Rapid and synchronous chemical induction of replicative-like senescence via a small molecule inhibitor.

Cellular senescence is acknowledged as a key contributor to organismal ageing and late-life disease. Though popular, the study of senescence in vitro can be complicated by the prolonged and asynchronous timing of cells committing to it and by its paracrine effects. To address these issues, we repurposed a small molecule inhibitor, inflachromene (ICM), to induce senescence to human primary cells. Within 6 days of treatment with ICM, senescence hallmarks, including the nuclear eviction of HMGB1 and -B2, are uniformly induced across IMR90 cell populations. By generating and comparing various high throughput datasets from ICM-induced and replicative senescence, we uncovered a high similarity of the two states. Notably though, ICM suppresses the pro-inflammatory secretome associated with senescence, thus alleviating most paracrine effects. In summary, ICM rapidly and synchronously induces a senescent-like phenotype thereby allowing the study of its core regulatory program without confounding heterogeneity.

High-Fat Diet Rapidly Modifies Trafficking, Phenotype, and Function of Plasmacytoid Dendritic Cells in Adipose Tissue.

Plasmacytoid dendritic cells (pDCs) display an increased abundance in visceral adipose tissue (VAT) of humans with obesity. In the current study, we set out to decipher the molecular mechanisms of their recruitment to VAT and the functional relevance of this process. We observed increased pDC numbers in murine blood, liver, spleen, and VAT after feeding a high-fat diet (HFD) for 3 wk when compared with a standard diet. pDCs were enriched in fat-associated lymphoid clusters representing highly specific lymphoid regions within VAT. HFD led to an enlargement of fat-associated lymphoid clusters with an increased density and migratory speed of pDCs as shown by intravital multiphoton microscopy. For their recruitment into VAT, pDCs employed P-selectin with E-selectin and L-selectin being only critical in response to HFD, indicating that the molecular cues underlying pDC trafficking were dependent on the nutritional state. Subsequent recruitment steps required α4ß1 and α4ß7 integrins and engagement of CCR7. Application of fingolimod (FTY720) abrogated egress of pDCs from VAT, indicating the involvement of sphingosine-1-phosphate in this process. Furthermore, HFD altered pDC functions by promoting their activation and type 1 IFN expression. Blocking pDC infiltration into VAT prevented weight gain and improved glucose tolerance during HFD. In summary, a HFD fundamentally alters pDC biology by promoting their trafficking, retention, and activation in VAT, which in turn seems to regulate metabolism.

Methylation of histone H3 at lysine 37 by Set1 and Set2 prevents spurious DNA replication.

DNA replication initiates at genomic locations known as origins of replication, which, in S. cerevisiae, share a common DNA consensus motif. Despite being virtually nucleosome-free, origins of replication are greatly influenced by the surrounding chromatin state. Here, we show that histone H3 lysine 37 mono-methylation (H3K37me1) is catalyzed by Set1p and Set2p and that it regulates replication origin licensing. H3K37me1 is uniformly distributed throughout most of the genome, but it is scarce at replication origins, where it increases according to the timing of their firing. We find that H3K37me1 hinders Mcm2 interaction with chromatin, maintaining low levels of MCM outside of conventional replication origins. Lack of H3K37me1 results in defective DNA replication from canonical origins while promoting replication events at inefficient and non-canonical sites. Collectively, our results indicate that H3K37me1 ensures correct execution of the DNA replication program by protecting the genome from inappropriate origin licensing and spurious DNA replication.

RNase A Treatment Interferes With Leukocyte Recruitment, Neutrophil Extracellular Trap Formation, and Angiogenesis in Ischemic Muscle Tissue.

Background: RNase A (the bovine equivalent to human RNase 1) and RNase 5 (angiogenin) are two closely related ribonucleases. RNase 5 is described as a powerful angiogenic factor. Whether RNase A shares the same angiogenic characteristic, or interferes with vessel growth as demonstrated for arteriogenesis, has never been investigated and is the topic of this present study. Methods and Results: To investigate whether RNase A shows a pro- or anti-angiogenic effect, we employed a murine hindlimb model, in which femoral artery ligation (FAL) results in arteriogenesis in the upper leg, and, due to provoked ischemia, in angiogenesis in the lower leg. C57BL/6J male mice underwent unilateral FAL, whereas the contralateral leg was sham operated. Two and seven days after the surgery and intravenous injection of RNase A (50 µg/kg dissolved in saline) or saline (control), the gastrocnemius muscles of mice were isolated from the lower legs for (immuno-) histological analyses. Hematoxylin and Eosin staining evidenced that RNase A treatment resulted in a higher degree of ischemic tissue damage. This was, however, associated with reduced angiogenesis, as evidenced by a reduced capillary/muscle fiber ratio. Moreover, RNase A treatment was associated with a significant reduction in leukocyte infiltration as shown by CD45+ (pan-leukocyte marker), Ly6G+ or MPO+ (neutrophils), MPO+/CitH3 + [neutrophil extracellular traps (NETs)], and CD68+ (macrophages) staining. CD68/MRC1 double staining revealed that RNase A treated mice showed a reduced percentage of M1-like polarized (CD68+/MRC1-) macrophages whereas the percentage of M2-like polarized (CD68+/MRC1+) macrophages was increased. Conclusion: In contrast to RNase 5, RNase A interferes with angiogenesis, which is linked to reduced leukocyte infiltration and NET formation.