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.