Patients with Crohn disease (CD) and ulcerative colitis (UC) suffer from chronic relapsing intestinal inflammation. While many studies focused on adaptive immunity, less is known about the role of innate immune cells in these diseases. Innate lymphoid cells (ILCs) are recently identified cells with a high cytokine-producing capacity at mucosal barriers. The aim was to study the impact of biological treatment on ILC in CD and UC. Patients initiating anti-tumor necrosis factor (TNF), ustekinumab, or vedolizumab treatment were prospectively followed up and peripheral and intestinal ILCs were determined. In the inflamed gut tissue of patients with inflammatory bowel disease, we found an increase of ILC1 and in immature NKp44 − ILC3, whereas there was a decrease of mature NKp44 + ILC3 when compared to healthy controls (HCs). Similar but less pronounced changes in ILC1 were observed in blood, whereas circulating NKp44 − ILC3 were decreased. Fifteen percent of CD patients had NKp44 + ILC3 in blood and these cells were not detected in blood of HCs or UC patients. Therapy with three different biologicals (ustekinumab targeting the IL-12/23 cytokines, anti-TNF and vedolizumab) partly restored intestinal ILC subset equilibrium with a decrease of ILC1 (except for ustekinumab) and an increase of NKp44 + ILC3. Anti-TNF also mobilized more NKp44 + ILC3 in circulation. As ILC1 are proinflammatory cells and as NKp44 + ILC3 contribute to homeostasis of intestinal mucosa, the observed effects of biologicals on ILCs might contribute to their clinical efficacy.
Eosinophils are leukocytes which reside in the gastrointestinal tract under homeostatic conditions, except for the esophagus which is normally devoid of eosinophils. Research on eosinophils has primarily focused on anti-helminth responses and type 2 immune disorders. In contrast, the search for a role of eosinophils in chronic intestinal inflammation and fibrosis has been limited. With a shift in research focus from adaptive to innate immunity and the fact that the eosinophilic granules are filled with inflammatory mediators, eosinophils are becoming a point of interest in inflammatory bowel diseases. In the current review we summarize eosinophil characteristics and recruitment as well as the current knowledge on presence, inflammatory and pro-fibrotic functions of eosinophils in inflammatory bowel disease and other chronic inflammatory conditions, and we identify research gaps which should be covered in the future.
Disorders of gut-brain interaction (DGBI), formerly termed functional gastrointestinal disorders (FGID), are highly prevalent although exact pathophysiological mechanisms remain unclear. Intestinal immune activation has been recognized, but increasing evidence supports a pivotal role for an active inflammatory state in these disorders. In functional dyspepsia (FD), marked eosinophil and mast cell infiltration has been repeatedly demonstrated and associations with symptoms emphasize the relevance of an eosinophil-mast cell axis in FD pathophysiology. In this Review, we highlight the importance of immune activation in DGBI with a focus on FD. We summarize eosinophil biology in both homeostasis and inflammatory processes. The evidence for immune activation in FD is outlined with attention to alterations on both cellular and molecular level, and how these may contribute to FD symptomatology. As DGBI are complex and multifactorial conditions, we shed light on factors associated to, and potentially influencing immune activation, including bidirectional gut-brain interaction, allergy and the microbiota. Crucial studies reveal a therapeutic benefit of treatments targeting immune activation, suggesting that specific anti-inflammatory therapies could offer renewed hope for at least a subset of DGBI patients. Lastly, we explore the future directions for DGBI research that could advance the field. Taken together, emerging evidence supports the recognition of FD as an immune-mediated organic-based disorder, challenging the paradigm of a strictly functional nature.
Background Eosinophils might play a pro-inflammatory role in inflammatory bowel disease (IBD). However, the role of eosinophils in intestinal fibrosis remains poorly understood, although a pro-fibrotic function has been hypothesized based on data outside the gastrointestinal tract. Therefore, we aimed to unravel the role of eosinophils in chronic intestinal inflammation and fibrosis and to explore eosinophil depletion as a potential therapeutic target. Methods A 3-cycles chronic dextran sodium sulphate (DSS) model was induced in 6-8-week-old C57BL/6 wild type mice. Mice received 3 DSS cycles (1.5% - 2.00% - 2.00%) in which 1 DSS cycle consisted of 1 week of DSS administration followed by 2 weeks of recovery. During the 3 cycles, and starting 3 days prior to colitis induction, anti-CCR3 antibody or isotype injections were given twice weekly to study the effect of the eosinophil depletion (total of 18 injections). The disease activity index (DAI; weight loss, stool consistency and presence of blood) was determined twice weekly. At sacrifice, colonic damage was scored macroscopically (presence of hyperaemia, adhesions and length and degree of colon affected by inflammation) and colonic single cells were isolated and fluorescently stained for flow cytometry. Eosinophils were thereby identified as CD45+ CD11b+ Siglec-F+ CD117- cells. Lastly, a histological active disease score was determined comprising of the sum of neutrophil infiltration, mononuclear cell infiltration, changes in mucosal architecture, goblet cell loss and epithelial defects (Creyns et al., 2019). Intestinal fibrosis was assessed via colon weight/length ratio, COL1A1 gene expression and collagen deposition with a Martius Scarlet Blue (MSB) staining and a colorimetric hydroxyproline assay. Results Blood and colonic eosinophil depletion via anti-CCR3 injections was confirmed via flow cytometry (figure 1). The DAI in the eosinophil depleted group was decreased compared to the isotype injected group (area under the curve 115.4 ± 29 vs 160.6 ± 28; p=0.02). A similar trend was seen in the macroscopic damage score (2.0 ± 1.3 vs 3.9 ± 2.1; p=0.08). Furthermore, a lower histological active disease score was found in the mice in which the eosinophils were depleted compared to the isotype injected mice (8.8 ± 0.8 vs 12.8 ± 1.5; p=0.002) (figure 2). Lastly, no differences in the parameters for fibrosis between the anti-CCR3 injected and isotype injected groups were observed (figure 3). Conclusion Eosinophil depletion via intraperitoneal anti-CCR3 injections resulted in partial protection from intestinal inflammation in chronic DSS, and could therefore be further explored as a potential therapeutic agent. In contrast, eosinophil depletion does not seem to have any anti-fibrotic effect
Background About one third of patients with Crohn’s disease (CD) develop strictures during their disease course requiring surgical resection. The immune landscape involved in this process is poorly understood. Therefore, we aimed to characterise the fibroblast phenotype, immune cells and their mediators involved in intestinal strictures. Methods We included 25 CD patients with stricturing disease in the terminal ileum (TI) and 10 controls with colorectal cancer (CRC), all undergoing an ileocolonic resection. Transmural samples from the resection specimen of the TI were obtained. Macroscopically, CD tissue was divided into unaffected, fibrostenotic and inflamed regions by an experienced histopathologist. Next, mucosa was separated from deeper layers, after which single cells were isolated and fluorescently stained for flow cytometry. Protein levels were determined via the MesoScale Discovery (MSD) platform in the corresponding samples. Comparisons between CRC controls and CD patients were performed via an unpaired t-test or Mann-Whitney analysis and corrected for multiple testing. Results An increase in active fibroblasts and decrease in inactive fibroblasts in the fibrotic and inflamed mucosa (p=0.0002 and p<0.0001) and deeper layers (p=0.003 and p=0.02) when compared to the CRC controls was observed, confirming ongoing tissue remodelling. An enrichment of active eosinophils was only seen in the fibrotic deeper layers (p=0.02), although an increase in T helper 2 (Th2) cells was observed in both the fibrotic and inflamed deeper layers (p=0.02 and p=0.04). In contrast, T helper 1 (Th1) cells were decreased in both fibrotic and inflamed mucosa (p=0.03 and p=0.02) and deeper layers (p=0.01 for both). Regulatory T cells were significantly enriched in both fibrotic and inflamed mucosa (p<0.0001 and p=0.0005) and deeper layers (p=0.01 and p=0.006) (figure 1). Protein quantification confirmed a significant increase in transforming growth factor-β3 (TGF-β3) in the fibrotic (p=0.007) and inflamed (p=0.0002) layers, but not in the more superficial mucosa. Comparably, IL-1β was increased in the fibrotic (p=0.05) and inflamed (p=0.05) deeper layers. A similar observation was made for basic fibroblast growth factor (bFGF) (p=0.004), although only a trend could be seen in the fibrotic deeper layers (p=0.08) (figure 2). Conclusion The fibrotic and inflamed tissue of CD patients is characterized by increased activated eosinophils, Th2 and regulatory T cells and decreased Th1 cells, as well as many of their mediator cytokines. The current immunological characterisation can help to prioritise potential anti-fibrotic targets for stricturing CD.
Background Patients with Crohn’s disease (CD) often develop strictures, necessitating surgical intervention. The immune pathways expressed in the fibrotic regions, especially in the deeper intestinal layers, are poorly characterized hence hampering therapeutic development for anti-fibrotic agents. We performed a detailed analysis of the immune cell populations in both inflammation and fibrosis in the mucosa and deeper intestinal layers in CD patients. Methods Patients with stricturing CD (n=25) or colorectal cancer (CRC, n=10), undergoing ileocolonic resection were included. The resection specimen of CD patients was macroscopically subdivided into fibrotic, inflammatory but not fibrotic, and unaffected regions by an IBD-experienced histopathologist. The mucosal layer was furthermore separated from the deeper intestinal layers. Immune cells were isolated, followed by flow cytometry. Quantitative real time PCR (qRT-PCR) was performed for the eosinophil chemoattractant CCL11 coding for eotaxin-1 and normalized against the housekeeping genes RPL13a and PPIA. Wilcoxon matched-pairs sign rank test was performed on paired samples while Dunn’s multiple testing was performed for unpaired comparisons. Results Flow cytometric analysis in CD patients showed mainly B cell enrichment in fibrotic tissue compared to the unaffected tissue, both in the mucosa (30.2% vs 20.9% of CD45+ cells, p=0.001) as in deeper layers (34.4% vs 24.1% of CD45+ cells, p=0.01) (figure 1). Mature dendritic cells (mDC), alternatively-activated macrophages and eosinophils were also enriched but in the deeper layers of the fibrotic segment compared to the mucosa (4.3% vs 3.4%, 1.07% vs 0.04% and 2.3% vs 1.5% of CD45+ cells; p = 0.02, 0.008 and 0.02) (figure 2). Moreover, increased eosinophilic presence was also shown in the unaffected CD region compared to the fibrotic area, both in deeper layers (4.0% vs 2.3% of CD45+ cells; p=0.03) as the mucosa (2.4% vs 1.5% of CD45+ cells; p=0.01) as well as in the inflamed deeper layers compared to the superficial layers (3.3% vs 2.1% of CD45+ cells, p=0.04). These data were corroborated by increased CCL11 mRNA expression, in the deeper layers of fibrotic tissue compared to the mucosa (52.3% vs 31.0%, p=0.03). Conclusion Our results suggest a role for innate immune cells in CD complicated with fibrosis. Especially in the deeper layers, eosinophils, mature dendritic cells and alternatively-activated macrophages are implicated. The eosinophilic enrichment in the deeper layers may partly be attributed to an increased eosinophil recruitment by CCL11. Our results offer opportunities for anti-fibrotic drug development in CD.
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