Local Flavor: At Protein Level, Too, Human Microglia Are Diverse

Just last month, gene expression profiling of individual mouse microglia showcased diverse phenotypes that reflected the cells’ multifaceted roles in development and immune surveillance. Efforts to do the same with human microglia have lagged, but that is changing. In the latest advance, researchers led by Chotima Böttcher and Josef Priller at Charité-Universitätsmedizin Berlin cataloged the state of dozens of protein markers in human microglia. They found success by combining a new method to isolate and preserve microglia from postmortem tissue with antibody detection and single-cell mass spec analysis. Their profiling locates multiple types of human microglia in different regions of the brain. It also defines a set of antibodies for identifying microglia subsets by standard flow cytometry methods. The work appeared December 17 in Nature Neuroscience.

Markers All Around. Protein expression profiles define four subtypes of microglia in human brain. Each line shows expression level in one of 36 independent brain samples; each color represents one of four subsets. [Courtesy of Böttcher et al., 2018, Nature Neuroscience.]

“This kind of technology is exactly what the field needs to move forward with the study of microglia,” said Oleg Butovsky, Brigham and Women’s Hospital, Boston. The results mainly confirm previous work, but the method the authors introduce for isolating microglia and analyzing protein expression is exciting, he said. “We need to be able to look at microglia in human brain at the proteomics level, and this study presents the most advanced technology today for analyzing protein expression in single cells,” he told Alzforum. “We will be seeing a lot more papers like this,” he said.

Much of what researchers know about microglia diversity, they discovered using RNA sequencing approaches on easily accessible mouse tissues. A few groups are tackling human microglia (see Jul 2017 news), but availability and quality of cells are a challenge for transcriptomics studies.

In the new work, first authors Böttcher, Stephan Schlickeiser, and Marjolein Sneeboer set out to develop a method for harvesting and preserving microglia from postmortem tissue, and analyzing them using not RNAseq, but antibodies, to construct a protein phenotype of human microglia. To get the most from the precious cells, the researchers used a variation of flow cytometry, employing antibodies tagged with stable isotopes to bind to microglial proteins. The bound antibodies were then quantitated by mass spectrometry. The assay allows multiplexing of antibodies, because each can be linked to a different stable isotope. With this technique, the investigators were able to measure binding of 35 different antibodies in one go, using as few as 1,000 cells. They isolated microglia from up to five different brain regions from nine donors, four men and five women, between the ages of 23 and 80, who had died of various causes. The researchers analyzed the brain cells in tandem with immune cells from the blood and cerebrospinal fluid of healthy people. To do that, each cell preparation was uniquely bar-coded using combinations of palladium isotopes; samples were then pooled for analysis to reduce batch-to-batch assay variation. After incubation with two antibody panels, covering 57 different proteins, the cells were individually vaporized and analyzed by mass spec.

The analysis identified a familiar phenotypic signature for human microglia, marked by expression of the purine receptor P2Y12 and TMEM119. P2Y12 was previously identified as a marker of homoeostatic microglia in mice and humans (Butovsky et al., 2014; Mildner et al., 2017), and its presence differentiated the microglia from monocytes and other immune cells in blood and CSF. The microglia also had higher expression of TREM2 than peripheral immune cells.

Within the core microglia phenotype, the investigators identified four subsets, which distributed in different patterns across five brain regions analyzed. One subset predominated in the subventricular zone and thalamus, and expressed more activation markers than microglia from other regions. Two other subtypes, residing mainly in the temporal and frontal lobes, uniquely expressed the mannose receptor CD206, better known as a marker for perivascular macrophages. The fourth, and rarest, subtype was detected in all five regions studied, including the cerebellum. The investigators could distinguish three of the four subsets by FACS using just four of the markers. They also noted increased expression of several markers in microglia from older brains, including CD11b, CD68, CD64, HLA-DR, and TREM2.

How faithfully did microglia from postmortem tissue reflect the living state? To answer that question, Böttcher and colleagues compared postmortem microglia with fresh cells isolated from surgical resection tissue from epilepsy patients, under conditions previously optimized to prevent gross changes in the cells’ phenotypes (see Jun 2017 news). The fresh and postmortem cells were largely similar, but not identical. Key differences included higher expression of P2Y12 and IRF8. However, the investigators don’t know if the changes arise postmortem, or as a result of epilepsy. In addition, Boettcher said, there may be additional differences in proteins that were not part of the selected panel.

“Our study underscores the validity of using postmortem human microglia for deep immune profiling,” Böttcher wrote to Alzforum. The results complement data obtained from single-cell RNA sequencing, she said, and prove the protein expression levels of mRNAs previously reported in microglia (Gosselin et al., 2017; Galatro et al., 2017). Because RNA and protein do not always match, it is important to comprehensively study cell phenotypes and functions at the protein level, she said.

Butovsky told Alzforum that even more powerful multiplexed antibody-based systems are coming down the pike, which will enable detection of hundreds of proteins in situ, without requiring cell isolation.

“This paper provides an important technical advance, as it defines a reliable and standardized procedure for the analysis of human microglia from postmortem brain tissues, providing high resolution and preventing batch effects,” said Marco Colonna, Washington University, St. Louis. “Importantly, the study defines cell surface molecules that enable us to dissect some regional heterogeneity of microglia in human brain. Once again, this result underscores the impact of microenvironment in the diversification of tissue resident cells,” he said.

What about changes in disease? Böttcher told Alzforum that the group is already using the technique to study microglia from people with Alzheimer’s disease and multiple sclerosis.

Featured Paper

Böttcher C, Schlickeiser S, Sneeboer MA, Kunkel D, Knop A, Paza E, Fidzinski P, Kraus L, Snijders GJ, Kahn RS, Schulz AR, Mei HE, NBB-Psy, Hol EM, Siegmund B, Glauben R, Spruth EJ, de Witte LD, Priller J. Human microglia regional heterogeneity and phenotypes determined by multiplexed single-cell mass cytometry. Nat Neurosci. 2019 Jan;22(1):78-90. Epub 2018 Dec 17 PubMed.

References

Butovsky O, Jedrychowski MP, Moore CS, Cialic R, Lanser AJ, Gabriely G, Koeglsperger T, Dake B, Wu PM, Doykan CE, Fanek Z, Liu L, Chen Z, Rothstein JD, Ransohoff RM, Gygi SP, Antel JP, Weiner HL. Identification of a unique TGF-β-dependent molecular and functional signature in microglia. Nat Neurosci. 2014 Jan;17(1):131-43. Epub 2013 Dec 8 PubMed.

Mildner A, Huang H, Radke J, Stenzel W, Priller J. P2Y12 receptor is expressed on human microglia under physiological conditions throughout development and is sensitive to neuroinflammatory diseases. Glia. 2017 Feb;65(2):375-387. Epub 2016 Nov 12 PubMed.

Gosselin D, Skola D, Coufal NG, Holtman IR, Schlachetzki JC, Sajti E, Jaeger BN, O’Connor C, Fitzpatrick C, Pasillas MP, Pena M, Adair A, Gonda DD, Levy ML, Ransohoff RM, Gage FH, Glass CK. An environment-dependent transcriptional network specifies human microglia identity. Science. 2017 Jun 23;356(6344) Epub 2017 May 25 PubMed.

Galatro TF, Holtman IR, Lerario AM, Vainchtein ID, Brouwer N, Sola PR, Veras MM, Pereira TF, Leite RE, Möller T, Wes PD, Sogayar MC, Laman JD, den Dunnen W, Pasqualucci CA, Oba-Shinjo SM, Boddeke EW, Marie SK, Eggen BJ. Transcriptomic analysis of purified human cortical microglia reveals age-associated changes. Nat Neurosci. 2017 Aug;20(8):1162-1171. Epub 2017 Jul 3 PubMed.


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