Macrophage Migration Inhibitory Factor (MIF) and Its Homologue d-Dopachrome Tautomerase (DDT) Inversely Correlate with Inflammation in Discoid Lupus Erythematosus

Abstract

Discoid Lupus Erythematosus (DLE) is a chronic cutaneous disease of unknown etiology and of immunoinflammatory origin that is characterized by inflammatory plaques and may lead to disfiguring scarring and skin atrophy. Current treatments are limited, with a large proportion of patients either poorly or not responsive, which makes DLE an unmet medical need. Macrophage migration inhibitory factor (MIF) is the prototype of a pleiotropic family of cytokine that also includes the recently discovered homologue D-dopachrome tautomerase (DDT) or MIF2. MIF and DDT/MIF-2 exert several biological properties, primarily, but not exclusively of a proinflammatory nature. MIF and DDT have been suggested to play a key role in the pathogenesis of several autoimmune diseases, such as multiple sclerosis and type 1 diabetes, as well as in the development and progression of certain forms of cancers. In the present study, we have performed an immunohistochemistry analysis for the evaluation of MIF in DLE lesions and normal skin. We found high levels of MIF in the basal layer of the epidermis as well as in the cutaneous appendage (eccrine glands and sebocytes) of normal skin. In DLE lesions, we observed a significant negative correlation between the expression of MIF and the severity of inflammation. In addition, we performed an analysis of MIF and DDT expression levels in the skin of DLE patients in a publicly available microarray dataset. Interestingly, while these in silico data only evidenced a trend toward reduced levels of MIF, they demonstrated a significant pattern of expression and correlation of DDT with inflammatory infiltrates in DLE skins. Overall, our data support a protective role for endogenous MIF and possibly DDT in the regulation of homeostasis and inflammation in the skin and open up novel avenues for the treatment of DLE.

Keywords: d-dopachrome tautomerase; discoid lupus erythematosus; macrophage migration inhibitory factor.

Figure 1

Immunohistochemistry analysis of MIF expression in normal skin biopsies. High levels of MIF can be detected at the basal layer of the epidermis (red arrow) (A) and in the cutaneous appendages (eccrine glands and sebocytes) (black arrow) (B). Immunohistochemistry-positive staining was defined as the presence of brown chromogen detection within the cytoplasm. Representative microphotographs are shown.

Figure 2

Immunohistochemistry analysis of MIF expression in DLE skin biopsies. Thirty-seven patients with DLE were recruited for this study. Biopsies were performed after a six-month wash-out period from topical treatments. Hematoxylin and eosin staining was used for histopathological evaluation. Representative microphotographs are shown. (A–D) In the presence of a high inflammatory score (2+; 3+), MIF low expression (0; 1+) was observed in the epidermis (one arrow), appendages (two arrows), and inflammatory infiltrate (three arrows). ((A,B): patient no. 1; (C,D): patient no. 2). (E–H) In the presence of a low inflammatory score (0; 1+), MIF high expression (2+; 3+) was observed in the epidermis (one arrow), appendages (two arrows), and inflammatory infiltrate (three arrows). Immunohistochemistry positive staining was defined as the presence of brown chromogen detection within the cytoplasm. (E,F): patient no. 3; (G,H): patient no. 4).

Figure 3

Correlation analysis between MIF expression and inflammatory score in DLE skin biopsies. Thirty-seven patients with DLE were recruited for this study. Biopsies were performed after a six-month wash-out period from topical treatments. Hematoxylin and eosin staining was used for histopathological evaluation. (A) Correlation between total MIF expression and inflammatory score. (B) Correlation between MIF expresses in the basal layer of the epidermis and the inflammatory score. (C) Correlation between MIF expression in the infiltrate and the inflammatory score. (D) Correlation between MIF expression in the annexes and the inflammatory score. Correlation analysis was performed using the non-parametric Spearman’s test.

Figure 4

Relative proportions of DLE samples with different expression of MIF and increasing inflammatory score. Thirty-seven patients with DLE were recruited for this study. Biopsies were performed after a six-month wash-out period from topical treatments. Hematoxylin and eosin staining was used for histopathological evaluation. Statistical analysis was performed using the Fisher’s chi-square test. Infl score: Inflammatory score.

Figure 5

Relative expression analysis of MIF and DDT in DLE samples. Determination of the expression levels of MIF and DDT was performed by interrogating the GSE52471 microarray dataset, retrieved from the Gene Expression Omnibus databank (GEO; https://www.ncbi.nlm.nih.gov/gds). The GSE52471 dataset included whole genome expression profiles of skin biopsies from seven DLE patients, 13 healthy donors, and 18 psoriatic patients. The LIMMA (Linear Models for Microarray Analysis) R package was used for identifying differentially expressed genes. An adjusted p-value < 0.05 was considered as the threshold of significance.

Figure 6

Correlation between MIF and DDT and infiltrating immune cells in DLE. The relative proportion of infiltrating immune cells in DLE skin samples, as compared to normal and psoriatic samples was performed using xCell (http://xcell.ucsf.edu/) software on the gene expression data obtained from the GSE52471 dataset. Data are provided separately for the myeloid cell populations (A) and the lymphoid cell populations (B). Differential analysis of immune cell abundance among the groups of samples was performed using the non-parametric Kruskal–Wallis test, followed by Dunn’s post hoc test. (C) Correlation analysis between MIF and infiltrating immune cell populations in DLE samples. (D) Correlation analysis between DDT and infiltrating immune cell populations in DLE samples. Correlation analysis was performed using the non-parametric Spearman’s test. The dashed line represents the threshold of significance, i.e., p < 0.05.