Lamina Propria Lymphocytes Research Articles

Artificial intelligence-based quantification of lymphocytes in feline small intestinal biopsies.

Feline chronic enteropathy is a poorly defined condition of older cats that encompasses chronic enteritis to low-grade intestinal lymphoma. The histological evaluation of lymphocyte numbers and distribution in small intestinal biopsies is crucial for classification and grading. However, conventional histological methods for lymphocyte quantification have low interobserver agreement, resulting in low diagnostic reliability. This study aimed to develop and validate an artificial intelligence (AI) model to detect intraepithelial and lamina propria lymphocytes in hematoxylin and eosin-stained small intestinal biopsies from cats. The median sensitivity, positive predictive value, and F1 score of the AI model compared with the majority opinion of 11 veterinary anatomic pathologists, were 100% (interquartile range [IQR] 67%-100%), 57% (IQR 38%-83%), and 67% (IQR 43%-80%) for intraepithelial lymphocytes, and 89% (IQR 71%-100%), 67% (IQR 50%-82%), and 70% (IQR 43%-80%) for lamina propria lymphocytes, respectively. Errors included false negatives in whole-slide images with faded stain and false positives in misidentifying enterocyte nuclei. Semiquantitative grading at the whole-slide level showed low interobserver agreement among pathologists, underscoring the need for a reproducible quantitative approach. While semiquantitative grade and AI-derived lymphocyte counts correlated positively, the AI-derived lymphocyte counts overlapped between different grades. Our AI model, when supervised by a pathologist, offers a reproducible, objective, and quantitative assessment of feline intestinal lymphocytes at the whole-slide level, and has the potential to enhance diagnostic accuracy and consistency for feline chronic enteropathy.

Effects of parenteral nutrition supplemented with beta‐hydroxy‐beta‐methylbutyrate on gut‐associated lymphoid tissue and morphology in mice

AbstractBackgroundParenteral nutrition (PN) without enteral nutrition (EN) leads to marked atrophy of gut‐associated lymphoid tissue (GALT), causing mucosal defense failure in both the gut and the extraintestinal mucosal system. We evaluated the effects of beta‐hydroxy‐beta‐methylbutyrate (HMB) on GALT and gut morphology in PN‐fed mice.MethodsExperiment 1: male Institute of Cancer Research mice were assigned to the Chow (n = 12), Control (standard PN: n = 10), or H600 and H2000 (PN containing 600 mg/kg or H2000 mg/kg body weight of Ca‐HMB: n = 12 and 10, respectively) groups. After 5 days of dietary manipulation, all mice were killed and the whole small intestine was harvested. GALT lymphocyte cell numbers and phenotypes of Peyer patch (PP), intraepithelial space, and lamina propria lymphocytes were evaluated. Experiment 2: 47 mice (Chow: n = 12; Control: n = 14; H600: n = 11; and H2000: n = 10) were fed for 5 days as in experiment 1. Proliferation and apoptosis of gut immune cells and mucosa, and protein expressions (mammalian target of rapamycin [mTOR], caspase‐3, and Bcl2) were evaluated in the small intestine.ResultsCompared with the Controls, the Chow and HMB groups showed significantly higher PP cell numbers, prevented gut mucosal atrophy, inhibited apoptosis of gut cells, and increased their proliferation in association with increased mTOR activity and Bcl2 expression.ConclusionHMB‐supplemented PN is a potentially novel method of preserving GALT mass and gut morphology in the absence of EN.

Comparative analysis of antigen-presenting cells in gingival tissues in healthy and periodontitis patients

AimsMicrobial flora of dental plaque trigger innate and adaptive immune responses. The function of antigen-presenting cells (APCs) is to bridge the innate and adaptive immune systems. The human immune system...

Discordant effects of Janus kinases inhibition ex-vivo on inflammatory responses in colonic compared to ileal mucosa.

Janus kinase (JAK) inhibitors are used for treating inflammatory bowel diseases (IBD). We aimed to identify molecular effects of JAK inhibition in human intestinal mucosa, considering IBD location and phenotype. Colonic and ileal explants from patients with ulcerative colitis (UC), Crohn's disease (CD), and non-IBD controls (NC) were assessed for phosphorylated signal transducers and activators of transcription (p-STAT) levels and Inflammatory genes expression panel in response to ex-vivo JAK inhibitor (tofacitinib). Cytokine production by lamina propria lymphocytes in response to tofacitinib was assessed. Human intestinal organoids were used to investigate JAK inhibitors' effects on iNOS expression. Explants were collected from 68 patients (UC=20; CD=20; NC=28). p-STAT1\3\5 inhibition rates varied, being higher in colonic compared to ileal explants. p-STAT1\3 inhibition rates negatively correlated with CRP levels. While significant alterations in 120 of 255 inflammatory genes were observed in colonic explants, only 30 were observed in ileal NC explants. In colonic explants from UC, significant alterations were observed in 5 genes, including NOS2. JAK inhibition significantly decreased Th1\Th2\Th17-related cytokine production from lamina propria lymphocytes. Various JAK inhibitors reduced IFN-γ-induced increase in iNOS expression in organoids. Site-specific anti-inflammatory effect of JAK inhibition by tofacitinib was noticed, whereby the colon was more robustly affected than the ileum. Ex-vivo response to tofacitinib is individual. JAK inhibition may attenuate inflammation by decreasing iNOS expression. Ex-vivo mucosal platforms may be a valuable resource for studying personalized drug effects in patients with IBD.

Clinical Features and Histopathological Analysis of Oral Lichen Planus: An Analysis of 105 Chinese Patients.

To study the clinical and pathological characteristics of oral lichen planus (OLP) in a large sample. A comprehensive analysis was conducted on 105 patients with oral lichen planus (OLP), considering various factors including sex, age, disease site, lesion type, lesion area, morphological characteristics, self-reported symptoms, and history of systemic diseases. Histopathological examination was performed for each patient, and the pathology results were analysed according to sex and age group. 70.5% of the OLP patients were female, and OLP was most likely to occur in the cheek, followed by the tongue, lips, gums and palate. The patients with moderate pain according to the VAS score accounted for 60%. Thirty-nine percent of the OLP patients had a systemic disease, and the most common clinical type of OLP was nonerosive. Most of the pathological results showed liquefaction degeneration of basal cells and infiltration of lamina propria lymphocytes. There was no statistically significant difference in pathological manifestations between male and female patients, and there were statistically significant differences in pathological manifestations among different ages patients. This study analysed the sociodemographic data and clinical manifestations of 105 OLP patients to guide follow-up treatment planning and disease monitoring. Moreover, pathological manifestations should be analysed to avoid delayed treatment and to monitor for carcinogenesis. Furthermore, the correlation of pathological manifestations among OLP patients with different sexes and ages is conducive to further research on the specific differential manifestations and possible underlying mechanisms involved.

Update: Induction of Inflammatory Bowel Disease in Immunodeficient Mice by Injection of Naïve CD4+ T cells (T Cell Transfer Model of Colitis).

The intestinal inflammation induced by injection of naïve CD4+ T cells into lymphocyte-deficient hosts (more commonly known as the T cell transfer model of colitis) shares many features of idiopathic inflammatory bowel disease (IBD) in humans, such as epithelial cell hyperplasia, crypt abscess formation, and dense lamina propria lymphocyte infiltration. As such, it provides a useful tool for studying mucosal immune regulation as it relates to the pathogenesis and treatment of IBD in humans. In the IBD model described here, colitis is induced in Rag (recombination-activating gene)-deficient mice by reconstitution of these mice with naïve CD4+CD45RBhi T cells through adoptive T cell transfer. Although different recipient hosts of cell transfer can be used, Rag-deficient mice are the best characterized and support studies that are both flexible and reproduceable. As described in the Basic Protocol, in most studies the transferred cells consist of naïve CD4+ T cells (CD45RBhi T cells) derived by fluorescence-activated cell sorting from total CD4+ T cells previously purified using immunomagnetic negative selection beads. In a Support Protocol, methods to characterize colonic disease progression are described, including the monitoring of weight loss and diarrhea and the histological assessment of colon pathology. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Induction of IBD in Rag-deficient mice by the transfer of naïve CD4+CD45RBhi T cells Support Protocol: Monitoring development of colitis.

Method of storage impacts graft immune cell populations in intestinal transplantation

Intestinal transplantation (IT) is the only treatment for intestinal failure patients who cannot tolerate parenteral nutrition. Although cold storage (CS) is the gold standard for intestinal allograft preservation, normothermic machine perfusion (NMP) has improved transplantation success in other organs. Allograft immune modulation is thought to contribute to this success. The objective of this study was to assess the impact of NMP on intestinal immune cell populations as it relates to ultimate allograft and recipient health. We hypothesized that NMP would stabilize overall immune cell populations while reducing pro-inflammatory cohorts, thus improving IT success. Porcine intestines were stored for 6H at 4°C (CS-T6, n=6) or perfused at 34°C (NMP-T6, n=8) and transplanted. Samples were collected following intestinal procurement (T0), storage (T6), 1-hour post-transplant (T1PT), and euthanasia (T48). Flow cytometric profiling and quantification of intraepithelial (IEL) and lamina propria (LP) lymphocytes and NK cells were performed at T0, T6, and T48. Immunofluorescence (IF) identified CD3+ T-cells at T1PT. One-way ANOVA analyzed cell counts with significance set at p< 0.05. Flow cytometry revealed that NMP preserved CD3+ IELs in the jejunum at T6 (63.74% vs 32.79%, p< 0.0001), and in the ileum at T6 (51.31% vs 42.77%, p< 0.0001) and at T48 (57.73% vs 34.21%, p<0.0001). Immunofluorescent cell quantification confirmed this preservation of CD3+ IEL at T1PT in the crypts of the jejunum (4.9% vs 2.1 %, p< 0.05) and ileum (6.9% vs 2.8%, p< 0.001) compared to CS. However, upon further subpopulation analyses using flow cytometry, ileal IEL T-cells were reduced throughout NMP compared to CS (T6 28.7% vs 44.0%; T48 16.65% vs 22.37%; p< 0.0001). NK cells were also decreased in ileal and jejunal LP at NMP-T6 compared to T0 (3.6% vs 5.6%, p< 0.01; 2.4% vs 4.3% p< 0.05). NMP reduces NK cells, a cell type known to be associated with rejection. Interestingly, NMP preserved the overall CD3+ T cell populations in the jejunum and ileum at T6, T1PT, and T48. The subpopulation of IELs, however, were reduced in the ileum following NMP. The role of these populations, specifically in IT, has yet to be determined. Further characterization of T-cell subpopulations and dynamics is needed. Complimentary evaluations of the microbiome and proteomic responses to IT NMP are ongoing. Understanding immune cell dynamics will improve IT outcomes for patients requiring this critical, lifesaving surgery. U.S. Department of Defense PR181265; NIH K01OD010199 SERCA, T32OD011130-15. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Number of intraepithelial lymphocytes and presence of a subepithelial band in normal colonic mucosa differs according to stainings and evaluation method

Chronic watery diarrhea is a frequent symptom. In approximately 10% of the patients, a diagnosis of microscopic colitis (MC) is established. The diagnosis relies on specific, but sometimes subtle, histopathological findings. As the histology of normal intestinal mucosa vary, discriminating subtle features of MC from normal tissue can be challenging and therefore auxiliary stainings are increasingly used. The aim of this study was to determine the variance in number of intraepithelial lymphocytes (IELs) and presence of a subepithelial band in normal ileum and colonic mucosa, according to different stains and digital assessment. Sixty-one patients without diarrhea referred to screening colonoscopy due to a positive feacal blood test and presenting with endoscopically normal mucosa were included. Basic histological features, number of IELs, and thickness of a subepithelial band was manually evaluated and a deep learning-based algorithm was developed to digitally determine the number of IELs in each of the two compartments; surface epithelium and cryptal epithelium, and the density of lymphocytes in the lamina propria compartment. The number of IELs was significantly higher on CD3-stained slides compared with slides stained with hematoxylin-and-eosin (HE) (p<0.001), and even higher numbers were reached using digital analysis. No significant difference between right and left colon in IELs or density of CD3-positive lymphocytes in lamina propria was found. No subepithelial band was present in HE-stained slides while a thin band was visualized on special stains. Conclusively, in this cohort of prospectively collected ileum and colonic biopsies from asymptomatic patients, the range of IELs and detection of a subepithelial collagenous band varied depending on the stain and method used for assessment. As assessment of biopsies from patients with diarrhea constitute a considerable workload in the pathology departments digital image analysis is highly desired. Knowledge provided by the present study highlight important differences that should be considered before introducing this method in the clinic.

A254 CANAGLIFLOZIN EXACERBATES CHEMICALLY INDUCED COLITIS WHEN COMBINED WITH A HIGH SUGAR DIET

Abstract Background Consumption of high-sugar diets exacerbates inflammation and hinders the healing process in animal models of acute colitis. Canagliflozin (CANA) is a sodium glucose cotransporter-2 (SGLT2) inhibitor primarily used for the management of type 2 diabetes and cardiovascular disease. CANA has demonstrated the ability to suppress human T cell and macrophage effector function and to improve inflammation in rat colitis models. Based on these findings, we hypothesized that CANA would mitigate the deleterious effects of a high sugar diet in a DSS colitis model through its effects on immune effector cells. Aims The aim of this study was to examine the effects of a high sugar diet and CANA administration on colonic inflammation in an animal model of acute colitis. Methods Adult wild-type male and female 129/SvEv mice were placed on standard chow or a high-sugar diet (HSD) containing 50% sucrose (AIN76A) ± CANA (10mg/kg) gavaged daily (n=4-6 for each group). Following three days on the respective diet, mice received 1.8-2% dextran sodium sulfate (DSS) in their drinking water for 5 days, followed by water alone for 4 days. Disease Activity Index (DAI) was computed daily based on changes in animal weight, stool consistency, and blood in stool. Colonic tissues were evaluated for histological changes, weight-to-length ratio, and cytokine levels using the MesoScale discovery platform. Results Mice on the HSD had increased severity of colitis compared with chow fed mice as evidenced by enhanced DAI, increased weight loss, and increased levels of colonic TNFα (pampersand:003C0.02). Response to CANA treatment was both sex and diet dependent. Contrary to our hypothesis, CANA treatment increased severity of colitis in male mice on the HSD as evidenced by increased DAI and delayed healing with increased enterocyte injury and increased levels of lamina propria neutrophils and lymphocytes. CANA treatment did not exacerbate disease severity in females on the HSD but did delay healing as evidenced by increased lamina propria neutrophils and lymphocytes and worse enterocyte injury. These effects were not observed in mice that were fed standard chow diets with CANA. Conclusions A high-sugar diet significantly exacerbates chemically induced colitis and delays healing. This effect is intensified when combined with CANA, especially in male mice. These findings indicate that effects of SGLT2 inhibitors on colonic inflammation can be modulated by both dietary intake and sex. Funding Agencies CCC

A272 EFFECTS OF EMPAGLIFLOZIN ON CHEMICALLY INDUCED COLITIS IN A MOUSE MODEL ARE MODULATED BY SEX AND DIETARY INTERACTIONS

Abstract Background High sugar diets have been shown to dramatically increase disease severity in mouse models of colitis. Empagliflozin (EMPA) is a highly selective sodium glucose cotransporter-2 (SGLT2) inhibitor that is used therapeutically as an antihyperglycemic agent in the management of type 2 diabetes. In human trials EMPA treatment exerts potent anti-inflammatory effects independently of glycemic control. Further, we have previously demonstrated in a genetic mouse model of colitis that EMPA treatment was highly effective in improving colonic inflammation. Based on these findings, we hypothesized that EMPA treatment may also be effective in mitigating disease severity in chemically induced colitis in mice fed a high sugar diet. Aims The aim of this study was to examine the effects of treatment with EMPA on chemically induced colitis in mice fed a high sugar diet Methods At 6-8 weeks of age, wild-type 129/SvEv mice were placed on chow (CH) or high sugar diet (HS) (50% sucrose: AIN76A) ± EMPA (10mg/kg). After two days on the diet, mice were administered dextran sodium sulfate (DSS) for 5 days in drinking water followed by water alone for 2 days (n=4-6 mice for all groups). Disease activity index (DAI) was calculated daily from animal weight change, stool consistency, and stool hemoccult. As a measurement of degree of healing, colonic tissues were evaluated at day 9 for histological changes, weight-to-length ratio, and cytokine levels by ELISA. As a measure of EMPA functionality, urinary and blood glucose levels were measured. Results Mice on the HS diet demonstrated increased susceptibility to colitis compared with chow fed mice with increased colonic levels of IL-1β, weight loss, and DAI. Significant interactions between sex and diet were seen in responses to EMPA treatment. EMPA treatment did not alter severity of colitis but did significantly delay healing in male mice on both the HS and chow diets as evidenced by increased DAI and increased enterocyte injury with increased levels of lamina propria neutrophils and lymphocytes. EMPA treatment did not increase disease severity in females on either diet. No differences were seen in colonic TNFα, IL-10, IL-12p70, or IL-22 between any of the groups. Urinary glucose levels were elevated in mice receiving EMPA and significantly higher in males compared with females (pampersand:003C0.05). EMPA treatment did not alter fasting blood glucose levels. Conclusions A high sugar diet strongly exacerbates disease severity in a sex-dependent manner in a chemically induced model of colitis. Further, contrary to our hypothesis, EMPA treatment did not improve colitis in females and worsened disease in males suggesting that the documented anti-inflammatory effects of EMPA may be condition dependent. Funding Agencies CCC

P082 Downregulated apelin expression in T cells in the setting of Chronic Colitis

Abstract Background APJ is a G protein-coupled receptor and was originally found as an orphan receptor. Isolation of apelin (APL) from the alimentary tract was found to be the endogenous ligand for APJ. It has been reported that APL is upregulated in the colonic tissues of a murine model of acute colitis induced by dextran sodium sulfate (DSS), and thus it may be suggested to be associated with the pathogenesis of inflammatory bowel diseases (IBD) in humans. However, the mechanism and function of APL in the context of IBD are still not understood. Here, we analyzed APL expression in an animal model of chronic colitis to define its function in the pathogenesis of IBD. Methods Each cell type in the colonic tissue, including epithelial cells and lamina propria lymphocytes, were first isolated from wild type C57BL6 mice (WT) to assess APL expression. Next, naïve T cells isolated from WT were adoptively transferred into Rag deficient mice (Rag-/-) to induce chronic colitis, and then APL expression was measured either in the colonic tissue or in the isolated splenic and colonic CD4+ T cells from these T cell-reconstituted Rag-/- to compare with those of WT or Rag-/- without colitis. In addition, WT naïve T cells were differentiated into either Th1, Th2 or Th17 in vitro to analyze APL expression. Finally, the Rag-/- receiving naïve T cells were administered a synthetic APL peptide, APL-13, to assess the severity of colitis. Results Semi-quantitative PCR (qPCR) revealed that CD4+ T cells express relatively higher level of APL compared to other cell types including the epithelial cells in colonic tissue from WT. However, APL expression in the colonic tissues from the Rag-/- induced chronic colitis was unexpectedly downregulated compared to control groups without colitis; this is not consistent with the previous report using acute DSS colitis model. Subsequently, qPCR revealed significantly decreased APL expression in the splenic and colonic T cells from Rag-/- induced colitis compared to that of WT. APL expressions in the effecter T cells in vivo and all of the differentiated T cells in vitro were also significantly downregulated compared to naïve T cells and non-differentiated control T cells, respectively. Given these results, APL-13 was injected into the Rag-/- that underwent T cell reconstitution to antagonize the APL downregulation. This resulted in reduced severity of colitis compared to that of vehicle control-injected group. Conclusion These results suggest that T cells can be one of the major sources of APL in colonic tissues, and APL downregulation in effecter T cells may lead to the development of chronic colitis. In addition, this study also suggests that APL may be a novel therapeutic target for IBD.

P028 A pathogenic role of activated mucosal-associated invariant T cells in an animal model of Inflammatory Bowel Disease

Abstract Background It is known that the mucosal-associated invariant T (MAIT) cells are the innate-like T cells that are restricted by the major histocompatibility complex-related molecule 1 (MR1), and that these cells express a semi-invariant T cell receptor. We have previously reported that the activation status of the circulating MAIT cells in patients with Ulcerative Colitis (UC) is associated with disease activity, and these cells infiltrate the inflamed colonic mucosa. These findings imply that MAIT cells are involved in the pathogenesis of Inflammatory Bowel Disease (IBD). However, the role of MAIT cells in the setting of IBD has not been revealed. Therefore, we investigated the role of MAIT cells in an animal model of UC. Methods To this end, we utilized MR1 deficient mice (MR1-/-), which lack MAIT cells, and isobutyl 6-formyl pterin (i6-FP), which is a synthetic antagonistic MR1 ligand. MR1-/- on the C57BL/6 background, their littermate wild-type controls, and C57BL/6 mice were sensitized by rectal administration of oxazolone to induce colitis. These were then administered an oral i6-FP. Splenocytes (SPL) and colonic lamina propria lymphocytes (LPL) were isolated from mice receiving i6-FP to measure cytokine production. MR1-/-, i6-FP-treated mice and their controls were orally administered FITC-dextran to analyze intestinal permeability. The peripheral blood mononuclear cells (PBMC) from the patients with UC were also isolated to study the effect of i6-FP on cytokine production by MAIT cells. Results MR1 deficiency or i6-FP treatment resulted in reduced severity of oxazolone-induced colitis. Mice treated with i6-FP resulted in reduced MAIT cell production of pro-inflammatory cytokines such as IFN-g and TNF in both SPL and colonic LPL. Similar results were also observed in PBMC isolated from the patients with UC when incubated with i6-FP. Although MR1 deficiency resulted in increased intestinal permeability, i6-FP administration did not affect gut integrity in mice. Conclusion The current studies indicate that MAIT cells have a pathogenic role in colitis and suppressing activation of these cells may reduce the severity of colitis without affecting gut integrity. Thus, MAIT cells may be potential therapeutic targets for IBD including UC.

Single-Cell Transcriptional Analysis of Lamina Propria Lymphocytes in the Jejunum Reveals Innate Lymphoid Cell-like Cells in Pigs.

Pigs are the most suitable model to study various therapeutic strategies and drugs for human beings, although knowledge about cell type-specific transcriptomes and heterogeneity is poorly available. Through single-cell RNA sequencing and flow cytometry analysis of the types in the jejunum of pigs, we found that innate lymphoid cells (ILCs) existed in the lamina propria lymphocytes (LPLs) of the jejunum. Then, through flow sorting of live/dead-lineage (Lin)-CD45+ cells and single-cell RNA sequencing, we found that ILCs in the porcine jejunum were mainly ILC3s, with a small number of NK cells, ILC1s, and ILC2s. ILCs coexpressed IL-7Rα, ID2, and other genes and differentially expressed RORC, GATA3, and other genes but did not express the CD3 gene. ILC3s can be divided into four subgroups, and genes such as CXCL8, CXCL2, IL-22, IL-17, and NCR2 are differentially expressed. To further detect and identify ILC3s, we verified the classification of ILCs in the porcine jejunum subgroup and the expression of related hallmark genes at the protein level by flow cytometry. For systematically characterizing ILCs in the porcine intestines, we combined our pig ILC dataset with publicly available human and mice ILC data and identified that the human and pig ILCs shared more common features than did those mouse ILCs in gene signatures and cell states. Our results showed in detail for the first time (to our knowledge) the gene expression of porcine jejunal ILCs, the subtype classification of ILCs, and the markers of various ILCs, which provide a basis for an in-depth exploration of porcine intestinal mucosal immunity.

Prolonged administration of total glucosides of paeony improves intestinal immune imbalance and epithelial barrier damage in collagen-induced arthritis rats based on metabolomics-network pharmacology integrated analysis.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by synovial inflammation and joint damage with complex pathological mechanisms. In recent years, many studies have shown that the dysregulation of intestinal mucosal immunity and the damage of the epithelial barrier are closely related to the occurrence of RA. Total glucosides of paeony (TGP) have been used clinically for the treatment of RA in China for decades, while the pharmacological mechanism is still uncertain. The purpose of this study was to investigate the regulatory effect and mechanism of TGP on intestinal immunity and epithelial barrier in RA model rats. The results showed that TGP alleviated immune hyperfunction by regulating the ratio of CD3+, CD4+ and CD8+ in different lymphocyte synthesis sites of the small intestine, including Peyer's patches (PPs), intraepithelial lymphocytes (IELs), and lamina propria lymphocytes (LPLs). Specially, TGP first exhibited immunomodulatory effects on sites close to the intestinal lumen (IELs and LPLs), and then on PPs far away from the intestinal lumen as the administration time prolonged. Meanwhile, TGP restores the intestinal epithelial barrier by upregulating the ratio of villi height (V)/crypt depth (C) and expression of tight junction proteins (ZO-1, occludin). Finally, the integrated analysis of metabolomics-network pharmacology was also used to explore the possible regulation mechanism of TGP on the intestinal tract. Metabolomics analysis revealed that TGP reversed the intestinal metabolic profile disturbance in CIA rats, and identified 32 biomarkers and 163 corresponding targets; network pharmacology analysis identified 111 potential targets for TGP to treat RA. By intersecting the results of the two, three key targets such as ADA, PNP and TYR were determined. Pharmacological verification experiments showed that the levels of ADA and PNP in the small intestine of CIA rats were significantly increased, while TGP significantly decreased their ADA and PNP levels. In conclusion, purine metabolism may play an important role in the process of TGP improving RA-induced intestinal immune imbalance and impaired epithelial barrier.

Gut Inflammation in axial Spondyloarthritis patients is characterized by a marked Type 17 skewed mucosal Innate-like T cell signature.

Patients with spondyloarthritis (SpA) often present with microscopic signs of gut inflammation, a risk factor for progressive disease. We investigated whether mucosal innate-like T cells are involved in dysregulated interleukin-23 (IL-23)/IL-17 responses in the gut-joint axis in SpA. Ileal and colonic intraepithelial lymphocytes (IELs), lamina propria lymphocytes (LPLs), and paired peripheral blood mononuclear cells (PBMCs) were isolated from treatment-naive patients with nonradiographic axial SpA with (n=11) and without (n=14) microscopic gut inflammation and healthy controls (n=15) undergoing ileocolonoscopy. The presence of gut inflammation was assessed histopathologically. Immunophenotyping of innate-like T cells and conventional T cells was performed using intracellular flow cytometry. Unsupervised clustering analysis was done by FlowSOM technology. Serum IL-17A levels were measured via Luminex. Microscopic gut inflammation in nonradiographic axial SpA was characterized by increased ileal intraepithelial γδ-hi T cells, a γδ-T cell subset with elevated γδ-T cell receptor expression. γδ-hi T cellswere also increased in PBMCs of patients with nonradiographic axial SpA versus healthy controls and were strongly associated with Ankylosing Spondylitis Disease Activity Score. The abundance of mucosal-associated invariant T cells and invariant natural killer T cells was unaltered. Innate-like T cells in the inflamed gut showed increased RORγt, IL-17A, and IL-22 levels with loss of T-bet, a signature that was less pronounced in conventional T cells. Presence of gut inflammation was associated with higher serum IL-17A levels. In patients treated with tumor necrosis factor blockade, the proportion of γδ-hi cells and RORγt expression in blood was completely restored. Intestinal innate-like T cells display marked type 17 skewing in the inflamed gut mucosa of patients with nonradiographic axial SpA. γδ-hi T cells are linked to intestinal inflammation and disease activity in SpA.

Anti-Inflammatory Effects of Synthetic Peptides Based on Glucocorticoid-Induced Leucine Zipper (GILZ) Protein for the Treatment of Inflammatory Bowel Diseases (IBDs).

Glucocorticoids (GCs) are commonly used to treat autoimmune and inflammatory diseases, but their clinical effects and long-term use can lead to serious side effects. New drugs that can replace GCs are needed. Glucocorticoid-induced leucine zipper (GILZ) is induced by GCs and mediates many of their anti-inflammatory effects, such as inhibiting the pro-inflammatory molecule NF-κB. The GILZ C-terminal domain (PER region) is responsible for GILZ/p65NF-κB interaction and consequent inhibition of its transcriptional activity. A set of five short peptides spanning different parts of the PER region of GILZ protein was designed, and their anti-inflammatory activity was tested, both in vitro and in vivo. We tested the biological activity of GILZ peptides in human lymphocytic and monocytic cell lines to evaluate their inhibitory effect on the NF-κB-dependent expression of pro-inflammatory cytokines. Among the tested peptides, the peptide named PEP-1 demonstrated the highest efficacy in inhibiting cell activation in vitro. Subsequently, PEP-1 was further evaluated in two in vivo experimental colitis models (chemically induced by DNBS administration and spontaneous colitis induced in IL-10 knock-out (KO) mice (to assess its effectiveness in counteracting inflammation. Results show that PEP-1 reduced disease severity in both colitis models associated with reduced NF-κB pro-inflammatory activity in colon lamina propria lymphocytes. This study explored GILZ-based 'small peptides' potential efficacy in decreasing lymphocyte activation and inflammation associated with experimental inflammatory bowel diseases (IBDs). Small peptides have several advantages over the entire protein, including higher selectivity, better stability, and bioavailability profile, and are easy to synthesize and cost-effective. Thus, identifying active GILZ peptides could represent a new class of drugs for treating IBD patients.

Paeonol alleviates ulcerative colitis in mice by increasing short-chain fatty acids derived from Clostridium butyricum

BackgroundIncreasing evidence suggests that repairing the damaged intestinal epithelial barrier and restoring its function is the key to solving the problem of prolonged ulcerative colitis. Previous studies have shown that paeonol (pae) can alleviate colitis by down-regulating inflammatory pathways. In addition, pae also has a certain effect on regulating intestinal flora. However, it remains unclear whether pae can play a role in repairing the intestinal barrier and whether there is a relationship between the therapeutic effect and the gut microbiota. PurposesThe aim of this study is to investigate the effect of pae on intestinal barrier repair in UC mice and how the gut microbiota plays a part in it. Study Design and MethodsThe therapeutic effect of pae was evaluated in a 3% DSS-induced UC mouse model. The role of pae in repairing the intestinal barrier was evaluated by detecting colonic cupped cells by Alcian blue staining, the expression of colonic epithelial tight junction protein by immunofluorescence and western blot, and the proportion of IL-22+ILC3 cells in the lamina propria lymphocytes by flow cytometry. Subsequently, 16S rRNA sequencing was used to observe the changes in intestinal flora, GC-MS was used to detect the level of SCFAs, and qPCR was used to identify the abundance of Clostridium butyricum in the intestine to evaluate the effect of pae on the gut microbiota. The antibiotic-mediated depletion of the gut flora was then used to verify that pae depends on C. butyricum to play a healing role. Finally, non-targeted metabolomics was employed to investigate the potential pathways of pae regulating C. butyricum. ResultsPae could improve intestinal microecological imbalance and promote the production of short-chain fatty acids (SCFAs). Most importantly, we identified C. butyricum as a key bacterium responsible for the intestinal barrier repair effect of pae in UC mice. Eradication of intestinal flora by antibiotics abolished the repair of the intestinal barrier and the promotion of SCFAs production by pae, while C. butyricum colonization could restore the therapeutic effects of pae in UC mice, which further confirmed that C. butyricum was indeed the “driver bacterium” of pae in UC treatment. Untargeted metabolomics showed that pae regulated some amino acid metabolism and 2-Oxocarboxylic acid metabolism in C. butyricum. ConclusionsOur study showed that the restoration of the impaired intestinal barrier by pae to alleviate colitis is associated with increased C. butyricum and SCFAs production, which may be a promising strategy for the treatment of UC.

B- and T-cell lymphocytes and other immune cell infiltration in the duodenal and rectal mucosa of severe asthmatic horses.

The objectives of this study were to quantify lymphocytes and eosinophils in the mucosa of the duodenum and rectum in asthmatic horses. 8 healthy and 10 asthmatic horses. Asthmatic horses were evaluated in a symptomatic (after 6 weeks of exposure to moldy hay) and asymptomatic status (3 and 7 months after being fed alfalfa pellets [n = 4] or treated with inhaled fluticasone [6]). Duodenal and rectal biopsies were endoscopically (n = 4 to 6) taken in each horse. Eosinophils were counted on slides stained with hematoxylin, eosin, phloxine, and saffron, and immunohistochemistry was used to evaluate T and B lymphocytes using CD3 and CD20, respectively. The duodenal and rectal epithelium of asthmatic and control horses contained exclusively T lymphocytes (CD3). Symptomatic asthmatic horses, compared to controls, had a significantly higher number of T lymphocytes (CD3) in the duodenal epithelium (P = .016) and the adjacent lamina propria of the villi (P = .04). Compared to symptomatic asthmatic horses, the fluticasone-treated group had significantly fewer T lymphocytes in the total lamina propria of the rectal mucosa (P < .01). Taken together, these results suggest that asthmatic horses have greater infiltration of T lymphocytes in the duodenal and rectal mucosa, indicating a certain degree of inflammation, which could be due to a systemic inflammatory effect and/or a local effect of ingested hay allergens in asthmatic horses. Systemic markers of inflammation have not been investigated to better qualify if the infiltration noted is due to a local and/or systemic effect.

S1PR1-dependent migration of ILC3s from intestinal tissue to the heart in a mouse model of viral myocarditis.

Type 3 innate lymphocytes (ILC3s) have recently been reported as key factors in inflammatory diseases, however, their role in viral myocarditis is unclear. By flow cytometry, CVB3 (Coxsachievirus B3)-induced myocarditis mice were detected to increase the number of ILC3s, and their main type was NKp46 + ILC3. In contrast, application of CD90.2 neutralizing antibody in T-cell-deficient mice reduced the number of ILCs and improved myocarditis. ILCs from CD45.1 mouse intestinal lamina propria lymphocytes were adoptively transferred into recipient mice, and a comparable proportion of CD45.1+ cells were observed in the hearts of CVB3-infected recipient mice. The upregulation of S1PR1 (Recombinant Sphingosine 1 Phosphate Receptor 1), KLF2 (Kruppel-like factor 2), CXCR6, and CXCL16 in the hearts of CVB3-infected mice, as well as the greatly reduced numbers of ILCs infiltrating the hearts after S1PR1 inhibition, suggest that intestinal ILCs may migrate to the hearts via the CXCL16/CXCR6 axis. Taken together, our results demonstrate that increased ILC3 in the heart during viral myocarditis may contribute to inflammatory progression, and that this increased population of ILC3 likely originates from the intestine.

CD226 deficiency exacerbated intestinal immune dysregulation in mice with dinitrochlorobenzene-induced atopic dermatitis.

Intestinal mucosal immunity plays a pivotal role in host defence. In this study, we found that cluster of differentiation 226 (CD226) gene knockout (KO) led to more severe atopic dermatitis (AD)-related skin pathologies and bowel abnormalities in a 2,4-dinitrochlorobenzene (DNCB)-induced AD-like mouse model. Following DNCB administration, the expression of CD226 was elevated in intestinal mucosal tissues, including group 3 innate lymphoid cells (ILC3s) and CD4+ T cells of Peyer's patches (PPs). CD226 deficiency led to an overactive intestinal immune response in the AD-like mice, as evidenced by increased inflammation and Th1/Th2-related cytokine levels as well as increased Paneth cell numbers and antimicrobial peptide (AMP) expression, which was likely due to the higher interleukin (IL)-22 production in the lamina propria. Additionally, CD226 deficiency increased the production of IL-4 and IL-17 in mesenteric lymph nodes as well as the number of PPs and expression of immunoglobulin (Ig) A in B cells. Moreover, insufficient expression of CD226 affected the characterization of intraepithelial and lamina propria lymphocytes in the intestinal mucosa. Finally, the number of PPs was increased in CD4+ T cell-specific CD226 KO and regulatory T (Treg) cell-specific CD226 KO mice; thus, loss of CD226 in Treg cells resulted in impaired Treg cell-suppressive function. Therefore, our findings indicate that CD226 deficiency alters intestinal immune functionality in inflammatory diseases.