Intestinal Intraepithelial Lymphocytes Research Articles

The role of intestinal intraepithelial lymphocytes in resistance against coccidiosis in chickens

Abstract Coccidiosis is a critical enteric disease that costs more than $14.5 billion in annual cost to world poultry industry. It is caused by different Eimeriaspecies that invade and replicate within the intestinal epithelium in a site-specific manner. The Eimeriainfection induces local intestinal damage, reduces feed consumption, feed utilization, and growth performance, and increasing mortality rate. With the recent antibiotic ban including anticoccidial drugs in chicken production, the live vaccination become widely used to control coccidiosis. However, the live vaccines are expensive, require time to circulate in the host to induce a protective immunity, and process a high-risk factor for necrotic enteritis caused by clostridium perfringens. Therefore, there is a high demand for development of safe, protective, and inexpensive vaccines against coccidiosis which require better understanding of host immune responses to Eimeria parasites. The aim of this study is to elucidate the protective role of intestinal intraepithelial lymphocytes (IELs) during the Eimeriainfection. At 3 week of age, SPF white-leghorn chicks were divided into 3 groups. Each group (n=50) was orally inoculated with 20,000 oocytes of Eimeria (E) acervuline or E. maxima, or mock-inoculated with PBS. At 2 day post-infection (dpi), 7 dpi and 14 dpi, the frequency and the number of small intestinal IEL populations were determined by a flow cytometry. Eimeriainfection induced the number of TCRγδ +, TCRβ +, TCR neg, TCRβ +CD4 +, TCRβ +CD4 +CD8α +, TCRβ +CD8αβ +, TCRβ +CD8αα +, and iCD8α IELs compared to the control group at 14 dpi. However, there was no difference among groups at 2 and 7 dpi. More experiments are underway to determine the effector and function of each subset of IELs. Startup funding, The OSU

A novel method for isolation and flow cytometry analysis of intraepithelial lymphocytes from colon biopsies

Investigating the immune responses of the intestine in response to different insults is predominantly limited to indirect methods such as circulating markers of intestinal health or gene expression from dissections. We describe here a validated protocol for the isolation and subsequent flow cytometry analysis of intestinal intraepithelial lymphocytes (IEL) from colonic biopsy samples. Colon biopsy samples were collected with endoscopy forceps from Holstein dairy bull calves at d 2, 28, and 42 of life. The biopsies were put into an isolation solution of Hanks' balanced salt solution, and fetal bovine serum followed by digestion solution. The solution was filtered and the flow-through, containing IEL, was stained with fluorescent antibodies for flow cytometry analysis. Density gradient separation of the isolate yielded higher viability and cleaner samples for flow cytometry analysis. Anti-bovine γ chain of the T cell receptor was used to identify populations of gamma delta (γδ) T cells via flow cytometry. In addition, γδ T cell subsets were identified using an anti-bovine antibody against the coreceptor workshop cluster 1. This method allowed for the precise identification of lymphocyte populations and evaluation of the proportion of different subsets of γδ T cells from intestinal IEL over time. The technique described here will allow the research community to characterize intestinal immune function over time and improve our understanding of how different management and nutritional strategies affect intestinal health.

Metformin alleviates liver fibrosis in mice by enriching Lactobacillus sp. MF-1 in the gut microbiota

BackgroundLiver fibrosis is associated with gut dysbiosis. Metformin administration has emerged as a promising method for the treatment of organ fibrosis. We aimed to investigate whether metformin ameliorates liver fibrosis by enhancing the gut microbiota in mice with carbon tetrachloride (CCl4)-induced liver fibrosis and the underlying mechanism. Materials and methodsA liver fibrosis mouse model was established, and the therapeutic effects of metformin were observed. We administered antibiotic treatment and performed fecal microbiota transplantation (FMT), and 16S rRNA-based microbiome analysis to evaluate the effects of the gut microbiome on metformin-treated liver fibrosis. We isolated the bacterial strain preferably enriched by metformin and assessed its antifibrotic effects. ResultsMetformin treatment repaired the gut integrity of the CCl4-treated mice. It reduced the number of bacteria in colon tissues and reduced the portal vein lipopolysaccharide (LPS) levels. The FMT performed on the metformin-treated CCl4 mice alleviated their liver fibrosis and reduced their portal vein LPS levels. The markedly changed gut microbiota was screened out from the feces and named Lactobacillus sp. MF-1 (L. sp. MF-1). In the CCl4-treated mice, daily gavage of L. sp. MF-1 maintained gut integrity, inhibited bacterial translocation, and reduced liver fibrosis. Mechanistically, metformin or L. sp. MF-1 inhibited the apoptosis of intestinal epithelial cells and restored CD3+ intestinal intraepithelial lymphocytes in the ileum and CD4+Foxp3+ lamina propria lymphocytes in the colon. ConclusionsMetformin and its enriched L. sp. MF-1 can reinforce the intestinal barrier to alleviate liver fibrosis by restoring immune function.

Intestinal cell type-specific communication networks underlie homeostasis and response to Western diet.

The small intestine plays a key role in immunity and mediates inflammatory responses to high fat diets. We have used single-cell RNA-sequencing (scRNA-seq) and statistical modeling to examine gaps in our understanding of the dynamic properties of intestinal cells and underlying cellular mechanisms. Our scRNA-seq and flow cytometry studies of different layers of intestinal cells revealed new cell subsets and modeled developmental trajectories of intestinal intraepithelial lymphocytes, lamina propria lymphocytes, conventional dendritic cells, and enterocytes. As compared to chow-fed mice, a high-fat high-sucrose (HFHS) "Western" diet resulted in the accumulation of specific immune cell populations and marked changes to enterocytes nutrient absorption function. Utilizing ligand-receptor analysis, we profiled high-resolution intestine interaction networks across all immune cell and epithelial structural cell types in mice fed chow or HFHS diets. These results revealed novel interactions and communication hubs among intestinal cells, and their potential roles in local as well as systemic inflammation.

Repression of the aryl-hydrocarbon receptor prevents oxidative stress and ferroptosis of intestinal intraepithelial lymphocytes

The aryl-hydrocarbon receptor (AHR) is a ligand-activated transcription factor that buoys intestinal immune responses. AHR induces its own negative regulator, the AHR repressor (AHRR). Here, we show that AHRR is vital to sustaining intestinal intraepithelial lymphocytes (IELs). AHRR deficiency reduced IEL representation in a cell-intrinsic fashion. Single-cell RNA sequencing revealed an oxidative stress profile in Ahrr-/- IELs. AHRR deficiency unleashed AHR-induced expression of CYP1A1, a monooxygenase that generates reactive oxygen species, increasing redox imbalance, lipid peroxidation, and ferroptosis in Ahrr-/- IELs. Dietary supplementation with selenium or vitamin E to restore redox homeostasis rescued Ahrr-/- IELs. Loss of IELs in Ahrr-/- mice caused susceptibility to Clostridium difficile infection and dextran sodium-sulfate-induced colitis. Inflamed tissue of inflammatory bowel disease patients showed reduced Ahrr expression that may contribute to disease. We conclude that AHR signaling must be tightly regulated to prevent oxidative stress and ferroptosis of IELs and to preserve intestinal immune responses.

Dysregulation of CD177+ neutrophils on intraepithelial lymphocytes exacerbates gut inflammation via decreasing microbiota-derived DMF

ABSTRACT Neutrophils synergize with intestinal resident intraepithelial lymphocytes (IELs) to serve as the first-line defense and maintain intestinal homeostasis. However, the underlying mechanisms whereby neutrophils regulate IELs to inhibit intestinal inflammation are still not completely understood. Here, we found that depletion of neutrophils (especially CD177+ subset) caused expansion of colitogenic TCRγδ+CD8αα+ IELs, increased intestinal inflammation, and dysbiosis after dextran sulfate sodium exposure or Citrobacter rodentium infection in mice. scRNA-seq analysis revealed a pyroptosis-related gene signature and hyperresponsiveness to microbiota in TCRγδ+CD8αα+ IELs from colitic Cd177 −/− mice. Microbiota-derived fumarate and its derivative dimethyl fumarate (DMF), as well as fumarate-producing microbiotas, decreased in the feces of colitic Cd177 −/− mice. Elimination of dysbiosis by antibiotics treatment or co-housing procedure and DMF supplementation restrained TCRγδ+CD8αα+ IEL activation. Consistently, DMF significantly alleviated intestinal mucosal inflammation in mice through restricting gasdermin D (GSDMD)-induced pyroptosis of TCRγδ+CD8αα+ IELs. Therefore, our data reveal that neutrophils inhibit intestinal inflammation by promoting microbiota-derived DMF to regulate TCRγδ+CD8αα+ IEL activation in a GSDMD-mediated pyroptosis-dependent manner, and that DMF may serve as a therapeutic target for the management of intestinal inflammation.

High-throughput Raman spectroscopy allows ex vivo characterization of murine small intestinal intra-epithelial lymphocytes (IEL).

T cells are considered to be critical drivers of intestinal inflammation in mice and people. The so called intra-epithelial lymphocyte (IEL) compartment largely consist of T cells. Interestingly, the specific regulation and contribution of IELs in the context of inflammatory bowel disease remains poorly understood, in part due to the lack of appropriate analysis tools. Powerful, label-free methods could ultimately provide access to this cell population and hence give valuable insight into IEL biology and even more to their disease-related functionalities. Raman spectroscopy has demonstrated over the last few years its potential for reliable cell characterization and differentiation, but its utility in regard to IEL exploration remains unknown. To address this question experimentally, we utilized a murine, T cell-driven experimental model system which is accepted to model human gut inflammation. Here, we repopulated the small intestinal IEL compartment (SI IELs) of Rag1-deficient mice endogenously lacking T cells by transferring naïve CD4+ T helper cells intraperitoneally. Using multivariate statistical analysis, high-throughput Raman spectroscopy managed to define a cell subpopulation ex vivo within the SI IEL pool of mice previously receiving T cells in vivo that displayed characteristic spectral features of lymphocytes. Raman data sets matched flow cytometry analyses with the latter identifying T cell receptor (TCR)αβ+ CD4+ T cell population in SI IELs from T cell-transferred mice, but not from control mice, in an abundance comparable to the one detected by Raman spectroscopy. Hence, in this study, we provide experimental evidence for high-throughput Raman spectroscopy to be a novel, future tool to reliably identify and potentially further characterize the T cell pool of small intestinal IELs ex vivo.

Intravital Imaging of Intestinal Intraepithelial Lymphocytes.

Intestinal intraepithelial lymphocytes (IEL) are a numerous population of T cells located within the epithelium of the small and large intestines, being more numerous in the small intestine (SI). They surveil this tissue by interacting with epithelial cells. Intravital microscopy is an important tool for visualizing the patrolling activity of IEL in the SI of live mice. Most IEL express CD8α; therefore, here we describe an established protocol ofintravital imaging that tracks lymphocytes labeled with a CD8α-specific monoclonal antibody in the SI epithelium of live mice. We also describe data acquisition and quantification of the movement metrics, including mean speed, track length, displacement length, and paths for each CD8α+ IEL using the available software. The intravital imaging technique for measuring IEL movement will provide a better understanding of the role of IEL in homeostasis and protection from injury or infection in vivo.

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

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). 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. 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). 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.

Glutamine alleviates intestinal injury in a murine burn sepsis model by maintaining intestinal intraepithelial lymphocyte homeostasis

Intestinal intraepithelial lymphocytes (IELs) play a sentinel role in the mucosal immune system because of their unique anatomical location in the epithelial layer. The disruption of IEL homeostasis is implicated in driving the intestinal injury of many typical inflammatory disorders, such as inflammatory bowel disease (IBD) and sepsis. Therefore, it is meaningful to alleviate intestinal injury by restoring IEL homeostasis in disease conditions. This study explores the effects of glutamine on intestinal IEL homeostasis in a murine model of burn sepsis. We report that glutamine inhibits inflammatory response and reduces injury in the small intestine of burn septic mice. This effect is attributed to the maintaining of IEL homeostasis by suppressing apoptosis and restoring the disrupted subpopulation balance induced by burn sepsis. Mechanistically, we show that glutamine does not affect the IL-15 dependent mechanisms that drive the maintenance and differentiation of IELs. Instead, glutamine sustains IEL homeostasis by upregulate aryl hydrocarbon receptor (AHR) and interleukin (IL)-22 transcription and expression. Consistently, the protective roles of glutamine in burn septic mice were repressed by further supplement with an AHR antagonist CH-223191. Collectively, our study reveals a new role of glutamine to maintain IEL homeostasis by activating the AHR signaling pathway, which in turn ameliorates intestinal injury in burn sepsis.

A randomized, double-blind, placebo-controlled, Phase 1/2a trial protocol to assess the safety and efficacy of TAK-101 administered by microneedles in patients with celiac disease

Introduction: Celiac disease (CD) affects 0.2 to 2.0% of the worldwide population, causing significant enteropathy and systemic symptoms. There is no effective treatment for CD, and patients must follow a strict gluten-free diet lifelong. Recent studies investigated the efficacy of the non-immunosuppressive agent TAK-101 delivered intravenously in reducing immunological response to gluten in CD patients. However, no study has evaluated transdermal delivery, which initiates different immunological pathways. Objective: In a population of CD patients, we aim to assess the safety and pharmacokinetics of TAK-1 delivered by a Hollow Microstructured Transdermal microneedle (MN) (Phase 1) and the efficacy of MN-TAK-101 compared to placebo (Phase 2). Methods: This will be a Phase 1/2a trial lasting 180 days for each phase. Phase 1 will be an open-label, 2-part multicenter study: 1A, a group of patients will be divided into escalating dose cohorts. While, in 1B, another group of patients will be divided into repeated ascending dose cohorts until the maximum tolerated dose is reached. Phase 2 will be a double-blind multicentered randomized (stratified, block) placebo-controlled trial comparing TAK-101 and placebo, using different subjects than those in Phase 1. The primary outcome is the change in Interferon-γ production from baseline after MN-TAK-101 or MN-placebo administration. Secondary outcomes will assess the changes in the ratio of villous height to crypt depth, in the number of intestinal intraepithelial lymphocytes, in CD4, CD8, and γδ cells frequency, in the Celiac symptom index-modified questionnaire and safety measurements. The sample size is between 27-49 patients for phases 1A and 1B; 76 patients in phase 2. For statistical analysis, only descriptive analysis will be done for phase 1, while for Phase 2, the choice of parametric vs. nonparametric tests will be considered according to the normality of the data. This is a first Phase 1/2a placebo control randomized trial to assess the safety and efficacy of TAK-101 applied transdermally for patients with celiac disease. Keywords: Celiac Disease; TAK-101; Microneedle; Gluten intolerance; Gluten desensitization.

Interferon regulatory factor-2 is required for the establishment of the gut intraepithelial T-cell compartment.

CD8αα+ intestinal intraepithelial lymphocytes (iIELs) are known for their unique role in keeping the integrity of the intestinal epithelial barrier, but factors affecting the development of these cells have not been thoroughly understood. Here, we found that the transcriptional regulator interferon regulatory factor-2 (IRF-2) plays a cell-intrinsic, indispensable role in establishing iIEL populations. CD8αα+, but not CD8αβ+, iIELs bearing TCRαβ or TCRγδ were severely reduced in numbers in mice lacking this factor (Irf2-/- mice). Moreover, the majority of residual CD8αα+TCRαβ+ iIELs in these mice was immature as judged from their Thy1.2high phenotype and inefficient T-bet expression. Thymic IEL precursors isolated from Irf2-/- mice failed to efficiently generate CD8αα+TCRαβ+ and TCRγδ+ IELs upon transfer in vivo and CD8αα+TCRαβ+ cells in response to IL-15 in vitro. Double mutant mice lacking both interleukin-15 (IL-15) and IRF-2 showed an even more severe iIEL defect than in mice lacking IL-15 alone. Upon increasing agonistic TCR signal strength through OT-II TCR transgenesis, CD8αα+TCRαβ+ iIELs became more abundant but remained immature on the Irf2-/- background. Our current observations, thus, revealed the unique bimodal role that IRF-2 plays in promoting not only generation of IEL progenitors in the thymus but also maturation of iIELs in the periphery in IL-15-dependent and -independent manners.

Intraepithelial lymphocytes in the pig intestine: T cell and innate lymphoid cell contributions to intestinal barrier immunity.

Intraepithelial lymphocytes (IELs) include T cells and innate lymphoid cells that are important mediators of intestinal immunity and barrier defense, yet most knowledge of IELs is derived from the study of humans and rodent models. Pigs are an important global food source and promising biomedical model, yet relatively little is known about IELs in the porcine intestine, especially during formative ages of intestinal development. Due to the biological significance of IELs, global importance of pig health, and potential of early life events to influence IELs, we collate current knowledge of porcine IEL functional and phenotypic maturation in the context of the developing intestinal tract and outline areas where further research is needed. Based on available findings, we formulate probable implications of IELs on intestinal and overall health outcomes and highlight key findings in relation to human IELs to emphasize potential applicability of pigs as a biomedical model for intestinal IEL research. Review of current literature suggests the study of porcine intestinal IELs as an exciting research frontier with dual application for betterment of animal and human health.

Development and function of natural TCR+ CD8αα+ intraepithelial lymphocytes.

The complexity of intestinal homeostasis results from the ability of the intestinal epithelium to absorb nutrients, harbor multiple external and internal antigens, and accommodate diverse immune cells. Intestinal intraepithelial lymphocytes (IELs) are a unique cell population embedded within the intestinal epithelial layer, contributing to the formation of the mucosal epithelial barrier and serving as a first-line defense against microbial invasion. TCRαβ+ CD4- CD8αα+ CD8αβ- and TCRγδ+ CD4- CD8αα+ CD8αβ- IELs are the two predominant subsets of natural IELs. These cells play an essential role in various intestinal diseases, such as infections and inflammatory diseases, and act as immune regulators in the gut. However, their developmental and functional patterns are extremely distinct, and the mechanisms underlying their development and migration to the intestine are not fully understood. One example is that Bcl-2 promotes the survival of thymic precursors of IELs. Mature TCRαβ+ CD4- CD8αα+ CD8αβ- IELs seem to be involved in immune regulation, while TCRγδ+ CD4- CD8αα+ CD8αβ- IELs might be involved in immune surveillance by promoting homeostasis of host microbiota, protecting and restoring the integrity of mucosal epithelium, inhibiting microbiota invasion, and limiting excessive inflammation. In this review, we elucidated and organized effectively the functions and development of these cells to guide future studies in this field. We also discussed key scientific questions that need to be addressed in this area.

A conserved Bacteroidetes antigen induces anti-inflammatory intestinal T lymphocytes.

The microbiome contributes to the development and maturation of the immune system. In response to commensal bacteria, intestinal CD4+ T lymphocytes differentiate into functional subtypes with regulatory or effector functions. The development of small intestine intraepithelial lymphocytes that coexpress CD4 and CD8αα homodimers (CD4IELs) depends on the microbiota. However, the identity of the microbial antigens recognized by CD4+ T cells that can differentiate into CD4IELs remains unknown. We identified β-hexosaminidase, a conserved enzyme across commensals of the Bacteroidetes phylum, as a driver of CD4IEL differentiation. In a mouse model of colitis, β-hexosaminidase-specific lymphocytes protected against intestinal inflammation. Thus, T cells of a single specificity can recognize a variety of abundant commensals and elicit a regulatory immune response at the intestinal mucosa.

Intravital Microscopy to Visualize Murine Small Intestinal Intraepithelial Lymphocyte Migration.

Intraepithelial lymphocytes (IELs) are critical sentinels involved in host defense and maintenance of the intestinal mucosal barrier. IELs expressing the γδ T-cell receptor provide continuous surveillance of the villous epithelium by migrating along the basement membrane and into the lateral intercellular space between adjacent enterocytes.Intravital imaging has furthered our understanding of the molecular mechanisms by which IELs navigate the epithelial compartment and interact with neighboring enterocytes at steady state and in response to infectious or inflammatory stimuli. Further, evaluating IEL migratory behavior can provide additional insight into the nature and extent of cellular interactions within the intestinal mucosa. Three protocols describe methodology to visualize small intestinal IEL motility in real time using fluorescent reporter-transgenic mice and/or fluorophore-conjugated primary antibodies and spinning-disk confocal microscopy. Using Imaris image analysis software, a fourth protocol provides a framework to analyze IEL migration and quantify lymphocyte/epithelial interactions. Together, these protocols for intravital imaging and subsequent analyses provide the basis for elucidating the spatiotemporal dynamics of mucosal immune cells and interactions with neighboring enterocytes under physiological or pathophysiological conditions. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Mouse preparation and laparotomy Support Protocol: Antibody labeling of cell surface markers Basic Protocol 2: Image acquisition by spinning-disk confocal microscopy Basic Protocol 3: 4D analysis of images.

Protective effect of ghrelin on intestinal I/R injury in rats.

This study aimed to investigate whether ghrelin affected the autophagy and inflammatory response of intestinal intraepithelial lymphocytes (IELs) by regulating the NOD2/Beclin-1 pathway in an intestinal ischemia–reperfusion (I/R) injury model. Twenty hours after implementing the intestinal I/R injury rat model, the small intestine and both lungs were collected for histological analysis. The morphological changes in the intestinal mucosa epithelium and lung tissues were evaluated using hematoxylin-eosin staining. The activity of autophagic vacuoles and organ injury were evaluated using electron microscopy. The cytokine levels (IL-10 and TNF-α) in IEL cells and lung tissue were determined using enzyme-linked immunosorbent assay. RT-qPCR and western blot assays were conducted to check the NOD2, Beclin-1, and ATG16 levels. Ghrelin relieved the I/R-induced destruction of the intestinal mucosa epithelium and lung tissues. Moreover, ghrelin enhanced autophagy in the intestinal epithelium and lungs of I/R rats. In addition, the levels of autophagy-associated proteins (Beclin-1, ATG16, and NOD2) were higher in the ghrelin treatment group than in rats with I/R. Ghrelin reduced significantly the IL-10 and TNF-α levels. However, these changes were reversed by the NOD2 antagonist. In conclusion, ghrelin may relieve I/R-induced acute intestinal mucosal damage, autophagy disorder, and inflammatory response in IELs by regulating the NOD2/Beclin-1 pathway.

The dephosphorylation of FADD at S191 induces an excessive expansion of TCRαβ+ IELs in the intestinal mucosa.

Intestinal intraepithelial lymphocytes (IELs) play a crucial role in host defence against pathogens in the intestinal mucosa. The development of intestinal IELs is distinct from peripheral T lymphocytes and remains elusive. Fas-associated protein with death domain (FADD) is important for T cell development in the thymus. Here we describe a novel function of FADD in the IEL development. FADD (S191A), a mouse FADD mutant at Ser191 to Ala mimicking constitutively unphosphorylated FADD, promoted a rapid expansion of TCRαβ+ IELs, not TCRγδ+ IELs. Mechanism investigation indicated that the dephosphorylation of FADD was required for cell activation mainly in TCRαβ+ CD8+ T cells. Consistently, FADD (S191A) as dephosphorylated FADD led to a high NF-κB activation in the TCR-dependent cell expansion. In addition, The FADD (S191A)-induced abnormal IEL populations resulted in the increased incidence and severity of colitis in mice. In summary, FADD signalling is involved in the intestinal IEL development and might be a regulator for intestinal mucosal homeostasis.

Common and Exclusive Features of Intestinal Intraepithelial γδ T Cells and Other γδ T Cell Subsets.

Murine peripheral lymph node TCR γδ T cells have been divided into type 1 and type 17 functional categories based on phenotypic and functional markers. Localized in the gut epithelial barrier, intestinal intraepithelial lymphocytes (iIEL) γδ T cells constitute a peculiar subset of T lymphocytes involved in intestinal homeostasis. However, whether iIEL γδ T cells obey the type 1/type 17 dichotomy is unclear. Using both global transcriptional signatures and expression of cell surface markers, we reveal that murine iIEL γδ T cells compose a distinct population, expressing ∼1000 specific genes, in particular genes that are responsible for cytotoxicity and regulatory functions. The expression of the transcription factor Helios is a feature of iIEL γδ T cells, distinguishing them from the other TCR γδ T subsets, including those present in the epithelia of other tissues. The marked expression of Helios is also shared by the other iIELs, TCRαβCD8αα lymphocytes present within the intestinal epithelium. Finally, we show that Helios expression depends in part on TGF-β signaling but not on the microbiota. Thus, our study proposes iIEL γδ T cells as a distinct subset and identifies novel markers to differentiate them from their peripheral counterparts.

Histological and neuronal changes in the duodenum of hamsters infected with Leishmania (Leishmania) infantum

Visceral leishmaniasis is a neglected tropical disease caused by parasites belonging to the Leishmania genus that infect macrophages in different tissues such as the spleen, liver, lymph nodes, bone marrow, and intestine. Therefore, this study aimed to investigate the integrity of the intestinal tract and the nitrergic (NADPH-dp) and metabolically active (NADH-dp) myenteric neurons of the duodenum of golden hamsters infected with L. (L.) infantum. Therefore, thirty golden hamsters were divided into six groups (n = 5); three of them were infected with 2 × 107 promastigote forms of L. (L.) infantum by intraperitoneal route (Infected Group - IG) and three were inoculated with saline solution (control group - CG). After 30, 60 and 90 days post-infection (DPI) infected animals were euthanized and the liver, spleen and duodenum were collected to analyze tissue parasitism. The duodenum was processed using usual histological techniques to analyze the main changes that occurred during infection and histochemical techniques to phenotype myenteric neurons. Amastigote forms were observed in the spleen, liver, and duodenum during all experimental periods, and tissue parasitism in these organs increased significantly over time. At 30 DPI, reduction in muscle tunic, increase in the total intestinal wall and the number of goblet cells PAS+ was observed. At 60 DPI, an increase in intestinal crypts and intraepithelial lymphocytes was observed, and a reduction in intestinal villi was observed at 90 DPI, along with an increase in crypt size. Regarding neurons, an increase in the density of the NADPH-dp population was observed at 30 DPI, but at 60 and 90 DPI a significant reduction of this population was observed. In general, infection progression was observed to cause significant morphofunctional changes in the duodenum of infected hamsters.