Humanized NSG Mouse Models as a Preclinical Tool for Translational Research in Inflammatory Bowel Diseases.

The development of animal models reflecting the pathologies of ulcerative colitis (UC) and Crohn's disease (CD) remains a major challenge. The NOD/SCID/IL2rγnull (NSG) mouse strain, which is immune-compromised, tolerates the engraftment of human peripheral blood mononuclear cells (PBMC) derived from patients with UC (NSG-UC) or CD (NSG-CD). This offers the opportunity to examine the impact of individual immunological background on the development of pathophysiological manifestations. When challenged with ethanol, NSG-UC mice exhibited a strong pro-inflammatory response, including the development of edemas, influx of human T cells, B cells and monocytes into the mucosa and submucosa, and elevated expression of the inflammatory markers CRP and CCL-7. Fibrotic alterations were characterized by an influx of fibroblasts and a thickening of the muscularis mucosae. In contrast, the development of pathological manifestations in NSG-CD mice developed without challenge and was signified by extensive collagen deposition between the muscularis propria and muscularis mucosae, as observed in the areas of strictures in CD patients. Vimentin-expressing fibroblasts supplanting colonic crypts and elevated expression of HGF and TGFß corroborated the remodeling phenotype. In summary, the NSG-UC and NSG-CD models partially reflect these human diseases and are powerful tools to examine the mechanism underlying the inflammatory processes in UC and CD.

NOD/scid IL-2Rγnull mice reconstituted with peripheral blood mononuclear cells from patients with Crohn's disease reflect the human pathological phenotype.

INTRODUCTION: Crohn's disease (CD) is characterized by pronounced intestinal fibrosis and severe mucosal damage and conventional animal models are limited to reflect these pathological manifestations. The aim of this study was to examine whether the combination of patient immune-profiling and preclinical studies in a mouse model based on NOD/scid IL-2Rγnull (NSG) reconstituted with peripheral blood mononuclear cells (PBMC) from CD patients has the capacity to harmonize ex vivo human and in vivo animal studies. METHODS: Immunological profiles of CD (n = 24) and ulcerative colitis (UC) patients (n = 47) were established by flow cytometry of subgroups of immune cells and subjected to hierarchical cluster and estimation graphics analyses. Pathological phenotypes of NSG mice, which were reconstituted with PBMC from CD, UC, and non-IBD donors (NSG-CD, NSG-UC, and NSG-non-IBD) were compared. Readouts were the clinical, colon, and histological scores; subtypes of immune cells from spleen and colon; and levels of inflammatory markers, such as c-reactive protein (CRP), monocyte chemotactic protein (MCP)-3, transforming growth factor-beta (TGFß), and hepatocyte growth factor (HGF). Fibrocytes were identified by immunohistochemistry in colonic sections. RESULTS: CD patients were significantly clustered in a group characterized by increased levels of TH1, TH2 cells, and decreased levels of CD14+ CD163+ monocytes (p = .003). In contrast to NSG-UC mice, NSG-CD mice exhibited an immune-remodeling phenotype characterized by enhanced collagen deposition, elevated levels of CD14+ CD163+ monocytes, HGF, and TGFß. This phenotype was further corroborated by the presence of human fibrocytes as components of fibrotic areas. CONCLUSION: The NSG-CD model partially reflects the human disease and allows for studying the development of fibrosis.

The PI3K pathway as a therapeutic intervention point in inflammatory bowel disease.

With glucose being the preferred source of energy in activated T cells, targeting glycolysis has become an attractive therapeutic intervention point for chronic inflammatory bowel diseases (IBD). The switch to glycolysis is mediated by phosphoinositide-3-kinases (PI3K) which relay signals from surface receptors to the AKT pathway. We first confirmed by analysis of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) that metabolism is shifted towards glycolysis in IBD patients as compared to non-IBD donors. In contrast to non-IBD donors, OCR correlated with ECAR (IBD: cor = 0.79, p = 2E-10; non-IBD: cor = 0.37, p = n.s.), in IBD patients. Second, we tested the PI3K inhibitor copanlisib as a potential therapeutic. Ex vivo, copanlisib suppressed the ECAR significantly in T cells activated by anti-CD3 antibodies and significantly decreased ECAR rates in the presence of copanlisib (anti-CD3: 58.24 ± 29.06; copanlisib: 43.16 ± 20.23, p < .000. In addition, copanlisib impaired the activation of CD4+ CD25+ T cells (anti-CD3: 42.15 ± 21.46; anti-CD3 + copanlisib: 26.06 ± 21.82 p = .013) and the secretion of cytokines (IFNγ: anti-CD3: 6332.0 ± 5707.61 pmol/ml; anti-CD3 + copanlisib: 6332.0 ± 5707.61, p = .018). In vivo, copanlisib significantly improved the histological scores (ethanol: 8.5 ± 3.81; copanlisib: 4.57 ± 2.82, p = .006) in the NSG-UC mouse model. Orthogonal partial least square analysis confirmed the efficacy of copanlisib. These data suggest that the PI3K pathway provides an attractive therapeutic intervention point in IBD for patients in relapse. Targeting metabolic pathways have the potential to develop phase dependent therapies.

Suppressing Kv1.3 Ion Channel Activity with a Novel Small Molecule Inhibitor Ameliorates Inflammation in a Humanised Mouse Model of Ulcerative Colitis.

BACKGROUND AND AIMS: The potassium channel Kv1.3 is a potentially attractive therapeutic target in T cell-mediated inflammatory diseases, as the activity of antigen-activated T cells is selectively impeded by Kv1.3 inhibition. In this study, we examined Kv1.3 as a potential therapeutic intervention point for ulcerative colitis [UC], and studied the efficacy of DES1, a small-molecule inhibitor of Kv1.3, in vitro and in vivo. METHODS: Kv1.3 expression on T cells in peripheral blood mononuclear cells [PBMCs] isolated from donors with and without UC was examined by flow cytometry. In biopsies from UC patients, Kv1.3-expressing CD4+ T cells were detected by flow cytometry and immunohistochemistry. In vitro, we determined the ability of DES1 to inhibit anti-CD3-driven activation of T cells. In vivo, the efficacy of DES1 was determined in a humanised mouse model of UC and compared with infliximab and tofacitinib in head-to-head studies. RESULTS: Kv1.3 expression was elevated in PBMCs from UC patients and correlated with the prevalence of TH1 and TH2 T cells. Kv1.3 expression was also detected on T cells from biopsies of UC patients. In vitro, DES1 suppressed anti-CD3-driven activation of T cells in a concentration-dependent manner. In vivo, DES1 significantly ameliorated inflammation in the UC model and most effectively so when PBMCs from donors with higher levels of activated T cells were selected for reconstitution. The efficacy of DES1 was comparable to that of either infliximab or tofacitinib. CONCLUSION: Inhibition of Kv1.3 [by DES1, for instance] appears to be a potential therapeutic intervention strategy for UC patients.

Head-to-head study of oxelumab and adalimumab in a mouse model of ulcerative colitis based on NOD/Scid IL2Rγnull mice reconstituted with human peripheral blood mononuclear cells.

This study's aim was to demonstrate that the combination of patient immune profiling and testing in a humanized mouse model of ulcerative colitis (UC) might lead to patient stratification for treatment with oxelumab. First, immunological profiles of UC patients and non-UC donors were analyzed for CD4+ T cells expressing OX40 (CD134; also known as TNFRSF4) and CD14+ monocytes expressing OX40L (CD252; also known as TNFSF4) by flow cytometric analysis. A significant difference was observed between the groups for CD14+ OX40L+ (UC: n=11, 85.44±21.17, mean±s.d.; non-UC: n=5, 30.7±34.92; P=0.02), whereas no significant difference was detected for CD4+ OX40+. CD14+ OX40L+ monocytes were correlated significantly with T helper 1 and 2 cells. Second, NOD/Scid IL2Rγ null mice were reconstituted with peripheral blood mononuclear cells from UC donors exhibiting elevated levels of OX40L, and the efficacy of oxelumab was compared with that of adalimumab. The clinical, colon and histological scores and the serum concentrations of IL-6, IL-1ß and glutamic acid were assessed. Treatment with oxelumab or adalimumab resulted in significantly reduced clinical, colon and histological scores, reduced serum concentrations of IL-6 and reduced frequencies of splenic human effector memory T cells and switched B cells. Comparison of the efficacy of adalimumab and oxelumab by orthogonal partial least squares discrimination analysis revealed that oxelumab was slightly superior to adalimumab; however, elevated serum concentrations of glutamic acid suggested ongoing inflammation. These results suggest that oxelumab addresses the pro-inflammatory arm of inflammation while promoting the remodeling arm and that patients exhibiting elevated levels of OX40L might benefit from treatment with oxelumab.