Potential of AKR1B10 as a Biomarker and Therapeutic Target in Type 2 Leprosy Reaction

The AKR1B10 (aldo-keto reductase family 1 member B10) gene has important functions in carcinogen-induced neoplasia. AKR1B10 is also expressed in type 2 reaction leprosy patients (R2). We measured the expression of AKR1B10 in the skin lesions of patients with leprosy by immunohistochemistry from biopsies that encompassed the spectrum of types of leprosy, based on the Ridley and Jopling classification [10 samples each of tuberculoid (TT), borderline tuberculoid (BT), mid-borderline (BB), and borderline lepromatous (BL) lesions; four samples of lepromatous lesions (LL)], reactional leprosy [14 samples of type 1 Reaction (R1) and 10 samples of type 2 Reaction (R2)], and biopsies from 9 healthy control (HC) subjects. In addition, 46 lepromatous lesions (BL and LL), 45 lepromatous lesions in regression, and 115 R2 lesions were included. Eight of 10 R2 samples (80%), 3 of 46 active BL and LL samples (6%), 23 of 45 BL and LL samples in regression (51%), and 107 of 115 R2 samples (93%) were positive for AKR1B10, differing significantly between all groups (p < 0.05). AKR1B10 expression was highest in the cytoplasm of macrophages. Thus, AKR1B10 is overexpressed on the lepromatous side (BL and LL) in samples that are in regression, especially type 2 reaction-associated lesions, rendering it a potential marker of type 2 reactional episodes of leprosy and a target of drugs against reactional episodes

Differential Expression of MicroRNAs in Leprosy Skin Lesions

Leprosy, a chronic infectious disease caused by Mycobacterium leprae, is a major public health problem in poor and developing countries of the Americas, Africa, and Asia. MicroRNAs (miRNAs), which are small non-coding RNAs (18–24 nucleotides), play an important role in regulating cell and tissue homeostasis through translational downregulation of messenger RNAs (mRNAs). Deregulation of miRNA expression is important for the pathogenesis of various neoplastic and non-neoplastic diseases and has been the focus of many publications; however, studies on the expression of miRNAs in leprosy are rare. Herein, an extensive evaluation of differentially expressed miRNAs was performed on leprosy skin lesions using microarrays. Leprosy patients, classified according to Ridley and Jopling’s classification or reactional states (R1 and R2), and healthy controls (HCs) were included. Punch biopsies were collected from the borders of leprosy lesions (10 tuberculoid, 10 borderline tuberculoid, 10 borderline borderline, 10 borderline lepromatous, 4 lepromatous, 14 R1, and 9 R2) and from 9 HCs. miRNA expression profiles were obtained using the Agilent Microarray platform with miRBase, which consists of 1,368 Homo sapiens (hsa)-miRNA candidates. TaqMan quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) was used to validate differentially expressed miRNAs. Sixty-four differentially expressed miRNAs, including 50 upregulated and 14 downregulated (fold change ≥2.0, p-value ≤ 0.05) were identified after comparing samples from patients to those of controls. Twenty differentially expressed miRNAs were identified exclusively in the reactional samples (14 type 1 and 6 type 2). Eight miRNAs were validated by RT-PCR, including seven upregulated (hsa-miR-142-3p, hsa-miR-142-5p, hsa-miR-146b-5p, hsa-miR-342-3p, hsa-miR-361-3p, hsa-miR-3653, and hsa-miR-484) and one downregulated (hsa-miR-1290). These miRNAs were differentially expressed in leprosy and several other diseases, especially those related to the immune response. Moreover, the integration of analysis of validated mi/mRNAs obtained from the same samples allowed target pairs opposite expression pattern of hsa-miRNA-142-3p and AKR1B10, hsa-miRNA-342-3p and FAM180b, and hsa-miRNA-484 and FASN. This study identified several miRNAs that might play an important role in the molecular pathogenesis of the disease. Moreover, these deregulated miRNAs and their respective signaling pathways might be useful as therapeutic markers, therapeutic targets, which could help in the development of drugs to treat leprosy

Linear regression and significance among Th1, Th2, Th17 and Treg profile targets based on ΔC_T values, total number of fungal cells, and number of viable fungal cells.

<p>Analysis of T cell subsets: Treg (A to E), Th1 (F and G), Th2 (H to L), Th17 (M to P). <b>#</b>: in front of the regression lines represents significant correlation (p and r² values). Regression analysis was performed using patient data.</p

Gene expression analysis of Th1 profile targets.

<p>(A) Comparison of T-bet transcription factor expression between JLD patients and healthy controls. (B) Comparison of T-bet transcription factor expression between patients with single lesion and multiple lesions. (C) Comparison of IFN-γ cytokine expression between JLD patients and healthy controls. (D) Comparison of IFN-γ cytokine expression between patients with a single lesion and patients with multiple lesions. Statistical analysis: Mann-Whitney U test (#p≤0.05)</p

Gene expression analysis of Th2 profile targets.

<p>(A) Comparison of the GATA3 transcription factor between JLD patients and healthy controls. (B) Comparison of GATA3 transcription factor between patients with a single lesion and patients with multiple lesions. (C) Comparison of IL-4 cytokine expression between JLD patients and healthy controls. (D) Comparison of IL-4 cytokine expression between patients with a single lesion and patients with multiple lesions. (E) Comparison of IL-5 cytokine between JLD patients and healthy controls. (F) Comparison of IL-5 cytokine expression between patients with a single lesion and patients with multiple lesions. (G) Comparison of IL-13 cytokine expression between JLD patients and healthy controls. (H) Comparison of IL-13 cytokine expression between patients with a single lesion and patients with multiple lesions. (I) Comparison of IL-33 cytokine expression between JLD patients and healthy controls. (J) Comparison of IL-33 cytokine expression between patients with a single lesion and patients with multiple lesions. Statistical analysis: Mann-Whitney U test (#p≤ 0.05)</p

Gene expression analysis of Th17 profile targets.

<p>(A) Comparison of RORC transcription factor between JLD patients and healthy controls. (B) Comparison of RORC transcription factor between patients with a single lesion and patients with multiple lesions. (C) Comparison of IL-17A cytokine expression between JLD patients and healthy controls. (D) Comparison of IL-17A cytokine expression between patients with a single lesion and patients with multiple lesions. (E) Comparison of IL-17F cytokine expression between JLD patients and healthy controls. (F) Comparison of IL-17F cytokine expression between patients with a single lesion and patients with multiple lesions. (G) Comparison of IL-22 cytokine expression between JLD patients and healthy controls. (H) Comparison of IL-22 cytokine expression between patients with single lesion and patients with multiple lesions. Statistical analysis: Mann-Whitney U test (#p≤0.05)</p

Gene expression analysis of Treg profile targets.

<p>(A) Comparison of transcription factor FoxP3 between JLD patients and healthy controls. (B) Comparison of FoxP3 transcription factor between patients with a single lesion and patients with multiple lesions. (C) Comparison of CTLA4 marker expression between JLD patients and healthy controls. (D) Comparison of CTLA4 marker expression between patients with a single lesion and patients with multiple lesions. (E) Comparison of IL-10 cytokine expression between JLD patients and healthy controls. (F) Comparison of IL-10 cytokine expression between patients with a single lesion and patients with multiple lesions. (G) Comparison of TGF-β1 cytokine expression between JLD patients and healthy controls. (H) Comparison of TGF-x1 cytokine expression between patients with a single lesion and patients with multiple lesions. (I) Comparison of IKZF2 marker expression between JLD patients and healthy controls. (J) Comparison of IKZF2 marker expression between patients with a single lesion and patients with multiple lesions. Statistical analysis: Mann-Whitney U test (#p≤0.05)</p

A genome wide association study identifies a lncRna as risk factor for pathological inflammatory responses in leprosy

Submitted by Sandra Infurna ([email protected]) on 2018-02-08T11:09:47Z No. of bitstreams: 1 milton_moraes_etal_IOC_2017.pdf: 3018090 bytes, checksum: 4f1884a5b459722255f6434f0d7dfe23 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2018-02-08T11:28:23Z (GMT) No. of bitstreams: 1 milton_moraes_etal_IOC_2017.pdf: 3018090 bytes, checksum: 4f1884a5b459722255f6434f0d7dfe23 (MD5)Made available in DSpace on 2018-02-08T11:28:23Z (GMT). No. of bitstreams: 1 milton_moraes_etal_IOC_2017.pdf: 3018090 bytes, checksum: 4f1884a5b459722255f6434f0d7dfe23 (MD5) Previous issue date: 2017Research Institute of the McGill University Health Centre. Program in Infectious Diseases and Immunity in Global Health. Montreal, Quebec, canada / McGill University. The McGill International TB Centre, Departments of Human Genetics and Medicine. Montreal, Quebec, Canada.Research Institute of the McGill University Health Centre. Program in Infectious Diseases and Immunity in Global Health. Montreal, Quebec, canada / McGill University. The McGill International TB Centre, Departments of Human Genetics and Medicine. Montreal, Quebec, Canada.Institut National de la Santé et de la Recherche Médicale. Laboratory of Human Genetics of Infectious Diseases. Necker Branch. Paris, France / University Paris Descartes. Imagine Institute. Paris, France / Rockefeller University. Giles Laboratory of Human Genetics of Infectious Diseases. Rockefeller Branch. New York, USA.Research Institute of the McGill University Health Centre. Program in Infectious Diseases and Immunity in Global Health. Montreal, Quebec, canada / McGill University. The McGill International TB Centre, Departments of Human Genetics and Medicine. Montreal, Quebec, Canada.Hospital for Dermato-Venerology. Ho Chi Minh City, Vietnam.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ. Brasil.Universidade Federal de Goiás. Instituto de Saúde Pública e Patologia Tropical. Goiânia, GO, BrasilInstituto Lauro de Souza Lima. Bauru, SP. Brasil.Instituto Lauro de Souza Lima. Bauru, SP. Brasil.Hospital for Dermato-Venerology. Ho Chi Minh City, Vietnam.Institut National de la Santé et de la Recherche Médicale. Laboratory of Human Genetics of Infectious Diseases. Necker Branch. Paris, France / University Paris Descartes. Imagine Institute. Paris, France / Rockefeller University. Giles Laboratory of Human Genetics of Infectious Diseases. Rockefeller Branch. New York, USA.Institut National de la Santé et de la Recherche Médicale. Laboratory of Human Genetics of Infectious Diseases. Necker Branch. Paris, France / University Paris Descartes. Imagine Institute. Paris, France / Rockefeller University. Giles Laboratory of Human Genetics of Infectious Diseases. Rockefeller Branch. New York, USA.Research Institute of the McGill University Health Centre. Program in Infectious Diseases and Immunity in Global Health. Montreal, Quebec, canada / McGill University. The McGill International TB Centre, Departments of Human Genetics and Medicine. Montreal, Quebec, Canada.Leprosy Type-1 Reactions (T1Rs) are pathological inflammatory responses that afflict a sub-group of leprosy patients and result in peripheral nerve damage. Here, we employed a family-based GWAS in 221 families with 229 T1R-affect offspring with stepwise replication to identify risk factors for T1R. We discovered, replicated and validated T1R-specific associations with SNPs located in chromosome region 10p21.2. Combined analysis across the three independent samples resulted in strong evidence of association of rs1875147 with T1R (p = 4.5x10-8; OR = 1.54, 95% CI = 1.32-1.80). The T1R-risk locus was restricted to a lncRNA-encoding genomic interval with rs1875147 being an eQTL for the lncRNA. Since a genetic overlap between leprosy and inflammatory bowel disease (IBD) has been detected, we evaluated if the shared genetic control could be traced to the T1R endophenotype. Employing the results of a recent IBD GWAS meta-analysis we found that 10.6% of IBD SNPs available in our dataset shared a common risk-allele with T1R (p = 2.4x10-4). This finding points to a substantial overlap in the genetic control of clinically diverse inflammatory disorders

Enrichment of shared risk-alleles associated with T1R leprosy and IBD phenotypes.

<p>GWAS loci for (A) Inflammatory Bowel Disease [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006637#pgen.1006637.ref024" target="_blank">24</a>], (B) Schizophrenia [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006637#pgen.1006637.ref025" target="_blank">25</a>], (C) Human height [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006637#pgen.1006637.ref026" target="_blank">26</a>] and (D) Human blood metabolites [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006637#pgen.1006637.ref027" target="_blank">27</a>] are represented by a single SNP with the strongest evidence for association. The count of SNPs is given below each central blue pie (panels A—D) for each disease. The light blue pie slices indicate the numbers of SNPs that were not available for comparisons with the leprosy families. The proportions of shared SNPs with T1R-free leprosy (left light green pie) and T1R leprosy (right light orange pie) are indicated as pies for each of the GWAS sets (panel A—D). The pies in panel E represents the totality of SNPs tested in the present study for T1R-free and T1R-affected families. The yellow slices indicate the proportion of SNPs with nominal evidence for association with T1R-free leprosy or T1R/leprosy. The darker shade of yellow represent the proportion of SNPs that are significantly heterogeneous between T1R free and affected leprosy patients. The hypergeometric test used as baseline the proportion of nominally associated GWAS SNPs to estimate the significance of enrichment of the T1R/leprosy and T1R-free leprosy GWAS SNPs among the four selected disease phenotypes.</p

Variants on chromosome 10 are preferentially associated with T1R.

<p>(A) Manhattan plot of the TDT results for the T1R-affected families in the discovery phase. Genetic variants represented by dots are plotted according to their chromosomal position on the x-axis against the negative log <i>p</i> values on the y-axis. The blue horizontal line indicates suggestive association (<i>p</i> = 1.0x10^-06), and the red horizontal line represents significant association (<i>p</i> = 5.0x10^-08). (B) Quantiles-Quantiles plots of genotyped and imputed variants for T1R-affected families in the discovery phase. Variants were plotted according to the observed <i>p</i> values on the y-axis and the expected <i>p</i> values on the x-axis. The 95% confidence interval dispersion is presented by grey shades. The slight deflation of the Q-Q plot is likely due to the sample size employed. (C) Visualization of a 220 kb region on chromosome region 10p21.2. Evidence of association for SNPs located in the interval is plotted for the T1R-free families and the T1R-affected families. The negative log of the <i>p</i>-value on the left y-axis is plotted against the chromosomal SNP position on the x-axis. The recombination rate in centimorgan per mega base is displayed on the right y-axis. The solid horizontal line indicates the suggestive association (<i>p</i> = 1.0 x 10⁻⁰⁶) while the doted horizontal line represents nominal association (<i>p</i> = 0.05). The diamond plot at the bottom of panel C indicates the <i>r</i>² linkage disequilibrium for variants nominally associated with T1R in 763 leprosy unaffected parents from both T1R-affected and T1R-free families. Highlighted in red are the leading SNPs in seven LD bins (<i>r</i>² > 0.9). (D) Evidence for association of seven tag SNPs for the SNPs associated with T1R in the Chromosome10p21.2 region is shown for the discovery phase only (red dots) and for the combined analysis of all three samples (orange lozenges). The negative log of the <i>p</i>-value is indicated on the left y-axis. The recombination rate in centimorgan per mega base is displayed on the right y-axis and indicated by a blue line. The blue horizontal line indicates suggestive association (<i>p</i> = 1.0x10^-06), and the red horizontal line represents significant association (<i>p</i> = 5.0x10^-08). The genomic locations of RefSeq or ENSEMBL genes are given at the bottom.</p