The complexity of the Microglia Uncovered..Nine different states...so good, it was published twice.

I seldom read a paper and think wow.....This study from Harvard reveals the signature of microglia as mice age and they look at over 76,000 individual cells and get their whole transcriptomes........M1 and M2 are out of the window here comes M1-M9




Hammond TR, Dufort C, Dissing-Olesen L, Giera S, Young A, Wysoker A, Walker AJ, Gergits F, Segel M, Nemesh J, Marsh SE, Saunders A, Macosko E, Ginhoux F, Chen J, Franklin RJM, Piao X, McCarroll SA, Stevens B.Single-Cell RNA Sequencing of Microglia throughout the Mouse Lifespan and in the Injured Brain Reveals Complex Cell-State Changes. Immunity. 2018 Nov 21. pii: S1074-7613(18)30485-0. doi: 10.1016/j.immuni.2018.11.004. [Epub ahead of print]

Microglia, the resident immune cells of the brain, rapidly change states in response to their environment, but we lack molecular and functional signatures of different microglial populations. Here, we analyzed the RNA expression patterns of more than 76,000 individual microglia in mice during development, in old age, and after brain injury. Our analysis uncovered at least nine transcriptionally distinct microglial states, which expressed unique sets of genes and were localized in the brain using specific markers. The greatest microglial heterogeneity was found at young ages; however, several states-including chemokine-enriched inflammatory microglia-persisted throughout the lifespan or increased in the aged brain. Multiple reactive microglial subtypes were also found following demyelinating injury in mice, at least one of which was also found in human multiple sclerosis lesions. These distinct microglia signatures can be used to better understand microglia function and to identify and manipulate specific subpopulations in health and disease.

This was so good you may just publish it twice (read it here)

Complex cell-state changes revealed by single cell RNA sequencing of 76,149 microglia throughout the mouse lifespan and in the injured brain. Timothy R Hammond, Connor Dufort, Lasse Dissing-Olesen, Stefanie Giera, Adam Young, Alec Wysoker, Alec J Walker, Michael Segel, James Nemesh, Arpiar Saunders, Evan Macosko, Robin JM Franklin, Xianhua Piao, Steve McCarroll, Beth Stevensdoi: https://doi.org/10.1101/406140

Microglia, the resident immune cells of the brain, rapidly change states in response to their environment, but we lack molecular and functional signatures of different microglial populations. In this study, we analyzed the RNA expression patterns of more than 76,000 individual microglia during development, old age and after brain injury. Analysis uncovered at least nine transcriptionally distinct microglial states, which expressed unique sets of genes and were localized in the brain using specific markers. The greatest microglial heterogeneity was found at young ages; however, several states including chemokine-enriched inflammatory microglia persisted throughout the lifespan or increased in the aged brain. Multiple reactive microglial subtypes were also found following demyelinating injury in mice, at least one of which was also found in human MS lesions. These unique microglia signatures can be used to better understand microglia function and to identify and manipulate specific subpopulations in health and disease.

Microglia are the resident macrophages of the brain, comprising 10% of brain cells. Not only are microglia active in injury and disease, but they also play critical roles in brain maintenance and development. 

In early development, microglia assume a variety of different morphologies and are distributed unevenly in the brain (Karperien et al., 2013). They congregate in specific areas, including the ventricular zone and around growing axon tracts, and not in other areas, like the developing cortex (Squarzoni et al., 2014), suggesting that transcriptionally and functionally different subpopulations of microglia exist. 

Clustering analysis revealed nine unique microglial states across all ages and conditions, including injury . Cluster sizes ranged from 0.2% of all microglia to as many as 24% of all microglia

They found the greatest microglial diversity at the youngest ages (E14.5 (embryonic day 14.5. A mouse is born on E20-E21and P5 (5 days after birth) and considerably less diversity in juveniles (P30 a month old. Mice mature at 5-6 weeks) and adults (P100 about 3 moths old)

Gene expression analysis showed that certain genes (Fcrls, P2ry12, Cx3cr1, Trem2, and C1qa) were highly expressed by most of the analyzed cells, but interestingly, only three (C1qa, Fcrls, Trem2) were uniformly expressed in all clusters (Fig 1e), suggesting existing tools and marker definitions need to be updated. 

Many microglial marker definitions were previously established in adult animals and they found that P2ry12, Cx3cr1 (often used in conditional knockouts), and Tmem119 were expressed at much lower levels or not at all in certain clusters of microglia from the developing brain. 

Gene expression patterns show that each microglial state reflects a specific and definable transcriptional program, rather than a simple modulation of commonly expressed microglial genes.

In aged mice, the most enriched subpopulation was defined by the gene chemokine (C-C motif) ligand 4 (Ccl4, 

Non-microglial macrophages and monocytes uniquely expressed the genes coagulation factor XIII, A1 subunit (F13a1, macrophage), histocompatibility 2, class II antigen A, alpha (H2-Aa, macrophage), chemokine (C-C motif) receptor 2 (Ccr2, monocyte), lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1, macrophage), and macrophage galactose N-acetylgalactosamine specific lectin 2 (Mgl2, macrophage), genes that were barely expressed, if at all, by microglia

Four microglia clusters were identified as being enriched in aging mice

They found that Injury Cluster–specific genes were variably upregulated among the microglia , suggesting the existence of subpopulations within the cluster. To delineate these genes, they created three categories: broadly responsive genes that were upregulated in greater than 60% of Injury Cluster 2 microglia, responsive genes that were upregulated in 30–60%, and selectively responsive genes that were upregulated in less than 30%. Broadly responsive genes included apolipoprotein E (Apoe), Ifi27l2a, and the major histocompatibility complex II (MHC-II) genes H2-Aa and H2-K1 (Fig 6d). Responsive genes included several interferon response genes (Irf7, Oasl2, and Ifit3), Ccl3, and lipoprotein lipase (Lpl) (Fig 6d). Selectively responsive genes included AXL receptor tyrosine kinase (Axl), Ccl4, chemokine (C-X-C motif) ligand 10 (Cxcl10), and Birc5 (Fig 6d). 

They showed these microglial subsets in MS.

I am sorry I don't have the time to fully explore this paper. Maybe when it is printed, it will have an editorial. We have seen how M1 become M2 as cell subsets are not static, etc. This shows that the simplistic one-two subsets of cells is far off the biology.

The sad thing about this however, is who is going to repeat this because the cost of this study must have been phenomenal.