Inflammatory bowel disease (IBD) is a chronic relapsing inflammation of the intestinal tract with currently not well-understood pathogenesis. In addition to the involvement of immune cells, ...increasing studies show an important role for fibroblasts in the pathogenesis of IBD. Previous work showed that glycolysis is the preferred energy source for fibroblasts in fibrotic diseases. 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3) is a key kinase supporting glycolysis. Increased expression of PFKFB3 in several cancers and inflammatory diseases has been previously reported, but the metabolic status of fibroblasts and the role of PFKFB3 in patients with IBD are currently unknown. Therefore, in this study, we evaluated the role of glycolysis and PFKFB3 expression in IBD. Single-sample gene set enrichment analysis (ssGSEA) revealed that glycolysis was significantly higher in IBD intestinal samples, compared to healthy controls, which was confirmed in the validation cohorts of IBD patients. Single-cell sequencing data indicated that PFKFB3 expression was higher in IBD-derived stromal cells.
In vitro
, PFKFB3 expression in IBD-derived fibroblasts was increased after the stimulation with pro-inflammatory cytokines. Using seahorse real-time cell metabolic analysis, inflamed fibroblasts were shown to have a higher extracellular acidification rate and a lower oxygen consumption rate, which could be reversed by inhibition of JAK/STAT pathway. Furthermore, increased expression of pro-inflammatory cytokines and chemokines in fibroblasts could be reverted by PFK15, a specific inhibitor of PFKFB3.
In vivo
experiments showed that PFK15 reduced the severity of dextran sulfate sodium (DSS)- and Tcell transfer induced colitis, which was accompanied by a reduction in immune cell infiltration in the intestines. These findings suggest that increased stromal PFKFB3 expression contributes to inflammation and the pathological function of fibroblasts in IBD. Inhibition of PFKFB3 suppressed their inflammatory characteristics.
Abstract
Background and Aims
The long-term safety and efficacy of allogeneic bone marrow-derived mesenchymal stromal cell bmMSC therapy in perianal Crohn’s disease CD fistulas is unknown. We aimed to ...provide a 4-year clinical evaluation of allogeneic bmMSC treatment of perianal CD fistulas.
Methods
A double-blind dose-finding study for local bmMSC therapy in 21 patients with refractory perianal fistulising Crohn’s disease was performed at the Leiden University Medical Center in 2012–2014. All patients treated with bmMSCs 1 x 107 bmMSCs cohort 1, n = 5; 3 × 107 bmMSCs cohort 2, n = 5; 9 × 107 bmMSCs cohort 3, n = 5 were invited for a 4-year evaluation. Clinical events were registered, fistula closure was evaluated, and anti-human leukocyte antigen HLA antibodies were assessed. Patients were also asked to undergo a pelvic magnetic resonance imaging MRI and rectoscopy.
Results
Thirteen out of 15 patients 87% treated with bmMSCs were available for long-term follow-up. Two non-MSC related malignancies were observed. No serious adverse events thought to be related to bmMSC therapy were found. In cohort 2 n = 4, all fistulas were closed 4 years after bmMSC therapy. In cohort 1 n = 4 63%, and in cohort 3 n = 5 43%, of the fistulas were closed, respectively. In none of the patients anti-HLA antibodies could be detected 24 weeks and 4 years after therapy. Pelvic MRI showed significantly smaller fistula tracts after 4 years.
Conclusions
Allogeneic bmMSC therapy for CD-associated perianal fistulas is also in the long-term a safe therapy. In bmMSC-treated patients, fistulas with closure at Week 24 were still closed after 4 years.
Chronic liver damage leads to the onset of fibrogenesis. Rodent models for liver fibrosis have been widely used, but are less suitable for screening purposes. Therefore the aim of our study was to ...design a novel model for liver fibrosis in zebrafish embryos, suitable for high throughput screening. Furthermore, we evaluated the efficacy of mesenchymal stromal cells (MSCs) to inhibit the fibrotic process and thereby the applicability of this model to evaluate therapeutic responses. Zebrafish embryos were exposed to TAA or CCL4 and mRNA levels of fibrosis-related genes (Collagen-1α1, Hand-2, and Acta-2) and tissue damage-related genes (TGF-β and SDF-1a, SDF-1b) were determined, while Sirius-red staining was used to estimate collagen deposition. Three days after start of TAA exposure, MSCs were injected after which the fibrotic response was determined. In contrast to CCL4, TAA resulted in an upregulation of the fibrosis-related genes, increased extracellular matrix deposition and decreased liver sizes suggesting the onset of fibrosis. The applicability of this model to evaluate therapeutic responses was shown by local treatment with MSCs which resulted in decreased expression of the fibrosis-related RNA markers. In conclusion, TAA induces liver fibrosis in zebrafish embryos, thereby providing a promising model for future mechanistic and therapeutic studies.
Perianal fistulas are a debilitating complication of Crohn's disease (CD). Due to unknown reasons, CD-associated fistulas are in general more difficult to treat than cryptoglandular fistulas ...(non-CD-associated). Understanding the immune cell landscape is a first step towards the development of more effective therapies for CD-associated fistulas. In this work, we characterized the composition and spatial localization of disease-associated immune cells in both types of perianal fistulas by high-dimensional analyses.
We applied single-cell mass cytometry (scMC), spectral flow cytometry (SFC), and imaging mass cytometry (IMC) to profile the immune compartment in CD-associated perianal fistulas and cryptoglandular fistulas. An exploratory cohort (CD fistula, n = 10; non-CD fistula, n = 5) was analyzed by scMC to unravel disease-associated immune cell types. SFC was performed on a second fistula cohort (CD, n = 10; non-CD, n = 11) to comprehensively phenotype disease-associated T helper (Th) cells. IMC was used on a third cohort (CD, n = 5) to investigate the spatial distribution/interaction of relevant immune cell subsets.
Our analyses revealed that activated HLA-DR+CD38+ effector CD4+ T cells with a Th1/17 phenotype were significantly enriched in CD-associated compared with cryptoglandular fistulas. These cells, displaying features of proliferation, regulation, and differentiation, were also present in blood, and colocalized with other CD4+ T cells, CCR6+ B cells, and macrophages in the fistula tracts.
Overall, proliferating activated HLA-DR+CD38+ effector Th1/17 cells distinguish CD-associated from cryptoglandular perianal fistulas and are a promising biomarker in blood to discriminate between these 2 fistula types. Targeting HLA-DR and CD38-expressing CD4+ T cells may offer a potential new therapeutic strategy for CD-related fistulas.
Perianal fistulas are defined as pathological connections between the anorectal canal and the perianal skin. Most perianal fistulas are cryptoglandular fistulas, which are thought to originate from ...infected anal glands. The remainder of the fistulas mainly arises as complications of Crohn's disease (CD), trauma, or as a result of malignancies. Fistulas in CD are considered as a consequence of a chronic and transmural inflammatory process in the distal bowel and can, in some cases, even precede the diagnosis of CD. Although both cryptoglandular and CD-associated fistulas might look similar macroscopically, they differ considerably in their complexity, treatment options, and healing rate. Therefore, it is of crucial importance to differentiate between these two types of fistulas. In this review, the differences between CD-associated and cryptoglandular perianal fistulas in epidemiology, pathogenesis, and clinical management are discussed. Finally, a flow chart is provided for physicians to guide them when dealing with patients displaying their first episode of perianal fistulas.
BACKGROUNDDue to their immunoregulatory and tissue regenerative features, mesenchymal stromal cells (MSCs) are a promising novel tool for the management of ulcerative proctitis (UP). Here we report ...on a phase IIa clinical study that evaluated the impact of local MSC therapy on UP.METHODSThirteen refractory UP patients, with an endoscopic Mayo score (EMS) of 2 or 3, were included. Seven patients received 20-40 million allogeneic MSCs (cohort 1), while 6 patients received 40-80 million MSCs (cohort 2). Adverse events (AEs) were assessed at baseline and on weeks 2, 6, 12, and 24. Clinical, endoscopic, and biochemical parameters were assessed at baseline and on weeks 2 and 6. Furthermore, we evaluated the engraftment of MSCs, the presence of donor-specific human leukocyte antigen (HLA) antibodies (DSAs), and we determined the impact of MSC therapy on the local immune compartment.RESULTSNo serious AEs were observed. The clinical Mayo score was significantly improved on weeks 2 and 6, and the EMS was significantly improved on week 6, compared with baseline. On week 6, donor MSCs were still detectable in rectal biopsies from 4 of 9 patients and DSAs against both HLA class I and class II were found. Mass cytometry showed a reduction in activated CD8+ T cells and CD16+ monocytes and an enrichment in mononuclear phagocytes and natural killer cells in biopsies after local MSC therapy.CONCLUSIONLocal administration of allogeneic MSCs is safe, tolerable, and feasible for treatment of refractory UP and shows encouraging signs of clinical efficacy and modulation of local immune responses. This sets the stage for larger clinical trials.TRIAL REGISTRATIONEU Clinical Trials Register (EudraCT, 2017-003524-75) and the Dutch Trial Register (NTR7205).FUNDINGECCO grant 2020.
Abstract
Background
Fistulas represent a frequent and severe complication in patients with Crohn disease (CD). Tumor necrosis factor-alpha (TNF-α), transforming growth factor-beta, and interleukin ...(IL)-13 are known to trigger epithelial-mesenchymal transition (EMT), promoting fistula formation. Here, we investigated the role of T-lymphocytes (T cells) in fistula pathogenesis.
Methods
CD3+CD8-, CD3+CD8+, or CD45+CD3- cells from healthy volunteers, patients with CD, and patients with CD with perianal fistula were co-cultured with HT-29 cells. The EMT, cytokine production, and mRNA expression were analyzed. Perianal CD fistula specimens were immunohistochemically stained for cytokines and their receptors. The effect of cytokines on EMT induction was investigated using an EMT spheroid model.
Results
Patients with CD with fistula revealed more CD3+CD8- and less CD3+CD8+ T cells in blood than healthy control patients and patients with CD without fistula. In perianal fistula specimens, CD4+ cells—and to a lesser extent CD8+ cells—were highly present around fistula tracts. When co-cultured with HT-29 cells, both cell subsets promoted EMT-related gene expression and TNF-α production in a time-dependent manner. The CD3+CD8- T cells from patients with CD with fistula also produced higher amounts of IL-13 than cells from healthy control patients or patients with CD without a fistula. We found that IL-22 and IL-22Rα1 were highly expressed in perianal CD fistula specimens and that IL-22 cotreatment potentiated TNF-α-induced EMT in HT-29 spheroids.
Conclusions
Our data indicate that both CD3+CD8- and CD3+CD8+ T cells play an important role in the pathogenesis of perianal CD fistulas by the secretion of TNF-α. Our data support clinical evidence indicating that anti-TNF-α therapy is effective in fistula treatment and identify IL-13 and IL-22 as possible novel therapeutic targets for fistula therapy.
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