Gut microbiota of the young ameliorates physical fitness of the aged in mice.

BACKGROUND: Aging is a natural process that an organism gradually loses its physical fitness and functionality. Great efforts have been made to understand and intervene in this deteriorating process. The gut microbiota affects host physiology, and dysbiosis of the microbial community often underlies the pathogenesis of host disorders. The commensal microbiota also changes with aging; however, the interplay between the microbiota and host aging remains largely unexplored. Here, we systematically examined the ameliorating effects of the gut microbiota derived from the young on the physiology and phenotypes of the aged. RESULTS: As the fecal microbiota was transplanted from young mice at 5 weeks after birth into 12-month-old ones, the thickness of the muscle fiber and grip strength were increased, and the water retention ability of the skin was enhanced with thickened stratum corneum. Muscle thickness was also marginally increased in 25-month-old mice after transferring the gut microbiota from the young. Bacteria enriched in 12-month-old mice that received the young-derived microbiota significantly correlated with the improved host fitness and altered gene expression. In the dermis of these mice, transcription of Dbn1 was most upregulated and DBN1-expressing cells increased twice. Dbn1-heterozygous mice exhibited impaired skin barrier function and hydration. CONCLUSIONS: We revealed that the young-derived gut microbiota rejuvenates the physical fitness of the aged by altering the microbial composition of the gut and gene expression in muscle and skin. Dbn1, for the first time, was found to be induced by the young microbiota and to modulate skin hydration. Our results provide solid evidence that the gut microbiota from the young improves the vitality of the aged. Video Abstract.

Enterotypical Prevotella and three novel bacterial biomarkers in preoperative stool predict the clinical outcome of colorectal cancer.

BACKGROUND: A significant proportion of colorectal cancer (CRC) patients suffer from early recurrence and progression after surgical treatment. Although the gut microbiota is considered as a key player in the initiation and progression of CRC, most prospective studies have been focused on a particular pathobionts such as Fusobacterium nucleatum. Here, we aimed to identify novel prognostic bacteria for CRC by examining the preoperative gut microbiota through 16S ribosomal RNA gene sequencing. RESULTS: We collected stool samples from 333 patients with primary CRC within 2 weeks before surgery and followed up the patients for a median of 27.6 months for progression and 43.6 months for survival. The sequence and prognosis data were assessed using the log-rank test and multivariate Cox proportional hazard analysis. The gut microbiota was associated with the clinical outcomes of CRC patients (Pprogress = 0.011, Pdecease = 0.007). In particular, the high abundance of Prevotella, a representative genus of human enterotypes, indicated lower risks of CRC progression (P = 0.026) and decease (P = 0.0056), while the occurrence of Alistipes assigned to Bacteroides sp., Pyramidobacter piscolens, Dialister invisus, and Fusobacterium nucleatum indicated a high risk of progression. A microbiota-derived hazard score considering the five prognostic bacteria accurately predicted CRC progression in 1000 random subsamples; it outperformed widely accepted clinical biomarkers such as carcinoembryonic antigen and lymphatic invasion, after adjustment for the clinicopathological stage (adjusted HR 2.07 [95% CI, 1.61-2.64], P = 7.8e-9, C-index = 0.78). PICRUSt2 suggested that microbial pathways pertaining to thiamine salvage and L-histidine degradation underlie the different prognoses. CONCLUSIONS: The enterotypical genus Prevotella was demonstrated to be useful in improving CRC prognosis, and combined with the four pathobionts, our hazard score based on the gut microbiota should provide an important asset in predicting medical outcomes for CRC patients. Video Abstract.

N-glycosylation of UNC93B1 at a Specific Asparagine Residue Is Required for TLR9 Signaling.

Toll-like receptors (TLRs) play critical roles in the first line of host defense against pathogens through recognition of pathogen-associated molecular patterns and initiation of the innate immune responses. The proper localization of TLRs in specific subcellular compartments is crucial for their ligand recognition and downstream signaling to ensure appropriate responses against pathogens while avoiding erroneous or excessive activation. Several TLRs, including TLR7 and TLR9 but not TLR4, depend on UNC93B1 for their proper intracellular localization and signaling. Accumulating evidence suggest that UNC93B1 differentially regulates its various client TLRs, but the specific mechanisms by which UNC93B1 controls individual TLRs are not well understood. Protein N-glycosylation is one of the most frequent and important post-translational modification that occurs in membrane-localized or secreted proteins. UNC93B1 was previously shown to be glycosylated at Asn251 and Asn272 residues. In this study, we investigated whether N-glycosylation of UNC93B1 affects its function by comparing wild type and glycosylation-defective mutant UNC93B1 proteins. It was found that glycosylation of Asn251 and Asn272 residues can occur independently of each other and mutation of neither N251Q or N272Q in UNC93B1 altered expression and localization of UNC93B1 and TLR9. In contrast, CpG DNA-stimulated TLR9 signaling was severely inhibited in cells expressing UNC93B1(N272Q), but not in cells with UNC93B1(N251Q). Further, it was found that glycosylation at Asn272 of UNC93B1 is essential for the recruitment of MyD88 to TLR9 and the subsequent downstream signaling. On the other hand, the defective glycosylation at Asn272 did not affect TLR7 signaling. Collectively, these data demonstrate that the glycosylation at a specific asparagine residue of UNC93B1 is required for TLR9 signaling and the glycosylation status of UNC93B1 differently affects activation of TLR7 and TLR9.

Opportunistic detection of Fusobacterium nucleatum as a marker for the early gut microbial dysbiosis.

BACKGROUND: The essential roles of gut microbiome have been emphasized in modulating human health and disease. Fusobacterium nucleatum (F. nucleatum), an obligate Gram-negative microorganism residing in oral cavity, gastrointestinal tract and elsewhere, has been recently considered as a potential oncobacterium associated with human cancers. However, the consequence of its enrichment was not extensively explored in terms of microbial homeostasis and stability at the early stage of disease development. RESULT: Our analysis on longitudinal metagenomic data generated by the Integrative Human Microbiome Project (iHMP) showed that F. nucleatum was frequently found in inflammatory bowel diseases (IBD) subjects with reduced microbial diversity. Using non-parametric logarithmic linear discriminant analysis (LDA) effect size (LEfSe) algorithm, 12 IBD- and 14 non-IBD-specific bacterial species were identified in the fecal metagenome and the IBD-specific ones were over-represented in the F. nucleatum-experienced subjects during long-term surveillance. In addition, F. nucleatum experience severely abrogated intra-personal stability of microbiome in IBD patients and induced highly variable gut microbiome between subjects. From the longitudinal comparison between microbial distributions prior and posterior to F. nucleatum detection, 41 species could be proposed as indicative "classifiers" for dysbiotic gut state. By multiple logistic regression models established on these classifiers, the high probability of experiencing F. nucleatum was significantly correlated with decreased alpha-diversity and increased number of biomarker species for IBD and colorectal cancer (CRC). Finally, microbial clustering confirmed that biomarker species for IBD and non-IBD conditions as well as CRC signature markers were well distinguishable and could be utilized for explaining gut symbiosis and dysbiosis. CONCLUSION: F. nucleatum opportunistically appeared under early dysbiotic condition in gut, and discriminative classifier species associated with F. nucleatum were successfully applied to predict microbial alterations in both IBD and non-IBD conditions. Our prediction model and microbial classifier biomarkers for estimating gut dysbiosis should provide a novel aspect of microbial homeostasis/dynamics and useful information on non-invasive biomarker screening.

Flagellin-Stimulated Production of Interferon-ß Promotes Anti-Flagellin IgG2c and IgA Responses.

Flagellin, a major structural protein of the flagellum found in all motile bacteria, activates the TLR5- or NLRC4 inflammasomedependent signaling pathway to induce innate immune responses. Flagellin can also serve as a specific antigen for the adaptive immune system and stimulate anti-flagellin antibody responses. Failure to recognize commensal-derived flagellin in TLR5-deficient mice leads to the reduction in antiflagellin IgA antibodies at steady state and causes microbial dysbiosis and mucosal barrier breach by flagellated bacteria to promote chronic intestinal inflammation. Despite the important role of anti-flagellin antibodies in maintaining the intestinal homeostasis, regulatory mechanisms underlying the flagellin-specific antibody responses are not well understood. In this study, we show that flagellin induces interferon-ß (IFN-ß) production and subsequently activates type I IFN receptor signaling in a TLR5- and MyD88-dependent manner in vitro and in vivo . Internalization of TLR5 from the plasma membrane to the acidic environment of endolysosomes was required for the production of IFN-ß, but not for other proinflammatory cytokines. In addition, we found that antiflagellin IgG2c and IgA responses were severely impaired in interferon-alpha receptor 1 (IFNAR1)-deficient mice, suggesting that IFN-ß produced by the flagellin stimulation regulates anti-flagellin antibody class switching. Our findings shed a new light on the regulation of flagellin-mediated immune activation and may help find new strategies to promote the intestinal health and develop mucosal vaccines.

Disrupted-in-schizophrenia 1 (DISC1) and Syntaphilin collaborate to modulate axonal mitochondrial anchoring.

In neuronal axons, the ratio of motile-to-stationary mitochondria is tightly regulated by neuronal activation, thereby meeting the need for local calcium buffering and maintaining the ATP supply. However, the molecular players and detailed regulatory mechanisms behind neuronal mitochondrial movement are not completely understood. Here, we found that neuronal activation-induced mitochondrial anchoring is regulated by Disrupted-in-schizophrenia 1 (DISC1), which is accomplished by functional association with Syntaphilin (SNPH). DISC1 deficiency resulted in reduced axonal mitochondrial movement, which was partially reversed by concomitant SNPH depletion. In addition, a SNPH deletion mutant lacking the sequence for interaction with DISC1 exhibited an enhanced mitochondrial anchoring effect than wild-type SNPH. Moreover, upon neuronal activation, mitochondrial movement was preserved by DISC1 overexpression, not showing immobilized response of mitochondria. Taken together, we propose that DISC1 in association with SNPH is a component of a modulatory complex that determines mitochondrial anchoring in response to neuronal activation.

Development of antibody-based c-Met inhibitors for targeted cancer therapy.

Signaling pathways mediated by receptor tyrosine kinases (RTKs) and their ligands play important roles in the development and progression of human cancers, which makes RTK-mediated signaling pathways promising therapeutic targets in the treatment of cancer. Compared with small-molecule compounds, antibody-based therapeutics can more specifically recognize and bind to ligands and RTKs. Several antibody inhibitors of RTK-mediated signaling pathways, such as human epidermal growth factor receptor 2, vascular endothelial growth factor, epidermal growth factor receptor or vascular endothelial growth factor receptor 2, have been developed and are widely used to treat cancer patients. However, since the therapeutic options are still limited in terms of therapeutic efficacy and types of cancers that can be treated, efforts are being made to identify and evaluate novel RTK-mediated signaling pathways as targets for more efficacious cancer treatment. The hepatocyte growth factor/c-Met signaling pathway has come into the spotlight as a promising target for development of potent cancer therapeutic agents. Multiple antibody-based therapeutics targeting hepatocyte growth factor or c-Met are currently in preclinical or clinical development. This review focuses on the development of inhibitors of the hepatocyte growth factor/c-Met signaling pathway for cancer treatment, including critical issues in clinical development and future perspectives for antibody-based therapeutics.

UNC93B1 is essential for the plasma membrane localization and signaling of Toll-like receptor 5.

The proper trafficking and localization of Toll-like receptors (TLRs) are important for specific ligand recognition and efficient signal transduction. The TLRs sensing bacterial membrane components are expressed on the cell surface and recruit signaling adaptors to the plasma membrane upon stimulation. On the contrary, the nucleotide-sensing TLRs are mostly found inside cells and signal from the endolysosomes in an acidic pH-dependent manner. Trafficking of the nucleotide-sensing TLRs from the endoplasmic reticulum to the endolysosomes strictly depends on UNC93B1, and their signaling is completely abolished in the 3d mutant mice bearing the H412R mutation of UNC93B1. In contrast, UNC93B1 was considered to have no role for the cell surface-localized TLRs and signaling via TLR1, TLR2, TLR4, and TLR6 is normal in the 3d mice. Unexpectedly, we discovered that TLR5, a cell surface receptor for bacterial protein flagellin, also requires UNC93B1 for plasma membrane localization and signaling. TLR5 physically interacts with UNC93B1, and the cells from the 3d or UNC93B1-deficient mice not only lack TLR5 at the plasma membrane but also fail to secret cytokines and to up-regulate costimulatory molecules upon flagellin stimulation, demonstrating the essential role of UNC93B1 in TLR5 signaling. Our study reveals that the role of UNC93B1 is not limited to the TLRs signaling from the endolysosomes and compels the further probing of the mechanisms underlying the UNC93B1-assisted differential targeting of TLRs.

Acidic amino acid residues in the juxtamembrane region of the nucleotide-sensing TLRs are important for UNC93B1 binding and signaling.

TLRs are divided into two groups based on their subcellular localization patterns. TLR1, 2, 4, 5, and 6 are expressed on the cell surface, whereas the nucleotide-sensing TLRs, such as TLR3, 7, 8, and 9 stay mainly inside cells. The polytopic membrane protein UNC93B1 physically interacts with the nucleotide-sensing TLRs and delivers them from the endoplasmic reticulum to endolysosomes, where the TLRs recognize their ligands and initiate signaling. In cells with nonfunctional UNC93B1, the nucleic acid-sensing TLRs fail to exit the endoplasmic reticulum and consequently do not signal. However, the detailed molecular mechanisms that underlie the UNC93B1-mediated TLR trafficking remain to be clarified. All nucleotide-sensing TLRs contain acidic amino acid residues in the juxtamembrane region between the leucine-rich repeat domain and the transmembrane segment. We show that the D812 and E813 residues of TLR9 and the D699 and E704 residues of TLR3 help to determine the interaction of these TLRs with UNC93B1. Mutation of the acidic residues in TLR3 and TLR9 prevents UNC93B1 binding, as well as impairs TLR trafficking and renders the mutant receptors incapable of transmitting signals. Therefore, the acidic residues in the juxtamembrane region of the nucleotide-sensing TLRs have important functional roles.

CRL4(CDT2) targets CHK1 for PCNA-independent destruction.

CDT2 targets proteins involved in replication licensing (CDT1), cell cycle control (p21), and chromatin modification (SET8) for destruction by the CUL4-based E3 ligase (CRL4). CRL4(CDT2) recruits these substrates through interactions with chromatin-bound PCNA and ubiquitinates them exclusively on chromatin. Rereplication and G(2) cell cycle arrest are observed in CDT2-depleted cells. The rereplication phenotype has been attributed to an inability to destroy CDT1, but the molecular target important for G(2) cell cycle arrest in CDT2-depleted cells has not been identified. Here we identify CHK1 as a novel CRL4(CDT2) substrate and demonstrate that CHK1 activity is required for maintaining G(2) arrest in CDT2-depleted cells. We demonstrate that CRL4(CDT2) targets the activated form of CHK1 for destruction in the nucleoplasm rather than on chromatin and that this occurs in a PCNA-independent manner. Although both CRL1 and CRL4 ubiquitinate CHK1, we report that they bind CHK1 in distinct cellular compartments. Our study provides insight into how elevated CDT2 expression levels may provide tumors with a proliferative advantage.

DDB1 targets Chk1 to the Cul4 E3 ligase complex in normal cycling cells and in cells experiencing replication stress.

The Chk1 protein kinase preserves genome integrity in normal proliferating cells and in cells experiencing replicative and genotoxic stress. Chk1 is currently being targeted in anticancer regimens. Here, we identify damaged DNA-binding protein 1 (DDB1) as a novel Chk1-interacting protein. DDB1 is part of an E3 ligase complex that includes the cullin proteins Cul4A and Cul4B. We report that Cul4A/DDB1 negatively regulates Chk1 stability in vivo. Chk1 associates with Cul4A/DDB1 during an unperturbed cell division cycle and both Chk1 phosphorylation and replication stress enhanced these interactions. Cul4A/DDB1 regulates Chk1 ubiquitination in vivo and Chk1 is directly ubiquitinated in vitro in a Cul4A/DDB1-dependent manner. Furthermore, Chk1 is stabilized in cells deficient for Cul4A/DDB1. This study shows that Chk1 abundance is regulated by the Cul4A/DDB1 ubiquitin ligase during an unperturbed cell division cycle, in response to replicative stress and on heat shock protein 90 inhibition, and that deregulation of the Chk1/Cul4A/DDB1 pathway perturbs the ionizing radiation-induced G(2) checkpoint.