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SARS-CoV-2, the causative virus of COVID-19, continues to threaten global public health. COVID-19 is a multi-organ disease, causing not only respiratory distress, but also extrapulmonary manifestations, including gastrointestinal symptoms with SARS-CoV-2 RNA shedding in stool long after respiratory clearance. Despite global vaccination and existing antiviral treatments, variants of concern are still emerging and circulating. Of note, new Omicron BA.5 sublineages both increasingly evade neutralizing antibodies and demonstrate an increased preference for entry via the endocytic entry route. Alternative to direct-acting antivirals, host-directed therapies interfere with host mechanisms hijacked by viruses, and enhance cell-mediated resistance with a reduced likelihood of drug resistance development. Here, we demonstrate that the autophagy-blocking therapeutic berbamine dihydrochloride robustly prevents SARS-CoV-2 acquisition by human intestinal epithelial cells via an autophagy-mediated BNIP3 mechanism. Strikingly, berbamine dihydrochloride exhibited pan-antiviral activity against Omicron subvariants BA.2 and BA.5 at nanomolar potency, providing a proof of concept for the potential for targeting autophagy machinery to thwart infection of current circulating SARS-CoV-2 subvariants. Furthermore, we show that autophagy-blocking therapies limited virus-induced damage to intestinal barrier function, affirming the therapeutic relevance of autophagy manipulation to avert the intestinal permeability associated with acute COVID-19 and post-COVID-19 syndrome. Our findings underscore that SARS-CoV-2 exploits host autophagy machinery for intestinal dissemination and indicate that repurposed autophagy-based antivirals represent a pertinent therapeutic option to boost protection and ameliorate disease pathogenesis against current and future SARS-CoV-2 variants of concern.

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Deletion of endoplasmic reticulum resident chaperone Grp78 results in activation of the unfolded protein response and causes rapid depletion of the entire intestinal epithelium. Whether modest reduction of Grp78 may affect stem cell fate without compromising intestinal integrity remains unknown. Here, we employ a model of epithelial-specific, heterozygous Grp78 deletion by use of VillinCreERT2-Rosa26ZsGreen/LacZ-Grp78+/fl mice and organoids. We examine models of irradiation and tumorigenesis, both in vitro and in vivo. Although we observed no phenotypic changes in Grp78 heterozygous mice, Grp78 heterozygous organoid growth was markedly reduced. Irradiation of Grp78 heterozygous mice resulted in less frequent regeneration of crypts compared with nonrecombined (wild-type) mice, exposing reduced capacity for self-renewal upon genotoxic insult. We crossed mice to Apc-mutant animals for adenoma studies and found that adenomagenesis in Apc heterozygous-Grp78 heterozygous mice was reduced compared with Apc heterozygous controls (1.43 vs. 3.33; P < 0.01). In conclusion, epithelium-specific Grp78 heterozygosity compromises epithelial fitness under conditions requiring expansive growth such as adenomagenesis or regeneration after γ-irradiation. These results suggest that Grp78 may be a therapeutic target in prevention of intestinal neoplasms without affecting normal tissue.Significance: Heterozygous disruption of chaperone protein Grp78 reduces tissue regeneration and expansive growth and protects from tumor formation without affecting intestinal homeostasis. Cancer Res; 78(21); 6098–106. ©2018 AACR.

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Background Previous studies have indicated that the appendix may be a priming site of ulcerative colitis (UC). Appendectomy is inversely associated with the development of UC, and is suggested to have a beneficial effect on the disease course in patients with refractory disease. Objective The aim of the current study was to assess histological features of appendices from patients with UC and their clinical relevance. Methods Patients with UC in remission and active UC (therapy refractory) that underwent appendectomy between 2012 and 2019 were included. Histological features of UC appendices were compared to those of patients with acute appendicitis and colon carcinoma. The Robarts Histopathology Index (RHI) was used to assess appendiceal inflammation. In patients with active UC, histological and clinical characteristics were compared between patients with and without endoscopic response following 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 presence of active appendiceal inflammation was comparable between patients in remission versus active disease (53.7% vs. 46.3%, p = 0.45) and limited versus extensive disease (58.5% vs. 41.5%, p = 0.50). Endoscopic response (Mayo 0-1) following appendectomy, assessed in 28 therapy refractory patients, was more frequently seen in patients with higher RHI scores (RHI > 6: 81.8% vs. RHI ≤ 6: 9.1%, p = 0.03) and limited disease (proctitis/left sided 63.6% vs. pancolitis 36.4%, p = 0.02). Conclusion The presence of active appendiceal inflammation is common in UC and does not correlate with colonic disease activity. More than 50% of UC patients in remission showed 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 pivotal organ in UC.

Indian Hedgehog (Ihh) is a morphogen expressed by epithelial cells in the small intestine and colon that signals in a paracrine manner to gp38+ stromal cells. The loss of Ihh signaling results in increased epithelial proliferation, lengthening and multiplication of intestinal crypts and the activation of a stromal cell immune response. How Ihh controls epithelial proliferation through the stroma and how it affects colorectal cancer development remains poorly defined. To study the influence of Ihh signaling on the earliest stage of colorectal carcinogenesis, we used a well characterized mouse model in which both alleles of the Adenoma Polyposis Coli (Apc) gene could be inducibly deleted, leading to instant transformation of the colonic epithelium to an adenomatous phenotype. Concurrent deletion of Ihh from the adenomatous colonic epithelium of Apc inducible double mutant mice resulted in a remarkable increase in the hyperproliferative epithelial phenotype and increased accumulation of Lgr5+ stem cells. Transcriptional profiling of sorted colonic gp38+ fibroblasts showed upregulation of three ErbB pathway ligands (EREG, BTC, and NRG1) in Apc−/−Ihh−/− double mutant mice. We found that recombinant EREG, BTC, and NRG1 but not Lgr5 ligand R-Spondin promoted growth and proliferation of Apc double mutant colonic organoids. Thus, the loss of Ihh enhances Apc-driven colonic adenomagenesis via upregulation of ErbB pathway family members in colonic stromal cells. Our findings highlight the critical role of epithelium-derived Indian Hedgehog as a stromal tumor suppressor in the intestine.

Background: Myeloid cells are critical determinants of the sustained inflammation in Crohn’s Disease (CD). Targeting such cells may be an effective therapeutic approach for refractory CD patients. Bromodomain and extra-terminal domain protein inhibitors (iBET) are potent anti-inflammatory agents; however, they also possess wide-ranging toxicities. In the current study, we make use of a BET inhibitor containing an esterase sensitive motif (ESM-iBET), which is cleaved by carboxylesterase-1 (CES1), a highly expressed esterase in mononuclear myeloid cells. Methods: We profiled CES1 protein expression in the intestinal biopsies, peripheral blood, and CD fistula tract (fCD) cells of CD patients using mass cytometry. The anti-inflammatory effect of ESM-iBET or its control (iBET) were evaluated in healthy donor CD14+ monocytes and fCD cells, using cytometric beads assay or RNA-sequencing. Results: CES1 was specifically expressed in monocyte, macrophage, and dendritic cell populations in the intestinal tissue, peripheral blood, and fCD cells of CD patients. ESM-iBET inhibited IL1β, IL6, and TNFα secretion from healthy donor CD14+ monocytes and fCD immune cells, with 10- to 26-fold more potency over iBET in isolated CD14+ monocytes. Transcriptomic analysis revealed that ESM-iBET inhibited multiple inflammatory pathways, including TNF, JAK-STAT, NF-kB, NOD2, and AKT signaling, with superior potency over iBET. Conclusions: We demonstrate specific CES1 expression in mononuclear myeloid cell subsets in peripheral blood and inflamed tissues of CD patients. We report that low dose ESM-iBET accumulates in CES1-expressing cells and exerts robust anti-inflammatory effects, which could be beneficial in refractory CD patients.

Primary sclerosing cholangitis (PSC) is an inflammatory disease of the biliary tree, characterised by stricturing bile duct disease and progression to liver fibrosis. The pathophysiology of PSC is still unknown. The concurrence with inflammatory bowel disease (IBD) in about 70% of cases has led to the hypothesis that gut-homing lymphocytes aberrantly traffic to the liver, contributing to disease pathogenesis in patients with both PSC and IBD (PSC-IBD). The discovery of mutual trafficking pathways of lymphocytes to target tissues, and expression of gut-specific adhesion molecules and chemokines in the liver has pointed in this direction. There is now increasing interest in using drugs that intervene with these trafficking pathways (e.g. vedolizumab, etrolizumab) for the treatment of PSC-IBD. In this review we discuss what is currently known about the immunological interactions between the gut and the liver in concomitant PSC and IBD, as well as potential therapeutic options for intervening in these mechanisms.

Background and aims Immortalized cell lines have been long used as substitute for ex vivo murine and human material, but exhibit features that are not found in healthy tissue. True human dendritic cells (DC) cannot be cultured or passaged as opposed to immortalized cell lines. Research in the fields of immunogenic responses and immunotolerance in DCs has increased over the last decade. Autophagy has gained interest in these fields as well, and has been researched extensively in many other cell types as well. Here we have studied the applicability of cell line-derived dendritic cell-like cells of six myeloid cell lines aimed at research focussed on autophagy. Methods Six myeloid leukaemia cell lines were differentiated towards cell line-derived dendritic cell-like cells (cd-DC) using GM-CSF, IL-4, Ionomycine and PMA: HL60, KG1, MM6, MV-4-11, THP1 and U937. Autophagy was modulated using Rapamycin, Bafilomycin A1 and 3MA. Cell lines were genotyped for autophagy-related SNPs using RFLP. Marker expression was determined with FACS analysis and cytokine profiles were determined using Human Cytometric Bead Assay. Antigen uptake was assessed using Fluoresbrite microspheres. Results and discussion All researched cell lines harboured SNPs in the autophagy pathways. MM6 and THP1 derived cd-DCs resembled monocyte-derived DCs (moDC) most closely in marker expression, cytokine profiles and autophagy response. The HL60 and U937 cell lines proved least suitable for autophagy-related dendritic cell research. Conclusion The genetic background of cell lines should be taken into account upon studying (the effects of) autophagy in any cell line. Although none of the studied cell lines recapitulate the full spectrum of DC characteristics, MM6 and THP1 derived cd-DCs are most suitable for autophagy-related research in dendritic cells.

Background Inflammatory bowel disease (IBD) is characterised by substantial heterogeneity in treatment response. With an expanding number of therapeutic agents, identifying optimal treatment at the patient level remains a major challenge. Aim To systematically review the available literature on predictive biomarkers of therapeutic response in IBD. Methods An electronic literature search was performed on 30 January 2018 using MEDLINE, EMBASE and the Cochrane Library. Retrospective, prospective, uncontrolled and controlled studies reporting on biomarkers predicting therapeutic response in paediatric and adult IBD populations were eligible for inclusion. The methodological quality of the included studies was assessed using the QUIPS tool. Due to anticipated heterogeneity and limited data, a qualitative, rather than quantitative, assessment was planned. Results Of the 10 638 citations identified, 92 articles met the inclusion criteria. Several potential DNA, mRNA and protein markers were evaluated as predictive biomarkers. Most studies focused on predicting response to anti-TNF agents. Substantial between-study heterogeneity was identified with respect to both the biomarkers studied and the definition of response. None of the included studies received a low risk of bias rating for all six domains. Currently, none of the biomarkers is sufficiently predictive for clinical use. Conclusions The search for predictive biomarkers is still in its infancy and current evidence is limited. Future research efforts should take into account the high patient heterogeneity within prospective trials with objective response assessment. Predictive models will most likely comprise a combination of several molecular markers from integrated omics-levels and clinical characteristics.

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.

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to digest solid food. This process is termed suckling-to-weaning transition and results in the replacement of neonatal epithelium with adult epithelium which goes hand in hand with metabolic and morphological adjustments. These complex developmental changes are the result of a genetic program that is intrinsic to the intestinal epithelial cells but can, to some extent, be modulated by extrinsic factors. Prolonged culture of mouse primary intestinal epithelial cells from late fetal period, recapitulates suckling-to-weaning transition in vitro. Here, we describe a detailed protocol for mouse fetal intestinal organoid culture best suited to model this process in vitro. We describe several useful assays designed to monitor the change of intestinal functions associated with suckling-to-weaning transition over time. Additionally, we include an example of an extrinsic factor that is capable to affect suckling-to-weaning transition in vivo, as a representation of modulating the timing of suckling-to-weaning transition in vitro. This in vitro approach can be used to study molecular mechanisms of the suckling-to-weaning transition as well as modulators of this process. Importantly, with respect to animal ethics in research, replacing in vivo models by this in vitro model contributes to refinement of animal experiments and possibly to a reduction in the use of animals to study gut maturation processes.

Indian Hedgehog (Ihh) is a morphogen expressed by epithelial cells in the small intestine and colon that signals in a paracrine manner to gp38+ stromal cells. The loss of Ihh signaling results in increased epithelial proliferation, lengthening and multiplication of intestinal crypts and the activation of a stromal cell immune response. How Ihh controls epithelial proliferation through the stroma and how it affects colorectal cancer development remains poorly defined. To study the influence of Ihh signaling on the earliest stage of colorectal carcinogenesis, we used a well characterized mouse model in which both alleles of the Adenoma Polyposis Coli (Apc) gene could be inducibly deleted, leading to instant transformation of the colonic epithelium to an adenomatous phenotype. Concurrent deletion of Ihh from the adenomatous colonic epithelium of Apc inducible double mutant mice resulted in a remarkable increase in the hyperproliferative epithelial phenotype and increased accumulation of Lgr5+ stem cells. Transcriptional profiling of sorted colonic gp38+ fibroblasts showed upregulation of three ErbB pathway ligands (EREG, BTC, and NRG1) in Apc

MiR-511-3p is embedded in intron 5 of the CD206/MRC1 gene Mrc1, expressed by macrophage and dendritic cell populations. CD206 and miR-511-3p expression are co-regulated, and their contribution to intestinal inflammation is unclear. We investigated their roles in intestinal inflammation in both mouse and human systems. Colons of CD206-deficient mice displayed normal numbers of monocytes, macrophage, and dendritic cells. In experimental colitis, CD206-deficient mice had attenuated inflammation compared with wild-type (WT) mice. However, neither a CD206 antagonist nor a blocking antibody reproduced this phenotype, suggesting that CD206 was not involved in this response. Macrophages isolated from CD206-deficient mice had reduced levels of miR-511-3p and Tlr4 compared with WT, which was associated with reduced pro-inflammatory cytokine production upon lipopolysaccharides (LPS) and fecal supernatant stimulation. Macrophages overexpressing miR-511-3p showed 50% increase of Tlr4 mRNA, whereas knockdown of miR-511-3p reduced Tlr4 mRNA levels by 60%, compared with scrambled microRNA (miRNA)-transduced cells. Response to anti-tumor necrosis factor (TNF) treatment has been associated with elevated macrophage CD206 expression in the mucosa. However, in colon biopsies no statistically significant change in miR-511-3p was detected. Taken together, our data show that miR-511-3p controls macrophage-mediated microbial responses and is involved in the regulation of intestinal inflammation.

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 Janus kinases (JAKs) mediate cytokine signaling involved in inflammatory bowel disease. The pan-JAK inhibitor tofacitinib has shown efficacy in the treatment of ulcerative colitis. However, concerns regarding adverse events due to their wide spectrum inhibition fueled efforts to develop selective JAK inhibitors. Given the crucial role of myeloid cells in intestinal immune homeostasis, we evaluated the effect of pan-JAK and selective JAK inhibitors on pro- and anti-inflammatory macrophage polarization and function (M1/M2) and in experimental colitis. Methods Murine bone marrow–derived macrophages or human monocytes were treated using JAK1 and JAK3 selective inhibitors (JAK1i;JAK3i) and tofacitinib and were evaluated by transcriptional, functional, and metabolic analyses. In vivo, oral administration of JAK1i and tofacitinib (10 or 30 mg/kg) was tested in both acute and acute rescue dextran sodium sulfate (DSS) colitis. Results Both tofacitinib and JAK1i but not JAK3i effectively inhibited STAT1 phosphorylation and interferon gamma–induced transcripts in M1 polarized macrophages. Strikingly, transcriptional profiling suggested a switch from M1 to M2 type macrophages, which was supported by increased protein expression of M2-associated markers. In addition, both inhibitors enhanced oxidative phosphorylation rates. In vivo, JAK1i and tofacitinib did not protect mice from acute DSS-induced colitis but ameliorated recovery from weight loss and disease activity during acute rescue DSS-induced colitis at the highest dose. Conclusion JAK1i and tofacitinib but not JAK3i induce phenotypical and functional characteristics of anti-inflammatory macrophages, suggesting JAK1 as the main effector pathway for tofacitinib in these cells. In vivo, JAK1i and tofacitinib modestly affect acute rescue DSS-induced colitis.

CD103+ dendritic cells (DC) are crucial for regulation of intestinal tolerance in humans. However, upon infection of the lamina propria this tolerogenic response is converted to an inflammatory response. Here we show that immunoglobulin A (IgA) immune complexes (IgA-IC), which are present after bacterial infection of the lamina propria, are important for the induction of inflammation by the human CD103+SIRPα+ DC subset. IgA-IC, by recognition through FcαRI, selectively amplify the production of proinflammatory cytokines TNF, IL-1β and IL-23 by human CD103+ DCs. These cells then enhance inflammation by promoting Th17 responses and activating human intestinal innate lymphoid cells 3. Moreover, FcαRI-induced cytokine production is orchestrated via upregulation of cytokine translation and caspase-1 activation, which is dependent on glycolytic reprogramming mediated by kinases Syk, PI3K and TBK1-IKKε. Our data suggest that the formation of IgA-IC in the human intestine provides an environmental cue for the conversion of a tolerogenic to an inflammatory response., Dendritic cells (DC) are important for maintaining immune homeostasis in the gut, but how they promote intestinal inflammation upon bacterial infection is still unclear. Here the authors show that IgA immune complexes induce proinflammatory cytokine production by metabolic reprogramming of otherwise tolerogenic human CD103+ DCs.

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 Crohn's disease is a chronic inflammatory disorder of the intestine and often leads to fibrosis, characterized by excess extracellular matrix [ECM] deposition, increased tissue stiffness, and stricture formation. Here we evaluated the contribution of myofibroblast-ECM interactions to the development of intestinal fibrosis in Crohn's disease. Methods Matched primary human myofibroblasts were isolated from stenotic, inflamed and normal-appearing small intestine within the same Crohn's disease patient [n = 10]. Cells were analyzed by gene expression profiling, microscopy and functional assays, including matrix metalloproteinase [MMP] production and ECM contraction. Results We demonstrated that myofibroblasts isolated from stenotic intestine differed both in phenotype and function from those isolated from purely inflammatory or normal-appearing intestine of the same patient. Stenotic myofibroblasts displayed increased expression of genes associated with ECM modulation and collagen deposition. Upon culture in a fibrotic environment, normal myofibroblasts increased expression of MMPs to counteract the mechanical force exerted by the matrix. Interestingly, stenotic myofibroblasts showed a paradoxical response with decreased expression of MMP3. In addition, stenotic myofibroblasts expressed increased levels of the collagen crosslinking enzyme lysyl oxidase [LOX] and induced significantly more ECM contraction than both normal and inflamed myofibroblasts. Importantly, LOX inhibition completely restored MMP3 activity in stenotic myofibroblasts grown in a fibrotic environment, and prevented excessive ECM contraction. Conclusions Together these data indicate aberrancies in the myofibroblast-ECM interaction in Crohn's disease, and identify LOX inhibition as a potential anti-fibrotic agent in this condition.

Deletion of endoplasmic reticulum resident chaperone Grp78 results in activation of the unfolded protein response and causes rapid depletion of the entire intestinal epithelium. Whether modest reduction of Grp78 may affect stem cell fate without compromising intestinal integrity remains unknown. Here, we employ a model of epithelial-specific, heterozygous Grp78 deletion by use of VillinCreERT2-Rosa26ZsGreen/LacZ-Grp78+/fl mice and organoids. We examine models of irradiation and tumorigenesis, both in vitro and in vivo. Although we observed no phenotypic changes in Grp78 heterozygous mice, Grp78 heterozygous organoid growth was markedly reduced. Irradiation of Grp78 heterozygous mice resulted in less frequent regeneration of crypts compared with nonrecombined (wild-type) mice, exposing reduced capacity for self-renewal upon genotoxic insult. We crossed mice to Apc-mutant animals for adenoma studies and found that adenomagenesis in Apc heterozygous-Grp78 heterozygous mice was reduced compared with Apc heterozygous controls (1.43 vs. 3.33; P < 0.01). In conclusion, epithelium-specific Grp78 heterozygosity compromises epithelial fitness under conditions requiring expansive growth such as adenomagenesis or regeneration after γ-irradiation. These results suggest that Grp78 may be a therapeutic target in prevention of intestinal neoplasms without affecting normal tissue. Significance: Heterozygous disruption of chaperone protein Grp78 reduces tissue regeneration and expansive growth and protects from tumor formation without affecting intestinal homeostasis. Cancer Res; 78(21); 6098–106. ©2018 AACR.

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.

Thiopurines are commonly used drugs in the therapy of Crohn's disease, but unfortunately only show a 30% response rate. The biological basis for the thiopurine response is unclear, thus hampering patient selection prior to treatment. A genetic risk factor associated specifically with Crohn's disease is a variant in ATG16L1 that reduces autophagy. We have previously shown that autophagy is involved in dendritic cell (DC)-T-cell interactions and cytoskeletal regulation. Here we further investigated the role of autophagy in DC cytoskeletal modulation and cellular trafficking. Autophagy-deficient DC displayed loss of filopodia, altered podosome distribution, and increased membrane ruffling, all consistent with increased cellular adhesion. Consequently, autophagy-deficient DC showed reduced migration. The cytoskeletal aberrations were mediated through hyperactivation of Rac1, a known thiopurine target. Indeed thiopurines restored the migratory defects in autophagy-deficient DC. Clinically, the ATG16L1 risk variant associated with increased response to thiopurine treatment in patients with Crohn's disease but not ulcerative colitis. These results suggest that the association between ATG16L1 and Crohn's disease is mediated at least in part through Rac1 hyperactivation and subsequent defective DC migration. As this phenotype can be corrected using thiopurines, ATG16L1 genotyping may be useful in the identification of patients that will benefit most from thiopurine treatment.

Summary: Intestinal epithelial cells have a defined hierarchy with stem cells located at the bottom of the crypt and differentiated cells more at the top. Epithelial cell renewal and differentiation are strictly controlled by various regulatory signals provided by epithelial as well as surrounding cells. Although there is evidence that stromal cells contribute to the intestinal stem cell niche, their markers and the soluble signals they produce have been incompletely defined. Using a number of established stromal cell markers, we phenotypically and functionally examined fibroblast populations in the colon. CD90+ fibroblasts located in close proximity to stem cells in vivo support organoid growth in vitro and express crucial stem cell growth factors, such as Grem1, Wnt2b, and R-spondin3. Moreover, we found that CD90+ fibroblasts express a family of proteins—class 3 semaphorins (Sema3)—that are required for the supportive effect of CD90+ fibroblasts on organoid growth. : Stem cell proliferation is dependent on regulatory signals from surrounding cells, including stromal cells. Karpus et al. identify a membrane marker, CD90, that specifically marks stem cell niche fibroblasts in the colon. CD90+ fibroblasts produce class 3 semaphorins that promote stem cell proliferation via the Nrp2 receptor. Keywords: colon, intestine, stem cell, niche, fibroblast, stroma, Gli1, CD90, semaphorin, Nrp2

The glucocorticoid receptor is present in a TCR-associated complex, which includes the Src family tyrosine kinase Lck. Glucocorticoids rapidly dissociate this complex, resulting in the inhibition of canonical Lck-phospholipase C (PLC)γ–dependent TCR signaling. The relative importance of this nongenomic role for the glucocorticoid receptor compared with its direct transcriptional effects is not known. Superantigens induce a state of steroid resistance in activated T cells. It was reported that, in addition to canonical Lck-PLCγ signaling, superantigens can activate a noncanonical G protein–PLCβ–dependent signaling pathway. In this study, we show that staphylococcal enterotoxin B activates a Gαq and PLCβ2–dependent pathway in human T cells. We find that this pathway bypasses the need for canonical Lck-PLCγ signaling in T cell activation and renders superantigen-stimulated T cells insensitive to glucocorticoids in vitro. We show that the PLCβ inhibitor U-73122 sensitizes staphylococcal enterotoxin B–treated mice to dexamethasone in vivo. In conclusion, we find that effects of glucocorticoids on TCR-induced T cell proliferation are mainly nongenomic and can be bypassed by the activation of an Lck-independent signaling pathway.

Background: The repertoire of immunomodulators that can be used for the treatment of inflammatory bowel disease is limited. The use of these drugs is further restricted by the occurrence of side effects in a proportion of patients. Miltefosine (hexadecylphosphocholine) is a lipid drug developed in the 1980s for the treatment of cancer but is nowadays best known for its application in the oral treatment of leishmaniasis. Although the exact mechanism of action of miltefosine has yet to be elucidated, the drug has previously been shown to inhibit phospholipases and protein kinase C, both key components of proproliferative signal transduction in T cells. Methods: Stimulated peripheral blood lymphocyte were treated with miltefosine, and proliferation was measured. We use the CD45RBhighT-cell transfer colitis model to investigate the effect of miltefosine treatment on intestinal inflammation. Effects on the severity of colitis were studied by histochemical and immunohistochemical staining, and cytokine levels were determined using a cytokine bead array. Results: Miltefosine inhibited T-cell proliferation in vitro. In the transfer model, miltefosine significantly ameliorated the severity of colitis as measured by clinical, (immuno)histochemical, and biochemical parameters. Conclusions: Miltefosine inhibits T-cell proliferation and effectively reduces inflammation in the T-cell transfer model. The drug may therefore be a candidate immunomodulator for inflammatory bowel disease. Copyright

Objective To determine the prevalence of upregulation of interferon (IFN) type I inducible genes, the so called ‘IFN type I signature’, in CD14 monocytes in 69 patients with primary Sjögren's syndrome (pSS) and 44 healthy controls (HC) and correlate it with disease manifestations and expression of B cell activating factor (BAFF). Methods Expression of IFI44L, IFI44, IFIT3, LY6E and MX1 was measured using real time quantitative PCR in monocytes. Expression values were used to calculate IFN type I scores for each subject. pSS patients positive for the IFN type I signature (IFN score≥10) and patients negative for the signature (IFN score

Background and aim Mesenchymal stromal cells (MSCs) are pluripotent cells that have immunosuppressive effects both in vitro and in experimental colitis. Promising results of MSC therapy have been obtained in patients with severe graft versus host disease of the gut. Our objective was to determine the safety and feasibility of autologous bone marrow derived MSC therapy in patients with refractory Crohn9s disease. Patients and intervention 10 adult patients with refractory Crohn9s disease (eight females and two males) underwent bone marrow aspiration under local anaesthesia. Bone marrow MSCs were isolated and expanded ex vivo. MSCs were tested for phenotype and functionality in vitro. 9 patients received two doses of 1–2×10 6 cells/kg body weight, intravenously, 7 days apart. During follow-up, possible side effects and changes in patients9 Crohn9s disease activity index (CDAI) scores were monitored. Colonoscopies were performed at weeks 0 and 6, and mucosal inflammation was assessed by using the Crohn9s disease endoscopic index of severity. Results MSCs isolated from patients with Crohn9s disease showed similar morphology, phenotype and growth potential compared to MSCs from healthy donors. Importantly, immunomodulatory capacity was intact, as Crohn9s disease MSCs significantly reduced peripheral blood mononuclear cell proliferation in vitro. MSC infusion was without side effects, besides a mild allergic reaction probably due to the cryopreservant DMSO in one patient. Baseline median CDAI was 326 (224–378). Three patients showed clinical response (CDAI decrease ≥70 from baseline) 6 weeks post-treatment; conversely three patients required surgery due to disease worsening. Conclusions Administration of autologous bone marrow derived MSCs appears safe and feasible in the treatment of refractory Crohn9s disease. No serious adverse events were detected during bone marrow harvesting and administration.

Objectives:In the salivary glands of patients with primary Sjögren Syndrome (pSjS) an accumulation of dendritic cells (DCs) is seen, which is thought to play a role in stimulating local inflammation. Aberrancies in subsets of monocytes, generally considered the blood precursors for DCs, may play a role in this accumulation of DCs. This study is aimed at determining the level of mature CD14lowCD16+ monocytes in pSjS and their contribution to the accumulation of DCs in pSjS.Methods:Levels of mature and immature monocytes in patients with pSjS (n = 19) and controls (n = 15) were analysed by flow cytometry. The reverse transmigration system was used for generation of DCs generated from monocyte subsets. The phenotype of DCs in pSjS salivary glands was analysed using immunohistochemistry. In vivo tracking of monocyte subsets was performed in a mouse model.Results:Increased levels of mature CD14lowCD16+ monocytes were found in patients with pSjS (mean (SD) 14.5 (5.5)% vs 11.4 (3.4)%). These cells showed normal expression of chemokine receptor and adhesion molecules. Mature monocytes partly developed into DC-lysosome-associated membrane glycoprotein (LAMP)+ (19.6 (7.5)%) and CD83+ (16 (9)%) DCs, markers also expressed by DCs in pSjS salivary glands. Monocyte tracking in the non-obese diabetic (NOD) mouse showed that the homologue population of mature mouse monocytes migrated to the salivary glands, and preferentially developed into CD11c+ DCs in vivo.Conclusions:Mature monocytes are increased in pSjS and patient and mouse data support a model where this mature monocyte subset migrates to the salivary glands and develops into DCs.

In the salivary glands of primary Sjögren's syndrome (pSjS) patients, type I IFN activity is increased, but systemic levels of type I IFN proteins are rarely detected. This study focused on the systemic activity of type I IFN in pSjS, as well as the role of peripheral plasmacytoid dendritic cells (pDC). Monocytes obtained from pSjS patients showed an increased expression of 40 genes. Twenty-three of these genes (58%), including IFI27, IFITM1, IFIT3 and IFI44, were inducible by type I IFN. pSjS serum had an enhanced capability of inducing IFI27, IFITM1, IFIT3 and IFI44 in the monocytic cell line THP-1, likely due to the action of IFN-beta. This effect could be inhibited by blocking the type I IFN receptor, supporting a high type I IFN bioactivity in pSjS serum. In addition, circulatory pDC showed increased expression of CD40. This expression was correlated to the expression level of the type I IFN-regulated genes IFI27 and IFITM1 in monocytes of the same individual. This study indicates that the increased type I IFN activity observed in pSjS patients is not only a local but also a systemic phenomenon and points to pDC as a possible source of this activity.