Immune checkpoint inhibitors (ICIs) have improved the prognosis in multiple cancer types. However, ICIs can induce immune-related adverse events such as immune-mediated enterocolitis (IMC). The gut... Show moreImmune checkpoint inhibitors (ICIs) have improved the prognosis in multiple cancer types. However, ICIs can induce immune-related adverse events such as immune-mediated enterocolitis (IMC). The gut microbiota may be implicated in IMC development. Therefore, we investigated fecal microbiota transplantation (FMT) as a treatment option for 2 patients with metastatic cancer suffering from refractory IMC. The patients were treated with, respectively, 1 and 3 FMTs after vancomycin pre-treatment. We monitored defecation frequency, fecal calprotectin, and microbiota composition. After FMT, both patients improved in defecation frequency, were discharged from the hospital, and received lower dosage of immunosuppressive therapy. Patient 1 developed an invasive pulmonary aspergillosis deemed to be related to prolonged steroid exposure. Patient 2 suffered from a Campylobacter jejuni infection after the first FMT and was treated with meropenem, resulting in a low-diversity microbiota profile and increased calprotectin levels and defecation frequency. After a second and third FMT, bacterial diversity increased and defecation frequency and calprotectin levels decreased. Pre-FMT, both patients showed low bacterial richness, but varying bacterial diversity. After FMT, diversity and richness were similar to healthy donor levels. In conclusion, FMT resulted in improvement of IMC symptoms and corresponding microbial changes in 2 cancer patients with refractory IMC. While more research is warranted, microbiome-modulation could be a promising new therapeutic option for IMC. Show less
Ouboter, L.F.; Barnhoorn, M.C.; Verspaget, H.W.; Plug, L.; Pool, E.S.; Szuhai, K.; ... ; Meulen-de Jong, A.E. van der 2023
BACKGROUND. Due 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... Show moreBACKGROUND. Due 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. METHODS. Thirteen 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. RESULTS. No 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. CONCLUSION. Local 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 REGISTRATION. EU Clinical Trials Register (EudraCT, 2017-003524-75) and the Dutch Trial Register (NTR7205). Show less
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... Show morePerianal 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. Show less
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,... Show moreInflammatory 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. Show less
Barnhoorn, M.C.; Meulen-de Jong, A.E. van der; Schrama, E.C.L.M.; Plug, L.G.; Verspaget, H.W.; Fibbe, W.E.; ... ; Schepers, K. 2022
Locally applied mesenchymal stromal cells (MSCs) have the capacity to promote the healing of perianal fistulas in Crohn's disease (CD) and are under clinical development for the treatment of... Show moreLocally applied mesenchymal stromal cells (MSCs) have the capacity to promote the healing of perianal fistulas in Crohn's disease (CD) and are under clinical development for the treatment of proctitis in ulcerative colitis (UC). Despite these clinical advances, the mechanism of action of local MSC therapy in inflammatory bowel disease (IBD) is largely unknown. We hypothesized that the local cytokine environment in IBD patients affects the immunomodulatory properties of MSCs. To evaluate this, 11 cytokines were analyzed in inflamed tissues obtained from CD and UC patients. Based on the identified cytokine profiles 4 distinct cytokine mixtures that mimic various inflammatory IBD environments were established. Next, MSCs were cultured in the presence of either of these 4 cytokine mixtures after which the expression of immunomodulatory and tissue regenerative molecules and the capacity of MSCs to modulate T-cell proliferation and dendritic cell (DC) differentiation were assessed. Our data show that MSCs respond, in a cytokine-specific manner, by upregulation of immunomodulatory and tissue regenerative molecules, including cyclooxygenase-2, indoleamine 2,3-dioxygenase, and transforming growth factor-beta 1. Functional studies indicate that MSCs exposed to a cytokine profile mimicking one of the 2 UC cytokine milieus were less effective in inhibition of DC differentiation. In conclusion, our data indicate that cytokine mixes mimicking the local cytokine milieus of inflamed UC colonic or CD fistulas tissues can differentially affect the immunomodulatory and tissue regenerative characteristics of MSCs. These data support the hypothesis that the local intestinal cytokine milieu serves as a critical factor in the efficacy of local MSC treatment. Show less
Barnhoorn, M.C.; Storm, B.N.; Boer, N.K. de; Voorn, M.M.P.J.A. van der 2021
Background: Fistulas represent a frequent and severe complication in patients with Crohn disease (CD). Tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta, and interleukin (IL)... Show moreBackground: Fistulas represent a frequent and severe complication in patients with Crohn disease (CD). Tumor necrosis factor-alpha (TNF-alpha), 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-alpha 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-22R(alpha 1) were highly expressed in perianal CD fistula specimens and that IL-22 cotreatment potentiated TNF-alpha-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-alpha. Our data support clinical evidence indicating that anti-TNF-alpha therapy is effective in fistula treatment and identify IL-13 and IL-22 as possible novel therapeutic targets for fistula therapy. Show less
Background Fistulas represent a frequent and severe complication in patients with Crohn disease (CD). Tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta, and interleukin (IL)... Show moreBackground Fistulas represent a frequent and severe complication in patients with Crohn disease (CD). Tumor necrosis factor-alpha (TNF-alpha), 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-alpha 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-22R(alpha 1) were highly expressed in perianal CD fistula specimens and that IL-22 cotreatment potentiated TNF-alpha-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-alpha. Our data support clinical evidence indicating that anti-TNF-alpha therapy is effective in fistula treatment and identify IL-13 and IL-22 as possible novel therapeutic targets for fistula therapy. Show less
Schoonderwoerd, M.J.A.; Koops, M.F.M.; Angela, R.A.; Koolmoes, B.; Toitou, M.; Paauwe, M.; ... ; Hawinkels, L.J.A.C. 2020
Purpose: Endoglin is a coreceptor for TGFb ligands that is highly expressed on proliferating endothelial cells and other cells in the tumor microenvironment. Clinical studies have noted increased... Show morePurpose: Endoglin is a coreceptor for TGFb ligands that is highly expressed on proliferating endothelial cells and other cells in the tumor microenvironment. Clinical studies have noted increased programmed cell death (PD)-1 expression on cytotoxic T cells in the peripheral blood of patients with cancer treated with TRC105, an endoglin-targeting antibody. In this study, we investigated the combination of endoglin antibodies (TRC105 and M1043) with an anti-PD1 antibody.Experimental Design: The combination anti-endoglin/anti-PD1 antibodies was tested in four preclinical mouse models representing different stages of cancer development. To investigate the underlying mechanism, Fc-receptor-knockout mice were used complemented with depletion of multiple immune subsets in mice. Tumor growth and the composition of immune infiltrate were analyzed by flow cytometry. Finally, human colorectal cancer specimens were analyzed for presence of endoglin-expressing regulatory T cells (Treg).Results: In all models, the combination of endoglin antibody and PD1 inhibition produced durable tumor responses, leading to complete regressions in 30% to 40% of the mice. These effects were dependent on the presence of Fcg receptors, indicating the involvement of antibody-dependent cytotoxic responses and the presence of CD8(+) cytotoxic T cells and CD4(+) Th cells. Interestingly, treatment with the endoglin antibody, TRC105, significantly decreased the number of intratumoral Tregs. Endoglin-expressing Tregs were also detected in human colorectal cancer specimens.Conclusions: Taken together, these data provide a rationale for combining TRC105 and anti-PD1 therapy and provide additional evidence of endoglin's immunomodulatory role. Show less
Up till now, research on inflammatory bowel disease [IBD] has mainly been focused on the immune cells present in the gastrointestinal tract. However, recent insights indicate that stromal cells... Show moreUp till now, research on inflammatory bowel disease [IBD] has mainly been focused on the immune cells present in the gastrointestinal tract. However, recent insights indicate that stromal cells also play an important and significant role in IBD pathogenesis. Stromal cells in the intestines regulate both intestinal epithelial and immune cell homeostasis. Different subsets of stromal cells have been found to play a role in other inflammatory diseases [e.g. rheumatoid arthritis], and these various stromal subsets now appear to carry out also specific functions in the inflamed gut in IBD. Novel potential therapies for IBD utilize, as well as target, these pathogenic stromal cells. Injection of mesenchymal stromal cells [MSCs] into fistula tracts of Crohn's disease patients is already approved and used in clinical settings. In this review we discuss the current knowledge of the role of stromal cells in IBD pathogenesis. We further outline recent attempts to modify the stromal compartment in IBD with agents that target or replace the pathogenic stroma. Show less
This thesis is about the role of stromal cells in inflammatory bowel disease (IBD) and to assess the therapeutic potential of local mesenchymal stromal cell (MSC)-therapy. First, we discuss the... Show moreThis thesis is about the role of stromal cells in inflammatory bowel disease (IBD) and to assess the therapeutic potential of local mesenchymal stromal cell (MSC)-therapy. First, we discuss the recent insights in the function of stromal cells in the healthy and inflamed gut and characterize fibroblasts in perianal fistulas from patients with IBD. Furthermore, we study the long-term effects of MSC-therapy for the treatment of perianal fistulas and the efficacy of locally injected MSCs in the inflamed intestines of in vivo IBD models. Lastly, we investigate the cytokine environment of inflamed tissue from IBD patients and how this could affect local MSC-therapy. Show less
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... Show moreBackground 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 10(7) bmMSCs cohort 1, n = 5; 3 x 10(7) bmMSCs cohort 2, n = 5; 9 x 10(7) 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. Show less
Helm, D. van der; Barnhoorn, M.C.; Jonge-Muller, E.S.M. de; Molendijk, I.; Hawinkels, L.J.A.C.; Coenraad, M.J.; ... ; Verspaget, H.W. 2019
Intraluminally injected MSC spheroids at least partially attenuate experimental colitis, with fewer phagocytes and proinflammmatory cytokines, when a high dose of MSCs in spheroids was administered.
