Activation of metabolic signalling by IL-15 is required for natural killer (NK) cell development. Here we show that Tsc1, a repressor of mTOR, is dispensable for the terminal maturation, survival and function of NK cells but is critical to restrict exhaustive proliferation of immature NK cells and activation downstream of IL-15 during NK cell development. Tsc1 is expressed in immature NK cells and is upregulated by IL-15. Haematopoietic-specific deletion of Tsc1 causes a marked decrease in the number of NK cells and compromises rejection of ‘missing-self' haematopoietic tumours and allogeneic bone marrow. The residual Tsc1-null NK cells display activated, pro-apoptotic phenotype and elevated mTORC1 activity. Deletion of Raptor, a component of mTORC1, largely reverses these defects. Tsc1-deficient NK cells express increased levels of T-bet and downregulate Eomes and CD122, a subunit of IL-15 receptor. These results reveal a role for Tsc1-dependent inhibition of mTORC1 activation during immature NK cell development.
Classical signaling lymphocyte activating molecule (SLAM) family receptors are abundant within many types of immune cells, whereas the nonclassical SLAM family receptors SLAMF8 and SLAMF9, which uniquely lack cytoplasmic signaling motifs, are highly expressed by myeloid cells. Due to the potential redundancy, whether these two receptors regulate macrophage function remains largely unknown. Here, we show that SLAMF8 and SLAMF9 co-regulate macrophage-mediated liver inflammation. To overcome the redundancy, we generated mice that simultaneously lacked SLAMF8 and SLAMF9 using CRISPR-Cas9 technology. Although macrophage differentiation was not altered by the combined deficiency of SLAMF8 and SLAMF9, the loss of these two receptors significantly protected against lipopolysaccharide (LPS)-induced liver injury. SLAMF8 and SLAMF9 double-deficient mice had a prolonged survival rate and less infiltration of inflammatory cells. The depletion of macrophages using clodronate liposomes abolished the effects of SLAMF8 and SLAMF9 deficiencies on LPS-induced liver injury, which demonstrates that these receptors are required for macrophage activation following LPS challenge. Moreover, the deficiency of SLAMF8 and SLAMF9 suppressed the secretion of inflammatory cytokines by downregulating the expression of Toll-like receptor-4 (TLR4), a receptor that specifically binds LPS, which led to decreased mitogen-activated protein kinases (MAPK) signaling activation. Notably, combined injections of truncated extracellular SLAMF8 and SLAMF9 proteins significantly alleviated LPS-induced liver injury. Thus, our findings provide insights into the role of SLAMF8 and SLAMF9 in endotoxin-induced liver injury and suggest that SLAMF8 and SLAMF9 are potential therapeutic targets for acute hepatic injury.
SHARPIN is a tumor‐associated gene involved in the growth and proliferation of many tumor types. A function of SHARPIN in cholangiocarcinoma (CCA) is so far unclear. Here, we studied the role and function of SHARPIN in CCA and revealed its relevant molecular mechanism. The expression of SHARPIN was analyzed in cholangiocarcinoma tissues from patients using immunohistochemistry, quantitative PCR, and western blot analysis. Expression of SHARPIN was suppressed/overexpressed by siRNA silencing or lentiviral overexpression vector, and the effect on cell proliferation was determined by the CCK‐8 assay and flow cytometry. Accumulation of reactive oxygen species was measured with MitoTracker, and JC‐1 staining showed mitochondrial fission/fusion and mitochondrial membrane potential changes as a result of the silencing or overexpression. The ferroptosis marker solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), and the antioxidant enzymes superoxide dismutase 1 (SOD‐1) and SOD‐2 were analyzed by western blot. The results showed that SHARPIN expression was increased in CCA tissue, and this was involved in cell proliferation. SHARPIN silencing resulted in accumulated reactive oxygen species, reduced mitochondrial fission, and a reduced mitochondrial membrane potential. Silencing of SHARPIN inhibited the ubiquitination and degradation of p53, and downregulated levels of SLC7A11, GPX4, SOD‐1, and SOD‐2, all of which contributed to excessive oxidative stress that leads to ferroptosis. Overexpression of SHARPIN would reverse the above process. The collected data suggest that in CCA, SHARPIN‐mediated cell ferroptosis via the p53/SLC7A11/GPX4 signaling pathway is inhibited. Targeting SHARPIN might be a promising approach for the treatment of CCA.
Natural killers (NKs) are a type of innate immune cell that can directly kill tumour cells without pre-activation. It is well-known that NK cell-mediated immune monitoring not only kills tumour cells directly, but also indirectly releases cytokines to regulate other leucocytes such as macrophages, T cells and dendritic cells (DCs). 1 NK cells can destroy infected and tumour cells by releasing cytolytic granules and interferon gamma (IFN-γ). Among these molecular mechanisms, the release of granzyme B and perforin is the most effective way for NK cells to kill tumour cells. 2 Another important function of NK cells is the release of pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNF-α). 3 NK cells are derived from bone marrow and are tightly regulated by IL-15
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