Identifying macrophages in Rett Syndrome Mouse Models
I started my PhD in immunology at the Weizmann Institute of Science, Israel, way back in 2010, in the lab of Prof. Steffen Jung. At the time I was very enthusiastic on investigating a certain type of macrophages called microglia, which are the main immune cell population in the CNS. These cells were known since the 19th century but were (and still are) very tricky to study, and their research lacked genetic tools to isolate them, or knocking down specific microglial genes.
Prof. Jung had set up these exact tools, with mice (CX3CR1-CreERT2) that upon tamoxifen treatment were supposed to knock-down those elusive microglial genes. I crossed these mice to many floxed mice of choice, and started to look for in vivo phenotypes, mainly in a multiple sclerosis (MS) model. I tested, I think, maybe 10 different strains, for any difference in the MS phenotype. One mouse had no microglial TNF, the other had no microglial MHCII, and so on, and so on…
I have wasted months of work looking for a phenotype. I had found none. And this was very disappointing. What else could we do? What would come out of my PhD?? Am I wasting my life here, or what???
Since, at the time (circa 2012), the macrophage community had started to gain interest in Rett Syndrome, a devastating neurodevelopmental disorder that is triggered by mutations in the gene MECP2, we decided to cross the mice to MECP2 f/f mice, as well. It was, for me, yet another cross to do, and probably - yet another failed project.
It was well known that neuronal, and even astroglia-specific deletion of MECP2 can cause symptoms in mice which are very similar to those in humans (i.e. growth retardation, locomotor deficits, respiratory problems and premature death). But what about macrophages, and microglia? And so we crossed our mice to the MECP2 floxed mice and started to monitor them for symptoms, which should appear at around 6 weeks of age.
At first, again, there was nothing.
All my mice seem completely normal. No growth retardation- they grew just fine. No locomotor problems…. certainly, no premature death…same old story. Oh well, I thought, just another lousy project. But for some reason, out of utter despair I suppose, I kept some cages around.
Obese phenotype mice in cre crossed MECP2 mice
One day I came down to the animal facility, and looked at those neglected cages with the Cre mice crossed to MECP2 flox again. There was one specific cage with two mice: one was a control, the other was a mutant.
I could not believe my eyes.
These mice were 6 months old. The control mouse was your regular, everyday mouse. The other one was also normal in any way except that it was FAT. Obese, actually. As if it was fed the whole time with high fat or “western type” diet. Except that its diet had been regular chow. Then I weighted those mice. The first one was 25 gram, lean and slim. The other mouse weighed 45 grams!
From that day, a completely new journey started. I looked at more and more mice and it turned out that all my mutants had this obesity phenotype. Obviously, I was still thinking about microglia and the CNS. The mice are fat? Why, of course, they probably eat more! Because their feeding circuits are all screwed up, because of altered microglia!
But no, that was actually not the case. The mice ate the same as controls. And their feeding circuits were perfectly fine. No sign of leptin resistance…no changes in feeding related neuropeptides…and finally, no change in microglia transcriptomics. Is this some sort of a sick joke? I found, totally by accident, this incredible phenotype, but it is not mediated by microglia?? Who does mediate it, then?
Brown Fat Morphology
And then, we (myself, Prof. Jung, and Dr. Sigalit Boura-Halfon) found a clue. The mice, as it turned out, did not eat more, but they combusted less energy. They simply burn less calories. And at this point, 2014, I learnt that there is a specific tissue, of which I knew nothing about at the time, that can contribute to energy expenditure: brown fat. Indeed, these mice of mine had a defect in brown fat morphology and function. What was most striking was that these brown fat deficits appeared to occur before the obesity kicked in.
So, was it possible that my supposedly microglia-only Cre system also targets these brown fat macrophages? I had to switch gears to a completely unknown direction, and started to read and understand what is the brown fat tissue, how does it function and what cells it includes. I have learnt how to isolate the brown fat tissue, how to isolate the macrophages from it, and how to stain them for FACS. And indeed- they were targeted by my system! As well as those damned, no-good microglia…
OK, so what is known about brown fat macrophages? I opened up the literature and found almost nothing. Isn’t that nice, I thought! This is the ultimate immunologist’s dream: to find a new cell type that no one had ever characterized before. Now I was really thrilled- I had these cells to sink my teeth in. All aboard the brown fat macrophage train!
I used every tool I had in my box, everything I have learned in my PhD, for this endeavor. Using fate labeling we could see that these cells are long lived, that they are in very close proximity to sympathetic neurons which activate the brown fat and mediate the energy expenditure, and that the very cause of the brown fat deficit was reduced sympathetic innervation, caused by these MECP2-null macrophages.
This small change- reduced sympathetic input to the brown fat, due to the involvement of neighboring macrophages- becomes, with time, a gigantic snow ball, as secondary effects such as insulin resistance and ectopic fat accumulation in the liver later develop. Mice which become slightly fat, will eventually gain more and more fat because of these secondary phenotypes.
So we now had found the cells that can explain what happens to the mice. But since almost nobody talked about these cells, it was very hard to convince the community that these cells could have such vast influence. And so we submitted the manuscript in early 2016…
…We spent over a year in review, which was very exhaustive. At one point, I finished work in the lab of Prof. Jung, and moved to another lab at the Institute for a post doc. I had to split my time between the new lab, doing revision work for the MECP2 paper, and revision work for another publication from the Jung’s lab…the two labs were two floors apart at the same building, which helped of course. At some point I was not sure which lab I was in, and in which floor... But sometimes good things happen, and this work, for which I am so proud, finally was accepted for Nature Immunology and was published last year.
And so, out of sheer luck, and some mixture of both despair and determination, I was drawn to a completely uncharted territory in science, and we were the first ones to describe the brown fat macrophages in detail, and show their function. It was a hard a long journey, but the satisfaction is immense. I would like to think that I might have contributed a little to the understanding of macrophage biology…and if someone would have told me back in 2010 that I would publish a paper on brown fat macrophages, I would have laughed so hard and said: brown what?!?