Indirect effects of immunological tolerance to a regular dietary protein reduce cutaneous scar formation.

Oral tolerance refers to the specific inhibition of immune responsiveness to T-cell-dependent antigens contacted through the oral route before parenteral immunization. Oral tolerance to one protein does not inhibit immune responses to other unrelated proteins, but parenteral injection of tolerated antigens plus adjuvant into tolerant, but not normal, mice inhibits immune responses to antigens injected concomitantly or soon thereafter. The inhibitory effect triggered by parenteral injection of tolerated proteins is known as bystander suppression or indirect effects of oral tolerance. Intraperitoneal injection of ovalbumin (OVA) plus alum adjuvant in OVA-tolerant mice soon before skin injury inhibits inflammation and improves cutaneous wound healing. However, as OVA is not a regular component of mouse chow, we tested whether indirect effects could be triggered by zein, the main protein of corn that is regularly present in mouse chow. We show that intraperitoneal injection of a single dose (10 µg) of zein plus alum adjuvant soon before skin injury in mice reduces leucocyte infiltration but increase the number of T cells and the expression of resistin-like molecule-α (a marker of alternatively activated macrophages) in the wound bed, increases the expression of transforming growth factor-ß3 in the newly formed epidermis and reduces cutaneous scar formation. These results suggest that indirect effects of oral tolerance triggered by parenteral injection of regular dietary components may be further explored as one alternative way to promote scarless wound healing.

Systemic effects of oral tolerance reduce the cutaneous scarring.

BACKGROUND: Immunological tolerance refer to the inhibition of specific immune responsiveness and the ingestion of proteins previous to immunization is a reliable method to induce (oral) tolerance. Parenteral exposure to tolerated antigens, in adjuvant, trigger indirect and systemic effects that inhibits concomitant immune responses to other unrelated antigens and also decrease unrelated inflammatory responses. Interesting, intraperitoneal (i.p.) exposure to orally-tolerated proteins soon before an incisional linear skin wound improves the healing by primary intention in mice. An important clinical and surgical objective is to identify strategies to improve wound healing and reduce scarring. OBJECTIVE: To evaluate whether i.p. injection of an orally-tolerated protein improves wound healing by secondary intention and reduce scarring of full-thickness excisional skin injury. METHODS: C57Bl/6 mice were turned tolerant to ovalbumin (OVA) by drinking a solution containing OVA; seven days later, they received an i.p. injection of OVA plus Al(OH)3 adjuvant immediately before two full-thickness excisional skin wounds, under anesthesia. The wound healing process was evaluated macro and microscopically after H&E, toluidine blue and Gomori's Trichrome staining. The presence of granulocytes, macrophages, miofibroblasts, fibronectin, collagen I and collagen III was investigated by immunofluorescence and the levels of cytokines by flow cytometry or ELISA. Mice not tolerant to OVA were included as controls. RESULTS: The i.p. injection of OVA+Al(OH)3 in mice orally tolerant to OVA reduced the subsequent inflammatory response in the wound bed and the cutaneous scarring. There was a change in the pattern of collagen deposition making it more similar to the pattern observed in intact skin. In tolerant mice, mast cells and granulocytes (Ly-6C/G+), were reduced, while lymphocytes (CD3+) were increased in the wound bed. Time course analysis of Th1/Th2/Th17 cytokines and growth factors showed slightly differences between tolerant and control groups. CONCLUSION: Parenteral injection of an orally-tolerated protein has systemic consequences that impair the inflammatory response triggered by skin injury and reduce the cutaneous scarring.

Improved cutaneous wound healing after intraperitoneal injection of alpha-melanocyte-stimulating hormone.

Skin wound healing is a complex process involving many types of cells and molecules and often results in scar tissue formation in adult mammals. However, scarless healing occurs in foetal skin and minimal scars may occur after cutaneous healing in the adult with reduced inflammation. Alpha-melanocyte-stimulating hormone (α-MSH) is widely distributed within the central nervous system and in other body regions, such as the skin, and has strong anti-inflammatory activity. The aim in the present experiments was to learn whether intraperitoneal (i.p) injection of α-MSH just before skin wounds antagonize inflammation and improves skin wound healing in adult mice. C57BL/6 young adult mice received an i.p. injection of 1 mg/kg of α-MSH and, 30 min later, two circular through-and-through holes (6.5 mm diameter) were made in their dorsal skin under anaesthesia. Control mice were wounded after vehicle injection. The wound healing process was analysed macroscopically and microscopically at 3, 7, 40 and 60 days. Skin samples were fixed in formalin, embedded in paraffin, sectioned at 5 µm, stained with H&E or toluidine blue for cell analysis or Gomori's trichrome for extracellular matrix (ECM) analysis. Other samples were fixed in DMSO+methanol, embedded in paraplast and incubated with anti-CD45, antismooth muscle actin, anticollagen-I and anticollagen-III for immunofluorescence analysis. Alpha-MSH significantly reduced the number of leucocytes, mast cells and fibroblasts at 3 and 7 days after injury. On days 40 and 60, α-MSH reduced scar area and improved the organization of the collagen fibres indicating that it may direct the healing into a more-regenerative/less-scarring pathway.

On the origin of immunopathology.

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".

The autoimmune nature of post-infarct myocardial healing: oral tolerance to cardiac antigens as a novel strategy to improve cardiac healing.

Following a myocardial infarction, lymphocytes have been suggested to react with the damaged heart tissue, which can impair proper tissue healing. In the present work, we investigate whether ingestion of a myocardial homogenate and the consequent development of immunological tolerance can modify the course of post-infarction myocardial repair. Infarction-like myocardial lesions were induced in Wistar rats by injecting high doses of isoproterenol. The healing process was evaluated morphologically and functionally for 60 days. Cardiac function was evaluated using isolated and perfused heart (Langendorff) preparations. At day 14 after isoproterenol treatment, lymphocytes from the mediastinal lymph nodes proliferated when exposed in vitro to myocardial homogenate. Moreover, TNF-α, IFN-γ and CCL-5, but not FOXP3+ expression, was increased in draining lymph nodes in isoproterenol-injured animals, indicating that the observed lymphocyte population that proliferated in response to cardiac components presented a pro-inflammatory and pro-fibrotic profile. In contrast, lymphocytes from draining lymph nodes of rats given a heart homogenate by gavage 7 days before isoproterenol did not proliferate. Furthermore, the group rendered tolerant expressed cardiac FOXP3+ earlier than did the control group, and showed a milder inflammatory infiltrate, lower MMP-9 expression, less collagen deposition, and improved cardiac performance when compared to animals that received only isoproterenol administration. The present findings suggest that the establishment of oral tolerance to heart components prior to myocardial infarction may drive the cardiac healing process to proceed with less inflammation and fibrosis, thus preserving contractile organ function.

Indirect effects of oral tolerance inhibit pulmonary granulomas to Schistosoma mansoni eggs.

Parenteral injection of tolerated proteins into orally tolerant mice inhibits the initiation of immunological responses to unrelated proteins and blocks severe chronic inflammatory reactions of immunological origin, such as autoimmune reactions. This inhibitory effect which we have called "indirect effects of oral tolerance" is also known as "bystander suppression." Herein, we show that i.p. injection of OVA + Al(OH)(3) minutes before i.v. injection of Schistosoma mansoni eggs into OVA tolerant mice blocked the increase of pulmonary granulomas. In addition, the expression of ICAM-1 in lung parenchyma in areas outside the granulomas of OVA-orally tolerant mice was significantly reduced. However, at day 18 after granuloma induction there was no difference in immunofluorescency intensity to CD3, CD4, F4/80, andα-SMA per granuloma area of tolerant and control groups. Reduction of granulomas by reexposure to orally tolerated proteins was not correlated with a shift in Th-1/Th-2 cytokines in serum or lung tissue extract.

Oral tolerance correlates with high levels of lymphocyte activity.

Oral tolerance is defined as an inhibition of specific immune responsiveness to a previously ingested antigen. Paradoxically, we found an increased lymphocyte activity in tolerant mice alongside the specific inhibition. Orally-tolerant mice presented higher number of immunoglobulin secreting cells (ISC) in spleen and bone marrow; showed a greater variety of Ig classes being produced: IgM and IgA in the spleen and IgG and IgM in the bone marrow. ISC from immunized mice produced mainly IgG. Despite having the same number of regulatory and activated T cells in the spleen after immunization, these cells appeared earlier in tolerant mice, right after the primary immunization. Also, tolerant mice showed a prompt expression of regulatory cytokines (TGF-ß and IL-10) and a transient expression of effector cytokines (IL-2 and IFN-γ). Thus, in addition to an inhibited specific responsiveness, orally-tolerant mice displayed an early and widespread mobilization of activated and regulatory lymphocytes.

Indirect effects of oral tolerance improve wound healing in skin.

Tissue injury in adult mammalian skin frequently results in scarring while fetal mammalian skin heals with complete regeneration. Inflammatory reactions are among the factors thought to impair regeneration. Previous studies have shown that the injection of an immunologically tolerated protein blocks immune responses to unrelated antigens and is also able to inhibit inflammation in mice. This phenomenon, which we refer to as the indirect effects of oral tolerance, does not require the simultaneous injection of the tolerated antigen and the second antigen, and also occurs when the two antigens are given by separate routes of immunization. Herein, we investigated whether the i.p. injection of an orally tolerated antigen (ovalbumin, OVA) would inhibit inflammatory reactions at an incisional lesion and influence healing of adult mouse skin. In OVA-tolerant mice, the injection of OVA minutes before wounding altered inflammation: it reduced the numbers of mast cells, neutrophils, and lymphocytes but increased the number of macrophages around the lesion area. Tolerant mice also showed fewer myofibroblasts and reduced scar area. Furthermore, tolerant mice displayed a pattern of extracellular matrix deposition similar to that observed in intact skin, plus characteristics of regeneration, such as an increased deposition of fibronectin and tenascin-C. These observations suggest that the indirect effects of oral tolerance can alter the process of wound healing in skin and reduce scar formation.

Different requirements for the adoptive transfer of oral tolerance and its indirect effects assessed by DTH and antibody responses in mice.

Oral tolerance inhibits T-cell dependent reactions to antigens previously contacted by oral route. Parenteral re-exposure to orally-tolerated antigens inhibits immune responses to unrelated antigens, a phenomenon we have called "indirect effects" of oral tolerance. We examined the requirements of previous irradiation of C57BL/6 and BALB/c recipients to successful transfer of oral tolerance and its indirect effects using 1x10(8) splenocytes. When DTH reactions were evaluated, irradiation was not required to transfer both oral tolerance and its indirect effects. C57BL/6, but not BALB/c recipients, required irradiation to adopt suppressed antibody responses to tolerizing antigen. In BALB/c recipients, the indirect effect was transferred only if serum from the tolerant donors was added to the transferred splenocytes. CFSE labeled donor cells were not eliminated from non-irradiated C57BL/6, although unable to suppress antibody responses. Our results provide further evidences on the existence of a functional barrier in immunocompetent recipients that hinders the adoptive transfer of different immunological activities. Interactions between cells and serum components may be necessary to bypass this barrier.

Cell-mediated immune response to unrelated proteins and unspecific inflammation blocked by orally tolerated proteins.

Oral tolerance promotes a generalized decrease in specific immune responsiveness to proteins previously encountered via the oral route. In addition, parenteral immunization with a tolerated protein also triggers a significant reduction in the primary responsiveness to a second unrelated antigen. This is generally explained by 'innocent bystander suppression', suggesting that the transient and episodic effects of inhibitory cytokines released by contact with the tolerated antigen would block responses to the second antigen. In disagreement with this view, we have previously shown that: (i) these inhibitory effects do not require concomitance or contiguity of the injections of the two proteins; (ii) that intravenous or intragastric exposures to the tolerated antigen are not inhibitory; and (iii) that the inhibitory effect, once triggered, persists in the absence of further contact with the tolerated protein, possibly by inhibition of secondary responsiveness (immunological memory). The present work confirms that immunological memory of the second unrelated antigen is hindered by exposure to the tolerated antigen and, in addition, shows that this exposure: (i) inhibits the inflammation triggered by an unrelated antigen through the double effect of inhibiting production of leucocytes in the bone marrow and blocking their migration to inflammed sites; and (ii) significantly blocks footpaw swelling triggered by carrageenan. Taken together, these results conclusively demonstrate that inhibitory effects of parenteral injection of tolerated antigens are much more general than suggested by the 'innocent bystander suppression' hypothesis.

Dynamic stability in random and scale-free B-lymphocyte networks.

One of the most intriguing features of the immune system is regulation: a limited response when perturbed repeatedly. We propose a minimal network model for immune regulation in a lymphocyte network containing two types of elements: B lymphocytes and ligands that bind to their receptors. Effective interactions between B cells, mediated by other components of the immune system can be excitatory or inhibitory. In our model, B cell clones and ligand species are represented by nodes, and interactions by links. We expect that, as in many complex systems, the connectivity distribution is broad, motivating study of the model on a scale-free network; for comparison we study the same dynamics on a random graph. We characterize the dynamics of the model and its response to perturbations. Our model reproduces several key features of immune system dynamics: regulation (saturation of response), and more rapid response upon repeated perturbation with the same agents. Our results suggest that a scale-free network of interactions contributes to the regulation and dynamics of the immune system.

The conservative physiology of the immune system. A non-metaphoric approach to immunological activity.

Historically, immunology emerged as a biomedical science, concerned with host defense and production of anti-infectious vaccines. In the late 50s, selective theories were proposed and from then on, immunology has been based in a close association with the neo-Darwinian principles, such as random generation of variants (lymphocyte clones), selection by extrinsic factors (antigens) - and, more generally, on genetic determinism and functionalism. This association has had major consequences: (1) immunological jargon is full of "cognitive" metaphors, founded in the idea of "foreignness"; (2) the immune system is described with a random clonal origin, coupled to selection by random encounters; and (3) physiological events are virtually absent from immunological descriptions. In the present manuscript, we apply systemic notions to bring forth an explanation including systemic mechanisms able to generate immunological phenomena. We replace "randomness plus selection" and the notion of foreignness by a history of structural changes which are determined by the coherences of the system internal architecture at any given moment. The importance of this systemic way of seeing is that it explicitly attends to the organization that defines the immune system, within which it is possible to describe the conservative physiology of the immune system. Understanding immune physiology in a systemic way of seeing also suggests mechanisms underlying the origin of immunopathogeny and therefore suggests new insights to therapeutic approaches. However, if seriously acknowledged, this systemic/historic approach to immunology goes along with a global conceptual change which modifies virtually everything in the domain of biology, as suggested by Maturana.

Immunological activities are modulated by enteral administration of an elemental diet in mice.

BACKGROUND & AIMS: Elemental diets (EDs) have been used successfully in treatment of some intestinal inflammatory diseases; however, the mechanism that mediates their effects is still unclear. In this study we evaluated the immunological effect of enteral administration of an ED in mice. METHODS: C57BL/6 mice were fed an ED (El-Diet) from weaning up to adulthood and immunological parameters were analyzed. RESULTS: El-Diet-fed mice presented an underdeveloped gut-associated-lymphoid tissue with lower numbers of TCRalphabeta+IELs and lamina propria cells and low levels of secretory IgA when compared to chow-fed mice. They showed a systemic decrease in the production of IgG and IgA as well as a skewing towards a Th2 profile of cytokine production upon in vitro stimulation with an increase in IL-4 and a reduction in IFN-gamma and IL-6 secretion. CONCLUSION: Our study demonstrated the role of EDs in modulating immunological activities in mice and proposes a rational for their successful use in treatment of some intestinal inflammatory diseases.

Systemic effects of oral tolerance on inflammation: mobilization of lymphocytes and bone marrow eosinopoiesis.

Oral tolerance is a T-cell mediated phenomenon defined by inhibition of immune responsiveness to a protein previously contacted by the oral route. Oral tolerance may prevent autoimmune and allergic diseases that involve the recruitment and/or activation of different cell types including mast cells, neutrophils, eosinophils, monocytes and lymphocytes. The mechanisms by which oral tolerance avoids these immunological disorders are still controversial. Herein we used a murine model of ovalbumin (OVA)-induced peritonitis to investigate the effect of oral tolerance on allergic inflammation. Frequency of leucocyte subpopulations was evaluated by global and differential cell counts in peritoneal lavage fluid, peripheral blood, and bone marrow. Changes on lymphocyte subsets and adhesion molecules expression by these cells were analysed by flow cytometry. As compared with OVA-immune mice, intraperitoneal challenge of tolerant animals with OVA resulted in a significantly milder peritonitis, mostly affecting neutrophils and eosinophils; a concomitant reduction in total white blood cell counts was also observed, mainly because of lower neutrophil and eosinophil counts. Eosinophils, but not neutrophils, were also reduced in the bone-marrow of OVA-challenged tolerant mice. No changes occurred in total peritoneal lymphocyte counts in OVA-tolerant mice, however, there was a significant decrease in CD3+ CD8+ T cells and an increase in B cells (CD45R+) in these animals as compared to immune OVA-challenged animals. Altered expression of CD18 and CD54, respectively, in blood and peritoneal lymphocytes was also noted. These results suggest that, in addition to local specific effects, oral tolerance has systemic effects on the mobilization of leucocytes and bone-marrow eosinopoiesis.

Increased polyclonal immunoglobulin reactivity toward human and bacterial proteins is associated with clinical protection in human Plasmodium infection.

BACKGROUND: Polyclonal B-cell activation is well known to occur in Plasmodium infections, but its role in pathogenesis or protection remains unclear. However, protective properties of natural antibodies have previously been demonstrated in other contexts. METHODS: Sera from asymptomatic and symptomatic Plasmodium-infected subjects locally detected in a survey study in the Brazilian Amazon, and from unexposed and exposed but presently uninfected control subjects, were assayed by a standardized quantitative immunoblot method allowing simultaneous detection of IgG or IgM reactivity to a large number of parasite-unrelated proteins. RESULTS: In subjects free of coinfection with hepatitis B virus, IgG reactivity to human brain antigens and Escherichia coli proteins was strikingly enhanced in asymptomatic Plasmodium-infected individuals when compared to such with clinical malaria symptoms, or to uninfected control subjects. This difference was most characteristic for limited exposure times (less than ten years locally, or 20 years in endemic areas). It was more significant than a similar trend found for IgG to Plasmodium falciparum antigens, and unrelated to parasitaemia levels. Asymptomatic subjects with comparatively short exposure characteristically showed relatively elevated IgG versus IgM reactivity. Polyclonal IgG reactivity appears triggered by previous P. falciparum but not Plasmodium vivax malaria. CONCLUSION: The observed difference in polyclonal antibody production seems related to intrinsic activation states of infected individuals, rather than to parasite-antigen specific immune responses. However, it appears influenced by preceding stimuli. This supports the idea that acquired clinical immunity may not exclusively depend on antigen-specific responses, but also on the individual polyclonal reaction.