During skin injury, immune response and repair mechanisms have to be coordinated for rapid skin regeneration and the prevention of microbial infections. Natural Killer (NK) cells infiltrate hypoxic skin lesions and Hypoxia-inducible transcription factors (HIFs) mediate adaptation to low oxygen. We demonstrate that mice lacking the Hypoxia-inducible factor (HIF)-1α isoform in NK cells show impaired release of the cytokines Interferon (IFN)-γ and Granulocyte Macrophage - Colony Stimulating Factor (GM-CSF) as part of a blunted immune response. This accelerates skin angiogenesis and wound healing. Despite rapid wound closure, bactericidal activity and the ability to restrict systemic bacterial infection are impaired. Conversely, forced activation of the HIF pathway supports cytokine release and NK cell-mediated antibacterial defence including direct killing of bacteria by NK cells despite delayed wound closure. Our results identify, HIF-1α in NK cells as a nexus that balances antimicrobial defence versus global repair in the skin.
Gut innate lymphoid cells (ILCs) show remarkable phenotypic diversity, yet microenvironmental factors that drive this plasticity are incompletely understood. The balance between NKp46+, IL-22–producing, group 3 ILCs (ILC3s) and interferon (IFN)-γ–producing group 1 ILCs (ILC1s) contributes to gut homeostasis. The gut mucosa is characterized by physiological hypoxia, and adaptation to low oxygen is mediated by hypoxia-inducible transcription factors (HIFs). However, the impact of HIFs on ILC phenotype and gut homeostasis is not well understood. Mice lacking the HIF-1α isoform in NKp46+ ILCs show a decrease in IFN-γ–expressing, T-bet+, NKp46+ ILC1s and a concomitant increase in IL-22–expressing, RORγt+, NKp46+ ILC3s in the gut mucosa. Single-cell RNA sequencing revealed HIF-1α as a driver of ILC phenotypes, where HIF-1α promotes the ILC1 phenotype by direct up-regulation of T-bet. Loss of HIF-1α in NKp46+ cells prevents ILC3-to-ILC1 conversion, increases the expression of IL-22–inducible genes, and confers protection against intestinal damage. Taken together, our results suggest that HIF-1α shapes the ILC phenotype in the gut.
Knee osteoarthritis (OA) is a degenerative knee joint disease that results from the breakdown of joint cartilage and underlying bone, affecting about 3.3% of the world's population. As OA is a multifactorial disease, the underlying pathological process is closely associated with genetic changes in articular cartilage and bone. Many studies have focused on the role of small noncoding RNAs in OA and identified numbers of microRNAs that play important roles in regulating bone and cartilage homeostasis. The connection between other types of small noncoding RNAs, especially tRNA-derived fragments and knee osteoarthritis is still elusive. The observation that there is limited information about small RNAs different than miRNAs in knee OA was very surprising to us, especially given the fact that tRNA fragments are known to participate in a plethora of human diseases and a portion of them are even more abundant than miRNAs. Inspired by these findings, in this review we have summarized the possible involvement of microRNAs and tRNA-derived fragments in the pathology of knee osteoarthritis.
Regulation of gene expression by premature termination of transcription has been well described in all domains of life, including metazoans, yeast, plants, and bacteria. Although methods for identification of such regulatory events by sequencing are available, the focused biochemical studies of the mechanism are hampered by lack of highly sensitive and accurate experimental methods. Here, we propose a new method for absolute quantification of premature transcription termination events, PTT-quant. It is based on highly sensitive two-step digital droplet PCR protocol, coupled with normalized cDNA synthesis attained by site-specific pre-cleavage of investigated transcripts with RNase H. As a consequence, our method enables the reliable and sensitive quantification of both, prematurely terminated and full-length transcripts. By application of our method to investigation of transcriptional riboswitches in Bacillus subtilis, we were able to precisely measure the dynamics of S-adenosylmethionine (SAM) riboswitch induction, which turned to be ~ 23% higher in comparison the results obtained without cDNA synthesis normalization.Key points• A novel method for quantification of premature transcription termination events was established.• PTT-quant measures absolute concentration of full-length and terminated transcripts.• RNase H and the digital droplet PCR technique is used in PTT-quant.
Regulation of gene expression by premature termination of transcription has been well described in all domains of life, including metazoans, yeast, plants and bacteria. Although methods for identification of such regulatory events by sequencing are available, the focused biochemical studies of the mechanism is hampered by lack of highly sensitive and accurate experimental methods. Here we propose a new method for absolute quantification of premature transcription termination events, PTT-quant. It is based on highly sensitive two-step digital droplet PCR protocol, coupled with normalized cDNA synthesis attained by site-specific pre-cleavage of investigated transcripts with RNase H. As a consequence, our method enables the reliable and sensitive quantification of both, prematurely terminated and full-length transcripts. By application of our method to investigation of transcriptional riboswitches in Bacillus subtilis, we were able to precisely measure the dynamics of SAM riboswitch induction, which turned to be ~23% higher in comparison the results obtained without cDNA synthesis normalization.
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