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Perforin, produced by natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), is one of the effectors of cell-mediated cytotoxicity (CMC) in vertebrates, playing a paramount role in killing target cells. However, whether and how perforin is involved in adaptive immune responses in early vertebrates remains unclear. Using Nile tilapia (Oreochromis niloticus) as a model, we investigated the characteristics of perforin in early vertebrates. Oreochromis niloticus perforin (OnPRF) possesses 2 conserved functional domains, membrane attack complex/perforin (MACPF) and protein kinase C conserved region 2 (C2) domains, although they share low amino acid sequence similarity with other homologs. OnPRF was widely expressed in various immune tissues and could respond to lymphocyte activation and T-cell activation in vitro at both the transcriptional and protein levels, indicating that it may be involved in adaptive immune responses. Furthermore, after infection with Edwardsiella piscicida and Aeromonas hydrophila, the mRNA and protein levels of OnPRF were significantly up-regulated within the adaptive immune response period. Additionally, we revealed that many transcription factors were involved in the transcriptional regulation of OnPRF, including p65, c-Fos, c-Jun, STAT1 and STAT4, and there was a synergy among these transcription factors. Overall, these findings demonstrate the involvement of OnPRF in T-cell activation and adaptive immune response in tilapia, thus providing new evidence for comprehending the evolution of immune response in early vertebrates., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Nile tilapia (Oreochromis niloticus) is one of the important economic fish species cultured worldwide. However, Streptococcus agalactiae has emerged as a significant bacterial threat, severely impacting the economy of tilapia industry. The immune response underlying the resistance of tilapia to S. agalactiae are not well understood, hindering the reasonable evaluation of breeding and the formulation of effective strategies. In this study, we investigated the differences in T-cell immunity between S. agalactiae-resistant and -susceptible tilapia. Compared with susceptible tilapia, resistant tilapia exhibited a higher percentage of T cells and BrdU + T cells during infection, indicating a superior proliferative capacity. Whether infected or not, T cells from resistant fish demonstrated a greater ability to resist apoptosis. Additionally, T cell effector genes, including interleukin (IL)-2, interferon (IFN)-γ, perforin A, and granzyme B were expressed at higher levels in resistant tilapia after infection. Along with these T-cell immune responses, resistant fish showed more effective clearance of infection. Our study elucidates the T-cell immune responses in resistant tilapia, which may contribute to the high resistance of tilapia to S. agalactiae, and provide valuable theoretical references for the selection and evaluation of disease-resistant fish strains in the future., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Secreted by natural killer cells and cytotoxic T lymphocytes, Granzyme B is involved in regulating the adaptive immune response in vertebrates and plays a pivotal role in resisting virus invasion and removing pathogens. Although it had been extensively studied in mammals, the involvement of Granzyme B in adaptive immune response of early vertebrates remained elusive. In this study, we investigated the Granzyme B in Oreochromis niloticus (OnGrB), found that its function domain was conserved. Additionally, OnGrB was widely expressed in various tissues and could respond to T-cell activation in vitro at the transcriptional level. Furthermore, we prepared the recombinant OnGrB (rOnGrB) as an immunogen to develop a mouse anti-OnGrB monoclonal antibody (mAb). Using this anti-OnGrB mAb as a tool, we explored the expression of OnGrB in the adaptive immune response of tilapia. Our findings revealed that T cell was a significant source of OnGrB production, the expression of OnGrB at the protein level and the proportion of OnGrB  +  T cells increased after both T cell activation in vitro and infection with Edwardsiella piscicida in vivo. More importantly, our findings also preliminarily illuminated that p65 could regulate the transcriptional activity of OnGrB. These results indicated that OnGrB was involved in the adaptive immunity of tilapia and played a critical role in T cell function in teleost. Our study provided theoretical support and new perspectives for understanding adaptive immunity in teleost., (Copyright © 2024. Published by Elsevier Ltd.)

As one of subunits for interleukin-2 receptor (IL-2R), CD122 can bind to IL-2 and then activate downstream signal transduction to participate in adaptive immune response. Although CD122 has been identified and investigated from several teleost species, studies on its function at T-cell level are still scarce for lack of specific antibodies. In this study, a typical CD122 in Nile tilapia (Oreochromis niloticus) was characterized by bioinformatics analysis, cloned to produce retrovirus infected NIH/3T3 cells for mouse immunization. After cell fusion and screening, we successfully developed a mouse anti-tilapia CD122 monoclonal antibody (mAb), which could specifically recognize CD122 and identify CD122-producing T cells of tilapia. Using the mAb to detect, CD122 was found to widely distribute in immune-related tissues, and significantly elevate post Edwardsiella piscicida infection or T-cell activation. More importantly, the expansion of CD122 + T cells and up-regulation of CD122 occurred both in total T cells and T-cell subsets during T-cell activation upon in vitro stimulation or in vivo infection. These results indicate that CD122 can be used as a T-cell activation marker in tilapia. Notably, CD122 mAb blocking blunted the activation of MAPK/Erk and mTORC1 pathways, and inhibited T-cell proliferation, suggesting a critical role of CD122 in ensuring proper proliferation of tilapia T cells. Therefore, this study enriches the knowledge of T-cell responses in fish and provides new evidence for understanding the evolution of lymphocyte-mediated adaptive immunity., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The transforming growth factor beta-activated kinase 1 (TAK1)/c-Jun N-terminal kinase (JNK) axis is an essential MAPK upstream mediator and regulates immune signaling pathways. However, whether the TAK1/JNK axis harnesses the strength in regulation of signal transduction in early vertebrate adaptive immunity is unclear. In this study, by modeling on Nile tilapia (Oreochromis niloticus), we investigated the potential regulatory function of TAK1/JNK axis on lymphocyte-mediated adaptive immune response. Both OnTAK1 and OnJNK exhibited highly conserved sequences and structures relative to their counterparts in other vertebrates. Their mRNA was widely expressed in the immune-associated tissues, while phosphorylation levels in splenic lymphocytes were significantly enhanced on the 4th day post-infection by Edwardsiella piscicida. In addition, OnTAK1 and OnJNK were significantly up-regulated in transcriptional level after activation of lymphocytes in vitro by phorbol 12-myristate 13-acetate plus ionomycin (P + I) or PHA, accompanied by a predominant increase in phosphorylation level. More importantly, inhibition of OnTAK1 activity by specific inhibitor NG25 led to a significant decrease in the phosphorylation level of OnJNK. Furthermore, blocking the activity of OnJNK with specific inhibitor SP600125 resulted in a marked reduction in the expression of T-cell activation markers including IFN-γ, CD122, IL-2, and CD44 during PHA-induced T-cell activation. In summary, these findings indicated that the conserved TAK1/JNK axis in Nile tilapia was involved in adaptive immune responses by regulating the activation of lymphocytes. This study enriched the current knowledge of adaptive immunity in teleost and provided a new perspective for understanding the regulatory mechanism of fish immunity., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Despite the advances in study on osmotic physiology in bony fish, the mechanism by which the immune system, especially T-cell immunity, adapts and responds to osmotic stress remains unknown. In the current study, we investigated the response of T cells to hyperosmotic stress in the bony fish Nile tilapia (Oreochromis niloticus). As a euryhaline fish, tilapia was able to adapt to a wide range of salinities; however, hypertonic stress caused inflammation and excessive T-cell activation. Furthermore, hypertonic stress increased the expression of IL-17A in T cells, upregulated the transcription factor RORα, and activated STAT3 signaling, along with IL-6- and TGF-β1-mediated pathways, revealing an enhanced Th17 response in this early vertebrate. These hypertonic stress-induced events collectively resulted in an impaired antibacterial immune response in tilapia. Hypertonic stress elevated the intracellular ROS level, which in turn activated the p38-MK2 signaling pathway to promote IL-17A production by T cells. Both ROS elimination and the p38-MK2 axis blockade diminished the increased IL-17A production in T cells under hypertonic conditions. Moreover, the produced proinflammatory cytokines further amplified the hypertonic stress signaling via the MKK6-p38-MK2 axis-mediated positive feedback loop. To our knowledge, these findings represent the first description of the mechanism by which T-cell immunity responds to hypertonic stress in early vertebrates, thus providing a novel perspective for understanding the adaptive evolution of T cells under environmental stress., (Copyright © 2024 by The American Association of Immunologists, Inc.)

As a multipotent cytokine, interleukin (IL)-2 plays important roles in activation, differentiation and survival of the lymphocytes. Although biological characteristics and function of IL-2 have been clarified in several teleost species, evidence regarding IL-2 production at the cellular and protein levels is still scarce in fish due to the lack of reliable antibody. In this study, we developed a mouse anti-Nile tilapia IL-2 monoclonal antibody (mAb), which could specifically recognize IL-2 protein and identify IL-2-producing lymphocytes of tilapia. Using this mAb, we found that CD3 + T cells, but not CD3 - lymphocytes, are the main cellular source of IL-2 in tilapia. Under resting condition, both CD3 + CD4-1 + T cells and CD3 + CD4-1 - T cells of tilapia produce IL-2. Moreover, the IL-2 protein level and the frequency of IL-2 + T cells significantly increased once T cells were activated by phytohemagglutinin (PHA) or CD3 plus CD28 mAbs in vitro. In addition, Edwardsiella piscicida infection also induces the IL-2 production and the expansion of IL-2 + T cells in the spleen lymphocytes. These findings demonstrate that IL-2 takes part in the T-cell activation and anti-bacterial adaptive immune response of tilapia, and can serve as an important marker for T-cell activation of teleost fish. Our study has enriched the knowledge regarding T-cell response in fish species, and also provide novel perspective for understanding the evolution of adaptive immune system., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

As an immune checkpoint, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) suppresses the activation, proliferation, and effector function of T cells, thus preventing an overexuberant response and maintaining immune homeostasis. However, whether and how this immune checkpoint functions in early vertebrates remains unknown. In the current study, using a Nile tilapia (Oreochromis niloticus) model, we investigated the suppression of T cell response by CTLA-4 in bony fish. Tilapia CTLA-4 is constitutively expressed in lymphoid tissues, and its mRNA and protein expression in lymphocytes are upregulated following PHA stimulation or Edwardsiella piscicida infection. Blockade of CTLA-4 signaling enhanced T cell activation and proliferation but inhibited activation-induced T cell apoptosis, indicating that CTLA-4 negatively regulated T cell activation. In addition, blocking CTLA-4 signaling in vivo increased the differentiation potential and cytotoxicity of T cells, resulting in an enhanced T cell response during E. piscicida infection. Tilapia CTLA-4 competitively bound the B7.2/CD86 molecule with CD28, thus antagonizing the CD28-mediated costimulatory signal of T cell activation. Furthermore, inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling, c-Myc, or glycolysis markedly impaired the CTLA-4 blockade-enhanced T cell response, suggesting that CTLA-4 suppressed the T cell response of tilapia by inhibiting mTORC1/c-Myc axis-controlled glycolysis. Overall, the findings indicate a detailed mechanism by which CTLA-4 suppresses T cell immunity in tilapia; therefore, we propose that early vertebrates have evolved sophisticated mechanisms coupling immune checkpoints and metabolic reprogramming to avoid an overexuberant T cell response., (Copyright © 2024 by The American Association of Immunologists, Inc.)

The van der Waals (vdW) heterostructures of Z-scheme PbI 2 /g-C 3 N 4 with an indirect bandgap have gained much attention in recent years due to their unique properties and potential applications in various fields. However, the optoelectronic characteristics and strain-modulated effects are not yet fully understood. By considering this, six stacking models of PbI 2 /g-C 3 N 4 are proposed and the stablest structure is selected for further investigation. The uniaxial and biaxial strains (-10%-10%) regulated band arrangement, charge distribution, optical absorption in the framework of density functional theory are systematically explored. The compressive uniaxial strain of -8.55% changes the band type from II→I, and the biaxial strains of -7.12%, -5.25%, 8.91% change the band type in a way of II→I→II→I, acting like the 'band-pass filter'. The uniaxial strains except -10% compressive strain, and the -6%, -4%, 2%, 4%, 10% biaxial strains will enhance the light absorption of PbI 2 /g-C 3 N 4 . The exerted strains on PbI 2 /g-C 3 N 4 generate different power conversion efficiency (ηPCE) values ranging from 3.64% to 25.61%, and the maximumηPCEis generated by -6% biaxial strain. The results of this study will pave the way for the development of new electronic and optoelectronic materials with customized properties in photocatalytic field and optoelectronic devices., (© 2024 IOP Publishing Ltd.)

Mammalian T-cell responses require synergism between the first signal and co-stimulatory signal. However, whether and how dual signaling regulates the T-cell response in early vertebrates remains unknown. In the present study, we discovered that the Nile tilapia ( Oreochromis niloticus ) encodes key components of the LAT signalosome, namely, LAT, ITK, GRB2, VAV1, SLP-76, GADS, and PLC-γ1. These components are evolutionarily conserved, and CD3ε mAb-induced T-cell activation markedly increased their expression. Additionally, at least ITK, GRB2, and VAV1 were found to interact with LAT for signalosome formation. Downstream of the first signal, the NF-κB, MAPK/ERK, and PI3K-AKT pathways were activated upon CD3ε mAb stimulation. Furthermore, treatment of lymphocytes with CD28 mAbs triggered the AKT-mTORC1 pathway downstream of the co-stimulatory signal. Combined CD3ε and CD28 mAb stimulation enhanced ERK1/2 and S6 phosphorylation and elevated NFAT1, c-Fos, IL-2, CD122, and CD44 expression, thereby signifying T-cell activation. Moreover, rather than relying on the first or co-stimulatory signal alone, both signals were required for T-cell proliferation. Full T-cell activation was accompanied by marked apoptosis and cytotoxic responses. These findings suggest that tilapia relies on dual signaling to maintain an optimal T-cell response, providing a novel perspective for understanding the evolution of the adaptive immune system.

Aim: The management of uterine prolapse poses a significant clinical challenge, with surgical intervention often necessary for symptom relief and restoration of pelvic floor function. However, the optimal surgical approach for uterine prolapse remains uncertain, prompting a comprehensive meta-analysis to compare the efficacy of various surgical methods. This study aims to assess the effectiveness of different surgical methods for treating uterine prolapse., Methods: We used computer search to retrieve relevant literature to compare the therapeutic effects of different surgical methods for treating uterine prolapse. The search was conducted in the Web of Science and PubMed databases, and articles published until October 2023 were obtained. We employed random effects and fixed effects models and performed a meta-analysis using the R software., Results: This study included 40 standard papers covering 25,896 patients with uterine prolapse. We used random and fixed effects models to conduct a meta-analysis of hysterectomy and uterine fixation procedures. The findings indicated that different surgical approaches had no significant impact on surgical success rates (I2 = 69%, p < 0.01; risk ratio (RR) (95% confidence intervals (CI)): 1.00 [0.98; 1.03]) or postoperative adverse reactions (I2 = 54%, p < 0.01; RR (95% CI), 1.10 [0.83; 1.45]). However, the durations of the surgical procedure for hysterectomy (I2 = 91%, p < 0.01; standardized mean difference (SMD) (95% CI), 0.78 [0.49; 1.07]), surgical blood loss (I2 = 97%, p < 0.01, SMD (95% CI): 1.14 [0.21; 2.07]), and intraoperative adverse reactions (I2 = 0%, p = 0.61, RR (95% CI): 1.37 [1.10; 1.71]) were statistically significant between hysterectomy and uterine fixation procedures. Additionally, publication bias and sensitivity tests showed no publication bias in this meta-analysis and no literature causing significant sensitivity., Conclusions: In the treatment of uterine prolapse, both hysterectomy and uterine fixation are similar in terms of surgical success rates and postoperative adverse reactions. However, hysterectomy is associated with longer duration of the surgical procedure, increased blood loss and higher incidence of intraoperative adverse reactions compared to uterine fixation.

Highly efficient catalysts for the oxygen evolution/reduction reaction (OER/ORR) have attracted great attention in research for energy devices with high conversion efficiency. Herein, systematic first-principles investigations are performed to explore the catalytic performance of graphitic C 4 N 3 loaded with single transition metal atoms (TM@g-t-C 4 N 3 ) for the OER/ORR. The results show that Fe, Co, Ni and Rh@g-t-C 4 N 3 exhibit fascinating bifunctional catalytic activities for both the OER and ORR. Moreover, it is observed that better activities are easily achieved when the valence d orbitals of doped TM atoms are nearly fully occupied. Further analysis reveals the volcano relationship between the OER/ORR performance and the adsorption Gibbs free energy. The adsorption free energy of intermediates in the OER/ORR process is also found to highly correlate with the electronic structures of TM@g-t-C 4 N 3 , which are mainly characterized by two quantities, one is the descriptor φ related to the electronegativity and the number of valence electrons in d orbitals, and the other is the projected d band center. The results indicate that it is possible to predict the catalytic performance of TM@g-t-C 4 N 3 by a detailed examination of the electronic properties of the doped TM atoms to some extent. This research not only provides several highly active g-t-C 4 N 3 -based single-atom catalysts (SACs) for the OER/ORR, but also reveals some potential regularities of SAC systems.

Immunosuppressive cytokines are a class of cytokines produced by immune cells and certain non-immune cells that have a suppressive effect on immune function. Currently known immunosuppressive cytokines include interleukin (IL)-10, transforming growth factor beta (TGF-β), IL-35, and IL-37. Although latest sequencing technologies have facilitated the identification of immunosuppressive cytokines in fish, IL-10 and TGF-β were the most well-known ones that have been widely studied and received continuous attention. Fish IL-10 and TGF-β have been identified as anti-inflammatory and immunosuppressive factors, acting on both innate and adaptive immune systems. However, unlike mammals, teleost fish underwent a third or fourth whole-genome duplication event, which significantly expanded the gene family associated with the cytokine signaling pathway, making the function and mechanism of these molecules need further investigation. In this review, we summarize the advances of studies on fish immunosuppressive cytokines IL-10 and TGF-β since their identification, mainly focusing on production, signaling transduction, and effects on the immunological function. This review aims to expand the understanding of the immunosuppressive cytokine network in fish., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The indirect immunofluorescence assay (IIFA) on HEp-2 cells has been widely used for screening anti-nuclear antibodies (ANA) that are associated with systemic autoimmune rheumatic diseases (SARD). Sera containing ANA display multiple distinct fluorescence patterns on HEp-2 cells. Among them, a dense fine speckled (DFS) pattern caused by anti-DFS70 antibodies has been reported to have higher prevalence in healthy individuals than in patients with SARD. This DFS pattern is often difficult to distinguish amongst other SARD-associated ANA patterns, in particular a mixed homogeneous and speckled pattern. Furthermore, a strong DFS pattern can mask other SARD-associated patterns. Hence, we developed a novel immunoprecipitation method using magnetic beads to remove anti-DFS70 antibodies in serum prior to running IIFA. We also aimed to confirm the presence of anti-DFS70 and to uncover any SARD-associated ANA patterns masked by a strong DFS pattern. The sera used in our study were from 70 individuals who had routine ANA screen, of which 35 sera had an isolated DFS pattern with monospecific anti-DFS70 antibodies confirmed by a complementary assay, and 35 were control sera without a DFS pattern. An immunoprecipitation method using magnetic beads coated with recombinant human full length DFS70 protein was developed. The performance of this new method was evaluated in comparison to an immunoadsorption method using the same DFS70 protein. Our newly developed immunoprecipitation method demonstrated excellent sensitivity (91.4%) and specificity (100%) in confirming the DFS pattern associated with anti-DFS70 in sera. Additionally, our method was able to remove anti-DFS70 and uncover SARD-associated ANA patterns masked by a strong DFS pattern. It also showed a clearer background on IIFA than that of the immunoadsorption method. The novel magnetic bead-based immunoprecipitation method demonstrated satisfactory performance in removing anti-DFS70 without interfering with the detection of other antibodies. It can be easily integrated with IIFA to confirm anti-DFS70 associated DFS pattern. Additionally, it can simultaneously unmask other ANA patterns, which cannot be achieved by a conventional protocol that requires a complementary anti-DFS70 specific assay to be performed. Therefore, the novel method provides a more effective and accurate solution for ANA screening., (Copyright © 2023 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

As a pleiotropic cytokine consisting of IL-12p35 and IL-12p40, Interleukin-12 (IL-12) features in inflammation regulation and anti-bacterial immunity. While IL-12 homologs have been identified in non-mammalian species, the precise mechanisms by which IL-12 contributes to early adaptive immune responses in vertebrates remain incompletely understood. Herein, an evolutionary conserved Oreochromis niloticus IL-12 (defined as OnIL-12) was identified by synteny characterization, structural comparisons and phylogenetic pattern of IL-12p35b and IL-12p40a. IL-12p35b and IL-12p40a exhibited widespread expression in lymphoid-related tissues of tilapia, while their mRNA expression in head-kidney demonstrated a significant increase after Edwardsiella piscicida infection. Compared with other lymphocytes, recombinant OnIL-12 (rOnIL-12) displayed stronger affinity binding to T cells. Although stimulation of lymphocytes with the p35b or p40a subunit resulted in a significant induction of IFN-γ expression, rOnIL-12 showed stronger potential to promote IFN-γ expression than these subunits. rOnIL-12 not only elevated the mRNA expression level Th1 cell-associated transcription factor T-bet in lymphocytes, but also increased the proportion of CD4-1 + IFN-γ + lymphocytes. Moreover, the mRNA and phosphorylation levels of STAT1, STAT3, STAT4 and STAT5 were enhanced by rOnIL-12. These findings will offer previous evidence for further exploration into the regulatory mechanisms of Th1 cellular immunity in early vertebrates., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Recent advances highlight a key role of transient fasting in optimizing immunity of human and mouse. However, it remains unknown whether this strategy is independently acquired by mammals during evolution or instead represents gradually evolved functions common to vertebrates. Using a tilapia model, we report that T cells are the main executors of the response of the immune system to fasting and that dietary restriction bidirectionally modulates T cell immunity. Long-term fasting impaired T cell immunity by inducing intense autophagy, apoptosis, and aberrant inflammation. However, transient dietary restriction triggered moderate autophagy to optimize T cell response by maintaining homeostasis, alleviating inflammation and tissue damage, as well as enhancing T cell activation, proliferation and function. Furthermore, AMPK is the central hub linking fasting and autophagy-controlled T cell immunity in tilapia. Our findings demonstrate that dietary restriction to optimize immunity is an ancient strategy conserved in vertebrate evolution, providing novel perspectives for understanding the adaptive evolution of T cell response., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

As the lowest organisms possessing T cells, fish are instrumental for understanding T cell evolution and immune defense in early vertebrates. This study established in Nile tilapia models suggests that T cells play a critical role in resisting Edwardsiella piscicida infection via cytotoxicity and are essential for IgM + B cell response. CD3 and CD28 monoclonal antibody crosslinking reveals that full activation of tilapia T cells requires the first and secondary signals, while Ca 2+ -NFAT, MAPK/ERK, NF-κB, and mTORC1 pathways and IgM + B cells collectively regulate T cell activation. Thus, despite the large evolutionary distance, tilapia and mammals such as mice and humans exhibit similar T cell functions. Furthermore, it is speculated that transcriptional networks and metabolic reprogramming, especially c-Myc-mediated glutamine metabolism triggered by mTORC1 and MAPK/ERK pathways, underlie the functional similarity of T cells between tilapia and mammals. Notably, tilapia, frogs, chickens, and mice utilize the same mechanisms to facilitate glutaminolysis-regulated T cell responses, and restoration of the glutaminolysis pathway using tilapia components rescues the immunodeficiency of human Jurkat T cells. Thus, this study provides a comprehensive picture of T cell immunity in tilapia, sheds novel perspectives for understanding T cell evolution, and offers potential avenues for intervening in human immunodeficiency., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

As a pleiotropic cytokine in the interleukin (IL)-12 family, IL-27β plays a significant role in regulating immune cell responses, eliminating invading pathogens, and maintaining immune homeostasis. Although non-mammalian IL-27β homologs have been identified, the mechanism of whether and how it is involved in adaptive immunity in early vertebrates remains unclear. In this study, we identified an evolutionarily conserved IL-27β (defined as OnIL-27β) from Nile tilapia ( Oreochromis niloticus ), and explored its conserved status through gene collinearity, gene structure, functional domain, tertiary structure, multiple sequence alignment, and phylogeny analysis. IL-27β was widely expressed in the immune-related tissues/organ of tilapia. The expression of OnIL-27β in spleen lymphocytes increased significantly at the adaptive immune phase after Edwardsiella piscicida infection. OnIL-27β can bind to precursor cells, T cells, and other lymphocytes to varying degrees. Additionally, IL-27β may be involved in lymphocyte-mediated immune responses through activation of Erk and JNK pathways. More importantly, we found that IL-27β enhanced the mRNA expression of the Th1 cell-associated cytokine IFN-γ and the transcription factor T-bet. This potential enhancement of the Th1 response may be attributed to the activation of the JAK1/STAT1/T-bet axis by IL-27β, as it induced increased transcript levels of JAK1, STAT1 but not TYK2 and STAT4. This study provides a new perspective for understanding the origin, evolution and function of the adaptive immune system in teleost., Competing Interests: The authors have stated explicitly that there are no conflicts of interest in connection with this article., (© 2023 The Authors. Published by Elsevier Ltd.)

Transforming growth factor-β1 (TGF-β1) can suppress the activation, proliferation, and function of many T-cell subsets, protecting organisms from inflammatory and autoimmune disease caused by an overexuberant immune response. However, whether and how TGF-β1 regulates T-cell immunity in early vertebrates remain unknown. Here, using a Nile tilapia (Oreochromis niloticus) model, we investigated suppression of the T-cell response by TGF-β1 in teleost species. Tilapia encodes an evolutionarily conserved TGF-β1, the expression of which in lymphocytes is significantly induced during the immune response following Edwardsiella piscicida infection. Once activated, tilapia T cells increase TGF-β1 production, which in turn suppresses proinflammatory cytokine expression and inhibits T-cell activation. Notably, we found administration of TGF-β1 cripples the proliferation of tilapia T cells, reduces the potential capacity of Th1/2 differentiation, and impairs the cytotoxic function, rendering the fish more vulnerable to bacterial infection. Mechanistically, TGF-β1 initiates the TGF-βR/Smad signaling pathway and triggers the phosphorylation and nuclear translocation of Smad2/3. Smad3 subsequently interacts with several transcriptional partners to repress transcription of cytokines IL-2 and IFN-γ but promote transcription of immune checkpoint regulator CTLA4 and transcription factor Foxp3. Furthermore, TGF-β1/Smad signaling further utilizes Foxp3 to achieve the cascade regulation of these T-cell genes. Taken together, our findings reveal a detailed mechanism by which TGF-β1 suppresses the T cell-based immunity in Nile tilapia and support the notion that TGF-β1 had already been employed to inhibit the T-cell response early in vertebrate evolution, thus providing novel insights into the evolution of the adaptive immune system., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The braking mechanisms to protect the host from tissue damage and inflammatory disease caused by an overexuberant immune response are common in many T cell subsets. However, the negative regulation of T cell responses and detailed mechanisms are not well understood in early vertebrates. In the current study, using a Nile tilapia (Oreochromis niloticus) model, we investigated the suppression of T cell immunity by IL-10. Tilapia encodes an evolutionarily conserved IL-10, whose expression in lymphocytes is markedly induced during the primary adaptive immune response against Aeromonas hydrophila infection. Activated T cells of tilapia produce IL-10, which in turn inhibits proinflammatory cytokine expression and suppresses PHA-induced T cell activation. Moreover, administration of IL-10 impairs the proliferation of tilapia T cells, reduces their potential to differentiate into Th subsets, and cripples the cytotoxic function, rendering the animals more vulnerable to pathogen attack. After binding to its receptor IL-10Ra, IL-10 activates the JAK1/STAT3 axis by phosphorylation and enhances the expression of the suppressor of cytokine signaling 3 (SOCS3), which in turn attenuates the activation of the NF-κB and MAPK/ERK signaling pathways, thus suppressing the T cell response of tilapia. Our findings elucidate a negative regulatory mechanism of T cell immunity in a fish species and support the notion that the braking mechanism of T cells executed through IL-10 existed prior to the divergence of the tetrapod lineage from teleosts. Therefore, this study, to our knowledge, provides a novel perspective on the evolution of the adaptive immune system., (Copyright © 2023 by The American Association of Immunologists, Inc.)

The presence of monospecific dense fine speckled 70 (DFS70) pattern in indirect immunofluorescence assay (IFA) without concomitant systemic autoimmune related diseases (SARD)-associated antibodies could be an exclusion biomarker for SARD. Since interpretation of DFS pattern on IFA is subjective, an assay for confirming the presence of anti-DFS70 is required. We evaluated the performance of two commercial assays [fluorescence enzyme immunoassay (FEIA) and line immunoassay (LIA)] for detecting anti-DFS70. Sera with monospecific DFS (n=176) and without DFS (n=179) pattern from referred patients for ANA testing, in two independent laboratories and healthy donors, were investigated for anti-DFS70 by FEIA (Phadia EliA) and LIA (Euroimmun). The assay performance including sensitivity and specificity at different cut-offs was evaluated, and optimal cut-offs were determined. The newly established optimal cut-offs (2.7 U/mL on EliA, band intensity of 28 on LIA) showed significantly better assay performance in detecting anti-DFS70 and confirming monospecific DFS pattern. A relative sensitivity of 93.7% with relative specificity of 100% on EliA and a relative sensitivity of 96.6% with relative specificity of 95.3% on LIA were achieved. Superior Cohen's Kappa agreements with IFA were also achieved for both assays (0.936 for EliA and 0.906 for LIA). Application of an optimal cut-off can significantly increase the assay performance for anti-DFS70 and enhance the accuracy in reporting DFS pattern by IFA., (Copyright © 2022 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Utilization of specialized Th1 cells to resist intracellular pathogenic infection represents an important innovation of adaptive immunity. Although transcriptional evidence indicates the potential presence of Th1-like cells in some fish species, the existence of CD3+CD4+IFN-γ+ T cells, their detailed functions, and the mechanism determining their differentiation in these early vertebrates remain unclear. In the present study, we identified a population of CD3+CD4-1+IFN-γ+ (Th1) cells in Nile tilapia upon T-cell activation in vitro or Edwardsiella piscicida infection in vivo. By depleting CD4-1+ T cells or blocking IFN-γ, Th1 cells and their produced IFN-γ were found to be essential for tilapia to activate macrophages and resist the E. piscicida infection. Mechanistically, activated T cells of tilapia produce IL-2, which enhances the STAT5 and mTORC1 signaling that in turn trigger the STAT1/T-bet axis-controlled IFN-γ transcription and Th1 cell development. Additionally, mTORC1 regulates the differentiation of these cells by promoting the proliferation of CD3+CD4-1+ T cells. Moreover, IFN-γ binds to its receptors IFNγR1 and IFNγR2 and further initiates a STAT1/T-bet axis-mediated positive feedback loop to stabilize the Th1 cell polarization in tilapia. These findings demonstrate that, prior to the emergence of tetrapods, the bony fish Nile tilapia had already evolved Th1 cells to fight intracellular bacterial infection, and support the notion that IL-2-mTORC1 signaling coordinates the STAT1/T-bet axis to determine Th1 cell fate, which is an ancient mechanism that has been programmed early during vertebrate evolution. Our study is expected to provide novel perspectives into the evolution of adaptive immunity., Competing Interests: The authors have declared that no competing interests exist.

The reduced stress resistance and increased disease risk associated with high-fat diet (HFD) in animals have attracted increasing attention. However, the effects of HFD on adaptive immunity in early vertebrates, especially non-tetrapods, remain unknown. In this study, using Nile tilapia (Oreochromis niloticus) as a model, we investigated the effects of HFD on the primordial T-cell response in fish. Tilapia fed with an HFD for 8 weeks showed impaired lymphocyte homeostasis in the spleen, as indicated by the decreased number of both T and B lymphocytes and increased transcription of proinflammatory cytokines interferon-γ and interleukin-6. Moreover, lymphocytes isolated from HFD-fed fish or cultured in lipid-supplemented medium exhibited diminished T-cell activation in response to CD3ε monoclonal antibody stimulation. Moreover, HFD-fed tilapia infected by Aeromonas hydrophila showed decreased T-cell expansion, increased T-cell apoptosis, reduced granzyme B expression, and impaired infection elimination. Additionally, HFD attenuated adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) activity in tilapia lymphocytes, which in turn upregulated fatty acid synthesis but downregulated fatty acid β-oxidation. Altogether, our results suggest that HFD impairs lymphocyte homeostasis and T cell-mediated adaptive immune response in tilapia, which may be associated with the abnormal lipid metabolism in lymphocytes. These findings thus provide a novel perspective for understanding the impact of HFD on the adaptive immune response of early vertebrates., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

As a pleiotropic cytokine mainly secreted by CD4 + T cells, interleukin (IL)-22 plays an important role in immune regulation and infection elimination. Despite IL-22 homologues have been identified in non-mammal, whether and how IL-22 participates in the adaptive immune response of early vertebrates have not been fully addressed. In this study, we identified an evolutionarily conserved IL-22 from Nile tilapia Oreochromis niloticus (defined as OnIL-22), proved by its properties regarding sequence, gene structure, functional domain, tertiary structure and phylogeny. IL-22 was broadly expressed in lymphoid-related tissues of tilapia, and with relatively higher levels in skin, gill, intestine and liver. The expression of OnIL-22 in spleen lymphocytes was markedly induced at the adaptive immune stage after Streptococcus agalactiae infection. Moreover, once lymphocytes were activated by PMA plus ionomycin or T-cell specific mitogen PHA in vitro, OnIL-22 expression was obviously up-regulated at both mRNA and protein levels. These results thus suggest that activated T cells produce IL-22 to take part in the adaptive immune response of tilapia. Furthermore, treatment of lymphocytes with recombinant OnIL-22 increased the expression of genes related to proliferation and survival, and further promoted the proliferation and reduced the apoptosis of lymphocytes during bacterial infection or T-cell activation. These cellular effects of IL-22 seem to be associated with JAK1/STAT3 axis downstream of IL-22, because IL-22 application not only elevated the mRNA expression of JAK1 and STAT3, but also enhanced their phosphorylation in lymphocytes. Altogether, we suggest that activated T cells produce IL-22 to promote lymphocyte proliferation and survival probability via JAK1/STAT3 signaling pathway, thus participating in adaptive immune response of Nile tilapia. Our study therefore provides helpful perspective for understanding the function and mechanism of adaptive immune system in teleost., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Objectives: Myositis autoantibodies (MAs) were traditionally used as a diagnostic biomarker for idiopathic inflammatory myopathy (IIM). Its clinical utility had recently expanded to include interstitial lung disease (ILD) diagnosis. Depending on the patient cohort, MAs false positives can be common. Correlation between ANA indirect immunofluorescent (IIF) pattern and MAs may improve its positive predictive value (PPV). The aim of our study was to determine the PPV of MAs in IIM and ILD in a real-world patient cohort. We also assessed whether concordance between MAs and ANA IIF pattern can improve the PPV of positive MA results., Methods: Patients with positive MAs and corresponding ANA IIF pattern were identified from Sutherland Centre of Immunology, New South Wales Health Pathology, Australia. The corresponding health records were reviewed to identify each patient's primary diagnosis. χ2 test was used to compare the PPV between MA-ANA concordant and discordant groups., Results: Between January 2016 and July 2019, 118 patients were positive for at least one MA (mean age 66.7 years, 55% female). The most frequently detected autoantibodies were Ro52, anti-synthetase antibodies and PM-Scl. The PPV of MAs for IIM or ILD was 47.4%. The overall concordance rate of MAs and ANA IIF pattern was 70.2%. Patients with concordant MA-ANA results were more likely to have true clinical disease (64.1% vs 17.8%, P <0.001)., Conclusion: Myositis autoantibodies have a low PPV for IIM and ILD in a real-world patient cohort. A positive concordance with ANA IIF pattern can improve MA test accuracy., (© The Author(s) 2022. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Interleukin-2 inducible T cell kinase (ITK) plays a predominant role in the T-cell receptor (TCR) signaling cascade to ensure valid T-cell activation and function. Nevertheless, whether it regulates T-cell response of early vertebrates remains unknown. Herein, we investigated the involvement of ITK in the lymphocyte-mediated adaptive immune response, and its regulation to T-cell activation in the Nile tilapia Oreochromis niloticus. Both sequence and structure of O. niloticus ITK (OnITK) were remarkably conserved with its homologues from other vertebrates, implying its potential conserved function. OnITK mRNA was extensively expressed in lymphoid-related tissues, and with the relative highest level in peripheral blood. Once Nile tilapia was infected by Edwardsiella piscicida, OnITK in splenic lymphocytes was significantly up-regulated on 7-day post infection at both transcription and translation levels, suggesting that OnITK might involve in the primary adaptive immune response of teleost. Furthermore, upon splenic lymphocytes were stimulated by T-cell specific mitogen PHA, OnITK mRNA and protein levels were dramatically elevated. More importantly, treatment of splenic lymphocytes with specific inhibitor significantly crippled OnITK expression, which in turn impaired the inducible expression of T-cell activation markers IFN-γ, IL-2 and CD122, indicating the critical roles of ITK in regulating T-cell activation of Nile tilapia. Taken together, our results suggest that ITK takes part in the lymphocyte-mediated adaptive immunity of tilapia, and is indispensable for T-cell activation of teleost. Our findings thus provide novel evidences for understanding the mechanism regulating T-cell immunity of early vertebrates, as well as the evolution of adaptive immune system., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Nowadays, the electrocatalytic nitrogen reduction reaction (NRR) still faces great challenges. It's significant to design the electrocatalysts with excellent activity and high selectivity. Herein, the 28 single atom catalysts of transition metal atoms anchored on defective silicene (TM@D-silicene) are designed for electrocatalytic ammonia synthesis under ambient conditions. Two independent screening schemes are proposed to screen the potential SAC candidate. The criteria of E bin < 0 eV, Δ G *N2 < -0.1 eV, Δ G *H > Δ G *N2 , and the lowest Δ G *NNH in scheme I, as well as Δ G *N2 < -0.1 eV, smaller G *NNH and larger G *NH2 in scheme II are utilized in the screening procedure. Finally, Cr@D-silicene is picked out since it performs well in the aspects of N 2 adsorption, selectivity and catalytic activity of NRR. Moreover, the electronic properties are systematically investigated to clarify why the Cr@D-silicene is qualified for NRR from the perspective of the strong interaction between N 2 and Cr, the continuous activation of the N 2 molecule, charge transfer and distribution. This work provides a new idea for electrocatalytic ammonia synthesis by using single-atom catalysts.

The traditional Haber-Bosch method of ammonia (NH 3 ) synthesis has low production efficiency and can lead to greenhouse gas emission due to high temperature and pressure dependent reactions. Hence, the nitrogen reduction reaction (NRR) in a mild environment has been developed. However, the inert NN triple bond and the competition with the hydrogen evolution reaction (HER) limit its wide application. In order to find an effective way of reducing N 2 into NH 3 , in this work, PC 6 monolayers with good electro-optical properties and eight transition metals (V, Cr, Mn, Fe, Co, Ni, Cu, Zn) are chosen to construct PC 6 -TM 3 and PC 6 -TM 4 single cluster catalysts (SCCs), which are proved to have low overpotential, multiple active-sites and superior activity. The thermodynamic stability, N 2 adsorption, reaction paths, selectivity for the NRR and catalytic mechanism are systematically investigated. (PC 6 -Co 3 , PC 6 -Fe 4 )/(PC 6 -V 3 , PC 6 -Cr 3 )/(PC 6 -V 4 , PC 6 -Mn 4 ) prefer to adsorb N 2 rather than H in the end-on/side-on I/side-on III mode. PC 6 -Fe 4 and PC 6 -Cr 3 are finally screened out which have excellent catalytic activity with an overpotential of -0.46 V and -0.26 V in the consecutive path of side-on III and I modes, respectively. Moreover, both of them have 100% faradaic efficiency and present high selectivity for the NRR. The catalytic mechanism is elucidated by discussing the electronic properties of PC 6 -Cr 3 , where the back-donation behaviors of Cr atoms play an important role during the formation of NH 3 . This research may provide theoretical guidance for finding potential NRR catalysts with excellent performance and high selectivity.

ZAP70 is essential for initiating the early events of T-cell antigen receptor (TCR) signaling cascade to ensure proper T cell activation and function. However, whether this molecule takes part in the T cell immune response of early vertebrates remains unclear. In the present study, using a teleost model Nile tilapia (Oreochromis niloticus), we investigated the potential involvement of ZAP70 in the T cell activation and adaptive immunity of fish species. Both primary and tertiary structures of O. niloticus ZAP70 (On-ZAP70) are highly conserved with those from other vertebrates. On-ZAP70 protein was widely expressed in lymphoid tissues, and with the highest level in thymus. Once Nile tilapia was infected by Aeromonas hydrophila, mRNA of On-ZAP70 in spleen lymphocytes was induced on day 5 and 8 after infection; meanwhile, phosphorylation of On-ZAP70 was also enhanced, suggesting that On-ZAP70 potentially participated in primary adaptive immune response of Nile tilapia. Furthermore, the frequency of ZAP70 positive lymphocytes was increased during the anti-bacterial adaptive immune response. More importantly, when spleen lymphocytes were activated by T cell specific mitogen PHA, a dramatical augment of On-ZAP70 could be observed at transcription, phosphorylation and cellular level, indicating the involvement of this molecule in T cells activation of Nile tilapia. Altogether, our results demonstrated that ZAP70 activation is an early event of T cell immunity that involved in the anti-bacterial adaptive immune response of Nile tilapia, and thus provided a new evidence to understand the evolution of the lymphocyte-mediated adaptive immunity., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Serving as a significant signaling molecule, RAC-alpha serine/threonine-protein kinase (Akt1) plays indispensable roles in cell cycle, growth, survival, metabolism, as well as immune response. However, how Akt1 regulates adaptive immune response in early vertebrate, especially the teleost, is largely unknown. Here, using a Nile tilapia Oreochromis niloticus model, we investigated the regulatory role of Akt1 in adaptive immunity of teleost. Both sequence and structure of the O. niloticus Akt1 (OnAkt1), were evolutionarily conserved comparing with the counterparts from other vertebrates. mRNA of OnAkt1 was widely expressed in lymphoid organs/tissues of Nile tilapia, with relative higher level in PBL. After Nile tilapia was infected by Aeromonas hydrophila, both transcription and phosphorylation levels of OnAkt1 were obviously elevated in spleen lymphocytes at the adaptive immune stage, suggesting Akt1 participated in primary adaptive immune response of Nile tilapia. Furthermore, OnAkt1 transcript or phosphorylation was dramatically augmented after spleen lymphocytes were activated by T cell specific mitogen PHA or lymphocyte agonist PMA. More critically, inhibition of Akt1 by specific inhibitor crippled the activation of downstream mTORC1 signaling, and impaired the up-regulation of T cell activation markers CD44, IFN-γ and CD122 in spleen lymphocytes upon PHA-induced T cell activation. Meanwhile, blockade of Akt1-activated mTORC1 signaling also decreased the frequency of BrdU + lymphocytes during A. hydrophila infection, indicating the critical role of Akt1 in regulating lymphocyte proliferation of Nile tilapia. Together, our results demonstrated that Akt1 modulated adaptive immune response of Nile tilapia by promoting lymphocyte activation and proliferation via mTORC1 signaling. Our study enriched the regulatory mechanism of lymphocyte-mediated adaptive immunity in teleost, and thus provided novel insights into the evolution of adaptive immune system., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Killer cell lectin-like receptor G subfamily 1 (KLRG1) is a receptor generally expressed on effector CD8 + T cells or NK cells at terminal differentiation stage, and it will be highly induced for lymphocyte cytotoxicity upon pathogen infection or lymphocyte activation. However, little is known about the character or function of KLRG1 in lower vertebrates. In present study, we reappraised a molecule that previously defined as KLRG1 in the genomic sequence of Nile tilapia Oreochromis niloticus, and identified it as an atypical KLRG1-like molecule (defined as On-KLRG1-L), and illustrated its potential function serving as a marker representing effector T lymphocytes of fish species. On-KLRG1-L consists of two C-type lectin-like domains (CTLDs) without transmembrane region, and the tertiary structure of the CTLD is highly alike to that in mouse KLRG1. As a CTLD-containing protein, the recombinant On-KLRG1-L could bind PGN and several microbes in vitro. On-KLRG1-L was widely expressed in immune-associated tissues, with the highest expression level in the gill. Once Nile tilapia is infected by Aeromonas hydrophila, mRNA level of On-KLRG1-L in spleen lymphocytes were significantly up-regulated on 5 days after infection. Meanwhile, On-KLRG1-L protein was also induced on 5 or 8 days after A. hydrophila infection. Furthermore, we found both mRNA and protein levels of On-KLRG1-L were dramatically enhanced within several hours after spleen lymphocytes were activated by T cell-specific mitogen PHA in vitro. More importantly, the ratio of On-KLRG1-L + T cells was also augmented after PHA stimulation. The observations suggested that the KLRG1-like molecule from Nile tilapia participated in lymphocyte activation and anti-bacterial adaptive immune response, and could serve as an activation marker of T lymphocytes. Our study thus provided new evidences to understand lymphocyte-mediated adaptive immunity of teleost., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

As fish constitute the first evolutionary group with primordial T cells, they are of importance for understanding the origin and evolution of adaptive immunity. Yet, the knowledge about how ancestral T cells function remains limited. Therefore, the teleost model Nile tilapia (Oreochromis niloticus) was used in this study to investigate the regulatory mechanisms of T-cell immunity in fish. We identified an evolutionarily conserved canonical NF-κB signaling pathway in Nile tilapia, which participates in primary adaptive immune response during Streptococcus agalactiae infection. Blockade of NF-κB activity severely impairs T-cell activation and expansion, rendering the animals more vulnerable to pathogen attack. Meanwhile, NF-κB signaling is indispensable for fish T cells to produce IL-17A during the antibacterial immune response. Moreover, IL-17A binds its receptor IL-17RA, initiates the ACT1-TRAF6-TAK1 axis, and triggers NF-κB-dependent T-cell activation, thus forming a positive feedback loop of T-cell immunity in Nile tilapia. Furthermore, IL-17A seems to promote innate immunity by regulating pro-inflammatory cytokines via TRAF6-NF-κB axis, indicating the presence of an NF-κB-dependent IL-17A signaling pathway for coordinating adaptive and innate immunity in fish. Our results suggest that fish NF-κB couples TCR and IL-17 signals to modulate ancestral T-cell immunity against bacterial infection, and the regulation of T-cell immunity by NF-κB and IL-17 is a strategy that existed prior to the divergence of the tetrapod lineage from teleost fish. This study, therefore, provides a new perspective on the evolution of adaptive immunity., (© 2021 Federation of American Societies for Experimental Biology.)

Eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) is a translation repressor downstream of mTORC1 pathway, and plays a pivotal role in the adaptive immune response. However, whether 4E-BP1 participates in the regulation of adaptive immunity of early vertebrates is still unclear. In present study, using Nile tilapia Oreochromis niloticus as a model, we investigated the regulatory roles of 4E-BP1 (On-4E-BP1) on lymphocyte-mediated adaptive immune response of fish species. On-4E-BP1 is highly conserved compared with the homologues from other vertebrates, and it is widely distributed in the immune-related tissues of Nile tilapia with the highest expression level in the gill. Once the animals were infected by Aeromonas hydrophila , transcription level of On-4E-BP1 in spleen lymphocytes was significantly induced on day 5 after infection. Meanwhile, phosphorylation of On-4E-BP1 in lymphocytes was also enhanced during the primary adaptive immune response, suggesting that 4E-BP1 is involved in the adaptive immunity of Nile tilapia. Furthermore, when lymphocytes were activated by agonist PMA or T cell specific mitogen PHA in vitro , phosphorylation of On-4E-BP1 was obviously up-regulated. More importantly, once mTORC1/4E-BP1 activity was blocked by specific inhibitor, the inducible expression of T cell activation markers IFN-γ and CD122 was severely impaired during PHA-induced T cell activation, suggesting this signaling regulates T lymphocyte activation of Nile tilapia. In addition, blockade of mTORC1/4E-BP1 axis also resulted in a crippled proliferation of lymphocyte during the primary response of anti-bacterial adaptive immunity. Collectively, we found that mTORC1/4E-BP1 signaling participates in the adaptive immune response by regulating lymphocyte activation and proliferation in Nile tilapia. This study enriches our current knowledge on teleost adaptive immunity, and provides novel perspective to understand the evolution of adaptive immune system., Competing Interests: The authors have stated explicitly that there are no conflicts of interest in connection with this article., (© 2021 The Author(s). Published by Elsevier Ltd.)

Tumor necrosis factors (TNFs) are pleiotropic cytokines with important functions in homeostasis and disease pathogenesis. Recent advances have shown that TNFs are also involved in the regulation of adaptive immune responses. However, the knowledge about how TNF participates in and regulates adaptive immune response in early vertebrates is still limited. In present study, we identified two isoforms of TNF, TNF-α and TNF-β, from Nile tilapia Oreochromis niloticus (On-TNF-α and β). After analyzing the sequence characteristics, we investigated their regulatory roles in adaptive immune response of this fish species. On-TNF-α and β are evolutionarily conserved compare with their homologs from other vertebrates. Both TNFs were distributed in a wide range of tissues in O. niloticus, and with relative higher expression level in gill. After the animals were infected by Streptococcus agalactiae, mRNA levels of On-TNF-α and TNF-β in spleen lymphocytes were significantly upregulated during the primary response stage of adaptive immunity. Meanwhile, both TNF proteins in spleen lymphocytes were also dramatically elevated during the adaptive immune stage after bacterial infection. These results indicate the potential participation of On-TNF-α and TNF-β in adaptive immune response of Nile tilapia. Furthermore, On-TNF-α and β transcripts were obviously augmented, once spleen lymphocytes were activated by T cell-specific mitogen PHA. More importantly, both recombinant On-TNF-α and β could induce the apoptosis of head-kidney leukocytes of Nile tilapia. And On-TNF-β but not On-TNF-α promoted the apoptosis by activating caspase-8 in the target cells. Altogether, our study revealed that TNF-α and TNF-β participated in the lymphocyte-mediated adaptive immune response of Nile tilapia by initiating the apoptosis, and thus shed novel perspective for the regulatory mechanism of adaptive immunity in teleost., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Ribosomal protein S6 kinase beta-1 (S6K1) is a serine/threonine kinase downstream of the mechanistic target of rapamycin (mTOR) pathway, and plays crucial roles in immune regulation. Although remarkable progress has been achieved with a mouse model, how S6K1 regulates adaptive immunity is largely unknown in early vertebrates. In this study, we identified an S6K1 from Nile tilapia Oreochromis niloticus (OnS6K1), and further investigated its potential regulatory role on the adaptive immunity of this fish species. Both sequence and structure of OnS6K1 were highly conserved with its homologs from other vertebrates and invertebrates. OnS6K1 was widely expressed in immune tissues, and with a relative higher expression level in the liver, spleen and head kidney. At the adaptive immune stage of Nile tilapia that infected with Aeromonas hydrophila, mRNA expression of OnS6K1 and its downstream effector S6 was significantly up-regulated in spleen lymphocytes. Meanwhile, their phosphorylation level was also enhanced during this process, suggesting that S6K1/S6 axis participated in the primary response of anti-bacterial adaptive immunity in Nile tilapia. Furthermore, after spleen lymphocytes were activated by the T cell-specific mitogen PHA or lymphocytes agonist PMA in vitro, mRNA and phosphorylation levels of S6K1 were elevated, and phosphorylation of S6 was also enhanced. Once S6K1 activity was blocked by a specific inhibitor, both mRNA and phosphorylation levels of S6 were severely impaired. More importantly, blockade of S6K1/S6 axis reduced the expression of T cell activation marker IFN-γ and CD122 in PHA-activated spleen lymphocytes, indicating the essential role of S6K1/S6 axis in regulating T cell activation of Nile tilapia. Together, our study suggests that S6K1 and its effector S6 regulate lymphocyte activation of Nile tilapia, and in turn promote lymphocyte-mediated adaptive immunity. This study enriched the mechanism of adaptive immune response in teleost and provided useful clues to understand the evolution of adaptive immune system., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The mitogen-activated protein kinase (MAPK) cascade is an ancient and evolutionarily conserved signaling pathway involved in numerous physiological processes. Despite great advances in understanding MAPK-mediated regulation of adaptive immune responses in mammals, its contribution to T-cell immunity in early vertebrates remains unclear. Herein, we used Nile tilapia ( Oreochromis niloticus ) to investigate the regulatory roles of MAPK/extracellular signal-regulated kinase (Erk) signaling in ancestral T-cell immunity of jawed fish. We found that Nile tilapia possesses an evolutionarily conserved MAPK/Erk axis that is activated through a classical three-tier kinase cascade, involving sequential phosphorylation of RAF proto-oncogene serine/threonine-protein kinase (Raf), MAPK/Erk kinase 1/2 (Mek1/2), and Erk1/2. In Nile tilapia, MAPK/Erk signaling participates in adaptive immune responses during bacterial infection. Upon T-cell activation, the MAPK/Erk axis is robustly activated, and MAPK/Erk blockade by specific inhibitors severely impairs T-cell activation. Furthermore, signals from MAPK/Erk were indispensable for primordial T cells to proliferate and exert their effector functions. Mechanistically, activation of the MAPK/Erk axis promoted glycolysis via induction of the transcriptional regulator proto-oncogene c-Myc (c-Myc), to ensure the proper activation and proliferation of fish T cells. Our results reveal the regulatory mechanisms of MAPK/Erk signaling in T-cell immunity in fish and highlight a close link between immune signals and metabolic programs. We propose that regulation of T-cell immunity by MAPK/Erk is a basic and sophisticated strategy that evolved before the emergence of the tetrapod lineage. These findings shed light on the evolution of the adaptive immune system., (© 2020 Wei et al.)

Calcium ion (Ca 2+ ) is a widespread and primitive second messenger that regulates physiological cell functions in almost all life beings. Ca 2+ influx-induced NFAT activation is essential for T cell function and adaptive immunity. However, whether and how Ca 2+ signaling modulates T cell immunity in early vertebrates, especially in nontetrapods, remains largely unknown. To address these questions, a Nile tilapia ( Oreochromis niloticus ) model was employed to investigate the regulation of ancestral T cell immunity by Ca 2+ -NFAT signaling in jawed fish. In Nile tilapia, an evolutionarily conserved Ca 2+ -NFAT signaling pathway is involved in the primary adaptive immune response during Streptococcus agalactiae infection. Meanwhile, T cell signals trigger several events along the Ca 2+ -NFAT axis in this early vertebrate, including Ca 2+ influx, calcineurin activation, and NFAT nuclear import. More critically, suppression of Ca 2+ -NFAT signaling by the calcineurin inhibitor cyclosporine A impairs primordial T cell activation, clonal expansion, and infection clearance. Mechanistically, Nile tilapia NFAT interacts with several other transcription factors for potent gene expression, and T cells in this nontetrapod employ Cabin1 and DYRK1A to regulate NFAT nuclear import and export, respectively. To the best of our knowledge, this study is the first to demonstrate the regulatory mechanism of Ca 2+ -NFAT signaling on T cell immunity in a nontetrapod species. We suggest that modulation of T cell immunity by Ca 2+ -NFAT signaling is a primitive strategy that already existed prior to the divergence of bony fish from the tetrapod lineage. The findings of this study provide valuable perspectives for understanding the evolution of adaptive immune system., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Inhibitory protein IκBα plays a crucial role in the inflammatory process and immune response by regulating the activity of transcription factor NF-κB. In teleost, great progress has been achieved regarding NF-κB signaling for innate immunity, but whether this pathway modulates adaptive immunity, and how, remains largely unclear. In this study, after characterizing the sequence, structure, and phylogeny of Nile tilapia Oreochromis niloticus IκBα (defined as On-IκBα), we investigated the association between IκBα-regulated NF-κB activation and the lymphocyte-mediated adaptive immune response in Nile tilapia. We found that On-IκBα was evolutionarily conserved, and its mRNA was expressed widely in various tissues, with most abundance in the trunk kidney. mRNA expression of On-IκBα was significantly upregulated in spleen at both innate and adaptive immune stages after Aeromonas hydrophila infection. Moreover, phosphorylation of On-IκBα and the downstream On-NF-κB p65 was obviously elevated in spleen leukocytes at 3, 5, or 8 days after A. hydrophila infection, indicating the activation of NF-κB signaling. Correlating with the augmented protein phosphorylation, leukocyte proliferation was enhanced during the same immune stage, suggesting the potential association of IκBα and IκBα-regulated NF-κB signaling in the primary adaptive immune response. Although lymphocyte activation by the T cell-specific mitogen PHA did not alter On-IκBα mRNA expression significantly, lymphocyte activation by the agonist PMA obviously elevated On-IκBα and OnNF-κB p65 phosphorylation in spleen leukocytes. Together, the results suggest that IκBα phosphorylation and its regulated NF-κB activation are essential events associated with lymphocyte activation, proliferation, and anti-bacterial adaptive immune response in Nile tilapia. Our study aids to understand the regulatory mechanism of adaptive immunity in teleost., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The mammalian target of rapamycin (mTOR) is an evolutionarily highly conserved atypical serine/threonine protein kinase, which regulates cell growth, proliferation, apoptosis, autophagy, and metabolism. As a regulatory protein, Raptor is awfully important for the stability and function of mTOR complex 1 (mTORC1). However, the studies about how Raptor/mTORC1 participates in and regulates immune response in lower vertebrates are still limited. In this study, we investigated the regulation of immune response by the Raptor/mTORC1 signaling pathway in rockfish Sebastes schlegelii . Sebastes schlegelii Raptor (Ss-Raptor) is a highly conserved protein during the evolution, in both primary and tertiary structure. Ss-Raptor mRNA was widely distributed in various tissues of rockfish and has a relative higher expression in spleen and blood. After infected by Micrococcus luteus or Listonella anguillarum , mRNA expression of Ss-Raptor rapidly increased within 48 h. Once Raptor/mTORC1 signaling was blocked by rapamycin, expression of the pro-inflammatory cytokines IL-1β and IL-8 was severely impaired, suggesting potential regulatory role of Raptor/mTORC1 signaling in the innate immune response of rockfish. In addition, Raptor/mTORC1 pathway participated in lymphocyte activation of rockfish through promoting 4EBP1 and S6 phosphorylation. Inhibition of Raptor/mTORC1 signaling crippled the lymphocyte expansion during primary adaptive immune response, manifesting by the decrease of lymphoid organ weight and lymphocyte numbers. More importantly, inhibition of Raptor/mTORC1 signaling impaired the lymphocyte mediated cytotoxic response, and made the fish more vulnerable to the bacterial infection. Together, our results suggested that Raptor and its tightly regulated mTORC1 signaling acts as modulatory center to regulate both innate and lymphocyte-mediated adaptive immune response during bacterial infection. This research has shed new light on regulatory mechanism of teleost immune response, and provide helpful evidences to understand the evolution of immune system., (Copyright © 2019 Li, Wei, Zhang, Chi and Yang.)

As an important economic mollusk in coastal areas, Octopus ocellatus dependents on innate immune system to resist the invasion of microorganisms. Lysozyme is a crucial effector owing to its significant lytic activity against bacterial pathogens during the immune responses. In this study, characteristic and immune function of an I-type lysozyme from O. ocellatus (OoLyz) was investigated. OoLyz shared a close relationship with the lysozymes from other bivalve mollusks. The mRNA of OoLyz exhibited a broad transcript in different tissues/organs, and with the greatest expression in hepatopancreas. The expression of OoLyz was significantly raised when O. ocellatus was infected by Vibrio anguillarum or Micrococcus luteus, suggesting OoLyz participated in innate immune response of host. Prokaryotic recombinant OoLyz (rOoLyz) exhibited obvious bacteriolysis ability towards both gram-negative bacteria V. anguillarum and Escherichia coli, and gram-positive bacteria M. luteus and Staphylococcus aureus. The bacteriolysis activities of rOoLyz towards gram-negative but not gram-positive bacteria was heat stable, indicating that OoLyz might clear gram-positive bacterium by enzyme-dependent mechanisms, but eliminate gram-negative microbe via enzymatic activity independent way. Scanning electron microscopy analysis showed that rOoLyz destroyed microbes by damaging cell wall. More importantly, the fact that rOoLyz could directly degrade the peptidoglycan, further revealed its bactericidal mechanism as a muramidase. Our results revealed the essential role of I-type lysozyme in the innate immunity of O. ocellatus, and shed new light to understand the mechanism of immune defense of mollusks., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

T cells suddenly appeared in jawed fish ∼450 million years ago. Biological studies of fish T cells may provide helpful evidence to understand evolution of adaptive immune systems. To this end, using a Nile tilapia ( Oreochromis niloticus ) model, we revealed the regulatory mechanism of adaptive immunity mediated by ancestral T cells in jawed fish. Nile tilapia T cells as well as a tightly regulated mammalian/mechanistic target of rapamycin complex 1 (mTORC1) pathway participate in the cellular adaptive immune response during Streptococcus agalactiae infection. Blockade of mTORC1 signaling by rapamycin impairs T cell activation and Ag-induced proliferation in this early vertebrate. More critically, we show that signals from mTORC1 are indispensable for primordial effector T cells to eliminate infection by promoting the expression of proinflammatory cytokines, cytotoxic-related molecules, and proapoptotic genes. Mechanistically, teleost mTORC1 directs effector T cell function by coordinating multiple metabolic programs, including glycolysis, glutaminolysis, and lipogenesis through activating key transcription factors c-Myc, HIF-1α, and sterol regulatory element-binding proteins, and thus links immune signals to metabolic reprogramming in jawed fish. To our knowledge, these results represent the first description of the regulatory mechanism for T cell-mediated adaptive immunity in a fish species. From an evolutionary viewpoint, our study suggests that primordial T cells are armed with sophisticated regulatory strategies like those in modern T cells prior to the divergence of bony fish from the tetrapod lineage. Therefore, our findings fill in an important gap regarding evolution of the adaptive immune system., (Copyright © 2019 by The American Association of Immunologists, Inc.)

H-Ras is a guanosine triphosphatase (GTPase), which acts as a molecular switch and controls multiple important cellular processes including lymphocyte activation and function. However, regulatory mechanism of adaptive immune response by H-Ras remains unclear in non-mammalian animals. In the present study, we investigated the involvement of H-Ras in lymphocyte activation with a teleost model Oreochromis niloticus. H-Ras from O. niloticus (On-H-Ras) is highly conserved with those from other vertebrates. The mRNA of On-H-Ras showed a wide expression pattern in the lymphoid-tissues and with the highest level in liver. After Aeromonas hydrophila infection, transcription of On-H-Ras was significantly induced on day 8 but came back to basal level on day 16, suggesting that On-H-Ras potentially participated in primary response during the adaptive immunity. Furthermore, On-H-Ras mRNA was obviously up-regulated when leukocytes were activated by T lymphocyte mitogen PHA in vitro. Meanwhile, protein level of H-Ras was also augmented once leukocytes were stimulated with lymphocyte receptor signaling agonist PMA and ionomycin. More importantly, once Ras activity was inhibited by specific inhibitor, the up-regulation of lymphocyte activation marker CD122 was obviously impaired during lymphocyte activation process. Therefore, On-H-Ras regulated lymphocyte activation through both mRNA and protein level. Altogether, our results illustrated the involvement of H-Ras in teleost adaptive immunity via controlling lymphocyte activation, and thus provided a novel perspective to understand evolution of the lymphocyte-mediated adaptive immunity., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

RAF proto-oncogene serine/threonine-protein kinase (c-Raf) is a MAP kinase kinase kinase (MAPKKK) that participates in the Erk1/2 pathway and plays an important role in lymphocyte activation. However, the study on how c-Raf regulates adaptive immunity in non-mammal is still limited. In present study, based on analysis of sequence characteristics of c-Raf from Oreochromis niloticus (On-c-Raf), we investigated its regulation roles on teleost lymphocyte activation. The On-c-Raf was highly conserved during evolution, which was composed of a Raf-like Ras-binding domain (RBD), a protein kinase C conserved region 1 (C1) domain and a serine/threonine protein kinase catalytic (S&#95;TKc) domain. Its mRNA showed a wide distribution in tissues of O. niloticus and with the highest expression in gill. After Aeromonas hydrophila infection, during the adaptive immune stage transcription level of On-c-Raf was significantly upregulated on day 8, but came back to original level on day 16 and 30, suggesting the potential involvement of On-c-Raf in primary response but not memory formation. Furthermore, On-c-Raf mRNA in leukocytes of Nile tilapias was obviously induced by in vitro stimulation of T cell mitogen PHA. More importantly, in vitro stimulation of lymphocytes agonist PMA augmented phosphorylation level of On-c-Raf in leukocytes detected by western-blot and immunofluorescent. Thus, c-Raf regulated lymphocyte activation of Nile tilapia on both mRNA and phosphorylation level. Together, our results revealed that the c-Raf from teleost Nile tilapia engaged in adaptive immune response by regulating lymphocytes activation. Since the regulatory mechanism of lymphocyte-mediated adaptive immunity is largely unknown in teleost, our study provided important evidences to understand teleost adaptive immunity, and also shed a novel perspective for the evolution of adaptive immune system., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Galactoside-binding lectin (galectin) is a type of pathogen recognition molecule that occupies an important position in the invertebrate innate immunity system. Our previous study has identified a galectin gene in mollusk Solen grandis (SgGal-1) and illustrated its potential roles in innate immunity. By the functional study using recombinant protein and specific antibody, here, we confirmed the pivotal roles of SgGal-1 in immune defense of S. grandis. SgGal-1 protein was expressed in many tested tissues including gill, mantle, hepatopancreas and gonad, except hemocytes and muscle. The recombinant SgGal-1 (rSgGal-1) bound PGN and β-glucan instead of LPS in vitro, and it further caused significant agglutination of five different microbes, suggesting SgGal-1 served as a pattern recognition receptor (PRR) involved in immune defense of mollusk. Furthermore, SgGal-1 recruited hemocytes to encapsulate, which was blocked by anti-rSgGal-1 serum. In the meantime, rSgGal-1 as well as promoted the phagocytosis of hemocytes against Escherichia coli in vitro. All these results suggested that SgGal-1 in S. grandis not only acted as a PRR recognizing microbes but also directly participated in the process of immune opsonization to protect the host from pathogenic infection., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Scavenger receptors are crucial for innate immunity owing to their prominent role in clearance of harmful endogenous factors, immune recognition, and more importantly, as co-receptors of Toll-like receptors (TLRs) to initiate downstream responses. At present, invertebrate scavenger receptors, especially their role in immune mechanisms, are largely unknown. We report here that scavenger receptors form a diverse superfamily in Octopus ocellatus, including at least five different members with distinct tissue expression patterns. Two members, OoSR-B and OoSR-I, are grouped into class B and I scavenger receptors, respectively. OoSR-B and OoSR-I are located on the hemocyte membrane, and both recombinant scavenger receptors could serve as pattern recognition receptors to bind a broad range of pathogen-associated molecular patterns. Although OoSR-B and OoSR-I expression was induced by bacterial stimulation, only OoSR-B promoted hemocyte phagocytosis. Moreover, OoSR-B, but not OoSR-I, could act as a co-receptor of TLR to activate TLR-NF-κB signaling and initiate TNF-α production during anti-bacterial response. As the first report on an invertebrate scavenger receptor acting as a co-receptor of TLR, our study reveals the immune mechanism mediated by scavenger receptors in O. ocellatus, and provides new insight into the evolution of this important receptor family., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Ag 2 O nanoparticles-loaded Bi 5 O 7 I microspheres forming a three dimensional Ag 2 O/Bi 5 O 7 I p-n heterojunction photocatalyst with wide-spectrum response were synthesized in this study. The results of transmission electron microscopy observations revealed that the Ag 2 O nanoparticles with the diameter of ca. 10-20nm were distributed on the surfaces of Bi 5 O 7 I nanosheets. The as-synthesized Ag 2 O/Bi 5 O 7 I exhibited an excellent wide-spectrum response to wavelengths ranging from ultraviolet (UV) to near-infrared (NIR), indicating its potential for effective utilization of solar energy. Compared with pure Bi 5 O 7 I, the Ag 2 O/Bi 5 O 7 I composite also demonstrated excellent photocatalytic activity for the degradation of Bisphenol A and phenol in aqueous solution under visible LED light irradiation. Among samples, the 20% Ag 2 O/Bi 5 O 7 I composite photocatalyst showed the highest photocatalytic activity for the degradation of Bisphenol A and phenol in aqueous solution. In addition, the 20% Ag 2 O/Bi 5 O 7 I composite also exhibited a photocatalytic activity for the degradation of Bisphenol A under NIR light irradiation. The improved photocatalytic activity is attributed to the formation of a p-n heterojunction between Ag 2 O and Bi 5 O 7 I, allowing the efficient utilization of solar energy (from UV to NIR) and high separation efficiency of photogenerated electron-hole pairs. The present work is desirable to explore a possible avenue for the full utilization of solar energy., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Peptidoglycan recognition proteins (PGRPs) are indispensable molecules in innate immunity due to their prominent function in sensing and eliminating invading microorganisms. In the present study, a short type PGRP from razor clam Solen grandis (SgPGRP-S1) was recombinantly expressed and purified to investigate its potential function in innate immunity. As a pattern recognition receptor, recombinant SgPGRP-S1 (rSgPGRP-S1) specifically bind Lys-type and Dap-type peptidoglycan in vitro, but not lipopolysaccharide or β-glucan. The peptidoglycan binding ability of rSgPGRP-S1 resulted in significant agglutination activity against Gram-negative Escherichia coli and Listonella anguillarum, as well as Gram-positive Micrococcus luteus. Furthermore, rSgPGRP-S1 was bactericidal, significantly suppressing the growth of both E. coli and Gram-positive Staphylococcus aureus. The protein also exhibited strong amidase activity and degraded bacterial peptidoglycan in the presence of Zn 2+ , suggesting amidase activity might contribute to SgPGRP-S1 antibacterial activity. These results indicate SgPGRP-S1 is multifunctional in innate immunity, mediating both immune recognition and bacteria elimination., (Copyright © 2017 Elsevier Ltd. All rights reserved.)