Macrophages Showing Markers of Cellular Senescence may not be Senescent Cells

Cellular senescence is one of the causes of aging: rising numbers of cells fall into a harmful senescent state and then linger there. The activities of these cells directly contribute to loss of tissue function and the progression of many age-related diseases. You might recall last year's investigations into possible cellular senescence in the immune system, focused on macrophages that exhibit some of the markers used to identify senescent cells. Does this mean that part of the macrophage population is in fact senescent in older people, and they would benefit from the removal of those cells, as is the case for other senescent cell types, or does it mean something else entirely, and these cells may not be harmful? In the open access paper here, the author's of last year's study suggest that the latter situation is the case, though whether or not these cells are damaging to an individual remains to be determined.

The markers in question here are p16 (also known as p16ink4a) and senescence-associated β-galactosidase (SAβG). If we approach this from the point of view of concern that treatments might be destroying cells unnecessarily, then SAβG isn't all that relevant, as I'm not aware of any group that actually targets that signal, versus only using it for assessment purposes. The companies that are developing pharmaceuticals to destroy senescent cells are not doing so in a way that specifically targets raised expression of these genes: drug development starts with a drug found in the compound libraries that is somewhat useful in killing the cells you want it to kill, and then you try to improve upon whatever it turns out to do under the hood. That mechanism doesn't have any necessary connection to the markers that the research community has developed to identify senescent cells.

On the other hand p16 is one of the targets used by Oisin Biotechnologies, and their gene therapy absolutely does recognize specific genes and and their expression levels, and selects cells for destruction on that basis. The Oisin team will in the course of their development find out one way or another whether or not removal of p16-expressing macrophages is useful. We might also recall that the earlier studies of mice genetically engineered to clear senescent cells used p16 as the identifying marker for cell destruction. Clearly the benefits there were achieved with a clean sweep of p16-expressing macrophages as well as other senescent cells, even if it can be argued that those macrophages are not senescent in the way we'd consider other cell types to be senescent.

The broader point raised in the paper here is that a refinement is needed in the current taxonomy of cellular senescence, especially in how it relates to markers that are coming to be understood as perhaps less specific than was originally hoped. That seems a fair enough comment on the current state of the research. I think that this desired progress will arrive, and fairly quickly now that senescent cells - as defined by various measures and markers - can be destroyed reliably and effectively. The size of the effects on health and life span in rodents obtained so far are large enough that future animal studies should fairly conclusively settle whether or not certain cell populations are bad and should be removed.

p16(Ink4a) and senescence-associated β-galactosidase can be induced in macrophages as part of a reversible response to physiological stimuli

The accumulation of p16Ink4a-positive cells is observed in aged mice, and their eradication has been linked to certain improvements in the health state of older animals consistent with rejuvenation. Even though p16Ink4a-positive cells in vivo have been assumed to be senescent, little evidence exists to directly support this assumption. Our previous work identifying macrophage subtypes that co-express markers conventionally assigned to senescent cells (SCs), p16Ink4a/SAβG, has prompted additional interpretations of previously published experimental data regarding the role of p16Ink4a-positive cells in aging and age-related diseases.

As such, defining the exact nature of p16Ink4a-positive cells is crucial for proper development of therapeutics for the prevention and treatment of aging and age-related diseases. Today, the field of aging is focused on the development of senolytic compounds that are isolated for their ability to selectively kill SCs generated in vitro. If these cells are different from p16Ink4a-positive cells accumulating in vivo with age, this could misdirect both academic studies of senescence as a phenomenon, as well as practical efforts to develop anti-aging therapeutics. These considerations motivated our present work, which was aimed at defining the nature of p16Ink4a-positive cells found in mouse tissues in vivo and their relation to the phenomenon of cellular senescence.

What is "cellular senescence"? Currently, all definitions agree that SCs cease to proliferate. However, this parameter is not sufficient to define SCs since this is also the property of terminally differentiated cells. One apparent difference is that terminal differentiation occurs in response to various physiological stimuli, while induction of senescence almost always occurs in response to genotoxic stress. Accordingly, the onset of senescence commonly involves p53, a major universal genotoxic stress response mechanism that triggers cell cycle arrest, the first step in conversion to senescence. Another intrinsic property of the senescent phenotype is that it is not reversible through known physiological stimuli, only occurring through the acquisition of genetic mutation or epigenetic modulations. Thus, a more precise definition of SCs should include those cells that irreversibly cease to proliferate following genotoxic stress. Currently, none of the other properties of SCs that are being used for their recognition, such as p16Ink4a- or SAβG-positivity, are sufficiently specific for SCs as to be essential components of this definition.

We previously demonstrated that a significant proportion of p16Ink4a/SAβG-positive cells in the fat tissue of older mice are of hematopoietic origin, express surface markers of macrophages and are capable of phagocytosis. Here, we demonstrate that these cells appear and accumulate independently of their p53 status. Furthermore, induction of p16Ink4a/SAβG markers can be significantly modulated (in both directions) by physiological stimuli known to polarize macrophages. In recent literature, a role for p16Ink4a has been implicated in macrophage physiology with no relation to other properties of senescence. For example, p16Ink4a expression is induced during monocyte differentiation into macrophages in vitro without affecting the cell cycle, and macrophages from p16Ink4a-deficient mice are skewed towards an M2 phenotype, exhibiting defects in M1 polarization response.

In summary, we conclude that a significant proportion of p16Ink4a/SAβG-positive cells accumulating in aging mice are macrophages that acquired this phenotype as part of their physiological reprogramming towards an M2-like phenotype. This interpretation is consistent with reports that tumor-associated macrophages (TAMs), which possess also an M2 phenotype, were shown to express p16Ink4a. It is highly unlikely that senolytic compounds isolated for their ability to eradicate bona fide SCs would be equally potent and selective against cells that simply resemble SCs by two unreliable biomarkers (p16Ink4a/SAβG) yet lack the most definitive properties of senescence. However, several molecules identified with anti-SC activities, including ruxolitinib, dasatinib, and quercetin, have documented anti-inflammatory effects on macrophages that may contribute to improvements in healthspan. We believe that the assumptions made in a series of recent works - that p16Ink4a/SAβG-positive cells are SCs - needs to be carefully re-evaluated, and that the effects of anti-SC therapies on macrophages needs to be evaluated.

Importantly, our results do not overthrow the significance of the SC's role in aging or disprove the rationale for the development of senolytic compounds. Nevertheless, they do question the accuracy of interpretation of the reasons for the improvement of the health of mice following the eradication of p16Ink4a-positive cells, raising the possibility that SCs may not be the only ones implicated in age-related frailty and that other players may be involved that could require different approaches to target.

Comments

The more information we get about cellular senescence the more it seems to point to a conclusion I've been trying to formulate for some time.

When it comes to stem cell therapies for instance it is clear the benefit being produced in a lot of those comes from the cells having a certain effect on the signaling environment. The transplanted stem cells don't linger for long but the changes last long after they have been removed by the immune system or programmed death.

Inversely - senescent cells shouldn't linger for long either and if not for apoptosis then at the least necrosis will get them sooner rather than later. And yet they do seem to accumulate with age - whether it is actually an accumulation or a slower rate of removal is an open question but one I believe is irrelevant to the point I'm trying to make.

Senescence - It all comes down to signaling. The amount of cells which exhibit classical "senescence" might be small but there is no cell in an aged body that communicates as it should, so you might as well consider any cell in a senior a senescent cell.
A terrifying idea if you expected a simple - Pill A deals with Damage/Hallmark A type of deal.
To me it seems like we'll need a complex combination therapy which makes use of the effects we get from stem cell transplants, parabiosis, senolytics and more.

And whenever we have it, it won't do much for any damage done to structures with limited regenerative potential like joints, muscles, the nervous system and so on. Those will require intervention beyond what is considered homeostasis.

My biggest concern as of now is a simple deduction that comes from the information as it is at present - for "senolytic" therapies to truly "work" they will need to be used early on in old adult life to be effective. Otherwise they will be a glorified anti-inflamatory aid with little to no effect on the outcome and trajectory of aging and lifespan. They might open up the door for further therapy in individuals who are too old but that is about all they will do on their own.

I've experienced some unintelligible screeching when I vocalized my observations recently. I can draw some parallels if I compare it to the reactions I've seen people putting way too much hope in telomerase therapies for instance.
It really makes me wonder if we'll ever be rid of the silver bullet mentality in "our" "community".

Posted by: Anonymoose at August 17th, 2017 12:23 AM

"My biggest concern as of now is a simple deduction that comes from the information as it is at present - for "senolytic" therapies to truly "work" they will need to be used early on in old adult life to be effective. Otherwise they will be a glorified anti-inflamatory aid with little to no effect on the outcome and trajectory of aging and lifespan. They might open up the door for further therapy in individuals who are too old but that is about all they will do on their own."

Didn't removing p16Ink4a cells in already aged mice (including their marcrophages) still have a positive effect, and led to an increase in lifespan vs controls? I remember it not removing some secondary damage such as cataracts.

Posted by: Jim at August 17th, 2017 1:48 AM

@Jim
Mice are not humans as it has been pointed out very often.
We don't share many age related pathologies.
As you pointed out yourself yesterday or the day before, stem cell therapies which work wonderfully in rats and mice do not in humans.

Posted by: Anonymoose at August 17th, 2017 3:04 AM

Cellular energy production (NAD+) declines markedly with age resulting in a whole host of aging related conditions. So I think it is necessary to amp up our NAD+ with aging to provide the necessary energy to allow our cells to function as they did in our youth. You can start a rejuvenation program by taking some nicotinamide riboside with resveratrol to restore SIR 1 activity. Then your body will have more available energy to repair DNA, restore full proteasome activity, keep youthful genes turned on, and detrimental genes of aging turned off. It all starts with having adequate NAD+ levels to Fight Aging.

Posted by: Biotechy at August 17th, 2017 10:13 AM

@Anonymoose - do you think you are being a bit overly pessimistic in this case? It can be fun to naysay and come and nay in the comments of biotech articles, but in the case of senescent cell removal things look pretty good. Being cautious and being a wet blanket are not the same thing.

Posted by: Jim at August 17th, 2017 10:54 AM

While there is clear evidence that telomere dynamics is very different in mice and humans, that cancer rate is much higher in mice, that mice don't develop AD, PD... senescent cells properties/effects/etc. don't seem to differ to a great extent in mice an humans, with current evidence, AFAIK. So I think pessimism based on analogy can't be applied here.

Posted by: Antonio at August 17th, 2017 11:58 AM

It is huge first step.
Idea that we will hopefully never have to experience senescent levels of 80 year old is very exciting even if it's not full rejuvenation.
Can't wait for AGE breakers, next huge step.

Posted by: RS at August 17th, 2017 1:27 PM

@Antonio
I wasn't being pessimistic on the contrary I'm being realistic.
The same reason mice are bad for studying telomere dynamics and cancer makes them bad for studying senescent cells as well.
Amusingly enough telomere elongation in mice did increase lifespan in one experiment if I remember it correctly, so really, there are very few things that do not extend the lifespan of mice, even things that shouldn't.

While we do not know why and how senescent cells accumulate with age it's hard to predict what a senolytic therapy for the whole body will do in a human. And while there is compelling data of senescent cells playing a role in a lot of degenerative diseases, they seem to be far from the root of the problem.

Posted by: Anonymoose at August 19th, 2017 3:54 PM

"there are very few things that do not extend the lifespan of mice, even things that shouldn't."

Nope, there are a lot of things that do not extend the lifespan of mice. You can find a lot of them in the NIA's Intervention Testing Program. Lend me a hammer and I will show you one more.

"The same reason mice are bad for studying telomere dynamics and cancer makes them bad for studying senescent cells as well."

As I said, telomere dynamics and cancer are very different in humans and mice, contrary to what happens to cell senescence. You can't disprove an argument by simply ignoring it.

Posted by: Antonio at August 19th, 2017 4:32 PM

@Antonio
Antonio, I'm not ignoring it.
I think you don't understand what I'm getting at. Mice have different telomere dynamics yes, and that is a part of the problem - it is quite possible to get a benefit in mice by using senolytics simply because they have this significant wiggle-way when it comes to the Hayflick limit and their significant cellular proliferation at all ages. Blast a bunch of their cells away from an earyl age? Doesn't matter, they can handle it. Us? I'm not so sure.

Another side of it is - studying disease and senescence in mice is not easy. We do not share a lot of diseases and the best we can do is building disease models based on inflicted injury. And we do know senescence plays a role in injury and healing. So that's another aspect of the research I roll my eyes at - does it actually help with osteoarthritis or does it simply help with the healing in the surgical procedure they use to emulate the condition in a mouse?

The lifespan data from Unity is significant, but the treatment was started early on when the mice were 12 months of age - it's hard to define how that correlates to human age but a lot of those mice reached 24 months (the last dying at around 36) and beyond so maybe ~40 or even bellow that, though you know as good as me that is not an exact estimate. Either way my point is - they started the treatments early.

There are many outstanding questions and even though optimism is not bad, it should always be cautious when it comes to biology. I prefer to stand on the side of realism and admit mice are bad for studying old age and age related pathology and to fully expect any treatment coming from mice experimentation to work differently in a heterogeneous human population.

Posted by: Anonymoose at August 19th, 2017 8:49 PM

"I think you don't understand what I'm getting at. Mice have different telomere dynamics yes, and that is a part of the problem - it is quite possible to get a benefit in mice by using senolytics simply because they have this significant wiggle-way when it comes to the Hayflick limit and their significant cellular proliferation at all ages."

1) As I said, again, cell senescence is similar in mice and humans. At least, much more similar than telomere dynamics and cancer. You don't need theory to guess it, it's experimental data.

2) What's the matter with the Hayflick limit? It's not a main cause of senescence on humans nor mice.

"So that's another aspect of the research I roll my eyes at - does it actually help with osteoarthritis or does it simply help with the healing in the surgical procedure they use to emulate the condition in a mouse?"

I assume they apply the senolytics after the wound is healed. Anyway, whether senolytics cure osteoarthritis or not, they produce a 25% LE in old mice, so they matter for aging. Also, we do know from experimental data in humans that senescent cells play an important role in human osteoarthritis.

12 months is not early for mice. Usual LE interventions for mice, like CR or gene therapy, are started at birth. Anyway, most probably, senolytics alone will not extend human lifespan too much, even if started early.

"There are many outstanding questions and even though optimism is not bad, it should always be cautious when it comes to biology. I prefer to stand on the side of realism"

I still think that that is not realism but ignoring the data. Mice are regularly used to develop cancer treatments, with some success rate. Senescence is more similar than cancer for mice and us, so the realistic guess is that senolytics will have a better success rate than oncology. Thinking that the translation will be very bad is the pessimistic way of thinking.

Posted by: Antonio at August 20th, 2017 1:06 AM

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