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By Jennifer Sokolowski, MD, PhD.
Microglia play important roles in the brain in both homeostatic and pathological conditions, acting to clear debris and dying cells. There is evidence to suggest that microglial dysfunction contributes to neurodegenerative diseases such as Alzheimer's and modulation of microglial activity may be a method to treat such diseases.1 Cell-based therapies represent a novel approach whereby the introduction of microglia that possess the desired phenotype could potentially be used to remediate defects in clearance. In a paper by Han et al., they use antibody-induced differentiation of stem cells to create microglia-like cells that traffic to the brain, localize to beta amyloid plaques and reduce their number.2
Immunohistochemistry-Paraffin: beta Amyloid Antibody (MOAB-2) [NBP2-13075] - IHC analysis of a formalin fixed paraffin embedded tissue section of human brain (Alzheimer's disease, hippocampus) using 1:40 dilution of anti-beta Amyloid antibody (clone MOAB-2). The staining was developed with HRP labeled anti-mouse secondary antibody and DAB reagent, and nuclei of cells were counter-stained with hematoxylin. This beta Amyloid antibody specifically stained the cells with Abeta 42/ Abeta aggregates while the normal cells were negative for abeta peptide.
In order to identify antibodies that induce microglia differentiation and migration, they use a schema that allows phenotypic selection in vivo. Namely, they created a phage library, transduced bone marrow, performed a bone marrow transplant, and then harvested brain tissue to isolate cells and identify any of the antibody genes that promoted trafficking of bone marrow cells to the brain. They identified the B1 gene as present in the cells that had migrated to the brain, and these cells expressed the microglial marker TMEM119. This implies that the B1 gene encodes an antibody that acts in an autocrine fashion to induce microglia-like cells, and they confirm that adding the exogenous antibody can also induce microglial phenotype and lead to expression of genes normally enriched in microglia (as opposed to other immune or neural cell types), such as CD11b/ITGAM, IBA1, TREM2, APOE, CD33, ITGB2, ADORA3, LGMN, PROS1, C1QA, GPR34, TGFBR1, SELPLG, HEXB, LTC4S, and CCL2. Next, immunoprecipitation and mass spectrometry identified vimentin as the target of the B1 antibody. They showed incubation with the B1 antibody led to vimentin phosphorylation and activation of downstream signaling cascades, ultimately leading to an M2-like phenotype and increased phagocytic clearance of beta amyloid plaques.
CD11b/Integrin alpha M was detected in immersion fixed paraffin-embedded sections of human brain (cerebral cortex) using Mouse Anti-Human/Equine CD11b/Integrin alpha M Monoclonal Antibody (Catalog # MAB16991). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (Catalog # CTS013). Tissue was stained using the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (red; Catalog # NL007) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm of microglia. Tissue was co-stained using a Sheep Anti-Human/Mouse/Rat Neurogranin Antigen Affinity-Purified Polyclonal Antibody (Catalog # AF7947) and an Alexa Fluor® 488-conjugated Donkey Anti-Sheep IgG Secondary Antibody (green).
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To identify effective antibodies, different selection criteria can be used depending on the context; migration of microglia-like cells from the bone marrow is one example, as in this case. However, other methods have been previously used, as exemplified by the selection of antibodies able to differentiate or transdifferentiate leukemic cells into natural killer-like cells. The phenotypic readout could alternatively be related to cell morphology or generation of a fluorescent reporter that reflects activation of a specific pathway.3,4 Overall, this innovative technique could be used in a variety of contexts to identify antibodies that induce specific cell-types which could replace defective cell populations or regenerate organ systems.
Jennifer Sokolowski, MD, PhD
University of Virginia, Department of Neurosurgery
Jennifer is doing a postdoc while completing her residency in Neurosurgery and has background in basic science, specifically neuroscience, cell death, and immunology, as well as background in medicine and translational and clinical research.
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