I spoke with Dr. Stefan Rose John, Institute of Biochemistry in Kiel Germany, a few years ago. Work he published in 2012 explains the importance of interlukin 6 (IL6) trans signaling by the soluble IL6 receptor and the importance to pro-inflammatory activities of IL6 (see the graphic from his paper above). He used his discovery to make medications for rheumatoid arthritis that are beneficial in autoimmune diseases, induce regeneration of the intestine epithelial cells and support the innate immune response in infections.
We spoke about the ability of S. neurona surface antigens to be involved in the trans-signaling processes. There are specific molecular structures on the surface of some S. neurona organisms that would lead scientists educated in the field to think that they would induce a inflammatory inducing process called trans-signaling, did he think so, I asked.
To understand why we asked him the question you have to understand the plethora of international work that went into myelin protein P2 (MP2) research. MP2 is a structural protein that stabilizes myelin membranes. It’s found in the peripheral nervous system (PNS) myelin, and in small amounts in the central nervous system (CNS) myelin. Horses have more MP2 protein in their CNS than other animals, by the way.
In the 1980-1990’s Myasthenia Gravis researchers were concerned with the outbreak of Mad Cow Disease in Europe. They had been using the spinal cords of cows to get MP2 proteins-there was a risk they would get Mad Cow. Scientists switched to equine tissues and they spent considerable time elucidating the equine MP2 protein structure, association with experimental allergic encephalitis (EAE) and sequencing important peptide fragments of the protein. All very useful to us.
Several scientists realized that cauda equina neuritis, later called polyneuritis equi, was associated with neuromuscular disease in horses and most importantly— disease could be detected with an ELISA test. Of course, the state-of-art at that time was using minced nerve tissues that contained MP2 in the assays. Improvements were made when we used recombinant proteins in our assays. We developed a recombinant MP2 and the fragment that contains a T-cell epitope. An epitope is a short run of amino acids.
The MP2 protein had amino acids that correspond to a T-cell epitope and many immunologically exciting observations were made about this epitope and what it did in experiments in animals. It could be used to stimulate inflammation and induce EAE— and the sequence was homologous to the IL6 cytokine. By the way, our work showed that a surface S. neurona SAG 6 has homology with this IL6 region of MP2. We wondered if the SnSAG 6 epitope could act like the MP2 T-cell epitope. We knew that recombinant SnSAG 1 is transiently inflammatory—we had immunized horses with recombinant SnSAG 1 in a clinical study and found it protected horses against S. neurona infections that lead to EPM.
Interleukin-6 is an important cytokine that has many activities. It regulates the immune as well as the nervous system, it controls liver regeneration and is involved in metabolic control of the body. Interleukin-6 binds a membrane bound receptor (IL6r) and the complex IL6-IL6r binds a third protein to initiate intracellular signaling. The associated membrane bound protein is ubiquitous, it is expressed on all cells. However, IL6r is only present on a few cells, includeing some white blood cells.
The location of these molecules, IL6 and IL6r, is important because cells that don’t express IL6r can’t respond to IL6. The ubiquitous protein to initiate intracellular signaling can’t trigger the IL6 cytokine cascade by itself. There is a soluble form of IL6r (called sIL6r) that can bind IL6, the soluble form can trigger intracellular signaling. The soluble complex (sIL6r-IL6) that binds the ubiquitous protein proceeds by a mechanism that is called trans-signaling. The cytokine IL6 is highly active in the central nervous system and the IL6-sIl6r crosses the blood brain barrier to elicit proinflammatory pathways.
Here is the punch line: the classic IL6-IL6r is regenerative or anti-inflammatory while IL6 trans-signaling is pro-inflammatory. The biology of IL6 has important consequence for therapeutic strategies that block the cytokine IL6. Block too much— for too long, and you affect the metabolism and innate immune responses of the animal. Block too little or not long enough and inflammation can return. It is a delicate balance to know in an individual how much to block and for how long. Some drugs are highly pro-inflammatory and work through trans-signalling. When some drugs are out of date or degrade in water they can initiate trans-signaling.
C-reactive protein (CRP) is an acute phase protein made in response to infection and is a pro-inflammatory molecule. We know from the literature that CRP is a physiological activator of IL6 receptor shedding. Remember that IL6r is found only on the surface membrane of liver cells, macrophages, neutrophils, some T-cells, and some specialized cells of the kidney (podocytes). When CRP (made by the liver) is elevated, IL6r is released from cells that express it, the sIL6r binds IL6 and the soluble complex can land on any cell and signal an inflammatory cascade. We have previously reported that as CRP values increase the likelihood of a positive polyneuritis equi ELISA test result also increases.
Back to my question to Dr. Rose-John, is it structurally likely S. neurona epitopes induce inflammation by binding and activating the trans-signaling cascade in horses? The answer is no. We do know, by experiment that S. neurona does bind and enter cells that express IL6r…perhaps this is the mechanism that sets off the inflammation. It is possible the epitope on the organism that corresponds to IL6 can bind IL6r on white blood cells and allows the cell to be infected by the protozoa.
The message to horse folks is that in the presence of S. neurona exposure it is important to monitor CRP and be very mindful of how much one decreases the levels of Il6.