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.

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.

Expressed sequence tags of Aspergillus fumigatus: Extension of catalogue and their evaluation as putative drug targets and/or diagnostic markers.

Aspergillus fumigatus a fungal pathogen is implicated in a spectrum of allergic and invasive disorders in humans. Validation of transcriptome of pathogen is essential for understanding its virulence mechanism and to identify new therapeutic targets/diagnostic markers. In order to rapidly identify genes of Aspergillus fumigatus we adopted sequencing of cDNA clones. Our earlier effort has lead to identification of 68 expressed sequence tags of Aspergillus fumigatus. Present study describes 52 more expressed sequence tags generated by sequencing 200 phage clones of a non-normalized cDNA library. One of the cDNA clones comprised of the complete coding region for tetratricopeptide repeat domain protein gene. Various homology search algorithms were employed to assign functions to expressed sequence tags coding for hypothetical proteins, and relevance of these expressed sequence tags or their protein products as drug targets/diagnostic markers was examined by searching for homologues in fungi and human.

Immunoproteomic analysis of secretory proteins ofAspergillus fumigatus with specific IGE immunoreactivity.

Allergenic/antigenic proteins are known to induce Type I and Type III hypersensitivity reactions leading to allergic bronchopulmonary aspergillosis (ABPA) in immunocompetent host. The common structural features or intrinsic properties of the allergens/antigens leading to allergenicity in a host are not well understood. In the current report, comparative analysis of proteins on two dimensional gel electrophoresis (2D-3) and specific IgE immunoblots ofA. fumigatus secretory proteins (1(st), 2(nd) and 3(rd) week culture filtrate proteins) was carried out. We observed a total of 159 proteins in 1(st), 2(nd) and 3(rd) week culture filtrates ofA. fumigatus. Specific IgE immunoreactivity was observed in 75 proteins with different intensity. Third week culture filtrate showed maximum number of proteins, 142, and specific IgE immunoreactive proteins, 65. MALDI-TOF analysis resulted in putative identification of two allergens as hypothetical protein YBL057c fromSaccharomyces cereviseae and unnamed protein product fromDebaryomyces hansenii (similar to IPF14568 ofCandida albicans). Identification of a repertoire of specific IgE immunoreactive proteins will facilitate the studies on structure-function relationship of these proteins relevant for diagnosis and pathogenesis.

Phylogenetic and Structural Analysis of Polyketide Synthases in Aspergilli.

Polyketide synthases (PKSs) of Aspergillus species are multidomain and multifunctional megaenzymes that play an important role in the synthesis of diverse polyketide compounds. Putative PKS protein sequences from Aspergillus species representing medically, agriculturally, and industrially important Aspergillus species were chosen and screened for in silico studies. Six candidate Aspergillus species, Aspergillus fumigatus Af293, Aspergillus flavus NRRL3357, Aspergillus niger CBS 513.88, Aspergillus terreus NIH2624, Aspergillus oryzae RIB40, and Aspergillus clavatus NRRL1, were selected to study the PKS phylogeny. Full-length PKS proteins and only ketosynthase (KS) domain sequence were retrieved for independent phylogenetic analysis from the aforementioned species, and phylogenetic analysis was performed with characterized fungal PKS. This resulted into grouping of Aspergilli PKSs into nonreducing (NR), partially reducing (PR), and highly reducing (HR) PKS enzymes. Eight distinct clades with unique domain arrangements were classified based on homology with functionally characterized PKS enzymes. Conserved motif signatures corresponding to each type of PKS were observed. Three proteins from Protein Data Bank corresponding to NR, PR, and HR type of PKS (XP_002384329.1, XP_753141.2, and XP_001402408.2, respectively) were selected for mapping of conserved motifs on three-dimensional structures of KS domain. Structural variations were found at the active sites on modeled NR, PR, and HR enzymes of Aspergillus. It was observed that the number of iteration cycles was dependent on the size of the cavity in the active site of the PKS enzyme correlating with a type with reducing or NR products, such as pigment, 6MSA, and lovastatin. The current study reports the grouping and classification of PKS proteins of Aspergilli for possible exploration of novel polyketides based on sequence homology; this information can be useful for selection of PKS for polyketide exploration and specific detection of Aspergilli.

Surfactant proteins SP-A and SP-D in human health and disease.

Surfactant proteins A (SP-A) and D (SP-D) are lung surfactant-associated hydrophilic proteins that have been implicated in surfactant homeostasis and pulmonary innate immunity. They are collagen-containing C-type (calcium-dependent) lectins, called collectins, and are structurally similar to mannose-binding protein of the lectin pathway of the complement system. Being carbohydrate pattern-recognition molecules, they recognize a broad spectrum of pathogens and allergens via the lectin domain, with subsequent activation of immune cells via the collagen region, thus offering protection against infection and allergenic challenge. SP-A and SP-D have been shown to be involved in viral neutralization, clearance of bacteria, fungi, and apoptotic and necrotic cells, down-regulation of allergic reaction, and resolution of inflammation. Studies on single-nucleotide polymorphism, protein levels in broncho-alveolar lavage, and gene knock-out mice have clearly indicated an association between SP-A and SP-D and a range of pulmonary diseases. In addition, recent studies using murine models of allergy and infection have raised the possibility that the recombinant forms of SP-A and SP-D may have therapeutic potential in controlling pulmonary infection, inflammation, and allergies in humans.

Identification and characterization of polyubiquitin gene from cDNA library of aspergillus fumigatus.

Aspergillus fumigatus (Afu) causes allergic and invasive forms of diseases in humans. In order to identify genes relevant for pathogenesis, a total of 235 cDNA clones were randomly selected and sequenced from cDNA library of Afu. One of the partially sequenced cDNA clones was homologous to polyubiquitin. Sequencing of the complete cDNA clone showed an open reading frame of 912 bases. Comparison with genomic sequence of Afu using BlastN program, revealed that polyubiquitin gene comprises of 992 bases and contains one intron of 80 bases. The recombinant expression of fusion protein showed an approximately molecular weight of 43-kDa on SDS-PAGE. The translation product of the cDNA sequence showed four tandem repeats of 76 amino acid residues in a single polyubiquitin protein and showed 100% identity with polyubiquitin protein sequences of S. cerevisiae, N. crassa, C. albicans, S. pombe, and M. grisae. Polyubiquitin gene is known to play important role in a variety of cellular processes and recently have been implicated in fungal pathogenesis. Identification of polyubiquitin gene of Afu has opened up scope to study its role in understanding Aspergillus biology and pathogenesis.

Protective effects of a recombinant fragment of human surfactant protein D in a murine model of pulmonary hypersensitivity induced by dust mite allergens.

Lung surfactant protein D (SP-D) is a carbohydrate pattern recognition immune molecule. It can interact with a range of pathogens, stimulate immune cells and manipulate cytokine profiles during host's immune response. SP-D has also been shown to interact, via its carbohydrate recognition domains, with glycoprotein allergens of house dust mite (Dermatophagoides pteronyssinus, Derp), inhibiting specific IgE isolated from mite-sensitive asthmatic patients from binding these allergens, and blocking subsequent histamine release from sensitized basophils. In the present study, we have examined the protection offered by various doses of intranasal administration of a recombinant fragment of human SP-D (rhSP-D) in a murine model of pulmonary hypersensitivity to Derp allergens which showed characteristic high levels of specific IgE antibodies, peripheral blood eosinophilia, pulmonary infiltrates and a Th2 cytokine response. Treatment of Derp mice with rhSP-D led to significant reduction in Derp-specific IgE levels, blood eosinophilia and pulmonary cellular infiltration. The levels of IL-4 and IL-5 were decreased, while those of IL-12 and IFN-gamma were raised in the supernatant of the cultured splenocytes, indicating a Th2 to Th1 polarization. These results suggest that SP-D has a protective role in the modulation of allergic sensitization and in the development of allergic reactions to Derp allergens and highlight potential of the rhSP-D as a therapeutic for pulmonary hypersensitivity.

Protective roles of pulmonary surfactant proteins, SP-A and SP-D, against lung allergy and infection caused by Aspergillus fumigatus.

Pulmonary surfactant proteins, SP-A and SP-D, are immune molecules which can directly interact with pathogens and allergens, stimulate immune cells and manipulate cytokine and chemokine profiles during host's immune response. Using an opportunistic fungal pathogen Aspergillus fumigatus (Afu), we have attempted to understand participation of SP-A and SP-D in the host immunity. Afu causes a systemic infection via lungs, called invasive aspergillosis (IPA) in immunocompromised subjects. In the immunocompetent subjects, it can cause an allergic disorder, called allergic bronchopulmonary aspergillosis (ABPA). Therapeutic administration of these proteins in a murine model of IPA can rescue mice from death. Treating mice, having ABPA, can suppress IgE levels, eosinophilia, pulmonary cellular infiltration and cause a marked shift from a pathogenic Th2 to a protective Th1 cytokine profile. These results highlight the potential of SP-A, SP-D and their recombinant forms, as novel therapeutics for lung allergy and infection.

Altered immune response to liposomal allergens of Aspergillus fumigatus in mice.

Aspergillus fumigatus has been implicated as the major pathogenic fungus causing Aspergillus-mediated disorders. It secretes complex glycoprotein antigens and allergens, which induce type I and type III mediated hypersensitivity reactions. The immune response to these allergens/antigens in allergic disorders is characterized by elevated levels of specific IgE, Th2 cytokines and eosinophilia. In the current study, the ability of negatively charged liposomes entrapped with glycoprotein antigens and allergens of A. fumigatus to modulate the immune response was studied. Immune response in mice was evaluated with both free and liposomal formulations. Liposome entrapped glycoprotein antigens/allergens of A. fumigatus elicited a Th1 type response with increased levels of TNF-alpha (5.5-folds), IFN-gamma (four-folds), specific IgG (three-folds) and IgG2a (2.4-folds), low titers of specific IgG1 (2.2-folds decrease) and IgE (three-folds decrease), and decreased peripheral eosinophilia by four-folds in comparison to mice receiving free glycoprotein allergens/antigens of A. fumigatus. Histopathological examination of lung tissue sections clearly indicated reduced eosinophil infiltration in mice immunized with liposomal formulations. These results suggest potential of liposomal formulations for A. fumigatus allergens/antigens for exploration in immunotherapy.

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.

Susceptibility of mice genetically deficient in SP-A or SP-D gene to invasive pulmonary aspergillosis.

Pulmonary surfactant proteins, SP-A and SP-D, are carbohydrate pattern recognition molecules of innate immunity, which significantly enhance phagocytosis and killing of Aspergillus fumigatus, a pathogenic fungus, by neutrophils and macrophages. The present study examined the susceptibility of immunosuppressed SP-A gene deficient (SP-A(-/-)) or SP-D gene deficient (SP-D(-/-)) mice to A. fumigatus conidia challenge compared to wild-type (WT) mice. A. fumigatus-challenged SP-A(-/-) (SP-A(-/-) IPA) mice showed less mortality (40%) than the WT-IPA mice (100%) and increased mortality (60%) following administration of SP-A with decreased TNF-alpha and IFN-gamma to IL-4 ratio than SP-A(-/-) IPA mice. The SP-D(-/-) IPA mice (57.14%) showed similar mortality as WT-IPA mice (60%). However, the SP-D (-/-) IPA mice (42.86% mortality on day 2) died earlier than the WT-IPA mice (20% mortality on day 2), showed a higher hyphal density and tissue injury in lungs. Treatment with SP-D or a recombinant fragment of human SP-D rhSP-D reduced the mortality to 50% and 33%, respectively, concomitant with higher IFN-gamma to IL-4 ratios in treated SP-D(-/-) mice, compared to untreated control group. The results showed that SP-D gene deficient mice are more susceptible to IPA while SP-A gene deficient mice acquire resistance to IPA.

Therapeutic effects of recombinant forms of full-length and truncated human surfactant protein D in a murine model of invasive pulmonary aspergillosis.

Aspergillus fumigatus (Afu) is an opportunistic fungal pathogen that can cause fatal invasive pulmonary aspergillosis (IPA) in immunocompromised individuals. Previously, surfactant protein D (SP-D), a surfactant-associated innate immune molecule, has been shown to enhance phagocytosis and killing of Afu conidia by phagocytic cells in vitro. An intranasal treatment of SP-D significantly increased survival in a murine model of IPA. Here we have examined mechanisms via which recombinant forms of full-length (hSP-D) or truncated human SP-D (rhSP-D) offer protection in a murine model of IPA that were immunosuppressed with hydrocortisone and challenged intranasally with Afu conidia prior to the treatment. SP-D or rhSP-D treatment increased the survival rate to 70% and 80%, respectively (100% mortality on day 7 in IPA mice), with concomitant reduction in the growth of fungal hyphae in the lungs, and increased levels of TNF-alpha and IFN-gamma in the lung suspension supernatants, as compared to untreated IPA mice. The level of macrophage inflammatory protein-1 alpha (MIP-1 alpha) in the lung cell suspension was also raised considerably following treatment with SP-D or rhSP-D. Our results appear to reaffirm the notion that under immunocompromised conditions, human SP-D or its truncated form can offer therapeutic protection against fatal challenge with Afu conidia challenge. Taken together, the SP-D-mediated protective mechanisms include enhanced phagocytosis by recruited macrophages and neutrophils and fungistatic properties, suppression of the levels of pathogenic Th2 cytokines (IL-4 and IL-5), enhanced local production of protective Th1 cytokines, TNF-alpha and IFN-gamma, and that of protective C-C chemokine, MIP-1 alpha.

Distinct alleles of mannose-binding lectin (MBL) and surfactant proteins A (SP-A) in patients with chronic cavitary pulmonary aspergillosis and allergic bronchopulmonary aspergillosis.

BACKGROUND: Distinct host immune status predisposes to different forms of pulmonary aspergillosis. METHODS: Patients with chronic cavitary pulmonary aspergillosis (CCPA; n=15) or allergic bronchopulmonary aspergillosis (ABPA; n=7) of Caucasian origin were screened for single nucleotide polymorphisms (SNPs) in the collagen region of surfactant proteins A1 (SP-A1) and A2 (SP-A2) and mannose binding lectin (MBL). RESULTS: The T allele at T1492C and G allele at G1649C of SP-A2 were observed at slightly higher frequencies in ABPA patients (86% and 93%) than in controls (63% and 83%), and the C alleles at position 1492 and 1649 were found in higher frequencies in CCPA patients (33% and 25%) than in ABPA patients (14% and 7%) (all p>0.05). However, the CC genotype at position 1649 of SP-A2 was significantly associated with CCPA (chi(2)=7.94; p(corr)< or =0.05). Similarly, ABPA patients showed a higher frequency of the TT genotype (71%) at 1492 of SP-A2 than controls (43%) and CCPA patients (41%) (p>0.05). In the case of MBL, the T allele (OR=3.1, range 1.2-8.9; p< or =0.02) and CT genotype (chi(2)=6.54; p(corr)< or =0.05) at position 868 (codon 52) were significantly associated with CCPA, but not with ABPA. Further analysis of genotype combinations at position 1649 of SP-A2 and at 868 of MBL between patient groups showed that both CC/CC and CC/CT SP-A2/MBL were found only in CCPA patients, while GG/CT SP-A2/MBL was significantly higher in CCPA patients in comparison to ABPA patients (p< or =0.05). SNPs analysed in SP-A1 did not differ between cases and controls. CONCLUSIONS: Distinct alleles, genotypes and genotype combinations of SP-A2 and MBL may contribute to differential susceptibility of the host to CCPA or ABPA.

Mannan-binding lectin in asthma and allergy.

Mannan-binding lectin (MBL) is a vital and versatile component of innate immunity. It is present in serum and may bind to a plethora of microbial pathogens and mediate opsonization of these by complement-dependent and/or independent mechanisms. Low-MBL levels in serum, attributed to certain genetic polymorphisms, constitute a major factor predisposing to several infectious diseases. However, recent studies propose that MBL extends beyond its classic role as a first-line host-defense molecule to a modulator of inflammation. In this review, we summarize and explore this potential and a possible novel role of MBL in asthma and allergy.

Susceptibility of mice genetically deficient in the surfactant protein (SP)-A or SP-D gene to pulmonary hypersensitivity induced by antigens and allergens of Aspergillus fumigatus.

Lung surfactant protein A (SP-A) and D (SP-D) are innate immune molecules which are known to interact with allergens and immune cells and modulate cytokine and chemokine profiles during host hypersensitivity response. We have previously shown therapeutic effects of SP-A and SP-D using a murine model of lung hypersensitivity to Aspergillus fumigatus (Afu) allergens. In this study, we have examined the susceptibility of SP-A (AKO) or SP-D gene-deficient (DKO) mice to the Afu allergen challenge, as compared with the wild-type mice. Both AKO and DKO mice exhibited intrinsic hypereosinophilia and several-fold increase in levels of IL-5 and IL-13, and lowering of IFN-gamma to IL-4 ratio in the lungs, suggesting a Th2 bias of immune response. This Th2 bias was reversible by treating AKO or DKO mice with SP-A or SP-D, respectively. The AKO and DKO mice showed distinct immune responses to Afu sensitization. DKO mice were found more susceptible than wild-type mice to pulmonary hypersensitivity induced by Afu allergens. AKO mice were found to be nearly resistant to Afu sensitization. Intranasal treatment with SP-D or rhSP-D (a recombinant fragment of human SP-D containing trimeric C-type lectin domains) was effective in rescuing the Afu-sensitized DKO mice, while SP-A-treated Afu-sensitized AKO mice showed several-fold elevated levels of IL-13 and IL-5, resulting in increased pulmonary eosinophilia and damaged lung tissue. These data reaffirm an important role for SP-A and SP-D in offering resistance to pulmonary allergenic challenge.

Tryptophan residue is essential for immunoreactivity of a diagnostically relevant peptide epitope of A. fumigatus.

The role of tryptophan (Trp17) in immunoreactivity of P1, the diagnostically relevant peptide from a major allergen/antigen of Aspergillus fumigatus, was evaluated by chemically modifying tryptophanyl residue of P1. In BIAcore kinetic studies, unmodified P1 showed a 100-fold higher binding with ABPA (Allergic Bronchopulmonary Aspergillosis) patients' IgG [KD (equilibrium dissociation constant) = 2.74 e(-8) +/- 0.13 M] than the controls' IgG (KD = 2.97 e(-6) +/- 0.14 M), whereas chemically-modified P1 showed similar binding [KD patients' IgG = 3.25 e(-7) +/- 0.16 M, KD controls' IgG = 3.86 e(-7) +/- 0.19 M] indicating loss of specific immunoreactivity of P1 on tryptophan modification. Modified P1 showed loss of specific binding to IgE and IgG antibodies of ABPA patients in ELISA (Enzyme-Linked Immunosorbent Assay). The study infers that tryptophan residue (Trp17) is essential for immunoreactivity of P1.

Expression and characterization of Asp fI, an immunodominant allergen/antigen of A. fumigatus in insect cell.

Asp fI is a major allergen/antigen/cytotoxin of Aspergillus fumigatus and exhibits ribonuclease activity. This allergen plays a role in allergic and invasive Aspergillosis and reported as a major cytotoxin with ribonuclease activity. To express the protein in large quantity and to characterize the multifunctional nature of Asp fI, we have generated recombinant baculovirus by introducing the gene in pFastBac HTa expression vector and expressed in insect cell. The baculovirus expression vector system has been used as a versatile system for the efficient expression of proteins with most eukaryotic posttranslational modification. Recombinant Asp fI was expressed as approximately 1% of the total cellular protein in infected Sf9 insect cells. The protein was purified using Ni2+ affinity column chromatography and the yield of purified protein was approximately 10 mg/l g of total cellular protein. Immunoreactivity of the protein was determined by immunoblot analysis using both poly His monoclonal antibody, IgG and IgE antibodies present in the sera of ABPA patients. The protein was glycosylated as revealed by the glycoprotein staining and was observed to retain both ribonuclease and cytotoxic activities. These results suggest that Asp fI expressed in insect cell was post translationally modified and biologically active that can be used as a diagnostic marker for biochemical studies.