Effects of Huangqi Gancao Decoction on intestinal immunity and microbiota in immunocompromised mice models.

Background: The classical medicinal formula Huangqi Gancao Decoction (HQGCD), originating from the medical book" Yi Lin Gai Cuo". Up to now, the studies focusing on the immunoenhancement effects of HQGCD are few, and the actionpathway is not yet clear. Method: In this study, SPF male KM mice were utilized as a model for immunosuppression. Comprehensive observations were made regarding the general behavior and condition of the mice, in addition to monitoring fluctuations in body weight and food intake. The blood routine index was measured, and morphological changes in the ileum and colon tissues were examined. The level of secretory immunoglobulin A (sIgA), superoxide dismutase (SOD), and malondialdehyde (MDA) in ileum and colon tissues were quantified. Additionally, the bone marrow total DNA index was assessed. Flow cytometry analyzed the proportions of CD3⁺, CD4⁺, CD8⁺, and CD4+CD8+ double-positive (DP) T lymphocytes in small intestinal intraepithelial lymphocytes (IELs). Lastly, the composition and diversity of the cecal microbiota were evaluated using 16S rDNA sequencing technology. Results: After HQGCD intervention, there were no significant changes in the mice's feed intake and body weight. However, the tissue structures of the ileum and colon showed recovery. In the blood routine index, there was an increase in the total white blood cell count, lymphocyte count, red blood cell count, hematocrit, and hemoglobin content. Additionally, the bone marrow total DNA index was elevated. Level of SOD and sIgA in ileum and colon tissues increased, while the level of MDA decreased. The proportions of CD3⁺ and CD4⁺ T lymphocytes within IELs increased, along with an increase in DP T lymphocytes in IELs (DP IELs), whereas the proportion of CD8⁺ T lymphocytes decreased. The cecal microbiota underwent changes, with an increase in the variety and number of beneficial microbiota. Conclusion: HQGCD could restore the intestinal immune function of immunocompromised mice, and had a certain positive effect on cecal microbiota.

Activation of mucosal insulin receptor exacerbates intestinal inflammation by promoting tissue resident memory T cells differentiation through EZH2.

BACKGROUND: Inflammatory Bowel Diseases (IBD), an autoimmune disease characterised by abnormal intestinal immunity, are related to vital morbidity around the world. However, therapeutic agents for IBD have not achieved desired benefit. Exploring new therapeutic targets for IBD, especially based on its abnormally intestinal immunity, could alleviate the flare-up and worsening of IBD. Tissue resident memory T cells (TRM) are core of multiple autoimmune diseases, including IBD. However, the mechanism of TRM differentiation remains to be investigated. METHODS: The alterations in mRNA and lncRNA profile of intestinal intraepithelial lymphocytes (IELs), the largest component of intestinal TRM, were analyzed in DSS-induced chronic colitis. Based on it, we examined the function of rectal insulin instillation in a dextran sodium sulfate (DSS) induced chronic colitis. Furthermore, we investigated the downstream-target of the insulin pathway-EZH2 and the crucial role of EZH2 in intestinal tissue resident memory T cell differentiation by utilizing EZH2fl/flCD4cre mice. RESULTS: Insulin receptor (INSR) expression was found to be significantly reduced. Activation of mucosal insulin pathway by rectal insulin instillation exacerbated colitis by disrupting IELs subgroups and up-regulating TNF-ɑ and IL-17 expression. Rectal insulin instillation promoted EZH2 expression and EZH2 inhibition alleviated chronic colitis. EZH2fl/flCD4cre mice restored the normal IEL subgroups and suppressed TNF-ɑ and IL-17 expression, exhibiting alleviated colitis. IELs from EZH2fl/flCD4cre mice exhibit significant changes in TRM related phenotype. CD4+TRM was significantly increased in chronic colitis and decreased in EZH2fl/flCD4cre mice. CONCLUSION: Insulin receptor of intestinal mucosal T-cells could promote intestinal TRM differentiation via EZH2. Our discoveries suggest that therapies targeting colonic INSR and EZH2 could be potential treatment for IBD based on its regulatory effects on TRM. Insulin receptor inhibitors rather than insulin should be applied during colitis-active phase. In addition, EZH2 shows to be a downstream signal of the insulin pathway and EZH2 inhibitor could alleviating intestinal inflammation. However, the critical role of EZH2 in TRM differentiation restricts the anti-tumor effects of EZH2 inhibitor in vivo.

Gut immune microenvironment and autoimmunity.

A variety of immune cells or tissues are present in the gut to form the gut immune microenvironment by interacting with gut microbiota, and to maintain the gut immune homeostasis. Accumulating evidence indicated that gut microbiota dysbiosis might break the homeostasis of the gut immune microenvironment, which was associated with many health problems including autoimmune diseases. Moreover, disturbance of the gut immune microenvironment can also induce extra-intestinal autoimmune disorders through the migration of intestinal pro-inflammatory effector cells from the intestine to peripheral inflamed sites. This review discussed the composition of the gut immune microenvironment and its association with autoimmunity. These findings are expected to provide new insights into the pathogenesis of various autoimmune disorders, as well as novel strategies for the prevention and treatment against related diseases.

Ablation of CD226 on CD4+ T cells modulates asthma progress associated with altered IL-10 response and gut microbiota.

To investigate the role of the costimulatory molecule CD226 in asthma pathogenesis, we produced a CD4+ T-cell-specific CD226 knockout mice model (Cd226ΔCD4) and induced airway allergic inflammation by administering ovalbumin (OVA). Our results revealed alleviated lung inflammation, decreased levels of OVA-specific IgE, and increased levels of IL-10 in the serum of Cd226ΔCD4 mice (P < 0.05). Moreover, IL-10 levels in CD4+ T cells were significantly elevated in the mediastinal lymph node, spleen, and Peyer's patches in the Cd226ΔCD4 mice compared with those in controls (P < 0.05 to P < 0.01). Notably, there was a significantly higher IL-10 mRNA levels in the large intestine of the mice (P < 0.05). The protective effect of CD226 deficiency is also associated with the accumulation of gut TCRγδ+ intraepithelial lymphocytes and reversion of the gut microbiome dysbiosis. The Bacteroidetes-to-Firmicutes ratio and the abundance of Akkermansia increased in the absence of CD226 after OVA treatment. Our data reveal the synchronous changes in the lung and intestine in OVA-treated CD226-knockout mice, supporting the gut-lung axis concept and providing evidence for novel therapeutic approaches for asthma.

Divergent roles for the gut intraepithelial lymphocyte GLP-1R in control of metabolism, microbiota, and T cell-induced inflammation.

Gut intraepithelial lymphocytes (IELs) are thought to calibrate glucagon-like peptide 1 (GLP-1) bioavailability, thereby regulating systemic glucose and lipid metabolism. Here, we show that the gut IEL GLP-1 receptor (GLP-1R) is not required for enteroendocrine L cell GLP-1 secretion and glucose homeostasis nor for the metabolic benefits of GLP-1R agonists (GLP-1RAs). Instead, the gut IEL GLP-1R is essential for the full effects of GLP-1RAs on gut microbiota. Moreover, independent of glucose control or weight loss, the anti-inflammatory actions of GLP-1RAs require the gut IEL GLP-1R to selectively restrain local and systemic T cell-induced, but not lipopolysaccharide-induced, inflammation. Such effects are mediated by the suppression of gut IEL effector functions linked to the dampening of proximal T cell receptor signaling in a protein-kinase-A-dependent manner. These data reposition key roles of the L cell-gut IEL GLP-1R axis, revealing mechanisms linking GLP-1R activation in gut IELs to modulation of microbiota composition and control of intestinal and systemic inflammation.

Dihydromyricetin Attenuates High-Intensity Exercise-Induced Intestinal Barrier Dysfunction Associated with the Modulation of the Phenotype of Intestinal Intraepithelial Lymphocytes.

BACKGROUND: Exercise-induced gastrointestinal syndrome (GIS) has symptoms commonly induced by strenuous sports. The study aimed to determine the effect of dihydromyricetin (DHM) administration on high-intensity exercise (HIE)-induced intestinal barrier dysfunction and the underlying mechanism involved with intestinal intraepithelial lymphocytes (IELs). METHODS: The HIE model was established with male C57BL/6 mice using a motorized treadmill for 2 weeks, and DHM was given once a day by oral gavage. After being sacrificed, the small intestines of the mice were removed immediately. RESULTS: We found that DHM administration significantly suppressed HIE-induced intestinal inflammation, improved intestinal barrier integrity, and inhibited a HIE-induced increase in the number of IELs and the frequency of CD8αα+ IELs. Meanwhile, several markers associated with the activation, gut homing and immune functions of CD8αα+ IELs were regulated by DHM. Mechanistically, luciferase reporter assay and molecular docking assay showed DHM could activate the aryl hydrocarbon receptor (AhR). CONCLUSIONS: These data indicate that DHM exerts a preventive effect against HIE-induced intestinal barrier dysfunction, which is associated with the modulation of the quantity and phenotype of IELs in the small intestine. The findings provide a foundation to identify novel preventive strategies based on DHM supplementation for HIE-induced GIS.

Enterovirus 71 Antagonizes Antiviral Effects of Type III Interferon and Evades the Clearance of Intestinal Intraepithelial Lymphocytes.

Enterovirus 71 (EV71) is the major pathogen causing severe neurological complications and hand, foot, and mouth disease. The intestinal mucosal immune system has a complete immune response and immune regulation mechanism, consisting of densely arranged monolayer intestinal epithelial cells (IECs) and intestinal intraepithelial lymphocytes (iIELs) distributed among the IECs, which constitute the first line of intestinal mucosa against infection of foreign pathogens. As an enterovirus, EV71 is transmitted by the intestinal tract; however, the mechanisms it uses to evade the immunosurveillance of the intestinal mucosal immune system are still incompletely clarified. The present study investigated how EV71 evades from recognizing and eliminating IECs, iIELs, and iNK cells. We found that EV71 infection induced a higher level of type III interferons (IFN-λ) than type I interferons (IFN-ß) in IECs, and the addition of IFN-λ markedly restricted EV71 replication in IECs. These results indicate that IFN-λ plays a more important role in anti-EV71 intestinal infection. However, EV71 infection could markedly attenuate the antiviral responses of IFN-λ. Mechanistically, 2A protease (2Apro) and 3C protease (3Cpro) of EV71 inhibited the IFN-λ production and IFN-λ receptor expression and further decreased the response of IECs to IFN-λ. In addition, we found that EV71-infected IECs were less susceptible to the lysis of intestinal NK (iNK) cells and CD3+iIELs. We revealed that the viral 2Apro and 3Cpro could significantly reduce the expression of the ligands of natural killer group 2D (NKG2D) and promote the expression of PD-L1 on IECs, rendering them to evade the recognition and killing of iNK and CD3+iIELs. These results provide novel evasion mechanisms of EV71 from intestinal mucosal innate immunity and may give new insights into antiviral therapy.

T Cell Receptor Is Required for Differentiation, but Not Maintenance, of Intestinal CD4+ Intraepithelial Lymphocytes.

The gut epithelium is populated by intraepithelial lymphocytes (IELs), a heterogeneous T cell population with cytotoxic and regulatory properties, which can be acquired at the epithelial layer. However, the role of T cell receptor (TCR) in this process remains unclear. Single-cell transcriptomic analyses revealed distinct clonal expansions between cell states, with CD4+CD8αα+ IELs being one of the least diverse populations. Conditional deletion of TCR on differentiating CD4+ T cells or of major histocompatibility complex (MHC) class II on intestinal epithelial cells prevented CD4+CD8αα+ IEL differentiation. However, TCR ablation on differentiated CD4+CD8αα+ IELs or long-term cognate antigen withdraw did not affect their maintenance. TCR re-engagement of antigen-specific CD4+CD8αα+ IELs by Listeria monocytogenes did not alter their state but correlated with reduced bacterial invasion. Thus, local antigen recognition is an essential signal for differentiation of CD4+ T cells at the epithelium, yet differentiated IELs are able to preserve an effector program in the absence of TCR signaling.

Recent advances in roles of G-protein coupled receptors in intestinal intraepithelial lymphocytes.

Intestinal intraepithelial lymphocytes (IELs) potentially provide the first line of immune defense against enteric pathogens. In addition, there is growing evidence supporting the involvement of IELs in the pathogenesis of gut disorders such as inflammatory bowel diseases. Various kinds of molecules are involved in the dynamics of IELs, such as homing to the intestinal epithelium and retention in the intestinal mucosa. G protein-coupled receptors (GPCRs) comprise the largest family of cell surface receptors and regulate many biological responses. Although some GPCRs, like CCR9, have been implicated to have roles in IEL homing, little is still known regarding the functional roles of GPCRs in IEL biology. In this review, we provide a concise overview of recent advances in the roles of novel GPCRs like GPR55 and GPR18 in the dynamics of IELs.

CD4CD8αα IELs: They Have Something to Say.

The intraepithelial lymphocytes (IELs) that reside within the epithelium of the intestine play a critical role in maintaining the immune balance of the gut. CD4CD8αα IELs are one of the most important types of IELs, and they play an irreplaceable role in maintaining the balance of the intestinal immune system. CD4CD8αα IELs are often regarded as a special subtype of CD4+ IELs that can express CD8αα on their cytomembrane. Hence, CD4CD8αα IELs not only have the ability to modulate the functions of immune cells but also are regarded as cytotoxic T lymphocytes (CTLs). Transcription factors, microbes, and dietary factors have a substantial effect on the development of CD4CD8αα IELs, which make them exert immunosuppression and cytotoxicity activities. In addition, there is an intimate relationship between CD4CD8αα IELs and inflammatory bowel disease (IBD), whereas it is still unclear how CD4CD8αα IELs influence IBD. As such, this review will focus on the unparalleled differentiation of CD4CD8αα IELs and discuss how these cells might be devoted to tolerance and immunopathological responses in the intestinal tract. In addition, the role of CD4CD8αα IELs in IBD would also be discussed.

Dynamics and clinical significance of intestinal intraepithelial lymphocytes.

Intestinal intraepithelial lymphocytes (IELs) are one of the largest populations of lymphocytes and comprised of heterogeneous populations with varying phenotypes and physiological/pathological functions. IELs located between the basolateral surfaces of the epithelial cells and then potentially provide a first line of immune defense against enteric pathogens, although, the precise roles of each IEL populations are not well defined. A variety of molecules are involved in the IEL-homing to the intestinal epithelium. Conventional IELs originate from circulating T cells activated in lymphoid organs and imprinted for gut homing. On the other hand, unconventional IELs derive from thymocytes and migrate to the intestinal epithelium, although, some of them may arise extrathymically. Regarding the interaction between IELs and epithelial cells, IELs are known to be highly motile and actively migrate along the basement membrane, suggesting their roles in immune surveillance. In addition, there has been growing evidence to support that IELs are involved in the pathogenesis of gut disorders such as celiac disease and inflammatory bowel diseases. In this review, we provide a comprehensive overview of IEL dynamics and their clinical significance.

Intestinal Epithelial Cells-Derived Hypoxia-Inducible Factor-1α Is Essential for the Homeostasis of Intestinal Intraepithelial Lymphocytes.

Hif-1α is a master regulator which involved in the transcriptional regulation of anti-inflammatory or cellular responding to hypoxia. Previous work shows that the absence of Hif-1α results in the destruction of intestinal epithelial cell (IEC) and abnormalities of intestinal barrier function. However, we know very little about other functions of Hif-1α on intestinal intraepithelial lymphocyte (IEL). Therefore, we generated a transgenic mouse (Hif1-α ΔIEC mice), which was knocked out Hif1-α specifically in IECs, to study the effect of Hif1-α on IEL. IELs were isolated from the small intestine and colon of mice, respectively, and examined by flow cytometry and quantitative real-time PCR. All the cytokines expression was detected by quantitative real-time PCR. The NSAID enteropathy was induced by gavaged with 5 mg/kg indomethacin and the experimental colitis was induced by administration of 2.5% DSS. We found that the number of IELs is increased in Hif1-α ΔIEC mice. It is showed that knockout of Hif1-α in IECs led to significant changes in IEL phenotype, including a marked decline in the CD8αα+ and TCRγδ+ population. The reduction of CD8αα+ IELs is accompanied by increased apoptosis, decreased proliferation and weakened migration in Hif1-α ΔIEC mice. Moreover, absence of intestinal epithelial Hif1-α markedly changed the population of IELs in NSAID-induced small intestinal injury and increased susceptibility to dextran sulfate sodium-induced colitis. In summary, our results first time demonstrate that IEC-derived Hif1-α is essential for maintaining IELs homeostasis and intestinal microbiota.

The cross-talk between enterocytes and intraepithelial lymphocytes.

The gut mucosa is continuously exposed to food and microbial antigens. Both enterocytes and intraepithelial lymphocytes have a pivotal role in maintaining the integrity of intestinal mucosa, as these cells guarantee a first line of defense against pathogens and toxic molecules. Enterocytes maintain a physical barrier against microbes and directly contribute to the gut homeostasis by sampling the luminal agents through several pattern recognition receptors or presenting antigen to mucosa T cells. Similarly, due to a close physical contact with the intestinal epithelial cells, the intraepithelial lymphocytes represent an important part of the gut lymphoid tissue, contrasting the entry and spread of pathogens. An alteration of the cross-talk between intestinal epithelial cells and intraepithelial lymphocytes might actively contribute to the development of intestinal immune disorders, as occurring in patients with celiac disease. In genetically predisposed individuals, the gluten exposure results in a massive production of interleukin-15, activation of intraepithelial lymphocytes, and modification of small intestinal mucosa architecture and function. We will review the recent studies on the pathophysiology of cross-talk between enterocytes and intraepithelial T cells, and how this interaction is crucial for intestinal integrity and homeostasis.

Molecular control of CD4(+) T cell lineage plasticity and integrity.

CD4(+) helper T cells and CD8(+) cytotoxic T cells form the two major subsets of peripheral T lymphocytes. Helper T cells fulfill crucial roles in the activation and coordination of the immune response, while cytotoxic T cells kill virus-infected or tumor cells. Recent data suggest that the lineage identify of helper T cells is not fixed and that CD4(+) T cells under certain physiological conditions can be reprogrammed to express CD8 lineage genes and to develop into intestinal intraepithelial CD4(+) cytotoxic T lymphocytes that lack the expression of the key helper T cell lineage commitment factor ThPOK. Moreover, the analysis of mice with a conditional deletion of the transcription factor ThPOK or the histone deacetylases HDAC1 and HDAC2 indicated that CD8 lineage genes are actively repressed in CD4(+) T cells in order to maintain the lineage integrity of helper T cells. In this review I summarize recent studies that indicate plasticity of CD4(+) T cells towards a CTL program and that demonstrate that ThPOK and HDAC1-HDAC2 are part of a transcriptional regulatory circuit that counteracts the activity of the transcription factor Runx3 to maintain CD4(+) T cell lineage integrity.

γδ T-cell-deficient mice show alterations in mucin expression, glycosylation, and goblet cells but maintain an intact mucus layer.

Intestinal homeostasis is maintained by a hierarchy of immune defenses acting in concert to minimize contact between luminal microorganisms and the intestinal epithelial cell surface. The intestinal mucus layer, covering the gastrointestinal tract epithelial cells, contributes to mucosal homeostasis by limiting bacterial invasion. In this study, we used γδ T-cell-deficient (TCRδ(-/-)) mice to examine whether and how γδ T-cells modulate the properties of the intestinal mucus layer. Increased susceptibility of TCRδ(-/-) mice to dextran sodium sulfate (DSS)-induced colitis is associated with a reduced number of goblet cells. Alterations in the number of goblet cells and crypt lengths were observed in the small intestine and colon of TCRδ(-/-) mice compared with C57BL/6 wild-type (WT) mice. Addition of keratinocyte growth factor to small intestinal organoid cultures from TCRδ(-/-) mice showed a marked increase in crypt growth and in both goblet cell number and redistribution along the crypts. There was no apparent difference in the thickness or organization of the mucus layer between TCRδ(-/-) and WT mice, as measured in vivo. However, γδ T-cell deficiency led to reduced sialylated mucins in association with increased gene expression of gel-secreting Muc2 and membrane-bound mucins, including Muc13 and Muc17. Collectively, these data provide evidence that γδ T cells play an important role in the maintenance of mucosal homeostasis by regulating mucin expression and promoting goblet cell function in the small intestine.

Chinese goose (Anser cygnoides) CD8a: cloning, tissue distribution and immunobiological in splenic mononuclear cells.

CD8 molecule is a cell membrane glycoprotein, which plays an important role in cell-mediated immunity. Here, we identified Chinese goose CD8α (goCD8α) gene for the first time. The full-length cDNA of goCD8α is 1459bp in length and contains a 711bp open reading frame. Phylogenetic analysis shows that the waterfowl CD8α formed a monophyletic group. Semi-quantitative RT-PCR analysis showed that transcripts of goCD8α mRNA were high in the immune-related organs and mucosal immune system in gosling, and high in thymus and spleen comparing to other immune-related tissues in goose. The obvious increase of CD8α expression was observed in spleen of acute new type gosling viral enteritis virus (NGVEV) infected bird, while the increase of CD8α were observed in the thymus, bursa of fabricius, and cecum of chronic infected bird. The CD8α mRNA transcription level in spleen mononuclear cells was significantly up-regulated when stimulated by phytohemagglutinin, but not by lipopolysaccharide in vitro.

IL-15 modulates the balance between Bcl-2 and Bim via a Jak3/1-PI3K-Akt-ERK pathway to promote CD8αα+ intestinal intraepithelial lymphocyte survival.

IL-15 is an essential survival factor for CD8αα(+) intestinal intraepithelial lymphocytes (iIELs) in vitro and in vivo. However, the IL-15-induced survival signals in primary CD8αα(+) iIELs remains elusive. Although Bcl-2 level in CD8αα(+) iIELs positively correlates with IL-15Rα expression in the intestinal epithelial cells, overexpression of Bcl-2 only moderately restores CD8αα(+) γδ iIELs in Il15(-/-) mice. Here, we found that IL-15 promptly activated a Jak3-Jak1-PI3K-Akt pathway that led to the upregulation of Bcl-2 and Mcl-1. This pathway also induced a delayed but sustained ERK1/2 activation, which not only was necessary for the maintenance of Bcl-2 but also resulted in the phosphorylation of extra-long Bim at Ser(65) . The latter event facilitated the dissociation of Bim from Bcl-2 without affecting Bim abundance in IL-15-treated CD8αα(+) iIELs. Using an adoptive cell transfer approach, we found that either overexpression of Bcl-2 or removal of Bim from CD8αα(+) iIELs promoted their survival in Il15ra(-/-) mice. Taken together, IL-15 promotes CD8αα(+) iIEL survival by both increasing Bcl-2 levels and dissociating Bim from Bcl-2 through activation of a Jak3-Jak1-PI3K-Akt-ERK1/2 pathway, which differs from a previously reported IL-15-induced survival signal.

Chicken NK cell receptors.

Natural killer cells are innate immune cells that destroy virally infected or transformed cells. They recognize these altered cells by a plethora of diverse receptors and thereby differ from other lymphocytes that use clonally distributed antigen receptors. To date, several receptor families that play a role in either activating or inhibiting NK cells have been identified in mammals. In the chicken, NK cells have been functionally and morphologically defined, however, a conclusive analysis of receptors involved in NK cell mediated functions has not been available. This is partly due to the low frequencies of NK cells in blood or spleen that has hampered their intensive characterization. Here we will review recent progress regarding the diverse NK cell receptor families, with special emphasis on novel families identified in the chicken genome with potential as chicken NK cell receptors.