Colin de Haar

CD64 distinguishes macrophages from dendritic cells in the gut and reveals the Th1-inducing role of mesenteric lymph node macrophages during colitis

Dendritic cells (DCs) and monocyte‐derived macrophages (MΦs) are key components of intestinal immunity. However, the lack of surface markers differentiating MΦs from DCs has hampered understanding of their respective functions. Here, we demonstrate that, using CD64 expression, MΦs can be distinguished from DCs in the intestine of both mice and humans. On that basis, we revisit the phenotype of intestinal DCs in the absence of contaminating MΦs and we delineate a developmental pathway in the healthy intestine that leads from newly extravasated Ly‐6Chi monocytes to intestinal MΦs. We determine how inflammation impacts this pathway and show that T cell‐mediated colitis is associated with massive recruitment of monocytes to the intestine and the mesenteric lymph node (MLN). There, these monocytes differentiate into inflammatory MΦs endowed with phagocytic activity and the ability to produce inducible nitric oxide synthase. In the MLNs, inflammatory MΦs are located in the T‐cell zone and trigger the induction of proinflammatory T cells. Finally, T cell‐mediated colitis develops irrespective of intestinal DC migration, an unexpected finding supporting an important role for MLN‐resident inflammatory MΦs in the etiology of T cell‐mediated colitis.

Disease-related expression of the IL6/STAT3/SOCS3 signalling pathway in ulcerative colitis and ulcerative colitis-related carcinogenesis

Background Mouse models have shown that interleukin (IL)6 stimulates survival, proliferation and progression to cancer of intestinal epithelial cells via activation of signal transducers and activators of transcription 3 (STAT3). Objective To investigate the expression of IL6/phosphorylated STAT3 (p-STAT3)/suppressor of cytokine signalling 3 (SOCS3) in biopsy specimens from patients with ulcerative colitis (UC) and UC-related colorectal cancer (CRC) progression. Methods Biopsy specimens from patients with inactive UC (n=18), active UC (n=28), UC with low-grade dysplasia (LGD) (n=9), UC with high-grade dysplasia (HGD) (n=7), UC-CRC (n=11) and sporadic CRC (n=14) were included. Biopsy specimens (n=9) from patients without colonic abnormalities served as control. The protein expression of IL6, p-STAT3 and SOCS3 was determined immunohistochemically. Results Patients with active UC had significantly more IL6 and p-STAT3-positive epithelial cells than both patients with inactive UC and controls (strong positive IL6: 53.6%, 11.1% and 0%, respectively; p-STAT3: 64.3%, 22.2% and 11.1%, respectively; all p≤0.012). SOCS3-positive cells were significantly increased in colonic epithelium of both inactive and active UC compared with controls (strong positive: 94.4%, 96.4% and 11.1%, respectively; both p<0.001). In dysplasia and cancer, significantly more epithelial cells expressed IL6 and p-STAT3 compared with controls (strong positive IL6: 72.7% and 0% respectively; p-STAT3: 54.5% and 11.1%, respectively; both p<0.05), whereas the proportion of SOCS3-positive cells in this progression reduced (LGD 33.3%; HGD 14.3%; UC-CRC 9.1%). In addition, methylation of the SOCS3 gene was detected in epithelial cells from UC-CRC biopsy specimens. Conclusion The importance of IL6/p-STAT3 in patients with inflammation-induced CRC was demonstrated. Moreover, SOCS3 may be involved in UC pathogenesis and the absence of SOCS3 seems critical for CRC progression.

Genomic ATG16L1 risk allele-restricted Paneth cell ER stress in quiescent Crohn's disease

Objective Although genome wide association studies have partly uncovered the genetic basis of Crohns disease (CD), it remains a challenge to link genetic polymorphisms to functional intestinal phenotypes. Paneth cells are specialised antimicrobial epithelial cells localised to the small-intestinal crypt-base. Here, we investigate whether genomic variations in ATG16L1 affect Paneth cell function. Design Genomic variation of ATG16L1 (T300A, rs2241880) was determined in DNA from 78 patients with CD and 12 healthy controls. Paraffin-embedded ileal biopsies from patients with genotype AA (n=17), GA (n=38) and patients with the GG allele (n=23) were stained for GRP78, phospho-EIF2α, lysozyme, cleaved-caspase 3, phosphohistone H3, phospho-IκB, p65, phospho-p38MAPK and PHLDA1. Microbial composition of biopsies was assessed by PCR. Disease phenotype was scored. Results In patients with quiescent disease but with an ATG16L1 risk allele, the endoplasmic reticulum (ER) stress markers GRP78 and pEIF2α were highly expressed in Paneth cells. Other CD risk gene variations did not correlate with Paneth cell ER stress. Functionally, patients with ER-stressed Paneth cells showed no changes in intestinal epithelial cells proliferation or apoptosis, Paneth cell or stem cell numbers, p65, phospho-IκB and phospho-p38 staining. However, a significantly increased presence of adherent-invasive Escherichia coli was observed in biopsies from patients with ER-stressed Paneth cells. Phenotypically, patients with GRP78 positive Paneth cells have relatively less colonic disease over ileal disease (−21%, p=0.04), more fistulas (+21%, p=0.05) and an increased need for intestinal surgery (+38%, p=0.002). Conclusions The ATG16L1 T300A polymorphism defines a specific subtype of patients with CD, characterised by Paneth cell ER stress even during quiescent disease. Paneth cell ER stress correlates with bacterial persistence, and is thus likely to modulate antimicrobial functionality of this cell type in patients with CD.

Results of the 2nd scientific workshop of the ECCO (III): basic mechanisms of intestinal healing.

The second scientific workshop of the European Crohns and Colitis Organization (ECCO) focused on the relevance of intestinal healing for the disease course of inflammatory bowel disease (IBD). The objective was to better understand basic mechanisms, markers for disease prediction, detection and monitoring of intestinal healing, impact of intestinal healing on the disease course of IBD as well as therapeutic strategies. The results of this workshop are presented in four separate manuscripts. This section describes basic mechanisms of intestinal healing, identifies open questions in the field and provides a framework for future studies.

New insights into the role of STAT3 in IBD

Abstract Abstract: Although it is clear that inflammatory bowel disease (IBD) involves an inappropriate immune response to floral components, the molecular determinants that mediate the gene transcription underlying and aggravating disease remain poorly understood. There is building momentum, however, that implicates differential regulation of the signal transducer and activator of transcription (STAT) 3 as an important factor in mediating pathogenic gene transcription in IBD, and this notion was reinforced by studies presented at the recent 2011 Digestive Disease Week (DDW) conference in Chicago. In the present report we integrate the existing body of literature with the novel data presented at this meeting to present a kaleidoscopic scheme so as to provide further insight into the role of STAT3 in IBD. A genetic propensity to its overactivation in the monocyte and epithelial compartment compromises the innate defense to allow low‐level bacterial infection to fester and eventually initiate disease. The subsequent STAT3 activation in various relevant mucosal immune compartments, in particular epithelial cell proliferation and survival, and the function of the regulatory T cells and Th17 cells allow the mucosal immune system to fight the infection and return to steady state. As such, the action of STAT3 in IBD is highly context‐dependent but always important. (Inflamm Bowel Dis 2012)

SOCS3 in immune regulation of inflammatory bowel disease and inflammatory bowel disease-related cancer

Inflammatory bowel disease (IBD) has unclear pathogenesis and it is related to the increasing risk of developing colorectal cancer (CRC). Recent studies have uncovered the molecular mechanism of intracellular signaling pathways of inflammatory cytokines such as tumor necrosis factor (TNF)-α, interferon (IFN)-γ and interleukin (IL)-6. The major transcription factors including STAT3 have been shown to play a major role in transmitting inflammatory cytokine signals to the nucleus. The suppressors of cytokine signaling (SOCS) 3 protein is the key physiological regulators of cytokine-mediated STAT3 signaling. As such it influences the development of inflammatory and malignant disorders like this associated with IBD. Here we review the complex function of SOCS3 in innate and adaptive immunity, different cell types (macrophages, neutrophils, dendritic cells, B cells, T cells and intestinal epithelial cells) and the role of SOCS3 on the pathogenesis of inflammatory bowel disease (IBD) and IBD-related cancer. Finally, we explore how this knowledge may open novel avenues for the rational treatment of IBD and IBD-related cancer.

Suppression of p21Rac Signaling and Increased Innate Immunity Mediate Remission in Crohn’s Disease

Overactivation of p21Rac1 is a rate-limiting step for innate immune function in Crohn’s disease and prevents remission. Crohn’s Disease on the Rac Crohn’s disease is a type of inflammatory bowel disease (IBD), wherein the body’s immune system attacks the gastrointestinal tract. In patients with Crohn’s, there are areas of apparently healthy tissue right next to damaged intestine, but it remains unclear what differentiates healthy and inflamed regions. Now, Parikh et al. examine signal transduction differences in healthy and inflamed tissue to find targets that may be protective in Crohn’s. The authors performed a comparative kinome profile in healthy controls as well as healthy and inflamed tissues from Crohn’s patients. They found that p21Rac1 GTPase signaling is suppressed in noninflamed tissue. What’s more, blocking p21Rac1 correlated with clinical improvement of IBD, potentially by boosting innate immune responses. These data suggest that blocking p21Rac1 may be protective for IBD. In inflammatory bowel disease (IBD), large areas of apparently healthy mucosa lie adjacent to ulcerated intestine. Knowledge of the mechanisms that maintain remission in an otherwise inflamed intestine could provide important clues to the pathogenesis of this disease and provide rationale for clinical treatment strategies. We used kinome profiling to generate comprehensive descriptions of signal transduction pathways in inflamed and noninflamed colonic mucosa in a cohort of IBD patients, and compared the results to non-IBD controls. We observed that p21Rac1 guanosine triphosphatase (GTPase) signaling was strongly suppressed in noninflamed colonic mucosa in IBD. This suppression was due to both reduced guanine nucleotide exchange factor activity and increased intrinsic GTPase activity. Pharmacological p21Rac1 inhibition correlated with clinical improvement in IBD, and mechanistically unrelated pharmacological p21Rac1 inhibitors increased innate immune functions such as phagocytosis, bacterial killing, and interleukin-8 production in healthy controls and patients. Thus, suppression of p21Rac activity assists innate immunity in bactericidal activity and may induce remission in IBD.

The cell biology of the intestinal epithelium and its relation to inflammatory bowel disease.

The epithelial layer of our intestines must meet two opposing requirements. On one hand it must allow for efficient uptake of nutrients and fluids, on the other hand it is a vital defence barrier between the milieu interior and the milieu exterior. In contrast to the lung that by virtue of cilia movement is kept virtually sterile, the gut epithelium is confronted by a stupendous microbiological load and a substantial xenobiotic challenge. The efficiency by which our intestinal epithelium manages to deal with the challenge of efficient nutrient absorption while simultaneously fulfilling its barrier function is testimony to what the forces of evolution can accomplish. Importantly, our understanding as to how our gut epithelial compartment manages this balancing act is now rapidly emerging, answering one of the oldest questions in cell biology. Importantly, when aberrations in this balance occur, for instance as a consequence genetic polymorphisms, increased propensity to develop chronic inflammation and inflammatory bowel disease is the result. Thus the knowledge on intestinal cell biology and biochemistry is not only of academic interest but may also aid design of novel avenues for the rational treatment of mucosal disease.

Impeded protein folding and function in active inflammatory bowel disease.

The intestinal tract is covered by a total of 300 square metres of IECs (intestinal epithelial cells) that covers the entire intestinal mucosa. For protection against luminal xenobiotics, pathogens and commensal microbes, these IECs are equipped with membrane-bound transporters as well as the ability to secrete specific protective proteins. In patients with active IBD (inflammatory bowel disease), the expression of these proteins, e.g. ABC (ATP-binding cassette) transporters such as ABCG2 (ABC transporter G2) and defensins, is decreased, thereby limiting the protection against various luminal threats. Correct ER (endoplasmic reticulum)-dependent protein folding is essential for the localization and function of secreted and membrane-bound proteins. Inflammatory triggers, such as cytokines and nitric oxide, can impede protein folding, which causes the accumulation of unfolded proteins inside the ER. As a result, the unfolded protein response is activated which can lead to a cellular process named ER stress. The protein folding impairment affects the function and localization of several proteins, including those involved in protection against xenobiotics. In the present review, we discuss the possible inflammatory pathways affecting protein folding and eventually leading to IEC malfunction in patients with active IBD.

Alcohol Facilitates CD1d Loading, Subsequent Activation of NKT Cells, and Reduces the Incidence of Diabetes in NOD Mice

Background Ethanol (‘alcohol’) is a partly hydrophobic detergent that may affect the accessibility of glycolipids thereby influencing immunological effects of these molecules. Methods The study included cellular in vitro tests using α-galactosylceramide (αGalCer), and in vivo NOD mice experiments detecting diabetes incidence and performing behavioural and bacterial analyses. Results Alcohol in concentrations from 0.6% to 2.5% increased IL-2 production from NKT cells stimulated with αGalCer by 60% (p<0.05). CD1d expressed on HeLa cells contained significantly increasing amounts of αGalCer with increasing concentrations of alcohol, suggesting that alcohol facilitated the passive loading of αGalCer to CD1d. NOD mice were found to tolerate 5% ethanol in their drinking water without signs of impairment in liver function. Giving this treatment, the diabetes incidence declined significantly. Higher numbers of CD3+CD49b+ NKT cells were found in spleen and liver of the alcohol treated compared to the control mice (p<0.05), whereas the amount of CD4+Foxp3+ regulator T cells did not differ. Increased concentrations of IFN-γ were detected in 24-hour blood samples of alcohol treated mice. Behavioural studies showed no change in attitude of the ethanol-consuming mice, and bacterial composition of caecum samples was not affected by alcohol, disqualifying these as protective mechanisms. Conclusion Alcohol facilitates the uptake of glycolipids and the stimulation of NKT cells, which are known to counteract Type 1 diabetes development. We propose that this is the acting mechanism by which treatment with alcohol reduces the incidence of diabetes in NOD mice. This is corroborated by epidemiology showing beneficial effect of alcohol to reduce the severity of atherosclerosis and related diseases.