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1.  Stable Expression and Characterization of an Optimized Mannose Receptor 
The mannose receptor (MR) is a macrophage surface receptor that recognizes pathogen associated molecular patterns (PAMPs) from a diverse array of bacterial, fungal and viral pathogens. Functional studies of the MR are hampered by the scarcity of human cell lines that express the receptor. Current model systems available for the study of MR biology often demonstrate low levels of expression and do not retain many of the classical MR properties. Although several laboratories have reported transient and stable expression of MR from plasmids, preliminary data from our laboratory suggests that these plasmids produce a protein that lacks critical domains and is often not stable over time. In this current report we describe the generation and characterization of a novel human codon-optimized system for transient and stable MR expression. Rare codons and sequences that contribute to mRNA instability were modified to produce mRNA that is qualitatively and quantitatively improved. Confocal imaging of the transient and stably expressed optimized receptor demonstrates a distribution consistent with previous reports. To demonstrate the functional characteristics of the optimized receptor, we further show that the introduction of codon-optimized MR plasmid can confer MR-associated phagocytosis of S. aureus to non-phagocytic HeLa cells. We show that three molecules participate in the engagement and internalization of S. aureus. MR was found to colocalize with Toll-like receptor 2 (TLR2) and Rab5 following exposure to pHrodo-stained S. aureus, suggesting cooperation among the three molecules to engage and internalize the bacterial particle. This study describes a transfection capable, optimized MR receptor with functional characteristics similar to the wild type receptor and further demonstrates a new system for the continued study of MR biology and function.
PMCID: PMC4645283  PMID: 26581716
Macrophages; Mannose receptor
2.  Characterization of functional mannose receptor in a continuous hybridoma cell line 
BMC Immunology  2012;13:51.
The mannose receptor is the best described member of the type I transmembrane C-type lectins; however much remains unanswered about the biology of the receptor. One difficulty has been the inability to consistently express high levels of a functional full length mannose receptor cDNA in mammalian cells. Another difficulty has been the lack of a human macrophage cell line expressing a fully functional receptor. Commonly used human macrophage cell lines such as U937, THP-1, Mono-Mac and HL60 do not express the mannose receptor. We have developed a macrophage hybridoma cell line (43MR cells) created by fusion of U937 cells with primary human monocyte-derived macrophages, resulting in a non-adherent cell line expressing several properties of primary macrophages. The purpose of this study was to identify and select mannose receptor-expressing cells using fluorescence-activated cell sorting and to characterize the expression and function of the receptor.
In the current study we show that the mannose receptor found on this novel cell has endocytic characteristics consistent with and similar to the mannose receptor found on the surface of monocyte-derived human macrophages and rat bone marrow-derived macrophages. In addition, we demonstrate that these cells engage and internalize pathogen particles such as S. aureus and C. albicans. We further establish the transfectability of these cells via the introduction of a plasmid expressing influenza A hemagglutinin.
The 43MR cell line represents the first naturally expressed MR-positive cell line derived from a human macrophage background. This cell line provides an important cell model for other researchers for the study of human MR biology and host-pathogen interactions.
PMCID: PMC3495026  PMID: 22967244
3.  Mitogen-activated protein kinases and NFκB are involved in SP-A-enhanced responses of macrophages to mycobacteria 
Respiratory Research  2009;10(1):60.
Surfactant protein A (SP-A) is a C-type lectin involved in surfactant homeostasis as well as host defense in the lung. We have recently demonstrated that SP-A enhances the killing of bacillus Calmette-Guerin (BCG) by rat macrophages through a nitric oxide-dependent pathway. In the current study we have investigated the role of tyrosine kinases and the downstream mitogen-activated protein kinase (MAPK) family, and the transcription factor NFκB in mediating the enhanced signaling in response to BCG in the presence of SP-A.
Human SP-A was prepared from alveolar proteinosis fluid, and primary macrophages were obtained by maturation of cells from whole rat bone marrow. BCG-SP-A complexes were routinely prepared by incubation of a ratio of 20 μg of SP-A to 5 × 105 BCG for 30 min at 37°C. Cells were incubated with PBS, SP-A, BCG, or SP-A-BCG complexes for the times indicated. BCG killing was assessed using a 3H-uracil incorporation assay. Phosphorylated protein levels, enzyme assays, and secreted mediator assays were conducted using standard immunoblot and biochemical methods as outlined.
Involvement of tyrosine kinases was demonstrated by herbimycin A-mediated inhibition of the SP-A-enhanced nitric oxide production and BCG killing. Following infection of macrophages with BCG, the MAPK family members ERK1 and ERK2 were activated as evidence by increased tyrosine phosphorylation and enzymatic activity, and this activation was enhanced when the BCG were opsonized with SP-A. An inhibitor of upstream kinases required for ERK activation inhibited BCG- and SP-A-BCG-enhanced production of nitric oxide by approximately 35%. Macrophages isolated from transgenic mice expressing a NFκB-responsive luciferase gene showed increased luciferase activity following infection with BCG, and this activity was enhanced two-fold in the presence of SP-A. Finally, lactacystin, an inhibitor of IκB degradation, reduced BCG- and SP-A-BCG-induced nitric oxide production by 60% and 80% respectively.
These results demonstrate that BCG and SP-A-BCG ingestion by macrophages is accompanied by activation of signaling pathways involving the MAP kinase pathway and NFκB.
PMCID: PMC2717924  PMID: 19566962
4.  N-Linked Glycosylation Attenuates H3N2 Influenza Viruses▿  
Journal of Virology  2007;81(16):8593-8600.
Over the last four decades, H3N2 subtype influenza A viruses have gradually acquired additional potential sites for glycosylation within the globular head of the hemagglutinin (HA) protein. Here, we have examined the biological effect of additional glycosylation on the virulence of H3N2 influenza viruses. We created otherwise isogenic reassortant viruses by site-directed mutagenesis that contain additional potential sites for glycosylation and examined the effect on virulence in naïve BALB/c, C57BL/6, and surfactant protein D (SP-D)-deficient mice. The introduction of additional sites was consistent with the sequence of acquisition in the globular head over the past 40 years, beginning with two sites in 1968 to the seven sites found in contemporary influenza viruses circulating in 2000. Decreased morbidity and mortality, as well as lower viral lung titers, were seen in mice as the level of potential glycosylation of the viruses increased. This correlated with decreased evidence of virus-mediated lung damage and increased in vitro inhibition of hemagglutination by SP-D. SP-D-deficient animals displayed an inverse pattern of disease, such that more highly glycosylated viruses elicited disease equivalent to or exceeding that of the wild type. We conclude from these data that increased glycosylation of influenza viruses results in decreased virulence, which is at least partly mediated by SP-D-induced clearance from the lung. The continued exploration of interactions between highly glycosylated viruses and surfactant proteins may lead to an improved understanding of the biology within the lung and strategies for viral control.
PMCID: PMC1951338  PMID: 17553891

Results 1-4 (4)