Excessive apoptosis of Rip1-deficient T cells leads to premature aging.

In mammals, the most remarkable T cell variations with aging are the shrinking of the naïve T cell pool and the enlargement of the memory T cell pool, which are partially caused by thymic involution. However, the mechanism underlying the relationship between T-cell changes and aging remains unclear. In this study, we find that T-cell-specific Rip1 KO mice show similar age-related T cell changes and exhibit signs of accelerated aging-like phenotypes, including inflammation, multiple age-related diseases, and a shorter lifespan. Mechanistically, Rip1-deficient T cells undergo excessive apoptosis and promote chronic inflammation. Consistent with this, blocking apoptosis by co-deletion of Fadd in Rip1-deficient T cells significantly rescues lymphopenia, the imbalance between naïve and memory T cells, and aging-like phenotypes, and prolongs life span in T-cell-specific Rip1 KO mice. These results suggest that the reduction and hyperactivation of T cells can have a significant impact on organismal health and lifespan, underscoring the importance of maintaining T cell homeostasis for healthy aging and prevention or treatment of age-related diseases.

Role of CXCR5+ CD8+ T cells in human hepatitis B virus infection.

The replication of HBV in hepatocytes can be effectively inhibited by lifelong antiviral therapy. Because of the long-term presence of HBV reservoirs, the virus rebound frequently occurs once the treatment is stopped, which poses a considerable obstacle to the complete removal of the virus. In terms of gene composition, regulation of B cell action and function, CXCR5+ CD8+ T cells are similar to CXCR5+ CD4+ T follicular helper cells, while these cells are characterized by elevated programmed cell death 1 and cytotoxic-related proteins. CXCR5+ CD8+ T cells are strongly associated with progression in inflammatory and autoimmune diseases. In addition, CXCR5 expression on the surface of CD8+ T cells is mostly an indicator of memory stem cell-like failure in progenitor cells in cancer that are more responsive to immune checkpoint blocking therapy. Furthermore, the phenomena have also been demonstrated in some viral infections, highlighting the duality of the cellular immune response of CXCR5+ CD8+ T cells. This mini-review will focus on the function of CXCR5+ CD8+ T cells in HBV infection and discuss the function of these CD8+ T cells and the potential of associated co-stimulators or cytokines in HBV therapeutic strategies.

Caspase-8 Promotes Pulmonary Hypertension by Activating Macrophage-Associated Inflammation and IL-1ß (Interleukin 1ß) Production.

BACKGROUND: Macrophages are involved in the pathogenesis of pulmonary arterial hypertension (PAH). Caspase-8, an apical component of cell death pathways, is significantly upregulated in macrophages of PAH animal models. However, its role in PAH remains unclear. Caspase-8 plays a critical role in regulating inflammatory responses via inflammasome activation, cell death, and cytokine induction. This study investigated the mechanism of regulation of IL-1ß (interleukin 1ß) activation in macrophages by caspase-8. METHODS: A hypoxia + SU5416-induced PAH mouse model and monocrotaline-induced rat model of PAH were constructed and the role of caspase-8 was analyzed. RESULTS: Caspase-8 and cleaved-caspase-8 were significantly upregulated in the lung tissues of SU5416 and hypoxia-treated PAH mice and monocrotaline-treated rats. Pharmacological inhibition of caspase-8 alleviated PAH compared with wild-type mice, observed as a significant reduction in right ventricular systolic pressure, ratio of right ventricular wall to left ventricular wall plus ventricular septum, pulmonary vascular media thickness, and pulmonary vascular muscularization; caspase-8 ablated mice also showed significant remission. Mechanistically, increased proliferation of pulmonary arterial smooth muscle cellss is closely associated with activation of the NLRP3 (NOD [nucleotide oligomerization domain]-, LRR [leucine-rich repeat]-, and PYD [pyrin domain]-containing protein 3) inflammasome and the IL-1ß signaling pathway. Although caspase-8 did not affect extracellular matrix synthesis, it promoted inflammatory cell infiltration and pulmonary arterial smooth muscle cell proliferation via NLRP3/IL-1ß activation during the development stage of PAH. CONCLUSIONS: Taken together, our study suggests that macrophage-derived IL-1ß via caspase-8-dependent canonical inflammasome is required for macrophages to play a pathogenic role in pulmonary perivascular inflammation.

CRISPR/Cas12a-Triggered Chemiluminescence Enhancement Biosensor for Sensitive Detection of Nucleic Acids by Introducing a Tyramide Signal Amplification Strategy.

CRISPR-based biosensors have attracted increasing attention in accurate and sensitive nucleic acid detection. In this work, we report a CRISPR/Cas12a-triggered chemiluminescence enhancement biosensor for the ultrasensitive detection of nucleic acids by introducing tyramide signal amplification for the first time (termed CRICED). The hybrid chain DNA (crDNA) formed by NH2-capture DNA (capDNA) and biotin-recognition DNA (recDNA) was preferentially attached to the magnetic beads (MBs), and the streptavidin-HRP was subsequently introduced to obtain MB@HRP-crDNA. In the presence of the DNA target, the activated CRISPR/Cas12a is capable of randomly cutting initiator DNA (intDNA) into vast short products, and thus the fractured intDNA could not trigger the toehold-mediated DNA-strand displacement reaction (TSDR) event with MB@HRP-crDNA. After the addition of tyramine-AP and H2O2, abundant HRP-tyramine-AP emerges through the covalent attachment of HRP-tyramine, exhibiting enhanced chemiluminescence (CL) signals or visual image readouts. By virtue of this biosensor, we achieved high sensitivity of synthetic DNA target and amplified DNA plasmid using recombinase polymerase amplification (RPA) as low as 17 pM and single-copy detection, respectively. Our proposed CRICED was further evaluated to test 20 HPV clinical samples, showing a superior sensitivity of 87.50% and specificity of 100.00%. Consequently, the CRICED platform could be an attractive means for ultrasensitive and imaging detection of nucleic acids and holds a promising strategy for the practical application of CRISPR-based diagnostics.

ABIN1 (Q478) is Required to Prevent Hematopoietic Deficiencies through Regulating Type I IFNs Expression.

A20-binding inhibitor of NF-κB activation (ABIN1) is a polyubiquitin-binding protein that regulates cell death and immune responses. Although Abin1 is located on chromosome 5q in the region commonly deleted in patients with 5q minus syndrome, the most distinct of the myelodysplastic syndromes (MDSs), the precise role of ABIN1 in MDSs remains unknown. In this study, mice with a mutation disrupting the polyubiquitin-binding site (Abin1Q478H/Q478H ) is generated. These mice develop MDS-like diseases characterized by anemia, thrombocytopenia, and megakaryocyte dysplasia. Extramedullary hematopoiesis and bone marrow failure are also observed in Abin1Q478H/Q478H mice. Although Abin1Q478H/Q478H cells are sensitive to RIPK1 kinase-RIPK3-MLKL-dependent necroptosis, only anemia and splenomegaly are alleviated by RIPK3 deficiency but not by MLKL deficiency or the RIPK1 kinase-dead mutation. This indicates that the necroptosis-independent function of RIPK3 is critical for anemia development in Abin1Q478H/Q478H mice. Notably, Abin1Q478H/Q478H mice exhibit higher levels of type I interferon (IFN-I) expression in bone marrow cells compared towild-type mice. Consistently, blocking type I IFN signaling through the co-deletion of Ifnar1 greatly ameliorated anemia, thrombocytopenia, and splenomegaly in Abin1Q478H/Q478H mice. Together, these results demonstrates that ABIN1(Q478) prevents the development of hematopoietic deficiencies by regulating type I IFN expression.

CRISPR/Cas12a-Enabled Multiplex Biosensing Strategy Via an Affordable and Visual Nylon Membrane Readout.

Most multiplex nucleic acids detection methods require numerous reagents and high-priced instruments. The emerging clustered regularly interspaced short palindromic repeats (CRISPR)/Cas has been regarded as a promising point-of-care (POC) strategy for nucleic acids detection. However, how to achieve CRISPR/Cas multiplex biosensing remains a challenge. Here, an affordable means termed CRISPR-RDB (CRISPR-based reverse dot blot) for multiplex target detection in parallel, which possesses the advantages of high sensitivity and specificity, cost-effectiveness, instrument-free, ease to use, and visualization is reported. CRISPR-RDB integrates the trans-cleavage activity of CRISPR-Cas12a with a commercial RDB technique. It utilizes different Cas12a-crRNA complexes to separately identify multiple targets in one sample and converts targeted information into colorimetric signals on a piece of accessible nylon membrane that attaches corresponding specific-oligonucleotide probes. It has demonstrated that the versatility of CRISPR-RDB by constructing a four-channel system to simultaneously detect influenza A, influenza B, respiratory syncytial virus, and SARS-CoV-2. With a simple modification of crRNAs, the CRISPR-RDB can be modified to detect human papillomavirus, saving two-thirds of the time compared to a commercial PCR-RDB kit. Further, a user-friendly microchip system for convenient use, as well as a smartphone app for signal interpretation, is engineered. CRISPR-RDB represents a desirable option for multiplexed biosensing and on-site diagnosis.

Enhanced chemiluminescence imaging sensor for ultrasensitive detection of nucleic acids based on HCR-CRISPR/Cas12a.

CRISPR/Cas systems have ignited increasing attention in accurate and sensitive nucleic acids detection. In this work, we proposed the first CRISPR/Cas12a-based chemiluminescence enhancement biosensor by employing HCR amplifying strategy (CLE-CRISPR) for nucleic acids detection, which shows the advantages of high sensitivity and specificity, low-cost, visual imaging by comparison to reported biosensors. Upon the DNA target recognition, the activated CRISPR/Cas12a enabled randomly cutting initiator DNA (intDNA) into vast short products, which could not trigger the toehold-mediated DNA-strand displacement reaction (TSDR) with MB@crDNA. Thereby, the terminus of crDNA induced the hybridization chain reaction (HCR) with the coexistence of two hairpins (H1 and H2), forming a long double-stranded DNA framework. The attached streptavidin-AP yielded a conspicuous CL signal or visual imaging directly related to the DNA target concentration. The proposed CLE-CRISPR platform exhibited excellent sensitivity, with a relatively low detection limit at 3 pM for synthetic DNA target and single copy detection for plasmid by combining recombinase polymerase amplification (RPA) kit. We further validated the practical application of this platform using HPV clinical samples, achieving superior sensitivity and specificity of 88.89% and 100%, respectively. We believe that this work not only extends the application scope of CRISPR/Cas12a, but also devotes a new approach for clinical diagnosis.

Caspase-8 Blocks Receptor-Interacting Protein Kinase-1 Kinase-Independent Necroptosis during Embryogenesis.

Caspase-8 (Casp8) suppresses receptor-interacting protein kinase-3 (RIPK3)/mixed lineage kinase domain-like protein (MLKL)-dependent necroptosis, demonstrated by the genetic evidence that deletion of Ripk3 or Mlkl prevented embryonic lethality of Casp8-deficient mice. However, the detailed mechanisms by which Casp8 deficiency triggers necroptosis during embryonic development remain unclear. In this article, we show that Casp8 deletion caused formation of the RIPK1-RIPK3 necrosome in the yolk sac, leading to vascularization defects, prevented by MLKL and RIPK3 deficiency, or RIPK3 RHIM mutant (RIPK3 V448P), but not by the RIPK1 kinase-dead mutant (RIPK1 K45A). In addition, Ripk1K45A/K45ACasp8 -/- mice died on embryonic day 14.5, which was delayed to embryonic day 17.5 by ablation of one allele in Ripk1 and was completely rescued by ablation of Mlkl Our results revealed an in vivo role of RIPK3 RHIM and RIPK1K45A scaffold-mediated necroptosis in Casp8 deficiency embryonic development and suggested that the Casp8-deficient yolk sac might be implicated in identifying novel regulators as an in vivo necroptotic model.

Caspase-8 auto-cleavage regulates programmed cell death and collaborates with RIPK3/MLKL to prevent lymphopenia.

Caspase-8 is an initiator of death receptor-induced apoptosis and an inhibitor of RIPK3-MLKL-dependent necroptosis. In addition, caspase-8 has been implicated in diseases such as lymphoproliferation, immunodeficiency, and autoimmunity in humans. Although auto-cleavage is indispensable for caspase-8 activation, its physiological functions remain poorly understood. Here, we generated a caspase-8 mutant lacking E385 in auto-cleavage site knock-in mouse (Casp8ΔE385/ΔE385). Casp8ΔE385/ΔE385 cells were expectedly resistant to Fas-induced apoptosis, however, Casp8ΔE385/ΔE385 cells could switch TNF-α-induced apoptosis to necroptosis by attenuating RIPK1 cleavage. More importantly, CASP8(ΔE385) sensitized cells to RIPK3-MLKL-dependent necroptosis through promoting complex II formation and RIPK1-RIPK3 activation. Notably, Casp8ΔE385/ΔE385Ripk3-/- mice partially rescued the perinatal death of Ripk1-/- mice by blocking apoptosis and necroptosis. In contrast to the Casp8-/-Ripk3-/- and Casp8-/-Mlkl-/- mice appearing autoimmune lymphoproliferative syndrome (ALPS), both Casp8ΔE385/ΔE385Ripk3-/- and Casp8ΔE385/ΔE385Mlkl-/- mice developed transplantable lymphopenia that could be significantly reversed by RIPK1 heterozygosity, but not by RIPK1 kinase dead mutation. Collectively, these results demonstrate previously unappreciated roles for caspase-8 auto-cleavage in regulating necroptosis and maintaining lymphocytes homeostasis.

Ubiquitin-binding domain in ABIN1 is critical for regulating cell death and inflammation during development.

ABIN1 is a polyubiquitin-binding protein known to regulate NF-κB activation and cell death signaling. Mutations in Abin1 can cause severe immune diseases in human, such as psoriasis, systemic lupus erythematosus, and systemic sclerosis. Here, we generated mice that disrupted the ubiquitin-binding domain of ABIN1 (Abin1UBD/UBD) died during later embryogenesis owing to TNFR1-mediated cell death, similar to Abin1-/- mice. Abin1UBD/UBD cells were rendered sensitive to TNF-α-induced apoptosis and necroptosis as the inhibition of ABIN1UBD and A20 recruitment to the TNF-RSC complex leads to attenuated RIPK1 deubiquitination. Accordingly, the embryonic lethality of Abin1UBD/UBD mice was rescued via crossing with RIPK1 kinase-dead mice (Ripk1K45A/K45A) or the co-deletion of Ripk3 and one allele of Fadd, but not by the loss of Ripk3 or Mlkl alone. Unexpectedly, Abin1UBD/UBD mice with the co-deletion of Ripk3 and both Fadd alleles died at E14.5. This death was caused by spontaneous RIPK1 ubiquitination-dependent multiple inflammatory cytokines over production and could be rescued by the co-deletion of Ripk1 or Tnfr1 combined with Ifnar. Collectively, these data demonstrate the importance of the ABIN1 UBD domain, which mediates the ABIN1-A20 axis, at limiting RIPK1 activation-dependent cell death during embryonic development. Furthermore, our findings reveal a previously unappreciated ubiquitin pathway that regulates RIPK1 ubiquitination by FADD/Casp8 to suppress spontaneous IKKε/TBK1 activation.

Red-emitting GSH-Cu NCs as a triplet induced quenched fluorescent probe for fast detection of thiol pollutants.

Thiol compounds exist widely on the Earth and have certain significance in the fields of the circulation of the sulfur element and industrial production. However, the odor and biological toxicity of thiol compounds make them pollutants that seriously threaten the environmental safety and the living quality of human. In this study, a novel triplet induced fluorescence "turn-off" strategy was designed for the detection of thiol pollutants via a glutathione-stabilized copper nanocluster (GSH-Cu NC) probe. The as-prepared GSH-Cu NCs not only have small size and good water-solubility, but also exhibit strong red-emitting fluorescence at 630 nm, which could be quenched quantitatively with the increase of the concentration of thiol pollutants. So they were employed to detect thioglycolic acid (TGA), 3-mercaptopropionic acid (MPA), 2-mercaptoethanol (ME) and 2-(diethylamino)ethanethiol (2-AT) in a wide linear range of 1-100 µM with detection limits of 0.73 µM, 0.43 µM, 0.37 µM, and 0.69 µM, respectively. This method was successfully applied to detect the above thiol pollutants in lake water with good recoveries. Moreover, their further application was also expanded as luminous test strips based on the excellent fluorescence characteristics of GSH-Cu NCs for fast real-time detection of thiol pollutants.

[Receptor-binding ability of fragments 260-600 and 397-796 of SARS-associated coronavirus spike protein].

BACKGROUND: To investigate the interaction between the host cell and the truncated S fragments to identify the receptor-binding domain of the spike (S) protein of SARS-associated coronavirus (SARS-CoV). METHODS: Two different fragments S260-600 and S397-796 of the SARS-CoV S protein were expressed in Escherichia coli (E.coli) using a pET expression vector, respectively. The two recombinant proteins were separately verified by Western blot, purified by nickel-affinity chromatography, and incubated with Vero cells, a susceptible cell line of SARS-CoV infection, for cell binding assay. After the sequential probing with sera from convalescent SARS-patients and FITC-labeled anti-human IgG, the cells were analyzed by flow cytometry. The NIH 3T3 cell, a non-permissive cell line of SARS-CoV infection, was used as controls. RESULTS: The recombinant proteins S260-600 and S397-796 were efficiently expressed in an insoluble form in E.coli. The appropriate expression of the proteins was confirmed by Western blotting using both SARS patients' sera and anti-6 x histidine antibody. The flow cytometry results showed that the both proteins were able to bind Vero cells, but the binding ability of S260-600 was somewhat stronger than that of S397-796. In contrast, the S260-600 protein did not bind NIH3T3 cells. CONCLUSION: Both S260-600 and S397-796 exhibited different receptor binding activity. The S260-600 fragment probably contains the important receptor binding domain and could be a potential candidate for the development of SARS vaccine and anti-SARS therapeutics.