Single-cell RNA sequencing unveils the hidden powers of zebrafish kidney for generating both hematopoiesis and adaptive antiviral immunity.

The vertebrate kidneys play two evolutionary conserved roles in waste excretion and osmoregulation. Besides, the kidney of fish is considered as a functional ortholog of mammalian bone marrow that serves as a hematopoietic hub for generating blood cell lineages and immunological responses. However, knowledge about the properties of kidney hematopoietic cells, and the functionality of the kidney in fish immune systems remains to be elucidated. To this end, our present study generated a comprehensive atlas with 59 hematopoietic stem/progenitor cell (HSPC) and immune-cells types from zebrafish kidneys via single-cell transcriptome profiling analysis. These populations included almost all known cells associated with innate and adaptive immunity, and displayed differential responses to viral infection, indicating their diverse functional roles in antiviral immunity. Remarkably, HSPCs were found to have extensive reactivities to viral infection, and the trained immunity can be effectively induced in certain HSPCs. In addition, the antigen-stimulated adaptive immunity can be fully generated in the kidney, suggesting the kidney acts as a secondary lymphoid organ. These results indicated that the fish kidney is a dual-functional entity with functionalities of both primary and secondary lymphoid organs. Our findings illustrated the unique features of fish immune systems, and highlighted the multifaced biology of kidneys in ancient vertebrates.

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.

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.

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.

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.

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.

Occurrences and Functions of Ly6Chi and Ly6Clo Macrophages in Health and Disease.

Macrophages originating from the yolk sac or bone marrow play essential roles in tissue homeostasis and disease. Bone marrow-derived monocytes differentiate into Ly6Chi and Ly6Clo macrophages according to the differential expression of the surface marker protein Ly6C. Ly6Chi and Ly6Clo cells possess diverse functions and transcriptional profiles and can accelerate the disease process or support tissue repair and reconstruction. In this review, we discuss the basic biology of Ly6Chi and Ly6Clo macrophages, including their origin, differentiation, and phenotypic switching, and the diverse functions of Ly6Chi and Ly6Clo macrophages in homeostasis and disease, including in injury, chronic inflammation, wound repair, autoimmune disease, and cancer. Furthermore, we clarify the differences between Ly6Chi and Ly6Clo macrophages and their connections with traditional M1 and M2 macrophages. We also summarize the limitations and perspectives for Ly6Chi and Ly6Clo macrophages. Overall, continued efforts to understand these cells may provide therapeutic approaches for disease treatment.

Extensive involvement of CD40 and CD154 costimulators in multiple T cell-mediated responses in a perciform fish Larimichthys crocea.

CD40 and CD154 are well-characterized costimulatory molecules involved in adaptive humoral immunity in humans and other mammals. These two costimulatory molecules were found to be originated from teleost fish during vertebrate evolution. However, the functionality of fish CD40 and CD154 remains to be explored. In this study, we identified the CD40 and CD154 homologs (LcCD40 and LcCD154) from large yellow croaker (Larimichthys crocea), a marine species of the perciform fish family. The LcCD40 and LcCD154 share conserved structural features to their mammalian counterparts, and are widely expressed in immune-relevant tissues and leukocytes at different transcriptional levels. Immunofluorescence staining and FCM analysis showed that LcCD40 and LcCD154 proteins are distributed on MHC-II+ APCs and CD4-2+ T cells, and are significantly upregulated in response to antigen stimulation. Co-IP assay exhibited strong association between LcCD40 and LcCD154 proteins. Blockade of LcCD154 with anti-LcCD154 antibody (Ab) or recombinant soluble LcCD40-Ig fusion protein remarkably decreased the MHC-II+ APC-initiated CD4+ T cell response upon Aeromonas hydrophila stimulation, and alloreactive T cell activation as examined by mixed lymphocyte reaction (MLR). These findings highlight the costimulatory role of LcCD40 and LcCD154 in T cell activities in Larimichthys crocea. Thus, the CD40 and CD154 costimulators may extensively participate in the regulation of multiple T cell-mediated immune responses in teleost fish. It is anticipated that this study would provide a cross-species understanding of the evolutionary history of CD40 and CD154 costimulatory signals from fish to mammals.

Essential Role of RIG-I in Hematopoietic Precursor Emergence in Primitive Hematopoiesis during Zebrafish Development.

Retinoic acid-inducible gene I (RIG-I) is an important cytosolic pattern recognition receptor crucial for sensing RNA virus infection and initiating innate immune responses. However, the participation of RIG-I in cellular development under physiological conditions remains limited. In this study, the regulatory role of RIG-I in embryonic hematopoiesis was explored in a zebrafish model. Results showed that rig-I was ubiquitously expressed during embryogenesis at 24 h postfertilization (hpf). A defect in RIG-I remarkably disrupted the emergence of primitive hematopoietic precursors and subsequent myeloid and erythroid lineages. In contrast, RIG-I deficiency did not have an influence on the generation of endothelial precursors and angiogenesis and the development of mesoderm and adjacent tissues. The alteration in these phenotypes was confirmed by whole-mount in situ hybridization with lineage-specific markers. In addition, immunostaining and TUNEL assays excluded the abnormal proliferation and apoptosis of hematopoietic precursors in RIG-I-deficient embryos. Mechanistically, RIG-I regulates primitive hematopoiesis through downstream IFN signaling pathways, as shown by the decline in ifnφ2 and ifnφ3 expression, along with rig-I knockdown, and rescue of the defects of hematopoietic precursors in RIG-I-defective embryos after administration with ifnφ2 and ifnφ3 mRNAs. Additionally, the defects of hematopoietic precursors in RIG-I morphants could be efficiently rescued by the wild-type RIG-I but could not be restored by the RNA-binding-defective RIG-I with site mutations at the RNA-binding pocket, which are essential for association with RNAs. This finding suggested that endogenous RNAs may serve as agonists to activate RIG-I-modulated primitive hematopoiesis. This study revealed the functional diversity of RIG-I under physiological conditions far beyond that previously known.

LncRNA CASC15, MiR-23b Cluster and SMAD3 form a Novel Positive Feedback Loop to promote Epithelial-Mesenchymal Transition and Metastasis in Ovarian Cancer.

Cancer Susceptibility Candidate 15 (CASC15), which is a newly identified long noncoding RNA crucial for epigenetic regulation in human tumors, was found to be associated with poor prognosis of the patients with ovarian cancer by utilizing The Cancer Genome Atlas and Gene Expression Omnibus database. Therefore, the purpose of this paper was to explore the functional role and latent molecular mechanism of CASC15 in the progression of ovarian cancer. In vitro and in vivo experiments validated CASC15 as an oncogenic lncRNA in ovarian cancer, which could enhance metastasis through TGF-ß-induced epithelial-mesenchymal transition progress. MiR-23b-3p and miR-24-3p, which are members of the miR-23b cluster, were identified to directly target CASC15 through luciferase assays. Further mechanistic investigations indicated that CASC15-mediated miR-23b-3p/miR-24-3p sequestration cooperatively upregulated SMAD3 expression, which, in turn, would permit increased CASC15 mRNA level as a transcription activation factor. This study first described a miR-23b-3p/miR-24-3p-mediated positive feedback loop between CASC15 and SMAD3, which may reflect the underlying molecular mechanism of CASC15's oncogenic function in ovarian cancer.

Regulatory role of BTLA and HVEM checkpoint inhibitors in T cell activation in a perciform fish Larimichthys crocea.

The BTLA and HVEM are two well-characterized immune checkpoint inhibitors in humans and other mammalian species. However, the occurrence and functionality of these two molecules in non-mammalian species remain poorly understood. In the present study, we identified the BTLA and HVEM homologs from large yellow croaker (Larimichthys crocea), an economically important marine species of the perciform fish family. The Larimichthys crocea BTLA and HVEM (LcBTLA and LcHVEM) share conserved structural features to their mammalian counterparts, and they were expressed in various tissues and cells examined at different transcriptional levels, with particular abundance in immune-relevant tissues and splenic leukocytes. Immunofluorescence staining and flow cytometry analysis showed that LcHVEM and LcBTLA proteins were distributed on MHC-II+ APCs and CD4-2+ T cells, and a strong interaction between LcBTLA and LcHVEM was detected in splenic leukocytes in the mixed lymphocyte reaction (MLR). By blockade assays using anti-LcBTLA and anti-LcHVEM Abs as well as recombinant soluble LcBTLA and LcHVEM proteins in different combinations, it was found that LcBTLA-LcHVEM interactions play an important inhibitory role in the activation of alloreactive T cells using MLR as a model, and APC-initiated antigen-specific CD4-2+ T cells in response to A. hydrophila (A. h) stimulation. These observations highlight the extensive functional roles of LcBTLA and LcHVEM immune-checkpoint inhibitors in allogeneic T cell reactions, and CD4-2+ T cell-mediated adaptive immune responses in Larimichthys crocea. Thus, the BTLA-HVEM checkpoint may represent an ancient coinhibitory pathway, which was originated in fish and was conserved from fish to mammals throughout the vertebrate evolution.

Effect of psychological capital and social support on job satisfaction among workers in a natural gas field / 中国职业医学

@#To explore the effects of psychological capital social support and their interaction on job satisfaction in Methods natural gas field workers. A total of 1 473 workers from a natural gas field were selected as the research subjects ， using convenient sampling method. Job Satisfaction Questionnaire Psychological Capital Questionnaire and Social Support ， Results Scale were used to investigate the scores of job satisfaction psychological capital and social support level. The ， ， detection rates of job satisfaction psychological capital and social support in the high level group were 55.9% 52.5% and ， 48.1% respectively. The detection rates of job satisfaction of workers in the high level psychological capital group and high level （ social support group were higher than those in the low level psychological capital group and low level social support group 67.4% vs ， vs ， P ） 43.3% 71.9% 41.2% all <0.01 . The results of multivariate logistic regression analysis showed that psychological capital ［ （CI） （ - ） and social support had positive effects on job satisfaction odds ratio and 95% confidence interval were 1.58 1.17 2.41 （ - ）， ， P ］ ， and 2.53 1.82 3.52 respectively all <0.01 . Moreover the psychological capital and social support had additive effect on ［ CI （ - ）， CI job satisfaction relative excess risk of interaction and 95% was 3.07 1.02 5.12 attributable proportion and 95% was （ - ）， CI （ - ）］， 0.48 0.35 0.61 synergy index and 95% was 2.34 1.72 3.16 but there was no multiplication interaction between （P ）Conclusion psychological capital and social support >0.05 . Psychological capital and social support can positively affect job satisfaction of natural gas workers. There is an additive interaction between psychological capital and social support on job ， satisfaction but no multiplicative interaction is found. Keywords: ； ； ； ； ； Psychological capital Social support Job satisfaction Interaction Natural gas Worker

Genome-Wide Characterization of Zebrafish Endogenous Retroviruses Reveals Unexpected Diversity in Genetic Organizations and Functional Potentials.

Endogenous retroviruses (ERVs) occupy a substantial fraction of mammalian genomes. However, whether ERVs extensively exist in ancient vertebrates remains unexplored. Here, we performed a genome-wide characterization of ERVs in a zebrafish (Danio rerio) model. Approximately 3,315 ERV-like elements (DrERVs) were identified as Gypsy, Copia, Bel, and class I-III groups. DrERVs accounted for approximately 2.3% of zebrafish genome and were distributed in all 25 chromosomes, with a remarkable bias on chromosome 4. Gypsy and class I are the two most abundant groups with earlier insertion times. The vast majority of the DrERVs have varied structural defects. A total of 509 gag and 71 env genes with coding potentials were detected. The env-coding elements were well-characterized and classified into four subgroups. A ERV-E4.8.43-DanRer element shows high similarity with HERV9NC-int in humans and analogous sequences were detected in species spanning from fish to mammals. RNA-seq data showed that hundreds of DrERVs were expressed in embryos and tissues under physiological conditions, and most of them exhibited stage and tissue specificity. Additionally, 421 DrERVs showed strong responsiveness to virus infection. A unique group of DrERVs with immune-relevant genes, such as fga, ddx41, ftr35, igl1c3, and tbk1, instead of intrinsic viral genes were identified. These DrERVs are regulated by transcriptional factors binding at the long terminal repeats. This study provided a survey of the composition, phylogeny, and potential functions of ERVs in a fish model, which benefits the understanding of the evolutionary history of ERVs from fish to mammals. IMPORTANCE Endogenous retroviruses (ERVs) are relics of past infection that constitute up to 8% of the human genome. Understanding the genetic evolution of the ERV family and the interplay of ERVs and encoded RNAs and proteins with host function has become a new frontier in biology. Fish, as the most primitive vertebrate host for retroviruses, is an indispensable integral part for such investigations. In the present study, we report the genome-wide characterization of ERVs in zebrafish, an attractive model organism of ancient vertebrates from multiple perspectives, including composition, genomic organization, chromosome distribution, classification, phylogeny, insertion time, characterization of gag and env genes, and expression profiles in embryos and tissues. The result helps uncover the evolutionarily conserved and fish-specific ERVs, as well as the immune-relevant ERVs in response to virus infection. This study demonstrates the previously unrecognized abundance, diversification, and extensive activity of ERVs at the early stage of ERV evolution.

Mesenchymal stem cells attenuate liver fibrosis by targeting Ly6Chi/lo macrophages through activating the cytokine-paracrine and apoptotic pathways.

Mesenchymal stem cell (MSC) therapy has become a promising treatment for liver fibrosis due to its predominant immunomodulatory performance in hepatic stellate cell inhibition and fibrosis resolution. However, the cellular and molecular mechanisms underlying these processes remain limited. In the present study, we provide insights into the functional role of bone marrow-derived MSCs (BM-MSCs) in alleviating liver fibrosis by targeting intrahepatic Ly6Chi and Ly6Clo macrophage subsets in a mouse model. Upon chronic injury, the Ly6Chi subset was significantly increased in the inflamed liver. Transplantation of BM-MSCs markedly promoted a phenotypic switch from pro-fibrotic Ly6Chi subset to restorative Ly6Clo subpopulation by secreting paracrine cytokines IL-4 and IL-10 from the BM-MSCs. The Ly6Chi/Ly6Clo subset switch significantly blocked the source of fibrogenic TGF-ß, PDGF, TNF-α, and IL-1ß cytokines from Ly6Chi macrophages. Unexpectedly, BM-MSCs experienced severe apoptosis and produced substantial apoptotic bodies in the fibrotic liver during the 72 h period of transplantation. Most apoptotic bodies were engulfed by Ly6Clo macrophages, and this engulfment robustly triggered MMP12 expression for fibrosis resolution through the PtdSer-MerTK-ERK signaling pathway. This paper is the first to show previously unrecognized dual regulatory functions of BM-MSCs in attenuating hepatic fibrosis by promoting Ly6Chi/Ly6Clo subset conversion and Ly6Clo macrophage restoration through secreting antifibrogenic-cytokines and activating the apoptotic pathway.

Inhibitory Role of an Aeromonas hydrophila TIR Domain Effector in Antibacterial Immunity by Targeting TLR Signaling Complexes in Zebrafish.

The Toll/interleukin-1 receptor (TIR) domain is a structural unit responsible for the assembly of signal protein complexes in Toll-like receptor (TLR) and interleukin-1 receptor signaling pathways. TIR domain homologs are found in a considerable number of bacteria and enhance bacterial infection and survival in host organisms. However, whether TIR domain homologs exist in Aeromonas hydrophila, a ubiquitous waterborne bacterium in aquatic environments, remains poorly understood. In this study, a TIR domain protein (TcpAh) was identified from A. hydrophila JBN2301. TIR domain of TcpAh is highly homologous to the counterpart domains in TLRs and myeloid differentiation factor 88 (MyD88). The zebrafish infected with mutant A. hydrophila with tcpAh deletion had a remarkably lower mortality than those infected with the wild-type strain. This result suggests that TcpAh is a crucial virulence factor for A. hydrophila infection. TcpAh exhibited a strong ability to associate with MyD88, tumor necrosis factor receptor-associated factor 3 (TRAF3) and TRAF-associated NF-κB activator-binding kinase 1 (TBK1) in TIR-TIR, TIR-Death domain (DD), and other alternative interactions. This finding suggests that TcpAh extensively interferes with MyD88 and TIR domain-containing adapter inducing interferon (IFN)-ß (TRIF) signaling pathways downstream of TLRs. Consequently, CD80/86 expression was suppressed by TcpAh via attenuating TLR-stimulated NF-κB activation, which ultimately led to the impairment of the major costimulatory signal essential for the initiation of adaptive humoral immunity against A. hydrophila infection. We believe that this study is the first to show a previously unrecognized mechanism underlying A. hydrophila evades from host antibacterial defense by intervening CD80/86 signal, which bridges innate and adaptive immunity. The mechanism will benefit the development of therapeutic interventions for A. hydrophila infection and septicemia by targeting TcpAh homologs.

A phosphatidic acid-binding lncRNA SNHG9 facilitates LATS1 liquid-liquid phase separation to promote oncogenic YAP signaling.

Long noncoding RNAs (lncRNAs) are emerging as a new class of important regulators of signal transduction in tissue homeostasis and cancer development. Liquid-liquid phase separation (LLPS) occurs in a wide range of biological processes, while its role in signal transduction remains largely undeciphered. In this study, we uncovered a lipid-associated lncRNA, small nucleolar RNA host gene 9 (SNHG9) as a tumor-promoting lncRNA driving liquid droplet formation of Large Tumor Suppressor Kinase 1 (LATS1) and inhibiting the Hippo pathway. Mechanistically, SNHG9 and its associated phosphatidic acids (PA) interact with the C-terminal domain of LATS1, promoting LATS1 phase separation and inhibiting LATS1-mediated YAP phosphorylation. Loss of SNHG9 suppresses xenograft breast tumor growth. Clinically, expression of SNHG9 positively correlates with YAP activity and breast cancer progression. Taken together, our results uncover a novel regulatory role of a tumor-promoting lncRNA (i.e., SNHG9) in signal transduction and cancer development by facilitating the LLPS of a signaling kinase (i.e., LATS1).

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.

Functional role of CD40 and CD154 costimulatory signals in IgZ-mediated immunity against bacterial infection.

CD40 and CD154 are one of the best-characterized costimulatory molecules essential for adaptive immunity, which extensively involved in T and B cell activation, IgM Ab production, isotype class switching, germinal center formation and affinity maturation. However, the functionality of CD40 and CD154 in IgZ-mediated immunity remains limited. In this study, we explored the regulatory role of Cd40-Cd154 interaction in IgZ-mediated antibacterial immunity in zebrafish. The results showed that the IgZ-mediated antibacterial response can be significantly induced in response to A. hydrophila infection. The percentage of Cd40+IgZ+ B cells and the production of IgZ Ab were substantially increased upon A. hydrophila stimulation, but these reactions were markedly declined in Cd154 blockade fish by administering anti-Cd154 Ab or recombinant sCd40-Ig protein, accompanied with the impairment of the vaccine-initiated IgZ-mediated immunoprotection of fish against A. hydrophila infection. These observations suggested the essential role of Cd40-Cd154 interaction in IgZ-mediated bacterial immunity. Notably, the Cd40 and Cd154 costimulatory signals are required for a TD antigen-induced IgZ immunity, but are not indispensable for a TI antigen-induced IgZ immune response. These findings indicated the differential role of Cd40-Cd154 interaction in bacterial TD and TI antigen-induced IgZ immunity, which suggested the existence of diverse regulatory mechanisms underlying IgZ-mediated antibacterial immune reactions. To our knowledge, this is the first report to show the functional role of Cd40-Cd154 costimulatory signaling pathway in IgZ-mediated immune defense against bacterial infection. We hope this study will improve the current understanding of the coevolution between the IgZ/IgT immunoglobins and CD40/CD154 costimulatory molecules.

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.