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

Abstract

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-β₃ 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.

Keywords: cutaneous scar; immunological tolerance; inflammation; mucosa; skin wound healing.

Figure 1

Intraperitoneal injection of zein soon before skin injury reduces scab. (a) Macroscopic appearance of wounds immediately after and at days 1, 3, 5 and 7 after skin injury in mice injected with saline, adjuvant or zein + adjuvant immediately before wounds. (b) Wound healing area at days 1, 3, 5 and 7 after skin injury. Data represent mean ± SEM. *P ≤ 0·05 compared with adjuvant group; n = 6 mice, 12 wounds. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2

Injection of zein before skin injury reduces granulation tissue and preserves angiogenesis. (a) Haematoxylin & eosin staining of intact skin and wounds at day 7 after skin injury in mice injected with saline, adjuvant or zein + adjuvant. The arrows in upper panels indicate the limits of granulation tissue area and small letters represent: d, dermis; e, epidermis; g, granulation tissue; he, hypertrophic epidermis; sc, scab; pc, panniculus carnosus. Boxes in the upper panels indicate the area where the regions are shown in higher magnification to illustrate leucocytes (black arrows), fibroblasts (white arrows) and microvessels (green arrows). Scale bars: upper panels, 500 µm; lower panels, 20 μm. Morphometric analysis of leucocytes (b), fibroblasts (c) and microvessels (d). Data represent mean ± SEM *P ≤ 0·05 compared with saline group, n = 6. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3

Injection of zein before skin injury reduces the expression of CD45 (leucocytes), α‐smooth muscle actin (SMA) (myofibroblasts) and the number of mast cells in the wound bed. (a,b) Immunostaining of intact skin and wounds, at day 7 after skin injury, with anti‐CD45 or with anti‐α‐SMA; nuclear counterstaining with 4′6‐diamidino‐2‐phenylindol (blue); (c) toluidine blue‐stained sections. The wounds are from mice injected with saline, adjuvant or zein + adjuvant minutes before skin injury. Data represent mean ± SEM of fluorescence intensity (in μm² × 10⁻³) in sections immunostained with CD45 (d) or α‐SMA (e); and morphometric analysis of mast cells (f). *P ≤ 0·05 compared with saline group, n = 6. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4

Kinetic analysis of cytokines in skin wound bed. Cytokines were analysed in intact skin (day 0) and in wounds from mice injected with saline (open triangle) or mice injected with zein (black circle), at 12 hr, and days 1, 3 and 7 after skin injury. Data represent mean ± SEM (six mice/group/time point). *P ≤ 0·05 compared with saline group at the same time after skin injury.

Figure 5

Injection of zein before skin injury increases the expression of resistin‐like molecule α (RELM‐α; alternatively activated macrophages), CD3 and transforming growth factor β ₃ (TGF‐β ₃) in the wound bed. (a) Immunostaining of intact skin and wounds, at day 7 after skin injury, with specific antibodies and nuclear counterstaining with 4′6‐diamidino‐2‐phenylindol (blue). Photomicrographs in the bottom line illustrate typical sections of each group imaged using differential interference contrast (DIC) and one insert with a picture of control staining lacking the primary antibody. Data represent mean ± SEM of fluorescence intensity (in μm² × 10⁻³) in sections immunostained with anti‐F4/80 (b), anti‐RELM‐α (c), anti‐CD3 (d) or anti‐TGF‐β ₃ (e). *P ≤ 0·05 compared with saline group, n = 6. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 6

Zein reduces cutaneous scarring and improves extracellular matrix remodelling. (a) Scar area and (b) representative pictures of dorsal skin 40 days after skin injury in control mice (saline and adjuvant groups) and in mice injected with zein. (c) Sections of intact skin or wounds harvested 40 days after skin injury stained with Gomori's trichrome. Scale bar 20 μm. n = 6. [Colour figure can be viewed at wileyonlinelibrary.com].