- Associate Professor, Cellular and Molecular Medicine
- Associate Professor, Immunobiology
- Associate Professor, BIO5 Institute
- Associate Professor, Medicine
- Associate Professor, Clinical Translational Sciences
- Ph.D. Genetics and Molecular Biology
- University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States
- Determining the Role of Onzin in Immunity.
- B.S. Genetics
- University of Georgia, Athens, Georgia, United States
- Duke University, Durham, North Carolina (2011 - 2015)
- Shark Tank Winner
- COM University of Arizona, Spring 2019
- Eureka Certificate in Translational Medicine
- Eureka Institute for Translational Medicine, Spring 2017
- Jo Rae Wright Award for Outstanding Science
- American Thoracic Society, Spring 2012
No activities entered.
DissertationPS 920 (Spring 2021)
ResearchCMM 900 (Spring 2021)
ResearchMCB 900 (Spring 2021)
ResearchPS 900 (Spring 2021)
ThesisMCB 910 (Spring 2021)
Directed ResearchMCB 792 (Fall 2020)
Honors Independent StudyMCB 399H (Fall 2020)
ResearchCMM 900 (Fall 2020)
ResearchPS 900 (Fall 2020)
ThesisCTS 910 (Fall 2020)
ThesisMCB 910 (Fall 2020)
Cell Biology of DiseaseCMM 504 (Summer I 2020)
ThesisCMM 910 (Summer I 2020)
Directed ResearchMCB 792 (Spring 2020)
DissertationCTS 920 (Spring 2020)
Honors Independent StudyMCB 399H (Spring 2020)
ResearchPS 900 (Spring 2020)
ThesisCMM 910 (Spring 2020)
DissertationCTS 920 (Fall 2019)
Honors Independent StudyMCB 199H (Fall 2019)
Honors ThesisMCB 498H (Fall 2019)
ResearchPS 900 (Fall 2019)
Rsrch Meth Psio SciPS 700 (Fall 2019)
Cell Biology of DiseaseCMM 404 (Summer I 2019)
Cell Biology of DiseaseCMM 504 (Summer I 2019)
Master's ReportABS 909 (Summer I 2019)
DissertationCTS 920 (Spring 2019)
Honors Independent StudyMCB 499H (Spring 2019)
Internship in Applied BiosciABS 593A (Spring 2019)
ThesisCMM 910 (Spring 2019)
ResearchPS 900 (Winter 2018)
Rsrch Meth Psio SciPS 700 (Winter 2018)
Honors Independent StudyMCB 499H (Fall 2018)
ResearchCTS 900 (Fall 2018)
Cell Biology of DiseaseCMM 504 (Summer I 2018)
Honors Independent StudyMCB 399H (Spring 2018)
ResearchCTS 900 (Spring 2018)
Honors Independent StudyMCB 399H (Fall 2017)
ResearchCTS 900 (Fall 2017)
Directed ResearchECOL 492 (Spring 2017)
Directed ResearchECOL 392 (Fall 2016)
Directed ResearchECOL 492 (Fall 2016)
Directed ResearchECOL 392 (Summer I 2016)
Directed ResearchECOL 392 (Spring 2016)
- Dakhama, A., Al Mubarak, R., Pavelka, N., Voelker, D., Seibold, M., Ledford, J. G., Kraft, M., Li, L., & Chu, H. W. (2020). Tollip Inhibits ST2 Signaling in Airway Epithelial Cells Exposed to Type 2 Cytokines and Rhinovirus. Journal of innate immunity, 12(1), 103-115.More infoThe negative immune regulator Tollip inhibits the proinflammatory response to rhinovirus (RV) infection, a contributor to airway neutrophilic inflammation and asthma exacerbations, but the underlying molecular mechanisms are poorly understood. Tollip may inhibit IRAK1, a signaling molecule downstream of ST2, the receptor of IL-33. This study was carried out to determine whether Tollip downregulates ST2 signaling via inhibition of IRAK1, but promotes soluble ST2 (sST2) production, thereby limiting excessive IL-8 production in human airway epithelial cells during RV infection in a type 2 cytokine milieu (e.g., IL-13 and IL-33 stimulation). Tollip- and IRAK1-deficient primary human tracheobronchial epithelial (HTBE) cells and Tollip knockout (KO) HTBE cells were generated using the shRNA knockdown and CRISPR/Cas9 approaches, respectively. Cells were stimulated with IL-13, IL-33, and/or RV16. sST2, activated IRAK1, and IL-8 were measured. A Tollip KO mouse model was utilized to test if Tollip regulates the airway inflammatory response to RV infection in vivo under IL-13 and IL-33 treatment. Following IL-13, IL-33, and RV treatment, Tollip-deficient (vs. -sufficient) HTBE cells produced excessive IL-8, accompanied by decreased sST2 production but increased IRAK1 activation. IL-8 production following IL-13/IL-33/RV exposure was markedly attenuated in IRAK1-deficient HTBE cells, as well as in Tollip KO HTBE cells treated with an IRAK1 inhibitor or a recombinant sST2 protein. Tollip KO (vs. wild-type) mice developed exaggerated airway neutrophilic responses to RV in the context of IL-13 and IL-33 treatment. Collectively, these data demonstrate that Tollip restricts excessive IL-8 production in type 2 cytokine-exposed human airways during RV infection by promoting sST2 production and inhibiting IRAK1 activation. sST2 and IRAK1 may be therapeutic targets for attenuating excessive neutrophilic airway inflammation in asthma, especially during RV infection.
- Dy, A. B., Arif, M. Z., Addison, K. J., Que, L. G., Boitano, S., Kraft, M., & Ledford, J. G. (2019). Genetic Variation in Surfactant Protein-A2 Delays Resolution of Eosinophilia in Asthma. Journal of immunology (Baltimore, Md. : 1950), 203(5), 1122-1130.More infoSurfactant protein-A (SP-A) is an important mediator of pulmonary immunity. A specific genetic variation in , corresponding to a glutamine (Q) to lysine (K) amino acid substitution at position 223 of the lectin domain, was shown to alter the ability of SP-A to inhibit eosinophil degranulation. Because a large subgroup of asthmatics have associated eosinophilia, often accompanied by inflammation associated with delayed clearance, our goal was to define how SP-A mediates eosinophil resolution in allergic airways and whether genetic variation affects this activity. Wild-type, SP-A knockout (SP-A KO) and humanized (SP-A2 223Q/Q, SP-A2 223K/K) C57BL/6 mice were challenged in an allergic OVA model, and parameters of inflammation were examined. Peripheral blood eosinophils were isolated to assess the effect of SP-A genetic variation on apoptosis and chemotaxis. Five days postchallenge, SP-A KO and humanized SP-A2 223K/K mice had persistent eosinophilia in bronchoalveolar lavage fluid compared with wild-type and SP-A2 223Q/Q mice, suggesting an impairment in eosinophil resolution. In vitro, human SP-A containing either the 223Q or the 223K allele was chemoattractant for eosinophils whereas only 223Q resulted in decreased eosinophil viability. Our results suggest that SP-A aids in the resolution of allergic airway inflammation by promoting eosinophil clearance from lung tissue through chemotaxis, independent of SP-A2 Q223K, and by inducing apoptosis of eosinophils, which is altered by the polymorphism.
- Dy, A. C., Arif, M. Z., Addison, K. J., Que, L. G., Boitano, S. A., Kraft, M., & Ledford, J. (2019). Genetic Variation in Surfactant Protein-A2 Delays Resolution of Eosinophilia in Asthma. J Immunol, 203(5), 1122-1130.More infoSurfactant protein-A (SP-A) is an important mediator of pulmonary immunity. A specific genetic variation in SP-A2, corresponding to a glutamine (Q) to lysine (K) amino acid substitution at position 223 of the lectin domain, was shown to alter the ability of SP-A to inhibit eosinophil degranulation. Because a large subgroup of asthmatics have associated eosinophilia, often accompanied by inflammation associated with delayed clearance, our goal was to define how SP-A mediates eosinophil resolution in allergic airways and whether genetic variation affects this activity. Wild-type, SP-A knockout (SP-A KO) and humanized (SP-A2 223Q/Q, SP-A2 223K/K) C57BL/6 mice were challenged in an allergic OVA model, and parameters of inflammation were examined. Peripheral blood eosinophils were isolated to assess the effect of SP-A genetic variation on apoptosis and chemotaxis. Five days postchallenge, SP-A KO and humanized SP-A2 223K/K mice had persistent eosinophilia in bronchoalveolar lavage fluid compared with wild-type and SP-A2 223Q/Q mice, suggesting an impairment in eosinophil resolution. In vitro, human SP-A containing either the 223Q or the 223K allele was chemoattractant for eosinophils whereas only 223Q resulted in decreased eosinophil viability. Our results suggest that SP-A aids in the resolution of allergic airway inflammation by promoting eosinophil clearance from lung tissue through chemotaxis, independent of SP-A2 Q223K, and by inducing apoptosis of eosinophils, which is altered by the polymorphism.
- Sandalova, E., Ledford, J. G., Baskaran, M., & Dijkstra, S. (2019). Translational Medicine in the Era of Social Media: A Survey of Scientific and Clinical Communities. Frontiers in medicine, 6, 152.More infoThe integration of new scientific discoveries into clinical practice costs considerable time and resources. With the increased use of social media for scientific communication, new opportunities arise to "bridge the gap" in translational medicine. The present study aimed to investigate how medical professionals access scientific information and understand their view on the role of social media in translational medicine. A questionnaire regarding (i) the use of social media for scientific updates, (ii) the opportunities and challenges of social media for translational medicine, (iii) social media function , and (iv) participant demographics was developed. The survey link was posted online from February, 2018, until April, 2018. A total of 555 professionals responded to the survey. Respondents identified themselves predominantly as researcher/scientists (27%) or medical/biomedical students (15%). The majority of participants was employed at a university or research institute (59%), and most practiced either in Europe (48%) or in Asia (37%). Seventy-eight percent of respondents reported receiving most of scientific news and updates via non-social media options, such as journal websites and newspapers. Fifty-one percent of respondents believed that social media could contribute to closing the gap between scientific discovery and translation to medical application. The most crucial opportunity created by social media was found to be "connecting the right scientist to the right clinician." Participants rated "the translation of scientific finding to clinical practice is too fast before the safety is properly demonstrated" as the most crucial challenge. Half of the respondents were aware of their institutions policy on the professional use of social media. Only 2% of respondents had previously used . Overall, medical professionals were positive about the idea that social media could contribute to the progress of translational medicine. However, it is clear that they are still being cautious about using social media for professional purposes. To fully harness the potential of social media on translational medicine, the medical community needs to be provided with educational programs, guidelines, and support infrastructure within social media.
- Takezaki, A., Tsukumo, S. I., Setoguchi, Y., Ledford, J. G., Goto, H., Hosomichi, K., Uehara, H., Nishioka, Y., & Yasutomo, K. (2019). A homozygous SFTPA1 mutation drives necroptosis of type II alveolar epithelial cells in patients with idiopathic pulmonary fibrosis. The Journal of experimental medicine, 216(12), 2724-2735.More infoIdiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by scattered fibrotic lesions in the lungs. The pathogenesis and genetic basis of IPF remain poorly understood. Here, we show that a homozygous missense mutation in caused IPF in a consanguineous Japanese family. The mutation in disturbed the secretion of SFTPA1 protein. Sftpa1 knock-in (Sftpa1-KI) mice that harbored the same mutation as patients spontaneously developed pulmonary fibrosis that was accelerated by influenza virus infection. Sftpa1-KI mice showed increased necroptosis of alveolar epithelial type II (AEII) cells with phosphorylation of IRE1α leading to JNK-mediated up-regulation of Ripk3. The inhibition of JNK ameliorated pulmonary fibrosis in Sftpa1-KI mice, and overexpression of Ripk3 in Sftpa1-KI mice treated with a JNK inhibitor worsened pulmonary fibrosis. These findings provide new insight into the mechanisms of IPF in which a mutation in promotes necroptosis of AEII cells through JNK-mediated up-regulation of Ripk3, highlighting the necroptosis pathway as a therapeutic target for IPF.
- Younis, U. S., Vallorz, E., Addison, K. J., Ledford, J. G., & Myrdal, P. B. (2019). Preformulation and Evaluation of Tofacitinib as a Therapeutic Treatment for Asthma. AAPS PharmSciTech, 20(5), 167.More infoPreformulation studies on tofacitinib citrate, a small molecule JAK3 specific inhibitor, have not been previously reported in literature. We therefore conducted several preformulation studies on tofacitinib citrate, and its free base, to better understand factors that affect its solubility, stability, and solid-state characteristics. Further, the results of the preformulation studies helped facilitate the development of a nebulized formulation of tofacitinib citrate for inhalational delivery to house dust mite allergen-challenged, BALB/c mice as a potential treatment for eosinophilic asthma. The preformulation results indicated tofacitinib having a basic pKa of 5.2, with its stability dependent on pH, ionic strength, and temperature. Degradation of tofacitinib follows apparent first-order kinetics. In order to maximize stability of the drug, ionic strength and temperature should be minimized, with an optimal range pH between 2.0 and 5.0. Additionally, our findings demonstrate that tofacitinib citrate can successfully be nebulized at a suitable droplet size for inhalation (1.2 ± 0.2 μm MMAD) through a nose-only chamber. Animals dosed with tofacitinib citrate demonstrated marked reductions in BAL eosinophils and total protein concentrations following HDM challenge. These data suggest that tofacitinib citrate represents the potential to be an effective therapy for eosinophilic asthma.
- Dijkstra, S., Kok, G., Ledford, J. G., Sandalova, E., & Stevelink, R. (2018). Possibilities and Pitfalls of Social Media for Translational Medicine. Frontiers in medicine, 5, 345.More infoWe live in an age where the sharing of scientific findings and ideas is no longer confined to people with access to academic libraries or scientific journals. Social media have permitted for knowledge and ideas to be shared with an unprecedented speed and magnitude. This has made it possible for research findings to have a greater impact and to be rapidly implemented in society. However, the spread of unfiltered, unreferenced, and non-peer-reviewed articles through social media comes with dangers as well. In this perspective article, we aim to address both the possibilities and pitfalls of social media for translational medicine. We describe how social media can be used for patient engagement, publicity, transparency, sharing of knowledge, and implementing findings in society. Moreover, we warn about the potential pitfalls of social media, which can cause research to be misinterpreted and false beliefs to be spread. We conclude by giving advice on how social media can be harnessed to combat the pitfalls and provide a new avenue for community engagement in translational medicine.
- Dy, A. B., Tanyaratsrisakul, S., Voelker, D. R., & Ledford, J. G. (2018). The Emerging Roles of Surfactant Protein-A in Asthma. Journal of clinical & cellular immunology, 9(4).More infoAsthma remains one of the most common respiratory diseases in both children and adults affecting up to 10% of the US population. Asthma is characterized by persistent symptoms, airway inflammation, airflow limitation and frequent exacerbations. Eosinophils are a key immune cell present in a large majority of asthmatics and their presence and dysregulation are clinically associated with more severe asthma. Surfactant protein A (SP-A) provides a first-line of defense in pulmonary innate immunity by virtue of its role in pathogen opsonization. SP-A is known to specifically bind to (Mp), a pathogen associated with asthma exacerbations, and functions to attenuate Mp pathogenicity and abrogate lung inflammation. In addition, SP-A has been shown to inhibit Mp-induced eosinophil peroxidase (EPO) release, a toxic product that can compromise the integrity of the delicate airway epithelia. We have determined that genetic variation in SP-A2 at position 223 that results in a glutamine (Q) to a lysine (K) substitution alters the ability of SP-A to inhibit EPO release and may offer a mechanistic explanation as to why some SP-A extracted from subjects with asthma is unable to carry out normal immune regulatory functions.
- Ito, Y., Schaefer, N., Sanchez, A., Francisco, D., Alam, R., Martin, R. J., Ledford, J. G., Stevenson, C., Jiang, D., Li, L., Kraft, M., & Chu, H. W. (2018). Toll-Interacting Protein, Tollip, Inhibits IL-13-Mediated Pulmonary Eosinophilic Inflammation in Mice. Journal of innate immunity, 10(2), 106-118.More infoToll-interacting protein (Tollip) is a key negative regulator of innate immunity by preventing excessive proinflammatory responses. Tollip genetic variation has been associated with airflow limitation in asthma subjects and Tollip expression. Whether Tollip regulates lung inflammation in a type 2 cytokine milieu (e.g., IL-13) is unclear. Our goal was to determine the in vivo role of Tollip in IL-13-mediated lung eosinophilic inflammation and the underlying mechanisms. Tollip-knockout (KO) and wild-type (WT) mice were inoculated intranasally with recombinant mouse IL-13 protein to examine lung inflammation. To determine how Tollip regulates inflammation, alveolar macrophages and bone marrow-derived macrophages from Tollip KO and WT mice were cultured with or without IL-13 and/or IL-33. IL-13-treated Tollip KO mice significantly increased lung eosinophilic inflammation and eotaxin-2 (CCL24) levels compared with the WT mice. IL-13- treated Tollip KO (vs. WT) macrophages, in the absence and particularly in the presence of IL-33, increased expression of the IL-33 receptor ST2L and CCL24, which was in part dependent on enhanced activation of interleukin (IL)-1 receptor-associated kinase 1 (IRAK1) and signal transducer and activator of transcription 6 (STAT6). Our results suggest that Tollip downregulates IL-13-mediated pulmonary eosinophilia in part through inhibiting the activity of the ST2L/IL-33/IRAK1 axis and STAT6.
- Kummarapurugu, A. B., Zheng, S., Ledford, J., Karandashova, S., & Voynow, J. A. (2018). High-Mobility Group Box 1 Upregulates MUC5AC and MUC5B Expression in Primary Airway Epithelial Cells. American journal of respiratory cell and molecular biology, 58(1), 126-128.
- Zhai, J., Insel, M., Addison, K. J., Stern, D. A., Pederson, W., Dy, A., Rojas-Quintero, J., Owen, C. A., Sherrill, D. L., Morgan, W., Wright, A. L., Halonen, M., Martinez, F., Kraft, M., Guerra, S., & Ledford, J. G. (2018). Club Cell Secretory Protein Deficiency Leads to Altered Lung Function. American journal of respiratory and critical care medicine.More infoClub cell secretory protein-16 (CC16), a member of the secretoglobin family, is one of the most abundant proteins in normal airway secretions and has been described as a serum biomarker for obstructive lung diseases.
- Addison, K. J., Morse, J., Robichaud, A., Daines, M. O., & Ledford, J. G. (2017). A Novel in vivo System to Test Bronchodilators. Journal of infectious pulmonary diseases, 3(1).More infoThe incidence and severity of asthma continue to rise worldwide. β-agonists are the most commonly prescribed therapeutic for asthma management but have less efficacy for some subsets of asthmatic patients and there are concerns surrounding the side effects from their long-term persistent use. The demand to develop novel asthma therapeutics highlights the need for a standardized approach to effectively screen and test potential bronchoprotective compounds using relevant in vivo animal models. Here we describe a validated method of testing potential therapeutic compounds for their fast-acting efficacy during the midst of an induced bronchoconstriction in a house dust mite challenged animal model.
- Lugogo, N., Francisco, D., Addison, K. J., Manne, A., Pederson, W., Ingram, J. L., Green, C. L., Suratt, B. T., Lee, J. J., Sunday, M. E., Kraft, M., & Ledford, J. G. (2017). Obese asthmatic patients have decreased surfactant protein A levels: Mechanisms and implications. The Journal of allergy and clinical immunology.More infoEosinophils are prominent in some patients with asthma and are increased in the submucosa in a subgroup of obese patients with asthma (OAs). Surfactant protein A (SP-A) modulates host responses to infectious and environmental insults.
- Noutsios, G. T., Willis, A. L., Ledford, J. G., & Chang, E. H. (2017). Novel role of surfactant protein A in bacterial sinusitis. International forum of allergy & rhinology, 7(9), 897-903.More infoChronic rhinosinusitis (CRS) is a common inflammatory disorder of the upper airway characterized by chronic inflammation and significant sinonasal remodeling. CRS is comprised of 2 major subgroups, based on whether polyps are present or absent. In some cases, it is characterized by colonization with opportunistic pathogens such as Pseudomonas aeruginosa (PA), Staphylococcus aureus, and other bacteria. The innate immune system of the sinonasal epithelium is the first line of defense against inhaled pathogens. Surfactant protein A (SP-A) is a member of the collectin family secreted by the airway epithelia and plays a critical role in airway innate immunity, as it can aggregate bacteria. We hypothesized that SP-A plays a role in bacterial CRS.
- Hsia, B. J., Ledford, J. G., Potts-Kant, E. N., Nikam, V. S., Lugogo, N. L., Foster, W. M., Kraft, M., Abraham, S. N., & Wright, J. R. (2016). Correction notice for TNF-R on mast cells regulate airway responses to Mycoplasma pneumoniae. The Journal of allergy and clinical immunology, 137(1), 336.
- Huang, C., Jiang, D., Francisco, D., Berman, R., Wu, Q., Ledford, J. G., Moore, C. M., Ito, Y., Stevenson, C., Munson, D., Li, L., Kraft, M., & Chu, H. W. (2016). Tollip SNP rs5743899 modulates human airway epithelial responses to rhinovirus infection. Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology, 46(12), 1549-1563.More infoRhinovirus (RV) infection in asthma induces varying degrees of airway inflammation (e.g. neutrophils), but the underlying mechanisms remain unclear.
- Ledford, J. G., Addison, K. J., Francisco, D., Foster, M. W., Voelker, D. R., Que, L. G., & Kraft, M. (2016). Genetic Variation in Surfactant Protein-A2 Results in Altered Regulation of Eosinophil Activities and Enhanced Eosinophilia in Patients with Asthma. Annals of the American Thoracic Society, 13 Suppl 1, S101.
- Stein, M. M., Hrusch, C. L., Gozdz, J., Igartua, C., Pivniouk, V., Murray, S. E., Ledford, J. G., Marques dos Santos, M., Anderson, R. L., Metwali, N., Neilson, J. W., Maier, R. M., Gilbert, J. A., Holbreich, M., Thorne, P. S., Martinez, F. D., von Mutius, E., Vercelli, D., Ober, C., & Sperling, A. I. (2016). Innate Immunity and Asthma Risk in Amish and Hutterite Farm Children. The New England journal of medicine, 375(5), 411-21.More infoThe 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.
- Ledford, J. G., Goto, H., Potts, E. N., Degan, S., Wei Chu, H., Voelker, D. R., Sunday, M. E., Cianciolo, G. J., Foster, W. M., Kraft, M., & Wright, J. R. (2015). Correction: SP-A Preserves Airway Homeostasis during Mycoplasma pneumoniae Infection in Mice. Journal of immunology (Baltimore, Md. : 1950), 195(6), 2917-8.
- Ledford, J. G., Voelker, D. R., Addison, K. J., Wang, Y., Nikam, V. S., Degan, S., Kandasamy, P., Tanyaratsrisakul, S., Fischer, B. M., Kraft, M., & Hollingsworth, J. W. (2015). Genetic variation in SP-A2 leads to differential binding to Mycoplasma pneumoniae membranes and regulation of host responses. Journal of immunology (Baltimore, Md. : 1950), 194(12), 6123-32.More infoMycoplasma pneumoniae is an extracellular pathogen that colonizes mucosal surfaces of the respiratory tract and is associated with asthma exacerbations. Previous reports demonstrate that surfactant protein-A (SP-A) binds live M. pneumoniae and mycoplasma membrane fractions (MMF) with high affinity. Humans express a repertoire of single-amino acid genetic variants of SP-A that may be associated with lung disease, and our findings demonstrate that allelic differences in SP-A2 (Gln223Lys) affect the binding to MMF. We show that SP-A(-/-) mice are more susceptible to MMF exposure and have significant increases in mucin production and neutrophil recruitment. Novel humanized SP-A2-transgenic mice harboring the hSP-A2 223K allele exhibit reduced neutrophil influx and mucin production in the lungs when challenged with MMF compared with SP-A(-/-) mice. Conversely, mice expressing hSP-A2 223Q have increased neutrophil influx and mucin production that are similar to SP-A(-/-) mice. Using tracheal epithelial cell cultures, we show that enhanced mucin production to MMF occurs in the absence of SP-A and is not dependent upon neutrophil recruitment. Increased phosphorylation of the epidermal growth factor receptor (EGFR) was evident in the lungs of MMF-challenged mice when SP-A was absent. Pharmacologic inhibition of EGFR prior to MMF challenge dramatically reduced mucin production in SP-A(-/-) mice. These findings suggest a protective role for SP-A in limiting MMF-stimulated mucin production that occurs through interference with EGFR-mediated signaling. SP-A interaction with the EGFR signaling pathway appears to occur in an allele-specific manner that may have important implications for SP-A polymorphisms in human diseases.
- Foster, M. W., Thompson, J. W., Ledford, J. G., Dubois, L. G., Hollingsworth, J. W., Francisco, D., Tanyaratsrisakul, S., Voelker, D. R., Kraft, M., Moseley, M. A., & Foster, W. M. (2014). Identification and Quantitation of Coding Variants and Isoforms of Pulmonary Surfactant Protein A. Journal of proteome research, 13(8), 3722-32.More infoPulmonary surfactant protein A (SP-A), a heterooligomer of SP-A1 and SP-A2, is an important regulator of innate immunity of the lung. Nonsynonymous single nucleotide variants of SP-A have been linked to respiratory diseases, but the expressed repertoire of SP-A protein in human airway has not been investigated. Here, we used parallel trypsin and Glu-C digestion, followed by LC-MS/MS, to obtain sequence coverage of common SP-A variants and isoform-determining peptides. We further developed a SDS-PAGE-based, multiple reaction monitoring (GeLC-MRM) assay for enrichment and targeted quantitation of total SP-A, the SP-A2 isoform, and the Gln223 and Lys223 variants of SP-A, from as little as one milliliter of bronchoalveolar lavage fluid. This assay identified individuals with the three genotypes at the 223 position of SP-A2: homozygous major (Gln223/Gln223), homozygous minor (Lys223/Lys223), or heterozygous (Gln223/Lys223). More generally, our studies demonstrate the challenges inherent in distinguishing highly homologous, copurifying protein isoforms by MS and show the applicability of MRM mass spectrometry for identification and quantitation of nonsynonymous single nucleotide variants and other proteoforms in airway lining fluid.
- Holmer, S. M., Evans, K. S., Asfaw, Y. G., Saini, D., Schell, W. A., Ledford, J. G., Frothingham, R., Wright, J. R., Sempowski, G. D., & Perfect, J. R. (2014). Impact of surfactant protein D, interleukin-5, and eosinophilia on Cryptococcosis. Infection and immunity, 82(2), 683-93.More infoCryptococcus 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.
- Ledford, J. G., Addison, K. J., Foster, M. W., & Que, L. G. (2014). Eosinophil-associated lung diseases. A cry for surfactant proteins A and D help?. American journal of respiratory cell and molecular biology, 51(5), 604-14.More infoSurfactant proteins (SP)-A and SP-D (SP-A/-D) play important roles in numerous eosinophil-dominated diseases, including asthma, allergic bronchopulmonary aspergillosis, and allergic rhinitis. In these settings, SP-A/-D have been shown to modulate eosinophil chemotaxis, inhibit eosinophil mediator release, and mediate macrophage clearance of apoptotic eosinophils. Dysregulation of SP-A/-D function in eosinophil-dominated diseases is also not uncommon. Alterations in serum SP-A/-D levels are associated with disease severity in allergic rhinitis and chronic obstructive pulmonary disease. Furthermore, oligimerization of SP-A/-D, necessary for their proper function, can be perturbed by reactive nitrogen species, which are increased in eosinophilic disease. In this review, we highlight the associations of eosinophilic lung diseases with SP-A and SP-D levels and functions.
- Ogawa, H., Ledford, J. G., Mukherjee, S., Aono, Y., Nishioka, Y., Lee, J. J., Izumi, K., & Hollingsworth, J. W. (2014). Surfactant protein D attenuates sub-epithelial fibrosis in allergic airways disease through TGF-β. Respiratory research, 15, 143.More infoSurfactant protein D (SP-D) can regulate both innate and adaptive immunity. Recently, SP-D has been shown to contribute to the pathogenesis of airway allergic inflammation and bleomycin-induced pulmonary fibrosis. However, in allergic airways disease, the role of SP-D in airway remodeling remains unknown. The objective of this study was to determine the contribution of functional SP-D in regulating sub-epithelial fibrosis in a mouse chronic house dust mite model of allergic airways disease.
- Li, Z., Tighe, R. M., Feng, F., Ledford, J. G., & Hollingsworth, J. W. (2013). Genes of innate immunity and the biological response to inhaled ozone. Journal of biochemical and molecular toxicology, 27(1), 3-16.More infoAmbient 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.
- Mitsuhashi, A., Goto, H., Kuramoto, T., Tabata, S., Yukishige, S., Abe, S., Hanibuchi, M., Kakiuchi, S., Saijo, A., Aono, Y., Uehara, H., Yano, S., Ledford, J. G., Sone, S., & Nishioka, Y. (2013). Surfactant protein A suppresses lung cancer progression by regulating the polarization of tumor-associated macrophages. The American journal of pathology, 182(5), 1843-53.More infoSurfactant 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.
- Aono, Y., Ledford, J. G., Mukherjee, S., Ogawa, H., Nishioka, Y., Sone, S., Beers, M. F., Noble, P. W., & Wright, J. R. (2012). Surfactant protein-D regulates effector cell function and fibrotic lung remodeling in response to bleomycin injury. American journal of respiratory and critical care medicine, 185(5), 525-36.More infoSurfactant 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.
- Hsia, B. J., Ledford, J. G., Potts-Kant, E. N., Nikam, V. S., Lugogo, N. L., Foster, W. M., Kraft, M., Abraham, S. N., & Wright, J. R. (2012). Mast cell TNF receptors regulate responses to Mycoplasma pneumoniae in surfactant protein A (SP-A)-/- mice. The Journal of allergy and clinical immunology, 130(1), 205-14.e2.More infoMycoplasma pneumoniae (Mp) frequently colonizes the airways of patients with chronic asthma and likely contributes to asthma exacerbations. We previously reported that mice lacking surfactant protein A (SP-A) have increased airway hyperresponsiveness (AHR) during M pneumoniae infection versus wild-type mice mediated by TNF-α. Mast cells (MCs) have been implicated in AHR in asthma models and produce and respond to TNF-α.
- Ledford, J. G., Kovarova, M., Jania, L. A., Nguyen, M., & Koller, B. H. (2012). ONZIN deficiency attenuates contact hypersensitivity responses in mice. Immunology and cell biology, 90(7), 733-42.More infoONZIN is abundantly expressed in immune cells of both the myeloid and lymphoid lineage. Expression by lymphoid cells has been reported to further increase after cutaneous exposure of mice to antigens and haptens capable of inducing contact hypersensitivity (CHS), suggesting that ONZIN has a critical role in this response. Here, we report that indeed ONZIN-deficient mice develop attenuated CHS to a number of different haptens. Dampened CHS responses correlated with a significant reduction in pro-inflammatory IL-6 at the challenge site in ONZIN-deficient animals, compared with wild-type controls. Together the study of these animals indicates that loss of ONZIN impacts the effector phase of the CHS response through the regulation of pro-inflammatory factors.
- Ledford, J. G., Mukherjee, S., Kislan, M. M., Nugent, J. L., Hollingsworth, J. W., & Wright, J. R. (2012). Surfactant protein-A suppresses eosinophil-mediated killing of Mycoplasma pneumoniae in allergic lungs. PloS one, 7(2), e32436.More infoSurfactant protein-A (SP-A) has well-established functions in reducing bacterial and viral infections but its role in chronic lung diseases such as asthma is unclear. Mycoplasma pneumoniae (Mp) frequently colonizes the airways of chronic asthmatics and is thought to contribute to exacerbations of asthma. Our lab has previously reported that during Mp infection of non-allergic airways, SP-A aides in maintaining airway homeostasis by inhibiting an overzealous TNF-alpha mediated response and, in allergic mice, SP-A regulates eosinophilic infiltration and inflammation of the airway. In the current study, we used an in vivo model with wild type (WT) and SP-A(-/-) allergic mice challenged with the model antigen ovalbumin (Ova) that were concurrently infected with Mp (Ova+Mp) to test the hypothesis that SP-A ameliorates Mp-induced stimulation of eosinophils. Thus, SP-A could protect allergic airways from injury due to release of eosinophil inflammatory products. SP-A deficient mice exhibit significant increases in inflammatory cells, mucus production and lung damage during concurrent allergic airway disease and infection (Ova+Mp) as compared to the WT mice of the same treatment group. In contrast, SP-A deficient mice have significantly decreased Mp burden compared to WT mice. The eosinophil specific factor, eosinophil peroxidase (EPO), which has been implicated in pathogen killing and also in epithelial dysfunction due to oxidative damage of resident lung proteins, is enhanced in samples from allergic/infected SP-A(-/-) mice as compared to WT mice. In vitro experiments using purified eosinophils and human SP-A suggest that SP-A limits the release of EPO from Mp-stimulated eosinophils thereby reducing their killing capacity. These findings are the first to demonstrate that although SP-A interferes with eosinophil-mediated biologic clearance of Mp by mediating the interaction of Mp with eosinophils, SP-A simultaneously benefits the airway by limiting inflammation and damage.
- Mukherjee, S., Giamberardino, C., Thomas, J., Evans, K., Goto, H., Ledford, J. G., Hsia, B., Pastva, A. M., & Wright, J. R. (2012). Surfactant protein A integrates activation signal strength to differentially modulate T cell proliferation. Journal of immunology (Baltimore, Md. : 1950), 188(3), 957-67.More infoPulmonary surfactant lipoproteins lower the surface tension at the alveolar-airway interface of the lung and participate in host defense. Previous studies reported that surfactant protein A (SP-A) inhibits lymphocyte proliferation. We hypothesized that SP-A-mediated modulation of T cell activation depends upon the strength, duration, and type of lymphocyte activating signals. Modulation of T cell signal strength imparted by different activating agents ex vivo and in vivo in different mouse models and in vitro with human T cells shows a strong correlation between strength of signal (SoS) and functional effects of SP-A interactions. T cell proliferation is enhanced in the presence of SP-A at low SoS imparted by exogenous mitogens, specific Abs, APCs, or in homeostatic proliferation. Proliferation is inhibited at higher SoS imparted by different doses of the same T cell mitogens or indirect stimuli such as LPS. Importantly, reconstitution with exogenous SP-A into the lungs of SP-A(-/-) mice stimulated with a strong signal also resulted in suppression of T cell proliferation while elevating baseline proliferation in unstimulated T cells. These signal strength and SP-A-dependent effects are mediated by changes in intracellular Ca(2+) levels over time, involving extrinsic Ca(2+)-activated channels late during activation. These effects are intrinsic to the global T cell population and are manifested in vivo in naive as well as memory phenotype T cells. Thus, SP-A appears to integrate signal thresholds to control T cell proliferation.
- Peters-Golden, M., Klinger, J. R., Carson, S. S., & , A. R. (2012). The case for increased funding for research in pulmonary and critical care. American journal of respiratory and critical care medicine, 186(3), 213-5.More infoThe current economic and political climate places future funding of the National Institutes of Health (NIH) and other federal biomedical research programs in jeopardy. This Pulmonary Perspective seeks to arm the diverse membership of the American Thoracic Society with the information necessary to understand and articulate the value of biomedical research in their respective communities. We provide a historical overview of NIH funding in general and of allocations directed at respiratory-related research in particular. We argue that this is in fact an opportune time to expand investments in biomedical research and that doing so makes sense from the perspectives of improving health, curtailing health care expenditures, and job creation and economic growth. We further argue that current levels of allocation toward respiratory research are incommensurate with the medical, economic, and societal burden of respiratory disease in the United States. Respiratory disease currently is the only leading cause of death that has risen, rather than fallen, in recent decades. Declines in the burden of cardiovascular disease and cancer followed substantial increases in research funding, and slowing the rising burden of respiratory disease will likewise require a greatly expanded investment in pulmonary, critical care, and sleep research.
- Jimenez-Preitner, M., Berney, X., Uldry, M., Vitali, A., Cinti, S., Ledford, J. G., & Thorens, B. (2011). Plac8 is an inducer of C/EBPβ required for brown fat differentiation, thermoregulation, and control of body weight. Cell metabolism, 14(5), 658-70.More infoBrown adipocytes oxidize fatty acids to produce heat in response to cold or to excessive energy intake; stimulation of brown fat development and function may thus counteract obesity. Brown adipogenesis requires activation of the transcription factor C/EBPβ and recruitment of the zinc finger protein Prdm16, but upstream inducers of these proteins are incompletely defined. Here, we show that genetic inactivation of Plac8, a gene encoding an evolutionarily conserved protein, induces cold intolerance, and late-onset obesity, as well as abnormal morphology and impaired function of brown adipocytes. Using brown preadipocyte lines we show that Plac8 is required for brown fat differentiation, that its overexpression induces C/EBPβ and Prdm16, and that upon induction of differentiation Plac8 associates with C/EBPβ and binds to the C/EBPβ promoter to induce its transcription. Thus, Plac8 is a critical upstream regulator of brown fat differentiation and function that acts, at least in part, by inducing C/EBPβ expression.
- Goto, H., Ledford, J. G., Mukherjee, S., Noble, P. W., Williams, K. L., & Wright, J. R. (2010). The role of surfactant protein A in bleomycin-induced acute lung injury. American journal of respiratory and critical care medicine, 181(12), 1336-44.More infoSurfactant 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.
- Ledford, J. G., Lo, B., Kislan, M. M., Thomas, J. M., Evans, K., Cain, D. W., Kraft, M., Williams, K. L., & Wright, J. R. (2010). Surfactant protein-A inhibits mycoplasma-induced dendritic cell maturation through regulation of HMGB-1 cytokine activity. Journal of immunology (Baltimore, Md. : 1950), 185(7), 3884-94.More infoDuring pulmonary infections, a careful balance between activation of protective host defense mechanisms and potentially injurious inflammatory processes must be maintained. Surfactant protein A (SP-A) is an immune modulator that increases pathogen uptake and clearance by phagocytes while minimizing lung inflammation by limiting dendritic cell (DC) and T cell activation. Recent publications have shown that SP-A binds to and is bacteriostatic for Mycoplasma pneumoniae in vitro. In vivo, SP-A aids in maintenance of airway homeostasis during M. pneumoniae pulmonary infection by preventing an overzealous proinflammatory response mediated by TNF-α. Although SP-A was shown to inhibit maturation of DCs in vitro, the consequence of DC/SP-A interactions in vivo has not been elucidated. In this article, we show that the absence of SP-A during M. pneumoniae infection leads to increased numbers of mature DCs in the lung and draining lymph nodes during the acute phase of infection and, consequently, increased numbers of activated T and B cells during the course of infection. The findings that glycyrrhizin, a specific inhibitor of extracellular high-mobility group box-1 (HMGB-1) abrogated this effect and that SP-A inhibits HMGB-1 release from immune cells suggest that SP-A inhibits M. pneumoniae-induced DC maturation by regulating HMGB-1 cytokine activity.
- Ledford, J. G., Pastva, A. M., & Wright, J. R. (2010). Review: Collectins link innate and adaptive immunity in allergic airway disease. Innate immunity, 16(3), 183-90.More infoAlthough 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.
- Ledford, J. G., Goto, H., Potts, E. N., Degan, S., Chu, H. W., Voelker, D. R., Sunday, M. E., Cianciolo, G. J., Foster, W. M., Kraft, M., & Wright, J. R. (2009). SP-A preserves airway homeostasis during Mycoplasma pneumoniae infection in mice. Journal of immunology (Baltimore, Md. : 1950), 182(12), 7818-27.More infoThe 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-alpha. 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-alpha response.
- Ledford, J. G., Kovarova, M., & Koller, B. H. (2007). Impaired host defense in mice lacking ONZIN. Journal of immunology (Baltimore, Md. : 1950), 178(8), 5132-43.More infoONZIN 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.
- Allen, I. C., Pace, A. J., Jania, L. A., Ledford, J. G., Latour, A. M., Snouwaert, J. N., Bernier, V., Stocco, R., Therien, A. G., & Koller, B. H. (2006). Expression and function of NPSR1/GPRA in the lung before and after induction of asthma-like disease. American journal of physiology. Lung cellular and molecular physiology, 291(5), L1005-17.More infoA genetic contribution to asthma susceptibility is well recognized, and linkage studies have identified a large number of genes associated with asthma pathogenesis. Recently, a locus encoding a seven-transmembrane protein was shown to be associated with asthma in founder populations. The expression of the protein GPRA (G protein-coupled receptor for asthma susceptibility) in human airway epithelia and smooth muscle, and its increased expression in a mouse model of asthma, suggested that a gain-of-function mutation in this gene increased the disease risk. However, we report here that the development of allergic lung disease in GPRA-deficient mice is unaltered. A possible explanation for this finding became apparent upon reexamination of the expression of this gene. In contrast to initial studies, our analyses failed to detect expression of GPRA in human lung tissue or in mice with allergic lung disease. We identify a single parameter that distinguishes GPRA-deficient and wild-type mice. Whereas the change in airway resistance in response to methacholine was identical in control and GPRA-deficient mice, the mutant animals showed an attenuated response to thromboxane, a cholinergic receptor-dependent bronchoconstricting agent. Together, our studies fail to support a direct contribution of GPRA to asthma pathogenesis. However, our data suggest that GPRA may contribute to the asthmatic phenotype by altering the activity of other pathways, such as neurally mediated mechanisms, that contribute to disease. This interpretation is supported by high levels of GPRA expression in the brain and its recent identification as the neuropeptide S receptor.
- Rabinowitz, J. E., Bowles, D. E., Faust, S. M., Ledford, J. G., Cunningham, S. E., & Samulski, R. J. (2004). Cross-dressing the virion: the transcapsidation of adeno-associated virus serotypes functionally defines subgroups. Journal of virology, 78(9), 4421-32.More infoFor 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.
- Ledford, J., Langlais, P. R., & Dy, A. B. (2019, August/Summer). Identification and functional testing of a novel receptor for SP-A on eosinophils.. Gordon Conference – Lung Development, Injury and Repair.
- Ledford, J., Langlais, P. R., Barker, N. K., & Dy, A. B. (2019, Sept/Fall). Identification and functional testing of a novel receptor for SP-A on eosinophils.. 2019 European Respiratory Society Congress.