All posts tagged mitochondria

ME/CFS Seahorse Energy Production Study Shows Surprises

Dr. Maureen Hanson leads one of the three NIH funded ME/CFS research centers, but her ME/CFS research doesn’t stop there.  Using samples from Dr. Daniel Peterson provided by the Simmaron Research Foundation, she’s also been assessing the metabolism of one of the most important cells in our immune systems: our T-cells.

T-cells affect a large part of our adaptive immune response that clears out infections. They do this by regulating our immune response (CD-4 or Helper T-cells) and/or by killing off pathogens that have infected other cells (CD-8 or cytotoxic T-cells).

Hanson and Mandarano and Seahorse machine

Maureen Hanson, Alexandra Mandarano and the Seahorse machine

Prior to getting activated, T-cells are primarily on sentry duty.  Once activated by dendritic cells presenting little bits of pathogens to them things change dramatically, however. The T-cells rev up their cellular engines to order to start pumping out cytokines or clones en masse in order to stop the infection. Both parts of energy production – glycolysis and oxidative phosphorylation –  have to jump into action.

In short, assessing the energy production of activated T-cells is a perfect way to determine if their energy metabolism has been affected in ME/CFS – and that’s just what Maureen Hanson’s group did.

Alexandra Mandarano, a graduate student in Hanson’s lab, took T-cells from 53 healthy controls and 45 pretty long duration (avg. a@ 12.7 years) ME/CFS patients and healthy controls and tricked them into going into high alert with antibodies plus IL-2. Then, using the Seahorse Flux Analyzer, she examined how well the two parts of their cellular energy production system did in both unactivated and activated T-cells:  glycolysis – the anaerobic part which takes place outside the mitochondria, and oxidative phosphorylation – the aerobic part which takes place inside the mitochondria (and produces far more ATP) .

Dr. Hanson presented on her results at the recent Open Medicine Foundation sponsored Harvard Symposium

Results

Whether they were activated or not, mitochondrial energy production; i.e. oxidative phosphorylation (the main ATP producer) was normal for both the CD4 and CD8 cells in the ME/CFS group. When pushed, the mitochondria in the ME/CFS patients’ cells quickly got energy production up to speed. That was a surprise. Usually when you push a cell or system in ME/CFS it fails- but, in this case, the T-cells responded normally.

Then came the real surprise.  Systems in ME/CFS often test out fine or at least not strongly abnormal at baseline or rest, but in this case Hanson found low glycolysis activity in both the T-helper cells (CD4) and the CD8 cells at baseline.  Simply prowling around the body, they had considerably lower levels of glycolytic activity.  When pushed, though, their glycolytic activity was normal.  The pattern was opposite to what we usually see.

That wasn’t all. It’s possible with the Seahorse to turn off different energy production pathways in order to assess how effectively the other pathways are at compensating.  When the oxidative phosphorylation pathways were turned off experimentally, the ME/CFS patients’ glycolytic pathways failed to compensate as effectively as did those of healthy controls.

Mandarano did not find problems with mitochondrial ATP production but did find issues with glycolysis

Thus no problems with mitochondrial energy production were found but three potential issues with glycolysis popped up: low glycolytic activity in both forms of unactivated T-cells, and poor glycolytic compensation with the oxidative phosphorylation pathways were turned off.

Hanson’s group next examined a critical part of energy production called the mitochondrial membrane potential. Our mitochondria need to maintain a certain membrane potential to keep up the flow of positively charged ions into the mitochondria. It does this by keeping more positively charged ions outside of the mitochondria and more negatively charged ions inside the mitochondria. Her group used a flow cytometer to assess the levels of mitochondria present and to determine how strong the membrane potential was.

The mass and membrane potential of the ME/CFS patients’ CD4 T-cells and the mitochondrial mass of the CD8 cells was normal, but the membrane potential of the CD8 T-cells – whether activated or not – were significantly impaired in the ME/CFS patients.

Four potential problems, then, were found:

  • low glycolytic activity in unactivated CD4 and CD8 T-cells
  • poor glycolytic compensation when the oxidative phosphorylation pathways were turned off
  • The mitochondrial membrane potential was impaired in the CD8 T-cells

Dr. Hanson left her presentation with the  encouraging statement that we are starting to put the pieces of the puzzle together in ME/CFS and the tantalizing suggestion that ME/CFS might be something different than what we think it is right now; i.e. keep an open mind, don’t put all your eggs in one basket, and be prepared for surprises.

Overview

Hanson and her co-authors have submitted a paper and we will get more details when their paper is published but, with these preliminary results, we have a few more data points on cellular energy production in ME/CFS. While noting that several study results are pending, maybe it’s time for a look at what we have.

It should be noted that measuring energy production is very complex. Different researchers are doing it in different ways, and I am no judge of any of them.  Researchers are using different instruments, different criteria, different kinds and numbers of patients, and they are reporting things differently. Solving those problems is one of the reasons for the NIH funded ME/CFS research centers where larger studies can use proven technologies and rigorously defined patient populations.

Check out some of the different protocols which have assessed mitochondrial functioning in isolation from the blood in ME/CFS:

Study protocols

  • Hanson’s group activated her T-cells using antibodies and IL-2 and then tested activated and unactivated cells in the Seahorse Machine
  • Tomas took PBMC’s and stressed them in the Seahorse machine
  • Stanford took PBMC’s and then used laboratory assays to test each of the complexes and flow cytometry to assess mitochondrial membrane potential
  • Fisher (unpublished) appears to have taken PBMC’s and stressed them in the Seahorse machine
  • Vermeulen measured ATP PBMC’s etc. in the lab
  • Smits measured ATP production rate in muscle biopsies

The Land of Mixed Signals

We seem to find ourselves in a familiar place – the land of mixed signals! One encouraging unmixed signal is that everyone seems to be finding something wrong – just often different things.

MITOCHONDRIA

Mitochondria Mass – Normal

  • Hanson – CD4 and CD8 (T-helper cells)
  • Fisher

Mitochondria ATP production – Normal

  • Hanson (T-cells)
  • Stanford study (not a Ron Davis study) (PBMC’s)
  • Fisher (PBMC’s)
  • Vermoulen (PBMC’s)
  • Smits (muscle biopsy)

Increased ATP Production Overall

  • Stanford
  • Preliminary results from NIH Intramural study

Reduced ATP production

  • Tomas – under both low and high glucose conditions

Functioning of Complexes – Normal

  • Stanford (I-IV)
  • Vermeulen (I-II)

Functioning of Complex V – reduced

  • Fisher
GLYCOLYSIS

Increased Glycolysis at Baseline (PBMC’s)

  • Stanford

Reduced Glycolysis at Baseline (T-cells)

  • Hanson

Reduced Glycolysis (low glucose conditions)

  • Tomas

Reduced Compensatory Glycolysis

  • Hanson
  • Tomas (?)

Glycolysis Stress Test (Glycolysis, glycolytic reserve, glycolytic capacity)- normal

  • Tomas
  • Fisher

It’s quite a muddle.  Surprisingly, though, the most consistent finding thus far is normal (or in two cases) increased mitochondrial production (!) Not many studies have directly measured glycolysis, but in these early days the results are mixed.

Isolation

cells in the blood

The most consistent result so far is normal (or increased) mitochondrial function but none of the above studies tested cells in the blood – where an inhibiting factor may lurk. (Seahorse machine cannot test cells in the blood.)

Note that all these studies are assessing the energy production of the mitochondria in isolation. None tested cells in the blood where Davis, Fluge and Mella and Prushty have found evidence that some sort of inhibiting factor may be present. The metabolomic findings which suggest problems with glycolysis are present have been assessing factors in the blood and urine as well.

Adding an exercise stress test would, of course, add another important factor. At the NIH ME/CFS Conference, Brian Walitt reported that the NIH is finding that exercise causes mitochondrial oxygen consumption (ATP production) to increase in the healthy controls but to decrease in about half of the ME/CFS patients. Several recent studies have validated that exercise impairs energy production in ME/CFS (blog coming up). Where and how the energy depletions are occurring is unclear. (Note that most of these studies examined immune cells not muscle cells.)

We obviously have long way to go to fit all the different pieces of the energy production puzzle in ME/CFS together but the good news is that an increasing amount of research is now being aimed at deciphering what’s inhibiting energy production in this disease.

The Simmaron Research Foundation’s collaboration with Maureen Hanson – which paired rigorously diagnosed patients with a respected researcher –  is just one way the Foundation is contributing to solving that puzzle.

Simmaron Patient Day Part II: The Hanson Report

Maureen Hanson has been making waves.  An ace molecular plant biologist prior to entering the chronic fatigue syndrome (ME/CFS) field, Hanson has worked on mitochondrial and gene studies in plants dating back decades. Now, with her son ill from ME/CFS, she’s turned her talents to this field, and has made a difference in a hurry.

A trusted researcher, Hanson scored one of the few XMRV grants and in a short period of time has produced studies on the gut, mitochondrial DNA, exercise, and metabolomics in ME/CFS. Last year, Hanson, created one the few chronic fatigue syndrome (ME/CFS) research centers in the U.S. (the Cornell Center for Ennervating NeuroImmune Disease, ) and this year she and her colleagues received one of the three NIH ME/CFS Research Center grants. She’s also a member of the Simmaron Research Foundation’s Scientific Advisory Board.

Last year Hanson was awarded a smaller NIH grant (R21) to do preliminary work assessing the energy production in ME/CFS patients’ immune cells using the Seahorse XF Analyzer.  In this blog we take a closer look at the work underway.

A Breakthrough Technology

It’s safe to say that the Seahorse machine is changing how researchers do research. In the mid-2000s the Seahorse folks introduced something new to the medical world called “extracellular flux (XF)” technology. A monolayer of cells is placed in a very small, 10 ml sensor chamber and then stimulated.  Every few seconds a sensor placed 200 microns above the cell monolayer takes a measurement.  Where past techniques required hours to assess oxygen metabolism, the Seahorse can do it in minutes.

This technology allows researchers to determine the energy consumption of cells by analyzing changes in oxygen and acid levels occurring in the media outside of them.  The amount of oxygen present indicates how much energy is being produced through glycolysis and by the mitochondria.

The ability to place energy stimulating or inhibiting or other drugs in the sensor chamber brings the possibilities of the Seahorse machine to an entirely new level. If the inability to produce energy turns out to be a key factor in ME/CFS, the Seahorse machine’s ability to test how drugs and other substances effect the energy production of cells could be a big boon indeed

Agilent, the company that produces the Seahorse machine, reports the machine has been used in over 250 studies. HIV researchers, for instance, recently used the machine to determine the effectiveness of the immune response in HIV patients. It turns out that in order to meet a threat, many of our immune cells undergo a huge metabolic shift as they get transformed from a resting to an active state. That shift coincides with large increases in glycolysis in particular.

A similar approach is being used in chronic fatigue syndrome (ME/CFS). Tomas’ recent Seahorse study suggested that ME/CFS patients’ immune cells (PBMC’s (T, B and NK cells, monocytes))are having severe problems producing energy. Tomas’ study opened up an important possibility but it was limited by its inability to determine which cells were having problems.

https://www.healthrising.org/blog/2017/11/11/cellular-energy-hit-chronic-fatigue-study/

Hanson is taking the next step in assessing immune functioning in ME/CFS with her R21 NIH grant. That grant gave her the funds to assess the energy production of individual immune cells separately (T, B and NK cells).  (Isabel Barao is also examining energy production in NK cells).

Each of these cell types has been potentially implicated in ME/CFS. The T-cell problems Derya Unutmaz of Jackson Labs saw are what attracted him to ME/CFS, and Mark Davis of Stanford recently found signs of unusual clonal expansion in ME/CFS patients’ T-cells.  The success some ME/CFS patients have with Rituximab suggests B-cell issues are present, and the problems ME/CFS patients’ natural killer cells have with killing have been known for decades.

T-cells are a particularly good subject because springing into action to kill other cells or to produce clones of themselves to fight invaders requires enormous amounts of energy. If energy production is flawed in ME/CFS, it’s probably going to show up in patients’ immune cells.

Glycolysis OK

Metabolomic studies suggest glycolysis might be inhibited in ME/CFS, but at the OMF’s Stanford Symposium Hanson stated that she hasn’t found impaired glycolysis. When glucose was given to the immune cells to stimulate their glycolytic processes, the cells were able to use it, but their respiratory capacity (oxidative phosphorylation) was blunted.

In another study, which the SMCI helped to fund, Hanson’s Metabolon metabolomics study found lower glucose levels (a surprise) as well as differences in fat and lipid metabolism (i.e. energy production), and in the sphingolipids that play a big role in Naviaux’s findings.

Hanson noted that low glucose levels are not a good sign, either. Low glucose levels have been associated with increased cortisol responses (possibly leading to exhaustion) and inflammation. Plus they may be able to mess with a person’s endurance.

The last sport anyone with ME/CFS is going to engage in is an endurance race.  That might make sense given that athletes with lower glucose levels tend to do worse in endurance sports. Overall Hanson’s metabolite findings suggest increased inflammation and reduced recovery from metabolic stress are part and parcel of ME/CFS. Metabolic stress, of course, is exactly what she’s measuring in her Seahorse study.

Hanson’s finding of normal glycolysis in ME/CFS patients’ T-cells mirrors the findings of Tomas’ recent Seahorse study. However, Hanson’s early findings are suggesting that, at least in the immune cells, the mitochondria are the issue.

Hanson has found that ME/CFS patients’ T-cells use less of their “respiratory capacity” when provoked than do healthy controls’ cells. If I’m reading this right, the capacity to produce energy is there, but it’s not being used.  The next step is to determine if the T-cells, when they become activated, can produce enough energy to be effective. If Dr. Hanson finds they’re not up to the task of producing adequate energy, she said, “they may also be unable to effectively respond to an immune challenge.”

Lethargic T-cells could have major implications for the immune system, as T-cells are important in just about every immune system activity. At least four different kinds of T-cells exist:  T-helper cells activate B and NK cells, T-killer cells destroy virally infected and cancerous cells, T-memory cells alert the immune system to danger, and T-regulatory cells help keep the immune system humming.  Small studies suggest that cytotoxic or killer T-cells have the same problems with killing infected cells that NK cells do.

Whether or not something in ME/CFS patients’ blood is essentially putting their cells to sleep is one of the more fascinating questions facing this field. Several researchers including Ron Davis of the Open Medicine Foundation and Fluge and Mella in Norway believe something in the blood is doing just that. Energy production issues in ME/CFS patients’ cells that have been isolated from the blood suggest that something may be wrong with the cells themselves. It’s possible, therefore, that problems may lie in both the blood and the cells.

Since the Seahorse machine allows researchers to insert different substances in the medium the cells are bathed in, I asked Dr. Hanson if she could use the machine to determine the effects ME/CFS patients’ blood may be having on their immune cells.

Dr. Hanson replied that the Seahorse machine could determine if something in ME/CFS patients’ serum affects mitochondrial function in immune cells from healthy people, but the Seahorse technology would not be able to tease out what factors in the serum are responsible.

The Seahorse requires large samples of difficult to obtain immune cells. T-cells are relatively easy to obtain; B and NK cells – not so much.  Getting Maureen Hanson the resources she needs to do her work is where the Simmaron Research Foundation comes in: it’s supplying the cells she needs to do her work.  Dr. Hanson stated that, “We are grateful to Simmaron Research for supporting the collection of additional samples from which individual cell types— such as B and NK cells—can be purified for analysis of glycolysis and oxidative phosphorylation”.

Next up, Dr Hanson will analyze the cellular energetics of those NK and B cells. Despite the Rituximab failure, B-cells are still of great interest in chronic fatigue syndrome (ME/CFS). It’s still possible, for instance, that the drug works for a significant subset of patients. Plus B-cells are heavily involved in autoimmunity. Dr. Light has proposed that energy depleted B-cells may increase the risk of an autoimmune process beginning.

The desire to examine NK cells is obvious. Reduced NK cell cytotoxicity is a hallmark of ME/CFS, and reduced cytotoxicity of T-cells appears to be present as well. Could that poor killing power be caused by the most basic of all problems – the inability to generate enough energy? Given the high energy requirements of activated immune cells, that’s a distinct possibility. Dr. Hanson’s work will take us closer than any other yet to answering that most fundamental of all questions.