Julie G. Ledford

Innate Immunity and Asthma Risk in Amish and Hutterite Farm Children

BACKGROUND The Amish and Hutterites are U.S. agricultural populations whose lifestyles are remarkably similar in many respects but whose farming practices, in particular, are distinct; the former follow traditional farming practices whereas the latter use industrialized farming practices. The populations also show striking disparities in the prevalence of asthma, and little is known about the immune responses underlying these disparities. METHODS We studied environmental exposures, genetic ancestry, and immune profiles among 60 Amish and Hutterite children, measuring levels of allergens and endotoxins and assessing the microbiome composition of indoor dust samples. Whole blood was collected to measure serum IgE levels, cytokine responses, and gene expression, and peripheral-blood leukocytes were phenotyped with flow cytometry. The effects of dust extracts obtained from Amish and Hutterite homes on immune and airway responses were assessed in a murine model of experimental allergic asthma. RESULTS Despite the similar genetic ancestries and lifestyles of Amish and Hutterite children, the prevalence of asthma and allergic sensitization was 4 and 6 times as low in the Amish, whereas median endotoxin levels in Amish house dust was 6.8 times as high. Differences in microbial composition were also observed in dust samples from Amish and Hutterite homes. Profound differences in the proportions, phenotypes, and functions of innate immune cells were also found between the two groups of children. In a mouse model of experimental allergic asthma, the intranasal instillation of dust extracts from Amish but not Hutterite homes significantly inhibited airway hyperreactivity and eosinophilia. These protective effects were abrogated in mice that were deficient in MyD88 and Trif, molecules that are critical in innate immune signaling. CONCLUSIONS The results of our studies in humans and mice indicate that the Amish environment provides protection against asthma by engaging and shaping the innate immune response. (Funded by the National Institutes of Health and others.).

Cross-Dressing the Virion: the Transcapsidation of Adeno-Associated Virus Serotypes Functionally Defines Subgroups

ABSTRACT For all adeno-associated virus (AAV) serotypes, 60 monomers of the Vp1, Vp2, and Vp3 structural proteins assemble via an unknown mechanism to form an intact capsid. In an effort to better understand the properties of the capsid monomers and their role in viral entry and infection, we evaluated whether monomers from distinct serotypes can be mixed to form infectious particles with unique phenotypes. This transcapsidation approach consisted of the transfection of pairwise combinations of AAV serotype 1 to 5 helper plasmids to produce mosaic capsid recombinant AAV (rAAV). All ratios (19:1, 3:1, 1:1, 1:3, and 1:19) of these mixtures were able to replicate the green fluorescent protein transgene and to produce capsid proteins. A high-titer rAAV was obtained with mixtures that included either serotype 1, 2, or 3, whereas an rAAV of intermediate titer was obtained from serotype 5 mixtures. Only mixtures containing the AAV4 capsid exhibited reduced packaging capacity. The binding profiles of the mixed-virus preparations to either heparin sulfate (HS) or mucin agarose revealed that only AAV3-AAV5 mixtures at the 3:1 ratio exhibited duality in binding. All other mixtures displayed either an abrupt shift or a gradual alteration in the binding profile to the respective ligand upon increase of a capsid component that conferred either HS or mucin binding. The transduction of cell lines was used to further evaluate the phenotypes of these transcapsidated virions. Three transduction profiles were observed: (i) small to no change regardless of ratio, (ii) a gradual increase in transduction consistent with titration of a second capsid component, or (iii) an abrupt increase in transduction (threshold effect) dependent on the specific ratios used. Interestingly, an unexpected synergistic effect in transduction was observed when AAV1 helper constructs were combined with type 2 or type 3 recipient helpers. Further studies determined that at least two components contributed to this observed synergy: (i) heparin-mediated binding from AAV2 and (ii) an unidentified enhancement activity from AAV1 structural proteins. Using this procedure of mixing different AAV helper plasmids to generate “cross-dressed” AAV virions, we propose an additional means of classifying new AAV serotypes into subgroups based on functional approaches to analyze AAV capsid assembly, receptor-mediated binding, and virus trafficking. Exploitation of this approach in generating custom-designed AAV vectors should be of significant value to the field of gene therapy.

Surfactant protein-D regulates effector cell function and fibrotic lung remodeling in response to bleomycin injury.

RATIONALE Surfactant protein (SP)-D and SP-A have been implicated in immunomodulation in the lung. It has been reported that patients with idiopathic pulmonary fibrosis (IPF) often have elevated serum levels of SP-A and SP-D, although their role in the disease is not known. OBJECTIVES The goal of this study was to test the hypothesis that SP-D plays an important role in lung fibrosis using a mouse model of fibrosis induced by bleomycin (BLM). METHODS Triple transgenic inducible SP-D mice (iSP-D mice), in which rat SP-D is expressed in response to doxycycline (Dox) treatment, were administered BLM (100 U/kg) or saline subcutaneously using miniosmotic pumps. MEASUREMENTS AND MAIN RESULTS BLM-treated iSP-D mice off Dox (SP-D off) had increased lung fibrosis compared with mice on Dox (SP-D on). SP-D deficiency also increased macrophage-dominant cell infiltration and the expression of profibrotic cytokines (transforming growth factor [TGF]-β1, platelet-derived growth factor-AA). Alveolar macrophages isolated from BLM-treated iSP-D mice off Dox (SP-D off) secreted more TGF-β1. Fibrocytes, which are bone marrow-derived mesenchymal progenitor cells, were increased to a greater extent in the lungs of the BLM-treated iSP-D mice off Dox (SP-D off). Fibrocytes isolated from BLM-treated iSP-D mice off Dox (SP-D off) expressed more of the profibrotic cytokine TGF-β1 and more CXCR4, a chemokine receptor that is important in fibrocyte migration into the lungs. Exogenous SP-D administered intratracheally attenuated BLM-induced lung fibrosis in SP-D(-/-) mice. CONCLUSIONS These data suggest that alveolar SP-D regulates numbers of macrophages and fibrocytes in the lungs, profibrotic cytokine expression, and fibrotic lung remodeling in response to BLM injury.

The role of surfactant protein A in bleomycin-induced acute lung injury.

RATIONALE Surfactant protein A (SP-A) is a collectin family member that has multiple immunomodulatory roles in lung host defense. SP-A levels are altered in the bronchoalveolar lavage (BAL) fluid and serum of patients with acute lung injury and acute respiratory distress syndrome, suggesting the importance of SP-A in the pathogenesis of acute lung injury. OBJECTIVES Investigate the role of SP-A in the murine model of noninfectious lung injury induced by bleomycin treatment. METHODS Wild-type (WT) or SP-A deficient (SP-A(-/-)) mice were challenged with bleomycin, and various indices of lung injury were analyzed. MEASUREMENTS AND MAIN RESULTS On challenge with bleomycin, SP-A(-/-) mice had a decreased survival rate as compared with WT mice. SP-A(-/-) mice had a higher degree of neutrophil-dominant cell recruitment and the expression of the inflammatory cytokines in BAL fluid than did WT mice. In addition, SP-A(-/-) mice had increased lung edema as assessed by the increased levels of intravenously injected Evans blue dye leaking into the lungs. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling and active caspase-3 staining suggested the increased apoptosis in the lung sections from SP-A(-/-) mice challenged with bleomycin. SP-A also specifically reduced bleomycin-induced apoptosis in mouse lung epithelial 12 cells in vitro. Moreover, intratracheal administration of exogenous SP-A rescued the phenotype of SP-A(-/-) mice in vivo. CONCLUSIONS These data suggest that SP-A plays important roles in modulating inflammation, apoptosis, and epithelial integrity in the lung in response to acute noninfectious challenges.

Impaired Host Defense in Mice Lacking ONZIN

ONZIN is a small, cysteine-rich peptide of unique structure that is conserved in all vertebrates examined to date. We show that ONZIN is expressed at high levels in epithelial cells of the intestinal tract, the lung, and in cells of the immune system including macrophages and granulocytes. Because this pattern of expression is suggestive of a role in innate immune function, we have generated mice lacking this protein and examined their ability to respond to challenge with infectious agents. Onzin−/− mice show a heightened innate immune response after induction of acute peritonitis with Klebsiella pneumoniae. This increased response is consistent with an increased bacterial burden in the Onzin−/− mice. Ex vivo studies show that, whereas phagocytosis is not altered in Onzin−/− neutrophils, phagocytes lacking this protein kill bacteria less effectively. This result identifies ONZIN as a novel class of intracellular protein required for optimal function of the neutrophils after uptake of bacteria.

SP-A Preserves Airway Homeostasis During Mycoplasma pneumoniae Infection in Mice

The lung is constantly challenged during normal breathing by a myriad of environmental irritants and infectious insults. Pulmonary host defense mechanisms maintain homeostasis between inhibition/clearance of pathogens and regulation of inflammatory responses that could injure the airway epithelium. One component of this defense mechanism, surfactant protein-A (SP-A), exerts multifunctional roles in mediating host responses to inflammatory and infectious agents. SP-A has a bacteriostatic effect on Mycoplasma pneumoniae (Mp), which occurs by binding surface disaturated phosphatidylglycerols. SP-A can also bind the Mp membrane protein, MPN372. In this study, we investigated the role of SP-A during acute phase pulmonary infection with Mp using mice deficient in SP-A. Biologic responses, inflammation, and cellular infiltration, were much greater in Mp infected SP-A−/− mice than wild-type mice. Likewise, physiologic responses (airway hyperresponsiveness and lung compliance) to Mp infection were more severely affected in SP-A−/− mice. Both Mp-induced biologic and physiologic changes were attenuated by pharmacologic inhibition of TNF-α. Our findings demonstrate that SP-A is vital to preserving lung homeostasis and host defense to this clinically relevant strain of Mp by curtailing inflammatory cell recruitment and limiting an overzealous TNF-α response.

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.

Surfactant Protein A Suppresses Lung Cancer Progression by Regulating the Polarization of Tumor-Associated Macrophages

Surfactant protein A (SP-A) is a large multimeric protein found in the lungs. In addition to its immunoregulatory function in infectious respiratory diseases, SP-A is also used as a marker of lung adenocarcinoma. Despite the finding that SP-A expression levels in cancer cells has a relationship with patient prognosis, the function of SP-A in lung cancer progression is unknown. We investigated the role of SP-A in lung cancer progression by introducing the SP-A gene into human lung adenocarcinoma cell lines. SP-A gene transduction suppressed the progression of tumor in subcutaneous xenograft or lung metastasis mouse models. Immunohistochemical analysis showed that the number of M1 antitumor tumor-associated macrophages (TAMs) was increased and the number of M2 tumor-promoting TAMs was not changed in the tumor tissue produced by SP-A-expressing cells. In addition, natural killer (NK) cells were also increased and activated in the SP-A-expressing tumor. Moreover, SP-A did not inhibit tumor progression in mice depleted of NK cells. Taking into account that SP-A did not directly activate NK cells, these results suggest that SP-A inhibited lung cancer progression by recruiting and activating NK cells via controlling the polarization of TAMs.

Review: Collectins link innate and adaptive immunity in allergic airway disease

Although the lipoprotein complex of pulmonary surfactant has long been recognized as essential for reducing lung surface tension, its role in lung immune host defense has only relatively recently been elucidated. Surfactant-associated proteins A (SP-A) and D (SP-D) can attenuate bacterial and viral infection and inflammation by acting as opsonins and by regulating innate immune cell functions. Surfactant-associated protein A and D also interact with antigen-presenting cells and T cells, thereby linking the innate and adaptive immune systems. A recent study from our laboratory demonstrated that mice deficient in SP-A have enhanced susceptibility to airway hyper-responsiveness and lung inflammation induced by Mycoplasma pneumonia, an atypical bacterium present in the airways of approximately 50% of asthmatics experiencing their first episode, and further supports an important role for SP-A in the host response to allergic airway disease. Animal and human studies suggest that alterations in the functions or levels of SP-A and SP-D are associated with both infectious and non-infectious chronic lung diseases such as asthma. Future studies are needed to elucidate whether alterations in SP-A and SP-D are a consequence and/or cause of allergic airway disease.

Genes of innate immunity and the biological response to inhaled ozone

Ambient ozone has a significant impact on human health. We have made considerable progress in understanding the fundamental mechanisms that regulate the biological response to ozone. It is increasingly clear that genes of innate immunity play a central role in both infectious and noninfectious lung disease. The biological response to ambient ozone provides a clinically relevant environmental exposure that allows us to better understand the role of innate immunity in noninfectious airways disease. In this brief review, we focus on (1) specific cell types in the lung modified by ozone, (2) ozone and oxidative stress, (3) the relationship between genes of innate immunity and ozone, (4) the role of extracellular matrix in reactive airways disease, and (5) the effect of ozone on the adaptive immune system. We summarize recent advances in understanding the mechanisms that ozone contributes to environmental airways disease.