MicroRNAs Regulated by the LPS/TLR2 Immune Axis as Bona Fide Biomarkers for Diagnosis of Acute Leptospirosis

Abstract

Leptospirosis remains a significant human health issue due to its systemic complications. Therefore, biomarkers that are more effective are urgently needed for the early diagnosis of leptospirosis. MicroRNAs (miRNAs) are evolutionarily conserved regulatory RNAs that have shown the potential to be used as biomarkers for diagnosis, prognosis, and therapy of infectious diseases. In this study, we performed an unbiased screen using the miRNome miRNA array to identify circulating miRNAs with the potential to serve as authentic biomarkers for early diagnosis of leptospirosis. Because leptospiral lipopolysaccharide (LPS) is the predominant leptospiral antigen and plays a vital role in immunological and biological activities, we used LPS treated and untreated in vitro (THP1 cells) and in vivo (BALB/c mice) surrogate models to identify the LPS-specific miRNAs. Differential expression analysis revealed 18 miRNAs to be associated strongly with LPS stimulation in THP1 cells. Of these, three (miR-let-7b-5p, miR-144-3p, and miR-21-5p) were observed to be present at increased levels in vivo. The identified miRNAs were validated for their biomarker potential using serum samples from leptospirosis-negative patients and patients with confirmed cases of leptospirosis. Identified miRNAs were able to discriminate the acute leptospiral infection from other febrile diseases with a test sensitivity and specificity of 93.2% and 88.19%, respectively. Gene functional enrichment and protein-protein interaction (PPI) network analysis revealed that the identified miRNAs play important roles in disease signal transduction, signaling by interleukins, the stress-activated protein kinase signaling cascade, the mitogen-activated protein kinase (MAPK) signaling pathway, and the cellular response to a transforming growth factor β (TGF-β) stimulus with a notable interconnection between these biological processes. IMPORTANCE Here, we used miRNAs that are differentially regulated by the LPS/TLR2 immune axis to devise a miRNA-based diagnosis for leptospirosis. The study established the role of the circulating stable miRNAs (miR-21-5p, miR-144-3p, and miR-let-7b-5p) as an early diagnostic marker for leptospirosis. These miRNAs can be used to diagnose acute leptospirosis and also to differentiate leptospiral infection from other bacterial and spirochetal infections, as proved by the use of human clinical samples. Thus, our findings indicate that miRNAs can play a crucial role in the diagnosis of infectious diseases, like leptospirosis, that are generally misdiagnosed.

Study on the Correlation between Interleukin-27 and CXCL10 in Pulmonary Tuberculosis

“…In fact, miR-let-7b-5p research is very limited currently; however, some scholars have noticed its role in infection and inflammation [ 28 , 29 ]. Our study also suggests that miR-let-7b-5p can participate in the antibacterial process, through the STAT1 pathway ( Figure 5(e) ).…”

Identification and validation of circulating miRNAs as potential new biomarkers for severe liver disease in patients with leptospirosis

“…Regarding leptospirosis, in vitro data demonstrated that several miRNAs associated with immune regulation were involved in Leptospira infection in a virulence- and species-specific manner [ 20 ]. Moreover, a previous study identified specific miRNAs that were able to discriminate the infection from other acute febrile diseases with a high sensitivity and specificity [ 21 ]. Additionally, our group recently demonstrated distinct expression of circulating microRNAs as novel biomarkers for identifying severe leptospirosis [ 22 ].…”

Role of toll-like receptor 2 during infection ofLeptospiraspp.: A systematic review

“…NFκB [48], Fibronectin [30] and CXCL2/MIP2 [40] showed the inhibition or decreased level in TLR2 blocked/deficient cell lines. The data convey the involvement of TLR2 in triggering the immune response during the Leptospira infection.…”

“…The studies encompassed a range of experimental models, including 3 human [20][21][22], 5 in-vivo [23][24][25][26][27], 14 in-vitro [28][29][30][31][32][33][34][35][36][37][38][39][40][41], and 3 ex-vivo studies [42][43][44]. Additionally, four studies [9,10,45,46] used a combination of in-vivo and in-vitro models, while two studies [47,48] used a combination of human, in-vitro, and in-vivo models. Finally, one study [49] used both in-vivo and ex-vivo models.…”

“…Indirect response: Blocked TLR2 / TLR2 deficient cell lines that stimulated using pathogenic Leptospira organism/cell components showed inhibition or decreased level of cytokine/immune mediators compared to intact TLR2 cell lines. Even with the heterogeneous stimulants and the cell lines, IL6 [9,10,28,29,37,41,45,47], IL8 [9,38,[41][42][43], IL 1β [29,41,43,47,48], TNFα [9,28,29,37,38,41,43,45,47,48], IFNγ [29], IL10 [46,48], iNOS [31,33], CCL2/MCP-1 [29,31,33,38,39], hBD2 [41,43], CCL10 [29,[38][39][40], COX2 [29], CXCL1/KC [39],…”