Journal of Theoretical Biology 375 (2015) 61–70

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On the origin of immunopathology Nelson M. Vaz a,n, Claudia R. Carvalho b a b

Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, MG, Brazil Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, 31270-901, MG, Brazil

art ic l e i nf o

a b s t r a c t

Article history: Received 8 April 2014 Received in revised form 2 June 2014 Accepted 4 June 2014 Available online 14 June 2014

Stranded between medicine and experimental biology, immunology is buried in its own problems and remains distant from important areas of current biology, such as evolutionary theory, developmental biology and cognitive sciences. Immunology has treated the living system merely as the place or dimension in which immune activity takes place, inserted on a misleading axis (progressive responsiveness versus no response; memory versus tolerance) which neglects the analysis of a robustly stable dynamics which is always present and is neither tolerance nor immunity—a problem currently approached as one of “regulatory” activity. However, a regulatory response also demands regulation, leading to an endless recursion and the adoption of a stimulus–response framework inevitably drives us away from the physiological processes in which lymphocytes are involved. Herein, we propose that immunological physiology, like everything else in the body is dynamic and conservative. Immunopathology, including inherited immunodeficiencies, severe forms of infectious diseases, allergy and autoimmune diseases, are interferences with this stability which frequently include oligoclonal expansions of T lymphocytes. We suggest that this decrease in clonal diversity results from a loss of the stabilizing connectivity among lymphocytes and are not simply markers of immunopathology, but are rather expressions of basic pathogenic mechanisms. The so-called autoimmune diseases are examples of this disequilibrium. In the last decade the characterization of an enormous and diversified commensal microbiota has posed a new and pressing problem: how to explain the harmonic conviviality with trillions of foreign macromolecules. In addition, robustly stable relations towards macromolecular diet can be established by simple ingestion, a state presently labeled as “oral tolerance”, a problem that has been buffered for decades as anti-inflammatory protection of the gut. A major change in terminology is necessary to describe this new panorama. We focus on two important gaps in immunological discussions: (a) the organism, seen simultaneously as the medium with which the immune system is constantly in touch and as the entity that mediates the contact with external materials; and (b) the observer, the immunologist, who operates as a human being in human languaging with other human beings, and characterizes immunological specificity. We acknowledge that we are proposing radical departures from current dogma and that we should justify them. Most of what we propose stem form a way of seeing called Biology of Cognition and Language, that derives from ideas of the neurobiologist/ philosopher Humberto Maturana, also known as “autopoiesis theory”. & 2014 Elsevier Ltd. All rights reserved.

Keywords: Cognition Systems Organism Observer Oligoclonality

1. Introduction The immune system and the nervous system have been repeatedly compared because both may be seen as networks (of lymphocytes or neurons) and are relational systems, which, at the same time, separate and insert or place the living system in contact with its medium. But, while neurobiology is closer to cognitive sciences and epistemology and their questions, immunology is closer to medicine

n

Corresponding author. E-mail address: [email protected] (N.M. Vaz).

http://dx.doi.org/10.1016/j.jtbi.2014.06.006 0022-5193/& 2014 Elsevier Ltd. All rights reserved.

and its urgencies. However, in looking for explanations of autoimmune diseases, can we proceed without cognitive questions? The way of seeing determines what we ask and the criteria of accepting answers. Are we looking for answers, or for different questions? In our way of seeing, to explain autoimmunity we need a theoretical framework wider than current immunology can offer. The description of a robustly stable lymphocyte activity – that is neither immunity (memory) nor tolerance – requires a new terminology that is still unavailable. We are not dealing with the “regulation of immune responses”, but rather with a different understanding of lymphocyte activity, a search for its physiology that in our way of seeing is conservative and maintains steady

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sates of lymphocyte activation. Approaching this aim led us to face two subjects of huge dimensions carefully avoided in biological discussions: (a) the living organism; and (b) the immunologist operating as a human observer in human “languaging”. Most of our arguments stem from a way of seeing called Biology of Cognition and Language, deriving from ideas of the neurobiologist-philosopher Humberto Maturana and co-workers also known as “autopoiesis theory”—a less adequate term. We do not intend to provide a complete synopsis of his work and we are entirely responsible for misunderstandings that may arise from our partial account of it. Nothing is as effective as reading his original work, from which we suggest three references: a short book chapter which we find to be particularly clear (Maturana, 1987); and two accounts of the history of Maturana's basic concepts: a book with a long interview with Maturana and Poerksen (2004) and a more technical account by himself (Maturana, 2002). Describing living systems as molecular autopoietic (self-constructing, self-maintaining) systems, i.e., as living machines turned upon themselves, does not mean much. Maturana sees the human observer in his observation and his engagement in human “languaging” as the main problem. He defines “languaging” as a way of living (an ontogenic phenotype), rather than as the transmission of symbolic information (Maturana, 2002). He claims that the brain and the nervous system are not in direct contact with the external medium and that the brain does not acquire and process information. The medium in which nervous system operates as a component (a sub-system) of the organism, is the organism itself. The medium in which the organism operates is a meta-medium for the nervous system. The world (the structural domain) in which the nervous system operates (the living system) is very different from the world (the relational domain) in which we operate as whole organisms. There is a ceaseless dance of structural perturbations and compensations between the nervous system and the organism, but it is the organism, as a whole, not the nervous system that deals with the external medium. In applying these ideas to immunology, we propose the definition of a conservative (invariant, closed) organization for the immune system, which may be seen as a singular entity operating in the organism as the medium that makes it possible. As a component of the organism, the immune system never touches the external medium. As external observers, we are perfectly able to see that components of the external medium routinely and/or accidentally penetrate the organism. From our privileged position we may also detect significant changes in the molecular and cellular structure of the immune system concomitant or subsequently to these exposures. We may then be misled to believe that these changes were caused (instructed, informed, determined) by the interaction with these foreign, invading materials—a specific immune response. However, in our way of seeing self/nonself discrimination can only be done by the immunologist. We aimed to describe a model in which pathology and physiology are the two sides of the same coin and to propose a common basis for the different forms of immunopathology presently recognized: immunodeficiency, allergy and autoimmunity. The adoption of a stimulus–response framework inevitably drives us away from the physiological processes in which lymphocytes are involved. We propose that pathology derives from altered perturbations and compensations of the closed organization of the immune system. This is not easy to accept because our knowledge painfully lacks a physiology of which immunopathology is a defect or deviation. It is quite clear, however, that the so-called immune system is not idle in the absence of immunopathology. We will also outline other concepts that we think are important to develop our argument on the implication's of autopoiesis for immunology and allow the reader to follow it.

2. Part 1—Responsiveness or autonomy? The present understanding of immunological activity presently face five interconnected fallacies, which may be summarily described as follows. 2.1. The fallacy of instruction An important example of misleading argument is to believe that, in its changes, dynamic systems follow (obey) the changes of the medium in which they operate. However, changes in living (and non-living) systems are determined by their own structure. Actually, it is the structure of the system that determines with which aspect of the medium they may interact. This is the opposite of believing that the medium acts as a template, which specifies (guide, orient) what happens with the system (Maturana, 2002). In the 1950s, experimental evidence of different kinds led to the rejection of instructive theories of antibody formation in favor of “selective” theories (Jerne, 1955) according to which antibody formation precedes the contact with antigens. 2.2. The fallacy of selection The word selection may suggest a voluntary choice between multiple alternatives; facing changes in the medium, the organism would choose some components or characteristics to implement its actions. This is exactly the opposite of what happens because an interaction can only specify a structural change in a system if this is already made possible by the previous structure of the system, and not by the structure of the interacting element. The word “selection” is deeply connected to the history of biology due to the notion of natural selection proposed by Darwin as an important factor in the origin of the species. Darwin himself stressed that this was only an adequate metaphor, but in spite of his warning, selection is frequently used to denote an instructive (guiding, orienting) influence of the medium. Maturana and Mpodozis argued that differential survival could be the result of the evolutionary process and not the mechanism that generates it Maturana and Mpodozis (2000). 2.3. The fallacy of specificity This fallacy has a double interpretation. First, specificity is highly degenerated. A single TCR may bind a million of different peptides (Wooldridge et al., 2012). One of the consequences of the loose and heterogeneous binding affinity of lymphocyte receptors, is that self/nonself discrimination is no longer a tenable hypothesis (Wucherpfennig et al., 2007). Second, immunological specificity is actually observer-dependent, i.e., is created by the immunologist as a human being in interaction with other human beings in human languaging and projected into the living organism; specific antibodies are functional entities created by pasting functional labels onto natural immunoglobulins (Vaz, 2011). “As horrifying this may be to hyperempiricists who neglect the observer, physics is necessarily the study of the behavior of physicists, biology the study of biologists and so on” (Provine, 2013). Immunology is the description of what immunologist do as observers (Vaz, 2011). 2.4. The fallacy of isolation The present characterization of the native microbiota will necessarily eliminate the idea that the organism is isolated from the contact with “foreign” macromolecules, but this was already negated by the daily absorption of immunologically significant amounts of intact dietary macromolecules during regular feeding of adult normal organisms. We have never been macromolecular

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islands: there is an intense and continuous transit of macromolecules between the organism and the macromolecular medium in which it operates and which make it possible. 2.5. The fallacy of immunological memory which presupposes a progressive kind of reactivity that would rapidly drive clonal expansions to destructive limits when the process of eliminating the antigen failed, or when the organism is frequently met in the medium. This paradox motivated the present emphasis on regulatory mechanisms and regulatory components. 2.6. To respond or not respond: Is this the question? For many reasons, immunology remains stuck in a stimulus/ response/regulation framework. In part, this happens because an alternative framework is not clearly apparent. Living systems, however, are autonomous entities that specify (guide, orient) what happens to them, and the same may be said of their sub-systems, such as the nervous and the immune systems. To understand the autonomy of living systems we must accept that they are not simply responders to environmental stimuli (Maturana, 2002).

3. Part 2—A major gap: The organism 3.1. The organism and the living system Living systems may be described as molecular self-created/maintained (autopoietic) systems (Maturana, 2002), a structural definition that only applies to the molecular/cellular realms (Maturana, 2014). However, living systems are also described as whole entities (organisms) in interaction with other living and non-living entities in their medium—a relational domain. The structural and the relational domains of description are separate and non-intersecting; knowing what takes place in one of these domains does not allow us to predict what is taking place in the other domain. Many different structural variations may fulfill the same role in an interaction of the system as a whole; reciprocally, many different interactions of the system as an entity may be reflected in the same specific changes in the structural domain. Thus, what happens in one domain cannot be inferred by what is known to happen in the other domain. The same reasoning applies to subcomponents of living systems, such as the nervous and the immune system of vertebrate organisms, i.e., they may be described in a structural domain in which their components and interactions among these components are distinguished; and they also may be seen as whole entities interacting with the medium in which they operate and which makes them possible. The organism is the medium for the immune system. The medium in which the organism operates is a meta-medium inaccessible to the immune system. Self/nonself discrimination is a pseudo problem because the immune subsystem is in constant contact with organism and never met external materials. Another important misunderstanding is to believe that the immense antibody and cellular diversity of the immune system allows any kind of interaction with foreign materials to take place (Fig. 1). This is very different from what was found in the 1970s, for example, in the characterization of Ir-genes (McDevitt and Chinitz, 1969; Vaz et al., 1970; Benacerraf and McDevitt, 1972). The activation of lymphocytes and the consequent production of antibody are usually taken to be proportional to the immune responsiveness. The strongest responses (blast transformation) of T cells are seen when they are exposed to mononuclear cells of a MHC-incompatible individual of the same species—as in alloreactive mixed lymphocyte reactions used to select human donors of transplants. This in itself is very unexpected. Even more strange is that the exposure to

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mononuclear cells of another animal species, even closely related species (e.g., mouse to rat cells) result in weaker, not stronger lymphocyte activation; even this weak activation disappears when the responding lymphocytes come from a germ-free animal (Wilson and Fox, 1971). Thus, lymphocyte responses are not directed to any foreign materials. The activation of B cells is either dependent of T cell activation or depends on mitogenic (unspecific) activation—as in responses to bacterial LPS (Coutinho, Möller, 1973). 3.2. Epigenesis Self/nonself discrimination is unable to explain immunological activity because it is not its generative mechanism, although it may be described as a result of it. The formation of specific antibodies and/or the activation of T lymphocytes do not occur because they were destined to meet specific foreign epitopes. Nascent lymphocytes have no predetermined targets, although their survival is conditioned to signaling through the clonal receptors they display. The operation of the immune system is a prime example of epigenesis because the genes that codify the chains of clonal receptors are not inherited, but emerge de novo in each organism. Immunological activity is epigenetic: its future is not contained in its present and the present was not yet determined in its past. The clonal receptors invented by nascent lymphocytes have no defined target, but the survival and eventual expansion of the lymphocyte, or its solitary disintegration, depends on whether these receptors happen to bind to previously activated cells. Lymphocytes, which manage to mature after inventing a clonal receptor on its membrane, either survive by engaging on what is already going on, or die by apoptosis. The organization of lymphocyte networks in the embryo or newborn animal starts in the presence of large amounts of transferred maternal immunoglobulins (Lemke et al., 2004). From then on, it depends fundamentally on what the organism does as a whole entity. The difference between self-determination and selfignorance is depicted in Fig. 2. On Fig. 2, an “immune system” is utterly simplified to suggest that it has a circular organization closed upon itself. The idea that the organism is guided by changes in internally generated patterns is not new in biology. Many animals generate an electric field around their bodies and orient themselves by alterations in this field triggered by the presence of other organisms. Similarly, we suggest that the immune system maintain patterns of activity, which are expressed, for example, in robustly conserved profiles of reactivity of natural immunoglobulins (Nobrega et al., 2002; Cohen, 2013). Perturbations in these patterns in autoimmune (Ferreira et al., 1997) and parasite diseases (Vaz et al., 2001) derive from compensatory changes by activation of some components and inhibition of others. In this way of seeing, the immune system is a closed subsystem of a closed larger system, which is the organism as a whole. 3.3. Perturbations and compensations of a network of relations between lymphocytes The activation and eventual clonal expansion of lymphocytes happens as compensations of perturbations of the connectivity, which mediate the dynamic stability of a highly connected network of lymphocytes and lymphocyte products with the organism (Jerne, 1974; Vaz and Varela, 1978). The reconstruction of this network is a continuous process that maintains an invariant organization amid a ceaseless replacement of components. The invariance of the organization is expressed, for example, on robustly stable profiles of reactivity of natural immunoglobulins (Nobrega et al., 2002; Cohen, 2013). Perturbations of this organization arise through variations in the dynamic of the system itself and/or by the invasion of the organism by external materials.

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Fig. 1. The organism and the immune system. The immune system in the organism: (A) immunity (clonal selection) – The standard way of seeing: the immune system interacts with an external medium of antigens, which stimulate the production of antibodies which help their elimination from the organism. (B) Immunobiology (conservative physiology): The living system and some of its subsystems are shown as dashed circles with arrows indicating their internal closed dynamics. The immune system interacts with the living system and other subsystems, such as the nervous system. Living systems have also a closed internal dynamics indicated by an arrow in the circle. Only the living system as a whole entity (the organism) interacts with the external medium. The double arrows between the subsystems and between the organism and the medium represent structural perturbations and compensations, not stimuli and responses.

Fig. 2. Self-ignoring and self-determination: (A) self-ignoring. Environmental antigens (a, b, c) are recognized by unities (cells, cell clones, sets of cell clones) anti-a, anti-b, anti-c independent from each other, which expand and help to eliminate the antigens. (B) Self-determination: the recognizing unities are interdependent and generate a cycle of internal interactions, the system closure. Unity anti-a recognizes a structure (a) which is a normal component of unity anti-c; which recognizes (c) on unity anti-b; which recognizes (b) on unity anti-a, closing a circuit. Idiotypic connections (Jerne, 1974) are just one kind of link binding together these unities (cells, cell clones, sets of cell clones). The important point is that this circuit (this “closure”) is maintained invariant in spite of structural variations and represents the organization of the system (Vaz and Varela, 1978). Environmental antigens (a', b', c') are confounded (degenerate specificity) with determinants a, b,c. The two hypothesis are not incompatible, but the first hypothesis is incomplete, since it lacks the internal connectivity through which the system self-creates and self-maintains itself. (What is shown here as “B” stands for the circle labeled “immune system” in Fig. 1).

3.4. Immune physiology is conservative The organism is frequently penetrated by macromolecular compounds of the diet (Vaz et al., 1997; Faria and Weiner, 2005; Pabst and Mowat, 2012) and products of the native microbiome (Palmer et al., 2007; Grice et al., 2009). The immune system is also involved in the clearance of senile or damaged cells and molecules (Grabar, 1975) and in the maintenance of body structure as illustrated by the importance of gamma-delta T cells for epidermal integrity (Heath and Carbone, 2013; Jameson et al., 2002). The consequences of these contacts, which comprise the overwhelming majority of connections with external materials, however, are not progressive immune responses and the organism does not develop an immunological memory of such events. Born in the study of infectious diseases, immunology is still concerned in

explaining immunopathology, but has neglected the most frequent events of immune physiology. 3.5. On creation, destruction, preservation In the XIX century, Carl Weigert, Paul Ehrlich’s cousin, who influenced him to create the “side chains” theory of antibody production, proposed what he called the “Siva theory” of pathology. In a detailed review of allergy, autoimmunity and pathology in the first half of the twentieth century, Parnes (2003) writes, and we quote: “In Hinduism, Siva (read Shiva) is one of the three primary gods consisting of Brahma, the creator; Vishnu, the preserver; and Siva, the destroyer. However, Siva's destructiveness is a constructive one, as he destroys in order to create new entities. The destruction was thus aimed at regeneration…” Creation,

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destruction and preservation, the holy trinity of Hinduism, also rules in biological systems. Elsewhere, Parnes also claimed that immunological activity could be viewed as incorporation of new components instead of interception of foreign materials (Parnes, 2004), a concept compatible with our interpretation of “oral tolerance” (Vaz and Carvalho, 1994; Vaz et al., 1997). Parnes (2003) mentions that: “To Carl Weigert,…bacteria may be the cause of disease, but they did not explain it.” Weigert proposed that every disease process begins with a “primary lesion, but the disease itself was the body's reaction to this primary lesion. “The inflammation was also part of this reactivity, which was essentially reparative, but often overshot and caused more harm than the original, primary lesion” (Parnes, 2003). This is similar to the idea we are proposing by pointing to the pathogenic significance of oligoclonal expansions of lymphocytes.

4. Part 3—Oral tolerance The arguments briefly outlined above gave rise to a number of experiments and, more important, to our particular way to interpret them. To illustrate the consequences of applying another way of seeing in the proposal of experiments that we have been doing for the last 20-30 years, we will concisely discuss a phenomenon misnamed as oral tolerance (Vaz et al., 1977), mistakenly defined as inhibition of immune responses to proteins previously given by the oral route. Oral tolerance may be triggered by other mucosae, may be established by simple eating of drinking solutions containing a T-dependent antigen, such as ovalbumin, snail hemoglobin, or antigen containing materials, such as raw peanuts and It may be transferred to naive recipients with spleen cells (Richman et al., 1978; Brandtzaeg, 1996; Faria and Weiner, 2005; Pabst and Mowat, 2012). One of our important findings was that tolerance is not an inhibition, but a stabilizing of immune responsiveness. Tolerant organisms produce small amounts of specific antibodies to the tolerated antigens, but this residual responsiveness is different from that of naive animals, because it remains stable even after repeated boosters with the specific antigen in adjuvant (Verdolin et al., 2001). This is a significant finding because the same stability is observed in the responsiveness to autologous antigens. The existence of autoantibodies (Avrameas and Ternick, 1995; Coutinho et al., 1995) and selfreacting activated T cells (Pereira et al., 1986) in healthy animals is unquestionable, however, healthy organisms are not undertaking progressive secondary-type immune responses to self-components—except, perhaps in some autoimmune diseases. In healthy organism, self-responsiveness is present, but is stable in a similar way that responsiveness is stable in oral tolerance, after mucosal exposure to proteins. As we argued in the previous section, the medium in which the immune system operates is the organism of which it is a component. When the need to describe the immune system in two separate non-intersecting domains is acknowledged, self/ nonself discrimination, which is a central issue in traditional descriptions, becomes a pseudo-problem and no longer requires an explanation; the relevant problem is the maintenance of the dynamic stability of the immune system. The seminal experiments of Medawar and coworkers that created the notion of specific immunological tolerance by clonal deletion (Billingham et al., 1953) were crucially important in the proposal of the clonal selection theory a few years thereafter (Burnet, 1957; 1959). Coutinho and co-workers repeated these experiments and showed that there was no deletion of alloreactive clones in tolerant animals; the number of these cells was increased and they were activated (Bandeira et al., 1989). More recently, Castro-Junior et al. (2012) showed that something similar occurs during the induction

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of oral tolerance. When orally tolerant mice are injected with the tolerated antigen in adjuvant, the production of specific antibodies of all isotypes (IgG, IgM, IgA and IgE) is inhibited, but the production of unspecific IgM and IgA is stimulated. A second important and large set of observations pertains to the consequences of injecting tolerated antigens (with adjuvants) into tolerant animals. These injections trigger anti-inflammatory processes that is able to block the inflammatory action of carrageenan (Ramos et al., 2008). This inhibition is probably important in blocking the initiation of antibody responses to unrelated antigens and even the injection of self-component in adjuvants may trigger the inhibitory effects (Carvalho et al., 1994, 1997; Carvalho and Vaz, 1996). Diverse other phenomena are modified by injection of tolerated proteins, such as: (a) inhibition of parental-to-F1 Graft versus Host disease in mice (Vaz and Carvalho, 1994); (b) marked inhibition of granuloma formation around Schistosoma mansoni eggs (Carvalho et al., 2002; Azevedo et al., 2012); (c) improvement of wound healing in mouse skin (Costa et al., 2011); (d) reducing fibrosis in rat heart after myocardium infarction by overdoses of isoproterenol (Ramos et al., 2012). Experimental arguments contradict the hypothesis suggesting that these effects result from “antigen bystander suppression” (Vaz et al., 1981; Miller et al., 1991; Carvalho et al., 1997). These results clearly demand a reinterpretation of the nature of immunological tolerance and adopting ideas derived from the autopoiesis theory allows the attention to be focused on the closed, recurrent and continuous quality of immunological activity, rather than on the sporadic changes that, as observers, we register as specific immune responses, and their suppression or regulation. In adopting another way of seeing we could find that exposure to tolerated antigens under immunogenic conditions has broader (general) biological and medical significance through presently undefined systemic effects that interfere with a wide array of activities, among which cutaneous wound healing (Costa et al., 2011) and embryo implantation in mouse uterus (Galdino et al., 2013).

5. Part 4—Are autoimmune diseases misguided immune responses, which harm body tissues? Do autoimmune diseases result from progressive, harmful, misguided immune responses to tissue antigens, as usually interpreted in a stimulus/response/regulation framework? Or, shall we see them as destabilizing processes manifested by oligoclonal expansions derived from restrictions of clonal diversity? (Pordeus et al., 2009). The control of autoimmune responses, presently attempted by immunosuppression is inefficient and has severe undesirable side effects, including the promotion of oligoclonality. Other forms of therapeutic interventions aiming the control of oligoclonal expansions, by increasing connectivity and thus restoring clonal diversity, are conceivable. This is not usually acknowledged. 5.1. Oligoclonal expansions Oligoclonal expansions of T lymphocytes may be seen as markers of pathologic situations, including immunodeficiency (Wong and Roth, 2007), autoimmune (Jones et al., 2013), allergic (Davies and O’Hehir, 2004) and severe infectious diseases (Finger et al., 2005), and dozens of other examples of which a few are listed in Appendix A. A causal link between these oligoclonal expansions and autoimmunity, triggered by previously states of lymphopenia has been suggested by several authors (OlivaresVillagomez et al., 2000; Min et al., 2004; Baccala and Theofilopoulos, 2005; Khoruts and Fraser, 2005; Milner et al., 2007). However, this was neither based on a general interpretation of the nature of immunological activity as we proposed elsewhere (Vaz and Varela, 1978; Vaz et al., 2006; Pordeus et al., 2009; Vaz,

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2011), nor related to the creation and maintenance of states of dynamic stability, as we have shown to happen in “oral tolerance” (Verdolin et al., 2001; Pordeus et al., 2009; Azevedo et al., 2012). However, oligoclonality in itself should not be taken as the direct pathogenic mechanism of these conditions. Hundreds of monoclonal or near-monoclonal organisms have been generated by genetic manipulation with different research objectives, which cannot be said to live normal lives, but survive and reproduce without overt signs of immunopathology. The source of pathology is a skewed oligoclonal immune system, with the loss of its dynamic stability; the reduced clonal diversity in itself is a reflection of this potentially pathogenic situation. 5.2. Embryo development and autoimmunity Ongoing progress in the understanding of tissue architecture have suggested that mechanisms usually described as responsiveness to foreign invasions, may also be described as mechanisms of repair belonging to a dynamics of self-creation and selfmaintenance that does not rely on self/nonself discrimination. For example, the TCR on dendritic-like resident T cells in the skin are of very limited diversity and are activated when they bind to peptides generated on damaged keratinocytes and this triggers inflammatory reactions (Jameson et al., 2002; Mueller et al., 2013). In the last few decades, significant advances were made in the understanding of biological development. These advances allowed the demonstration that diseases classically ascribed to autoimmune mechanisms, such as type-1 diabetes, may derive from defects in the expression of transcription factors, such as Hox-11 during embryonic development (Lonyai et al., 2008). These alterations in morphogenic processes precede the pathogenic activity of lymphocytes upon the pancreas and determine as well other abnormalities in development, such as in salivary and tear glands and in the cochlea in the internal ear. For these reasons type1diabetes is frequently associated with Sjögren, or sicca syndrome and severe auditory deficits. NOD (Non-Obese Diabetic) mice, which have been extensively used as experimental models of type-1 diabetes, were recently found to be almost deaf (Lonyai et al., 2008). Thus, the chain of events that leads to type-1 diabetes is more complex and delicate than the simple emergence of autoreactive lymphocyte clones that damage the pancreas, as previously believed. Similarly, there is evidence that in experimental type-2 diabetes in rats, damage to the retina depend on neurologic alterations in the bone marrow and changes in circadian rhythms (Busik et al., 2009). Thus, the damage is not restricted to the vascular bed of the retina. Something even more delicate seems to happen in Crohn's disease, a disease of the human intestine supposed to be autoimmune. An experimental model of Crohn's disease suggests that the lesions depend on: (a) a mutation in a gene related to autophagy (ATG16L1); (b) from a viral infection; (c) from the intestinal microbiome; (d) from immunological activity; and, finally, of a concomitant damage to the intestine. The lesions derive from a failure in regeneration derived from three environmental factors associated with a mutation (Cadwell et al., 2010). 5.3. Revisiting anti-infectious vaccination A systemic understanding of immunological activity may provide an alternative explanation for the (also erratic) efficiency of antiinfectious vaccination. If vaccines owed their efficiency to immunological memory they would not be so difficult to invent. Something else is clearly at stake. Based on the idea that immunopathology stems mainly from ruptures in the immune system ‘closed’ organization, which allows pathogenic oligoclonal expansions with loss of

dynamic stability, we proposed that severe forms of infectious diseases occur only in those members of a population who were prone to develop oligoclonal expansions in contact with the wild infectious agent, and that anti-infectious vaccines work by previously expanding their clonal diversity, rather than by intensifying their responses to specific epitopes. If this were even partially true, research on the design of anti-infectious vaccine should follow quite different pathways (Pordeus et al., 2009). 5.4. Intravenous immunoglobulins in high doses Although developed empirically, and for anti-infectious protection, the use of intravenous immunoglobulins in high doses (IVIg) may be an example of interventions, which add lost connections among lymphocytes. The treatment may owe its erratic efficiency to our ignorance of which specific clones of lymphocytes should be introduced to restore lost connections. There is preliminary evidence that immunoglobulins selected by binding to antigens putatively involved in the autoimmune process have an enhanced efficiency (Svetlicky et al., 2013; Blank et al., 2014). This way of seeing could also help to devise new methods of diagnosis of autoimmune diseases by analysis of clonal diversity (pattern recognition), especially of T cells, by methods already available. The therapeutic effects of “vaccination” with T cells (Cohen, 2009) and with modified peptides derived from self-components (Raz et al., 2007; GershoniYahalom et al., 2010) are conceivably due to similar mechanisms.

6. Part 5: IgE production 6.1. IgE and immunopathology IgE is traditionally studied as a mediator of type I hypersensitivity reactions that contribute to the pathogenesis of allergic diseases such as asthma, allergic rhinitis and atopic dermatitis. It is also involved in protective immunity against some helminth parasites. Experimentally induced IgE responses are considered to be short-lived, but this depends on the adopted model. Many years ago, we have shown that immunization of mice from high responder strains with minute doses (0.1–1.0 mg) of potent T-dependent immunogens in Al(OH)3 elicits strong and persistent IgE responses, similar to those found in human allergic patients. Injected with higher doses (100 mg) of the same antigens, these same strains displayed only transient formation of IgE, in spite of prolonged IgG formation (Levine and Vaz, 1970). This model was not further investigated because, in those same experiments, we were able to characterize the first MHC-linked genes controlling immune responsiveness (Ir-genes) to protein antigens (Vaz et al., 1970,, 1971) and this drove our attention away from IgE production. At that time, we ascribed the transience of IgE responses to higher doses to the activation of suppressor T cells (Levine and Vaz, 1970), which would be presently called T regulatory cells (Tregs). However, we now interpret these results as indicating that minute doses of potent T-dependent immunogens evoke oligoclonal T cells responses in high responder animals; these same doses are unable to immunize low-responder animals, and higher doses evoke polyclonal responses in both high- and low-responder strains. This way of seeing is in line with many other unrelated observations linking IgE production to autoimmune diseases, GvH reactions, intoxication with heavy metals and a series of other conditions. 6.2. IgE and oligoclonality Increased IgE formation is frequently associated with oligoclonal expansions of T lymphocytes and, consequently, with

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immunopathology, in general, not only of allergic diseases as commonly understood (Cahenzli et al., 2014; Wu and Zarrin, 2014). As a component of the dynamics of the immune system, the formation of IgE may possibly contribute to reduce its own formation through a reduction in oligoclonality. IgE is very efficient in triggering powerful inflammatory reactions, therefore collaborates in expanding the variety of cellular interactions in the organism and, thus, favors activating interactions, which increase clonal diversity, and results in a reduction in oligoclonality. The formation of IgE may cyclically hinder its own formation. Mutant mice bearing monoclonal populations of B and T lymphocytes specific for influenza virus hemagglutinin (HA) and chicken ovalbumin (OVA), respectively, when injected with crosslinked OVA-HA, produced IgE concentrations up to 200 mg/ml. IgE production was lessened when the animals were adoptively transferred with polyclonal compatible T cells; the rate of reduction being proportional to the number, of regulatory T cells transferred, and thus their diversity (De Lafaille et al., 2001). However, subsequent findings of the same group showed that these quasi-monoclonal mice, when orally exposed to OVA, developed oral tolerance, even in the absence of naturally occurring regulatory T cells (Mucida et al., 2005; Bilate and Lafaille, 2012). Spontaneous experimental autoimmune encephalomyelitis arises in 100% of mutant mice exclusively harboring myelin basic protein-specific T cells, and can be prevented by a single injection of CD4 þ T cells obtained from normal donors. Transfer of monoclonal OVA-specific CD4 þ T cells did not confer protection from disease even when present at an 80% proportion. However, protection was conferred by cells bearing limited TCR diversity, including cells expressing a single V alpha4 TCR chain or cells lacking N nucleotides (Olivares-Villagomez et al., 2000).

7. Part 6—Self-defense or self-construction? Herein we have argued that immunologic protection, usually understood as a dedicated mechanism of detection and elimination of foreign materials, derives from physiologic (non-cognitive) mechanisms of self construction and maintenance of the organism, a way of seeing previously suggested by Grabar (1975). Schoolchildren are often shown that beans germinating inside a cardboard box with a small window on one side incline toward the light source. This happens because light slows cell division and plant cells in the darker side of the plant stem divide more rapidly. The inclination of the plant toward the light source is a result of differential cell division, not a cognitive decision of the plant, contradicting common-sense expectation. Many years ago, in the laboratory of Michel Rabinovitch, at NYU, one of us had the pleasure to watch experiments on phagocytosis in which free-living amoebas (Achantoamoeba) were exposed to mixture of sheep and horse red cells (Rabinovitch, 1970). The two types of red cells could be readily distinguished by size, the sheep red cells being significantly smaller. I can hardly forget what I saw under phase microscopy. The moving amoebas would repeatedly collide with horse red cells and apparently pushed them aside, as if ignoring them, but rapidly engulf the sheep red cells they touched, as if preferring them. However, the “choice” of sheep red blood cells, that we were observing, was never an option for the amoebas. The engulfment of sheep red blood cells depends on the presence of galactose residues on their membranes, which were lacking on horse red blood cells. The amoebas were blind to horse cells. As every other living system, amoebas are structurally determined. Ascribing intentions to a system is part of our descriptions of the system's behavior in the

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medium in which it operates; the intentions are not part of the structural dynamics of the system. These are examples of confounding mechanisms and results of their operation in the discussion of biological problems. Thus, when understood as a mechanism, specific immunological defense acquires a cognitive quality, which often is not acknowledged. To protect the body against foreign materials, a previous discrimination between foreign and familiar molecules must take place, and this requires the definition of a cognitive agent or agency. On the other hand, understood as result of the operation of a mechanism, which we call an “immune system”, the dynamic structure of the immune system is not a cognitive mechanism. In our way of seeing, immunological activity is not a cognitive activity; lymphocytes do not recognize antigens, or keep memories of these encounters. These comments might help to analyze again notions such as memory cells, or regulatory T cells, which are abundant in current discussions. Immunology still lacks a definition of the organization of the immune system, i.e., a set of relations among its components that remains invariant while everything else varies (Vaz and Varela, 1978; Vaz et al., 2006; Pordeus et al., 2009).

8. Part 7—Conclusions and predictions From its inception, immunology was inserted on a misleading axis (progressive responsiveness versus no response; memory versus tolerance) which neglects the analysis of its physiology. Our contention is that specific progressive immune responses, either naturally or artificially induced, are not aspects of immunological physiology, but rather of their derangement or pathology. In our way of seeing (Table 1) the physiology of the immune system is characterized by its invariant, robustly conserved patterns of activity that is always present but is neither insufficient (infectious diseases, immunodeficiency), nor is excessive (allergy), nor deviated from its normal targets (autoimmunity). Thus, the invariant aspects (the equilibrium, the organization) of the system are crucially dependent of the connectivity among lymphocytes themselves. We have proposed that immunopathology derives from perturbations and compensations of the closed organization of the immune system and that to explain autoimmunity as well as other kinds of immune deviation, we need a theoretical framework wider than current immunology can offer. This task is not easy to accomplish; major gaps exist between immunology and other important areas of biology such as developmental biology and evolution. However, it is impossible to understand mechanisms of autoimmunity without understanding the physiology of the immune system. Thus, an important area of investigation is to characterize the cellular interactions that in the course of an organism development bring forth what we name as the immune system. Directions for future research thus include further investigating crosstalk between lymphocytes and different cell types, not only professional antigen presenting cells, wherever lymphocytes are located. In the clinic, we should insist and improve diagnostic procedures based on pattern recognition, such as those allowed by immunoblotting (Nobrega et al., 2002), or micro-arrays (Cohen. 2013). In general terms, we should look for conservation of lymphocyte and immunoglobulin reactivity, for what is maintained invariant amid constant variation. Oral tolerance and the Injection of tolerated proteins can effectively prevent autoimmunity and inflammation even to antigens not specifically related and we believe it might be effective in medical conditions where the initial triggering of pathogeny is known as in bone marrow transplantation, in attempts to prevent graft-versus-host reactions. In experimental therapy, we should search for interventions able to bring back a lost connectivity. It is possible that the

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Table 1 Differences between the usual and proposed ways of seeing immunological activity and phenomena. Premises

Clonal selection

Conservative Physiology

Reality Immune activity Events Cellular/molecular structure Organization In the absence of antigens Main physiologic aspect Main pathologic aspect Clonal specifity Anti-infectious vaccination Infectious diseases Allergic diseases Autoimmune diseases Lymphopoiesis by lymphopenia Forms of prevention/therapy Possible new developments

Objective, observer-independent Sporadic, to external antigens Clonal expansions and contractions Permanently variable None acknowledged Clones remain iddle Regulation of clonal expansion Antigen-dependent, clonal stimulation High By memory, progressive responsiveness Deficit of specific immune responses Overreactive specific immune responses Deviated specific immune responses Homeostasis Vaccination, passive and adoptive transfers Unknown

Objective, observer-dependent (not solipsistic) (Vaz, 2011) Recurrent, plural, robustly conservative, low level Networkish, historic-systemic, observer-dependent Permanently variable Invariant, robustly conserved patterns of activity (e.g. natural IgM) Maintenance of internal activity Maintenance of internal activity, invariant organization Loss of clonal interconnectivity, oligoclonal T cell expansions Polyspecific (degenerate, plastic, versatile) By expanding clonal interconnectivity of susceptible individuals Perturbation/compensation of invariant organization Perturbation/compensation of invariant organization Perturbation/compensation of invariant organization Expansions result from lowering clonal interconnectivity Restoring lowered connectivity (IvIg, T-cell vaccines, injecting tolerated antigens) T-cell vaccines; injecting tolerated antigens

effectiveness of intravenous immunoglobulins (IvIG) and of T cell vaccines developed by Cohen's group, such as those using modified HSP-peptides in type-1 diabetes (Raz et al., 2014) is due to their modification of lymphocyte interconnectivity. If this is true, peptides derived from MHC products themselves, and other ubiquitous proteins of strong immunogenic relevance, will probably act similarly. Curiously, the growing awareness toward the importance of systemic ideas has had some peculiar consequences. One of the most striking is the treatment of severe human infections, to which no effective treatment is available, with fecal microbiota transplantation. The treatment is safe, inexpensive, and effective (Bakken et al., 2011). If compared with the sophistication of current immunological methods, it should make us humble.

Acknowledgements We thank Archimedes Barbosa de Castro-Junior, Gustavo Campos Ramos and Vitor Pordeus for permanent support and valuable suggestions to this manuscript. This work was supported by Fundação de Amparo à Pesquisa de Minas Gerais and Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil.

Appendix A Oligoclonal expansions of lymphocytes, mostly of T cells but also of B cells, have been reported in primary immunodeficiency, autoimmune, allergic and infectious diseases; in healthy and abnormal aging; in restoring miscellaneous lymphopenic conditions; in adoptive transfer of lymphocytes. In addition, oligoclonal expansions have been observed to be conducive to malignancies (e.g., liver); it is reduced during effective treatments, e.g., by IVIg; and was also unexpected observed in many odd pathological situations, such as: calcific aortic stenosis; chronic cigarette smoking; cutaneous lymphocytic infiltrates of various etiologies; intoxication by heavy plumb or mercury; inclusion body myositis; PEG-ADA treatment for adenosine deaminase (ADA) deficiency; periprosthetic inflammation (joint arthroplasty); post-polio syndrome; and, stiff person syndrome. Among others, it has been registered in the following autoimmune conditions: acute coronary syndrome; alopecia areata; aplastic anemia; active atherosclerosis plaques (human); atherosclerotic lesions of apolipoprotein E-deficient mice; autoimmune hepatitis; autoimmune myocarditis; Behcet's disease; chronic inflammatory demyelinating polyneuropathy (CIDP); type-1 and type-2 diabetes; experimental allergic encephalomyelitis (EAE in mice and rats); inflammatory bowel diseases (IBDs);

idiopathic thrombocytopenic purpura; inflammatory myopathies; Guillain-Barre and Fisher syndromes; unspecific inflammatory diseases of the nervous system; Kawasaki disease; human lymphoproliferative syndrome; multiple sclerosis; myasthenia gravis; pancreatitis (several forms); pemphigus foliaceus; primary biliary cirrhosis;psoriasis; rheumatic fever; rheumatoid arthritis and synovitis; sarcoidosis; Sjögren syndrome; spondylarthitis (with HLA-B27 involvement); systemic sclerosis (scleroderma); systemic lupus erythematosus (SLE); SLE nephritis; thyroid eye diseases; thyroid Grave's disease; thyroid Hashimoto disease; posterior; uveitis; vitiligo (associated to melanoma treatment).

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Richman, L.K., Chiler, J.M., Brown, W.R., Hanson, D.G., Vaz, N.M., 1978. Entericallyinduced immunological tolerance—I. Induction of suppressor T lymphocytes by intragastric administration of soluble protein antigens. J. Immunol. 121, 2429–2434 (no DOI). Svetlicky, N., Ortega-Hernandez, O.D., Moutho, L., Guillevin, L., Thiesen, H.J., Altman, A., Y., S.Please check author name in “Svetlicky et al. (2013)”., 2013. The advantage of specific intravenous immunoglobulin (sIVIG) on regular IVIG: experience of the last decade. J. Clin. Immunol. 33 (Suppl 1), http://dx.doi.org/ 10.1007/s10875-012-9842-5 (S27-32). Vaz, N.M., Vaz, E.M., Levine, B.B., 1970. Relationship between H-2 genotype and immune responsiveness to low doses of ovalbumin in the mouse. J. Immunol. 104, 1572–1578 (no DOI). Vaz, N.M., Phillips-Quagliatta, J.M., Vaz, E.M., Levine, B.B., 1971. Immune responsiveness of mice to ovalbumin and ovomucoid: relationship to H-2 type. J. Exp. Med. 134, 1335–1342 (no DOI). Vaz, N.M., Maia, L.C.S., Hanson, D.G., Lynch, J.M., 1977. Inhibition of homocitotropic antibody response in adult mice by previous feeding of the specific antigen. J. Allergy Clin. Immunol. 60, 110, http://dx.doi.org/10.1016/0091-6749(77)90035-5. Vaz, N.M., Varela, F.G., 1978. Self and nonsense: an organism-centered approach to immunology. Med. Hypothesis 4, 231–257, http://dx.doi.org/10.1016/03069877(78)90005-1. Vaz, N.M., Maia, L.C., Hanson, D.G., Lynch, J.M., 1981. Cross-suppression of specific immune responses after oral tolerance. Mem. Inst. Oswaldo Cruz 76 (1), 83–91, http://dx.doi.org/10.1590/S0074-02761981000100009. Vaz, N.M., Carvalho, C.R., 1994. Assimilation, tolerance and the end of innocence. Ciênc. Cult. 46 (5-6), 351–357 (no DOI). Vaz, N.M., Faria, A.M.C., Verdolin, B.A., Carvalho, C.R., 1997. Immaturity, ageing and oral tolerance. Scand. J. Immunol. 46, 225–229, http://dx.doi.org/10.1046/ j.1365-3083.1997.d01-117.x.

Vaz, N.M., Fesel C.A., Nóbrega, A., Silva Neto, A.F., Secor, W.E., Colley, D.G., 2001. Severity of schistosomiasis mansoni in male CBA mice is related to IgG profiles reacting with mouse liver extracts in Panama-blots. FESBE, Caxambu MG, Brasil. (Abstract 24.003). Vaz, N.M., Ramos, G.C., Pordeus, V., Carvalho, C.R., 2006. The conservative physiology of the immune system. A non-metaphoric approach to immunological activity. Clin. Dev. Immunol. 13 (2-4), 133–142, http://dx.doi.org/10.1080/ 17402520600877216. Vaz, N.M., 2011. The specificity of immunological observations. Constructivist Found. 6 (3), 334–351 (no DOI). Verdolin, B.A., Ficker, S.M., Faria, A.M.C., Vaz, N.M., Carvalho, C.R., 2001. Stabilization of serum antibody responses triggered by initial mucosal contact with the antigen independently of oral tolerance induction. Braz. J. Biol. Med. Res 34 (2), 211–219, http://dx.doi.org/10.1590/S0100-879  2001000200008. Wilson, D.B., Fox, D.H., 1971. Quantitative studies on the mixed lymphocyte interaction in rats. VI. Reactivity of lymphocytes from conventional and germfree rats to allogeneic and xenogeneic cell surface antigens. J. Exp. Med. 134, 857–870, http://dx.doi.org/10.1084/jem.134.4.857. Wong, S.-Y., Roth, D.B., 2007. Murine models of Omenn syndrome. J. Clin. Invest. 117, 1213–1216, http://dx.doi.org/10.1172/JCI32214. Wooldridge, L., Ekeruche-Makinde, J., van den Berg, H.A., Skowera, A., Miles, J.J., Tan, M.P., Sewell, A.K., 2012. A single autoimmune T-cell receptor recognizes over a million different peptides. J. Biol. Chem. 287 (2), 1168–1177, http://dx. doi.org/10.1074/jbc.M111.289488. Wu, L.C., Zarrin, A.A., 2014. The production and regulation of IgE by the immune system. Nat. Rev. Immunol. 14, 247–259, http://dx.doi.org/10.1038/nri3632. Wucherpfennig, K.W., Allen, P.M., Celada, F., Cohen, I.R., De Boer, R.J., Christopher Garcia, K., Sercarz, E.E., 2007. Polyspecificity of T cell and B cell receptor recognition. Semin. Immunol. 19 (4), 216–224, http://dx.doi.org/10.1016/j. smim.2007.02.012.