Emerging evidence is revealing that exosomes contribute to many aspects of physiology and disease through intercellular communication. However, the biological roles of exosome secretion in exosome-secreting cells have remained largely unexplored. Here we show that exosome secretion plays a crucial role in maintaining cellular homeostasis in exosome-secreting cells. The inhibition of exosome secretion results in the accumulation of nuclear DNA in the cytoplasm, thereby causing the activation of cytoplasmic DNA sensing machinery. This event provokes the innate immune response, leading to reactive oxygen species (ROS)-dependent DNA damage response and thus induce senescence-like cell-cycle arrest or apoptosis in normal human cells. These results, in conjunction with observations that exosomes contain various lengths of chromosomal DNA fragments, indicate that exosome secretion maintains cellular homeostasis by removing harmful cytoplasmic DNA from cells. Together, these findings enhance our understanding of exosome biology, and provide valuable new insights into the control of cellular homeostasis.

Cellular senescence prevents the proliferation of cells at risk for neoplastic transformation. However, the altered secretome of senescent cells can promote the growth of the surrounding cancer cells. Although extracellular vesicles (EVs) have emerged as new players in intercellular communication, their role in the function of senescent cell secretome has been largely unexplored. Here, we show that exosome-like small EVs (sEVs) are important mediators of the pro-tumorigenic function of senescent cells. sEV-associated EphA2 secreted from senescent cells binds to ephrin-A1, that is, highly expressed in several types of cancer cells and promotes cell proliferation through EphA2/ephrin-A1 reverse signalling. sEV sorting of EphA2 is increased in senescent cells because of its enhanced phosphorylation resulting from oxidative inactivation of PTP1B phosphatase. Our results demonstrate a novel mechanism of reactive oxygen species (ROS)-regulated cargo sorting into sEVs, which is critical for the potentially deleterious growth-promoting effect of the senescent cell secretome.

Accumulating evidence indicates that the senescence-associated secretory phenotype (SASP) contributes to many aspects of physiology and disease. Thus, controlling the SASP will have tremendous impacts on our health. However, our understanding of SASP regulation is far from complete. Here, we show that cytoplasmic accumulation of nuclear DNA plays key roles in the onset of SASP. Although both DNase2 and TREX1 rapidly remove the cytoplasmic DNA fragments emanating from the nucleus in pre-senescent cells, the expression of these DNases is downregulated in senescent cells, resulting in the cytoplasmic accumulation of nuclear DNA. This causes the aberrant activation of cGAS-STING cytoplasmic DNA sensors, provoking SASP through induction of interferon-β. Notably, the blockage of this pathway prevents SASP in senescent hepatic stellate cells, accompanied by a decline of obesity-associated hepatocellular carcinoma development in mice. These findings provide valuable new insights into the roles and mechanisms of SASP and possibilities for their control.

Microglia are thought to play important roles in the maintenance of neuronal circuitry and the regulation of behavior. We found that the cortical microglia contain an intrinsic molecular clock and exhibit a circadian expression of cathepsin S (CatS), a microglia-specific lysosomal cysteine protease in the brain. The genetic deletion of CatS causes mice to exhibit hyperlocomotor activity and removes diurnal variations in the synaptic activity and spine density of the cortical neurons, which are significantly higher during the dark (waking) phase than the light (sleeping) phase. Furthermore, incubation with recombinant CatS significantly reduced the synaptic activity of the cortical neurons. These results suggest that CatS secreted by microglia during the dark-phase decreases the spine density of the cortical neurons by modifying the perisynaptic environment, leading to downscaling of the synaptic strength during the subsequent light-phase. Disruption of CatS therefore induces hyperlocomotor activity due to failure to downscale the synaptic strength.

The family Endornaviridae contains several members from diverse hosts, including plants, fungi and oomycetes. They are found as large dsRNA elements with a nick in the coding strand. All members encode a conserved RNA-dependent RNA polymerase, but no other domain that is conserved among all members. Based on the conserved domain database comparison the various domains have different origins, indicating a highly modular evolutionary history. In some cases, domains with similar putative functions are found that are derived from different protein families, indicating convergent evolution for a required function.

Bell peppers (Capsicum annuum) harbour a large dsRNA virus. The linear genome (14.7 kbp) of two isolates from Japanese and USA bell pepper cultivars were completely sequenced and compared. They shared extensive sequence identity and contained a single, long ORF encoding a 4815 aa protein. This polyprotein contained conserved motifs of putative viral methyltransferase (MTR), helicase 1 (Hel-1), UDP-glycosyltransferase and RNA-dependent RNA polymerase. This unique arrangement of conserved domains has not been reported in any of the known endornaviruses. Hence this virus, for which the name Bell pepper endornavirus (BPEV) is proposed, is a distinct species in the genus Endornavirus (family Endornaviridae). The BPEVencoded polyprotein contains a cysteine-rich region between the MTR and Hel-1 domains, with conserved CXCC motifs shared among several endornaviruses, suggesting an additional functional domain. In agreement with general endornavirus features, BPEV contains a nick in the positive-strand RNA molecule. The virus was detected in all bell pepper cultivars tested and transmitted through seed but not by graft inoculations. Analysis of dsRNA patterns and RT-PCR using degenerate primers revealed putative variants of BPEV, or closely related species, infecting other C. annuum genotypes and three other Capsicum species (C. baccatum, C. chinense and C. frutescens).

CD4(+) T cells have T(h) cell function and include two major functional subsets, T(h)1 and T(h)2. However, there are a restricted number of studies concerning phenotypic classification of human CD4(+) T cells. Here by using seven- and eight-color flow cytometric analysis, we investigated the function of the subsets classified by four markers, CD27, CD28, CD45RA and CCR7. Five major subsets were identified by using these markers. These subsets showed different patterns of cytokine production after they were stimulated with phorbol myristate acetate and ionomycin. The analyses of cytokine production suggested that CCR7(+)CD45RA(+)CD27(+)CD28(+), CCR7(+)CD45RA(-)CD27(+)CD28(+) and CCR7(-)CD45RA(-)CD27(+)CD28(+) subsets were naive, central memory and effector memory T cells, respectively, whereas CCR7(-)CD45RA(-)CD27(-)CD28(+) and CCR7(-)CD45RA(-)CD27(-)CD28(-) subsets included T(h)1 and T(h)2 cells. The analysis of cytokine production by these subsets stimulated with anti-CD3 and anti-CD28 mAbs or with human cytomegalovirus antigens showed that IFN-gamma production was significantly higher in the CCR7(-)CD45RA(-)CD27(-)CD28(-) subset than in other subsets and that both CCR7(-)CD45RA(-)CD27(-)CD28(+) and CCR7(-)CD45RA(-)CD27(-)CD28(-) subsets produced a higher level of IL-4 than did other subsets. Our analyses demonstrated that the CCR7(-)CD45RA(-)CD27(-)CD28(-) subset predominantly included T(h)1 effector cells and that CCR7(-)CD45RA(-)CD27(-)CD28(+) subsets included T(h)1 and T(h)2 effector memory/effector cells as well as unclassified cells. The analysis of classification by using these four markers also suggested the differentiation pathway of human CD4(+) T cells.

There is increasing evidence that CD4ϩ T-cell-dependent responses are associated with the maintenance of neuropathic pain. However, little is known about the precise mechanism(s) underlying the activation of CD4 ϩ T-cells. We herein show that inhibition of cathepsin S (CatS) activity, either through genetic deletion or via a pharmacological inhibitor, Z-Phe-Leu-COCHO (Z-FL), significantly attenuated the maintenance of tactile allodynia, splenic hypertrophy, increased number of splenic CD4ϩ T-cells and the final cleavage step of the MHC class II-associated invariant chain following peripheral nerve injury. It was also noted that splenectomy significantly attenuated the peripheral nerve injury-induced tactile allodynia, whereas the adoptive transfer of splenic CD4