Intestinal Intraepithelial Lymphocytes Research Articles

Carnosol Maintains Intestinal Barrier Function and Mucosal Immune Homeostasis in DSS-Induced Colitis.

Ulcerative colitis (UC) is a chronic inflammatory disease, characterized by recurrent flares of mucosal inflammation, which is limited in the colon and rectum. Compromised epithelial barrier functions have been indicated in the initiation of UC. Carnosol (CA), a natural active ortho-diphenol diterpene compound, is one of the active ingredients in plants such as rosemary and sage. The anti-inflammatory and anti-oxidative effects of CA have been reported in several animal models, but its effect on mucosal inflammation remains elusive. We established a mouse experimental colitis model characterized by epithelial barrier destruction using dextran sulfate sodium (DSS). CA was intraperitoneally administrated. Flow cytometry was performed to determine phenotypes of intraepithelial lymphocytes and lamina propria cells. qRT-PCR was used for gene expression. ER stress in the colon was determined by immunofluorescence staining and qRT-PCR. Thapsigargin was used to induce ER stress in HCT-116 cells in vitro. We found CA significantly alleviated DSS-induced colitis in mice marked by relieved clinical symptoms and colonic pathological damage. Inflammatory cell infiltration and cytokine expression in the colon were suppressed by CA during colitis. Furthermore, CA restored epithelial barrier functions and intestinal intraepithelial lymphocyte (IEL) homeostasis in mice with DSS insults. Mechanistically, we induced endoplasmic reticulum (ER) stress in HCT-116 cells (an intestinal epithelial cell line) with thapsigargin, and CA reversed this effect. In addition, we collected inflamed mucosal biopsies from 23 patients with UC, and cultured overnight with or without CA, showing CA significantly reduced expression of ER stress signaling molecule and pro-inflammatory agents. Our data demonstrate that CA acts as an effective drug for experimental colitis and maintains proper epithelial barrier functions via suppressing epithelial ER stress, providing new evidence that CA might be a promising therapeutic candidate for UC.

Type I interferon co‐stimulation enhances γδ intraepithelial lymphocyte IFNγ production

Intraepithelial lymphocytes (IEL) expressing the γδ T cell receptor (TCR) constantly survey the intestinal epithelium, facilitating a localized effector response to limit pathogen invasion. Despite constant γδ TCR triggering in vivo, these IELs remain immunologically quiescent, suggesting that a co‐stimulatory signal is required to overcome their activation threshold. Type I interferon (IFN) is a family of pro‐inflammatory cytokines that are critical for host defense against bacterial and viral infection. Activated γδ IELs limit viral replication in intestinal epithelial cells by producing type I IFNs, such as IFNα; however, the role of IFNα in mediating γδ IEL antiviral effector function remains unknown. To determine whether IFNα directly activates these sentinel lymphocytes, murine small intestinal γδ IELs were isolated, sorted, and stimulated ex vivo with 1μg/mL αCD3, 10 ng/mL IFNα, or both for 5 h. Following treatment with αCD3 and IFNα, we observed a 27%±9.3 increase in IFNγ+ γδ IELs (p<0.0001) and a 2.5‐fold increase in IFNγ mean fluorescence intensity (MFI)(p<0.0001) compared to αCD3 stimulation. Interestingly, IFNα treatment alone was not sufficient to induce IFNγ production. To determine the threshold of TCR signaling necessary for this enhanced IFNγ production, we treated γδ IELs with suboptimal concentrations of αCD3 (0.01 or 0.1 μg/mL), to mimic constant TCR triggering in vitro.At 0.1 μg/mL αCD3, IFNα induced a 27%±7.2 increase in frequency of IFNγ+ γδ IELs (p<0.01) and a significant increase in MFI of γδ IEL IFNγ production over αCD3 treatment alone (p<0.001). With a low level of TCR priming (0.1 μg/mL αCD3), a low dose of IFNα (0.1 ng/mL) was able to stimulate a 22%±6.8 increase in IFNγ+ γδ IELs compared to αCD3 treatment alone. However, higher concentrations of IFNα failed to further enhance γδ IEL IFNγ production. Stimulation of γδ IELs with IFNα ex vivo was sufficient to induce STAT1 and STAT4 phosphorylation; therefore, we asked whether STAT1 or STAT4 is required for IFNα‐induced IFNγ production. To this end, we treated WT, STAT1 KO, or STAT4 KO γδ IELs with 1 μg/mL αCD3, 10 ng/mL IFNα, or both and found that the IFNα‐induced increase in IFNγ production was abrogated in STAT4‐, but not STAT1‐deficient γδ IELs (p<0.0001). To assess changes in STAT1 and STAT4 expression in γδ IELs during acute viral infection, WT mice were infected orally with 1.5x106 PFU murine gamma herpesvirus (MHV). We observed a 16%±3 increase in the frequency of γδ IELs 3 days post‐infection compared to controls, which corresponded with a decrease in total STAT1 (MFI: 905±95 vs 638±102, p<0.01). Taken together, our data demonstrate that γδTCR signaling is required for IFNα‐induced IFNγ production in a STAT4‐dependent manner. These findings suggest that IFNα signaling in conjunction with constant TCR triggering promotes the rapid activation and expansion of γδ IELs, which may enhance their effector response to microbial pathogens.

Three Layers of Intestinal γδ T Cells Talk Different Languages With the Microbiota.

The mucosal surfaces of our body are the main contact site where the immune system encounters non-self molecules from food-derived antigens, pathogens, and symbiotic bacteria. γδ T cells are one of the most abundant populations in the gut. Firstly, they include intestinal intraepithelial lymphocytes, which screen and maintain the intestinal barrier integrity in close contact with the epithelium. A second layer of intestinal γδ T cells is found among lamina propria lymphocytes (LPL)s. These γδ LPLs are able to produce IL-17 and likely have functional overlap with local Th17 cells and innate lymphoid cells. In addition, a third population of γδ T cells resides within the Peyer´s patches, where it is probably involved in antigen presentation and supports the mucosal humoral immunity. Current obstacles in understanding γδ T cells in the gut include the lack of information on cognate ligands of the γδ TCR and an incomplete understanding of their physiological role. In this review, we summarize and discuss what is known about different subpopulations of γδ T cells in the murine and human gut and we discuss their interactions with the gut microbiota in the context of homeostasis and pathogenic infections.

Glutamate attenuates lipopolysaccharide induced intestinal barrier injury by regulating corticotropin-releasing factor pathway in weaned pigs.

ObjectiveThe purpose of this study was to evaluate the protection of glutamate (GLU) against the impairment in intestinal barrier function induced by lipopolysaccharide (LPS) stress in weaned pigs.MethodsTwenty-four weaned pigs were divided into four treatments containing: i) non-challenged control, ii) LPS-challenged control, iii) LPS+1.0% GLU, and iv) LPS+2.0% GLU. On day 28, pigs were treated with LPS or saline. Blood samples were collected at 0, 2, and 4 h post-injection. After blood samples collection at 4 h, all pigs were slaughtered, and spleen, mesenteric lymph nodes, liver and intestinal samples were obtained.ResultsDietary GLU supplementation inhibited the LPS-induced oxidative stress in pigs, as demonstrated by reduced malondialdehyde level and increased glutathione level in jejunum. Diets supplemented with GLU enhanced villus height, villus height/crypt depth and claudin-1 expression, attenuated intestinal histology and ultrastructure impairment induced by LPS. Moreover, GLU supplementation reversed intestinal intraepithelial lymphocyte number decrease and mast cell number increase induced by LPS stress. GLU reduced serum cortisol concentration at 4 h after LPS stress and downregulated the mRNA expression of intestinal corticotropin-releasing factor signal (corticotrophin-releasing factor [CRF], CRF receptor 1 [CRFR1], glucocorticoid receptor, tryptase, nerve growth factor, tyrosine kinase receptor A), and prevented mast cell activation. GLU upregulated the mRNA expression of intestinal transforming growth factor β.ConclusionThese findings indicate that GLU attenuates LPS-induced intestinal mucosal barrier injury, which is associated with modulating CRF signaling pathway.

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-β) 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.

The histone demethylase Kdm6b regulates the maturation and cytotoxicity of TCRαβ+CD8αα+ intestinal intraepithelial lymphocytes

Intestinal intraepithelial lymphocytes (IELs) are distributed along the length of the intestine and are considered the frontline of immune surveillance. The precise molecular mechanisms, especially epigenetic regulation, of their development and function are poorly understood. The trimethylation of histone 3 at lysine 27 (H3K27Me3) is a kind of histone modifications and associated with gene repression. Kdm6b is an epigenetic enzyme responsible for the demethylation of H3K27Me3 and thus promotes gene expression. Here we identified Kdm6b as an important intracellular regulator of small intestinal IELs. Mice genetically deficient for Kdm6b showed greatly reduced numbers of TCRαβ+CD8αα+ IELs. In the absence of Kdm6b, TCRαβ+CD8αα+ IELs exhibited increased apoptosis, disturbed maturation and a compromised capability to lyse target cells. Both IL-15 and Kdm6b-mediated demethylation of histone 3 at lysine 27 are responsible for the maturation of TCRαβ+CD8αα+ IELs through upregulating the expression of Gzmb and Fasl. In addition, Kdm6b also regulates the expression of the gut-homing molecule CCR9 by controlling H3K27Me3 level at its promoter. However, Kdm6b is dispensable for the reactivity of thymic precursors of TCRαβ+CD8αα+ IELs (IELPs) to IL-15 and TGF-β. In conclusion, we showed that Kdm6b plays critical roles in the maturation and cytotoxic function of small intestinal TCRαβ+CD8αα+ IELs.

Guanylin ligand protects the intestinal immune barrier by activating the guanylate cyclase-C signaling pathway

Inflammatory bowel disease (IBD) impacts patient quality of life significantly. The dysfunction of intestinal immune barrier is closely associated with IBD. The guanylate cyclase-C (GC-C) signaling pathway activated by the guanylin (Gn) ligand is involved in the occurrence and development of IBD. However, how it regulates the intestinal immune barrier is still unclear. To investigate the effect of the GC-C pathway on intestinal mucosal immunity and provide experimental basis for seeking new therapeutic strategies for IBD, we focused on Caco-2 cells and intestinal intra-epithelial lymphocytes (IELs), which displayed inflammatory responses induced by lipopolysaccharide (LPS). GC-C activity was modulated by transfection with Gn overexpression or GC-C shRNA plasmid. Levels of Gn, GC-C, and CFTR; transepithelial electrical resistance (TER); paracellula r permeability; and levels of IL-2, IFN-γ, and secretory IgA (sIgA) were examined. The study found that after stimulation with LPS, Gn, GC-C, CFTR, TER, and sIgA levels were all significantly reduced, IL-2 and IFN-γ levels as well as paracellular permeability were significantly increased. These indicators changed inversely and significantly after transfection with the Gn overexpression vector. Compared to the vector controls, GC-C-silenced cells displayed significantly decreased levels of GC-C, CFTR, and TER and increased levels of IL-2, IFN-γ, and paracellular permeability stimulated by LPS. The results show that Gn ligand can protect the intestinal immune barrier by activating the GC-C signaling pathway, which may be helpful for the development of new treatments for IBD. Data availability statementThe data used to support the findings of this study are available from the corresponding author upon request.

Microinflammation in the intestinal mucosa and symptoms of irritable bowel syndrome.

Potential etiological mechanisms of irritable bowel syndrome (IBS) have been reported, and emerging data suggest that immune activation is present in a major subset of IBS, especially in those with diarrhea. Intestinal mucosal mast cell and intraepithelial lymphocyte (IEL) infiltration and related factors were examined in patients with IBS. In addition, the correlations of symptoms and micro-inflammation were assessed. Intestinal biopsy specimens were obtained from patients with IBS and controls. Mast cells and IELs were stained with specific antibodies. The mRNA levels of cytokines and chemokines were assessed by quantitative reverse transcription polymerase chain reaction. Infiltration of mast cells in the duodenum was significantly higher in IBS patients than in control subjects. The infiltration of IELs was higher in the duodenum and terminal ileum of IBS patients compared to the control subjects. The numbers of duodenal and ileal IELs were significantly correlated. The number of IELs but not mast cells in the duodenum and terminal ileum was significantly correlated with diarrhea frequency in control subjects and IBS patients. The expression level of the chemotactic chemokine CXCL11 was significantly higher in the duodenum of IBS patients. Duodenal mast cells and IELs were increased in IBS patients. In addition, a positive correlation was found between the number of duodenal and ileal IELs and the frequency of diarrhea. Given that the present study was strictly observational, further studies are needed to clarify the pathophysiological functions associated with micro-inflammation in IBS.

The Diagnostic Utility of Flow Cytometry in Celiac Disease Presented Isolated Iron Deficiency Anemia.

Flow cytometric analysis of intestinal intraepithelial lymphocytes contributes to the diagnosis of celiac disease. Celiac disease may present with iron deficiency anemia alone which is considered as one of the forms of atypical celiac disease. In this study, we have aimed to investigate the diagnostic utility of flow cytometric analysis of intraepithelial lymphocytes in this atypical form. Three groups were formed: the patients with unexplained iron deficiency (group 1), the patients with celiac disease (group 2), and the patients who underwent gastroduodenoscopy for other reasons (group 0). Duodenal biopsy samples were used for flow cytometric analysis of intraepithelial lymphocytes. T cell receptor gammadelta intraepithelial lymphocytes and CD3-/CD103+ intraepithelial lymphocytes were determined with relevant monoclonal antibodies. Sensitivity-specificity calculation was performed to evaluate the usability of flow cytometric variables as diagnostic tests. Group 1 had 22 patients, group 2 had 14 patients, and group 0 had 56 patients. In the comparison of the 3 groups, CD3+/ TCRγδ+ intraepithelial lymphocytes were found to be higher in celiac patients than other cases. CD3+/TCRγδ+ intraepithelial lymphocyte was evaluated for its usability as a diagnostic test. The cut-off value of CD3+/TCRγδ+ intraepithelial lymphocyte as 16.39% according to receiver operating characteristics curve analysis determined celiac disease in 14 of 22 patients in group 1 with 91.7% sensitivity and 80.4% specificity. Although celiac disease is diagnosed with serologic tests and histologic examination, successively, the increase in intestinal CD3+/TCRγδ+ intraepithelial lymphocytes may be used as a diagnostic test, and it may assist in revealing atypical forms of celiac disease.

Evaluation of immune efficacy of Omp2 protein against Lawsonia intracellularis in mice

Porcine proliferative enteropathy (PPE) is caused by the obligate intracellular bacterium Lawsonia intracellularis. Infection results in an enteric disease characterised by decreased growth performance of pigs, and presents a major economic burden for swine industries worldwide. Since vaccination is an effective technique for controlling PPE, novel effective vaccine platforms are need to be developed. In this study, five proteins of L. intracellularis were screened through animal experiments and the highly immunoprotective Omp2 protein was identified. Then, the immune efficacy of Omp2 was further evaluated based on humoral and cell mediated immune (CMI) responses, faecal bacterial shedding, histopathological lesions, immune barrier function of intestinal mucosa as well as digestive and absorptive capacity following challenge of mice with L. intracellularis. Mice immunised with Omp2 had reduced faecal shedding, fewer histopathological lesions and reduced bacteria colonisation of the ileum. Additionally, Omp2 immunised mice showed stronger serum IgG and IFN-γ levels, up-regulated Occludin and zonula occludens-1 (ZO-1) mRNA levels, as well as increased numbers of intestinal intraepithelial lymphocytes (IELs) and levels of sIgA. On the contrary, the activities of LPS, α-AMS and AKP were significantly increased.Our investigation indicated that immunization with Omp2 reduced the severity of clinical signs and provided efficacious immunoprotection for target animals against L. intracellularis infection in mouse model.

Isolation and immunophenotyping by flow cytometry of canine peripheral blood and intraepithelial and lamina propria duodenal T lymphocytes

The gut associated lymphoid tissue (GALT) effector sites play a crucial role on the pathogenesis of many immune-mediated gastrointestinal diseases. The lymphocytes at these effector sites are principally T cells which present important morphological, phenotypical and functional differences. Flow cytometry (FC) is one of the most commonly used techniques to characterize intestinal lymphocytes in human and animal models. Published studies with a focus on dogs for intraepithelial lymphocytes (IEL) immunophenotyping exist in very limited numbers. Moreover, no lamina propria lymphocytes (LPL) isolation protocols in the canine species have been described for FC evaluation. In addition to immune intestinal dysregulation, imbalances in the peripheral blood immune system have been described in both human and animal gastrointestinal disorders. The aim of this study was to provide a protocol for canine IEL and LPL isolation for FC immunophenotyping of T cells subsets. Specifically, T helper, T cytotoxic, activated Th and Tc lymphocytes, regulatory, double negative, double positive, IFN-γ and IL-4 producing T cells, and to compare their respective populations between these effector sites and with the blood stream compartment in healthy dogs. The potential relationship of these cells distributions with age, sex and breed was also evaluated. This study included sixteen healthy dogs of different sexes and breeds with a mean age of 4.55 ± 2.93 years old. The selected protocols for the three immune compartments showed proper cell yield, purity, viability, and the absence of phenotypic and functional disturbances. Histologically, an adequate separation of the duodenal epithelium from the lamina propria was also observed. All the proposed T cells subsets were identified in the three immune compartments studied, showing some statistically significant differences in their distributions at these locations that highlight the importance of their individual evaluation. This study provides an adequate method for canine small intestine IEL and LPL isolation for FC immunophenotyping and is key for future studies on the gastrointestinal immune system associated with different canine diseases.

IL-15 and PIM kinases direct the metabolic programming of intestinal intraepithelial lymphocytes

Intestinal intraepithelial lymphocytes (IEL) are an abundant population of tissue-resident T cells that protect and maintain the intestinal barrier. IEL respond to epithelial cell-derived IL-15, which is complexed to the IL-15 receptor α chain (IL-15/Rα). IL-15 is essential both for maintaining IEL homeostasis and inducing IEL responses to epithelial stress, which has been associated with Coeliac disease. Here, we apply quantitative mass spectrometry to IL-15/Rα-stimulated IEL to investigate how IL-15 directly regulates inflammatory functions of IEL. IL-15/Rα drives IEL activation through cell cycle regulation, upregulation of metabolic machinery and expression of a select repertoire of cell surface receptors. IL-15/Rα selectively upregulates the Ser/Thr kinases PIM1 and PIM2, which are essential for IEL to proliferate, grow and upregulate granzyme B in response to inflammatory IL-15. Notably, IEL from patients with Coeliac disease have high PIM expression. Together, these data indicate PIM kinases as important effectors of IEL responses to inflammatory IL-15.

Granzyme B prevents aberrant IL-17 production and intestinal pathogenicity in CD4+ T cells.

CD4+ T cell activation and differentiation are important events that set the stage for proper immune responses. Many factors are involved in the activation and differentiation of T cells, and these events are tightly controlled to prevent unwanted and/or exacerbated immune responses that may harm the host. It has been well documented that granzyme B, a potent serine protease involved in cell-mediated cytotoxicity, is readily expressed by certain CD4+ T cells, such as regulatory T cells and CD4+CD8αα+ intestinal intraepithelial lymphocytes, both of which display cytotoxicity associated with granzyme B. However, because not all CD4+ T cells expressing granzyme B are cytotoxic, additional roles for this protease in CD4+ T cell biology remain unknown. Here, using a combination of in vivo and in vitro approaches, we report that granzyme B-deficient CD4+ T cells display increased IL-17 production. In the adoptive transfer model of intestinal inflammation, granzyme B-deficient CD4+ T cells triggered a more rapid disease onset than their WT counterparts, and presented a differential transcription profile. Similar results were also observed in granzyme B-deficient mice infected with Citrobacter rodentium. Our results suggest that granzyme B modulates CD4+ T cell differentiation, providing a new perspective into the biology of this enzyme.

TAK-101 Nanoparticles Induce Gluten-Specific Tolerance in Celiac Disease: A Randomized, Double-Blind, Placebo-Controlled Study

In celiac disease (CeD), gluten induces immune activation, leading to enteropathy. TAK-101, gluten protein (gliadin) encapsulated in negatively charged poly(dl-lactide-co-glycolic acid) nanoparticles, is designed to induce gluten-specific tolerance. TAK-101 was evaluated in phase 1 dose escalation safety and phase 2a double-blind, randomized, placebo-controlled studies. Primary endpoints included pharmacokinetics, safety, and tolerability of TAK-101 (phase 1) and change from baseline in circulating gliadin-specific interferon-γ-producing cells at day 6 of gluten challenge, in patients with CeD (phase 2a). Secondary endpoints in the phase 2a study included changes from baseline in enteropathy (villus height to crypt depth ratio [Vh:Cd]), and frequency of intestinal intraepithelial lymphocytes and peripheral gut-homing T cells. In phase 2a, 33 randomized patients completed the 14-day gluten challenge. TAK-101 induced an 88% reduction in change from baseline in interferon-γ spot-forming units vs placebo (2.01 vs 17.58, P= .006). Vh:Cd deteriorated in the placebo group (-0.63, P= .002), but not in the TAK-101 group (-0.18, P= .110), although the intergroup change from baseline was not significant (P= .08). Intraepithelial lymphocyte numbers remained equal. TAK-101 reduced changes in circulating α4β7+CD4+ (0.26 vs 1.05, P= .032), αEβ7+CD8+ (0.69 vs 3.64, P= .003), and γδ (0.15 vs 1.59, P= .010) effector memory T cells. TAK-101 (up to 8 mg/kg) induced no clinically meaningful changes in vital signs or routine clinical laboratory evaluations. No serious adverse events occurred. TAK-101 was well tolerated and prevented gluten-induced immune activation in CeD. The findings from the present clinical trial suggest that antigen-specific tolerance was induced and represent a novel approach translatable to other immune-mediated diseases. ClinicalTrials.gov identifiers: NCT03486990 and NCT03738475.

MHC Class I on murine hematopoietic APC selects Type A IEL precursors in the thymus.

TCRαβ+ CD8α+ CD8β- intestinal intraepithelial lymphocytes (CD8αα IEL) are gut T cells that maintain barrier surface homeostasis. Most CD8αα IEL are derived from thymic precursors (IELp) through a mechanism referred to as clonal diversion. In this model, self-reactive thymocytes undergo deletion in the presence of CD28 costimulation, but in its absence undergo diversion to the IEL fate. While previous reports showed that IELp were largely β2m dependent, the APC that drive the development of these cells are poorly defined. We found that both CD80 and CD86 restrain IELp development, and conventional DCs play a prominent role. We sought to define a CD80/86 negative, MHCI positive APC that supports the development to the IEL lineage. Chimera studies showed that MHCI needs to be expressed on hematopoietic APC for selection. As thymic hematopoietic APC are heterogeneous in their expression of MHCI and costimulatory molecules, we identified four thymic APC types that were CD80/86neg/low and MHCI+ . However, selective depletion of β2m in individual APC suggested functional redundancy. Thus, while hematopoietic APC play a critical role in clonal diversion, no single APC subset is specialized to promote the CD8αα IEL fate.

From Initiators to Effectors: Roadmap Through the Intestine During Encounter of Toxoplasma gondii With the Mucosal Immune System.

The gastrointestinal tract is a major portal of entry for many pathogens, including the protozoan parasite Toxoplasma gondii. Billions of people worldwide have acquired T. gondii at some point in their life, and for the vast majority this has led to latent infection in the central nervous system. The first line of host defense against Toxoplasma is located within the intestinal mucosa. Appropriate coordination of responses by the intestinal epithelium, intraepithelial lymphocytes, and lamina propria cells results in an inflammatory response that controls acute infection. Under some conditions, infection elicits bacterial dysbiosis and immune-mediated tissue damage in the intestine. Here, we discuss the complex interactions between the microbiota, the epithelium, as well as innate and adaptive immune cells in the intestinal mucosa that induce protective immunity, and that sometimes switch to inflammatory pathology as T. gondii encounters tissues of the gut.

Circadian Clock Disruption Suppresses PDL1+ Intraepithelial B Cells in Experimental Colitis and Colitis-Associated Colorectal Cancer.

Background & AimsThe circadian clock is crucial for physiological homeostasis including gut homeostasis. Disorder of the circadian clock may contribute to many diseases including inflammatory bowel disease (IBD). However, the role and the mechanisms of circadian clock involvement in IBD still are unclear.MethodsDisorder of the circadian clock including chronic social jet lag and circadian clock gene deficiency mice (Bmal1-/-, and Per1-/-Per2-/-) were established. Dextran sulfate sodium (DSS) and/or azoxymethane were used to induce mouse models of colitis and its associated colorectal cancer. Flow cytometry, immunohistochemistry, immunofluorescence, Western blot, and reverse-transcription quantitative polymerase chain reaction were used to analyze the characteristics of immune cells and their related molecules.ResultsMice with disorders of the circadian clock including chronic social jet lag and circadian clock gene deficiency were susceptible to colitis. Functionally, regulatory B (Breg) cells highly expressing Programmed cell death 1 ligand 1 (PDL1) in intestinal intraepithelial lymphocytes (IELs) helped to alleviate the severity of colitis after DSS treatment and was dysregulated in DSS-treated Bmal1-/- mice. Notably, interleukin 33 in the intestinal microenvironment was key for Bmal1-regulated PDL1+ Breg cells and interleukin 33 was a target of Bmal1 transcriptionally. Dysregulated PDL1+ B cells induced cell death of activated CD4+ T cells in DSS-treated Bmal1-/- mice. Consequently, circadian clock disorder was characterized as decreased numbers of Breg+ PDL1+ cells in IELs and dysfunction of CD4+ T cells promoted colitis-associated colorectal cancer (CRC) in mice. In clinical samples from CRC patients, low expression of Bmal1 gene in paracancerous tissues and center area of tumor was associated closely with a poorer prognosis of CRC patients.ConclusionsOur study uncovers the importance of the circadian clock regulating PDL1+ Breg+ cells of IELs in IBD and IBD-associated CRC.

The downregulation of IL-18R defines bona fide kidney-resident CD8+ Tcells.

SummaryStepwise induction of CD69 and CD103 marks distinct differentiation stages of mucosal Trms. But the majority of non-mucosal Trm lacks CD103 expression. The expression of CD69 alone cannot faithfully define Trm cells in heavily vascularized non-mucosal tissues, such as the kidney. Here, we found that a subset of kidney Trms downregulated IL-18 receptor during differentiation. Via global transcriptional analysis and parabiosis experiments, we have discovered that the downregulation of interleukin-18 receptor (IL-18R) is associated with the establishment of tissue residency. Together with the expression of CD69, IL-18Rlo exclusively identify tissue-resident cells whereas IL-18Rhi population contains both tissue-resident and migratory ones. Local cytokines including transforming growth factor β (TGF-β) and interferon α (IFN-α)/β as well as TGF-β-dependent suppression of transcription factor Tcf-1 are essential for IL-18R downregulation during kidney Trm differentiation. Together, we identified a convenient surface marker to distinguish bona fide kidney-resident CD8+ T cells as well as underlying molecular mechanisms controlling this differentiation process.

Unconventional Intestinal Intraepithelial Lymphocytes in Health and Disease.

The intestinal epithelium is constantly exposed to a myriad of antigenic stimuli derived from commensals, food particles and pathogens present in the lumen of the intestines. This complex environment requires a similarly complex immune system capable of preventing exacerbated responses against food particles and commensals, while at the same time eliminating potential pathogens. These functions are accomplished in part by the intraepithelial lymphocyte (IEL) compartment. IELs are a diverse group of immune cells that primarily reside in between intestinal epithelial cells, maintaining an intimate association with these cells. IELs are a diverse population of cells: some of them express a T cell receptor (TCR), while others do not, and within TCR+ and TCR- IELs there are many IEL subpopulations that represent different developmental pathways and functions. In this review, we will focus on "unconventional" T cells present in the intestinal epithelium, in particular TCRγδ+, TCRαβ+CD4+CD8αα+, and TCRαβ+CD8αα+ IELs. We will discuss their development and potential functions both in humans and in mice.

Using a Reporter Mouse to Map Known and Novel Sites of GLP-1 Receptor Expression in Peripheral Tissues of Male Mice.

Glucagon-like peptide-1 receptor (GLP-1R) activation is used in the treatment of diabetes and obesity; however, GLP-1 induces many other physiological effects with unclear mechanisms of action. To identify the cellular targets of GLP-1 and GLP-1 analogues, we generated a Glp1r.tdTomato reporter mouse expressing the reporter protein, tdTomato, in Glp1r-expressing cells. The reporter signal is expressed in all cells where GLP-1R promoter was ever active. To complement this, we histologically mapped tdTomato-fluorescence, and performed Glp-1r mRNA in situ hybridization and GLP-1R immunohistochemistry on the same tissues. In male mice, we found tdTomato signal in mucus neck, chief, and parietal cells of the stomach; Brunner's glands; small intestinal enteroendocrine cells and intraepithelial lymphocytes; and myenteric plexus nerve fibers throughout the gastrointestinal tract. Pancreatic acinar-, β-, and δ cells, but rarely α cells, were tdTomato-positive, as were renal arteriolar smooth muscle cells; endothelial cells of the liver, portal vein, and endocardium; aortal tunica media; and lung type 1 and type 2 pneumocytes. Some thyroid follicular and parafollicular cells displayed tdTomato expression, as did tracheal cartilage chondrocytes, skin fibroblasts, and sublingual gland mucus cells. In conclusion, our reporter mouse is a powerful tool for mapping known and novel sites of GLP-1R expression in the mouse, thus enhancing our understanding of the many target cells and effects of GLP-1 and GLP-1R agonists.