Hyperpigmentation Results in Aberrant Immune Development in Silky Fowl (Gallus gallus domesticus Brisson)

Abstract

The Silky Fowl (SF) is known for its special phenotypes and atypical distribution of melanocytes among internal organs. Although the genes associated with melanocyte migration have been investigated substantially, there is little information on the postnatal distribution of melanocytes in inner organs and the effect of hyperpigmentation on the development of SF. Here, we analyzed melanocyte distribution in 26 tissues or organs on postnatal day 1 and weeks 2, 3, 4, 6, 10, and 23. Except for the liver, pancreas, pituitary gland, and adrenal gland, melanocytes were distributed throughout the body, primarily around blood vessels. Interaction between melanocytes and the tissue cells was observed, and melanin was transported by filopodia delivery through engulfed and internalized membrane-encapsulated melanosomes. SFs less than 10 weeks old have lower indices of spleen, thymus, and bursa of Fabricius than White Leghorns (WLs). The expression levels of interferon-γ and interlukin-4 genes in the spleen, and serum antibody levels against H5N1 and infectious bursal disease virus were lower in SF than in WL. We also found immune organ developmental difference between Black-boned and non-Black- boned chickens from SFs and WLs hybrid F2 population. However, degeneration of the thymus and bursa of Fabricius occurred later in SF than in WL after sexual maturity. Analysis of apoptotic cells and apoptosis-associated Bax and Bcl-2 proteins indicated that apoptosis is involved in degeneration of the thymus and bursa of Fabricius. Therefore, these results suggest that hyperpigmentation in SF may have a close relationship with immune development in SF, which can provide an important animal model to investigate the roles of melanocyte.

Fig 1. Immunization and antibody detection in the serum of SF and WL.

Vaccines, injection routes, doses, and sample times are listed (a). Lower antibody levels against H5N1 Re4 were detected in the serum of SF, and obvious differences were calculated between SF and WL in females at 6 and 10 weeks of age (b). Antibody level against infectious bursal disease virus (IBDV) was low in the serum of SF, but no difference was detected between the SF and WL (c). *P < 0.05.

Fig 2. Tissue distribution of melanocytes in Silky Fowl (SF).

Spleen capsule and parenchyma (a). Thymus (b). Bursa of Fabricius (c). Testis (d). Oviduct (e). Ovary (f). Leg muscle (g). Dorsal skin (h). Leg skin (i). Brain (j). Trachea (k). Lung (l). Kidney (m). Stomach (n). Intestine (o). Melanocyte: green arrow. Lymph node: blue arrow. Hematoxylin and eosin (H&E) stain. Scale bar = 100 μm.

Fig 3. Melanocyte morphology.

Numerous melanocytes (indicated by a blue arrow), with the appearance of dendritic cells and with round nuclei and abundant melanin, were observed in the skin (a), testis (b), thymus (c), and ovary (d). Macrophages in the thymus were stained by 3, 4-dihydroxy-l-phenylalanine (DOPA; c, green arrow). Scale bar = 100 μm.

Fig 4. Tissue-dependent co-localization of melanocytes with heterophils and mast cells.

Melanocytes co-localized with heterophils in the bursa of Fabricius (a) and ovaries (b). H&E stain. Melanocytes co-localized with mast cells in the lungs (c) and ovaries (d). Toluidine blue stain. Scale bar = 100 μm.

Fig 5. Tissue-specific ultrastructural characteristics of melanocytes and neighboring cells.

Transmission electron microscopy was used to observe the melanocytes. Melanosomes were observed in the melanocytes (blue arrow) and the immune cells (green arrow) of the thymus (a). Melanosomes were present in other cells (green arrow) in the skin (b). Melanosomes were secreted by exocytosis from melanocytes in the skin (c). Melanosomes were observed in the interstitial cells (green arrow) in the ovaries (d). Melanocytes and neighboring cells were connected by dendrites in the ovaries (e). Long dendrites were detected in melanocytes and neighboring cells in the ovaries (f).

Fig 6. Organ indices and immune gene expression in the F2 population.

(a) Lower indices of spleen, thymus, and bursa of Fabricius were detected in Black-boned chickens, with a significance difference (P < 0.05) in the thymus. (b) Lower expression levels of IFN-γ and IL-4 were detected in Black-boned chickens, compared with those in non-Black-boned chickens (P < 0.05). Higher expression level of GAL-7 was detected in Black-boned chickens, compared with that in non-Black-boned chickens, but the difference was not statistically significant. B, Black-boned chicken. Non B, non-Black-boned chicken.

Fig 7. CD3⁺ cells in the spleen and thymus of SF and WL.

(A) CD3⁺ cells were observed in the spleen (a, b) and thymus (e, f) of SF and in the spleen (c, d) and thymus (g, h) of WL. Scale bar = 100 μm. (B) There were significant differences during early development between the numbers of CD3⁺ cells in the spleen and thymus of SF and WL. *P < 0.05, **P < 0.01.

Fig 8. Bu-1⁺ cells in the spleen, thymus, and bursa of Fabricius of SF and WL.

(A) Bu-1⁺ cells were observed in the spleen (a, b), thymus (e, f), and bursa of Fabricius (i, j) of SF and in the spleen (c, d), thymus (g, h), and bursa of Fabricius (k, l) of WL. Scale bar = 100 μm. (B) There were significant differences in the numbers of Bu-1⁺ cells in the spleen, thymus, and bursa of Fabricius of SF, compared with those in WL, during early development. *P < 0.05, **P<0.01.

Fig 9. Expression of IFN-γ, IL-4 and GAL-7 in the spleen of SF and WL.

Lower expression levels of IFN-γ (a) and IL-4 (b) were detected in SF at 10 and 6 weeks of age, respectively, but higher expression levels were detected in SF at 23 weeks of age. High expression level of GAL-7 was detected in SF before 6 weeks of age, but low expression levels were detected at 10 and 23 weeks of age. (c). *P < 0.05, **P < 0.01.

Fig 10. TUNEL analysis of apoptosis in SF and WL.

(A) Apoptotic cells were observed in the bursa of Fabricius (a) and thymus (b) of SF, and the bursa of Fabricius (c) and thymus (d) of WL. Scale bar = 100 μm. (B) There were significant differences between the numbers of apoptotic cells in the spleen, thymus, and bursa of Fabricius in SF, compared with those in WL. **P<0.01.

Fig 11. Immunohistochemical analysis of Bax expression.

(A) Bax expression (brown) was detected in the spleen (a), thymus (b), and bursa of Fabricius (c) and of SF and the spleen (d), thymus (e), and bursa of Fabricius (f) of WL. Scale bar = 100 μm. (B) There was a significant difference in the numbers of Bax⁺ cells in the spleen, thymus, and bursa of Fabricius of SF, compared with those of WL. **P<0.01.