Genome-Wide Screening of mRNA Expression in Leprosy Patients

Abstract

Leprosy, an infectious disease caused by Mycobacterium leprae, affects millions of people worldwide. However, little is known regarding its molecular pathophysiological mechanisms. In this study, a comprehensive assessment of human mRNA was performed on leprosy skin lesions by using DNA chip microarrays, which included the entire spectrum of the disease along with its reactional states. Sixty-six samples from leprotic lesions (10TT, 10BT, 10BB, 10BL, 4LL, 14R1, and 10R2) and nine skin biopsies from healthy individuals were used as controls (CC) (ages ranged from 06 to 83 years, 48 were male and 29 female). The evaluation identified 1580 differentially expressed mRNAs [Fold Change (FC) ≥ 2.0, p ≤ 0.05] in diseased lesions vs. healthy controls. Some of these genes were observed in all forms of the disease (CD2, CD27, chit1, FA2H, FAM26F, GZMB, MMP9, SLAMF7, UBD) and others were exclusive to reactional forms (Type "1" reaction: GPNMB, IL1B, MICAL2, FOXQ1; Type "2" reaction: AKR1B10, FAM180B, FOXQ1, NNMT, NR1D1, PTX3, TNFRSF25). In literature, these mRNAs have been associated with numerous pathophysiological processes and signaling pathways and are present in a large number of diseases. The role of these mRNAs maybe studied in the context of developing new diagnostic markers and therapeutic targets for leprosy.

Keywords: immunohistochemistry; leprosy; mRNA; microarray; signaling pathways.

Figure 1

Study design. Patients diagnosed with leprosy underwent two punch “5” biopsies on the border of skin lesions (A). One sample was fixed in 10% buffered formalin for routine histological processing and sections were stained with H&E, Fite-Faraco, and immunohistochemistry (B). The second sample was fixed in RNAlater solution for RNA extraction (C). Skin samples from nine healthy subjects were collected and processed in similarly. Samples with good RNA quality were hybridized on microarray plates for identification of differentially expressed mRNAs (FC ≥ 2.0, p ≤ 0.05) (C). Patients were classified according to Ridley and Jopling's criteria (D). Differentially expressed mRNAs were identified by comparison between the various groups (E).

Figure 2

Histological sections of skin biopsies stained with H&E and Faraco-Fite. CC showing skin and subcutaneous tissue without inflammatory process (A, H&E x2 and B, H&E x20). In TT, there are tuberculoid granulomas with epithelioid macrophages in the center and lymphocytes at the periphery (C, H&E x2 and D, H&E x20). In BT, the granulomas are similar to those of TT (E, H&E x2), in which granuloma involves a nerve branch (F, H&E x20). In BB, the inflammatory infiltrate is less intense (G, H&E x2) and granulomas are less defined with lymphocytes and macrophages involving neural branches (H, H&E x20). The inflammatory infiltrate in BL is similar to BB, but more extensive with a larger number of macrophages, lymphocytes and plasma cells (I, H&E x2 and J, H&E x20). In LL, the infiltrates are dense, occupying all segments of the skin and subcutaneous tissue (K, H&E x2), and they consist of multivacuolated macrophages with rare permeating lymphocytes (L, H&Ex40). In R1, granulomas are defined with little inflammatory infiltrate extending adjacent to the interstitium (M, H&E x2), and the pre-existing granulomas are permeated by young macrophages and lymphocytes and occasionally exhibit necrosis (N, H&E x40). In R2, the inflammatory infiltrates involve all layers of the dermis with clusters of neutrophils forming micro abscesses (O, H&E x2 and P, H&E x40). Bacilloscopy 0+ in TT sample (Q, Fite-Faraco x100) and 6+ in LL sample (R, Faraco-Fite x100).

Figure 3

Histological sections of skin biopsies stained by IHC. ANGPTL4 expression in neural branches (Schwann cell) and luminal in sweat glands of CC (A1, IHC x20); strong expression in macrophages in TT granulomas (A2, IHC x10). Weak expression of BAI1 in neural branches of CC (B1, IHC x20) and absence of granulomas R1 (B2, IHC x20). Weak expression of BCAT1 in sebaceous gland CC (C1, IHC x10) and strong in macrophages of R1 granulomas (C2, IHC x20). CD2 positive in rare lymphocytes around capillaries on the papillary dermis of CC (D1, IHCx40) and large numbers of lymphocytes in the periphery granuloma TT (D2, HCI x40). CD27 expression in rare lymphocytes around capillaries in the papillary dermis of CC (E1, IHC x100) and large numbers of lymphocytes in the periphery and inside some TT granulomas (E2, IHCx40). No expression of CD52 in lymphocytes of CC for (F1, IHCx40) and large numbers of lymphocytes in the periphery of R1 granuloma (F2, IHC x40). EML2 expression absent in all skin components in DC (G1, X4 IHC) and weak positivity in macrophages of R2 (G2, IHCx40). FA2H expressed in sebaceous glands of CC (H1, IHCx10) and macrophage of R1 granulomas (H2, IHCx 40). GZMB expression absent in perivascular lymphocyte of CC (I1, IHCx40) and finely granular and cytoplasmic positivity in rare lymphocytes in the periphery and inside TT granulomas (I2, IHCx100). Strong LIPA expression in sebaceous glands of CC (J1, IHC x2) and macrophages of TT granulomas (J2, IHC x 40). Strong MMP9 expression in macrophages of TT and R2 granulomas (K1, IHCx40, K2, IHCx40). Absent expression of NCF1 in all skin components in CC (L1, IHCx2) and positivity in macrophage of R2 granuloma (L2, IHCx 40). Weak immunostaining of PTX3 on perivascular cells in CC (M1, IHCx20) and in macrophages, neutrophils, and interstitial cells of R2 (M2, IHCx 40). SIGLEC15 expression in endothelial cells of CC (N1, IHCx 20) and macrophages in LL (N2, IHCx 40). UBD expression in the inner layer of the excretory duct of sweat gland cells (O1, IHCx 4) and in vacuoles of macrophage in LL (O2, IHCx 100).

Figure 4

Heat map of the 88 mRNAs submitted to RT-PCR validation. Hierarchical unsupervised cluster, with euclidean distance and average linkage of all samples and differentially expressed gene validated. All samples [controls, clinical forms (TT, BT, BB, BL, LL), R1, and R2] and the 69 genes validated were submitted to analysis of the unsupervised hierarchical groups, with correlation Pearson metric and average linkage. Samples selected for validation are marked with a ^*.