What Relevance does Myelin and Lymphocyte Protein have to ETX and MS?
Myelin and Lymphocyte Protein
We first need to introduce a protein called myelin and lymphocyte protein (MAL) as there is increasing evidence indicates that it is a key receptor for ETX i.e. a main binding site.MAL-slide5
In BBB cells and oligodendrocytes, MAL sorts apically associated membrane proteins to the surface of a cell shuttling between the Trans-Golgi-Network (TGN) apparatus in cells and the plasma membrane. It is therefore acts as a “transporter” protein in certain cells – much like an “Uber taxi”.
MAL is known to be expressed in various tissues in wide range of species from mice to dogs to sheep to humans as well as zebrafish. MAL, however, is not homogeneous across or within a species and different species have different forms and different “aromatic amino acid residues”, which make MAL more or less reactive to other proteins, such as ETX. For instance, it is known that rat and mouse MAL is approximately 100 times more sensitive to ETX than human MAL and zebrafish MAL does not bind at all (Rumah Thesis). In addition, within humans, there are four isomers of MAL i.e. four slightly different versions of the same protein.
MAL and Zebrafish
It is known that ETX is not toxic towards zebrafish MAL. As zebrafish are translucent, it is possible to see movement across membranes…..
There is an interesting paper published by D Adler “Clostridium perfringens Epsilon Toxin Compromises the Blood-Brain Barrier in a Humanized Zebrafish Model” (45) and (41), which found: “When zebrafish endothelial cells (ECs) are engineered to express human MAL (hMAL), proETX binding occurs in a time-dependent manner. Injection of activated toxin in hMAL zebrafish initiates BBB leakage, hMAL downregulation, blood vessel stenosis, perivascular edema, and blood stasis.” This provides strong evidence of MAL being required to breach the BBB.
MAL and Human Red Blood Cells
It is known that ETX haemolyses human erythrocytes (red blood cells (RBCs)) but not RBCs in mice, rabbits, sheep or monkeys from a paper by J Gao: “Hemolysis in human erythrocytes by Clostridium perfringens epsilon toxin requires activation of P2 receptors” (38). During active MS, numerous reports suggest that circulating RBCs are larger than normal and fragment more easily.
“In this study, the hemolytic ability of ETX was examined using erythrocytes from 10 species including murine, rabbit, sheep, monkey and human. We found that ETX caused hemolysis in human erythrocytes (HC50 = 0.2 μM) but not erythrocytes from the other test species. ” This is a very interesting finding.
Some very recent research in a paper by K Rumah; “Human blood exposure to Clostridium perfringens epsilon toxin may shed light on erythrocyte fragility during active multiple sclerosis” (53) has further confirmed the links between MAL, RBCs and ETX.
In essence, K Rumah has found that an isomer of MAL is expressed in human RBCs only – not sheep, goat, guinea pig, monkeys (rhesus macaques) or cows. He has also identified that in humans the impact of ETX is enhanced by the presence of CD4s expressing MAL (a different isomer which is more reactive than the MAL in RBCs).
It is also well-known that MS patients frequently experience “fatigue”. Now that research has been published, which links expression of MAL to RBCs, then this could explain the cause of the fatigue.
In other words, humans are “luckier” than other animals in that their RBCs and CD4s react with the toxin before it gets to the more sensitive areas such as the BBB or brain. It is also known that human MAL is much less reactive to ETX than sheep or mouse MAL. These factors may help to explain why humans are able to tolerate ETX exposure better than sheep, mice or rats.
Therefore it is hypothesised that the effect of ETX toxicity is significantly reduced in humans compared with sheep, mice and rats because the RBCs act as a buffer or “sponge” to mop up a significant proportion of the toxin and thereby reduce the impact of the toxin on the human brain compared to that inflicted on the brains of mice.
MAL and Lymphocytes
In sheep, mice, rats and humans it is well known that MAL is expressed in both BBB and oligodendrocytes.
However, in humans, but significantly not in mice or rats, MAL is also known to be expressed in a large number of lymphocytes, which are a subset of white blood cells (leukocytes) and the human immune system. MA Alonso, who discovered MAL, also identified that approximately 50% of CD4+ and 25% of CD8+ human cells also express MAL. Alonso (15) also concluded that MAL again acts as a transporter of a protein, Lck, that is responsible for the T helper cell’s activation, to specialized membranes in the cells, which suggests that it is in contact with the cell membrane. It is not yet known exactly which human lymphocytes express MAL and identifying this information would make a useful future study.
Why is MAL expression in human lymphocytes so significant?
We first need to digress again before addressing this question. The two principal constituents of the human adaptive immune system are special types of leukocytes, called B and T lymphocytes. Both major types of lymphocyte are initially derived from hematopoietic stem cells in the bone marrow. B cell lymphocytes mature in Bone marrow (hence they are termed B cells) and T cell lymphocytes mature in the Thymus (hence T cells). There are a number of sub-groups of T cell lymphocytes, such as killer T cells, cytotoxic T cells, T helper cells and T regulatory cells (Tregs). It is known that B cells do not express MAL but a large percentage of T cells do and further research is needed to identify exactly which T cells express MAL, as it is likely that they will be targeted by ETX (39).
T helper cells (also known as T effector cells) not only help activate B cells to secrete antibodies and macrophages to destroy ingested microbes but they also help activate cytotoxic T cells to kill infected target cells. The B cells identify pathogens and present fragments (antigens) to T helper cells, which release lymphokines and activate the B cells. Once activated, the B cell divides, its offspring (plasma cells) secrete millions of copies of the antibody that recognizes this antigen. These antibodies circulate in blood plasma and lymph and bind to pathogens expressing the antigen and mark them for destruction by complement activation or for uptake and destruction by phagocytes. Antibodies can also neutralize challenges directly, by binding to bacterial toxins or by interfering with the receptors that viruses and bacteria use to infect cells.
When B cells and T cells are activated and begin to replicate, some of their offspring become long-lived memory cells. Throughout the lifetime of an animal, these memory cells remember each specific pathogen encountered and can mount a strong response if the pathogen is detected again. This is “adaptive” because it occurs during the lifetime of an individual as an adaptation to infection with that pathogen and prepares the immune system for future challenges. Immunological memory can be in the form of either passive short-term memory or active long-term memory. T helper cells are therefore important cells in adaptive immunity, as they are required for almost all adaptive immune responses.
T reg cells (also known as suppressor T cells) on the other hand are crucial for the maintenance of immunological tolerance (9). Treg cells comprise about 5-10% of CD4+ cells. Their major role is to shut down T cell-mediated immunity toward the end of an immune reaction and to suppress auto-reactive T cells that escaped the process of negative selection in the thymus.
T cells and Multiple Sclerosis
Even in 1981, it was known that MS initiated a reduction in T cell numbers and that also that lymphocytes are enlarged and abnormal-looking. It is known that many MS patients experience flu like symptoms before an episode and it is speculated that these symptoms may be caused by ETX damage to MAL-producing lymphocytes. According to the MS Society’s website (48): “Over the last 15 years, much knowledge has been gained about the specific roles of T cells. Among the activities that have been observed are:
• Decreased regulatory T cell function in the peripheral blood of MS patients during an acute exacerbation (also known as an attack, relapse or flare);
• Increased numbers of helper T cells in the cerebral spinal fluid (CSF);
• Increased numbers of activated T cells passing into the brain from peripheral blood (which then attract other immune cells into the brain);
• Presence of T cells in MS plaques (sclerosis); and
• Increased frequency of activated T cells against the myelin seen in MS patients compared to healthy controls.”
• More recently it has been shown that patients with MS show loss of functional suppression by CD4+CD25+ T cells (i.e. Tregs cells are damaged) (6).
In essence, if ETX attacks human lymphocytes, which express MAL, then it is highly logical and plausible that this process is disrupting the proper functioning of the immune system. If it is the Treg cells and/or the T helper cells, which are damaged by ETX, this could be the cause of the autoimmune features of MS especially since Treg cells have a key role in modulating the immune system, maintaining tolerance to self-antigens, and preventing autoimmune disease.
It is further speculated that, if ETX is the trigger, it may be that initially during an MS attack not only are the human lymphocytes expressing MAL damaged by ETX but, at the same time, these lymphocytes may act as a buffer or “sponge” to mop up a proportion of the toxin and thereby reduce the impact of the toxin on the human brain compared to that inflicted on the brains of mice. This is clearly a line of investigation to be followed up by further research.
Any damage to T reg cells or T helper cells from ETX could be the cause of the memory B cells not being switched off properly and for the long-term persistence of “faulty” memory B cells.
It is therefore hypothesised that future research will identify that it is the CD4+CD25+ Treg lymphocytes (which are a subset of CD4+ lymphocytes) and the T helper cells which express MAL. Further, it may be the case that the “auto-immune” features of MS are in some way exacerbated by damage to the T cells expressing MAL.
Role of B cells in Multiple Sclerosis
Therefore, it was interesting to find that current MS treatments, believed to act via T cell inhibition, also depleted memory B cells (24). It is also known that T cell enhancement strategies can make the symptoms of MS worse. So perhaps it is the B cell depletion strategies that are successful as they knockout sufficient numbers of rogue memory B cells.
In an article by Baker et al (31) (2017) the abstract details the following drugs: “beta-interferons, glatiramer acetate, cytostatic agents, dimethyl fumarate, fingolimod, cladribine, daclizumab, rituximab/ocrelizumab physically, or functionally in the case of natalizumab, also depleted CD19+, CD27+ memory B cells. This depletion was substantial and long-term following CD52 and CD20-depletion (31) and both also induced long-term inhibition of MS with few treatment cycles, indicating induction-therapy activity. Importantly, memory B cells were augmented by B cell activating factor (atacicept) and tumor necrosis factor (infliximab) blockade that are known to worsen MS….(31) and (24).
There are a subset of t cells, T helper 17 cells and T17 cells which are not matured in the thymus and these may act like B cells. It is not known which cells switch them off but if it is the MAL expressing Treg cells, then again the T17 cells could behave like B cells and again not be switched off once an infection has subsided continuing the “auto-immune” response.
In humans, it is widely reported and well known that lymphocytes are damaged by MS although there is little research as yet determining which cells are affected. It is further speculated that long term damage to the lymphocytes expressing MAL (possibly Treg cells) may somehow lead to Secondary Progressive MS? These areas certainly need further research.
A further experiment shows that MAL is critical for ETX to affect mice. There is a video of two mice, one a normal mouse given a lethal dose of ETX and the other a “knock-out MAL” transgenic mouse (i.e. one which has been bred without the MAL gene) and given 15 times the lethal dose of ETX. The knock-out MAL mouse survives while the normal mouse dies. This experiment has been repeated at even higher doses and the MAL knock-out mice continue to survive.
A recent article published in July 2018 (39) provides evidence of a physical interaction between ETX and MAL, which was another missing piece of the jigsaw. A summary of the research follows: “Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein that crosses the Blood-Brain Barrier, binds to myelin and hence, has been suggested as a putative agent for the onset of multiple sclerosis, a demyelinating neuroinflammatory disease………..Whilst there is evidence of antibodies to ETX in controls, there is a significant increase in MS patients. Myelin and lymphocyte protein (MAL) has been identified as a key protein in the cytotoxic effect of Etx, however the association of Etx with the immune system remains a central question. Here, we show that Etx selectively recognizes and kills only human cell lines expressing MAL through a direct Etx-MAL interaction. Experiments on lymphocytic cell lines reveal that MAL expressing T cells, but not B cells, are sensitive to Etx, and revealed the toxin as a molecular tool to distinguishing sub-populations of lymphocytes. The overall results open the door to investigate the role of Etx and Clostridium perfringens on inflammatory and autoimmune diseases like multiple sclerosis.”
References can be found as follows:
Rumah et al (23) published a paper in 2015 showing that MAL is required for binding and activity (cytotoxicity) of ETX.
Furthermore, Dr J Linden et al. (27) found evidence in 2015 that ETX specifically targets the myelin-forming cells of the CNS – mature oligodendrocytes – leading to cell death. She found ETX was highly specific for oligodendrocytes, as other cells of the CNS are unaffected and ETX-induced oligodendrocyte death results in demyelination and is dependent on the expression of MAL.