Th17 Cell-Derived Amphiregulin Promotes Colitis-Associated Intestinal Fibrosis Through Activation of mTOR and MEK in Intestinal Myofibroblasts.

BACKGROUND & AIMS: Intestinal fibrosis is a significant complication of Crohn's disease (CD). Gut microbiota reactive Th17 cells are crucial in the pathogenesis of CD; however, how Th17 cells induce intestinal fibrosis is still not completely understood. METHODS: In this study, T-cell transfer model with wild-type (WT) and Areg-/- Th17 cells and dextran sulfate sodium (DSS)-induced chronic colitis model in WT and Areg-/- mice were used. CD4+ T-cell expression of AREG was determined by quantitative reverse-transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay. The effect of AREG on proliferation/migration/collagen expression in human intestinal myofibroblasts was determined. AREG expression was assessed in healthy controls and patients with CD with or without intestinal fibrosis. RESULTS: Although Th1 and Th17 cells induced intestinal inflammation at similar levels when transferred into Tcrßxδ-/- mice, Th17 cells induced more severe intestinal fibrosis. Th17 cells expressed higher levels of AREG than Th1 cells. Areg-/- mice developed less severe intestinal fibrosis compared with WT mice on DSS insults. Transfer of Areg-/- Th17 cells induced less severe fibrosis in Tcrßxδ-/- mice compared with WT Th17 cells. Interleukin (IL)6 and IL21 promoted AREG expression in Th17 cells by activating Stat3. Stat3 inhibitor suppressed Th17-induced intestinal fibrosis. AREG promoted human intestinal myofibroblast proliferation, motility, and collagen I expression, which was mediated by activating mammalian target of rapamycin and MEK. AREG expression was increased in intestinal CD4+ T cells in fibrotic sites compared with nonfibrotic sites from patients with CD. CONCLUSIONS: These findings reveal that Th17-derived AREG promotes intestinal fibrotic responses in experimental colitis and human patients with CD. Thereby, AREG might serve as a potential therapeutic target for fibrosis in CD.

Phospholipase A2-activating protein is associated with a novel form of leukoencephalopathy.

Leukoencephalopathies are a group of white matter disorders related to abnormal formation, maintenance, and turnover of myelin in the central nervous system. These disorders of the brain are categorized according to neuroradiological and pathophysiological criteria. Herein, we have identified a unique form of leukoencephalopathy in seven patients presenting at ages 2 to 4 months with progressive microcephaly, spastic quadriparesis, and global developmental delay. Clinical, metabolic, and imaging characterization of seven patients followed by homozygosity mapping and linkage analysis were performed. Next generation sequencing, bioinformatics, and segregation analyses followed, to determine a loss of function sequence variation in the phospholipase A2-activating protein encoding gene (PLAA). Expression and functional studies of the encoded protein were performed and included measurement of prostaglandin E2 and cytosolic phospholipase A2 activity in membrane fractions of fibroblasts derived from patients and healthy controls. Plaa-null mice were generated and prostaglandin E2 levels were measured in different tissues. The novel phenotype of our patients segregated with a homozygous loss-of-function sequence variant, causing the substitution of leucine at position 752 to phenylalanine, in PLAA, which causes disruption of the protein's ability to induce prostaglandin E2 and cytosolic phospholipase A2 synthesis in patients' fibroblasts. Plaa-null mice were perinatal lethal with reduced brain levels of prostaglandin E2 The non-functional phospholipase A2-activating protein and the associated neurological phenotype, reported herein for the first time, join other complex phospholipid defects that cause leukoencephalopathies in humans, emphasizing the importance of this axis in white matter development and maintenance.

Immunosuppression by Intestinal Stromal Cells.

This chapter summarizes evidence that intestinal myofibroblasts, also called intestinal stromal cells, are derived in the adult from tissue mesenchymal stem cells under homeostasis and may be replenished by bone marrow mesenchymal stromal (stem) cells that are recruited after severe intestinal injury. A comparison of mechanism of immunosuppression or tolerance by adult intestinal stromal cells (myofibroblasts) is almost identical with those reported for mesenchymal stem cells of bone marrow origin. The list of suppression mechanisms includes PD-L1 and PD-L2/PD-1 immune checkpoint pathways, soluble mediator secretion, toll-like receptor-mediated tolerance, and augmentation of Treg cells. Further, both mesenchymal stem cells and intestinal stromal cells express an almost identical repertoire of CD molecules. Lastly, others have reported that isolate intestinal stromal cells are capable of differentiating into bone and less well into chondrocyte, but not into adipocytes, a finding that we have confirmed. These findings suggest that intestinal stromal cells (myofibroblasts) are partially differentiated adult, tissue-resident stem cells which are capable of exerting immune tolerance in the intestine. Their role in repair of inflammatory bowel disease and immune suppression in colorectal cancer needs further investigation.

Impact of Colorectal Cancer Sidedness and Location on Therapy and Clinical Outcomes: Role of Blood-Based Biopsy for Personalized Treatment.

Colorectal cancer is one of the most common malignant diseases in the United States and worldwide, and it remains among the top three causes of cancer-related death. A new understanding of molecular characteristics has changed the profile of colorectal cancer and its treatment. Even controlling for known mutational differences, tumor side of origin has emerged as an independent prognostic factor, and one that impacts response to therapy. Left- and right-sided colon cancers differ in a number of key ways, including histology, mutational profile, carcinogenesis pathways, and microbiomes. Moreover, the frequency of certain molecular features gradually changes from the ascending colon to rectum. These, as well as features yet to be identified, are likely responsible for the ongoing role of tumor sidedness and colorectal subsites in treatment response and prognosis. Along with tumor molecular profiling, blood-based biopsy enables the identification of targetable mutations and predictive biomarkers of treatment response. With the application of known tumor characteristics including sidedness and subsites as well as the utilization of blood-based biopsy, along with the development of biomarkers and targeted therapies, the field of colorectal cancer continues to evolve towards the personalized management of a heterogeneous cancer.

Role of gastric epithelial cell-derived transforming growth factor beta in reduced CD4+ T cell proliferation and development of regulatory T cells during Helicobacter pylori infection.

Gastric epithelial cells (GECs) express the class II major histocompatibility complex (MHC) and costimulatory molecules, enabling them to act as antigen-presenting cells (APCs) and affect local T cell responses. During Helicobacter pylori infection, GECs respond by releasing proinflammatory cytokines and by increasing the surface expression of immunologically relevant receptors, including class II MHC. The CD4(+) T cell response during H. pylori infection is skewed toward a Th1 response, but these cells remain hyporesponsive. Activated T cells show decreased proliferation during H. pylori infection, and CD4(+) CD25(+) FoxP3(+) regulatory T cells (Tregs) are present at the site of infection. In this study, we examined the mechanisms surrounding the CD4(+) T cell responses during H. pylori infection and found that transforming growth factor ß (TGF-ß) plays a major role in these responses. GECs produced TGF-ß1 and TGF-ß2 in response to infection. Activated CD4(+) T cells in culture with H. pylori-treated GECs were decreased in proliferation but increased upon neutralization of TGF-ß. Naïve CD4(+) T cell development into Tregs was also enhanced in the presence of GEC-derived TGF-ß. Herein, we demonstrate a role for GEC-produced TGF-ß in the inhibition of CD4(+) T cell responses seen during H. pylori infection.

Degeneration of the pericryptal myofibroblast sheath by proinflammatory cytokines in inflammatory bowel diseases.

BACKGROUND & AIMS: Inflammatory bowel diseases (IBDs) are characterized by remodeling of the intestinal mucosa, which is associated with excessive cytokine release. Previous studies have shown that the epithelium in the crypt region of the mucosa in patients with Crohn's disease is susceptible to proinflammatory cytokines. We investigated whether the subepithelial myofibroblasts in this region were affected by these inflammatory conditions. METHODS: Immunofluorescence and immunohistochemistry were performed on inflamed and uninflamed specimens from patients with IBD to detect alpha-smooth muscle actin (alphaSMA), desmin, and tenascin-C. The effects of the proinflammatory cytokines interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma were analyzed in human intestinal myofibroblast cultures by immunoblotting and apoptosis assays. RESULTS: Immunofluorescence analysis revealed decreased levels of the extracellular matrix molecule tenascin-C in pericryptal sheaths and alphaSMA in the immediate vicinity of the crypts in the inflamed specimens, indicating that the myofibroblast pericryptal sheath is affected by proinflammatory cytokines. Although individual cytokines did not affect myofibroblast proliferation or survival, cytokine combinations triggered caspase-dependent apoptosis. alphaSMA levels were reduced significantly in cells exposed to cytokines, either alone or in combination, suggesting dedifferentiation of myofibroblasts. Proinflammatory cytokines did not affect tenascin-C expression, suggesting that the decrease observed in the inflamed mucosa resulted from myofibroblast apoptosis. CONCLUSIONS: The subepithelial myofibroblasts of the epithelial sheath are disrupted in the intestinal mucosa of patients with IBD. A loss of myofibroblasts appears to result from the susceptibility of these cells to proinflammatory cytokines.

Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens.

Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and promotion of the tumor microenvironment. While there are currently many techniques for the isolation of MFs from gastrointestinal (GI) tissues, this protocol introduces a novel element of isolation of these stromal cells from frozen tissue. Freezing GI tissue specimens not only allows the researcher to acquire samples from worldwide collaborators, biobanks, and commercial vendors, it also permits the delayed processing of fresh samples. The described protocol will consistently yield characteristic spindle-shaped cells with the MF phenotype that express the markers CD90, α-SMA and vimentin. As these cells are derived from patient samples, the use of primary cells also confers the benefit of closely mimicking MFs from disease states-namely cancer and inflammatory bowel diseases. This technique has been validated in gastric, small bowel, and colonic MF primary culture generation. Primary MF cultures can be used in a vast array of experiments over a number of passage and their purity assessed by both immunocytochemistry and flow cytometry analysis.

Peptide nanofiber-CaCO3 composite microparticles as adjuvant-free oral vaccine delivery vehicles.

To combat mucosal pathogens that cause gastrointestinal (GI) infections, local mucosal immunity is required which is best achieved through oral vaccination. Oral delivery of vaccines is also a safe and convenient alternative to injected vaccines due to its non-invasive nature and high compliance rate for all ages. However, the lack of effective and safe mucosal adjuvants, the selective permeability of the mucus barrier, and the harsh GI environment continue to pose a significant challenge for oral vaccine development. Microparticle-based strategies are attractive for oral vaccination due to their ability to efficiently penetrate the mucus barrier and have the added advantage of protecting the antigen in the harsh gastric environment. In this work, self-adjuvanting peptide nanofiber-CaCO3 composite microparticles were prepared and investigated for oral vaccine delivery. Compared to polymeric microparticles, inorganic CaCO3 microparticles have unique advantages due to the biocompatibility of CaCO3 as a natural mineral, mild preparation conditions, and its porous structure that is suitable for loading other materials. Particle size distribution, nanofiber loading efficiency, morphology, and degradation in simulated gastric fluid were characterized. The composite microparticles were efficient at penetrating the mucus barrier and were localized to immune inductive sites and elicited the production of mucosal antibody responses, particularly the protective IgA isotype following oral administration. The magnitude of the mucosal immune response was comparable to the gold-standard adjuvant cholera toxin B (CTB). Our results indicate that OVA-KFE8/CaCO3 composite microparticles are efficient self-adjuvanting oral vaccine delivery vehicles for induction of mucosal antibody responses.

The safety of two Bacillus probiotic strains for human use.

Probiotics based on Bacillus strains have been increasingly proposed for prophylactic and therapeutic use against several gastro-intestinal diseases. We studied safety for two Bacillus strains included in a popular East European probiotic. Bacillus subtilis strain that was sensitive to all antibiotics listed by the European Food Safety Authority. Bacillus licheniformis strain was resistant to chloramphenicol and clindamycin. Both were non-hemolytic and did not produce Hbl or Nhe enterotoxins. No bceT and cytK toxin genes were found. Study of acute toxicity in BALB/c mice demonstrated no treatment-related deaths. The oral LD(50) for both strains was more than 2 x 10(11) CFU. Chronic toxicity studies were performed on mice, rabbits, and pigs and showed no signs of toxicity or histological changes in either organs or tissues. We demonstrated that while certain risks may exist for the B. licheniformis strain considering antibiotic resistance, B. subtilis strain may be considered as non-pathogenic and safe for human consumption.