Study On The Mechanism Of Short-Term Fasting To Enhance T-Cell Immunity In Tilapia

The function of the immune system depends on energy supply,and energy deficiency can seriously affect the vital activity of immune cells.Fish often respond to pathogen infestation by reducing or even stopping feeding,and the mechanisms by which the immune system responds under such energy-limited conditions are unclear.Recent advances have highlighted the critical role of brief fasting in optimizing immunity in humans and mice.However,it remains unknown whether this strategy was independently acquired by mammals during evolution or represents a progressively evolved function common to vertebrates.T cells are an important weapon in vertebrate immune defense,playing a key role in anti-infection,cellular memory,and assisting in antibody production by B cells.T cell immunity is precisely regulated by many factors,among which metabolic programs are critical for T cell biological processes,fate determination,and immune function.In this study,we investigated the regulatory mechanism of fasting on fish T cells in Nile tilapia,which is important for understanding fish T cell metabolic immunity and exploring the evolution of T cell immunity.By analyzing the transcriptome of spleen leukocytes from tilapia with normal fed,short-term（3 days）fasting,and long-term（7 days）fasting,we found that T cells may be the main enforcer of the immune system response to fasting.Further studies showed that long-term fasting for 7 days significantly induced the transcriptional expression of pro-inflammatory cytokines IFN-γ,IL-1β,TNF-α,IL-6,the cytotoxic genes Perforin A and Granzyme B,and apoptosis of T cells in spleen leukocytes or T cells,resulting in a significant decrease in the frequency of T cells in lymphocytes and a severe deficiency in the absolute number of T cells,thus impairing T cell homeostasis in tilapia;whereas a 3-day short-term fasting had a significant effect on T cell homeostasis in tilapia.3-day short-term fasting had no significant effect on the inflammatory response,T-cell apoptosis and homeostasis of tilapia spleen lymphocytes.Notably,a 7-day fast also resulted in a decrease in the absolute number of Ig M⁺B cells and CD3^-Ig M^-lymphocytes in both spleen and head kidney,but the magnitude of the decrease was lower than that of T cells,and the frequency of CD3⁺T cells in head kidney was less affected than that in spleen,suggesting that spleen T cells are more susceptible to nutrient deprivation.Short-term fasting led to an increase in T-cell size;CD3ε,CD8,and CD4-1 m RNA levels;and CD3 protein expression,indicating that short-term fasting enhanced T cell activation.In addition,stimulation of spleen leukocytes using T-cell mitogen PHA resulted in higher expression of IL-2 and its receptor CD122 in short-term fasted tilapia compared to the fed group;AKT,NF-κB or ERK1/2 phosphorylation levels were significantly enhanced in leukocytes or T cells,confirming that short-term fasting enhances T-cell activation in tilapia.The m RNA expression of ULK1,Beclin-1,ATG5/7/12,and LC3B was upregulated in spleen leukocytes or T cells from short-term fasted tilapia,with enhanced phosphorylation of ULK1,increased Beclin-1,LC3B protein expression and P62 protein degradation;simultaneously stronger LC3 protein expression and autophagy-lysosomal staining were observed in leukocytes or CD3⁺T cells,indicating that short-term fasting induced autophagy in tilapia T cells.In vitro blockade of autophagy in short-term fasted tilapia leukocytes significantly increased T cell apoptosis;impaired PHA stimulation-triggered phosphorylation of NF-κB,AKT,S6,and upregulated expression of T cell activation marker genes CD122 and IFN-γm RNA,suggesting that short-term dietary restriction promotes T cell immunity in tilapia by initiating moderate autophagy.Inhibition of autophagy during Aeromonas hydrophila infection,a significant decrease in the proportion and absolute number of T cells in spleen lymphocytes,and reduced Brd U admixture indicated that blocking autophagy severely impaired pathogen-induced T cell proliferation.Meanwhile,inhibition of autophagy impaired pathogen-induced transcriptional expression of the pro-inflammatory cytokines and the cytotoxic genes,significantly increasing individual mortality in tilapia,indicating that autophagy is essential for the normal immune function of tilapia T cells.Furthermore,we suggest that the fasting-induced AMPK activation in tilapia leukocytes was associated with reduced blood glucose levels because our findings demonstrate that metformin administration activated the LKB1/AMPK axis.Similar to the autophagy inhibition,blocking AMPK signaling compromised T-cell activation and pathogen-induced T cell proliferation,downregulated expression of pro-inflammatory cytokines and cytotoxic genes,and exacerbated T cell apoptosis in tilapia,indicating that tilapia require AMPK activity to maintain T cell activation,proliferation,survival,and function.Upon energy restriction,blockade of AMPK signaling inhibited the increase in T cell autophagic lysosomal activity,impaired ULK1 phosphorylation,and upregulated P62 protein levels,suggesting that AMPK regulates T cell autophagy in tilapia.Thus,these results suggest that AMPK is a central hub linking fasting and autophagy-controlled T-cell immunity in tilapia and that short-term energy limitation can promote T-cell immunity in tilapia through AMPK-dependent autophagy.The expression of autophagy marker proteins and lysosomal activity in leukocytes showed increased autophagy levels after pathogen infection,which combined with the above results indicated that both fasting and pathogen infection could trigger autophagy.After pathogen infection,the expression of autophagy marker proteins and lysosomal activity in leukocytes showed increased autophagy levels,which combined with the above results indicated that both fasting and pathogen infection could trigger autophagy.Compared with the fed group,the expression of autophagy marker proteins and autophagy lysosomal activity in leukocytes or T cells of short-term fasted tilapia showed that a strong autophagic response had been initiated within 72 h after infection;normal levels were restored after 120 h of infection,indicating that short-term fasting could trigger AMPK-dependent T cell autophagy earlier and keep autophagy within a controlled range.This mode of autophagy regulation triggered by short-term fasting at the early stage of infection could rescue T cells,reduce inflammatory tissue damage,and promote cellular immune response.In our study,we also found that short-term fasting promoted AMPK activation while upregulating the lipocalin receptor AdipoR1.Antibody blockade of AdipoR1 in leukocytes significantly reduced fasting-induced AMPK phosphorylation activation,indicating that fasting could promote AMPK activation in tilapia leukocytes via AdipoR1.In contrast,in vitro agonist activation of AdipoR1 in leukocytes resulted in a significant increase in intracellular Ca²⁺concentration,protein of Ca M and Ca MKKβand phosphorylation of AMPKα,indicating that AdipoR1 can promote AMPK activation in tilapia through Ca²⁺-Ca MKKβsignaling.Further studies revealed that the AdipoR1-mediated Ca²⁺-Ca MKKβ-AMPK signaling pathway in tilapia leukocytes or T cells is involved in immune responses during pathogenic bacterial infections;blocking AdipoR1-mediated Ca²⁺-Ca MKKβ-AMPK signaling during pathogenic bacterial infections using AdipoR1 antibody or Ca MKKβinhibitors severely impaired the proliferation of lymphocytes and T cells,reduced the transcriptional expression of pro-inflammatory cytokines and cytotoxic genes,and exacerbated T cell apoptosis,indicating that AdipoR1-mediated Ca²⁺-Ca MKKβ-AMPK signaling has an integral role in the T cell immune response in tilapia.Therefore,the above results suggest that short-term fasting may promote T cell immunity through AdipoR1-mediated AMPK signaling.Interestingly,adiponectin（ADPN）is missing in the tilapia genome,and we tried to explore the activation mechanism of AdipoR1 in tilapia T cells.The presence of tilapia complement-C1q/tumor necrosis factor-related protein 9（CTRP9）recombinant protein triggered the activation of Ca²⁺-Ca MKKβ-AMPK signaling,promoted glucose uptake and expression of key proteins for glycolysis by T cells,and decreased apoptosis of T cells;while antibody blocking AdipoR1 impairs the promotion of glucose uptake and glycolysis by CTRP9 in tilapia T cells,suggesting that CTRP9 is able to regulate glucose uptake and glycolysis in tilapia T cells through AdipoR1-mediated intracellular signaling regulates the metabolic activity of tilapia T cells.These studies suggest that CTRP9,which is also an adipocytokine,may serve as a substitute to compensate for the absence of ADPN in tilapia.In summary,we elucidated the mechanisms by which AMPK-related signaling pathways regulate T cell immunity in tilapia,and 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.