LncRNA CamK-A Regulates Ca2+-Signaling-Mediated Tumor Microenvironment Remodeling.

Cancer cells entail metabolic adaptation and microenvironmental remodeling to survive and progress. Both calcium (Ca2+) flux and Ca2+-dependent signaling play a crucial role in this process, although the underlying mechanism has yet to be elucidated. Through RNA screening, we identified one long noncoding RNA (lncRNA) named CamK-A (lncRNA for calcium-dependent kinase activation) in tumorigenesis. CamK-A is highly expressed in multiple human cancers and involved in cancer microenvironment remodeling via activation of Ca2+-triggered signaling. Mechanistically, CamK-A activates Ca2+/calmodulin-dependent kinase PNCK, which in turn phosphorylates IκBα and triggers calcium-dependent nuclear factor κB (NF-κB) activation. This regulation results in the tumor microenvironment remodeling, including macrophage recruitment, angiogenesis, and tumor progression. Notably, our human-patient-derived xenograft (PDX) model studies demonstrate that targeting CamK-A robustly impaired cancer development. Clinically, CamK-A expression coordinates with the activation of CaMK-NF-κB axis, and its high expression indicates poor patient survival rate, suggesting its role as a potential biomarker and therapeutic target.

lncRNA BREA2 promotes metastasis by disrupting the WWP2-mediated ubiquitination of Notch1.

Notch has been implicated in human cancers and is a putative therapeutic target. However, the regulation of Notch activation in the nucleus remains largely uncharacterized. Therefore, characterizing the detailed mechanisms governing Notch degradation will identify attractive strategies for treating Notch-activated cancers. Here, we report that the long noncoding RNA (lncRNA) BREA2 drives breast cancer metastasis by stabilizing the Notch1 intracellular domain (NICD1). Moreover, we reveal WW domain containing E3 ubiquitin protein ligase 2 (WWP2) as an E3 ligase for NICD1 at K1821 and a suppressor of breast cancer metastasis. Mechanistically, BREA2 impairs WWP2-NICD1 complex formation and in turn stabilizes NICD1, leading to Notch signaling activation and lung metastasis. BREA2 loss sensitizes breast cancer cells to inhibition of Notch signaling and suppresses the growth of breast cancer patient-derived xenograft tumors, highlighting its therapeutic potential in breast cancer. Taken together, these results reveal the lncRNA BREA2 as a putative regulator of Notch signaling and an oncogenic player driving breast cancer metastasis.

Essential role of an ERV-derived Env38 protein in adaptive humoral immunity against an exogenous SVCV infection in a zebrafish model.

Endogenous retroviruses (ERVs) are the relics of ancient retroviruses occupying a substantial fraction of vertebrate genomes. However, knowledge about the functional association of ERVs with cellular activities remains limited. Recently, we have identified approximately 3,315 ERVs from zebrafish at genome-wide level, among which 421 ERVs were actively expressed in response to the infection of Spring viraemia of carp virus (SVCV). These findings demonstrated the previously unrecognized activity of ERVs in zebrafish immunity, thereby making zebrafish an attractive model organism for deciphering the interplay among ERVs, exogenous invading viruses, and host immunity. In the present study, we investigated the functional role of an envelope protein (Env38) derived from an ERV-E5.1.38-DanRer element in zebrafish adaptive immunity against SVCV in view of its strong responsiveness to SVCV infection. This Env38 is a glycosylated membrane protein mainly distributed on MHC-II+ antigen-presenting cells (APCs). By performing blockade and knockdown/knockout assays, we found that the deficiency of Env38 markedly impaired the activation of SVCV-induced CD4+ T cells and thereby led to the inhibition of IgM+/IgZ+ B cell proliferation, IgM/IgZ Ab production, and zebrafish defense against SVCV challenge. Mechanistically, Env38 activates CD4+ T cells by promoting the formation of pMHC-TCR-CD4 complex via cross-linking MHC-II and CD4 molecules between APCs and CD4+ T cells, wherein the surface subunit (SU) of Env38 associates with the second immunoglobin domain of CD4 (CD4-D2) and the first α1 domain of MHC-IIα (MHC-IIα1). Notably, the expression and functionality of Env38 was strongly induced by zebrafish IFNφ1, indicating that env38 acts as an IFN-stimulating gene (ISG) regulated by IFN signaling. To the best of our knowledge, this study is the first to identify the involvement of an Env protein in host immune defense against an exogenous invading virus by promoting the initial activation of adaptive humoral immunity. It improved the current understanding of the cooperation between ERVs and host adaptive immunity.

NF2 regulates IP3R-mediated Ca2+ signal and apoptosis in meningiomas.

Meningiomas are the most common primary intracranial tumors and account for nearly 30% of all nervous system tumors. Approximately half of meningioma patients exhibit neurofibromin 2 (NF2) gene inactivation. Here, NF2 was shown to interact with the endoplasmic reticulum (ER) calcium (Ca2+) channel inositol 1,4,5-trisphosphate receptor 1 (IP3R1) in IOMM-Lee, a high-grade malignant meningioma cell line, and the F1 subdomain of NF2 plays a critical role in this interaction. Functional assays indicated that NF2 promotes the phosphorylation of IP3R (Ser 1756) and IP3R-mediated endoplasmic reticulum (ER) Ca2+ release by binding to IP3R1, which results in Ca2+-dependent apoptosis. Knockout of NF2 decreased Ca2+ release and promoted resistance to apoptosis, which was rescued by wild-type NF2 overexpression but not by F1 subdomain deletion truncation overexpression. The effects of NF2 defects on the development of tumors were further studied in mouse models. The decreased expression level of NF2 caused by NF2 gene knockout or mutation affects the activity of the IP3R channel, which reduces Ca2+-dependent apoptosis, thereby promoting the development of tumors. We elucidated the interaction patterns of NF2 and IP3R1, revealed the molecular mechanism through which NF2 regulates IP3R1-mediated Ca2+ release, and elucidated the new pathogenic mechanism of meningioma-related NF2 variants. Our study broadens the current understanding of the biological function of NF2 and provides ideas for drug screening of NF2-associated meningioma.

PD-L1/BTLA Checkpoint Axis Exploited for Bacterial Immune Escape by Restraining CD8+ T Cell-Initiated Adaptive Immunity in Zebrafish.

Programmed death-ligand 1/programmed cell death 1 (PD-L1/PD-1) is one of the most important immune checkpoints in humans and other mammalian species. However, the occurrence of the PD-L1/PD-1 checkpoint in evolutionarily ancient vertebrates remains elusive because of the absence of a PD-1 homolog before its appearance in tetrapods. In this article, we identified, to our knowledge, a novel PD-L1/B and T lymphocyte attenuator (BTLA) checkpoint in zebrafish by using an Edwardsiella tarda-induced bacterial infection model. Results showed that zebrafish (Danio rerio) PD-L1 (DrPD-L1) and BTLA (DrBTLA) were differentially upregulated on MHC class II+ macrophages (Mϕs) and CD8+ T cells in response to E. tarda infection. DrPD-L1 has a strong ability to interact with DrBTLA, as shown by the high affinity (KD = 5.68 nM) between DrPD-L1/DrBTLA proteins. Functionally, the breakdown of DrPD-L1/DrBTLA interaction significantly increased the cytotoxicity of CD8+BTLA+ T cells to E. tarda-infected PD-L1+ Mϕ cells and reduced the immune escape of E. tarda from the target Mϕ cells, thereby enhancing the antibacterial immunity of zebrafish against E. tarda infection. Similarly, the engagement of DrPD-L1 by soluble DrBTLA protein diminished the tolerization of CD8+ T cells to E. tarda infection. By contrast, DrBTLA engagement by a soluble DrPD-L1 protein drives aberrant CD8+ T cell responses. These results were finally corroborated in a DrPD-L1-deficient (PD-L1-/-) zebrafish model. This study highlighted a primordial PD-L1/BTLA coinhibitory axis that regulates CD8+ T cell activation in teleost fish and may act as an alternative to the PD-L1/PD-1 axis in mammals. It also revealed a previously unrecognized strategy for E. tarda immune evasion by inducing CD8+ T cell tolerance to target Mϕ cells through eliciting the PD-L1/BTLA checkpoint pathway.

Bionic Structural Coloration of Textiles Using the Synthetically Prepared Liquid Photonic Crystals.

The structural coloration of textiles with bionic photonic crystals (PCs) is expected to become a critical approach to the ecological coloration of textiles. Rapid and large-area preparation of PC structurally colored textiles can be achieved via self-assembly of high mass fractions of liquid photonic crystals (LPCs). However, the rapid and large-scale manufacturing of LPCs remains a challenge. In this work, the pH regulator is added in the process of emulsion polymerization to solve the problem of phase transformation caused by the thermal decomposition of the initiator to produce H+ , directly achieving 40 wt.% PS nanospheres in the dispersion. Then oligomers and small-molecule salts are removed from the system via dialysis, and the pre-crystallized LPC system is efficiently prepared. Adjusting the particle size and the mass fraction of nanospheres is shown to be an efficient way to control the optical properties of LPCs. The rapid and large-area preparation of PC structural color fabric and the patterned PC structural color fabric with an iridescent effect is implemented by using LPCs as the assembly intermediate. By constructing the encapsulation layer on the surface of the PC structural color fabric, the consistency of high structural stability and high color saturation of the PC is realized.

Single-cell transcriptome profiling reveals diverse immune cell populations and their responses to viral infection in the spleen of zebrafish.

Teleost fish are indispensable model organisms for comparative immunology research that should lead to an improved understanding of the general principles of vertebrate immune system design. Although numerous studies on fish immunology have been conducted, knowledge about the cell types that orchestrate piscine immune systems remains limited. Here, we generated a comprehensive atlas of immune cell types in zebrafish spleen on the basis of single-cell transcriptome profiling. We identified 11 major categories from splenic leukocyte preparations, including neutrophils, natural killer cells, macrophages/myeloid cells, T cells, B cells, hematopoietic stem and progenitor cells, mast cells, remnants of endothelial cells, erythroid cells, erythroid progenitors, and a new type of serpin-secreting cells. Notably, we derived 54 potential subsets from these 11 categories. These subsets showed differential responses to spring viremia of carp virus (SVCV) infection, implying that they have diverse roles in antiviral immunity. Additionally, we landscaped the populations with the induced expression of interferons and other virus-responsive genes. We found that trained immunity can be effectively induced in the neutrophil and M1-macrophage subsets by vaccinating zebrafish with inactivated SVCV. Our findings illustrated the complexity and heterogeneity of the fish immune system, which will help establish a new paradigm for the improved understanding of fish immunology.

Zbtb46 Controls Dendritic Cell Activation by Reprogramming Epigenetic Regulation of cd80/86 and cd40 Costimulatory Signals in a Zebrafish Model.

The establishment of an appropriate costimulatory phenotype is crucial for dendritic cells (DCs) to maintain a homeostatic state with optimal immune surveillance and immunogenic activities. The upregulation of CD80/86 and CD40 is a hallmark costimulatory phenotypic switch of DCs from a steady state to an activated one for T cell activation. However, knowledge of the regulatory mechanisms underlying this process remains limited. In this study, we identified a Zbtb46 homolog from a zebrafish model. Zbtb46 deficiency resulted in upregulated cd80/86 and cd40 expression in kidney marrow-derived DCs (KMDCs) of zebrafish, which was accompanied with a remarkable expansion of CD4+/CD8+ T cells and accumulation of KMDCs in spleen of naive fish. Zbtb46 -/- splenic KMDCs exhibited strong stimulatory activity for CD4+ T cell activation. Chromatin immunoprecipitation-quantitative PCR and mass spectrometry assays showed that Zbtb46 was associated with promoters of cd80/86 and cd40 genes by binding to a 5'-TGACGT-3' motif in resting KMDCs, wherein it helped establish a repressive histone epigenetic modification pattern (H3K4me0/H3K9me3/H3K27me3) by organizing Mdb3/organizing nucleosome remodeling and deacetylase and Hdac3/nuclear receptor corepressor 1 corepressor complexes through the recruitment of Hdac1/2 and Hdac3. On stimulation with infection signs, Zbtb46 disassociated from the promoters via E3 ubiquitin ligase Cullin1/Fbxw11-mediated degradation, and this reaction can be triggered by the TLR9 signaling pathway. Thereafter, cd80/86 and cd40 promoters underwent epigenetic reprogramming from the repressed histone modification pattern to an activated pattern (H3K4me3/H3K9ac/H3K27ac), leading to cd80/86 and cd40 expression and DC activation. These findings revealed the essential role of Zbtb46 in maintaining DC homeostasis by suppressing cd80/86 and cd40 expression through epigenetic mechanisms.

Negative Regulatory Role of the Spring Viremia of Carp Virus Matrix Protein in the Host Interferon Response by Targeting the MAVS/TRAF3 Signaling Axis.

Spring viremia of carp virus (SVCV) is a severe infectious pathogen that causes high rates of mortality in cyprinids and other fish species. Despite numerous investigations of SVCV infection, the underlying molecular mechanisms remain poorly understood. In this study, we found that the SVCV matrix protein (SVCV-M) played an inhibitory role in the host interferon (IFN) response by targeting the MAVS/TRAF3 signaling axis, thereby uncovering a previously unrecognized mechanism of SVCV escape from host innate antiviral immunity. Mechanistically, SVCV-M was located at the mitochondria independent of MAVS, which allowed SVCV-M to build an arena for competition with the MAVS platform. A microscale thermophoresis assay showed that SVCV-M had a high affinity for TRAF3, as indicated by a lower equilibrium dissociation constant (KD) value than that of MAVS with TRAF3. Therefore, the association of MAVS with TRAF3 was competitively impaired by SVCV-M in a dose-dependent manner. Accordingly, SVCV-M showed a potent ability to inhibit the K63-linked polyubiquitination of TRAF3. This inhibition was accompanied by the impairment of the IFN response, as shown by the marked decline in IFN-φ1-promoter (pro) luciferase reporter activity. By constructing truncated TRAF3 and SVCV-M proteins, the RING finger, zinc finger, and coiled-coil domains of TRAF3 and the hydrophobic-pocket-like structure formed by the α2-, α3-, and α4-helices of SVCV-M may be the major target and antagonistic modules responsible for the protein-protein interaction between the TRAF3 and SVCV-M proteins. These findings highlighted the intervention of SVCV-M in host innate immunity, thereby providing new insights into the extensive participation of viral matrix proteins in multiple biological activities. IMPORTANCE The matrix protein of SVCV (SVCV-M) is an indispensable structural element for nucleocapsid condensation and virion formation during viral morphogenesis, and it connects the core nucleocapsid particle to the outer membrane within the mature virus. Previous studies have emphasized the architectural role of SVCV-M in viral construction; however, the potential nonstructural functions of SVCV-M in viral replication and virus-host interactions remain poorly understood. In this study, we identified the inhibitory role of the SVCV-M protein in host IFN production by competitively recruiting TRAF3 from the MAVS signaling complex and impairing TRAF3 activation via inhibition of K63-linked polyubiquitination. This finding provided new insights into the regulatory role of SVCV-M in host innate immunity, which highlighted the broader functionality of rhabdovirus matrix protein apart from being a structural protein. This study also revealed a previously unrecognized mechanism underlying SVCV immune evasion by inhibiting the IFN response by targeting the MAVS/TRAF3 signaling axis.

New Insights into IgZ as a Maternal Transfer Ig Contributing to the Early Defense of Fish against Pathogen Infection.

IgZ or its equivalent IgT is a newly discovered teleost specific Ig class that is highly specialized in mucosal immunity. However, whether this IgZ/IgT class participates in other biological processes remains unclear. In this study, we unexpectedly discovered that IgZ is highly expressed in zebrafish ovary, accumulates in unfertilized eggs, and is transmitted to offspring from eggs to zygotes. Maternally transferred IgZ in zygotes is found at the outer and inner layers of chorion, perivitelline space, periphery of embryo body, and yolk, providing different lines of defense against pathogen infection. A considerable number of IgZ+ B cells are found in ovarian connective tissues distributed between eggs. Moreover, pIgR, the transporter of IgZ, is also expressed in the ovary and colocalizes with IgZ in the zona radiata of eggs. Thus, IgZ is possibly secreted by ovarian IgZ+ B cells and transported to eggs through association with pIgR in a paracrine manner. Maternal IgZ in zygotes showed a broad bacteriostatic activity to different microbes examined, and this reactivity can be manipulated by orchestrating desired bacteria in water where parent fish live or immunizing the parent fish through vaccination. These observations suggest that maternal IgZ may represent a group of polyclonal Abs, providing protection against various environmental microbes encountered by a parent fish that were potentially high risk to offspring. To our knowledge, our findings provide novel insights into a previously unrecognized functional role of IgZ/IgT Ig in the maternal transfer of immunity in fish, greatly enriching current knowledge about this ancient Ig class.

Shark IgNAR: The Next Broad Application Antibody in Clinical Diagnoses and Tumor Therapies?

Antibodies represent a relatively mature detection means and serve as therapeutic drug carriers in the clinical diagnosis and treatment of cancer-among which monoclonal antibodies (mAbs) currently occupy a dominant position. However, the emergence and development of small-molecule monodomain antibodies are inevitable due to the many limitations of mAbs, such as their large size, complex structure, and sensitivity to extreme temperature, and tumor microenvironments. Thus, since first discovered in Chondroid fish in 1995, IgNAR has become an alternative therapeutic strategy through which to replace monoclonal antibodies, thus entailing that this novel type of immunoglobulin has received wide attention with respect to clinical diagnoses and tumor therapies. The variable new antigen receptor (VNAR) of IgNAR provides an advantage for the development of new antitumor drugs due to its small size, high stability, high affinity, as well as other structural and functional characteristics. In that respect, a better understanding of the unique characteristics and therapeutic potential of IgNAR/VNAR in clinical and anti-tumor treatment is needed. This article reviews the advantages of its unique biochemical conditions and molecular structure for clinical diagnoses and novel anti-tumor drugs. At the same time, the main advantages of the existing conjugated drugs, which are based on single-domain antibodies, are introduced here, thereby providing new ideas and methods for the development of clinical diagnoses and anti-tumor therapies in the future.

Dityrosine-inspired photocrosslinking technique for 3D printing of silk fibroin-based composite hydrogel scaffolds.

Photoinduced self-crosslinking technology is a great facilitator of 3D bioprinting of silk fibroin (SF) by allowing rapid solidification of a deliberately formulated SF-based photocrosslinkable bioink. An SF-based, photocrosslinked hydrogel was fabricated with tyramine-modified sodium carboxymethyl cellulose (CMC-Na) as a co-crosslinkable constituent and Ru(bpy)3Cl2 (Ru(II)) and potassium persulfate (KPS) as blue light photoinitiators. Photorheological studies demonstrated that the photocrosslinking and viscoelasticity of the composite could be tuned by varying the relative content of the two constituents. Xanthan gum (XG) was employed in formulating the SF-based photocrosslinkable bioink, and the improved rheological properties and printability were evidenced by the resulting tunable shear-thinning behavior and shear thixotropy. 3D SF-based hydrogel scaffolds with uniform pores with a size of approximately 550 µm × 1000 µm were constructed via extrusion-based printing and a simple 30 s post-photocrosslinking combined process. Furthermore, the CMC-Na incorporated 3D hydrogel scaffolds exhibited sufficient structural strength, adequate filament fineness, and tunable transparency, which shows a promising prospect in the application of tissue engineering and regenerative medicine.

The zebrafish NLRP3 inflammasome has functional roles in ASC-dependent interleukin-1ß maturation and gasdermin E-mediated pyroptosis.

The NLR family pyrin domain containing 3 (NLRP3) inflammasome is one of the best-characterized inflammasomes in humans and other mammals. However, knowledge about the NLRP3 inflammasome in nonmammalian species remains limited. Here, we report the molecular and functional identification of an NLRP3 homolog (DrNLRP3) in a zebrafish (Danio rerio) model. We found that DrNLRP3's overall structural architecture was shared with mammalian NLRP3s. It initiates a classical inflammasome assembly for zebrafish inflammatory caspase (DrCaspase-A/-B) activation and interleukin 1ß (DrIL-1ß) maturation in an apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC)-dependent manner, in which DrNLRP3 organizes DrASC into a filament that recruits DrCaspase-A/-B by homotypic pyrin domain (PYD)-PYD interactions. DrCaspase-A/-B activation in the DrNLRP3 inflammasome occurred in two steps, with DrCaspase-A being activated first and DrCaspase-B second. DrNLRP3 also directly activated full-length DrCaspase-B and elicited cell pyroptosis in a gasdermin E (GSDME)-dependent but ASC-independent manner. These two events were tightly coordinated by DrNLRP3 to ensure efficient IL-1ß secretion for the initiation of host innate immunity. By knocking down DrNLRP3 in zebrafish embryos and generating a DrASC-knockout (DrASC-/-) fish clone, we characterized the function of the DrNLRP3 inflammasome in anti-bacterial immunity in vivo The results of our study disclosed the origin of the NLRP3 inflammasome in teleost fish, providing a cross-species understanding of the evolutionary history of inflammasomes. Our findings also indicate that the NLRP3 inflammasome may coordinate inflammatory cytokine processing and secretion through a GSDME-mediated pyroptotic pathway, uncovering a previously unrecognized regulatory function of NLRP3 in both inflammation and cell pyroptosis.

Differential immune responses of immunoglobulin Z subclass members in antibacterial immunity in a zebrafish model.

Immunoglobulin Z (IgZ) or its equivalent immunoglobulin T (IgT) is a newly identified immunoglobulin (Ig) class from teleost fish. This Ig class is characterized by its involvement in mucosa-associated lymphoid tissues (MALTs) for mucosal defence against pathogen infection. Recently, several subclass members of IgZ/IgT, such as IgZ, IgZ2, Igτ1, Igτ2 and Igτ3, have been further identified from zebrafish, common carp and rainbow trout. However, the functional diversity and correlation among these subclasses remain uncertain. Here, we explored the differential immune reactions of the IgZ and IgZ2 subclasses in antibacterial immunity in a zebrafish model. IgZ was extensively distributed in the peripheral serum and skin/gill MALTs and showed a rapid induction upon bacterial infection. IgZ2 was specialized in skin/gill MALTs and showed a strong induction following IgZ production. Correspondingly, the IgZ+ B cells had a wider distribution in the systemic primary/secondary lymphoid tissues and MALTs than the IgZ2+ B cells, which were predominant in MALTs. IgZ and IgZ2 exhibited a complementary effect in antibacterial immunity by possessing differential abilities. That is, IgZ is preferentially involved in bactericidal reaction that is in part C1q-dependent, and IgZ2 participates in neutralization action through bacteria-coating activity. The production of IgZ largely depended on the αß T/CD4+ T cells, whereas that of IgZ2 did not, suggesting the different dependencies of IgZ and IgZ2 on systemic immunity. Our findings demonstrate that the functional behaviour and mechanism of the IgZ/IgT family are more diverse than previously recognized and thus improve the current knowledge about this ancient Ig class.

Characterization of cGAS homologs in innate and adaptive mucosal immunities in zebrafish gives evolutionary insights into cGAS-STING pathway.

Cyclic GMP-AMP synthase (cGAS) is one of the most-characterized cytoplasmic DNA sensors in humans and other mammals. However, knowledge about cGAS homologs in nonmammalian species remains limited. In this study, we report the molecular and functional identification of two cGAS homologs, namely, DrcGASa and DrcGASb, from a zebrafish (Danio rerio) model. DrcGASa and DrcGASb share the same overall conservative structural architectures and functional domains/residues to mammalian cGASs. Both homologs synthesized a 2'3'-cGAMP isomer but not a 3'3'-cGAMP isomer via oligomerization in response to DNA stimulation. Overexpression of DrcGASa/b in HEK293T cells and zebrafish embryos significantly activated NF-κB and IFN-I signaling pathways in a STING-dependent manner. Knockdown of DrcGASa or DrSTING impaired such activations, thereby reducing the host innate immunity against bacterial and viral infections. DrcGASa, but not DrcGASb, was involved in immunoglobulin Z-mediated mucosal immunity in gill-associated lymphoid tissue, suggesting differential functions between the two DrcGASs. This reaction was associated with the DrcGAS-DrSTING-IFNφ1 signaling axis in GALT's γδ T cells. Our findings provide experimental evidence that a modern cGAS-STING pathway that mainly participates in IFN-mediated immunity originated from teleost fish based on the functional constraint of cGAS and STING proteins during vertebrate evolution.

BTLA-HVEM Checkpoint Axis Regulates Hepatic Homeostasis and Inflammation in a ConA-Induced Hepatitis Model in Zebrafish.

The BTLA-HVEM checkpoint axis plays extensive roles in immunomodulation and diseases, including cancer and autoimmune disorders. However, the functions of this checkpoint axis in hepatitis remain limited. In this study, we explored the regulatory role of the Btla-Hvem axis in a ConA-induced hepatitis model in zebrafish. Results showed that Btla and Hvem were differentially expressed on intrahepatic Cd8+ T cells and hepatocytes. Knockdown of Btla or Hvem significantly promoted hepatic inflammation. Btla was highly expressed in Cd8+ T cells in healthy liver but was downregulated in inflamed liver, as evidenced by a disparate proportion of Cd8+Btla+ and Cd8+Btla- T cells in individuals without or with ConA stimulation. Cd8+Btla+ T cells showed minimal cytotoxicity to hepatocytes, whereas Cd8+Btla- T cells were strongly reactive. The depletion of Cd8+Btla- T cells reduced hepatitis, whereas their transfer enhanced hepatic inflammation. These observations indicate that Btla endowed Cd8+Btla+ T cells with self-tolerance, thereby preventing them from attacking hepatocytes. Btla downregulation deprived this tolerization. Mechanistically, Btla-Hvem interaction contributed to Cd8+Btla+ T cell tolerization, which was impaired by Hvem knockdown but rescued by soluble Hvem protein administration. Notably, Light was markedly upregulated on Cd8+Btla- T cells, accompanied by the transition of Cd8+Btla+Light- to Cd8+Btla-Light+ T cells during hepatitis, which could be modulated by Cd4+ T cells. Light blockade attenuated hepatitis, thereby suggesting the positive role of Light in hepatic inflammation. These findings provide insights into a previously unrecognized Btla-Hvem-Light regulatory network in hepatic homeostasis and inflammation, thus adding a new potential therapeutic intervention for hepatitis.

Blue Light Induced Photopolymerization and Cross-Linking Kinetics of Poly(acrylamide) Hydrogels.

Blue light induced photopolymerization and photo-cross-linking kinetics of acrylamide (AM), with camphorquinone/diphenyl iodonium hexafluorophosphate (CQ/DPI) as photoinitiators, were investigated. The effects of a number of parameters, including mass fraction of CQ, DPI, and AM (wCQ, wDPI, and wAM) and light intensity (I), on photopolymerization efficiency and photogelation process were systematically studied by photo-differential scanning calorimetry (DSC) and photo-rheometry. Photo-DSC indicated that the maximum photopolymerization rate (Rp, max) was proportional to wCQ0.5, wDPI0.5, I0.5, and wAM, while Photo-Rheometry showed linear relationships between gel time tgel and wCQ and I, respectively, and power law relationships between tgel and wDPI and wAM, respectively. In addition, both peak cross-linking rate Rc,max, and delay time td, which were both linearly proportional to wCQ0.5, wDPI0.5, and I0.5, showed power law relationships with wAM. Furthermore, exponential patterns were observed between all these factors, wCQ, wDPI, wAM, and I and plateau modulus G'∞. Combining such correlations obtained from experimental data, an empirical model was established describing the projected mechanical properties of poly(acrylamide) hydrogels from blue light initiated photopolymerization and photo-cross-linking.

Characterization of an NLRP1 Inflammasome from Zebrafish Reveals a Unique Sequential Activation Mechanism Underlying Inflammatory Caspases in Ancient Vertebrates.

NLRP1 inflammasome is one of the best-characterized inflammasomes in humans and other mammals. However, the existence of this inflammasome in nonmammalian species remains poorly understood. In this study, we report the molecular and functional identification of an NLRP1 homolog, Danio rerio NLRP1 (DrNLRP1) from a zebrafish (D. rerio) model. This DrNLRP1 possesses similar structural architecture to mammalian NLRP1s. It can trigger the formation of a classical inflammasome for the activation of zebrafish inflammatory caspases (D. rerio Caspase [DrCaspase]-A and DrCaspase-B) and maturation of D. rerio IL-1ß in a D. rerio ASC (DrASC)-dependent manner. In this process, DrNLRP1 promotes the aggregation of DrASC into a filament with DrASCCARD core and DrASCPYD cluster. The assembly of DrNLRP1 inflammasome depends on the CARD-CARD homotypic interaction between DrNLRP1 and DrASCCARD core, and PYD-PYD interaction between DrCaspase-A/B and DrASCPYD cluster. The FIIND domain in DrNLRP1 is necessary for inflammasome assembly. To understand the mechanism of how the two DrCaspases are coordinated in DrNLRP1 inflammasome, we propose a two-step sequential activation model. In this model, the recruitment and activation of DrCaspase-A/B in the inflammasome is shown in an alternate manner, with a preference for DrCaspase-A followed by a subsequent selection for DrCaspase-B. By using morpholino oligonucleotide-based knockdown assays, the DrNLRP1 inflammasome was verified to play important functional roles in antibacterial innate immunity in vivo. These observations demonstrate that the NLRP1 inflammasome originated as early as in teleost fish. This finding not only gives insights into the evolutionary history of inflammasomes but also provides a favorable animal model for the study of NLRP1 inflammasome-mediated immunology and diseases.

Cbfß governs osteoblast-adipocyte lineage commitment through enhancing ß-catenin signaling and suppressing adipogenesis gene expression.

The mechanism underlying how transcription factors regulate mesenchymal stem cell lineage commitment remains unclear. To determine the role of core-binding factor subunit beta (Cbfß) in osteoblast lineage commitment, we generated three mouse models by deleting Cbfß at different osteoblast lineage stages. We demonstrated that the Cbfßf/fPrx1-Cre, Cbfßf/fCol2α1-Cre, and Cbfßf/fOsx-Cre mice exhibited severe osteoporosis with substantial accumulation of marrow adipocytes resembling aged bone from enhanced adipogenesis, indicating that mesenchymal stem cells and osteoblasts can be programed and reprogramed, respectively, into adipocytes. Consistently, Cbfß-deficient calvarial cells and bone marrow mesenchymal stem cells displayed strong adipogenic potential, with 5- to ∼70-fold increased adipocyte gene expression, which can be rescued by Cbfß overexpression. Canonical Wnt signaling was impeded in the Cbfß-deficient cells, with ∼80% decrease of Wnt10b expression. Accordingly, ChIP and luciferase assays demonstrated that Cbfß/RUNX2 binds to Wnt10b promoter driving Wnt10b expression. Furthermore, Wnt3a suppressed adipogenesis but did not rescue osteoblastogenesis in Cbfß-deficient cells. Notably, mixing culture of Cbfß-deficient with normal cells demonstrates that Cbfß functions not only through WNT paracrine pathway but also through endogenous signaling. Further analysis shows that Cbfß/RUNX2 inhibits c/ebpα expression at transcriptional level. Our results show that, besides its osteogenic role, Cbfß governs osteoblast-adipocyte lineage commitment both cell nonautonomously through enhancing ß-catenin signaling and cell autonomously through suppressing adipogenesis gene expression to maintain osteoblast lineage commitment, indicating Cbfß may be a therapeutic target for osteoporosis.