Innate Biomarkers for EPM and PNE (polyneuritis)
The cute picture of Lucy is to remind you to get your horse tested, no matter how you get your horse to the vet. And to draw you in to read this important blog!
Note: Disease causes tissue damage and these disease-associated molecules are also biomarkers. Read this separate and important topic "Test Twice Treat Once".
We determined that serum amyloid A (SAM) is a marker for chronic inflammation that is associated with equine protozoal myeloencephalitis (EPM) in experimentally infected horses. We discarded SAM as a candidate serum marker, not because it is a non-specific indicator of inflammation, it was late in the disease process.
Serum amyloid A is detected late in S. neurona infections, our ideal test is a detectable marker that precedes clinical signs. We may go back and investigate SAM as an indicator of chronic inflammation in field cases of polyneuritis equi (PNE). For now, it’s on the back burner or, hopefully, someone else will take the challenge.
Common causes of an elevated CRP in horses
Three common causes of CRP in horses S. neurona, intestinal parasites, ulcers
Our attention for a candidate disease biomarker focused on C-reactive protein, CRP. It, like SAM, is not specific. Any infectious agent, bacteria/protozoa/virus, that sets off the inflammatory cascade, results in a rise in serum CRP concentration. Including infections with Sarcocystis neurona. You probably already know that S. neurona starts as an infection in the gut of the horse. If S. neurona is in the environment, the effect on CRP is due to chronic, even daily, exposure. Chronic elevation in CRP is a problem and we will explain why*.
Other common equine gut infections are intestinal parasites. Don’t forget encysted parasites, these infections don’t show up on fecal tests but they are insidious and will be important in CRP evaluations. One difference between this marker and SAM is that CRP is an acute phase protein. And after the inciting cause of inflammation is alleviated, the CRP will drop. If the trigger for CRP remains then the CRP will be chronically high.
Don’t forget that another common cause of chronically high CRP in horses can be hindgut ulcer disease. These horses have typical presentations, but this cause could be missed if it isn’t on everyone’s radar. Be sure and get this off the list before thinking of less common causes of elevated inflammatory markers.
What are acute phase proteins
Acute phase proteins are the first line defenses against infections
Acute phase proteins are found in the plasma and they increase following infection, inflammation or trauma. Tillet and Frances first recognized CRP in 1930. CRP is a protein synthesized in the liver and is normally present as a trace constituent of serum.
The pathway is well worked out, inflammatory conditions cause release of interleukin-6 and other cytokines that trigger the synthesis of CRP and fibrinogen by the liver. During the acute phase response, levels of CRP rapidly increase, within 2 hours of the insult, reaching a peak at 48 hours. With resolution of the acute phase response, CRP declines with a relatively short half-life of 18 hours. We find it is longer in horses and attribute the longer responses to common causes seen in horses.
The proposed function of CRP is to aid in complement activation, influence phagocytic cell function, and to augment cell mediated cytotoxicity. This triad of innate responses facilitate attacking and removing infectious organisms. Investigations over the past few years have shown that quantification of CRP and fibrinogen in plasma or serum can provide valuable diagnostic information in the detection, prognosis, and monitoring of disease.
A serum CRP level is a screen for infectious and inflammatory diseases. Because there are large numbers of disparate conditions that can increase CRP production, an elevated CRP level does not diagnose a specific disease. You may remember the term from previous blogs, CRP is not pathognomonic! However, an elevated CRP level can provide support for the presence of an inflammatory disease.
Data from Pathogenes bench, CRP in horses
The science behind using CRP to monitor PNE in horses
We evaluated CRP in normal horses and those with neuromuscular disease. In this study the serum cut off value was 16μg/ml. These data showed that an elevated CRP was present in almost three quarters of the horses with clinical signs of neuromuscular disease.
Significantly more horses with neuromuscular disease had an elevated CRP when compared to clinically normal horses. The data showed that Sarcocystis seropositive horses, irrespective of clinical status or serotype, were likely to have an elevated CRP. In the study we were evaluating responses to S. fayeri and the disease equine muscular sarcocystosis, EMS. We concluded that the toxin associated with EMS was inflammatory.
In another small study, we found no statistically significant relationship between the presence of the neuritogenic receptor antibodies (MPP) and CRP concentration. However, the comparison suggested as CRP values increased, the likelihood of antibodies against MPP also increased. In our PNE study we determined that MPP is associated with axon damage, measured by neurofilament light (NfL).
When taken together, we are saying that chronic inflammation, measured by serum levels of CRP, increases the chance of polyneuropathy and that, over time, myelin protein 2 is damaged. The damaged myelin P2 induces an immune reaction that becomes chronic resulting in polyneuritis equi. When the horse has PNE axons are damaged and there is a measurable increase in neurofilament light. Immune disease precedes axon damage.
The significance of the statements are that changes in CRP over time can give you a window on inflammatory disease. The reason that antibody biomarkers aren’t as good is that antibodies will take months to go down after the insult, however CRP will recede when the insult is gone.
Infection-->Inflammation-->IL6-->CRP CRP<-->Il6
*CRP can increase IL6 and dysregulated CRP can cause polyneuropathy
It is useful to understand how CRP is involved in chronic inflammation. Chronic inflammation, no matter the etiology, can result in PNE. Any infection, even in those not easily seen like encysted small strongyles or encysted tape worm larvae or hindgut ulcer disease, stimulates an innate immune response and that involves inflammatory cytokines. We concentrate on the IL-6 cytokine and its receptor, IL-6r.
The cytokine IL-6 binds membrane receptors present on target cells (cognate receptors) to initiate a cascade of responses. This is a normal signaling cascade, called classic IL-6 signaling, and is involved in regenerative and anti-inflammatory responses. There are also soluble IL-6 receptors, sIL-6r, circulating in the plasma. The circulating receptors can bind IL-6 and the complex activates pro- inflammatory pathways. To say it another way, IL-6 trans-signaling via the sIL-6r is important in the proinflammatory activities of IL-6. This is important because the trans-signaling pathway is active on cells that are unresponsive to IL-6.
Infection stimulates inflammation because circulating I-L6 receptors bind IL-6 and initiate CRP production by the liver. It is important to know that CRP can also stimulate IL-6 production. If the proper turn-off pathway isn’t initiated the system is dysregulated. Dysregulated IL-6-CRP can lead to polyneuropathy in the horse.
Biology is important…do no harm
Take the Baby Bear approach, treatment has to be just right
A detailed knowledge of IL-6 biology has important consequence for therapeutic strategies aimed at the blockade of the cytokine IL-6. We know how to turn off IL-6 receptors in a dose dependent way. Our wealth of information in PNE horses links IL-6 to PNE. There is a fine line to manipulate regenerative and anti-inflammatory IL-6 reactions that are desired over stimulating trans-signaling and pro-inflammation. It can be the difference between health and disease.