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

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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.




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MouseDoctor

5 comments

  • This article was submitted to a pre-print server in August. It made it through the reviewers and editors at Immunity in November. While the physical sciences have been far ahead of biological and medical sciences, this practice should be encouraged so that research can be disseminated more quickly.

    So it wasn't really published twice.

    http://www.sciencemag.org/news/2017/09/are-preprints-future-biology-survival-guide-scientists

    It's a good piece of work!

    • Nice to see that you understand the concept of Embargo. Maybe the Biological and Medical Sciences understand the concept of publication etiquette.

    • Why bother having papers reviewed you can just put them online and save yourself the hassle of peer review, which obviously changed the title.

  • I assume the comment above is not real otherwise they could could be in a mess if it was launched early.

    It is rather funny given that novelty is generally a reject feature of many biological papers including immunity I am sure. However, as most people can't afford a Hadron Collider for their physics stuff, no fear of been pipped to the post. I guess this is the same no-one is going to knock up 70,000 RNA seq in a few minutes without a wodge of cash.

    Also, why bother submitting to "Immunity" with your attitude you should have sent it to Faculty1000, online first and then reviewed no need to bother with a pre-print service.

    • The journal says
      "We will consider papers previously posted on preprint servers like arXiv, bioRxiv, BioRN, ChemRxiv, or ChemRN. We are very interested to hear from researchers about their motivations for posting and to discuss with them whether it is the best approach for their paper, so we strongly encourage authors who are planning to submit to us to contact the editors first to discuss their specific paper and situation".

      The paper was submitted on 15 June 2018

      It has a date in August for the pre-print service.

      The submission of revision was in September so presumably they informed the journal of their actions and it was accepted on 2 November 2018 and online a few weeks later.

      The journal says

      "This policy only applies to the original submitted version of the paper; we do not support posting of revisions that respond to editorial input and peer review or posting of the final published version to preprint servers. Also, our prepublication publicity policies with regard to coverage in the broader media still apply to studies posted on preprint servers. For more information about preprint servers and the discussions related to them, please see this blog post. http://crosstalk.cell.com/blog/what-were-hearing-from-the-community-about-preprints".

      I wonder what "Nature" thinks about the pre-print service?

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