| Part I The role of glucose in regulating intestinal inflammation.Background and aims:Inflammatory bowel disease(IBD)is a chronic inflammatory disease that occurs in the lower gastrointestinal tract,including Crohn's disease(CD)and ulcerative colitis(UC).According to incomplete statistics,more than 5 million people in the world suffer from UC or CD,especially in North America and Europe.However,the incidence of IBD in Asia(such as China,Japan,South Korea,etc.),which considered as a "low-risk zone",has significantly increased in recent years.The protracted and persistent nature of the disease has led a heavy economic and spiritual burden on patients.Previous studies have shown that the occurrence and development of IBD is affected by complex interactions between environmental factors,changes in flora,multiple genetic susceptibilities and changes in the immune system.With the continuous in-depth research on IBD in recent years,dietary factors may play an important role in the etiology and course of IBD.As the main energy source of the body,glucose are beneficial to maintain the structure and function of body tissues and cells.On the other hand,long-term intake of a large amount of glucose can cause the body's immune metabolism disorders,internal environment disorders,and induce a variety of autoimmune diseases such as diabetes,IBD and obesity.In recent years,high-sugar diet has been considered as a risk factor for the development of IBD.Previous studies have shown that glucose plays an important role in regulating the differentiation of naive T cells.However,how glucose at moderate levels regulates T cell function and intestinal inflammation is still poorly understood.Thus,our aim to study the effect of glucose intake on the T cell function and its role in intestinal inflammation.Methods:1.Chronic colitis in Rag-/-mice were induced via transferring CBirl TCR transgenic(CBirl Tg)CD4+T cells,which are specific for an immunodominant microbiota antigen CBirl flagellin.Recipient Rag-/-mice were administered normal drinking water or water contain 6%(w/v)and 20%(w/v)glucose on the same day of T cell transfer for the whole duration of the experiments.Six weeks later,the mice were sacrificed,and the intestinal histopathology was examined.CD4+T cell phenotypes in mesenteric lymph node(MLN)and spleen were analyzed by flow cytometry(FACS).2.We cultured CBirl Tg naive CD4+T cell with glucose at a series of doses(500ug/ml,2500ug/ml,7000ug/ml)under neutral,Th1,Th17,and Treg polarization conditions.5 days later,cells were harvested,and the frequency of related T cell subsets were analysis by FACS.3.C57BL/6(B6)mice or Rag-/-mice transferred with CBirl Tg naive CD4+T cells were treated with or without 6%(w/v)glucose in drinking water for two weeks.Two weeks later,the mice were sacrificed for analysis of different subsets of CD4+T cells in mice spleen and MLN.Result:1.Recipient mice that received 6%(w/v)glucose developed less severe colitis than the control group while the glucose fed at 20%(w/v)aggravated colitis development in mice.Feeding 6%(w/v)glucose increased the percentages of Foxp3+Treg cells but not IFN-?+ and IL-17+CD4+T cells in the MLN while the glucose fed at 20%(w/v)increased the percentages of IFN-?+ and IL-17+T cells in the MLN.2.Glucose did not affect CD4+T cell differentiation under neutral conditions and neither affected Thl nor Th 17 differentiation under Thl and Th 17 conditions,but promoted Treg development in a dose-dependent manner under Treg polarization condition,which peaked at a dose of 7000 ?g/ml.3.Mice were treated with 6%(w/v)glucose increased the percentage of Treg cells but not Thl and Th17 cells in mice MLN both in C57BL/6(B6)mice or Rag-/-mice transferred with CBirl Tg naive CD4+T cells.Conclusion:1.Glucose at different doses differentially regulates intestinal inflammation,which might be attributed to its effect on the balance between Treg cells and effector T cells.2.Moderate amounts of glucose promote Treg differentiation but do not affect Thl and Th17 cell development both in vitro and in vivo.Part II The role of glucose in regulating T cell function and intestinal homeostasis.Background and aims:CD4+T cells play an important role in regulating intestinal immunity.Recent studies have shown that the imbalance of pro-inflammatory Th17 and regulatory T cells(Treg)in the body ia a contributing factor for IBD,suggesting that the abnormality of the intestinal mucosal immune system plays a vital role in the occurrence,development and prognosis of IBD.Regulatory T cells(Treg)are a special group of CD4+T cells that regulate the intestinal immune response via inhibiting the proliferation and function of other effector T cells.Therefore,they are regarded as a key regulator of inflammation which play an important role in maintaining immune tolerance and homeostasis.It has been shown that peripheral naive T cells could be activated and differentiated into Treg cell upon sensing antigen signals.Thus gut,where contains rich commensal microbiota and dietary antigens,had been consider as the main site of peripheral differentiation of Foxp3+Treg cells.However,how gut microbiota and dietary antigens regulate the differentiation of intestinal Tregs have not yet been fully revealed.Recent studies shown that the aromatic hydrocarbon receptor(Ahr)can sense a variety of flora and dietary antigen signals and play an important role in regulating the differentiation and function of Treg cells.Thus,we aim to explore whether that glucose regulats Treg cell differentiation to maintain intestinal immune homeostasis through Ahr-mediated molecular pathway.Methods:1.CBirl Tg CD4+T cells(CD45.2)were treated with or without glucose(7000ug/ml)under Treg conditions for 5 days and then co-cultured with CFSE-labeled CD45.1+naive CD4+ T cells for 60h.CD45.1+ naive CD4+T cells proliferation were analysis by flow cytometry(FACS).2.CD45RBhi CD4+T cell adoptive transfer model,a well-established colitis model in which transfer of CD45RBhi CD4+T cells into Rag-/-mice induces chronic colitis in the recipient mice,was established.CD45.1+ CD45RBhi CD4+T cells were transferred into Rag-/-mice together with or without glucose-induced CD45.2+Treg cells or control CD45.2+Treg cells.Mice were sacrificed 4 weeks later,histopathology of the intestine was examined and the ratio of CD45.1+T cell in mouse mesenteric lymph node(MLN)and lamina propria(LP)were analyzed by FACS.3.Naive CD4+T cells were cultured with indicative dose of glucose(7000ug/ml)under Treg polarization condition for 2 days.Cyplal and Cypla2 expression were detected by qPCR.4.CD4+T cells were activated with anti-CD3/CD28 mAbs in the presence(Glu)or absence(Ctr)of 7000 ?g/ml glucose ąCH223191(3 ?M)under Treg polarization conditions.The frequency of Foxp3+Treg in CD4+cells after 5 days culture was analyzed by FACS.5.Rag-/-mice transferred with Cd4cre Ahrfl/fl(Ahr?Cd4)or Foxp3creAhrfl/fl(Ahr?Foxp3)naive T cells were treated with(Glu)or without(Ctr)6%glucose in drinking water for 2 weeks.The frequency of Foxp3+,IL-17+and IFN?+CD4+T cell in mouse MLN and spleen were determined by FACS.Result:1.Compared to the control Treg,glucose-induced Treg cells showed enhanced capacity in suppressing naive CD4+T cell proliferation.2.Recipient mice that received glucose-induced Tregs showed less severe intestinal inflammation than the mice that received control Tregs.The ratio of CD45.2+Treg cells and CD45.1+effector CD4+T cells was increased in mice that received glucose-induced Treg cells compared with the mice that received control Treg cells.3.Compared to the control,glucose enhanced Cyp1a1 and Cyp1a2 expression.4.Compared to the control,glucose promoted Treg differentiation.Blockade of AhR signaling inhibited glucose-induced Treg cell differentiation.5.Glucose treatment did not affect Treg generation and Thl and Th17 differentiation in mice transferred with CD4 T cell-specific or Treg cell-specific Ahr-deficient T cells.Conclusion:1.Glucose-induced Treg cells have enhanced capacity in suppressing T cell proliferation and induction of colitis.2.Glucose promotes Ahr activation in T cells.3.AhR mediates glucose induction of Treg cells.Part ? The role and mechanism of L-lactate in regulating intestinal inflammation.Background and aims:Inflammatory bowel disease(IBD)is a gut chronic inflammatory disease which pathogenesis seems to involve a primary defect in one or more elements for the maintenance of intestinal homeostasis.As one of the most important elements,intestinal epithelial barrier can protect the host from the external environment and lock up harmful microorganisms and toxins in the intestinal lumen to prevent them from invading the intestine.On the other hand,they can play the role of non-professional antigen-presenting cells by directly processing and presenting antigens to intestinal immune cells.The intestinal barrier is the complex system composed of intestinal epithelial cells(IEC).Under normal circumstances,epithelial cells can maintain the integrity of the epithelial barrier through their own proliferation,differentiation and migration.The damage and impairment of the intestinal barrier result in exposure to lumen pathogens and absorption of immunogenic factors,leading to increased inflammation and dysregulation of intestinal homeostasis.Recent studies have shown that changes in the intestinal microenvironment are closely related to the maintenance of the intestinal barrier function and integrity.It has been shown that the metabolites of the gut microbiota play an important role in regulating epithelial repair.However,the effect of host metabolites,which similar considered as an important factor in regulating intestinal microenvironment,on intestinal epithelium has not been fully revealed.Thus,our study aims to investigate whether L-lactate,the most common primary metabolite of host cells,participated in regulating epithelial repair and its role in intestinal inflammation.Methods:1.Mode-K cells were cultured in reduced serum(1%FBS)media,wounded,and treated with or without 5mM L-lactate.For video microscopy,images were taken every 15 minutes.Migration distance,cell speed,Mean Squared Displacement(MSD),a value,autocorrelation was analyzed and calculated using MRI Wound Healing Tool macro for FIJI software(NIH).2.Mode-K cells treated with or without 5mM L-lactate for 12 hours,migration relevant genes Cdc42 and Pakl were detected by qPCR.3.Mode-K cells were serum-starved then treated with or without 5mM L-lactate(n=5 samples per group)for 4 hours.A mito-stress test was performed.Oxygen consumption rate(OCR)over time,extracellular acidification rate(ECAR)over time,basal OCR,ATP-related OCR,Maximal OCR and basal ECAR were detected and analysis by Seahorse.4.Mode-K cells were cultured in reduced serum(1%FBS)media,wounded,and treated with or without 5mM L-lactate in the presence or absence of 2nM oligomycin,images were taken 15 minutes apart for video microscopy.Migration distance,cell speed,Mean Squared Displacement(MSD),? value,autocorrelation was analyzed and calculated using MRI Wound Healing Tool macro for FIJI software(NIH).5.Mice were fed DSS in drinking with or without 30 mM L-lactate for 7 days.After 7 days,mice were changed to regular drinking water with or with L-lactate for another 3 days.Mice were weighed daily and sacrificed on day 10.Mice colon and cecum were collected for histopathology and organ culture,colonic IEC were collected for mRNA isolation and Colonic IEC Cdc42 and Pakl gene expression were analyzed by qPCR.Result:1.Mode-K cells treated with L-lactate moved faster to the wound area over 24 hours than non-treated cells,cell tracking result showed that L-lactate treatment increased cell MSD,? value,direction autocorrelation and speed.2.After 5mM L-lactate treatment for 12 hours,Mode-k cell Cdc42 expression was significantly increased,and there is a slight increase in Pakl expression,but this difference was not significant.3.L-lactate promoted basal and ATP-related oxygen consumption,but there is no effect on maximal oxygen consumption and extracellular acidification rate.4.Oligomycin suppressed cell migration induced by L-lactate to the levels similar to the non-treated cells,besides,oligomycin reduced cell MSD,? value,direction autocorrelation and speed.5.Mice fed with L-lactate showed less weight loss and developed less severe colitis than the control mice.Colonic IL-6 production showed a decreased tendency in mice treated with L-lactate.Besides,L-lactate treatment promoted the expression of Cdc42 and Pakl.Conclusion:1.L-lactate treatment promotes intestinal epithelial cell migration by way of facilitating speed and persisting.2.L-lactate treatment promotes intestinal epithelial cell mitochondrial ATP production.3.mitochondrial ATP synthase is critical for L-lactate-induced intestinal epithelial cell migration in directional persistence and speed.4.L-lactate inhibits colitis development,possibly through regulation of IEC migration. |
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