Lea I. Kredel

Adipokines from local fat cells shape the macrophage compartment of the creeping fat in Crohn's disease

Objective The creeping fat in Crohns disease (CD) is infiltrated by macrophages; local adipokine levels are increased. This study aimed to link these observations to define a role for macrophages in the pathology of human CD. Methods Human peripheral blood CD14 cells were polarised in vitro into M1 and M2 macrophages. The effects on adipokine receptors, phenotypic surface markers, cytokines and chemokines were assessed after treatment with leptin and adiponectin. Immunohistochemistry visualised macrophage subtypes in samples of mesenteric fat tissue from patients with CD. The chemotactic potential of secreted macrophage products was determined by T cell migration and chemokine production in vitro. Results Although both adipokines altered the phenotype and function of M1 and M2 macrophages, M2 macrophages were more susceptible. M1 responded to leptin by increased cytokine production, but the stronger effect was seen in M2 macrophages with high expression of interleukin (IL)-10, IL-6 and tumour necrosis factor α. Adiponectin exerted similar effects and led to upregulated mannose receptor expression by M2 macrophages. Large macrophage numbers within the mesenteric fat tissue of patients with CD comprise a unique infiltration predominantly of M2 macrophages, leading to an IL-10-rich environment. While leptin increased the potency of both subtypes to attract CD3 T cells, adiponectin only affected M2 macrophages. Conclusion The adipocyte-dependent microenvironment within the creeping fat of patients with CD modulates the local macrophage compartment to a preference for the M2 subtype. The findings in this study with human cells suggest a protective role for the mesenteric fat in CD in terms of an enveloping barrier with the potential to limit intestinal inflammation.

Diversity of Intestinal Macrophages in Inflammatory Bowel Diseases.

Macrophages as innate immune cells and fast responders to antigens play a central role in protecting the body from the luminal content at a huge interface. Chronic inflammation in inflammatory bowel diseases massively alters the number and the subset diversity of intestinal macrophages. We here address the diversity within the human intestinal macrophage compartment at the level of similarities and differences between homeostasis and chronic intestinal inflammation as well as between UC and CD, including the potential role of macrophage subsets for intestinal fibrosis. Hallmark of macrophages is their enormous plasticity, i.e., their capacity to integrate signals from their environment thereby changing their phenotype and functions. Tissue-resident macrophages located directly beneath the surface epithelium in gut homeostasis are mostly tolerogenic. The total number of macrophages increases with luminal contents entering the mucosa through a broken intestinal barrier in ulcerative colitis (UC) as well as in Crohn’s disease (CD). Although not fully understood, the resulting mixtures of tissue-resident and tissue-infiltrating macrophages in both entities are diverse with respect to their phenotypes and their distribution. Macrophages in UC mainly act within the intestinal mucosa. In CD, macrophages can also be found in the muscularis and the mesenteric fat tissue compartment. Taken together, the present knowledge on human intestinal macrophages so far provides a good starting point to dig deeper into the similarities and differences of functional subsets and to finally use their phenotypical diversity as markers for complex local milieus in health and disease.

Adipose-Tissue and Intestinal Inflammation – Visceral Obesity and Creeping Fat

Obesity has become one of the main threats to health worldwide and therefore gained increasing clinical and economic significance as well as scientific attention. General adipose-tissue accumulation in obesity is associated with systemically increased pro-inflammatory mediators and humoral and cellular changes within this compartment. These adipose-tissue changes and their systemic consequences led to the concept of obesity as a chronic inflammatory state. A pathognomonic feature of Crohn’s disease (CD) is creeping fat (CF), a locally restricted hyperplasia of the mesenteric fat adjacent to the inflamed segments of the intestine. The precise role of this adipose-tissue and its mediators remains controversial, and ongoing work will have to define whether this compartment is protecting from or contributing to disease activity. This review aims to outline specific cellular changes within the adipose-tissue, occurring in either obesity or CF. Hence the potential impact of adipocytes and resident immune cells from the innate and adaptive immune system will be discussed for both diseases. The second part focuses on the impact of generalized adipose-tissue accumulation in obesity, respectively on the locally restricted form in CD, on intestinal inflammation and on the closely related integrity of the mucosal barrier.

Herpes Simplex Virus Sepsis in a Young Woman with Crohn’s Disease

We present the case of a herpes simplex virus-1 [HSV-1] sepsis with severe herpes hepatitis in a young female treated with triple immunosuppressive therapy [adalimumab, azathioprine, prednisolone] for refractory Crohns disease [CD]. The patient presented with high fever, generalised abdominal tenderness, strongly elevated transaminases, coagulopathy, and pancytopenia. Comprehensive diagnostics including blood HSV-1 polymerase chain reaction [PCR], liver biopsy, and immunohistochemistry revealed the diagnosis of fulminant herpes hepatitis. HSV-1 positivity of cutaneous lesions proved the disseminated nature of the infection. Early treatment with intravenous acyclovir led to a rapid improvement of the patients condition and resulted in a full recovery of her liver function. This is the first reported case of HSV-sepsis in a patient with CD. Physicians treating inflammatory bowel disease [IBD] patients with combined immunosuppressive therapy should be aware of the possibility of herpes hepatitis, and early empirical antiviral therapy should be considered in immunosuppressed patients presenting with fever and severe anicteric hepatitis.

Role of visceral fat in colonic inflammation: from Crohn's disease to diverticulitis.

Purpose of review The composition of activated adipose tissue with adipocytes secreting a broad spectrum of immune-modulatory adipokines next to adipose tissue-derived stromal cells and professional immune effector cells in the visceral fat creates a complex network of inflammatory processes shaping local immune responses in the adjacent inflamed intestinal mucosa. Recent findings In Crohns disease a particular phenomenon called ‘creeping fat’ can be observed. Here the hyperplastic mesenteric fat tissue not only grows around inflamed small intestinal segments but also furthermore affects the regulation of the mucosal immune system. Diverticular disease is highly prevalent in the western world but the knowledge about its immunopathology remains incomplete. Interestingly, adipose tissue also frequently covers the basolateral site of inflamed diverticula, hence locally reflecting the phenomenon seen in Crohns disease. Summary This review aims to summarize the current knowledge in which measures this intraabdominal fat participates in the regulation of intestinal inflammation with a particular focus on differences and possible parallels in Crohns disease and diverticulitis. The available data allow for suggesting that each inflamed diverticula mechanistically reflects Crohns disease on a miniature scale.

Accuracy of diagnostic tests and a new algorithm for diagnosing cytomegalovirus colitis in inflammatory bowel diseases: a diagnostic study

PurposeThe optimal method for detecting CMV colitis in patients with inflammatory bowel disease (IBD) has not been established. We wanted to investigate which diagnostic test would be most accurate when defining CMV colitis rather by the further clinical course than by using another diagnostic modality.MethodsAll consecutive patients with moderately or severely active IBD who had been tested for CMV by PCR, histology, or antigenemia assay at the two campuses CBF and CCM of the Charité - Universitätsmedizin Berlin between September 2006 and September 2009 were included in this retrospective study. During that time, in patients with a positive CMV test, immunosuppressive treatment of any kind was immediately reduced and antiviral treatment was started. This allowed identifying patients who responded to antiviral treatment and those who only responded to later escalation of immunosuppressive therapy.ResultsOne hundred and nine patients were identified, out of whom nine were considered to have clinically relevant CMV colitis. Sensitivity and specificity were 1 and 0.94 for CMV PCR and 0.5 and 1 for pp65 antigen immunofluorescence assay from peripheral blood, 0.67 and 0.98 for immunohistochemistry, and 0.17 and 0.98 for hematoxylin-eosin staining. When using absence of leukocytosis, splenomegaly, and steroid refractory disease as clinical parameters to test for CMV colitis, blood CMV PCR and immunohistochemistry were able to exclude CMV colitis in negative patients with a 75% likelihood of positive patients to have clinically relevant CMV colitis.ConclusionsBlood-based CMV PCR together with simple clinical parameters can exclude clinically relevant CMV colitis at a high specificity.

P014 The inflammasome induces macrophage-mediated disruption of the epithelial barrier

and ER-alpha expression, Ki-67 and Tunel were evaluated by immunohystochemical methods. Results: ER-beta, ER-alpha expression and their ratio, assessed in normal mucosa, in UC and in UC-dysplasia, did not show significant changes while in UC-carcinoma revealed a dramatic fall of ER-beta expression (p < 0.01) and still more ER-beta/ERalpha (p < 0.005). Apoptosis and Tunel/Ki-67 ratio demonstrated a statistically significant progressive increase from UC to UCcarcinoma through UC-dysplasia. Conclusions: Our experience assessed modifications of ERs expression, apoptosis and cell proliferation in dysplasiacarcinoma sequence in the course of UC. We observed a dramatic fall of ER-beta, ER-beta/ER-alpha and a progressive increase of apoptosis in progressive steps from long-lasting UC to UC-carcinoma. As observed in adenoma model, the ERbeta drop could be a biomarker of dysplasia progression and a relevant point in the study of carcinogenesis in the course of UC. Further studies are warranted.

Regulation of Macrophage Phenotype in Crohn's Disease Hypertrophied Adipose Tissue

Background: Mutations within the gene loci, encoding protein tyrosine phosphatase nonreceptor type 2 (PTPN2) and nucleotide oligomerization domain 2 (NOD2), have been associated with Crohns disease (CD). We have recently shown that PTPN2 limits proinflammatory effects in human intestinal epithelial cells (IEC) and monocytes. A dysregulated immune response to bacterial antigens, such as the NOD2-ligand, muramyl-dipeptide (MDP), has been strongly implicated in the pathogenesis of CD. The transcription factor T-bet is upregulated in NOD2-deficient mice, activated during CD and controls the secretion of proinflammatory cytokines, such as IFNγ. Here, we studied whether PTPN2 controls NOD2mediated effects in human THP-1 monocytes. Methods: Protein analysis was performed by Western blotting, mRNA analysis by real-time PCR, cytokine secretion by ELISA and visual imaging by immunofluorescence (IF) studies. PTPN2 or NOD2 knock-down was induced by siRNA. Primary intestinal colonic lamina propria fibroblasts (CLPF) were isolated from surgical specimens derived from patients with active CD (n=5) and were genotyped for the CD-associated PTPN2 variant. Results: MDP (100 ng/ml) increased PTPN2 mRNA (p<0.05; n=3) and protein expression (p<0.05; n=3) by 24 h treatment. This effect was absent in NOD2 knock-down cells (n=3). Levels of the transcription factor T-bet (p<0.01) were also enhanced by MDP stimulation and this effect was potentiated by PTPN2 knock-down (p<0.05; n=3). While MDP treatment induced secretion of TNF, IL-8 and IFNγ (p<0.01; n=3 each) in THP-1 cell supernatants, knock-down of PTPN2 by siRNA resulted in diminished secretion of all three cytokines in response to MDP (p<0.01; n=3 each). We next studied whether PTPN2 regulates NOD2-mediated autophagy. PTPN2 knock-down prevented the MDP-induced expression of microtubule-associated protein 1 light-chain 3 B-II (LC3B-II), a marker of autophagy activity, in THP-1 cells (n=3; p<0.05). Though MDP was able to increase PTPN2 and LC3B-II protein levels in PTPN2 wild-type (WT) CLPF (n=3), in CLPF carrying the CD-associated PTPN2 variant (n=2), MDP was unable to increase PTPN2 or LC3B-II expression. By IF studies we found elevated numbers of LC3B+ vesicles, indicating autophagosome assembly, in MDP-treated PTPN2-WT cells. However, in PTPN2-variant cells LC3B+ vesicles were less numerous, but larger in size, when compared to PTPN2WT cells, suggesting that defective autophagosome formation occurred. Conclusions: We demonstrate that PTPN2 is regulated by MDP via a NOD2-dependent mechanism and controls NOD2-mediated cytokine secretion and autophagy. These findings demonstrate for the first time a functional interaction between the two CD candidate genes, PTPN2 and NOD2, and suggest that the CD-associated PTPN2 variant could favour a dysregulated immune response to bacterial antigens.