Iryna V. Pinchuk

Intestinal myofibroblasts: targets for stem cell therapy

The subepithelial intestinal myofibroblast is an important cell orchestrating many diverse functions in the intestine and is involved in growth and repair, tumorigenesis, inflammation, and fibrosis. The myofibroblast is but one of several α-smooth muscle actin-positive (α-SMA(+)) mesenchymal cells present within the intestinal lamina propria, including vascular pericytes, bone marrow-derived stem cells (mesenchymal stem cells or hematopoietic stem cells), muscularis mucosae, and the lymphatic pericytes (colon) and organized smooth muscle (small intestine) associated with the lymphatic lacteals. These other mesenchymal cells perform many of the functions previously attributed to subepithelial myofibroblasts. This review discusses the definition of a myofibroblast and reconsiders whether the α-SMA(+) subepithelial cells in the intestine are myofibroblasts or other types of mesenchymal cells, i.e., pericytes. Current information about specific, or not so specific, molecular markers of lamina propria mesenchymal cells is reviewed, as well as the origins of intestinal myofibroblasts and pericytes in the intestinal lamina propria and their replenishment after injury. Current concepts and research on stem cell therapy for intestinal inflammation are summarized. Information about the stem cell origin of intestinal stromal cells may inform future stem cell therapies to treat human inflammatory bowel disease (IBD).

Role of Gastric Epithelial Cell-Derived Transforming Growth Factor β in Reduced CD4+ T Cell Proliferation and Development of Regulatory T Cells during Helicobacter pylori Infection

ABSTRACT 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 β (TGF-β) plays a major role in these responses. GECs produced TGF-β1 and TGF-β2 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-β. Naïve CD4+ T cell development into Tregs was also enhanced in the presence of GEC-derived TGF-β. Herein, we demonstrate a role for GEC-produced TGF-β 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 Crohns 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.

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.

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.

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.

Abstract 1857: Dysregulation of transsulfuration enzymes contribute to malignant transformation in a murine model of colitis-associated carcinogenesis

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Introduction: Ulcerative colitis (UC) is a highly morbid, chronic inflammatory disease characterized by mucosal ulceration of the colonic mucosa and is associated with the higher risk of colitis-associated cancer (CAC). The exact mechanism(s) causing UC progression to CAC is currently unknown. The balanced activity of transsulfuration pathway enzymes, cystathionine-gamma-lyase (CSE) and cystathionine-beta-synthase (CBS), and their production of endogenous hydrogen sulfide gas, are critical to maintenance of the colonic homeostasis. CSE activity is suggested to be important for wound healing and mucosal protection. Consistent with this, we have previously showed a decrease CSE expression in human colonic mucosa obtained from patients with chronic UC, compared to normal colonic mucosa by immunocytochemistry. By contrast, increased CBS expression is implicated in the progression of sporadic colorectal carcinoma. However, the role of these enzymes in CAC is unknown. We hypothesize that the dysregulation in CSE/CBS expression and activity is important to the progression from UC to CAC. Methods: CSE null mice and wild type Sv129/B6 (control) mice were used in azoxymethane-dextran sodium sulfate (AOM-DSS) colon cancer model which mimics human CAC. The disease development was followed up to day 80. Confocal microscopy and Western blot was used to assess the gene expression during cancer development. Size, number, and time interval to tumor formation, as well inflammation were assessed. Results: Abrogation of CSE expression using CSE null animals in AOM-DSS model of CAC accelerated the time to tumor development and resulted in an increase in both tumor size (p<0.001) and number (p<0.001) compared to wild-type controls. CBS protein expression was increased within the colonic tumor when compared to the normal margin in AOM-DSS treated animals by Western blot analysis and tissue immunostaining. Interestingly, CAC liver metastases, an exceedingly rare finding in this mouse model, were identified. Conclusion: Taken together, our human and murine data suggest that dysregulation in the transsulfuration pathway enzymes CSE and CBS expression/activity may be critical contributor to the CAC development in UC and may serve as a potential biomarker for disease progression in the future. Citation Format: Paul Johnson, Ches’Que M. Phillips, Carl Grim, John R. Zatarain, Aakash H. Gajjar, Suimin Qiu, Rui Wang, Celia Chao, Iryna V. Pinchuk, Mark R. Hellmich. Dysregulation of transsulfuration enzymes contribute to malignant transformation in a murine model of colitis-associated carcinogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1857.

S1762 TLR-3 Mediated Regulation of B7 Negative Co-Stimulators on Stromal Cells: A Potential Mechanism of Colonic Immune Evasion During Intestinal Viral Infection

BACKGROUND: Activation of TLR3 triggers specific signaling pathways leading to the activation of NF-kB and/or IRF3 transcription factors, enabling the host to mount innate immune responses against different dsRNA viruses and serve as a “danger” receptor in nonviral diseases. However, TLR3-mediated organ specific activation of the B7-coinhibitory pathway PD-L1/PD-1 can be exploited locally leading to the impairment in antiviral T effector cell responses promoting successful viral invasion of the host. Despite significant advances in understanding of the process of TLR3 signaling, the mechanisms by which TLR3 influence homeostasis and contribute to the B7 negative molecules-mediated viral invasion in the gut remain obscure. Human colonic stromal cells, A.k.a. myofibroblasts/fibroblasts (CMFs), are one of the important cell types taking part in innate immunity. CMFs express TLR3, and represent a major cell population expressing B7 negative costimulators PD-L1 and PD-L2 in normal colon. CMFs suppress activated CD4+ effector T cell responses in a PD-L1/2 dependent manner. We hypothesized that in human colonic mucosa, CMFs might be among the first cells to respond to TLR3 mediated stimuli via modulation of PD-L1/2 expression and, thus, may contribute to the impaired T effector cell responses associated with viral invasion. METHODS: TLR3, PD-L1/2 expression on human CMFs isolated from normal mucosa in response to the synthetic analog of TLR3 ligand, poly(I:C), was quantified using real-time RT-PCR, Western blot and FACS analysis. RESULTS: TLR3 stimulation on CMFs by poly(I:C) resulted in robust upregulation of PD-L1 and PD-L2 mRNA and cell surface protein. The expression of IFN-β precedes the upregulation of PD-L1 and PD-L2 mRNA. Stimulation of CMFs with IFN-β leads to the upregulation of the PD-L1 and PD-L2 protein expression (Westen blot analysis) suggesting involvement of IFN-β in the TLR3-mediated PD-L1/2 upregulation. Blocking of the MyD88 adaptor expression in CMFs by using specific siRNA did not affect PD-L2 upregulation in response to the TLR3 stimulation, but reduced the induction of the PD-L1. It is possible that TLR3mediated upregulation of PD-L1 on CMFs may be synergized by activation of heat shock proteins that are known to signal through the TLR4MyD88 adaptor pathway. CONCLUSION: The presented results indicate that CMFs are among the key players in innate immune responses in colonic mucosa. Our data suggest that TLR3 mediated PD-L1/2 upregulation on CMFs may favor suppression of T effector cells responses and this may allow the progression of host invasion during intestinal viral infection. Supported by AGA, CCFA foundations and NIDDK.

842 Cancer-Derived Myofibroblasts Promote Tumor Initiating Cell Expansion in Colorectal Cancer

G A A b st ra ct s Furthermore, when the exon 3 microsatellite was surrounded by 6 nucleotides that normally flank the exon 10 microsatellite, fully mutant exon 3 frameshifts appeared compared to none from the WT exon 3 sequence. Conclusions: The selectivity for specific coding microsatellite frameshift mutation within ACVR2 lies in part with the flanking nucleotides surrounding the microsatellite. Our observation may have implications for frequency of frameshifts at other coding and noncoding microsatellites, and for how MMR deficiency drives actual frameshift mutation.