Non-Coding RNAs in the Etiology and Control of Major and Neglected Human Tropical Diseases.

Non-coding RNAs (ncRNAs) including microRNAs (miRs) and long non-coding RNAs (lncRNAs) have emerged as key regulators of gene expression in immune cells development and function. Their expression is altered in different physiological and disease conditions, hence making them attractive targets for the understanding of disease etiology and the development of adjunctive control strategies, especially within the current context of mitigated success of control measures deployed to eradicate these diseases. In this review, we summarize our current understanding of the role of ncRNAs in the etiology and control of major human tropical diseases including tuberculosis, HIV/AIDS and malaria, as well as neglected tropical diseases including leishmaniasis, African trypanosomiasis and leprosy. We highlight that several ncRNAs are involved at different stages of development of these diseases, for example miR-26-5p, miR-132-3p, miR-155-5p, miR-29-3p, miR-21-5p, miR-27b-3p, miR-99b-5p, miR-125-5p, miR-146a-5p, miR-223-3p, miR-20b-5p, miR-142-3p, miR-27a-5p, miR-144-5p, miR-889-5p and miR-582-5p in tuberculosis; miR-873, MALAT1, HEAL, LINC01426, LINC00173, NEAT1, NRON, GAS5 and lincRNA-p21 in HIV/AIDS; miR-451a, miR-let-7b and miR-106b in malaria; miR-210, miR-30A-5P, miR-294, miR-721 and lncRNA 7SL RNA in leishmaniasis; and miR-21, miR-181a, miR-146a in leprosy. We further report that several ncRNAs were investigated as diseases biomarkers and a number of them showed good potential for disease diagnosis, including miR-769-5p, miR-320a, miR-22-3p, miR-423-5p, miR-17-5p, miR-20b-5p and lncRNA LOC152742 in tuberculosis; miR-146b-5p, miR-223, miR-150, miR-16, miR-191 and lncRNA NEAT1 in HIV/AIDS; miR-451 and miR-16 in malaria; miR-361-3p, miR-193b, miR-671, lncRNA 7SL in leishmaniasis; miR-101, miR-196b, miR-27b and miR-29c in leprosy. Furthermore, some ncRNAs have emerged as potential therapeutic targets, some of which include lncRNAs NEAT1, NEAT2 and lnr6RNA, 152742 in tuberculosis; MALAT1, HEAL, SAF, lincRNA-p21, NEAT1, GAS5, NRON, LINC00173 in HIV/AIDS; miRNA-146a in malaria. Finally, miR-135 and miR-126 were proposed as potential targets for the development of therapeutic vaccine against leishmaniasis. We also identify and discuss knowledge gaps that warrant for increased research work. These include investigation of the role of ncRNAs in the etiology of African trypanosomiasis and the assessment of the diagnostic potential of ncRNAs for malaria, and African trypanosomiasis. The potential targeting of ncRNAs for adjunctive therapy against tuberculosis, leishmaniasis, African trypanosomiasis and leprosy, as well as their targeting in vaccine development against tuberculosis, HIV/AIDS, malaria, African trypanosomiasis and leprosy are also new avenues to explore.

miRNome Expression Analysis Reveals New Players on Leprosy Immune Physiopathology.

Leprosy remains as a public health problem and its physiopathology is still not fully understood. MicroRNAs (miRNA) are small RNA non-coding that can interfere with mRNA to regulate gene expression. A few studies using DNA chip microarrays have explored the expression of miRNA in leprosy patients using a predetermined set of genes as targets, providing interesting findings regarding the regulation of immune genes. However, using a predetermined set of genes restricted the possibility of finding new miRNAs that might be involved in different mechanisms of disease. Thus, we examined the miRNome of tuberculoid (TT) and lepromatous (LL) patients using both blood and lesional biopsies from classical leprosy patients (LP) who visited the Dr. Marcello Candia Reference Unit in Sanitary Dermatology in the State of Pará and compared them with healthy subjects. Using a set of tools to correlate significantly differentially expressed miRNAs with their gene targets, we identified possible interactions and networks of miRNAs that might be involved in leprosy immunophysiopathology. Using this approach, we showed that the leprosy miRNA profile in blood is distinct from that in lesional skin as well as that four main groups of genes are the targets of leprosy miRNA: (1) recognition and phagocytosis, with activation of immune effector cells, where the immunosuppressant profile of LL and immunoresponsive profile of TT are clearly affected by miRNA expression; (2) apoptosis, with supportive data for an antiapoptotic leprosy profile based on BCL2, MCL1, and CASP8 expression; (3) Schwann cells (SCs), demyelination and epithelial-mesenchymal transition (EMT), supporting a role for different developmental or differentiation gene families, such as Sox, Zeb, and Hox; and (4) loss of sensation and neuropathic pain, revealing that RHOA, ROCK1, SIGMAR1, and aquaporin-1 (AQP1) may be involved in the loss of sensation or leprosy pain, indicating possible new therapeutic targets. Additionally, AQP1 may also be involved in skin dryness and loss of elasticity, which are well known signs of leprosy but with unrecognized physiopathology. In sum, miRNA expression reveals new aspects of leprosy immunophysiopathology, especially on the regulation of the immune system, apoptosis, SC demyelination, EMT, and neuropathic pain.

FoxP3 provides competitive fitness to CD4⁺CD25⁺ T cells in leprosy patients via transcriptional regulation.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae. FoxP3 have been shown to have important implications in various diseases. The present study describes the mechanism of action of FoxP3 in CD4⁺CD25⁺ T cells derived from leprosy patients. Increased molecular interactions of FoxP3 with histone deacetylases 7/9 in the nucleus of CD4⁺CD25⁺ T cells derived from borderline lepromatous leprosy/lepromatous leprosy (BL/LL) patients were found to be responsible for FoxP3-driven immune suppression activities during the progression of leprosy. Further, downregulation of CTLA-4 and CD25 genes in siFoxP3-treated PBMCs derived from BL/LL patients elucidated the transcription-activating nature of FoxP3. This observation was supported by direct binding of FoxP3 to the promoter region of the CTLA-4 and CD25 genes, and FoxP3's molecular interaction with histone acetyl transferases. The study also revealed that the increased expression of miR155 in CD4⁺CD25⁺ cells from BL/LL governs the competitive fitness of these cells. Again, reduced Annexin V & propidium iodide staining and Nur77 expression, and concomitantly increased Ki-67 positivity suggested that CD4⁺CD25⁺ cells derived from BL/LL patients are more competitively fit than those from borderline tuberculoid leprosy/tuberculoid leprosy and healthy controls. Taken together, the study shows the orchestration of FoxP3 leading to competitive fitness of Treg cells in leprosy.

Profiling microRNA expression in bovine alveolar macrophages using RNA-seq.

MicroRNAs (miRNAs) are important regulators of gene expression and are known to play a key role in regulating both adaptive and innate immunity. Bovine alveolar macrophages (BAMs) help maintain lung homeostasis and constitute the front line of host defense against several infectious respiratory diseases, such as bovine tuberculosis. Little is known, however, about the role miRNAs play in these cells. In this study, we used a high-throughput sequencing approach, RNA-seq, to determine the expression levels of known and novel miRNAs in unchallenged BAMs isolated from lung lavages of eight different healthy Holstein-Friesian male calves. Approximately 80 million sequence reads were generated from eight BAM miRNA Illumina sequencing libraries, and 80 miRNAs were identified as being expressed in BAMs at a threshold of at least 100 reads per million (RPM). The expression levels of miRNAs varied over a large dynamic range, with a few miRNAs expressed at very high levels (up to 800,000RPM), and the majority lowly expressed. Notably, many of the most highly expressed miRNAs in BAMs have known roles in regulating immunity in other species (e.g. bta-let-7i, bta-miR-21, bta-miR-27, bta-miR-99b, bta-miR-146, bta-miR-147, bta-miR-155 and bta-miR-223). The most highly expressed miRNA in BAMs was miR-21, which has been shown to regulate the expression of antimicrobial peptides in Mycobacterium leprae-infected human monocytes. Furthermore, the predicted target genes of BAM-expressed miRNAs were found to be statistically enriched for roles in innate immunity. In addition to profiling the expression of known miRNAs, the RNA-seq data was also analysed to identify potentially novel bovine miRNAs. One putatively novel bovine miRNA was identified. To the best of our knowledge, this is the first RNA-seq study to profile miRNA expression in BAMs and provides an important reference dataset for investigating the regulatory roles miRNAs play in this important immune cell type.

MicroRNA-21 targets the vitamin D-dependent antimicrobial pathway in leprosy.

Leprosy provides a model to investigate mechanisms of immune regulation in humans, given that the disease forms a spectrum of clinical presentations that correlate with host immune responses. Here we identified 13 miRNAs that were differentially expressed in the lesions of subjects with progressive lepromatous (L-lep) versus the self-limited tuberculoid (T-lep) disease. Bioinformatic analysis revealed a significant enrichment of L-lep-specific miRNAs that preferentially target key immune genes downregulated in L-lep versus T-lep lesions. The most differentially expressed miRNA in L-lep lesions, hsa-mir-21, was upregulated in Mycobacterium leprae-infected monocytes. By directly downregulating Toll-like receptor 2/1 heterodimer (TLR2/1)-induced CYP27B1 and IL1B expression as well as indirectly upregulating interleukin-10 (IL-10), hsa-mir-21 inhibited expression of the genes encoding two vitamin D-dependent antimicrobial peptides, CAMP and DEFB4A. Conversely, knockdown of hsa-mir-21 in M. leprae-infected monocytes enhanced expression of CAMP and DEFB4A and restored TLR2/1-mediated antimicrobial activity against M. leprae. Therefore, the ability of M. leprae to upregulate hsa-mir-21 targets multiple genes associated with the immunologically localized disease form, providing an effective mechanism to escape from the vitamin D-dependent antimicrobial pathway.