Rab17 mediates differential antigen sorting following efferocytosis and phagocytosis

Yin, Charles; Kim, Young S.; Argintaru, Dean; Heit, Bryan

doi:10.1038/cddis.2016.431

Cited by 45 publications

(64 citation statements)

References 73 publications

(109 reference statements)

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“…To illustrate the ability of FDM printing to produce complex chambers, we designed a reusable magnetic Leiden chamber designed to fit 18 mm circular coverslips. Using these Leiden chambers we performed phagocytosis assays with transgene-expressing macrophages ( Figure 5G-H), again producing results equivalent to those we have previously reported 32 .…”

Section: Easy Implementation Of Diverse Chamber Designssupporting

confidence: 83%

“…MerTK, Lifeact-RFP, mCherry-Rab17 and PM-GFP constructs were prepared previously 29,32,38 . #1.5 thickness glass coverslips, polydimethylsiloxane (PDMS), 16% paraformaldehyde (PFA) and glass slides were from Electron Microscopy Sciences.…”

Section: Methodsmentioning

confidence: 99%

“…Phagocytosis was quantified as described previously 32 . Briefly, J774.2 macrophages were split into the multiplex imaging chamber or onto 18 mm circular coverslips and allowed to recover for 24 hrs.…”

Section: Phagocytosis Assaysmentioning

confidence: 99%

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Customizable Live-Cell Imaging Chambers for Multimodal and Multiplex Fluorescence Microscopy

Tepperman

Zheng

Taka

et al. 2020

Preprint

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While new high-resolution microscopy techniques are continually developed, adoption of these methods is often difficult due to an inability to meet the experimental conditions required for an experiment in a format which also meets the demanding optical requirements of these microscopy techniques. Although specialized imaging chambers can meet these challenges, the difficulty of manufacturing customized chambers in-house and the relatively high cost and design inflexibility of commercial chambers has limited the incorporation of imaging chambers into fluorescence and super-resolution microscopy experiments. Herein, we demonstrate the use of fused deposition modeling (3D printing) for producing inexpensive, customized imaging chambers that are compatible with long-duration live-cell imaging using fluorescence and super-resolution microscopy techniques. In this approach, biocompatible 3D printing plastics are used to generate imaging chambers designed to meet the specific needs of an experiment, followed by adhesion of the printed chamber to a glass coverslip suitable for fluorescence and super-resolution imaging. This technique produces a chamber that is impermeant to liquids that can support the growth and imaging of cells over multiple days. The utility of these chambers is then demonstrated using designs for multiplex microscopy, imaging under shear, chemotaxis, and general cellular imaging. Together, this approach represents an inexpensive yet highly customizable approach to produce imaging chambers that are compatible with many modern microscopy techniques.

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Section: Easy Implementation Of Diverse Chamber Designssupporting

confidence: 83%

Section: Methodsmentioning

confidence: 99%

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Customizable Live-Cell Imaging Chambers for Multimodal and Multiplex Fluorescence Microscopy

Tepperman

Zheng

Taka

et al. 2020

Preprint

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“…The top band of cells was collected, washed once (300 × g, 6 min, 20 °C) with phosphate buffered saline (PBS, 137 mM NaCl, 10 mM Na2HPO4), and resuspended at 2 × 10 6 cells/ml in RPMI-1640 + 10% FBS + 1% antibiotic-antimycotic. 200 μl of this suspension was placed on sterile glass coverslips for 1 h at 37 °C, washed twice with PBS, and then differentiated into M0-polarized macrophages as per our published protocols 52,77 .…”

Section: Primary Macrophage Culturementioning

confidence: 99%

GATA2 Expression by Intima-Infiltrating Macrophages Drives Early Atheroma Formation

Akingbasote

et al. 2019

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Aberrant macrophage polarization is a major contributor to the onset and progression of atherosclerosis. Despite this, macrophage polarization during in early stages of human atherosclerotic disease is poorly understood. Using transcriptomic analysis of macrophages recovered from early-stage human atherosclerotic lesions, we have identified a unique gene expression profile dissimilar to that observed in later stages of disease that is characterized by upregulation of the hematopoietic transcription factor GATA2. GATA2 overexpression in vitro recapitulated defects observed in patient macrophages, including deficiencies in the uptake and processing of apoptotic cells, and in the catalysis of atherogenic protein modifications, with GATA2 knockdown abrogating these defects. Our data describe a previously unreported macrophage differentiation state present in early atheroma formation and identifies GATA2 as a driver of macrophage functional defects during the early stages of atherosclerosis in humans.

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“…Non-adherent cells were removed after a 1 h/37 °C incubation with two PBS washes. Monocytes were differentiated into M0, M1 or M2 polarized macrophages as per our published protocols (30,31).…”

Section: Primary Human Macrophage Culturementioning

confidence: 99%

Soluble CD93 is an Apoptotic Cell Opsonin Recognized by the α_xβ₂Integrin

Ellins

et al. 2018

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Abbreviations: ADAM -a disintegrin and metalloproteinase. BSA -bovine serum albumin. CHO -Chinese hamster ovary cells. CTLD -C-type lectin-like domain. FBS -fetal bovine serum. MMPmatrix metalloproteinase. PBMC -peripheral blood mononuclear cells. PFA -paraformaldehyde. sCD93 -soluble CD93. ABSTRACTEfferocytosis -the phagocytic removal of apoptotic cells -is essential for the maintenance of homeostasis and prevention of the inflammatory and autoimmune diseases which can follow the lysis of uncleared apoptotic cells. CD93 is a transmembrane glycoprotein previously implicated in efferocytosis and angiogenesis, and upon mutation, results in the onset of efferocytosis-associated diseases such as atherosclerosis and rheumatoid arthritis. CD93 is produced as a cell surface protein which is shed as soluble CD93, but it is unknown how CD93 mediates efferocytosis or whether its efferocytic activity is mediated by the soluble or membrane-bound form. Herein, we demonstrate that the membrane bound form of CD93 has no phagocytic, efferocytic, or tethering activity, whereas soluble CD93 potently opsonizes apoptotic cells but not a broad range of Gram-Negative, Gram-Positive or fungal microorganisms. Using mass spectrometry, we identified the αxβ2 integrin as the receptor required for soluble CD93-mediated efferocytosis, and via deletion mutagenesis determined that soluble CD93 binds to apoptotic cells via its C-Type Lectin-Like domain, and to αxβ2 by its EGF-like repeats. This bridging of apoptotic cells to the αxβ2 integrin markedly enhanced efferocytosis by macrophages, and could be abrogated by knockdown of αxβ2 integrin. Combined, these data elucidate the mechanism by which CD93 regulates efferocytosis and identify a previously unreported opsonin-receptor system utilized by the immune system for the efferocytic clearance of apoptotic cells.Clearly, there is data supporting three distinct and mutually exclusive models of CD93 efferocytic function. In this study, we directly asses these three models of CD93 function, using in vitro models of binding, efferocytosis and opsonization. These experiments disfavor the efferocytic receptor and tethering receptor models of CD93 function, and instead demonstrate a strong opsonic effect of sCD93 against apoptotic cells but not against a panel of microorganisms. Using mass-spectrometry we then identified αxβ2 integrin as the efferocytic receptor which recognizes sCD93 opsonized targets, with apoptotic cells "bridged" by the CD93 CTLD domain to αxβ2 integrin on phagocytes via the CD93 EGFlike domain. This represents a previously undescribed opsonin-receptor system which aids in the efferocytosis of apoptotic cell by phagocytes such as macrophages. MATERIALS AND METHODS MaterialsDH5a Escherichia coli was a gift from Dr. John McCormick (University of Western Ontario), Rhodotorula minuta and Candida glabrate were gifts from Dr. Andre Lachance (University of Western Ontario). Escherichia coli K29 + and K29 -, and Burkholderia cenocepacia were gifts from Dr. Susan Koval (University of Wester...

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Rab17 mediates differential antigen sorting following efferocytosis and phagocytosis

Cited by 45 publications

References 73 publications

Customizable Live-Cell Imaging Chambers for Multimodal and Multiplex Fluorescence Microscopy

Customizable Live-Cell Imaging Chambers for Multimodal and Multiplex Fluorescence Microscopy

GATA2 Expression by Intima-Infiltrating Macrophages Drives Early Atheroma Formation

Soluble CD93 is an Apoptotic Cell Opsonin Recognized by the α_xβ₂Integrin

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Rab17 mediates differential antigen sorting following efferocytosis and phagocytosis

Cited by 45 publications

References 73 publications

Customizable Live-Cell Imaging Chambers for Multimodal and Multiplex Fluorescence Microscopy

Customizable Live-Cell Imaging Chambers for Multimodal and Multiplex Fluorescence Microscopy

GATA2 Expression by Intima-Infiltrating Macrophages Drives Early Atheroma Formation

Soluble CD93 is an Apoptotic Cell Opsonin Recognized by the αxβ2Integrin

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Soluble CD93 is an Apoptotic Cell Opsonin Recognized by the α_xβ₂Integrin