Systemic effects of oral tolerance in bone healing.

Bone fractures cause acute inflammation that, despite being important for initial repair, may delay the healing of the damaged bone. Parenteral injection of dietary protein has been shown to decrease inflammation and accelerate the repair of skin wounds and other inflammatory pathologies. Thus, our aim was to evaluate whether the intraperitoneal (i.p.) immunization with zein, an abundant protein in rodent chow, would favor bone healing. Wistar rats received i.p. immunization: saline (SG), adjuvant (AG) and zein associated with adjuvant (ZG). Then, a 2 mm of defect bone was performed on the right tibia, and on days 7, 14, 28 and 45 thereafter, analyses were performed. The results showed that the injection of zein reduced inflammation without impairing bone mineralization. Moreover, biomechanical tests demonstrated higher levels of maximum force (N) in ZG, indicating better mechanical resistance in relation to the others. The computerized tomography also indicated lower levels of medullary content in the ZG than in the SG, suggesting the absence of trabeculae in the medullary region in the ZG. These findings suggest that the injection of zein in previously tolerated animals may improve bone repair, leading to mechanically functional bone formation.

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.

Effect of a protein-free diet in the development of food allergy and oral tolerance in BALB/c mice.

The aim of the present study was to investigate the effect of a protein-free diet in the induction of food allergy and oral tolerance in BALB/c mice. The experimental model used was mice that were fed, since weaning up to adulthood, a balanced diet in which all dietary proteins were replaced by amino acid diet (Aa). The absence of dietary proteins did not prevent the development of food allergy to ovalbumin (OVA) in these mice. However, Aa-fed mice produced lower levels of IgE, secretory IgA and cytokines. In addition, when compared with mice from control group, Aa-fed mice had a milder aversive reaction to the allergen measured by consumption of OVA-containing solution and weight loss during food allergy development. In addition, mice that did not have dietary proteins in their diets were less susceptible to induction of oral tolerance. One single oral administration was not enough to suppress specific serum Ig and IgG1 levels in the Aa-fed group, although it was efficient to induce suppression in the control group. The present results indicate that the stimulation by dietary proteins alters both inflammatory reactivity and regulatory immune reactivity in mice probably due to their effect in the maturation of the immune system.

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.

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.

Study of oral tolerance and its indirect effects in adoptive cell transfer experiments.

Parenteral exposure to antigens to which oral tolerance had been previously induced results in the inhibition of immune responses to other unrelated antigens. Herein we tested whether indirect effects of oral tolerance could be adoptively transferred. Anti-Ova- and antihemoglobin-specific responsiveness as well as oral tolernace to Ova were transferred to irradiated, but not to normal, nonirradiated recepients. Irradiation, thus, facilitated adoptive transfer of oral tolerance. However, the inhibitory (indirect) effects upon the unrelated immunogen were not adoptively transferred, even to irradiated recepients. In addition, we studied adoptively transferred CFSE-labeled spleen cells by flow cytometry in recipient spleen, inguinal lymph nodes, and bone marrow, both in irradiated and nonirradiated recipients, 1, 3, or 5 days after cell transfer. Comparing the percent and absolute number of CFSE-labeled cells in each organ displayed significant differences in the dynamics of decay of adoptively transferred cells from tolerant or immune donors.