A new stage in the development of immunology

The study of the immune system, a complex, multicomponent (with its own organs, cells and molecules) system of protecting the body from all foreign substances, has been carried out for about a century and a half. And I must say, the history of the development of immunology is not only a gradual accumulation of knowledge. Sometimes there were events that truly overturned the already established ideas. One of them is the discovery in the middle of the last century of T– and B-lymphocytes, which specifically respond to invading cells and molecules. This became the foundation for the creation of a new approach in clinical immunology, and it seemed that everything about the immune system had finally become known, all that remained was to deepen knowledge. However, at the turn of the century, a family of image–recognizing receptor proteins was discovered, which again fundamentally changed the understanding of the immune system. Undoubtedly, it will soon bring important results for practical medicine as well – it will help to choose the right drugs that increase the body’s resistance and achieve success in the treatment of serious and dangerous diseases.

Before we talk in more detail about the role of these receptors in the body’s defense system, let’s recall how events developed and ideas about the immune system changed.

In the second half of the 19th century, when L. Pasteur first developed the scientific principles of preventive vaccination against chicken cholera, anthrax and rabies, the immune system was understood only to protect the body from infections. Later it became clear that this system performs more complex functions. Important steps in understanding its mechanisms are primarily related to the discoveries of two outstanding scientists, Nobel laureates in 1908 – I.I. Mechnikov, who formulated the cellular theory of immunity and discovered the protective role of phagocytosis, and P.Ehrlich, who created the theory of antibody formation. This served as the basis for the division of immunity into natural (innate) and specific (adaptive). It was believed that natural immunity is nonspecific, it is provided primarily by phagocytosis, which is carried out by macrophages and neutrophils, and is triggered quickly – immediately after the microbe enters the body. In contrast, specific immunity is accompanied by the formation of antibodies and cells with equally specific receptors in the body. It is more effective, but develops later, because each time the body re-forms cells in response to the ingestion of an antigen, producing antibodies to it.

The rebirth of immunology dates back to the middle of the last century and is associated with the works of another Nobel laureate (1960), F. Burnet, who created the clonal breeding theory and defined immunology as the science of controlling the constancy of the molecular (antigenic) composition of the body. In other words, all cells and molecules that are foreign to the body, as well as damaged ones, must be destroyed by the immune system. However, this requires a whole arsenal of millions of different antibodies to virtually any antigen. Specific antibodies accumulate, as Burnett found, during embryonic development and are produced by cells that are formed from lymphocyte precursors. But at that stage of the research, it remained unclear how the immune system distinguishes a “stranger” from “its own”? All that was clear was that, according to Burnet’s theory of tolerance, normally all cells specific to the complex molecules of their own body are immediately selected and destroyed. If this does not happen, the destruction of tissues by their own antibodies begins. Burnet called these pathologies autoimmune. Indeed, we now know many such diseases and understand how to deal with them. From this theory, it also became clear why organs and tissues of the donor are rejected during transplantation. Thus, a new field of immunology was born – transplantation, which today quite successfully solves the problems of rejection of donor tissues.

Shortly after the appearance of the clonal selection theory of immunity, new technologies revealed that the population of lymphocytes – carriers of the specificity of immune reactions – is heterogeneous and includes two main subpopulations. One of them consists of B-lymphocytes, which are responsible for the formation of antibody molecules that bind foreign antigens in order to remove them from the body. The other consists of T-lymphocytes, which destroy foreign cells (cytotoxic T-lymphocytes, or T-killers), and also participate in the regulation of a specific immune response (T-helpers).

The study of the molecular mechanisms of antigen recognition and induction of the formation of antibodies and specific T-lymphocytes revealed that the system of formation of a specific response to a foreign antigen is complex and multi-stage. It turned out that macrophages and dendritic cells are involved in this process, which process the antigen and present it to lymphocytes in combination with certain molecules. And of course, at all stages of the development of the immune process, all cells constantly “talk” with the help of special information molecules, cytokines, which interact with receptors on the cell surface. Hundreds of different cytokines and receptors are now known, and scientists are constantly discovering new ones.

Specific immunity has become known as adaptive, because when a new antigen enters the body, the immune system adapts to it by producing antibodies or specific effector cells. Note that the specificity for each antigen is not recorded in the genome, but its carriers – the corresponding specific cells – are present in the body for virtually any possible antigen and are obtained as a result of mutations. Advances in the study of this type of immunity have led to a breakthrough in applied immunology. In particular, technologies have been developed based on the use of monoclonal antibodies (antibodies of the same specificity): enzyme immunoassay, cell phenotyping. They have fundamentally improved the diagnostic capabilities of infectious, autoimmune, oncological and other diseases associated with suppression or excessive activity of the immune system.

Immunodeficiency, as is known, can be congenital (diseases associated with congenital disorders in the immune system) and acquired (pathologies caused by damage to components of the immune system by radiation, chemicals, and most often infection, such as HIV). It has been shown that insufficient functioning of the immune system accompanies a wide variety of chronic diseases and prevents the elimination of the focus of inflammation. For the treatment of such diseases, a large number of immunomodulatory and immunostimulating drugs have been developed and are being produced based on active substances from the thymus and bone marrow, where T and B lymphocytes are formed, respectively, or from microorganisms; even synthetic analogues of these substances have been created. In fact, the same immunomodulators, but in gradually increasing doses, can also be used for the cardinal treatment of allergic diseases, which, as it became clear, are associated with hyperreactivity of the immune system.

The obvious successes in the study of adaptive immunity have pushed the study of innate and nonspecific resistance of the body to the periphery of the interests of immunology. For a long time, it was believed that the lymphocyte, with its pronounced specificity, was almost the only immunocompetent cell in the body, while the rest were assigned an auxiliary role at best. New generations of immunologists do not know or do not remember the work done on mammalian animals raised in sterile conditions and dying from microbial shock when they are transferred to a normal environment. Apparently, they also forgot about the observations concerning the prolonged stay of people in confined spaces (for example, polar explorers), which also prove the important role of natural immunity in protecting the body from infection. By the way, more than 98% of multicellular organisms do not have adaptive immunity with T and B lymphocytes, there are only natural resistance factors. Nevertheless, they not only successfully cope with infection, but also do not suffer from cancer and allergic diseases.

Meanwhile, in the last decade, theoretical and practical issues have clearly begun to accumulate in immunology, the solution of which is important for the clinic. They are extremely difficult to explain or solve in terms of the determining role of adaptive immunity. Let’s list just a few of them.

In recent decades, the incidence of various allergic diseases has been increasing, and these are often polyallergies to various allergens. The exact reasons for this growth are still unclear, although there are many hypotheses. The number of so-called pseudoallergic diseases has also increased dramatically, the clinical picture of which does not differ from true allergies, but they develop without the participation of specific antibodies. The mechanisms of pseudoallergia development are unclear, and approaches to its treatment depend on it.

The number of people suffering from chronic infections has increased significantly, and they are not caused by highly pathogenic microorganisms (such as hepatitis viruses), but by those that can be asymptomatic in the body’s microflora for a long time (on mucous membranes, skin, and various body tissues). The reasons for these processes also remained unclear for a long time.

Among the normal microflora of the body, there are numerous microorganisms to which it does not exhibit immune reactions (tolerant). The same is true for food products, many of which have high antigenicity and partially enter the body through the epithelial layer in undigested form (the so-called oral tolerance). Normally, a person does not form antibodies to such antigens. The mechanisms of formation of such tolerance are difficult to explain on the basis of the theory of T-, B-immunity. We have given only a few of the many questions that need to be answered for the further development of immunology and that cannot be answered from the point of view of the established approach.

In 1989, Carl Janoway suggested that genetically programmed image-recognizing receptors are located on the surface of human cells, which recognize the molecular structures that are most often repeated on the surface of microorganisms. Apparently, these receptors (initially called molecular forms associated with the pathogen) should determine the specificity of the innate immune system’s response to the introduction of pathogenic microflora into the body. At that time, Janoway could only cite mannozolectin receptors as proof of his hypothesis – proteins that bind to bacterial monosaccharide (mannose) and cause the activation of complement and macrophages. Logic dictated that in order to identify all microorganisms, there should be only a few hundred similar receptors on the cells of a macroorganism. Indeed, they were soon found, and now new ones are being discovered almost every month.

The most studied family of signaling proteins is Toll–like receptors (TLRs), so named by analogy with Toll receptors, which were first discovered in 1992 in the fruit fly (Drosophila melanogaster). Toll protein is involved in the embryonic development of drosophila, namely it regulates the dorsoventral (back-abdomen) polarity of the insect. Four years later, it was noticed that fruit flies mutated by this receptor die from a fungal infection, but are resistant to other infections. This was the first indication that Toll proteins are the first to notify the immune system of the appearance of a pathogen. Soon, drosophila was found to have Toll receptors responsible for resistance to other microorganisms. It became clear that these receptors are involved in protecting the body from infections.

Entering the epithelial layer, they attach to TLRs and activate them. Epithelial cells begin to produce chemokines that attract cells of the innate immune system – neutrophils and macrophages – to this site. They phagocytize (devour) invading microbes. If there are many microorganisms, they activate TLR on the cells of the innate immune system. On the one hand, it enhances the phagocytosis of microorganisms. On the other hand, dendritic cells transfer the processed microbial antigen in complex with the MNC2 molecule to T and B lymphocytes and produce a set of cytokines. As a result, an adaptive immune response develops according to the first type (cytotoxic T-lymphocytes mature, killing microorganisms) or according to the second type (plasma cells mature, which produce antibodies that bind microorganisms).

Subsequently, TLRs were also found in plants, amphibians, fish, and other animals. By now, 13 genes encoding TLR synthesis have already been identified in humans, and this is most likely just the beginning. Apparently, TLRs are the most ancient family of proteins among the image–recognizing receptors.

By chemical composition, TLRs are transmembrane glycoproteins characterized by repeats enriched in leucine. Such structures spatially form the shape of a horseshoe, which is believed to be formed taking into account the molecular forms associated with the pathogen. This ensures that each type of TLR is specifically linked to a specific type of molecule present in a large group of microorganisms but absent from the host body. The specificity of the attachment is probably determined by the tertiary structure of these molecules.

Most TLRs are located on the cell surface, less often in the cytoplasm, in the area of the Golgi apparatus. The TLRs that bind to microorganisms that develop outside the cells (bacteria, protozoa, fungi) are usually found on the cell membrane. Viruses and other intracellular microbes are targeted by TLRs sitting on the internal structures of cells. TLRs work, as it turned out, in pairs, which helps to recognize significantly more types of substances. And if at first it seemed that TLRs recognize only the molecular structures characteristic of various microorganisms, then recently evidence has been accumulating that they can react to a variety of allergens.

TLRs are found on many cells of the body, and primarily on those responsible for innate immunity: macrophages, dendritic cells, eosinophils, mast cells, and normal killers. They are also found on T and B lymphocytes. The outer part of the TLR, which looks like a horseshoe, specifically connects to the molecular structure characteristic of a certain type of microorganisms. The other part of the molecule undergoes changes, leading to the activation of one of the signal transmission pathways from the periphery of the cell to the nucleus. Further, as a result of the activation of nuclear factors, RNA transcription begins, followed by protein synthesis. The cell is activated – it actively synthesizes a variety of cytokines.

So, a hundred years after the discovery of natural immunity factors in the body, it turned out that all their reactions, which develop in response to the introduction of a pathogenic microorganism into the body, are highly specific. But unlike adaptive immunity, where specificity is manifested to each of the millions of possible antigens, innate immunity has a limited number of receptors specific to conservative microbial structures inherent in the entire class of pathogens. For example, all gram-negative bacteria have a lipopolysaccharide that binds to TLR4. Another important difference between innate immune responses is that they develop quickly – within one to two hours, while adaptive responses take more than two days. This is easily explained: mature cells of natural immunity are already present in the body, and it takes time for specific lymphoid cells of adaptive immunity to multiply and mature.

But the most unexpected thing was the detection of TLR on dividing cells of the epithelium (mainly cutaneous) and endothelium. After all, it was previously believed that the epithelial integument of the body is nothing more than a mechanical barrier to infection – in this sense, they were compared even with the shell of a turtle. It follows from this that, penetrating through the mucous membrane or skin, the infectious agent immediately encounters a powerful specific response from the innate immune system, which, instantly recognizing the type of invading aggressor, quickly deploys actions to destroy it either independently or by attracting adaptive immunity. It turns out that image-recognizing receptors conduct the orchestra not only of innate immunity, but also of adaptive immunity.

It is the innate immune system that reacts first to the introduction of a pathogenic microorganism into the body. This happens as follows. The pathogenic microorganism enters the mucous membrane. Its epithelial cells use TLR to recognize and identify it – determine which class of pathogens it belongs to (gram–negative or gram-positive bacteria, fungi, viruses, etc.). As a result, epithelial cells are appropriately activated and begin to synthesize many molecules, including signaling molecules – chemokines (cytokines that stimulate the movement of other cells to the place where they are formed). They ensure the attraction of various cells of the immune system to this site – macrophages, neutrophils, basophils, eosinophils and mast cells, which are also activated by substances of the invading pathogen. As a result, an inflammatory microphage is organized, which quickly copes with the invading pathogen. All this is constantly happening in the body, and we don’t even notice it, because there are no clinical manifestations that we perceive as a disease.

Microorganisms attach to epithelial cells through TLR, as a result of which they are activated. Different types of epithelial cells perform different functions. Activated epithelial cells of the Goblet produce mucus, in which microorganisms useful to the host’s body live and multiply – the true microflora, to which the body is tolerant, and conditionally pathogenic microorganisms, which are affected by factors of the immune system, sharply limiting their division. In the intestinal lumen, outside the mucus, microbes do not multiply and die. Activated epithelial cells of Panet produce antimicrobial substances that are released into the intestinal lumen and neutralize microorganisms. Activated epithelial M cells transfer a microbial antigen prepared by them in combination with a protein molecule to Peyer’s plaques and other lymphoid formations of the mucous membrane, which triggers the synthesis of specific antibodies belonging to the class of immunoglobulins A (IgA). Finally, activated epithelial cells attract neutrophils and stimulate their release into the intestinal lumen. In total, all these factors inhibit the reproduction of microorganisms.

If a lot of pathogenic microorganisms are introduced and the innate immune system cannot cope with them, adaptive immunity is activated. In this case, the inflammation usually goes into the stage of a detailed process with the corresponding symptoms of the disease (pain, fever, etc.).

Thus, TLRs guide the reactions of not only innate immunity, but also adaptive, i.e. they determine not only the onset and nature of the immune response to the appearance of a foreign body, but also the type of immune response.

Another property of TLRs located in the epithelial cells of the mucous membranes and dividing skin cells is extremely important, namely– the specific targeted regulation of microflora. There are a huge number of microorganisms of different classes in our body – viruses in the cells of various tissues and organs, microbes and fungi on the mucous membranes and in the skin. Some of them become normal and permanent microflora, and the body’s tolerance is formed to it – immune reactions do not develop. This microflora brings great benefits to the body: it synthesizes vitamins necessary for the body, regulates intestinal motility, etc. The mechanism of formation of such tolerance has not yet been clarified.

In addition, many opportunistic and pathogenic microorganisms constantly pass through our body – this is the so–called passing microflora. We have long known that a large number of antimicrobial substances are formed on the surface of the skin and especially the mucous membranes, which stop or inhibit the reproduction of these microorganisms. The discovery of TLR on epithelial cells largely clarifies the mechanisms of such antimicrobial action. Now it is clear how the release of more than 90% of neutrophils formed in the body through the mucous membranes into the intestinal lumen is activated and how the innate immune system performs targeted specific regulation of the passing microflora.

Until recently, the main attention of scientists and doctors was focused on the body’s fight against pathogenic and opportunistic microflora living on our mucous membranes and skin, and few people thought about the possible role of such microorganisms in regulating and maintaining the normal activity of the immune system. However, long-standing data indicate the crucial role of this diverse microflora in maintaining the normal activity of our body’s immune system. The existence of a mammal, including a human, without microbes is quite possible, as evidenced by the already mentioned work with animals that were kept in sterile chambers. However, their life requires a fundamentally different diet, and their removal from sterile conditions without prior adaptation ends in rapid death from microbial shock.

Something similar happens to humans. After a long stay in confined spaces, their microflora (for example, polar explorers or astronauts) is sharply depleted, leaving only those microorganisms to which the body is tolerant. Note that the total amount of microbial biomass does not change. After people return to normal conditions, they develop acute respiratory diseases and intestinal catarrh, which usually pass quickly. These conditions were called the “disease of the first port” (the name comes from the time when polar explorers, who spent many months wintering in Antarctica in a closed group, after the first stop of the icebreaker in Sydney, all, without exception, fell ill with acute respiratory infections and dyspepsia).

The sharp increase in the number of allergic diseases worldwide is obviously related to the same reasons. However, it is well known that people living in rural areas suffer from these diseases much less frequently than the urban population of developed countries, where sanitary and hygienic conditions are much better (which makes it possible to avoid epidemics). In rural residents, the body’s contamination with microorganisms is significantly higher and the immune system is in a “trained” state.

Since the time of Mechnikov, it has been proven that regular intake of probiotics (for example, fermented dairy products containing live microbes useful for the body) leads to a reduction in the incidence of allergies and increases the body’s resistance to infections. But these microbes are a passing microflora for the body, because after the end of their intake they quickly disappear. Everything points to the importance of microflora in maintaining the normal functioning of the body’s immune system, and now we can explain the mechanisms of this phenomenon.

Toll-like receptors are genetically determined, so they are present in small quantities on cells of the natural immune system (as well as on other cells of the body) already at the birth of a child. They are also present on the cells of antimicrobial animals, although in minimal quantities. Only after contact with the microflora does the number of receptors increase [14]. Consequently, the cells of our body can be in an active and inactive state, depending on the amount of TLRs that appear on them. To activate cells, i.e. to increase the amount of TLR on them, they must be constantly irritated by microbial products. However, the normal permanent microflora cannot perform this function of TLR activation on cells, since they are tolerant to it. And only the passing microflora, which includes conditionally pathogenic microorganisms, can support the cells of the innate immune system in an active state. That is why it is extremely important for the body.

At the turn of the century, immunology once again reached a fundamentally new level. A common TLR regulatory system has been discovered, which coordinates the entire immune system. When a small amount of a foreign antigen appears in the body, only the evolutionarily old innate immune system comes into play. If she can’t cope, TLR is connected and a new evolutionary acquisition, adaptive immunity, is regulated. It is equally important that due to TLR, the immune system, among other things, monitors a set of microorganisms that are optimal for human life.

It is clear that shifts in TLR composition and violations of their regulatory function can cause a wide variety of pathologies. Thus, a decrease in TLR activity affects the microbial biocenosis. And then the conditionally pathogenic microflora becomes the permanent microflora of the body, which leads to atypical forms of inflammatory processes caused by conditionally pathogenic microorganisms that do not develop in the normal state of TLR. Increased TLR activity, on the contrary, is associated with the development of allergic and autoimmune diseases. It is also likely that pathological conditions in the body caused by the decreased activity of some TLRs may lead to an increase in the activity of others with the development of corresponding ailments.

There is no doubt that the new immunology is only taking the first steps, and it is probably too early to draw concrete conclusions for practical medicine. Nevertheless, drugs that block or activate TLR are already being prepared for release. Probably, discoveries of more effective methods of treating chronic inflammatory processes, including allergic ones, and ways to prevent their occurrence will soon follow.

Authors: Konstantin Alekseevich Lebedev, MD, Professor, Head of the laboratory. Clinical Immunology of the Moscow State Medical and Dental University.

Inna Dmitrievna Ponyakina, Candidate of Biological Sciences, Deputy Head. the same laboratory.