Our awesome self defenses

Previously, we wrote an extensive review on how the immune system combats pathogens such as coronaviruses. The immune system responses can be divided into non-specific rapid responses that are initiated in mere hours post infection and subsequent specific responses (such as antibody production) that commence days after the original infection. One of these early responses to fight infection is the initiation of inflammation, a concept with which we are all familiar. So, what is inflammation, why (and how) is it invoked to protect you, and why can it be dangerous if not managed properly? In this particular story, we will focus especially on one grand aspect of inflammation: its ability to mutate genetic content.

Before we go into explaining inflammation itself, we will reproduce a table from our initial article on the different arms of the immune system and what specific aspect of virus existence is being targeted, so that we can quickly focus on all the early, non-specific responses. They are quite fascinating!

To review these non-specific early host defenses, we decided to go to the original source that we reviewed for the overview of the immune system response to viruses: the fourth edition of Medical microbiology edited by Samuel Baron.

Some of these early host defenses are always in place. These include anatomic barriers (for example, skin or mucosa at body surfaces or endothelial cells within the body); nonspecific inhibitors (compounds present in body fluids or tissues that can either interfere with viral attachment, directly inactivate viruses, and act defensively inside cells); or presence phagocytic cells (cells that engulf the virus to destroy it).

The ones of interest to us - fever, inflammation, and interferon - are induced by the infection.

It is somewhat challenging to map a logical progression of events, but suffice it to say, the first necessary step is the recognition of an insult, in this case, the viral infection. Recognition of the invaders can be through the discovery of the pathogen directly by the immune cells, or through recognition of its foreign genetic material that needs to be replicated to produce pathogen progeny. The outcome of that recognition though is a production of cytokines by either the pathogen-triggered immune cells or surrounding tissues. Cytokines, which are specific molecular signals, alert the immune system to take action and also induce both fever and inflammation. Cytokines that result in inflammation, are referred to as pro-inflammatory cytokines such as IL-1, TNF-α, IFN-γ, and IL-6.

Probably the most famous of these is the interferon.

But before we dive into how interferons can protect from insults such as virus infection, let us now steer into inflammation.

Inflammation – why such a burning topic?

Perhaps in the simplest terms, inflammation is a recognition by the body that some damage is occurring and then reacting to it. As we will see below, multiple events come together to stimulate an inflammatory response, in the case of a virus infection, those include cell damage caused by the virus or a reaction to specific virus-stimulated biological responses. The purpose of the inflammation of course is signal to bring in some help and in the case of virus infection, inflammation itself can also be anti-viral.

We always hear how inflammation is bad, so how is it good on a short-term acute basis?

The outcomes of inflammation are: alterations to the blood flow, edema (swelling due to fluid retention in the tissue) and accumulation of leukocyte immune cells. The consequences of these are increased temperature in the local tissue, thereby reducing oxygen levels in that tissue, and increasing levels of CO2 and organic acids. Let’s start with increasing temperature. During the early stages of inflammation increased blood flow, as well as altered energy metabolism of affected tissue, leads to increased temperature levels.

Virus replication can be significantly influenced by even a modest rise in temperature. Change from 37°C to 38°C can drastically decrease the production of many viruses, and this helps explain why fever is one of the bodily responses to fight viral infections. The pro-inflammatory cytokines mentioned above can all lead to an increase in temperature. The added benefit of fever (you never thought you would hear such a statement, did you?) is that it also promotes the generation of cytotoxic T-cells.

As inflammation continues, blood flow recedes, reducing the number of red blood cells carrying oxygen to the affected tissue. On top of that, the limited oxygen that is brought in also has a harder time diffusing through edema fluid. As a consequence of reduced oxygen levels available to the affected tissue, less energy can be produced (in the form of ATP), and hence reducing the energy available for viral synthesis.

To compensate, cells increase anaerobic glycolysis (production of energy when oxygen is limited), which increases the accumulation of CO2 and organic acids in these same tissues. The build-up of CO2 and organic acids, in turn, decreases the local pH which in turn can also inhibit the proper viral replication.

Therefore, local inflammation resulting from a viral infection can interfere with the virus being able to replicate efficiently in multiple different ways.

Interferons – masters of quick defense

Interferon cytokines are small proteins produced by the cells upon being triggered, and then secreted by the cell into the extracellular fluids. This allows the interferons to protect against the virus at a local tissue that is being infected but also allows the interferon to be spread around the body by the bloodstream thus allowing the protection of distant organs against a spreading infection.

Interferon does not inactivate viruses directly. Instead, it binds to specific receptors of surrounding cells and triggers these cells to activate the use of numerous antiviral genes that will help prevent viral replication in these surrounding cells. There are many ways in which the activation of the antiviral genes by the interferon can fight the virus but basically, any part of the virus life cycle could be targeted. For example, induced antiviral proteins can inhibit the production of viral proteins; inhibit the duplication of the virus genetic material or even destroy the virus genome; or interfere with the assembly of viral particles. Interferons are also produced at about the same time as the virus is replicating, to act in a protective manner simultaneously as the virus attempts to spread.

There are three main types of interferon that we will highlight: alpha, beta, and gamma.

Interferon alpha (INF-α although there are many subtypes) is produced by leukocytes’ immune cells (dendritic cells, macrophages, and B cells) induced by the presence of foreign cells, bacterial cells, viruses, virus-infected cells as well as tumor cells.

Interferon beta (INF-β) is produced in most body cells when induced by foreign genetic material.

Interferon gamma (IFN-γ) is produced by T-cells when induced by foreign antigens (fragments of foreign material presented to T-cells to activate them so they can kill cells presenting such antigens) and natural killer cells. IFN-γ is especially powerful in that it magnifies the actions of other interferons while its own effects appear to be more potent than other interferons as well.

So far everything sounds all good, so what’s the big deal? Accordingly, let us get to the thorniest of issues.

Innate immune response goes nuclear – mutagenesis

One of the more interesting aspects of early innate immune responses is that it can attack the virus by mutating it! In other words, attempting to kill it by compromising its genetic code. But it is a double-edged sword as we will soon find out. Also, it is a two-way attack. First, let’s continue with the interferons’ saga.

As mentioned, interferons can induce the expression of many genes - the outcome of which is to produce an antiviral environment. Interferons are also the best characterized activators of genes coding for proteins called deaminases, although these proteins are activated by all of the pro-inflammatory cytokines we mentioned above. Deaminases play a crucial role in antiviral innate immunity in a highly unique way. They are a large family of proteins consisting of APOBEC proteins (which stands for apolipoprotein B mRNA editing enzyme catalytic polypeptide-like protein, so you can see why scientists prefer to use acronyms in this case!) or AID proteins (activation-induced deaminases) and finally ADAR1 (adenosine deaminase acting on RNA 1).

But what is really important here is what these proteins do: they deaminate specific genetic compounds which means, chemically altering them and in the process converting them to another genetic compound, most commonly from cytidine to uridine. This means they instantly introduce a mutation in the genetic code of invading viruses and through hypermutation of the viral genome they can inhibit the proper use of the genetic code and thus limit the infection. In other words, we have proteins “for hire” in our cells that can mutate genetic material, and they are invoked by the inflammatory environment!

This is how they came to light as part of the innate immune system, through the discovery of the ability of deaminases to inhibit the replication of certain types of HIV virus.

Furthermore, these deaminases are observed in many cell types, not just immune cells, so their role is not just protection against invading viruses, but also against protection of viral elements already residing in our genomes (mobile retroelements). Such viral elements that jumped into human genomes in the past can sometimes still be duplicating themselves and this is another way of protection from that. Excitingly, there is also emerging evidence that these deaminase proteins might be manipulating mRNA templates that are used for the production of proteins, thus imprinting environmental influence on biological outcomes!

That is just one way how inflammation can lead to mutagenesis. There is one more.

Another key feature of inflammation is the production of reactive oxygen and nitrogen chemicals designed to destroy pathogens. Remember those accumulating leukocyte immune cells as one of the hallmarks of inflammation? Many reactive oxygen and nitrogen species are produced or derived from these innate immune cells.

These reactive oxygen and nitrogen species can act as signalling molecules but are also very destructive due to their high potential to take part in chemical reactions. As a consequence, these reactive chemicals can damage DNA and lead to mutagenesis, and hence constitute another way how inflammation contributes to mutagenesis. One of the most common ways of damaging and mutating the DNA is through DNA oxidation but these chemicals can also deaminate DNA bases, just like those proteins induced by inflammation can, as discussed directly above. Hypohalous acids (or oxyacids) that also belong in the repertoire of chemicals produced by the innate immune cells can also react with DNA, and we should mention that because one such genetic product of the chemical reaction, 5-chlorocytosine, is so persistently observed during inflammation that it has become a biomarker for chronic inflammation.

In addition, the oxidative chemicals that can damage DNA, can also damage and mutate DNA in an indirect manner by reacting with polyunsaturated fatty acids (if you ever wondered why they might be bad for you) and form species that can be used to then chemically alter DNA as well.

These mutations we are discussing, can arise in couple ways, like by leading to the mispairing of nucleotides causing a single point mutation (mutation where a single nucleotide base is altered). Alternatively, they can lead to the breakage of the DNA backbone. The breakage of DNA, if not fixed properly, can lead to much larger scale alterations of the genetic code because it can cause a large scale sequence rearrangement of mutations where many nucleotide bases at a time are either deleted out of place, or inserted where they do not belong, or fragments of genetic code are swapped from one place to another (this is termed translocations).

But there is a potentially heavy price to pay for such dangerous actions.

Inflammation double edge sword – carcinogenesis

One potentially dangerous consequence of inflammation is that it will not only contribute to accelerated mutagenesis and genomic instability of invading pathogens but also that of our own cells. Unfortunately, inflammation can cause high levels of mutagenic DNA damage along the way, and since the accumulation of DNA mutations is a hallmark of cancer development, inflammation could be a pro-tumorigenic process!

One way the body mitigates this side-effect of inflammation on our self is through the employment of a myriad of DNA damage repair mechanisms that are stimulated into action to a greater extent during inflammation. Here is where interferons come into place once again, as they can also promote DNA repair through the stimulation of appropriate genes.

However, these DNA repair processes are not infallible or can be overwhelmed if the amount of damage is just too excessive. Thus inflammation, if excessive enough can contribute to cancer development by increasing mutagenesis. In addition, the outcomes of inflammation, such as remodeling of the extracellular matrix and weakening of vascular barriers to facilitate immune cell migration, or the stimulation of cellular proliferation to regenerate damaged tissue, also promote cancer progression if an unfortunate combination of genetic mutations were to arise allowing cancer to start developing. Tumors can even generate their own inflammatory microenvironment to aid their own development, and in contrast, mitigating inflammation can be an effective strategy for slowing or even preventing cancer development.

For example, the deamination process of inducing mutations we discussed above is becoming more apparent in its importance in driving the evolution of certain cancers. One study showed that ~15% of genetically sequenced tumors contain mutations by APOBEC deaminases, and some cancer lines exhibit 50% or more, such as breast, lung, cervical, and head and neck cancers.

Blowing it all out of proportion

Furthermore, outcomes of inflammation can propagate further inflammation in a self-feeding loop system. For example, DNA damage can also exacerbate inflammation. This may have evolved because pathogens themselves can cause DNA damage.

Let’s bring up one example because it is so interesting (ah, the wonders of the molecular world! Ever so fascinating).

One protein, called PARP1 (poly(ADP-ribose) polymerase) detects and binds breaks in the DNA to help recruit other proteins to the site to fix the damage. It does so by attaching a specific chemical in a repeated fashion that is then recognized by these DNA repair proteins. But PARP1 can also attach these chains of chemicals to other proteins, including those that regulate gene use, and a modification of one such regulator of gene use increases the use of genes involved in the induction of an inflammatory state.

Thus, inhibition of PARP1 decreases inflammation including by decreasing the production of pro-inflammatory cytokines. The reason why we wanted to focus on this guy is because PARP inhibitors are also famous as cancer therapy in cancers that arise due to already existing defects in repairing DNA breaks (such as those due to BRCA gene mutations, for example). Thus, while one defect of not being able to properly fix broken DNA might have helped the development of cancer, the use of PARP inhibitors then prevents additional repair of DNA breakage. This leads to actually overwhelming cancers with too much DNA damage and leading to enough genomic instability that such cancer cells are forced to die. This double whammy process for targeting certain cancers is referred to as synthetic lethality.

Damage from inflammation, or even damage from pathogen insult, can result in cell death which can also be pro-inflammatory events (if you wondered how inflammation can just arise without some pathogenic insult).

But it appears that even certain of the previously discussed deaminase proteins could also promote inflammation. One study showed that liver cells made to have active APOBEC deaminase resulted in improved production and secretion of pro-inflammatory cytokine IL-6. Since IL-6 also promotes the activation of such deaminase proteins, this could result in high levels of both the deaminase protein and IL-6, forming a positive feedback loop. The authors commented that this could help explain how under certain conditions a prolonged non-resolving inflammation could further promote cancerous transformation of some of the deadly liver cancers.

Bringing it all together

The take home message is that inflammation and DNA damage contribute to each other in a dangerous self-feeding loop. Thus inflammation, which has important protective roles, should be short lived and not chronic. It is well understood that chronic inflammation is associated with many different disease states. If you ever wondered why, here are some of the dots connected for you in this article. This is significant because inflammation obviously does not have to be triggered solely by pathogens. Other sources of tissue trauma can be due to radiation, loss of blood supply, auto-immune self attack, toxins (including from diet or drugs), physical injury, and one that we might too often take for granted, the negative impacts of psychological stress.

So, what can be done to reduce inflammation? There is no point worrying about your inflammation state and inflaming your state further! Rather certain anti-inflammatory behaviours are recommended, and let us keep it simple for now.

Based on some recommendations, managing an inflammatory illness requires the following:

• Understand what is the underlying distress that leads to the inflammatory disease state;
• Reduce the chronic pro-inflammatory state by improving diet, increasing physical activity, and practicing regular relaxation and meditative exercises;
• Relief of certain symptoms may need short-term medical interventions.

If this sounds super familiar to you, that is probably because we all have been hearing the same type of advice our entire lives. We all know this stuff and we all try to do something about this to a smaller or greater degree, depending on life’s complexity and our ability to muster the discipline. Discipline is the tricky part and perhaps some of the underlying causes undermining our collective discipline to practice these simple choices might be underestimating the magnitude of what is at stake if we do not actively practice a healthy lifestyle.

Now we all have one extra reason to try just a tiny bit harder.

To stop the undesired mutagenesis taking place.

So let inflammation work for us only when our innate immunity selects it for us, and not because of our bad habits.

This article has been produced by Merogenomics Inc. and edited by Jason Chouinard, B.Sc. Reproduction and reuse of any portion of this content requires Merogenomics Inc. permission and source acknowledgment. It is your responsibility to obtain additional permissions from the third party owners that might be cited by Merogenomics Inc. Merogenomics Inc. disclaims any responsibility for any use you make of content owned by third parties without their permission.

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