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1.
J Immunol ; 206(9): 2135-2145, 2021 05 01.
Article in English | MEDLINE | ID: mdl-33858961

ABSTRACT

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.


Subject(s)
Membrane Glycoproteins/immunology , Microfilament Proteins/immunology , Serine/immunology , Spleen/immunology , Streptococcus pneumoniae/immunology , Animals , Mice , Mice, Inbred C57BL , Mice, Transgenic , Phosphorylation , Streptococcus pneumoniae/isolation & purification
2.
Sci Adv ; 5(8): eaax3387, 2019 08.
Article in English | MEDLINE | ID: mdl-31453341

ABSTRACT

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.


Subject(s)
Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Macrophages, Alveolar/cytology , Microfilament Proteins/genetics , Pneumonia, Bacterial/prevention & control , Administration, Inhalation , Animals , Animals, Newborn , Granulocyte-Macrophage Colony-Stimulating Factor/administration & dosage , Humans , Macrophages, Alveolar/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Pneumonia, Bacterial/immunology , Pneumonia, Bacterial/microbiology
3.
Vaccine ; 35(34): 4389-4395, 2017 08 03.
Article in English | MEDLINE | ID: mdl-28687405

ABSTRACT

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 0-17years, 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.


Subject(s)
Carrier State/epidemiology , Pneumococcal Infections/epidemiology , Streptococcus pneumoniae/growth & development , Streptococcus pneumoniae/isolation & purification , Adolescent , Carrier State/microbiology , Child , Child, Preschool , Female , Heptavalent Pneumococcal Conjugate Vaccine/administration & dosage , Humans , Infant , Infant, Newborn , Male , Microbial Sensitivity Tests , Missouri/epidemiology , Nasopharynx/microbiology , Pneumococcal Infections/microbiology , Pneumococcal Infections/prevention & control , Pneumococcal Vaccines/administration & dosage , Pneumococcal Vaccines/classification , Prevalence , Seroepidemiologic Studies , Serogroup , Streptococcus pneumoniae/classification , Streptococcus pneumoniae/immunology , Vaccination , Vaccines, Conjugate/administration & dosage
4.
J Leukoc Biol ; 102(3): 941-948, 2017 09.
Article in English | MEDLINE | ID: mdl-28637896

ABSTRACT

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.


Subject(s)
B-Lymphocytes/immunology , Cell Movement/immunology , Endothelial Cells/immunology , Integrin alpha4beta1/immunology , Phosphoproteins/immunology , Tumor Necrosis Factor-alpha/immunology , Animals , B-Lymphocytes/cytology , Cell Movement/genetics , Coculture Techniques/methods , Cytoskeletal Proteins , Endothelial Cells/cytology , Integrin alpha4beta1/genetics , Mice , Mice, Knockout , Microfilament Proteins , Phosphoproteins/genetics , Tumor Necrosis Factor-alpha/genetics
5.
Biores Open Access ; 5(1): 299-307, 2016.
Article in English | MEDLINE | ID: mdl-27843708

ABSTRACT

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.

6.
Blood ; 128(24): 2785-2796, 2016 12 15.
Article in English | MEDLINE | ID: mdl-27758872

ABSTRACT

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.


Subject(s)
Macrophages, Alveolar/metabolism , Membrane Glycoproteins/metabolism , Microfilament Proteins/metabolism , Pulmonary Alveoli/cytology , Pulmonary Alveoli/metabolism , Animals , Animals, Newborn , CD11 Antigens/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Mice, Inbred C57BL , Models, Biological , Monocytes/metabolism , PPAR gamma/metabolism , Pneumococcal Infections/pathology , Podosomes/metabolism , Protein Transport , Up-Regulation/drug effects
7.
Mol Immunol ; 78: 79-88, 2016 10.
Article in English | MEDLINE | ID: mdl-27614263

ABSTRACT

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.


Subject(s)
Cell Movement/physiology , Macrophages, Peritoneal/metabolism , Phosphoproteins/metabolism , Podosomes/metabolism , Animals , Cytoskeletal Proteins , Mice , Mice, Knockout , Microfilament Proteins , Microscopy, Confocal
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