Identifying quantitative sncRNAs signature using global sequencing as a potential biomarker for tuberculosis diagnosis and their role in regulating host response.

OBJECTIVES: The study aimed to identify a quantitative signature of circulating small non-coding RNAs (sncRNAs) as a biomarker for pulmonary tuberculosis disease (active-TB/ATB) and explore their regulatory roles in host-pathogen interactions and disease progression. METHODS: We conducted a cross-sectional study recruiting subjects diagnosed with active-TB (drug-sensitive and drug-resistant) and healthy controls. Sera samples were collected and utilized for preparing small RNA libraries. Quantitative patterns of circulating sncRNAs (miRNAs, piRNAs and tRFs) were identified via high-throughput sequencing and DeSeq2 analysis and validated in independent active-TB cohorts. Functional knockdown for two selected miRNAs were also performed. RESULTS: A diagnostic signature of four sncRNAs for both drug-sensitive and drug-resistant active-TB cases was validated, exhibiting an AUC of 0.96 (95% CI: 0.937-0.996, p < 0.001) with 86.7% sensitivity (95% CI: 0.775-0.932) and 91.7% specificity (95% CI: 0.730-0.990) in ROC analysis. Functional knockdown demonstrated regulatory roles of hsa-miR-223-5p and hsa-miR-10b-5p in Mycobacterium tuberculosis (Mtb) growth and pro-inflammatory cytokine expression (IL-6 and IL-8). CONCLUSION: The study identified a diagnostic tool utilizing a signature of four sncRNAs with high specificity and sensitivity, enhancing our understanding of sncRNAs as ATB diagnostic biomarker. Additionally, hsa-miR-223-5p and hsa-miR-10b-5p demonstrated potential roles in Mtb pathogenesis and host-response to infection.

Antisense Therapy for Infectious Diseases.

Infectious diseases, particularly Tuberculosis (TB) caused by Mycobacterium tuberculosis, pose a significant global health challenge, with 1.6 million reported deaths in 2021, making it the most fatal disease caused by a single infectious agent. The rise of drug-resistant infectious diseases adds to the urgency of finding effective and safe intervention therapies. Antisense therapy uses antisense oligonucleotides (ASOs) that are short, chemically modified, single-stranded deoxyribonucleotide molecules complementary to their mRNA target. Due to their designed target specificity and inhibition of a disease-causing gene at the mRNA level, antisense therapy has gained interest as a potential therapeutic approach. This type of therapy is currently utilized in numerous diseases, such as cancer and genetic disorders. Currently, there are limited but steadily increasing studies available that report on the use of ASOs as treatment for infectious diseases. This review explores the sustainability of FDA-approved and preclinically tested ASOs as a treatment for infectious diseases and the adaptability of ASOs for chemical modifications resulting in reduced side effects with improved drug delivery; thus, highlighting the potential therapeutic uses of ASOs for treating infectious diseases.

Host-Directed Targeting of LincRNA-MIR99AHG Suppresses Intracellular Growth of Mycobacterium tuberculosis.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) kills 1.6 million people worldwide every year, and there is an urgent need for targeting host-pathogen interactions as a strategy to reduce mycobacterial resistance to current antimicrobials. Noncoding RNAs are emerging as important regulators of numerous biological processes and avenues for exploitation in host-directed therapeutics. Although long noncoding RNAs (lncRNAs) are abundantly expressed in immune cells, their functional role in gene regulation and bacterial infections remains understudied. In this study, we identify an immunoregulatory long intergenic noncoding RNA, lincRNA-MIR99AHG, which is upregulated in mouse and human macrophages upon IL-4/IL-13 stimulation and downregulated after clinical Mtb HN878 strain infection and in peripheral blood mononuclear cells from active TB patients. To evaluate the functional role of lincRNA-MIR99AHG, we used antisense locked nucleic acid (LNA) GapmeR-mediated antisense oligonucleotide (ASO) lncRNA knockdown experiments. Knockdown of lincRNA-MIR99AHG with ASOs significantly reduced intracellular Mtb growth in mouse and human macrophages and reduced pro-inflammatory cytokine production. In addition, in vivo treatment of mice with MIR99AHG ASOs reduced the mycobacterial burden in the lung and spleen. Furthermore, in macrophages, lincRNA-MIR99AHG is translocated to the nucleus and interacts with high affinity to hnRNPA2/B1 following IL-4/IL-13 stimulation and Mtb HN878 infection. Together, these findings identify lincRNA-MIR99AHG as a positive regulator of inflammation and macrophage polarization to promote Mtb growth and a possible target for adjunctive host-directed therapy against TB.

Differential Targeting of c-Maf, Bach-1, and Elmo-1 by microRNA-143 and microRNA-365 Promotes the Intracellular Growth of Mycobacterium tuberculosis in Alternatively IL-4/IL-13 Activated Macrophages.

Mycobacterium tuberculosis (Mtb) can subvert the host defense by skewing macrophage activation toward a less microbicidal alternative activated state to avoid classical effector killing functions. Investigating the molecular basis of this evasion mechanism could uncover potential candidates for host directed therapy against tuberculosis (TB). A limited number of miRNAs have recently been shown to regulate host-mycobacterial interactions. Here, we performed time course kinetics experiments on bone marrow-derived macrophages (BMDMs) and human monocyte-derived macrophages (MDMs) alternatively activated with IL-4, IL-13, or a combination of IL-4/IL-13, followed by infection with Mtb clinical Beijing strain HN878. MiR-143 and miR-365 were highly induced in Mtb-infected M(IL-4/IL-13) BMDMs and MDMs. Knockdown of miR-143 and miR-365 using antagomiRs decreased the intracellular growth of Mtb HN878, reduced the production of IL-6 and CCL5 and promoted the apoptotic death of Mtb HN878-infected M(IL-4/IL-13) BMDMs. Computational target prediction identified c-Maf, Bach-1 and Elmo-1 as potential targets for both miR-143 and miR-365. Functional validation using luciferase assay, RNA-pulldown assay and Western blotting revealed that c-Maf and Bach-1 are directly targeted by miR-143 while c-Maf, Bach-1, and Elmo-1 are direct targets of miR-365. Knockdown of c-Maf using GapmeRs promoted intracellular Mtb growth when compared to control treated M(IL-4/IL-13) macrophages. Meanwhile, the blocking of Bach-1 had no effect and blocking Elmo-1 resulted in decreased Mtb growth. Combination treatment of M(IL-4/IL-13) macrophages with miR-143 mimics or miR-365 mimics and c-Maf, Bach-1, or Elmo-1 gene-specific GapmeRs restored Mtb growth in miR-143 mimic-treated groups and enhanced Mtb growth in miR-365 mimics-treated groups, thus suggesting the Mtb growth-promoting activities of miR-143 and miR-365 are mediated at least partially through interaction with c-Maf, Bach-1, and Elmo-1. We further show that knockdown of miR-143 and miR-365 in M(IL-4/IL-13) BMDMs decreased the expression of HO-1 and IL-10 which are known targets of Bach-1 and c-Maf, respectively, with Mtb growth-promoting activities in macrophages. Altogether, our work reports a host detrimental role of miR-143 and miR-365 during Mtb infection and highlights for the first time the role and miRNA-mediated regulation of c-Maf, Bach-1, and Elmo-1 in Mtb-infected M(IL-4/IL-13) macrophages.

Deletion of Interleukin-4 Receptor Alpha-Responsive Keratinocytes in BALB/c Mice Does Not Alter Susceptibility to Cutaneous Leishmaniasis.

The skin microenvironment at the site of infection plays a role in the early events that determine protective T helper 1/type 1 immune responses during cutaneous leishmaniasis (CL) infection. During CL in nonhealing BALB/c mice, early interleukin-4 (IL-4) can instruct dendritic cells for protective Th1 immunity. Additionally, keratinocytes, which are the principal cell type in the skin epidermis, have been shown to secrete IL-4 early after Leishmania major infection. Here, we investigated whether IL-4/IL-13 signaling via the common IL-4 receptor alpha chain (IL-4Rα) on keratinocytes contributes to susceptibility during experimental CL. To address this, keratinocyte-specific IL-4Rα-deficient (KRT14cre IL-4Rα-/lox) mice on a BALB/c genetic background were generated by gene targeting and site-specific recombination (Cre/loxP) under the control of the keratinocyte-specific krt14 locus. Following high-dose infection with L. major IL-81 and LV39 promastigotes subcutaneously in the footpad, footpad swelling, parasite burden, IFN-γ/IL-4/IL-13 cytokine production, and type 1 and type 2 antibody responses were similar between KRT14cre IL-4Rα-/lox and littermate control IL-4Rα-/lox BALB/c mice. An intradermal infection with low-dose L. major IL-81 and LV39 promastigotes in the ear showed results in infected KRT14cre IL-4Rα-/lox BALB/c mice similar to those of littermate control IL-4Rα-/lox BALB/c mice, with the exception of a significant decrease observed in parasite burden only at the site of LV39 infection in the ear. Collectively, our results show that autocrine and paracrine signaling of IL-4/IL-13 through the IL-4Rα chain on keratinocytes does not influence the establishment of a nonhealing Th2 immune response in BALB/c mice during L. major infection.