《列子》思想提供了一条精神超越的身体修行路径.身体修行如何能够提升精神境界是这一路径需要解决的问题.这就需要解决三个层次的问题:一是宇宙论与价值本体的非断裂,以作为身体修行通达本体境界的基本可能;二是保证身体修行所通达的非道德性的精神境界是正向的,以实现精神超越而非精神堕落;三是将不同的精神境界统一于身体修行,以保证身体修行通达本体境界的一致性.以《天瑞》和《黄帝》为例,列子实现精神超越的身体路径回答了以上三个问题.

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.

AbstractAs 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 Ca2+–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.

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.

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.

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-kappa B signaling pathway in Nile tilapia, which participates in primary adaptive immune response during Streptococcus agalactiae infection. Blockade of NF-kappa B activity severely impairs T-cell activation and expansion, rendering the animals more vulnerable to pathogen attack. Meanwhile, NF-kappa B 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-kappa B-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-kappa B axis, indicating the presence of an NF-kappa B-dependent IL-17A signaling pathway for coordinating adaptive and innate immunity in fish. Our results suggest that fish NF-kappa B couples TCR and IL-17 signals to modulate ancestral T-cell immunity against bacterial infection, and the regulation of T-cell immunity by NF-kappa B 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.

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.

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.

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.

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.