Publications

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Journal Articles

Novel coenzyme Q6 genetic variant increases susceptibility to pneumococcal disease

Published in Nature Immunology, 2024

Acute lower respiratory tract infection (ALRI) remains a major worldwide cause of childhood mortality, compelling innovation in prevention and treatment. Children in Papua New Guinea (PNG) experience profound morbidity from ALRI caused by Streptococcus pneumoniae. As a result of evolutionary divergence, the human PNG population exhibits profound genetic variation and diversity. To address unmet health needs of children in PNG, we tested whether genetic variants increased ALRI morbidity. Whole-exome sequencing of a pilot child cohort identified homozygosity for a novel single-nucleotide variant (SNV) in coenzyme Q6 (COQ6) in cases with ALRI. COQ6 encodes a mitochondrial enzyme essential for biosynthesis of ubiquinone, an electron acceptor in the electron transport chain. A significant association of SNV homozygosity with ALRI was replicated in an independent ALRI cohort (P = 0.036). Mice homozygous for homologous mouse variant Coq6 exhibited increased mortality after pneumococcal lung infection, confirming causality. Bone marrow chimeric mice further revealed that expression of variant Coq6 in recipient (that is, nonhematopoietic) tissues conferred increased mortality. Variant Coq6 maintained ubiquinone biosynthesis, while accelerating metabolic remodeling after pneumococcal challenge. Identification of this COQ6 variant provides a genetic basis for increased pneumonia susceptibility in PNG and establishes a previously unrecognized role for the enzyme COQ6 in regulating inflammatory-mediated metabolic remodeling.

Recommended citation: Walker, E.C., Javati, S., Todd, E.M., Matlam, J., Lin, X., Bryant, M., Krone, E., Ramani, R., Chandra, P., Green, T.P., Anaya, E.P., Zhou, J.Y., Alexander, K.A., Tong, R.S., Yuasi, L., Boluarte, S., Yang, F., Greenberg, L., Nerbonne, J.M., Greenberg, M.J., Clemens, R.A., Philips, J.A., Wilson, L.D., Halabi, C.M., DeBosch, B.J., Blyth, C.C., Druley, T.E., Kazura, J.W., Pomat, W.S., Morley, S.C. Novel coenzyme Q6 genetic variant increases susceptibility to pneumococcal disease. Nat Immunol 25, 2247–2258 (2024). https://doi.org/10.1038/s41590-024-01998-4
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Novel Mouse Model Reveals That Serine Phosphorylation of L-Plastin Is Essential for Effective Splenic Clearance of Pneumococcus

Published in The Journal of Immunology, 2021

Asplenia imparts susceptibility to life-threatening sepsis with encapsulated bacteria, such as the pneumococcus. However, the cellular components within the splenic environment that guard against pneumococcal bacteremia have not been defined. The actin-bundling protein L-plastin (LPL) is essential for the generation of marginal zone B cells and for anti-pneumococcal host defense, as revealed by a mouse model of genetic LPL deficiency. In independent studies, serine phosphorylation of LPL at residue 5 (S5) has been described as a key “switch” in regulating LPL actin binding and subsequent cell motility, although much of the data are correlative. To test the importance of S5 phosphorylation in LPL function, and to specifically assess the requirement of LPL S5 phosphorylation in anti-pneumococcal host defense, we generated the “S5A” mouse, expressing endogenous LPL bearing a serine-to-alanine mutation at this position. S5A mice were bred to homozygosity, and LPL was expressed at levels equivalent to wild-type, but S5 phosphorylation was absent. S5A mice exhibited specific impairment in clearance of pneumococci following i.v. challenge, with 10-fold-higher bacterial bloodstream burden 24 h after challenge compared with wild-type or fully LPL-deficient animals. Defective bloodstream clearance correlated with diminished population of marginal zone macrophages and with reduced phagocytic capacity of multiple innate immune cells. Development and function of other tested leukocyte lineages, such as T and B cell motility and activation, were normal in S5A mice. The S5A mouse thus provides a novel system in which to elucidate the precise molecular control of critical immune cell functions in specific host–pathogen defense interactions.

Recommended citation: Anaya, E.P., Lin, X., Todd, E.M., Szasz, T.P., Morley, S.C.; Novel Mouse Model Reveals That Serine Phosphorylation of L-Plastin Is Essential for Effective Splenic Clearance of Pneumococcus. J Immunol 1 May 2021; 206 (9): 2135–2145. https://doi.org/10.4049/jimmunol.2000899
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The actin-bundling protein L-plastin regulates NLRP3 inflammasome activation in macrophages

Published in The Journal of Immunology, 2020

In macrophages, inflammasome pathways induce IL-1β release and pyroptotic cell death to clear infections. For example, NLRP3 inflammasome activation in lung resident alveolar macrophages (AMs) is crucial in controlling pneumonia. We have found that mice lacking the actin-bundling protein L-plastin (LPL−/−) mice are highly susceptible to Streptococcus pnuemoniae lung infection and produce less IL-1β. LPL is also required for podosome formation. Podosomes are integrin-based sites of adhesion that anchor the actin cytoskeleton of AMs to the extracellular environment. As such, AMs can sense their mechanical environment via podosomes. We have found that AMs lacking LPL exhibit defective NLRP3 inflammasome activation in ex-vivo stimulation. Specifically, LPL is required for ASC oligomerization and downstream IL-1β processing after NLRP3 assembly. Since LPL also supports mechanosensitive integrin signaling, we hypothesized that LPL might link contact-based mechanosensation to inflammasome activation in macrophages. To test this hypothesis, we challenged LPL−/− mice with bleomycin, as bleomycin-induced lung injury and fibrosis is thought to be dependent upon NLRP3 activation. LPL−/− mice were resistant to bleomycin-induced lung injury, suggesting a previously unrecognized mechanosensitive mechanism regulating inflammasome activation in macrophages. We propose that the increased lung stiffness in fibrotic lung exerts mechanical stress on AMs and thus may contribute in inflammasome signaling. Increased understanding how mechanical signaling regulates may thus aid in developing therapies to treat lung pathologies i.e. acute respiratory distress syndrome (ARDS), COPD and lung fibrosis.

Recommended citation: Joshi, H., Anaya, E., Almgren-Bell, A., Szasz, T.P., Todd, E.M., Morley, S.C.; The actin-bundling protein L-plastin regulates NLRP3 inflammasome activation in macrophages. J Immunol 1 May 2020; 204 (1_Supplement): 144.24.
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Inhaled GM-CSF in neonatal mice provides durable protection against bacterial pneumonia

Published in Science Advances, 2019

Pneumonia poses profound health threats to preterm infants. Alveolar macrophages (AMs) eliminate inhaled pathogens while maintaining surfactant homeostasis. As AM development only occurs perinatally, therapies that accelerate AM maturation in preterms may improve outcomes. We tested therapeutic rescue of AM development in mice lacking the actin-bundling protein L-plastin (LPL), which exhibit impaired AM development and increased susceptibility to pneumococcal lung infection. Airway administration of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) to LPL−/− neonates augmented AM production. Airway administration distinguishes the delivery route from prior human infant trials. Adult LPL−/− animals that received neonatal GM-CSF were protected from experimental pneumococcal challenge. No detrimental effects on surfactant metabolism or alveolarization were observed. Airway recombinant GM-CSF administration thus shows therapeutic promise to accelerate neonatal pulmonary immunity, protecting against bacterial pneumonia.

Recommended citation: Todd, E. M., Ramani, R., Szasz, T. P., & Morley, S. C. (2019). Inhaled GM-CSF in neonatal mice provides durable protection against bacterial pneumonia. Science Advances, 5(8). https://doi.org/10.1126/sciadv.aax3387
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Inflammasome activation in macrophages is regulated by actin-bundling protein L-plastin

Published in The Journal of Immunology, 2019

Macrophage secretion of IL-1β is crucial to combat and clear bacterial infections. IL-1β release is tightly controlled by assembly of multimolecular inflammasomes (i.e. NLRP3, NLRC4, AIM2) and irregulated production can contribute to various pathologic conditions (i.e. acute respiratory disease and ventilator-associated lung injury). Prior research suggests that mechanical stress elevates macrophage IL-1β production. Because increased lung stiffness in diseases (e.g. acute respiratory distress syndrome) exerts mechanical stress on residing alveolar macrophages (AMs). Understanding how mechanical stress modulates AMs becomes crucial to elucidating the pathophysiology of pulmonary disease. We observed that IL-1β production in AMs was abrogated in absence of L-plastin (LPL), a hematopoietic restricted actin-bundling protein. LPL is also required for podosome formation. Podosomes are integrin-based, adhesive and mechanosensitive signaling organelles. LPL-deficient macrophages showed decreased IL-1β production when cultured on varying substrate stiffness. These observations suggest that LPL may link contact-based mechanosensation to inflammasome activation. Additionally, Pneumolysin toxin in Streptococcus pneumoniae infection activates NLRP3 and may exacerbate inflammatory pathogenesis in stiffened lungs. Further, we will investigate LPL mediated mechano-sensitive inflammasome activation in pneumococcal lung infections using mouse models. Our observations indicate previously unknown mechanosensitive pathway for inflammasome activation in macrophages. Understanding the molecular mechanism will aid in developing immunomodulatory therapies to treat inflammatory disorders and diseases.

Recommended citation: Joshi, H., Todd, B.E., Szasz, T., Anaya, E., Morley, S.C.; Inflammasome activation in macrophages is regulated by actin-bundling protein L-plastin. J Immunol 1 May 2019; 202 (1_Supplement): 117.14. https://doi.org/10.4049/jimmunol.202.Supp.117.14
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Colonization with 19F and other pneumococcal conjugate vaccine serotypes in children in St. Louis, Missouri, USA

Published in Vaccine, 2017

Background The epidemiology of nasopharyngeal (NP) pneumococcal carriage varies with geography and has changed in response to pneumococcal conjugate vaccine (PCV): a low prevalence (3% or less of colonizing isolates) of colonization by vaccine-type (VT) pneumococcal serotypes after PCV introduction has been reported. The primary goal of this study was to determine the VT serotype prevalence of NP pneumococcal colonization of children residing in the St. Louis, MO, USA metropolitan area following introduction of the 13-valent PCV in 2010. The secondary goal of this study was to identify characteristics associated with NP pneumococcal carriage of any serotype. Methods Between July 2013 and April 2016, we enrolled 397 healthy children, aged 0–17 years, who required sedation for procedures or minor surgeries at St. Louis Children’s Hospital. NP swabs were collected after sedation or anesthesia and cultured for pneumococcus. Vaccine records were obtained from primary care providers or from state immunization databases. Parents/guardians completed a questionnaire to provide demographics, past medical history and household characteristics. Results Of the 88 pneumococcal isolates recovered from 84 colonized subjects (21.2% of all enrolled subjects; 95% CI 17.2–25.2%), 16 were VT. Eleven isolates were serotype 19F (12.5%), four (4.5%) were 6A and one (1.1%) was 19A. Prevalence of VT among colonizing isolates was thus 18.2% (CI 10.1–26.1%) in our cohort, despite complete PCV vaccination in 87% of colonized children. Factors associated with pneumococcal colonization by any serotype included younger age and daycare attendance. Conclusion Children in St. Louis exhibit a higher prevalence of VT serotypes among pneumococcal carriage isolates than has been reported in other areas in the US, demonstrating the necessity of ongoing surveillance of local epidemiology and providing evidence that serotype 19F can remain prevalent in a pediatric population despite high vaccine uptake.

Recommended citation: McFarland, M., Szasz, T.P., Zhou, J.Y., Motley, K., Sivapalan, J.S., Isaacson-Schmid, M., Todd, E.M., Hogan, P.G., Fritz, S.A., Burnham, C.D., Hoffmann, S., Morley, S.C., Colonization with 19F and other pneumococcal conjugate vaccine serotypes in children in St. Louis, Missouri, USA, Vaccine, Volume 35, Issue 34, 2017, Pages 4389-4395, ISSN 0264-410X, https://doi.org/10.1016/j.vaccine.2017.06.047.
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Technical Advance: New in vitro method for assaying the migration of primary B cells using an endothelial monolayer as substrate

Published in Journal of Leukocyte Biology, 2017

Migration of B cells supports their development and recruitment into functional niches. Therefore, defining factors that control B cell migration will lead to a better understanding of adaptive immunity. In vitro cell migration assays with B cells have been limited by poor adhesion of cells to glass coated with adhesion molecules. We have developed a technique using monolayers of endothelial cells as the substrate for B cell migration and used this technique to establish a robust in vitro assay for B cell migration. We use TNF-α to up-regulate surface expression of the adhesion molecule VCAM-1 on endothelial cells. The ligand VLA-4 is expressed on B cells, allowing them to interact with the endothelial monolayer and migrate on its surface. We tested our new method by examining the role of L-plastin (LPL), an F-actin-bundling protein, in B cell migration. LPL-deficient (LPL−/−) B cells displayed decreased speed and increased arrest coefficient compared with wild-type (WT) B cells, following chemokine stimulation. However, the confinement ratios for WT and LPL−/− B cells were similar. Thus, we demonstrate how the use of endothelial monolayers as a substrate will support future interrogation of molecular pathways essential to B cell migration.

Recommended citation: Stewart-Hutchinson, P.J., Szasz, T.P., , Jaeger, E.R., Onken, M.D., Cooper, J.A., Morley, S.C., Technical Advance: New in vitro method for assaying the migration of primary B cells using an endothelial monolayer as substrate, Journal of Leukocyte Biology , Volume 102, Issue 3, September 2017, Pages 941–948, https://doi.org/10.1189/jlb.1TA0117-008R.
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Alveolar macrophage development in mice requires L-plastin for cellular localization in alveoli

Published in Blood, 2016

Alveolar macrophages are lung-resident sentinel cells that develop perinatally and protect against pulmonary infection. Molecular mechanisms controlling alveolar macrophage generation have not been fully defined. Here, we show that the actin-bundling protein L-plastin (LPL) is required for the perinatal development of alveolar macrophages. Mice expressing a conditional allele of LPL (CD11c.Crepos-LPLfl/fl) exhibited significant reductions in alveolar macrophages and failed to effectively clear pulmonary pneumococcal infection, showing that immunodeficiency results from reduced alveolar macrophage numbers. We next identified the phase of alveolar macrophage development requiring LPL. In mice, fetal monocytes arrive in the lungs during a late fetal stage, maturing to alveolar macrophages through a prealveolar macrophage intermediate. LPL was required for the transition from prealveolar macrophages to mature alveolar macrophages. The transition from prealveolar macrophage to alveolar macrophage requires the upregulation of the transcription factor peroxisome proliferator-activated receptor-γ (PPAR-γ), which is induced by exposure to granulocyte-macrophage colony-stimulating factor (GM-CSF). Despite abundant lung GM-CSF and intact GM-CSF receptor signaling, PPAR-γ was not sufficiently upregulated in developing alveolar macrophages in LPL−/− pups, suggesting that precursor cells were not correctly localized to the alveoli, where GM-CSF is produced. We found that LPL supports 2 actin-based processes essential for correct localization of alveolar macrophage precursors: (1) transmigration into the alveoli, and (2) engraftment in the alveoli. We thus identify a molecular pathway governing neonatal alveolar macrophage development and show that genetic disruption of alveolar macrophage development results in immunodeficiency.

Recommended citation: Elizabeth M. Todd, Julie Y. Zhou, Taylor P. Szasz, Lauren E. Deady, June A. D’Angelo, Matthew D. Cheung, Alfred H. J. Kim, Sharon Celeste Morley; Alveolar macrophage development in mice requires L-plastin for cellular localization in alveoli. Blood 2016; 128 (24): 2785–2796. doi: https://doi.org/10.1182/blood-2016-03-705962
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Prospective Preliminary In Vitro Investigation of a Magnetic Iron Oxide Nanoparticle Conjugated with Ligand CD80 and VEGF Antibody As a Targeted Drug Delivery System for the Induction of Cell Death in Rodent Osteosarcoma Cells

Published in BioResearch Open Access, 2016

Target drug deliveries using nanotechnology are a novel consideration in the treatment of cancer. We present herein an in vitro mouse model for the preliminary investigation of the efficacy of an iron oxide nanoparticle complex conjugated to vascular endothelial growth factor (VEGF) antibody and ligand cluster of differentiation 80 (CD80) for the purpose of eventual translational applications in the treatment of human osteosarcoma (OSA). The 35 nm diameter iron oxide magnetic nanoparticles are functionalized with an n-hydroxysuccinimide biocompatible coating and are conjugated on the surface to proteins VEGF antibody and ligand CD80. Combined, these proteins have the ability to target OSA cells and induce apoptosis. The proposed system was tested on a cancerous rodent osteoblast cell line (ATCCTMNPO CRL-2836) at four different concentrations (0.1, 1.0, 10.0, and 100.0 μg/mL) of ligand CD80 alone, VEGF antibody alone, and a combination thereof (CD80+VEGF). Systems were implemented every 24 h over different sequential treatment timelines: 24, 48, and 72 h, to find the optimal protein concentration required for a reduction in cell proliferation. Results demonstrated that a combination of ligand CD80 and VEGF antibody was consistently most effective at reducing aberrant osteoblastic proliferation for both the 24- and 72-h timelines. At 48 h, however, an increase in cell proliferation was documented for the 0.1 and 1 μg/mL groups. For the 24- and 72-h tests, concentrations of 1.0 μg/mL of CD80+VEGF and 0.1 μg/mL of VEGF antibody were most effective. Concentrations of 10.0 and 100.0 μg/mL of CD80+VEGF reduced cell proliferation, but not as remarkably as the 1.0 μg/mL concentration. In addition, cell proliferation data showed that multiple treatments (72-h test) induced cell death in the osteoblasts better than a single treatment. Future targeted drug delivery system research includes trials in OSA cell lines from greater phylum species having spontaneous OSA, such as the dog, and on a human OSA cell line model.

Recommended citation: Kovach, A. K., Gambino, J. M., Nguyen, V., Nelson, Z., Szasz, T., Liao, J., Williams, L., Bulla, S., & Prabhu, R. (2016). Prospective preliminary in vitro investigation of a magnetic iron oxide nanoparticle conjugated with ligand CD80 and VEGF antibody as a targeted drug delivery system for the induction of cell death in rodent osteosarcoma cells. BioResearch Open Access, 5(1), 299–307. https://doi.org/10.1089/biores.2016.0020
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L-Plastin promotes podosome longevity and supports macrophage motility

Published in Molecular Immunology, 2016

Elucidating the molecular regulation of macrophage migration is essential for understanding the pathophysiology of multiple human diseases, including host responses to infection and autoimmune disorders. Macrophage migration is supported by dynamic rearrangements of the actin cytoskeleton, with formation of actin-based structures such as podosomes and lamellipodia. Here we provide novel insights into the function of the actin-bundling protein l-plastin (LPL) in primary macrophages. We found that podosome stability is disrupted in primary resident peritoneal macrophages from LPL−/− mice. Live-cell imaging of F-actin using resident peritoneal macrophages from LifeACT-RFP+ mice demonstrated that loss of LPL led to decreased longevity of podosomes, without reducing the number of podosomes initiated. Additionally, macrophages from LPL−/− mice failed to elongate in response to chemotactic stimulation. These deficiencies in podosome stabilization and in macrophage elongation correlated with impaired macrophage transmigration in culture and decreased monocyte migration into murine peritoneum. Thus, we have identified a role for LPL in stabilizing long-lived podosomes and in enabling macrophage motility.

Recommended citation: Zhou, J.Y., Szasz, T.P., Stewart-Hutchinson, P.J., Sivapalan, J., Todd, E.M., Deady, L.E., Cooper, J.A., Onken, M.D., Morley, S.C., L-Plastin promotes podosome longevity and supports macrophage motility, Molecular Immunology, Volume 78, 2016, Pages 79-88, ISSN 0161-5890, https://doi.org/10.1016/j.molimm.2016.08.012.
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Defective monocyte motility disrupts alveolar macrophage development in mice deficient for L-plastin

Published in The Journal of Immunology, 2016

Alveolar macrophages, sentinel cells essential to pulmonary immunity, mature during perinatal development. Fetal monocytes arrive in the lungs during late embryonic development and mature into alveolar macrophages through a pre-alveolar macrophage intermediate. Here we identify a requirement for the actin-bundling protein L-plastin (LPL) in a key transition during neonatal alveolar macrophage development. LPL−/− mice demonstrated a two-fold reduction in alveolar macrophages. The requirement for LPL was cell-intrinsic, as mice expressing a CD11c-specific deletion of LPL (CD11c.Cre-LPLfl/fl mice) also exhibited a significant (> 75%) reduction in alveolar macrophages. Impaired alveolar macrophage development in LPL−/− mice resulted from a block in the transition of the intermediate pre-alveolar macrophages to mature alveolar macrophages. Differentiation of monocytes into alveolar macrophages requires transit to the alveolar space, which exposes precursor cells to the essential growth factor GM-CSF, which in turn upregulates the transcription factor PPAR-γ. LPL is required for monocyte migration into the alveoli, providing a mechanism by which LPL deficiency impairs alveolar macrophage maturation. As predicted by this model, pre-alveolar macrophages in LPL−/− mice did not upregulate PPAR-γ, indicating that exposure to GM-CSF is reduced. Finally, LPL−/−, CD11c.Cre-LPLfl/fl and CD11c.Cre-LPLfl/+ mice failed to efficiently clear pulmonary pneumococcal infection, revealing a physiological consequence of impaired alveolar macrophage development. In summary, we show that genetic disruption of monocyte motility can impair differentiation of a key cell type, subsequently impairing host immunity.

Recommended citation: Morley, S.C., Todd, E.M., Zhou, J.Y., Deady, L.E., D’Angelo, J.A., Szasz, T. ; Defective monocyte motility disrupts alveolar macrophage development in mice deficient for L-plastin. J Immunol 1 May 2016; 196 (1_Supplement): 119.9. https://doi.org/10.4049/jimmunol.196.Supp.119.9
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Taylor Szasz Green

Publications

Journal Articles