Impact of toll-like-receptor-9 (TLR9) deficiency on visceral adipose tissue adipokine expression during chronic DSS-induced colitis in mice.

BACKGROUND: Studies postulate an involvement of adipokines in inflammatory gastrointestinal diseases. Leptin-deficient ob/ob mice as well as TLR9-deficient mice have a more moderate course of chronic DSS-induced colitis (DSS-CC) and adipocytes do express functional TLR9 molecules. MATERIAL AND METHODS: Adipokine mRNA expression in visceral adipose tissue of mice before and after the induction of DSS-CC was investigated. Experiments were performed in both TLR9(wt/wt) and TLR9(-/-) mice. In vitro, the effect of TLR9 blocking peptide on leptin and visfatin protein secretion was studied in 3T3-L1 adipocytes. RESULTS: Induction of DSS-CC led to an upregulation of leptin mRNA expression in TLR9(wt/wt) mice, while TLR9(-/-) animals showed a significant reduction of leptin expression even below baseline. While visfatin expression remained unchanged in TLR9(wt/wt) animals, TLR9(-/-) mice exhibited a significant induction during DSS-CC. CTRP-3 expression was reduced after colitis induction only in TLR9(-/-) animals. Of note, IL-6 expression levels remained unchanged, while CXCL1/KC and cyclophilin A expression was reduced in DSS-CC. Inhibition of TLR9 signaling by using TLR9 blocking peptide led to reduced leptin protein secretion into cell culture supernatants in 3T3-L1 adipocytes, while visfatin protein secretion was enhanced. CONCLUSIONS: DSS-CC leads to differential adipokine expression profiles in the visceral fat pad in TLR9(wt/wt) vs. TLR9(-/-) mice. In vitro, inhibition of TLR9 signaling induces visfatin secretion while inhibiting leptin secretion in adipocytes. Thus, visceral adipokines are regulated by intact TLR9 signaling pathway and a specific interplay between the leptin- and the TLR9-pathways might be of pathophysiological importance in chronic intestinal inflammation.

Systemic metabolic signaling in acute and chronic gastrointestinal inflammation of inflammatory bowel diseases.

Acute and chronic intestinal inflammation stimulates innate and adaptive immune systems, thereby increasing energy demand of activated immune cells. Energy regulation by systemically released mediators is of critical importance for homeostasis. We wanted to find out how systemic metabolic mediators are affected during intestinal inflammation. A total of 123 patients suffering from Crohn's disease (CD), 76 patients with ulcerative colitis (UC), and 21 healthy controls were recruited. Patients receiving systemic steroids or therapy regimens including biologicals (anti-TNF) were excluded from the study. Serum levels of IL-6, CRP, insulin, glucose, free fatty acid, and RBP-4 were measured by ELISA and RIA. Intestinal inflammation was accompanied by elevated systemic inflammatory para-meters such as IL-6 and CRP in UC and CD and, concomitantly, with elevated insulin levels and increased insulin/glucose ratio in patients with UC. This indicates insulin resistance in liver, muscle, and fat. In addition, intestinal inflammation was associated with elevated levels of circulating free fatty acids in UC and CD, indicating an activation of the organism's appeal for energy-rich substrates (energy appeal reaction). RBP-4 serum levels were also high in acute and chronic intestinal inflammation in UC and CD, which can support insulin resistance. The organism's "energy appeal reaction" in response to acute and chronic inflammation provides free energy in the circulation, which is needed by inflammatory cells. A major mechanism of the redirection program is insulin resistance. New therapeutic strategies might be developed in the future, directly impacting on the storage and utilization of energy-rich fuels.

Downregulation of the ubiquitin-proteasome system in normal colonic macrophages and reinduction in inflammatory bowel disease.

BACKGROUND: In normal mucosa, intestinal lamina propria macrophages (IMACs) maintain tolerance against food antigens and the commensal bacterial flora. Several mechanisms have been identified that mediate tolerance. The ubiquitin-proteasome system (UPS) is a large multiprotein complex that degrades cellular proteins. As the UPS may modulate immune functions of IMACs, we performed a detailed investigation of UPS expression and function under normal conditions and in cells derived from patients suffering from inflammatory bowel disease (IBD). METHODS: IMACs were isolated from intestinal mucosa. mRNA expression of macrophages differentiated in vitro (i.v. MACs) and IMACs was compared by Affymetrix® oligonucleotide arrays. Quantitative Taqman-PCR was performed on five exemplary proteasomal and five ubiquitinylation genes each. Proteins were analyzed by immunohistochemistry and Western blotting. Proteasome function was assessed by a fluorimetric test. RESULTS: Affymetrix analysis showed downregulation of mRNA expression of almost all represented proteasomal and of 22 ubiquitination-associated genes in IMACs as compared to i.v. MACs and monocytes. By quantitative PCR, up to tenfold higher mRNA expression of 10 exemplary genes of the UPS (UBE2A, UBE2D2, UBE2L6, USP14, UBB and ATPase2, ß2, ß5, ß2i/MECL-1, ß5i/LMP7) was demonstrated in i.v. MACs as compared to IMACs. Immunohistochemistry and Western blots confirmed these findings in intestinal mucosa of controls and patients suffering from diverticulitis. In contrast, a significant increase in protein amounts was found in mucosa of patients with IBD. CONCLUSION: Reduced expression of subunits of the UPS in IMACs of normal mucosa supports the concept of the presence of a nonreactive, anergic macrophage phenotype in the gut under normal conditions. Reinduction in IMACs of IBD mucosa reflects activated IMACs which can present antigenic peptides and thus support inflammation.

Clinical relevance of IgG antibodies against food antigens in Crohn's disease: a double-blind cross-over diet intervention study.

BACKGROUND: Environmental factors are thought to play an important role in the development of Crohn's disease (CD). Immune responses against auto-antigens or food antigens may be a reason for the perpetuation of inflammation. METHODS: In a pilot study, 79 CD patients and 20 healthy controls were examined for food immunoglobulin G (IgG). Thereafter, the clinical relevance of these food IgG antibodies was assessed in a double-blind cross-over study with 40 patients. Based on the IgG antibodies, a nutritional intervention was planned. The interferon (IFN)gamma secretion of T cells was measured. Eosinophil-derived neurotoxin was quantified in stool. RESULTS: The pilot study resulted in a significant difference of IgG antibodies in serum between CD patients and healthy controls. In 84 and 83% of the patients, respectively, IgG antibodies against processed cheese and yeast were detected. The daily stool frequency significantly decreased by 11% during a specific diet compared with a sham diet. Abdominal pain reduced and general well-being improved. IFNgamma secretion of T cells increased. No difference for eosinophil-derived neurotoxin in stool was detected. CONCLUSION: A nutritional intervention based on circulating IgG antibodies against food antigens showed effects with respect to stool frequency. The mechanisms by which IgG antibodies might contribute to disease activity remain to be elucidated.

Recipient NOD2/CARD15 status affects cellular infiltrates in human intestinal graft-versus-host disease.

Nucleotide-binding oligomerization domain 2/caspase recruitment domain 15 (NOD2/CARD15) polymorphisms have been identified as risk factors of both Crohn's disease and graft-versus-host disease (GVHD) following allogeneic stem cell transplantation. However, the role of these receptors of innate immunity in the pathophysiology of gastrointestinal GVHD is still poorly defined. Immunohistological features of intestinal GVHD were analysed in gastrointestinal biopsies from 58 patients obtained at the time of first onset of intestinal symptoms. The observed changes were correlated with concomitant risk factors and the presence of polymorphisms within the pathogen recognition receptor gene NOD2/CARD15. Intestinal GVHD was associated with a stage-dependent decrease in CD4 T cell infiltrates and an increase in CD8 T cells in the lamina propria; CD8 infiltrates correlated with extent of apoptosis and consecutive epithelial proliferation. The presence of NOD2/CARD15 variants in the recipient was associated with a significant loss of CD4 T cells: in a semiquantitative analysis, the median CD4 score for patients with wild-type NOD2/CARD15 was 1.1 (range 3), but only 0.4 (range 2) for patients with variants (P = 0.002). This observation was independent from severity of GVHD in multivariate analyses and could not be explained by the loss of forkhead box P3(+) T cells. Our results suggest a loss of protective CD4 T cells in intestinal GVHD which is enhanced further by the presence of NOD2/CARD15 variants. Our study might help to identify more selective therapeutic strategies in the future.

CpG-oligodeoxynucleotides stimulate immunoglobulin A secretion in intestinal mucosal B cells.

Bacterial DNA motifs (such as CpG-oligodeoxynucleotides: CpG-ODN) induce innate immune responses via binding to Toll-like-receptor-9 (TLR-9). In murine intestinal mucosa treatment with CpG-ODN worsens chronic intestinal inflammation, whereas it prevents or ameliorates colitis when given in a prophylactic setting. In tonsils B cells have been reported to express TLR-9, especially after activation. Whether B cells in the human intestinal mucosa also express TLR-9 and whether their function can be influenced by CpG-ODN is, so far, unknown. Mucosal B cells were isolated according to a new protocol from surgical specimens of patients with inflammatory bowel disease and from controls by collagenase digestion followed by magnetic cell sorting using anti-CD19 antibody armed magnetic beads. TLR-9 mRNA and protein expression were quantified by real-time polymerase chain reaction (PCR) and Western blot, respectively. Immunoglobulin A (IgA) secretion was measured by enzyme-linked immunosorbent assay after stimulation of isolated B cells with CpG-ODN, control GpC-ODN or lipopolysaccharide (LPS). Flow cytometric analysis of the isolated lamina propria mononuclear cells showed a purification of 73% (+/-22%) CD19(+) cells. By quantitative reverse transcription-PCR and by Western blot TLR-9 expression in this cell population was evident. IgA secretion was increased significantly by CpG-ODN incubation compared with GpC-ODN and LPS. Compared with unstimulated controls, CpG-ODN up-regulated IgA secretion to 139% (+/-21%). These data demonstrate that CD19(+) mucosal B cells express TLR-9 and secrete increased levels of IgA upon stimulation with CpG-ODN, indicating an additional link between adaptive and innate intestinal immune responses.

[Partial splenic artery embolization for the treatment of a patient with hypersplenism due to liver cirrhosis]. / Partielle Milzarterienembolisation zur Behandlung eines Hypersplenismus bei Leberzirrhose.

HISTORY AND CLINICAL FINDINGS: A 52-year-old man with liver cirrhosis and ascites, hepatorenal syndrome and severe pancytopenia was admitted to hospital for treatment and consideration for future liver transplantation. INVESTIGATIONS: Laboratory tests revealed severe leukocytopenia and thrombocytopenia, which had persisted after otherwise successful treatment of spontaneous bacterial peritonitis and a hepatorenal syndrome. There was no evidence of a toxic cause such as drugs or alcohol nor of an underlying haematologic disease. The most likely diagnosis was hypersplenism. DIAGNOSIS, THERAPY AND CLINICAL COURSE: Because of the high risk of a splenectomy it was decided to perform a partial splenic arterial embolization. Both leukocyte and thrombocyte counts rose after the intervention and continued to stay within normal range. This made it possible to discharge the patient from inpatient treatment until he was admitted again for a subsequently successful liver transplantation. CONCLUSIONS: Partial splenic artery embolization can be successfully used to treat severe leukocytopenia and thrombocytopenia in patients with liver cirrhosis and hypersplenism.

Regulation of galectin-3 function in mucosal fibroblasts: potential role in mucosal inflammation.

Recently we identified galectin-3 (gal-3), which is secreted by colonic epithelial cells (CEC), to be a strong activator of colonic lamina propria fibroblasts (CLPF). Modulation of CLPF function may play a role during stricture and fistula formation in inflammatory bowel disease (IBD). Therefore, we investigated further the expression of gal-3 and effects on CLPF. The aim of this study is to perform a direct comparison of gal-3 between tissue from healthy controls and from patients with either Crohn's disease (CD) or ulcerative colitis (UC). CEC, CLPF and intestinal macrophages (IMAC) were isolated from control and IBD colonic tissue. Interleukin-8 secretion as a readout of CLPF activation was quantified by enzyme-linked immunosorbent assay. Gal-3 in cell cultures and tissue samples was evaluated by Western blot, immunofluorescence and immunohistochemistry. CLPF-migration was assayed in the 48-well modified Boyden chamber. Gal-3 expression was found in all segments of the colon. In the terminal ileum, less gal-3 was found compared with the colon. Immunohistochemistry and immunofluorescence revealed a homogenous distribution of gal-3 in CEC and IMAC of control mucosa and UC. However, significantly less gal-3 was found in IMAC from CD patients. In CD fistulae and stenoses, gal-3 expression was reduced significantly and barely detectable. In co-incubation studies lactose reduced significantly the CLPF-stimulatory potential of gal-3, indicating that the C-terminal domain of gal-3 is responsible for CLPF activation. Gal-3 stimulated CLPF migration in CLPF derived from fistulae. In conclusion, gal-3 expression is down-regulated in CD-fistulae and stenoses as well as in IMAC in CD patients. Gal-3 induces migration of CLPF derived from fistulae. Its role for stricture and fistula formation warrants further investigation.

Anti-inflammatory role of sympathetic nerves in chronic intestinal inflammation.

BACKGROUND: Substance P (SP) is a pro-inflammatory neuropeptide in colitis, whereas sympathetic neurotransmitters are anti-inflammatory at high concentrations. AIM AND METHODS: In all layers of the colon, nerve fibre densities of SP(+) and sympathetic nerve fibres were investigated (22 Crohn's disease, six diverticulitis, and 22 controls). In addition, the nerve fibre repellent factor semaphorin 3C (SEMA3C) was studied. The functional role of the sympathetic nervous system was tested in dextran sodium sulfate (DSS) and Il10(-/-) colitis. RESULTS: In all layers, Crohn's disease patients demonstrated a loss of sympathetic nerve fibres. Sprouting of SP(+) nerve fibres was particularly observed in the mucosa and muscular layer in Crohn's disease. SEMA3C was detected in epithelial cells, and there was a marked increase of SEMA3C-positive crypts in the mucosa of Crohn's disease patients compared to controls. In Crohn's disease, the number of SEMA3C-positive crypts was negatively related to the density of mucosal sympathetic nerve fibres. Sympathectomy reduced acute DSS colitis but increased chronic DSS colitis. Sympathectomy also increased chronic colitis in Il10(-/-) mice. CONCLUSIONS: This study demonstrated a loss of sympathetic and an increase of SP(+) nerve fibres in Crohn's disease. SEMA3C, a sympathetic nerve repellent factor, is highly expressed in the epithelium of Crohn's disease patients. In chronic experimental colitis, the sympathetic nervous system confers an anti-inflammatory influence. Thus, the loss of sympathetic nerve fibres in the chronic phase of the disease is most probably a pro-inflammatory signal, which might be related to repulsion of these fibres by SEMA3C and other repellents.