Kamlesh Pawar

Monocytes, a type of leukocytes, are key players in immune responses, transitioning to macrophages at infection sites. Differentiation, which is crucial for macrophage survival and resistance to apoptosis, is a tightly regulated process. Based on our earlier findings implicating the 5'-fragment of tRNA^HisGUG (5'-HisGUG) in macrophage biology, we specifically focused on these fragments to investigate its regulation during monocyte-to-macrophage differentiation and its role in macrophage survival. We performed small RNA sequencing during monocyte-to-macrophage differentiation using THP-1 cells and identified 5'-HisGUG as a prominently regulated tDR. Notably, transfection of 5'-HisGUG in macrophages decreased survival under apoptotic stress. We propose that 5'-HisGUG can also act as an obstructive RNA (oRNA), and its reduction is critical for macrophage survival under stress.

Toll-like receptors (TLRs) are crucial components of the innate immune system. Endosomal TLR7 recognizes single-stranded RNAs, yet its endogenous ssRNA ligands are not fully understood. We previously showed that extracellular (ex-) 5′-half molecules of tRNA ^HisGUG (the 5′-tRNA ^HisGUG half) in extracellular vesicles (EVs) of human macrophages activate TLR7 when delivered into endosomes of recipient macrophages. Here, we fully explored immunostimulatory ex-5′-tRNA half molecules and identified the 5′-tRNA ^ValCAC/AAC half, the most abundant tRNA-derived RNA in macrophage EVs, as another 5′-tRNA half molecule with strong TLR7 activation capacity. Levels of the ex-5′-tRNA ^ValCAC/AAC half were highly up-regulated in macrophage EVs upon exposure to lipopolysaccharide and in the plasma of patients infected with Mycobacterium tuberculosis . The 5′-tRNA ^ValCAC/AAC half-mediated activation of TLR7 effectively eradicated bacteria infected in macrophages. Mutation analyses of the 5′-tRNA ^ValCAC/AAC half identified the terminal GUUU sequence as a determinant for TLR7 activation. We confirmed that GUUU is the optimal ratio of guanosine and uridine for TLR7 activation; microRNAs or other RNAs with the terminal GUUU motif can indeed stimulate TLR7, establishing the motif as a universal signature for TLR7 activation. These results advance our understanding of endogenous ssRNA ligands of TLR7 and offer insights into diverse TLR7-involved pathologies and their therapeutic strategies.

Toll-like receptors (TLRs) play a crucial role in the innate immune response. Although endosomal TLR7 recognizes single-stranded RNAs, their endogenous RNA ligands have not been fully explored. Here, we report 5′-tRNA half molecules as abundant activators of TLR7. Mycobacterial infection and accompanying surface TLR activation up-regulate the expression of 5′-tRNA half molecules in human monocyte-derived macrophages (HMDMs). The abundant accumulation of 5′-tRNA halves also occur in HMDM-secreted extracellular vehicles (EVs); the abundance of EV-5′-tRNA^HisGUGhalf molecules is >200-fold higher than that of the most abundant EV-microRNA (miRNA). Sequence identification of the 5′-tRNA halves using cP-RNA-seq revealed abundant and selective packaging of specific 5′-tRNA half species into EVs. The EV-5′-tRNA^HisGUGhalf was experimentally demonstrated to be delivered into endosomes in recipient cells and to activate endosomal TLR7. Up-regulation of the 5′-tRNA half molecules was also observed in the plasma of patients infected withMycobacterium tuberculosis. These results unveil a novel tRNA-engaged pathway in the innate immune response and assign the role of “immune activators” to 5′-tRNA half molecules.

Post-transcriptional non-template additions of nucleotides to 3′-ends of RNAs play important roles in the stability and function of RNA molecules. Although tRNA nucleotidyltransferase (CCA-adding enzyme) is known to add CCA trinucleotides to 3′-ends of tRNAs, whether other RNA species can be endogenous substrates of CCA-adding enzyme has not been widely explored yet. Herein, we used YAMAT-seq to identify non-tRNA substrates of CCA-adding enzyme. YAMAT-seq captures RNA species that form secondary structures with 4-nt protruding 3′-ends of the sequence 5′-NCCA-3′, which is the hallmark structure of RNAs that are generated by CCA-adding enzyme. By executing YAMAT-seq for human breast cancer cells and mining the sequence data, we identified novel candidate substrates of CCA-adding enzyme. These included fourteen “CCA-RNAs” that only contain CCA as non-genomic sequences, and eleven “NCCA-RNAs” that contain CCA and other nucleotides as non-genomic sequences. All newly-identified (N)CCA-RNAs were derived from the mitochondrial genome and were localized in mitochondria. Knockdown of CCA-adding enzyme severely reduced the expression levels of (N)CCA-RNAs, suggesting that the CCA-adding enzyme-catalyzed CCA additions stabilize the expression of (N)CCA-RNAs. Furthermore, expression levels of (N)CCA-RNAs were severely reduced by various cellular treatments, including UV irradiation, amino acid starvation, inhibition of mitochondrial respiratory complexes, and inhibition of the cell cycle. These results revealed a novel CCA-mediated regulatory pathway for the expression of mitochondrial non-coding RNAs.

Main survival mechanism of pathogenic mycobacteria is to escape inimical phagolysosomal environment inside the macrophages. Many efforts have been made to unravel the molecular mechanisms behind this process. However, little is known about the involvement of microRNAs (miRNAs) in the regulation of phagolysosomal biosynthesis and maturation. Based on a bottom up approach, we searched for miRNAs that were involved in phagolysosomal processing events in the course of mycobacterial infection of macrophages. After infecting THP-1 derived macrophages with viable and heat killed Mycobacterium bovis BCG (BCG), early time points were identified after co-localization studies of the phagosomal marker protein LAMP1 and BCG. Differences in LAMP1 localization on the phagosomes of both groups were observed at 30 min and 4 h. After in silico based pre-selection of miRNAs, expression analysis at the identified time points revealed down-regulation of three miRNAs: miR-3619-5p, miR-637 and miR-324-3p. Consequently, most likely targets were predicted that were supposed to be mutually regulated by these three studied miRNAs. The lysosomal cysteine protease Cathepsin S (CTSS) and Rab11 family-interacting protein 4 (RAB11FIP4) were up-regulated and were considered to be connected to lysosomal trafficking and autophagy. Interaction studies verified the regulation of CTSS by miR-3619-5p. Down-regulation of CTSS by ectopic miR-3619-5p as well as its specific knockdown by siRNA affected the process of autophagy in THP-1 derived macrophages.

Small non-coding RNA play a major part in host response to bacterial agents. However, the role of long non-coding RNA (lncRNA) in this context remains unknown. LncRNA regulate gene expression by acting e.g. as transcriptional coactivators, RNA decoys or microRNA sponges. They control development, differentiation and cellular processes such as autophagy in disease conditions. Here, we provide an insight into the role of lncRNA in mycobacterial infections. Human macrophages were infected with Mycobacterium bovis BCG and lncRNA expression was studied early post infection. For this purpose, lncRNA with known immune related functions were preselected and a lncRNA specific RT-qPCR protocol was established. In addition to expression-based prediction of lncRNA function, we assessed strategies for thorough normalisation of lncRNA. Arrayed quantification showed infection-dependent repression of several lncRNA including MEG3. Pathway analysis linked MEG3 to mTOR and PI3K-AKT signalling pointing to regulation of autophagy. Accordingly, IFN-γ induced autophagy in infected macrophages resulted in sustained MEG3 down regulation and lack of IFN-γ allowed for counter regulation of MEG3 by viable M. bovis BCG. Knockdown of MEG3 in macrophages resulted in induction of autophagy and enhanced eradication of intracellular M. bovis BCG.

Salmonella are able to modulate host cell functions facilitating both uptake and resistance to cellular host defence mechanisms. While interactions between bacterial modulators and cellular proteins have been the main focus of Salmonella research, relatively little is known about mammalian gene regulation in response to Salmonella infection. A major class of mammalian gene modulators consists of microRNAs. For our study we examined interactions of microRNAs and regulated mRNAs in mammalian intestinal Salmonella infections using a piglet model. After performing microRNA as well as mRNA specific microarray analysis of ileal samples from Salmonella infected as well as control piglets, we integrated expression analysis with target prediction identifying microRNAs that mainly regulate focal adhesion as well as actin cytoskeleton pathways. Particular attention was given to miR-29a, which was involved in most interactions including Caveolin 2. RT-qPCR experiments verified up-regulation of miR-29a after infection while its predicted target Caveolin 2 was significantly down-regulated as examined by transcript and protein detection. Reporter gene assays as well as RNAi experiments confirmed Caveolin 2 to be a miR-29a target. Knock-down of Caveolin 2 in intestinal epithelial cells resulted in retarded proliferation as well as increased bacterial uptake. In addition, our experiments showed that Caveolin 2 regulates the activation of the small Rho GTPase CDC42 but apparently not RAC1 in human intestinal cells. Our study outlines for the first time important regulation pathways in intestinal Salmonella infection pointing out that focal adhesion and organisation of actin cytoskeleton are regulated by microRNAs. Functional relevance is shown by miR-29a mediated Caveolin 2 regulation, modulating the activation state of CDC42. Further analysis of examined interactions may support the discovery of novel strategies impairing the uptake of intracellular pathogens.

The present study has examined the effect of different concentrations (1 μg/ml, 10 μg/ml and 100 μg/ml) of titanium oxide (TiO₂) nanoparticles (NPs) (2 NP albeit at lower concentrations. In DNA fragmentation assay, there was dose-dependent increase in the DNA fragmentation ( r = 0.96). Ultrathin cross-sections revealed TiO₂ NPs inside head and plasma membrane of the buffalo spermatozoa as assessed by TEM. These studies suggest that TiO₂ NPs may have cytotoxic effect on buffalo spermatozoa by affecting sperm functionality and causing high amount of DNA fragmentations.