RNase L and Chronic Fatigue Syndrome (ME/CFS): An Overview from Patrick Englebienne

Englebienne, Patrick. 2003. RNase L in health and disease: what did we learn recently? Journal of Chronic Fatigue Syndrome 11 (2): 97-109.

Besides being an overview, this article presents intriguing new information about the ramifications of RNase L dysfunction in CFS and expands on two players that may play an important role in maintaining RNase L dysregulation.

Type I IFN’s and Immune Defense – Given our ever expanding knowledge of interferons Englebienne suggests that it is worthwhile to take a look again at the role type I interferons (IFN’s a/b) play in immune defense. Type I IFN’s are cytokines secreted within a cell when it comes under attack by pathogens. Viruses, bacteria, chlamydia and mycoplasma all induce IFN production. Although there is evidence that some cells produce IFN in response to extracellular infections, it is important to note that the IFN a/b signaling system primarily responds to intracellular infections. We will see in other papers that IFN’s are also induced by such as toxins and other agents associated with cellular stress. Upon binding to receptors on the cell membranes IFN’s signal for the production of enzymes (RNase L, PKR and 2-5OAS) central to cell defense. Some researchers believe that dysfunctional IFN a/b pathways play a important role in other poorly understood diseases such as lupus and rheumatoid arthritis.

First the primacy of RNase L, PKR and 2-5OAS in the IFN induced cellular defense system is emphasized. Our understanding of the scope of RNase L activity continues to broaden as researchers delve more deeply into the enzymes functions. Until recently it was thought that RNase L targeted only viral and ribosomal RNA’s. Recent studies suggest, however, that RNase L targets the mRNA involved in muscle cell differentiation.

The recognition that RNase L targets mitochondria mRNA involved in maintaining mitochondrial membrane permeability suggests that RNase L may play a role in cell apoptosis. Increased membrane permeability is one of the hallmarks of the apoptotic process. RNase L also plays a regulatory role in cell differentiation. A mutant RNase L gene (coding for underactive RNase L) has been implicated in prostrate cancer. Englebienne introduces a long list of immune system proteins that may, because they are downstream of RNase L, be disrupted by RNase L dysfunction.

Most interesting for our purposes are two genes, ISG-15 and ISG-43, induced by interferons (IFN’s) and regulated by RNase L. When intracellular ISG 15 is activated it targets cellular proteins for degradation by organelles called proteasomes that chew up damaged or unwanted proteins. When ISG-15 is secreted outside the cell it induces IFN-y production by T-cells and ultimately assists in NK cell proliferation. ISG-43 provides a counterweight to ISG 15 by removing it from proteins it is attached to. Interestingly it removes ‘kill me’ targets from serpine proteases – one of which is elastase. Degradation of ISG 43 mRNA could therefore lead to increased elastaste activity. Elastase is one of the proteases believed to fragment RNase L ISG 43 is also able to activate 25OAS.

IFN’s and RNase L dysregulation – The source of the RNase L dysregulation found in CFS is still unclear. It is clear, however, that the propensity of the 2-5OAS enzyme in CFS patients to produce 2-5A dimers instead of trimers comes near the heart of the problem. The production of 2-5A dimers is either due to an inappropriate induction of the 2-5OAS enzyme either by type I IFN’s or by atypical nucleotides.

Review: if intracellular pathogens are present IFN a/b is produced. Type I IFNS’s wake up, so to speak, 2-5OAS. Upon being prodded awake by Type I IFN’s 2-5OAS checks its environment to determine if any signs of pathogenic activity (dsRNA) are present. If they are then 2-5OAS becomes activated and starts producing 2-5A. Unfortunately for CFS patients 2-5OAS predominantly produces a type of 2-5A (dimers) that bind to RNase L but do not activate it and, more importantly, leave it vulnerable to fragmentation by inflammatory and/or apoptotic enzymes.

The system is more complicated than first seen. The 2-5OAS enzyme exists in several forms; the p69 form produces primarily trimers; the p100 form produces mostly dimers. Different signals apparently call for the activation of different forms. We are still not clear which oligonucleotides or IFN signals activate which form of 2-5OAS. We do know that unusually short sections of oligonucleotides appear to activate the p100 form of the enzyme – and thus initiate 2-5A dimer production. See More RNase L for more background information.

Our first hint that some progress has been made in identifying the culprit behind RNase L dysregulation comes when Englebienne reports ‘preliminary observations’ indicate that aberrant 2-5OAS production in CFS is probably due to high levels of oligonucleotides in CFS patients cells. The efficacy of Ampligen, a mismatched dsRNA product, in reregulating the 2-5OAS system in some CFS patients, suggests the IFN signaling pathways are functioning correctly in many CFS patients and that, if given the chance, the 2-5OAS enzyme in CFS is capable of producing 2-5A trimers.

(Because Ampligen, unfortunately, upregulates the activity of an enzyme, PKR, that is already upregulated in CFS patients, it is no longer considered a viable form of treatment for many CFS patients.) Absent any means of attacking 2-5OAS dysregulation, Englebienne suggests attempting to reduce the proteolytic activity (that results in RNase L fragmentation) found in CFS patients through calcium antagonists and elastase inhibitors. Both have shown some efficacy or promise in regulating RNase L functioning in CFS patients.

(Reducing the rate of proteolysis and therefore the rate of RNase L fragmentation in CFS patients should lead to reduced RNase L fragmentation. Given the emerging complexity of the RNase L enzyme, however, it is difficult to predict what the effects would be.

Stopping the fragmentation does not address the production of the aberrant 2-5A dimers and the inappropriate binding and inactivation of RNase L. Since 2-5A dimers inactivate RNase L when they bind to it one wonders if RNase L fragmentation might have at least one positive outcome – a still active (if poorly controlled) RNase L enzyme. Would RNase L be totally or partially inactivated if the fragmentation stops? No one knows exactly what the 37-kDa fragment does. Does it retain some of its old functions or are its actions purely negative? Could eliminating the fragment could result in no RNase L activity at all in CFS patients who are only producing 2-5A dimers? What about apoptosis? If the RNase L enzyme is bound by 2-5A dimers how is it able to contribute to apoptosis? Reduced apoptosis rates could lead to increased risk of autoimmune diseases, cancer, etc.???. This appears to be a tricky situation.

Englebienne next lays out a scenario describing a self-perpetuating RNase L activation. First, he suggests increased activity of the RNase L fragment results in the unregulated destruction of certain kinds of mRNA. Destruction of the mitochondrial mRNA that regulates mitochondrial membrane permeability could result in outflows from the mitochondria of a substance, cytochrome C, that initiates the apoptotic cell suicide program. Triggering the cells apoptotic programs results in the production of the two apoptotic and inflammatory proteases (calpain, elastase) known to fragment RNase L. An accompanying destruction of mRNA governing serine protease inhibitors (ISG 43) opens the floodgates for further elastase (a serine protease) production. This leads to further RNase L fragmentation, which in turn, leads to further elastase production, thus creating a vicious cycle. The patient in stuck in a vortex of inflammatory and apoptotic proteolysis powered by the inappropriate destruction of mRNA by a fragmented RNase L.

Furthermore the destruction of ISG 15 mRNA results in reduced IFN-y production because T-cells are not stimulated to induce the proliferation of two cells (NK and LAK cells) involved in innate immune defense.

Summary: Our increasing knowledge of RNase L’s activities indicate RNase L dysregulation has the potential to negatively effect a wide variety of cellular activities including immune defense, proteasome targeting, cellular differentiation, and proliferation, mitochondrial activity and apoptosis as well as the ability to disrupt both the innate immune (NK, LAK cell proliferation) and adaptive immune (T-cell activation) defenses. Because one of the mRNA’s RNase L regulates is involved in regulation of a protease (elastase) capable of fragmenting RNase L, RNase L dysregulation could be self-perpetuating. Similarly RNase L mediated disruption of mitochondrial membranes could result in increased production of a apoptotic protease (calpain) able to fragment RNase L as well. Thus two scenarios are presented that could result in a self-perpetuating cycle of RNase L fragmentation.

(Please note what follows is simply some unwarranted speculation from an uniformed laymen…Give it no special attention.) Having a background in ecology, I try to put things in a holistic framework. The presence of awholly negative (but completely natural?) isoform has a always been a source of irritation for me. Why would the body produce such a thing? It could be that a pathogen induces the 2-5OAS system to produce a dimer that dismantles the RNase L system but because this involves two steps involving two separate entities – a p100 enzyme and a 2-5A dimer – both of which must be co-opted or even manufactured by a pathogen, this seems highly unlikely. The p100/2-5A dimer production is a natural process – as such it must serve some useful function. There is some speculation that 2-5A dimers ‘wake up’ the system but this is only speculation. Come with me on a wild ride as I, unencumbered by medical training or background, take a different look at the p100 2-5OAS enzyme.

2-5A dimers appear to be produced regularly, if in low amounts, even in healthy people. They are simply produced in much larger amounts in people with CFS. What positive role could the p100 2-5OAS/2-5A dimer play? Remember that it binds with RNase L but inactivates it. It does not appear to directly harm RNase L but it does leave it in a vulnerable position.

What if the 2-5A dimers are induced in order to shut RNase L down? What if an overactive or otherwise dysfunctional RNase L is the culprit? Could the 2-5A dimer be one way the body shuts down an RNase L enzyme that is destroying the wrong kind of RNA? Remember that RNase L has many functions; it does not simply figure in pathogen defense, it also selectively filters out host mRNA at certain times in order to, for instance, ensure that muscle cells develop properly.

The p100 isoform could be part of a feedback system designed to ramp down RNase L production. It could be activated by types of RNA that signal system dysfunction. The problem in CFS occurs when this situation becomes chronic (reducing our antiviral defenses) and when protease activity becomes high enough that the unprotected RNase L is fragmented.)

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