The Smyth line (SL) of chicken is an excellent avian model for human autoimmune vitiligo [4–6], which is a multifactorial disease of complex etiology resulting in loss of melanocytes in the skin. In the current study, we took advantage of the unique attributes of the SL chicken model (outlined in the Introduction) and the power of gene expression analysis by microarray to investigate the pathomechanism of vitiligo in SL chickens (SLV). To our best knowledge, this is the first paper using microarray analysis to study transcriptomic expression profiles in evolving vitiligo lesions. Expression levels of genes from this microarray study were validated based on their comparability to those from qRT-PCR (Table 3), which was suggested to have higher detection sensitivity than microarray . While higher expression was observed for CR2 and POU2AF1 by qRT-PCR than with microarray, their expression trends were the same with both methodologies, supporting the validity of the microarray study. The expression difference may be due to the upper fluorescent detection limit of the microarray scanner. Differentially expressed (DE) genes identified by microarray analysis and the interpretations by Ingenuity Pathway Analysis http://www.ingenuity.com support the multifactorial nature of vitiligo. For the sake of simplicity, the discussion will focus on observations regarding the involvement of the immune system, melanocyte biology, redox status, neurology, apoptosis and other physiopathological factors identified to play a role in the spontaneous development of SLV.
The autoinflammatory/autoimmune nature of SLV was supported by expression profiling (Table 1 and 2) and IPA functional interpretations of DE genes (e.g. inflammatory response and immunological disease) (Figure 1 and 2). DE genes of both T and B cell markers (Table 1 and 2) and functions of humoral and cell-mediated immune response (Figure 1 and 2) in the current study are in line with previous studies demonstrating involvement of both types of immune responses in melanocyte loss in the etiology of SLV [6, 10, 12, 16]. Furthermore, it appears that the humoral immune response was particularly important in early SLV development, since DE genes in EV samples were significantly (1st network) associated with this function (Figure 2). In addition, the humoral immune response also appeared important in SLV progression (AV) and complete depigmentation (CV) as supported by significantly up-regulated DE genes related to B cell development and/or activation such as IGJ (immunoglobulin J chain), POU2AF1 (a protein essential for the B cell response and germinal center formation), BTK (Bruton's tyrosine kinase), mucolipin 2 (a target for BTK in B cells), SPI1 (a lymphoid specific enhancer involved in B cell differentiation and activation), and PIK3AP1 (B cell activation) in these samples (Table 1 and 2). On the other hand, DE genes associated with the cell-mediated immune response were identified only in CV samples and with lower significance than DE genes associated with the humoral immune response in EV samples. A more relevant role of the humoral immune response in the pathomechanism of SLV is contradictory to the demonstrated more direct role of the cell-mediated immune response in this disorder [6, 16]. Confirmation of this finding will definitely impact future direction of vitiligo research.
Aside from involvement of adaptive immunity in SLV, it is apparent that innate immunity also plays an important role in SLV development since the majority of immune-related DE genes were derived from this branch of the immune system (Table 1 and 2). Innate immunity has not received much attention in vitiligo studies in either the SL animal model or human patients. Elevated expression of genes for chemokines (e.g. CXCL13, CCL19), pattern recognition receptors (e.g. MMR, TLR15, MD-2 and PRAM-1), TNF and type 1 and 2 interferon cytokines (e.g. LITAF, TNFSF13B, ISG12-2, ITIF5, IRF1 and IRF8) (Table 1 and 2) suggested both anti-bacterial and anti-viral activity especially in early and active stages of SLV (EV and AV). The complement system also appears to participate in SLV development and progression as supported by significantly increased expression of genes for C3, C3AR1 and CR2 in AV samples (Table 1 and 2). Functional analysis of networks by IPA revealed that DE genes identified in EV and AV samples were significantly associated (1st and 3rd network) with the functions of anti-microbial response and infectious disease, respectively. These observations suggest a potential role of the innate immune response as a precipitating and effector factor in the development of the melanocyte-specific adaptive immune response in SLV. Initiation of antimicrobial activity in SLV is in agreement with the strong association of live herpesvirus of turkey administration at hatch (routine vaccination to protect SL and BL chickens from Marek's disease virus) as a reliable environmental trigger of SLV expression in vitiligo-susceptible SL chickens . Additionally, as innate immunity is also initiated in response to cellular stress, the observed alterations in melanocyte biology and redox status (see below) may also be responsible for activation of innate immune responses in SLV .
It was previously shown that intrinsic abnormalities of melanocytes, the target cell in vitiligo, were present in SL chickens compared to BL and LBL (vitiligo-resistant, similar plumage color pattern control) controls [7, 28]. In the current study, melanocyte loss appeared to be preceded by disturbances in melanosome structure, intracellular molecule transport and tyrosine synthesis as demonstrated by decreased expression of MMP115, SLC24A2, SLC24A5, GPR143, and Shikimate 5-dehydrogenase in EV samples (Table 1). Dysfunction and loss of melanogenesis became obvious in AV and CV samples, as suggested by significantly decreased gene expression in TYR and TRP1 in these samples compared to NV and EV samples (Table 2). This finding is consistent with findings in human studies, where down-regulation of TYR and TRP1 was revealed in lesional skin compared to non-lesional skin of vitiligo patients and skin from healthy donors [29–31].
Oxidative stress was previously demonstrated in vitiligo patients [32–35] and in SLV chickens . In humans, oxidative stress involvement was further supported by the repigmenting effect of antioxidant substances in the treatment of vitiligo lesions [36–38]. In line with findings from these studies, there were DE genes for all SL samples and those generated from within SL comparison identified by IPA to be associated with the function of free radical scavenging (including functional annotation of synthesis/production of oxidative species). NV samples had similar DE gene expression for both pro- and anti-oxidative proteins relative to the BL control samples whose expression was considered as the value of 1 (Table 2). Up-regulation of these DE genes in SLV samples, especially AV samples (Table 2) suggested association of SLV development with disturbances in redox status. Among these, remarkably elevated expression was noticed for Gp91-phox and cytochrome b (Table 2), suggesting an important role of these proteins in changing the redox status in SLV chickens. Gp91-phox is a subunit of NADPH oxidase containing the heme binding site important in reactive oxygen species (ROS) production . The source of NADPH-dependent ROS production in vitiligo development is not clear, however, after activation, phagocytes and B cells are capable of NAPDH-dependent release of ROS [40, 41]. With more DE genes related to B cell than phagocyte development and/or activation in the current study (Table 1 and 2) and the greater B cell than macrophage infiltration in feather tissues at all vitiligo states reported by Shi and Erf , it is likely that B cells could be an important source of ROS in SLV development. As revealed by IPA, decreased transmembrane potential of mitochondria and the mitochondrial membrane was one of the many functional interpretations of DE genes in EV, AV and CV samples suggesting mitochondrial involvement in ROS production. Elevated expression of cytochrome b and acetyl-CoA carboxylase 2, which is important in fatty acid oxidation, in EV, AV and CV compared to NV samples points to mitochondrial production of ROS probably due to premature electron leakage of oxygen at the location of cytochrome b in the electron transport chain .
Neural involvement in vitiligo was supported by appearance of localized vitiligo lesions following nerve damage in affected skin and by morphological changes in terminal cutaneous nerves in vitiligo patients . In line with observations in humans, neurological disease was presented as one of the functional interpretations by IPA for DE genes in SL samples and DE genes identified based on within SL comparisons in the current study. In addition, expression of TAC1 (gene for neurotransmitter substance P, neurokinin A, neuropeptide K and neuropeptide gamma), KCNE1L (a protein capable of regulating neurotransmitter release and neuronal excitability), ALS2 (plays a role in axon and dendrite development) and NPY (neuropeptide Y) were decreased in EV, AV, and CV samples compared to NV samples (Table 1 and 2), supporting neural involvement in SLV development. NPY expression was also investigated in human studies; the results, however, were not consistent [44, 45] and differed from the decreased expression with SLV progression in the current study. Other studies revealed an increased number of nerve growth factor receptor-, calcitonin gene-related peptide- and protein gene product 9.5-positive nerve fibers in involved skin from vitiligo patients compared to healthy controls [45, 46]. Moreover, increased serum and urinary levels of catecholamines and their relative metabolites were found in vitiligo patients, especially in early and active stages of vitiligo, than in controls [47–49]. It was speculated that a role of catecholamines in vitiligo onset was due to their potential to undergo oxidation, resulting in formation of quinones, semiquinone radicals and oxyradicals [47, 49].
While the mechanism by which melanocytes are destroyed in vitiligo is still in debate, it has been suggested that they disappear via apoptosis (presumably initiated by cytotoxic T cells) rather than necrosis . In the current study, significant up-regulation of apoptosis-related DE genes, especially in AV compared to NV, EV and/or CV samples (Table 2), indicated a close relationship of apoptosis with SLV development. These apoptosis-related up-regulated DE genes included GZMA, LITAF (induced by p53 and has been implied in p53 induced apoptosis), caspase-associated recruitment domain, member 11 (CARD11, a positive regulator of cell apoptosis), gasdermin 1 (involved in apoptosis induction), TNF receptor associated factor 5 (TRAF5) and programmed cell death 1 (PDCD1) (Table 2). In addition, participation of apoptosis in SLV etiology was also supported by the IPA association of functions such as cell death (Figure 1) and pathways of apoptosis signaling and cytotoxic T cell-mediated apoptosis of target cells with DE genes in SL samples and DE genes identified by within SL comparison. These findings are consistent with elevated levels of apoptotic (TUNEL+) cells in the melanocyte region of growing feathers from vitiliginous SL chickens (particularly during active depigmentation) compared to non-vitiliginous SL, BL and LBL controls . The mechanism of apoptosis was also indicated by decreased levels of anti-apoptotic protein Bcl2 in lesional skin compared to nonlesional skin of vitiligo patients [51, 52].
The decreased expression of the gene for keratin especially in EV and AV compared to NV samples (Table 1) may suggest disturbance in keratinocyte function in early stages of SLV development. Since keratinocytes play important roles in supporting melanocyte growth and regulating melanogenesis in melanocytes , keratinocyte malfunction can have detrimental effects on melanocyte functions. Constant and concurrent down-regulation in matrix metalloproteinase MMP9 and MMP13 in SL chickens could be due to aberrant keratinocyte function, based on the observations in human studies that keratinocyte-derived MMP9 was important in repigmentation by enhancing melanocyte migration [54, 55]. Three DE genes TMEM9, TMEM22 and sorting Nexin 10 may suggest abnormal inter/intracellular molecule/organelle trafficking in SLV chickens. The reason for noticeably decreased expression in lipoprotein VSAF in SL chickens compared to BL is not clear due to lack of functional information on this protein. Significant down-regulation of parathyroid hormone (PTH) in EV, AV and CV compared to NV samples may suggest a possible role of PTH in SLV expression as its biological function is to increase serum calcium levels and vitamin D synthesis, both of which are important in melanogenesis.