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Background and AimsHistone deacetylase inhibitors [HDACi] exert potent anti-inflammatory effects. Because of the ubiquitous expression of HDACs, clinical utility of HDACi is limited by off-target effects. Esterase-sensitive motif [ESM] technology aims to deliver ESM-conjugated compounds to human mononuclear myeloid cells, based on their expression of carboxylesterase 1 [CES1]. This study aims to investigate utility of an ESM-tagged HDACi in inflammatory bowel disease [IBD].MethodsCES1 expression was assessed in human blood, in vitro differentiated macrophage and dendritic cells, and Crohn’s disease [CD] colon mucosa, by mass cytometry, quantitative polymerase chain reaction [PCR], and immunofluorescence staining, respectively. ESM-HDAC528 intracellular retention was evaluated by mass spectrometry. Clinical efficacy of ESM-HDAC528 was tested in dextran sulphate sodium [DSS]-induced colitis and T cell transfer colitis models using transgenic mice expressing human CES1 under the CD68 promoter.ResultsCES1 mRNA was highly expressed in human blood CD14+ monocytes, in vitro differentiated and lipopolysaccharide [LPS]-stimulated macrophages, and dendritic cells. Specific hydrolysis and intracellular retention of ESM-HDAC528 in CES1+ cells was demonstrated. ESM-HDAC528 inhibited LPS-stimulated IL-6 and TNF-α production 1000 times more potently than its control, HDAC800, in CES1high monocytes. In healthy donor peripheral blood, CES1 expression was significantly higher in CD14++CD16- monocytes compared with CD14+CD16++ monocytes. In CD-inflamed colon, a higher number of mucosal CD68+ macrophages expressed CES1 compared with non-inflamed mucosa. In vivo, ESM-HDAC528 reduced monocyte differentiation in the colon and significantly improved colitis in a T cell transfer model, while having limited potential in ameliorating DSS-induced colitis.ConclusionsWe demonstrate that monocytes and inflammatory macrophages specifically express CES1, and can be preferentially targeted by ESM-HDAC528 to achieve therapeutic benefit in IBD.

Background A fistula is an abnormal tract connecting two epithelialized surfaces, for example the intestine and the skin. Perianal fistula are a common complication of patients suffering Crohn’s Disease (CD), but also occur in non-IBD patients in the form of cryptoglandular fistula. Around one third of all CD patients develop fistula at some point during their disease course. Fistula are often refractory to therapy, due to poor wound healing responses. In contrast, cryptoglandular fistula often respond to standard therapy. The biological background of this difference is unknown, and comparative studies between the two groups are lacking. The aim of this study was to characterize the cellular composition in fistula tracts of CD and cryptoglandular patients. Methods Curettage material of perianal fistula tracts was obtained during surgical intervention from patients with CD (n=15) and cryptoglandular fistulas (n=5). Single-cell suspensions were stained with a 35-antibody panel, focusing on myeloid and T-cell markers and were analyzed using mass cytometry (CyTOF). To visualize macrophages in the fistula tract we performed in situ hybridization with CD68 and TNF-α. Results The main cellular component of both fistula tracts consisted of CD66a+ granulocytes (64 +/- 24%). However, the remaining mononuclear compartment differed significantly between Crohn and cryptoglandular fistula. In CD, the majority was of lymphoid nature (CD3+ T cells 57 +/-21%, CD19+ B cells 14 +/-15%), while in cryptoglandular tracts, the majority consisted of myeloid origin (61+/- 15%). Within the T cell compartment, the majority of cells was CD45RO+, indicating activation. Presence of a seton increased the proportion of CD45RO+ T cells, in particular in CD4+ cells. In the myeloid compartment, CD14high/HLA-int monocytes, CD14int/HLA-high inflammatory macrophages and CD14high/CD163+ resident macrophages were identified. Interestingly, CD patient samples contained less monocyte-like cells, and substantially more resident macrophages compared to cryptoglandular samples. This feature tended to be even more enhanced in the presence of a seton, although this did not reach statistical significance. In situ hybridization showed a high production of TNF-α in epithelial-like cells in fistula tract of Crohn’s disease patients, but not in macrophages. Conclusion Despite granulocytes being the main contributor to the cellular composition of fistula tracts, striking differences were found between Crohns and cryptoglandular fistula, both in lymphoid/myeloid balance, and in the presence of resident macrophages. We also showed that epithelial-like cells in Crohns’s disease fistula tracts produce high amounts of TNF-α. These differences may contribute to the lack of response to therapy in CD.

Background The aim of the current study was to assess histological features of appendices from patients with UC and its clinical relevance. Methods Patients with UC in remission and active UC (therapy refractory) that underwent an appendectomy in the frame of clinical trials between 2012–2019 were included. Histological features of UC appendices were compared to those of patients with acute appendicitis and colon carcinoma. The Robarts Histopathology Index score (RHI) was used to assess appendiceal inflammation. In patients with active UC, clinical and histological characteristics were compared between patients with and without endoscopic response after appendectomy. Results In total, 140 appendix specimens were assessed (n=35 UC remission, n=35 active UC, n=35 acute appendicitis, n=35 colon carcinoma). Histological features of appendices from UC patients looked like UC rather than acute appendicitis. The incidence of active appendiceal inflammation was not different between UC patients in remission versus active disease (53.7% versus 46.3%, p=0.45) and limited versus extensive disease (58.5% versus 41.5%, p=0.50). Endoscopic response, assessed in 28 therapy refractory patients, was more frequently seen in patients with higher RHI scores (RHI>9 81.8% versus RHI≤9 9.1%, p=0.004) and limited disease (proctitis/left sided 63.6% versus pancolitis 36.4%, p=0.02). Conclusion The presence of active appendiceal inflammation is common in UC and not related to disease activity in the colon. More than 50% of UC patients in remission show active histological disease in the appendix. Favorable response to appendectomy for refractory UC was seen in cases with ulcerative appendicitis. These findings might support the role of the appendix as a driving force in UC.

Inflammatory bowel diseases, such as ulcerative colitis and Crohn’s disease, are disabling conditions characterised by chronic, relapsing inflammation of the gastrointestinal tract. Current treatments are not universally effective or, in the case of therapeutic antibodies, are hampered by immune responses. Janus kinase inhibitors are orally delivered small molecules that target cytokine signalling by preventing phosphorylation of Janus kinases associated with the cytokine receptor. Subsequently, phosphorylation of signal transducers and activators of transcription that relay Janus kinase signalling and transcription of cytokines in the nucleus will be diminished. Key cytokines in the pathogenesis of inflammatory bowel diseases are targeted by Janus kinase inhibitors. Several Janus kinase inhibitors are in development for the treatment of inflammatory bowel diseases. Tofacitinib, inhibiting signalling via all Janus kinase family members, was effective in phase 2 and 3 trials in moderate-severe ulcerative colitis. GSK2586184, a Janus kinase 1 selective inhibitor, induced clinical and endoscopic response in ulcerative colitis; however, the study was discontinued at an early stage due to liver toxicity observed in systemic lupus patients receiving the drug. Filgotinib, a Janus kinase 1 selective inhibitor investigated in treatment of Crohn’s disease, was superior to placebo. As adverse events associated with the broad immunological effect of these agents have been reported, the future application of these drugs is potentially limited. We will discuss the treatment efficacy of Janus kinase inhibition in inflammatory bowel diseases, how current Janus kinase inhibitors available target immune responses relevant in inflammatory bowel disease, and whether more specific kinase inhibition could be effective.

Background Alterations in the mesentery of Crohn’s disease (CD) patients have long been described, although the functional importance of this tissue is less clear. Some studies hypothesise an anti-inflammatory role for enhanced mesentery, whereas others consider a more pathologic function, given its close proximity to the inflamed intestine. Better phenotypic and functional evaluation of the cells present in this tissue is warranted to improve our understanding of its role in disease. We have previously shown a disproportionate presence of macrophages in CD mesorectum. In this study, we aimed to better characterise the phenotype and function of the macrophages in the mesentery in CD. Methods We collected mesenteric specimens from Crohn’s disease, ulcerative colitis (UC) and non-inflammatory bowel disease (IBD) patients, undergoing surgical bowel resections. In IBD patients, mesentery was collected contiguous to the inflamed region of the intestine, in non-IBD patients, mesentery adjacent to the resection margin. Macrophages were sorted by flow cytometry as CD45+ CD66b-CD14+CD11b+ cells. Sorted cells were analysed by expression profiling and functional analysis. Results Macrophages were present in all analysed tissues. Within the population, a discriminating expression pattern for CD11b was present, dividing CD14+ macrophages in a CD11bhigh and a CD11bdim subset. Transcriptional profiling and gene set enrichment showed the CD11bhigh population to be consistent with IFN-induced pro-inflammatory macrophages, while the CD11bdim population was most consistent with IL4-induced regulatory macrophages. Among the top upregulated genes in CD11bhigh macrophages were S100A8 and S100A9, the two heterodimeric subunits for calprotectin. Flow cytometry confirmed higher expression of calprotectin in the CD11bhigh population also on the protein level. Functionally, CD11bdim macrophages showed higher phagocytic capacity, again consistent a role in tissue repair. Strikingly, the CD11bdim subset was diminished significantly in the mesentery of CD patients, with a near absence in some patients, while the profile in UC was comparable to non-IBD patients. Conclusion Mesenteric macrophages contain two populations, CD11bhigh with a pro-inflammatory profile and CD11bdim with a regulatory profile. In the mesentery of CD patients, the CD11bdim population was strongly decreased, consistent with a pro-inflammatory role for this tissue.

Background To facilitate personalised treatment of inflammatory bowel disease (IBD), biomarkers for disease entity and disease severity in easily accessible samples are required. Therefore, we aimed to (i) identify proteins that discriminate Crohn’s disease (CD) from ulcerative colitis (UC), (ii) assess the association of proteomic profiles with disease severity and (iii) assess the potential to use serum samples as a proxy for intestinal biopsies. Methods In this prospective cross-sectional study, IBD patients with active endoscopic disease (presence of ≥1 ulcer (CD) or Mayo score ≥1 (UC)) and age matched non IBD controls (normal endoscopy) were included. In IBD, biopsies were taken from inflamed tissue; in CD from the edge of an ulcer and in UC and controls between 20 and 25 cm from the anal verge. Disease severity was dichotomised in mild vs. severe defined as SES CD Results Forty-one CD, 39 UC and 10 controls were included. Median SES-CD score [IQR] was 9 [7–16] in CD. In UC, 20 (51.3%), 15 (38.5%) and 4 (10.3%) patients had an endoscopic Mayo score of 3, 2 and 1, respectively. Three proteins differed significantly between UC and CD in tissue (IL-17A, IL13 and CCL4). However, this was not reflected in serum levels of these molecules. For prediction of disease severity in CD, elastic net regression identified a set of 11 serum proteins that discriminated mild from severe disease (accuracy 0.667). Top five proteins were IFNγ (OR 3.03), CCL23 (OR 1.28), Cystatin D (OR 0.61), TNF (OR 1.28) and HGF (OR 1.17) (Figure 1). In tissue, no discriminatory profile was identified. Conversely, in serum of UC, no proteins were selected, while a set of three tissue proteins was associated with disease severity (accuracy 0.725). These proteins were osteoprotegerin (OPG) (OR 1.66), IL8 (OR 1.09) and CCL25 (OR 0.97). The discrepancy between serum and tissue was further supported by correlation analyses. Of all 92 proteins, only three showed significant correlation between tissue and serum: IL17A (r = 0.44), TGF-α (r = 0.42) and IL6 (r = 0.39). Conclusion Proteomic profiles between tissue and serum in active IBD correlated poorly. Only in CD, serum markers differentiated mild from severe disease. In UC, no proteins in serum were selected whereas in tissue, expression of OPG, IL-8 and CCL25 was associated with disease severity. These data again emphasise the difference between CD and UC on a molecular level.

Background Histone deacetylases (HDACs) are a group of enzymes that control histone and non-histone deacetylation and influence inflammatory gene transcription. Certain members of the HDAC family control the function of macrophages and play an important role in immune response. In this study, we aimed to study the expression of HDACs in mucosal macrophages isolated from inflammatory bowel diseases (IBD) patients. Methods Both macroscopically inflamed and non-inflamed colon resection tissue were collected from 15 Crohn’s disease (CD) and nine ulcerative colitis (UC) patients operated on for therapy refractory disease. Of the CD patients, 53% had ileal and 47% ileocolonic disease. Of the UC patients, 44% had left-sided colitis and 56% pancolitis. Lamina propria was separated from the muscularis externa, and a targeted array for epigenetic enzymes was performed. To assess the relevance of HDAC9 gene expression in terms of protein level, immunofluorescence staining of HDAC9 protein was undertaken in tissue sections from inflamed and non-inflamed mucosa. CD68 was used as a pan-macrophage marker. Results From our array, expression of HDAC9 was significantly higher in the inflamed mucosa of CD patients compared with UC patients (p = 0.005). Gene expression of HDAC9 in non-inflamed mucosa from CD was elevated compared with non-inflamed mucosa from UC. In addition, in CD, HDAC9 mRNA level was increased in inflamed tissue in comparison to non-inflamed tissue (p = 0.046). In conjunction with the expression data, HDAC9 protein was found highly expressed in inflamed tissue. HDAC9 was predominantly localised in the cytoplasmic compartment of macrophages in non-inflamed tissue whilst HDAC9 localised to the nucleus of macrophages in inflamed tissue. Conclusion HDAC9 is member of class IIA HDAC superfamily that exerts pro-inflammatory properties. The inhibition of HDAC9 in experimental murine colitis clearly enhances regulatory T-cell function, suggesting a critical role for HDAC9 in breaching immune homeostasis (de Zoeten EF et al, 2009). We suggest here that HDAC9 can serve as an additional marker to distinguish CD from UC in tissue biopsies. Furthermore, we show for the first time that HDAC9 protein is expressed in mucosal macrophages of CD patients, indicating its potential in mediating macrophage inflammatory function in IBD. Further studies are currently being undertaken to elucidate the role of HDAC9 in CD pathogenesis.

Background: Intestinal fibrosis is a process driven by chronic inflammation leading to increased presence of myofibroblasts and collagen deposition. Although strictures are rarely seen in ulcerative colitis [UC], longstanding disease is believed to cause fibrosis resulting in altered bowel function. Methods: The presence of fibrosis was studied in colectomy specimens from patients with recent-onset UC refractory to medical treatment [ n = 13] and longstanding UC [ n = 16], and colon cancer patients without UC [ n = 7] as controls. Severity of inflammation was scored according to the Geboes score on haematoxylin and eosin stainings. Immunohistochemistry was performed to detect α-smooth muscle actin, fibronectin and collagen I and III. Results: Colectomy specimens from patients with acute UC showed significantly more inflammation than those with longstanding disease [19 vs 9 points, p = 0.01]. Both acute and longstanding UC showed a thicker muscularis mucosa than controls [0.10 vs 0.10 vs 0.05mm, respectively, p = 0.019]. An increase in collagen I and III deposition in the mucosa was observed in UC compared with controls (40% [30–75] vs 25% [10–25], p = 0.033), but this did not differ significantly among acute and longstanding UC patients. Conclusions: Collagen deposition is enhanced in UC compared with controls. However, UC collagen deposition does not increase significantly over time and does not seem to aggravate the entire fibrotic process.

Background and Aims Regulatory macrophages play a critical role in tissue repair, and we have previously shown that anti-tumour necrosis factor [TNF] antibodies induce these macrophages in vitro and in vivo in IBD patients. The induction of regulatory macrophages can be potentiated using the combination of anti-TNF and thiopurines, consistent with the enhanced efficacy of this combination therapy described in clinical trials. As thiopurines are unfortunately associated with significant side effects, we here aimed to identify alternatives for combination therapy with anti-TNF, using the macrophage induction model as a screening tool. Methods Mixed lymphocyte reactions were treated with anti-TNF and a library of 1600 drug compounds. Induction of CD14+CD206+ macrophages was analysed by flow cytometry. Positive hits were validated in vitro and in the T cell transfer model of colitis. Results Among the 98 compounds potentiating the induction of regulatory macrophages by anti-TNF were six benzimidazoles, including albendazole. Albendazole treatment in the presence of anti-TNF resulted in alterations in the tubulin skeleton and signalling though AMPK, which was required for the enhanced induction. Combination therapy also increased expression levels of the immunoregulatory cytokine IL-10. In vivo, albendazole plus anti-TNF combination therapy was superior to monotherapy in a model of colitis, in terms of both induction of regulatory macrophages and improvement of clinical symptoms. Conclusions Albendazole enhances the induction of regulatory macrophages by anti-TNF and potentiates clinical efficacy in murine colitis. Given its favourable safety profile, these data indicate that the repurposing of albendazole may be a novel option for anti-TNF combination therapy in IBD.

primed, but not IL8 stimulated PMN from CD patients was observed. Conclusions: Although initiation of apoptosis in CD PMN is normal as determined by Annexin V staining, an enhanced survival signal provided by GM-CSF-induced PKB activation may lead to a decreased cleaving of caspases, thereby rescuing PMN from the apoptotic pathway. In toto, our results suggest an enhanced GM-CSF-mediated PMN survival, which may play a role in the chronic intestinal inflammation in CD.

stiffness-mediated induction of aSMA protein expression, FAK phosphorylation, and MLCK and ET-1 gene expression. In addition, CARD-024 partially stimulated members of the COX2/IL-1b inflammatory pathway. Conclusions: In summary, CARD-024 attenuated the pro-fibrotic response of colonic myofibroblasts to either TGFb stimulation or high matrix stiffness, suggesting that vitamin D analogs such as CARD-024 may ameliorate intestinal fibrosis.