Tyrosine phosphatase SHP-2 mediates C-type lectin receptor-induced activation of the kinase Syk and anti-fungal TH17 responses.

Fungal infection stimulates the canonical C-type lectin receptor (CLR) signaling pathway via activation of the tyrosine kinase Syk. Here we identify a crucial role for the tyrosine phosphatase SHP-2 in mediating CLR-induced activation of Syk. Ablation of the gene encoding SHP-2 (Ptpn11; called 'Shp-2' here) in dendritic cells (DCs) and macrophages impaired Syk-mediated signaling and abrogated the expression of genes encoding pro-inflammatory molecules following fungal stimulation. Mechanistically, SHP-2 operated as a scaffold, facilitating the recruitment of Syk to the CLR dectin-1 or the adaptor FcRγ, through its N-SH2 domain and a previously unrecognized carboxy-terminal immunoreceptor tyrosine-based activation motif (ITAM). We found that DC-derived SHP-2 was crucial for the induction of interleukin 1ß (IL-1ß), IL-6 and IL-23 and anti-fungal responses of the TH17 subset of helper T cells in controlling infection with Candida albicans. Together our data reveal a mechanism by which SHP-2 mediates the activation of Syk in response to fungal infection.

TSC1/mTOR-controlled metabolic-epigenetic cross talk underpins DC control of CD8+ T-cell homeostasis.

Dendritic cells (DCs) play pivotal roles in T-cell homeostasis and activation, and metabolic programing has been recently linked to DC development and function. However, the metabolic underpinnings corresponding to distinct DC functions remain largely unresolved. Here, we demonstrate a special metabolic-epigenetic coupling mechanism orchestrated by tuberous sclerosis complex subunit 1 (TSC1)-mechanistic target of rapamycin (mTOR) for homeostatic DC function. Specific ablation of Tsc1 in the DC compartment (Tsc1DC-KO) largely preserved DC development but led to pronounced reduction in naïve and memory-phenotype cluster of differentiation (CD)8+ T cells, a defect fully rescued by concomitant ablation of mTor or regulatory associated protein of MTOR, complex 1 (Rptor) in DCs. Moreover, Tsc1DC-KO mice were unable to launch efficient antigen-specific CD8+ T effector responses required for containing Listeria monocytogenes and B16 melanomas. Mechanistically, our data suggest that the steady-state DCs tend to tune down de novo fatty acid synthesis and divert acetyl-coenzyme A (acetyl-CoA) for histone acetylation, a process critically controlled by TSC1-mTOR. Correspondingly, TSC1 deficiency elevated acetyl-CoA carboxylase 1 (ACC1) expression and fatty acid synthesis, leading to impaired epigenetic imprinting on selective genes such as major histocompatibility complex (MHC)-I and interleukin (IL)-7. Remarkably, tempering ACC1 activity was able to divert cytosolic acetyl-CoA for histone acetylation and restore the gene expression program compromised by TSC1 deficiency. Taken together, our results uncover a crucial role for TSC1-mTOR in metabolic programing of the homeostatic DCs for T-cell homeostasis and implicate metabolic-coupled epigenetic imprinting as a paradigm for DC specification.

Regulated intramembrane proteolysis of the AXL receptor kinase generates an intracellular domain that localizes in the nucleus of cancer cells.

Deregulation of the TAM (TYRO3, AXL, and MERTK) family of receptor tyrosine kinases (RTKs) has recently been demonstrated to predominately promote survival and chemoresistance of cancer cells. Intramembrane proteolysis mediated by presenilin/γ-secretase is known to regulate the homeostasis of some RTKs. In the present study, we demonstrate that AXL, but not TYRO3 or MERTK, is efficiently and sequentially cleaved by α- and γ-secretases in various types of cancer cell lines. Proteolytic processing of AXL redirected signaling toward a secretase-mediated pathway, away from the classic, well-known, ligand-dependent canonical RTK signaling pathway. The AXL intracellular domain cleavage product, but not full-length AXL, was further shown to translocate into the nucleus via a nuclear localization sequence that harbored a basic HRRKK motif. Of interest, we found that the γ-secretase-uncleavable AXL mutant caused an elevated chemoresistance in non-small-cell lung cancer cells. Altogether, our findings suggest that AXL can undergo sequential processing mediated by various proteases kept in a homeostatic balance. This newly discovered post-translational processing of AXL may provide an explanation for the diverse functions of AXL, especially in the context of drug resistance in cancer cells.-Lu, Y., Wan, J., Yang, Z., Lei, X., Niu, Q., Jiang, L., Passtoors, W. M., Zang, A., Fraering, P. C., Wu, F. Regulated intramembrane proteolysis of the AXL receptor kinase generates an intracellular domain that localizes in the nucleus of cancer cells.

Characterization of age-related immune features after autologous NK cell infusion: Protocol for an open-label and randomized controlled trial.

Background: Aging is usually accompanied by functional declines of the immune system, especially in T-cell responses. However, little is known about ways to alleviate this. Methods: Here, 37 middle-aged healthy participants were recruited, among which 32 were intravenously administrated with expanded NK cells and 5 with normal saline. Then, we monitored changes of peripheral senescent and exhausted T cells within 4 weeks after infusion by flow cytometry, as well as serum levels of senescence-associated secretory phenotype (SASP)-related factors. In vitro co-culture assays were performed to study NK-mediated cytotoxic activity against senescent or exhausted T cells. Functional and phenotypic alteration of NK cells before and after expansion was finally characterized. Results: After NK cell infusion, senescent CD28-, CD57+, CD28-CD57+, and CD28-KLRG1+ CD4+ and CD8+ T-cell populations decreased significantly, so did PD-1+ and TIM-3+ T cells. These changes were continuously observed for 4 weeks. Nevertheless, no significant changes were observed in the normal saline group. Moreover, SASP-related factors including IL-6, IL-8, IL-1α, IL-17, MIP-1α, MIP-1ß, and MMP1 were significantly decreased after NK cell infusion. Further co-culture assays showed that expanded NK cells specifically and dramatically eliminated senescent CD4+ T cells other than CD28+CD4+ T cells. They also showed improved cytotoxic activity, with different expression patterns of activating and inhibitory receptors including NKG2C, NKG2A, KLRG1, LAG3, CD57, and TIM3. Conclusion: Our findings imply that T-cell senescence and exhaustion is a reversible process in healthy individuals, and autologous NK cell administration can be introduced to alleviate the aging. Clinical Trial Registration: ClinicalTrials.gov, ChiCTR-OOh-17011878.

Overexpression of OsRAN2 in rice and Arabidopsis renders transgenic plants hypersensitive to salinity and osmotic stress.

Nucleo-cytoplasmic partitioning of regulatory proteins is increasingly being recognized as a major control mechanism for the regulation of signalling in plants. Ras-related nuclear protein (Ran) GTPase is required for regulating transport of proteins and RNA across the nuclear envelope and also has roles in mitotic spindle assembly and nuclear envelope (NE) assembly. However, thus far little is known of any Ran functions in the signalling pathways in plants in response to changing environmental stimuli. The OsRAN2 gene, which has high homology (77% at the amino acid level) with its human counterpart, was isolated here. Subcellular localization results showed that OsRan2 is mainly localized in the nucleus, with some in the cytoplasm. Transcription of OsRAN2 was reduced by salt, osmotic, and exogenous abscisic acid (ABA) treatments, as determined by real-time PCR. Overexpression of OsRAN2 in rice resulted in enhanced sensitivity to salinity, osmotic stress, and ABA. Seedlings of transgenic Arabidopsis thaliana plants overexpressing OsRAN2 were overly sensitive to salinity stress and exogenous ABA treatment. Furthermore, three ABA- or stress-responsive genes, AtNCED3, AtPLC1, and AtMYB2, encoding a key enzyme in ABA synthesis, a phospholipase C homologue, and a putative transcriptional factor, respectively, were shown to have differentially induced expression under salinity and ABA treatments in transgenic and wild-type Arabidopsis plants. OsRAN2 overexpression in tobacco epidermal leaf cells disturbed the nuclear import of a maize (Zea mays L.) leaf colour transcription factor (Lc). In addition, gene-silenced rice plants generated via RNA interference (RNAi) displayed pleiotropic developmental abnormalities and were male sterile.

Epigenetic regulator CXXC5 recruits DNA demethylase Tet2 to regulate TLR7/9-elicited IFN response in pDCs.

TLR7/9 signals are capable of mounting massive interferon (IFN) response in plasmacytoid dendritic cells (pDCs) immediately after viral infection, yet the involvement of epigenetic regulation in this process has not been documented. Here, we report that zinc finger CXXC family epigenetic regulator CXXC5 is highly expressed in pDCs, where it plays a crucial role in TLR7/9- and virus-induced IFN response. Notably, genetic ablation of CXXC5 resulted in aberrant methylation of the CpG-containing island (CGI) within the Irf7 gene and impaired IRF7 expression in steady-state pDCs. Mechanistically, CXXC5 is responsible for the recruitment of DNA demethylase Tet2 to maintain the hypomethylation of a subset of CGIs, a process coincident with active histone modifications and constitutive transcription of these CGI-containing genes. Consequently, CXXC5-deficient mice had compromised early IFN response and became highly vulnerable to infection by herpes simplex virus and vesicular stomatitis virus. Together, our results identify CXXC5 as a novel epigenetic regulator for pDC-mediated antiviral response.

The Small G Protein AtRAN1 Regulates Vegetative Growth and Stress Tolerance in Arabidopsis thaliana.

The evolutionarily conserved small G-protein Ran plays important role in nuclear translocation of proteins, cell cycle regulation, and nuclear envelope maintenance in mammalian cells and yeast. Arabidopsis Ran proteins are encoded by a family of four genes and are highly conserved at the protein level. However, their biological functions are poorly understood. We report here that AtRAN1 plays an important role in vegetative growth and the molecular improvement of stress tolerance in Arabidopsis. AtRAN1 overexpression promoted vegetative growth and enhanced abiotic tolerance, while the atran1 atran3 double mutant showed higher freezing sensitivity than WT. The AtRAN1 gene is ubiquitously expressed in plants, and the expression levels are higher in the buds, flowers and siliques. Subcellular localization results showed that AtRAN1 is mainly localized in the nucleus, with some present in the cytoplasm. AtRAN1 could maintain cell division and cell cycle progression and promote the formation of an intact nuclear envelope, especially under freezing conditions.

mTOR regulates TLR-induced c-fos and Th1 responses to HBV and HCV vaccines.

Although IL-12 plays a critical role in priming Th1 and cytotoxic T lymphocyte (CTL) responses, Toll-like receptor (TLR) signaling only induces low amounts of IL-12 in dendritic cells and macrophages, implying the existence of stringent regulatory mechanisms. In this study, we sought to uncover the mechanisms underlying TLR-induced IL-12 expression and the Th1 response. By systemic screening, we identified a number of protein kinases involved in the regulation of TLRinduced IL-12 expression. In particular, PI3K, ERK, and mTOR play critical roles in the TLR-induced Th1 response by regulating IL-12 and IL-10 production in innate immune cells. Moreover, we identified c-fos as a key molecule that mediates mTOR-regulated IL-12 and IL-10 expression in TLR signaling. Mechanistically, mTOR plays a crucial role in c-fos expression, thereby modulating NFκB binding to promoters of IL-12 and IL-10. By controlling the expression of a special innate gene program, mTOR can specifically regulate the TLR-induced T cell response in vivo. Furthermore, blockade of mTOR by rapamycin efficiently boosted TLR-induced antigen-specific T and B cell responses to HBV and HCV vaccines. Taken together, these results reveal a novel mechanism through which mTOR regulates TLR-induced IL-12 and IL-10 production, contributing new insights for strategies to improve vaccine efficacy.

Microarray analyses and comparisons of upper or lower flanks of rice shoot base preceding gravitropic bending.

Gravitropism is a complex process involving a series of physiological pathways. Despite ongoing research, gravitropism sensing and response mechanisms are not well understood. To identify the key transcripts and corresponding pathways in gravitropism, a whole-genome microarray approach was used to analyze transcript abundance in the shoot base of rice (Oryza sativa sp. japonica) at 0.5 h and 6 h after gravistimulation by horizontal reorientation. Between upper and lower flanks of the shoot base, 167 transcripts at 0.5 h and 1202 transcripts at 6 h were discovered to be significantly different in abundance by 2-fold. Among these transcripts, 48 were found to be changed both at 0.5 h and 6 h, while 119 transcripts were only changed at 0.5 h and 1154 transcripts were changed at 6 h in association with gravitropism. MapMan and PageMan analyses were used to identify transcripts significantly changed in abundance. The asymmetric regulation of transcripts related to phytohormones, signaling, RNA transcription, metabolism and cell wall-related categories between upper and lower flanks were demonstrated. Potential roles of the identified transcripts in gravitropism are discussed. Our results suggest that the induction of asymmetrical transcription, likely as a consequence of gravitropic reorientation, precedes gravitropic bending in the rice shoot base.