Comparative morphology and phagocytic capacity of primary human adult microglia with time-lapse imaging
Graphical abstract
Introduction
Microglia are the resident phagocytic cells of the CNS that actively scavenge and provide immune surveillance (Perry and Gordon, 1988, Glenn et al., 1992, Brown and Neher, 2014, Kettenmann et al., 2011). Seminal work by Pio del Rio-Hortega revealed that microglia have various morphological configurations that are accompanied by specialization of function. Ramified microglia have a small nucleus and cytoplasm with complex branched processes, amoeboid microglia have a large nucleus and are oval-shaped surrounded by ruffled membrane sheets, and pseudopodic microglia have oblong cell bodies with two protrusions containing fan-shaped ruffled membranes (Del Rio Hortega, 1920, Kernohan and Penfield, 1932, Morrison and Filosa, 2013, Karperien et al., 2015, Sivagnanam et al., 2010). Ramified microglia actively scan the environment, and upon activation may transform into amoeboid or pseudopodic morphologies to phagocytose particles (Kettenmann et al., 2011, Glenn et al., 1992). In multiple sclerosis, microglia can cause inflammation and injure neurons (González et al., 2014, Watanabe et al., 2016), but can also serve neuroprotective roles by cleaning up debris by phagocytosis and promoting cellular repair (Fu et al., 2014, Neumann et al., 2009). Microglia also protect the brain from intracerebral infections by phagocytosing bacteria into lysosomes (Schütze et al., 2014, Kaur et al., 2004, Ribes et al., 2009). Phagocytosis requires cytoskeletal network rearrangement and relies specifically on F-actin, a filamentous polymer of the cytoskeleton. The balance of actin polymerization and depolymerization enables cell motility, cytokinesis, and dynamic changes in cell shape. Particles that come in contact with transmembrane phagocytic receptors cause actin accumulation at the spot of contact, where the engulfment of the particle in a phagocytic cup follows membrane elongation (Gitik et al., 2010). Actin dynamics are often studied using fluorescent phalloidin staining (Miller et al., 2003). Studies on murine microglia suggest that impaired actin dynamics reduce the phagocytosis of bacteria particles (Uhlemann et al., 2016). The phagocytosis can be seen as membrane ruffling where F-actin accumulates in regions around the contact point with microspheres (Koizumi et al., 2007). Alteration to the F-actin filaments or to the directly-associated cytoskeletal proteins in microglia result in the increased probability of developing neurodegenerative diseases or neuro-inflammatory disorders. Improper actin dynamics decrease the phagocytic ability of microglia, resulting in the accumulation of extracellular debris and disease development, as exemplified by the onset of Alzheimer's disease due to amyloid-β accumulation (Daria et al., 2016). Macrophages, being similar to microglia in function, display dysfunctional phagocytosis when the organization of F-actin filaments is inhibited (de Oliveira and Mantovani, 1988). Dendritic cells (DCs) have properties similar to microglia. DCs are found throughout body tissues where they capture, process, and present antigens to T cells via MHC molecules (Steinman and Cohn, 1973, Banchereau and Steinman, 1998, Savina and Amigorena, 2007, Segura and Villadangos, 2009, Merad et al., 2013, González et al., 2014). DC morphology is characterized by long cytoplasmic extensions of asymmetric shapes. Primary human adult microglia and fetal microglia were comparable to DCs in their capacity to stimulate T cells and phagocytose E. coli bioparticles (Lambert et al., 2008). E. coli bioparticles are killed E. coli bacteria that are prepared with a fluorescent dye to track phagocytosis. Human DCs derived from monocytes in vitro (mDCs) are functionally-competent, capable of cytokine production and phagocytosis (Durafourt et al., 2012). In the current study, we evaluated the morphology and capacity of primary human adult microglia and mDCs to phagocytose E. coli bioparticles, and quantified the relative uptake of the bioparticles in acidic compartments.
Section snippets
Cell cultures
Primary human microglia were obtained from normal-appearing white matter resected tissue of a temporal lobe surgery for non-tumor-related intractable epilepsy using previously published procedures (Yong and Antel, 1997, Williams et al., 1992, Williams et al., 1994). The study was approved by the McGill University institutional review board and samples were procured with informed consent. Cells were cultured in MEM with 5% FBS, penicillin, streptomycin, and l-glutamine in tissue culture-treated
Comparative morphology of primary human microglia and mDCs
Cells purified from temporal lobe resected tissue of non-tumor-related intractable epilepsy were highly enriched in microglia. The majority of cells stained double positive for the microglia markers Iba-1 and CD68 (Supplemental figure). Three morphologically-distinct microglia populations were detected in the cultures, including amoeboid, pseudopodic, and ramified shapes (Fig. 1A–C). The proportion of microglia morphologies in the culture were 71% (2.6 SD) amoeboid, 16.9% (4.7 SD) pseudopodic,
Discussion
Our data show the distinctive amoeboid, pseudopodic, and ramified morphologies of microglia consistent with some of the types first described by Pio del Rio-Hortega. The pseudopodic microglia exhibited fan-like lamellipodia, as described by Sivagnanam et al. (2010), and were similar to the microglia once described as a ‘bipolar cell’ (Williams et al., 1992). There were also rare, small mobile cells among the microglia population that resembled the small mobile cells observed in the mDC
Conclusions
Pseudopodic and amoeboid microglia from human adult primary tissue had similar phagocytic capacity. Microglia took up particles in a multiple step fashion involving contact, retrograde transport on membrane sheets, and transfer into an acidic compartment. F-actin density was observed at the membrane edge and where E. coli bioparticles were internalized consistent with a phagocytic cup. These specializations could be useful for microglia in the brain tissue since it would increase their coverage
Acknowledgments
Research financial support was received from the Multiple Sclerosis Society of Canada, the Canadian Institutes of Health Research, and from NSERC (RGPIN 418522 2013). T.E.K. was supported by a Senior Bourses de Chercheurs-Boursiers Award, and Killam Foundation Scholar program. Thank you to Mahdieh Tabatabaei Shafiei and Elena Lin for editing and proof-reading the manuscript.
References (36)
- et al.
Neuroimmune regulation of microglial activity involved in neuroinflammation and neurodegenerative diseases
J. Neuroimmunol.
(2014) - et al.
Latrunculin A is a potent inhibitor of phagocytosis by macrophages
Life Sci.
(1988) - et al.
Macrophages and microglia in the nervous system
Trends Neurosci.
(1988) - et al.
Antigen presentation by dendritic cells in vivo
Curr. Opin. Immunol.
(2009) - et al.
Dominance of E. coli phagocytosis over LPS in the inflammatory response of microglia
J. Neuroimmunol.
(2010) - et al.
Immune regulatory and effector properties of human adult microglia studied in vitro and in situ
Adv. Neuroimmunol.
(1994) - et al.
Response of amoeboid and ramified microglial cells to lipopolysaccharide injections in postnatal rats-a lectin and ultrastructural study
Neurosci. Res.
(1997) - et al.
Dendritic cells and the control of immunity
Nature
(1998) - et al.
Microglial phagocytosis of live neurons
Nat. Neurosci.
(2014) - et al.
Young microglia restore amyloid plaque clearance of aged microglia
EMBO J.
(2016)
El ‘tercer elemento’ de los centros nerviosos. I. La microglıa en estado normal
Bol. Soc. Esp. Biol.
Comparison of polarization properties of human adult microglia and blood-derived macrophages
Glia
Phagocytosis of microglia in the central nervous system diseases
Mol. Neurobiol.
Cytoskeleton plays a dual role of activation and inhibition in myelin and zymosan phagocytosis by microglia
FASEB J.
Characterisation of ramified microglial cells: detailed morphology, morphological plasticity and proliferative capability
J. Anat.
MerTK is a functional regulator of myelin phagocytosis by human myeloid cells
J. Immunol.
Quantitating the subtleties of microglial morphology with fractal analysis
Front. Cell. Neurosci.
Phagocytosis of Escherichia coli by amoeboid microglial cells in the developing brain
Acta Neuropathol.
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These authors contributed equally, shared first author.