knockout mice show intrinsic hypereosinophilia and airway hyper-responsiveness that can be alleviated by rhSP-D. The rhSP-D can bind activated eosinophils, inhibit chemotaxis and degranulation, and selectively induce oxidative burst and apoptosis in sensitized eosinophils. A global proteomics study of rhSP-D-treated eosinophilic cell line AML14.3D10 identified large-scale molecular changes associated with oxidative burst, cell stress and survival-related proteins potentially responsible for apoptosis induction. The data also suggested an involvement of RNA binding- and RNA splicing-related proteins. Thus, the proteomics approach yielded a catalog of differentially expressed proteins that may be protein signatures defining mechanisms of SP-D-mediated maintenance of homeostasis during allergy.

Linking surfactant protein SP-D and IL-13: implications in asthma and allergy.

Surfactant protein D (SP-D) is an innate immune molecule that plays a protective role against lung infection, allergy, asthma and inflammation. In vivo experiments with murine models have shown that SP-D can protect against allergic challenge via a range of mechanisms including inhibition of allergen-IgE interaction, histamine release by sensitised mast cells, downregulation of specific IgE production, suppression of pulmonary and peripheral eosinophilia, inhibition of mechanisms that cause airway remodelling, and induction of apoptosis in sensitised eosinophils. SP-D can also shift helper T cell polarisation following in vivo allergenic challenge, from pathogenic Th2 to a protective Th1 cytokine response. Interestingly, SP-D gene deficient (-/-) mice show an IL-13 over-expressing phenotype. IL-13 has been shown to be involved in the development of asthma. Transgenic mice over-expressing IL-13 in the lung develop several characteristics of asthma such as pulmonary eosinophilia, airway epithelial hyperplasia, mucus cell metaplasia, sub-epithelial fibrosis, charcot-Leyden-Like crystals, airways obstruction, and non-specific airways hyper-responsiveness to cholinergic stimulation. Although both IL-4 and IL-13 are capable of inducing asthma like phenotype, the effector activity of IL-13 appears to be greater than that of IL-4. SP-D -/- mice seem to express considerably higher levels of IL-13, which is consistent with increased sensitivity and exaggerated immune response of the mice to allergenic challenge. Allergenic exposure also induces elevation in SP-D protein levels in an IL-4/IL-13-dependent manner, which prevents further activation of sensitised T cells. This negative feedback loop seems essential in protecting the airways from inflammatory damage after allergen inhalation. Here, we examine this link between IL-13 and SP-D, and its implications in the progression/regulation of asthma and allergy.

Human surfactant protein D alters oxidative stress and HMGA1 expression to induce p53 apoptotic pathway in eosinophil leukemic cell line.

Surfactant protein D (SP-D), an innate immune molecule, has an indispensable role in host defense and regulation of inflammation. Immune related functions regulated by SP-D include agglutination of pathogens, phagocytosis, oxidative burst, antigen presentation, T lymphocyte proliferation, cytokine secretion, induction of apoptosis and clearance of apoptotic cells. The present study unravels a novel ability of SP-D to reduce the viability of leukemic cells (eosinophilic leukemic cell line, AML14.3D10; acute myeloid leukemia cell line, THP-1; acute lymphoid leukemia cell lines, Jurkat, Raji; and human breast epithelial cell line, MCF-7), and explains the underlying mechanisms. SP-D and a recombinant fragment of human SP-D (rhSP-D) induced G2/M phase cell cycle arrest, and dose and time-dependent apoptosis in the AML14.3D10 eosinophilic leukemia cell line. Levels of various apoptotic markers viz. activated p53, cleaved caspase-9 and PARP, along with G2/M checkpoints (p21 and Tyr15 phosphorylation of cdc2) showed significant increase in these cells. We further attempted to elucidate the underlying mechanisms of rhSP-D induced apoptosis using proteomic analysis. This approach identified large scale molecular changes initiated by SP-D in a human cell for the first time. Among others, the proteomics analysis highlighted a decreased expression of survival related proteins such as HMGA1, overexpression of proteins to protect the cells from oxidative burst, while a drastic decrease in mitochondrial antioxidant defense system. rhSP-D mediated enhanced oxidative burst in AML14.3D10 cells was confirmed, while antioxidant, N-acetyl-L-cysteine, abrogated the rhSP-D induced apoptosis. The rhSP-D mediated reduced viability was specific to the cancer cell lines and viability of human PBMCs from healthy controls was not affected. The study suggests involvement of SP-D in host's immunosurveillance and therapeutic potential of rhSP-D in the eosinophilic leukemia and cancers of other origins.

Identification and characterization of a laminin-binding protein of Aspergillus fumigatus: extracellular thaumatin domain protein (AfCalAp).

Aspergillus fumigatus, an opportunistic fungal pathogen, infects the human host via inhalation of airborne conidia. Adhesion of fungal conidia, to host cells and extracellular matrix (ECM) components associated with host tissue surfaces, is thought to be the primary step in the pathogenesis and dissemination of infection. To identify novel adhesion proteins (adhesins) of A. fumigatus, we screened its proteome in silico using SPAAN (software program for prediction of adhesins and adhesin-like proteins using neural networks). One of the predicted adhesin-encoding genes with a P(ad) (probability of being adhesin) value >0.9, the gene encoding extracellular thaumatin domain protein (AfCalA), was cloned and expressed in Escherichia coli. Recombinant AfCalAp showed significant binding with laminin and murine lung cells. Anti-AfCalAp antibodies inhibited the binding of AfCalAp to laminin in a dose-dependent manner. Significant binding of anti-AfCalAp antibodies to 2 h swollen conidia suggests the presence of AfCalAp on the conidial surface. AfCalA transcript was not detectable in resting conidia but was detected in conidia incubated with RPMI 1640 medium in the presence and absence of lung epithelial cell line (A539)-derived ECM. Elevated levels of IgE antibodies specific to AfCalAp were observed in the sera of two out of seven patients with allergic bronchopulmonary aspergillosis. The study confirms the relevance of the bioinformatic approach for predicting fungal adhesins and establishes AfCalAp as a novel laminin-binding protein of A. fumigatus.

Prophylactic and Therapeutic Potential of Asp f1 Epitopes in Naïve and Sensitized BALB/c Mice.

BACKGROUND: The present study examines a hypothesis that short allergen-derived peptides may shift an Aspergillus fumigatus (Afu-) specific TH2 response towards a protective TH1. Five overlapping peptides (P1-P5) derived from Asp f1, a major allergen/antigen of Afu, were evaluated for prophylactic or therapeutic efficacy in BALB/c mice. METHODS: To evaluate the prophylactic efficacy, peptides were intranasally administered to naïve mice and challenged with Afu-allergens/antigens. For evaluation of therapeutic efficacy, the mice were sensitized with Afu-allergens/antigens followed by intranasal administration of peptides. The groups were compared for the levels of Afu-specific antibodies in sera and splenic cytokines evaluated by ELISA. Eosinophil peroxidase activity was examined in the lung cell suspensions and lung inflammation was assessed by histopathogy. RESULTS: Peptides P1-, P2- and P3 decreased Afu-specific IgE (84.5~98.9%) and IgG antibodies (45.7~71.6%) in comparison with Afu-sensitized mice prophylactically. P1- and P2-treated ABPA mice showed decline in Afu-specific IgE (76.4~88%) and IgG antibodies (15~54%). Increased IgG2a/IgG1 and IFN-gamma/IL-4 ratios were observed. P1-P3 prophylactically and P1 therapeutically decreased IL-5 levels and eosinophil peroxidase activity. P1 decreased inflammatory cells' infiltration in lung tissue comparable to non-challenged control. CONCLUSION: Asp f1-derived peptide P1, prophylactically and therapeutically administered to Balb/c mice, is effective in regulating allergic response to allergens/antigens of Afu, and may be explored for immunotherapy of allergic aspergillosis in humans.

Recombinant surfactant protein-D selectively increases apoptosis in eosinophils of allergic asthmatics and enhances uptake of apoptotic eosinophils by macrophages.

Pulmonary surfactant protein-D (SP-D) is a multifunctional, pattern recognition molecule involved in resistance to allergen challenge and pulmonary inflammation. In view of therapeutic effects of exogenous SP-D or recombinant fragment of human surfactant protein-D (rhSP-D) (composed of eight Gly-X-Y collagen repeat sequences, homotrimeric neck and lectin domains) in murine models of lung allergy and hypereosinophilic SP-D gene-deficient mice, we investigated the possibility of a direct interaction of purified rhSP-D with human eosinophils derived from allergic patients and healthy donors. rhSP-D showed a sugar- and calcium-dependent binding to human eosinophils, suggesting involvement of its carbohydrate recognition domain. While eosinophils from allergic patients showed a significant increase in apoptosis, oxidative burst and CD69 expression in presence of rhSP-D, eosinophils from healthy donors showed no significant change. However, these eosinophils from healthy donors when primed with IL-5 exhibited increase in apoptosis on incubation with rhSP-D. Apoptosis mediated by rhSP-D in primed eosinophils was not affected by the antioxidant, N-acetyl-L-cysteine. There was a manifold increase in binding of rhSP-D to apoptotic eosinophils than the normal eosinophils and rhSP-D induced a significant increase in uptake of apoptotic eosinophils by J774A.1 macrophage cells. The study suggests that rhSP-D mediated preferential increase of apoptosis of primed eosinophils while not affecting the normal eosinophils and increased phagocytosis of apoptotic eosinophils may be important mechanisms of rhSP-D and plausibly SP-D-mediated resolution of allergic eosinophilic inflammation in vivo.