Michael J. Rosen

L-arginine Supplementation Improves Responses to Injury and Inflammation in Dextran Sulfate Sodium Colitis

Inflammatory bowel disease (IBD), consisting of Crohns disease and ulcerative colitis (UC), results in substantial morbidity and is difficult to treat. New strategies for adjunct therapies are needed. One candidate is the semi-essential amino acid, L-arginine (L-Arg), a complementary medicine purported to be an enhancer of immunity and vitality in the lay media. Using dextran sulfate sodium (DSS) as a murine colonic injury and repair model with similarities to human UC, we assessed the effect of L-Arg, as DSS induced increases in colonic expression of the y + cationic amino acid transporter 2 (CAT2) and L-Arg uptake. L-Arg supplementation improved the clinical parameters of survival, body weight loss, and colon weight, and reduced colonic permeability and the number of myeloperoxidase-positive neutrophils in DSS colitis. Luminex-based multi-analyte profiling demonstrated that there was a marked reduction in proinflammatory cytokine and chemokine expression with L-Arg treatment. Genomic analysis by microarray demonstrated that DSS-treated mice supplemented with L-Arg clustered more closely with mice not exposed to DSS than to those receiving DSS alone, and revealed that multiple genes that were upregulated or downregulated with DSS alone exhibited normalization of expression with L-Arg supplementation. Additionally, L-Arg treatment of mice with DSS colitis resulted in increased ex vivo migration of colonic epithelial cells, suggestive of increased capacity for wound repair. Because CAT2 induction was sustained during L-Arg treatment and inducible nitric oxide (NO) synthase (iNOS) requires uptake of L-Arg for generation of NO, we tested the effect of L-Arg in iNOS−/− mice and found that its benefits in DSS colitis were eliminated. These preclinical studies indicate that L-Arg supplementation could be a potential therapy for IBD, and that one mechanism of action may be functional enhancement of iNOS activity.

A Randomized Prospective Trial of Endoscopic Ultrasound to Guide Combination Medical and Surgical Treatment for Crohn's Perianal Fistulas

AIMS:To prospectively determine if rectal endoscopic ultrasound (EUS) can guide combination medical and surgical therapy and improve outcomes for patients with perianal fistulizing Crohns disease.METHODS:Ten patients with perianal Crohns disease were prospectively enrolled in a randomized prospective pilot study. The patients were randomized to either the EUS cohort or the control group. All patients underwent a rectal EUS to delineate fistula anatomy followed by an examination under anesthesia by a colorectal surgeon with seton placement and/or incision and drainage, as indicated. The surgeon was blinded to the initial EUS results of patients in the control group. Medical treatment was maximized with 6-mercaptopurine (1.0–1.5 mg/kg) or azathioprine (2.0–2.5 mg/kg), ciprofloxacin (1,000 mg a day) or metronidazole (1,500 mg a day), and infliximab (5 mg/kg at 0, 2, and 6 wk and then every 8 wk). For patients in the control group, additional interventions (seton removal and repeat surgery) were at the discretion of the surgeon (without EUS guidance). Patients in the EUS cohort had EUS performed at weeks 22 and 38, with additional surgical interventions based on EUS findings. The primary end point was complete cessation of drainage at week 54. All patients had a repeat EUS performed at week 54 to determine the fistula status on EUS (secondary end point). The need for additional surgery was defined as a treatment failure.RESULTS:Ten patients were enrolled in the study. One of 5 (20%) in the control group and 4 of 5 (80%) in the EUS group had complete cessation of drainage. From the control group, 3 patients failed due to repeat surgery (2 for persistent/recurrent fistula and 1 for abscess), and 1 had a persistent drainage at week 54. In the EUS cohort, 1 patient had a recurrent abscess after his seton fell out prematurely.In the EUS cohort, the median time to cessation of drainage was 99 days, and the time to EUS evidence of fistula inactivity was 229 days.CONCLUSION:This pilot study suggests that using EUS to guide combination medical and surgical therapy for perianal fistulizing Crohns disease improves the outcomes.

Necrotising enterocolitis is characterised by disrupted immune regulation and diminished mucosal regulatory (FOXP3)/effector (CD4, CD8) T cell ratios

Background Necrotising enterocolitis (NEC) is the most common gastrointestinal emergency in premature infants. Immaturity of gastrointestinal immune regulation may predispose preterm infants to NEC as FOXP3 T regulatory cells (Treg) are critical for intestinal immune homoeostasis. Objective To investigate the hypothesis that abnormal developmental regulation of lamina propria Treg would define premature infants with NEC. Design Lamina propria mononuclear cell populations from surgically resected ileum from 18 patients with NEC and 30 gestational age-matched non-NEC surgical controls were prospectively isolated. Polychromatic flow cytometry was performed to phenotype and analyse lamina propria T cell populations. The cytokine gene expression profile in NEC tissue was compared with that of non-NEC controls. Results The total number of Treg, CD4, or CD8 T cells in each ileum section was independent of gestational age, age or postmenstrual age and similar between patients with NEC and controls. In contrast, the ratio of Treg to CD4 T cells or Treg to CD8 T cells was significantly lower in NEC ileum than in infants without NEC (medians 2.9% vs 6.6%, p=0.001 and medians 6.6% vs 25.9%, p<0.001, respectively). For any given number of CD4 or CD8 T cells, Treg were, on average, 60% lower in NEC ileum than in controls. NEC tissue cytokine gene expression profiles were characteristic of inhibited Treg development or function. Treg/CD4 and Treg/CD8 ratios recovered between initial resection for NEC and reanastomosis. Conclusion The proportion of lamina propria Treg is significantly reduced in the ileum of premature infants with NEC and may contribute to the excessive inflammatory state of this disease.

STAT6 activation in ulcerative colitis: A new target for prevention of IL‐13‐induced colon epithelial cell dysfunction

Background: Interleukin 13 (IL‐13) is upregulated in ulcerative colitis (UC) and increases colon epithelial permeability by inducing apoptosis and expression of the pore‐forming tight junction protein claudin‐2. IL‐13 induces activation of signal transducer and activator of transcription 6 (STAT6). However, the STAT6 phosphorylation status in patients with UC is unknown, as is the effect of STAT6 inhibition on colonic epithelium exposed to IL‐13. The study aims were to determine if mucosal STAT6 phosphorylation is increased in patients with UC, and if STAT6 inhibition attenuates IL‐13‐induced colon epithelial cell dysfunction. Methods: Immunohistochemical staining for phosphorylated (p) STAT6 was performed on colonic tissue from newly diagnosed pediatric subjects with UC (early UC) or Crohns disease (CD), colectomy tissue from adults with UC (advanced UC), and controls. Colon HT‐29 and T84 cells were transfected with STAT6 small interfering RNA (siRNA), or treated with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor that inhibits STAT6, prior to IL‐13 treatment. Results: The median score for epithelial pSTAT6 was 0 in control subjects, 2 in early UC (versus control P = 0.019), 4 in advanced UC (P = 0.003), and 0 in CD (P = 0.4). Cell transfection with STAT6 siRNA prevented IL‐13‐induced apoptosis and claudin‐2 expression. SAHA inhibited IL‐13‐induced STAT6 phosphorylation, apoptosis, and claudin‐2 expression, and mitigated IL‐13‐induced reductions in transepithelial resistance. Conclusions: UC is associated with increased colonic epithelial STAT6 phosphorylation, and STAT6 inhibition prevents IL‐13‐induced apoptosis and barrier disruption. These data identify STAT6 as a novel target for UC treatment and support further study of SAHA as a therapeutic agent. (Inflamm Bowel Dis 2011;)

Inflammatory Bowel Disease in Children and Adolescents

The inflammatory bowel diseases (IBDs), including ulcerative colitis and Crohn disease, are chronic inflammatory disorders of the gastrointestinal tract most often diagnosed in adolescence and young adulthood, with a rising incidence in pediatric populations. These disorders are common enough in children that most pediatricians and other pediatric clinicians will encounter children with IBD in their general practice. Inflammatory bowel disease is caused by a dysregulated mucosal immune response to the intestinal microflora in genetically predisposed hosts. Although children can present with the classic symptoms of weight loss, abdominal pain, and bloody diarrhea, many present with nonclassic symptoms of isolated poor growth, anemia, or other extraintestinal manifestations. Once IBD is diagnosed, the goals of therapy consist of eliminating symptoms, normalizing quality of life, restoring growth, and preventing complications while minimizing the adverse effects of medications. Unique considerations when treating children and adolescents with IBD include attention to the effects of the disease on growth and development, bone health, and psychosocial functioning. The purpose of this review is to provide a contemporary overview of the epidemiologic features, pathogenesis, diagnosis, and management of IBD in children and adolescents.

Transactivation of EGFR by LPS induces COX-2 expression in enterocytes

Necrotizing enterocolitis (NEC) is the leading cause of gastrointestinal morbidity and mortality in preterm infants. NEC is characterized by an exaggerated inflammatory response to bacterial flora leading to bowel necrosis. Bacterial lipopolysaccharide (LPS) mediates inflammation through TLR4 activation and is a key molecule in the pathogenesis of NEC. However, LPS also induces cyclooxygenase-2 (COX-2), which promotes intestinal barrier restitution through stimulation of intestinal cell survival, proliferation, and migration. Epidermal growth factor receptor (EGFR) activation prevents experimental NEC and may play a critical role in LPS-stimulated COX-2 production. We hypothesized that EGFR is required for LPS induction of COX-2 expression. Our data show that inhibiting EGFR kinase activity blocks LPS-induced COX-2 expression in small intestinal epithelial cells. LPS induction of COX-2 requires Src-family kinase signaling while LPS transactivation of EGFR requires matrix metalloprotease (MMP) activity. EGFR tyrosine kinase inhibitors block LPS stimulation of mitogen-activated protein kinase ERK, suggesting an important role of the MAPK/ERK pathway in EGFR-mediated COX-2 expression. LPS stimulates proliferation of IEC-6 cells, but this stimulation is inhibited with either the EGFR kinase inhibitor AG1478, or the selective COX-2 inhibitor Celecoxib. Taken together, these data show that EGFR plays an important role in LPS-induction of COX-2 expression in enterocytes, which may be one mechanism for EGF in inhibition of NEC.

STAT6 Deficiency Ameliorates Severity of Oxazolone Colitis by Decreasing Expression of Claudin-2 and Th2-Inducing Cytokines

Patients suffering from ulcerative colitis (UC) exhibit chronic colonic inflammation caused by a dysregulated mucosal immune response and epithelial barrier disruption. Th2 cytokines, including IL-13, have been implicated in the pathogenesis of UC. IL-13 induces phosphorylation of STAT6, and we previously demonstrated increased epithelial p-STAT6 in children with UC. In this study, we investigated the role of STAT6 in oxazolone colitis, a murine model of UC, by inducing colitis in STAT6-deficient (STAT6−/−) and wild type (WT) mice. We observed increased epithelial cell, T cell, macrophage, and NKT cell STAT6 phosphorylation, as well as increased p-STAT6+ IL-13–producing NKT cells, in colitic WT mice. Colitis was attenuated in STAT6−/− mice, with improvements in weight, colon length, and histopathology. There was decreased induction of the pore-forming tight junction protein claudin-2 in STAT6−/− mice. Similarly, short hairpin RNA STAT6 knockdown reduced claudin-2 induction and transepithelial resistance decrease in IL-13–treated human T84 cells. Tissue expression of IL-13, IFN-γ, IL-17, and IL-10 mRNA was similarly induced in WT and STAT6−/− colitic mice; however, we observed increased mRNA expression for the Th2-inducing cytokines IL-33 and thymic stromal lymphopoietin in WT mice with colitis, which was abrogated in STAT6−/− mice. Mesenteric lymph node cells from STAT6−/− mice with colitis exhibited reduced secretion of IL-4, IL-5, IL-13, and IFN-γ. IL-33 augmented mesenteric lymph node cell secretion of IL-5, IL-13, IL-6, and IFN-γ. These data implicate STAT6 in the pathogenesis of colitis in vivo with important roles in altering epithelial barrier function and regulating Th2-inducing cytokine production.

Review article: applying pharmacokinetics to optimise dosing of anti-TNF biologics in acute severe ulcerative colitis.

Acute severe ulcerative colitis (ASUC), the most aggressive presentation of ulcerative colitis (UC), occurs in 15% of adults and children with UC. First line therapy with intravenous corticosteroids is ineffective in half of adults and one‐third of children. Therapeutic monoclonal antibodies against TNF (anti‐TNF therapy) are emerging as a common treatment for ASUC due to their similar efficacy to calcineurin inhibitors and more favourable adverse effect profile.

Activation of the Epidermal Growth Factor Receptor in Macrophages Regulates Cytokine Production and Experimental Colitis

Macrophages regulate innate immunity to maintain intestinal homeostasis and play pathological roles in intestinal inflammation. Activation of the epidermal growth factor receptor (EGFR) promotes cellular proliferation, differentiation, survival, and wound closure in several cell types. However, the impact of EGFR in macrophages remains unclear. This study was to investigate whether EGFR activation in macrophages regulates cytokine production and intestinal inflammation. We found that EGFR was activated in colonic macrophages in mice with dextran sulfate sodium (DSS)–induced colitis and in patients with ulcerative colitis. DSS-induced acute colitis was ameliorated, and recovery from colitis was promoted in Egfrfl/flLysM-Cre mice with myeloid cell–specific deletion of EGFR, compared with LysM-Cre mice. DSS treatment increased IL-10 and TNF levels during the acute phase of colitis, and increased IL-10 but reduced TNF levels during the recovery phase in Egfrfl/flLysM-Cre mice. An anti–IL-10 neutralizing Ab abolished these effects of macrophage-specific EGFR deletion on DSS-induced colitis in Egfrfl/flLysM-Cre mice. LPS stimulated EGFR activation and inhibition of EGFR kinase activity enhanced LPS-stimulated NF-κB activation in RAW 264.7 macrophages. Furthermore, induction of IL-10 production by EGFR kinase-blocked RAW 264.7 cells, in response to LPS plus IFN-γ, correlated with decreased TNF production. Thus, although selective deletion of EGFR in macrophages leads to increases in both pro- and anti-inflammatory cytokines in response to inflammatory stimuli, the increase in the IL-10 level plays a role in suppressing proinflammatory cytokine production, resulting in protection of mice from intestinal inflammation. These results reveal an integrated response of macrophages regulated by EGFR in intestinal inflammatory disorders.

High-throughput multi-analyte Luminex profiling implicates eotaxin-1 in ulcerative colitis.

Accurate and high-throughput technologies are needed for identification of new therapeutic targets and for optimizing therapy in inflammatory bowel disease. Our aim was to assess multi-analyte protein-based assays of cytokines/chemokines using Luminex technology. We have reported that Luminex-based profiling was useful in assessing response to L-arginine therapy in the mouse model of dextran sulfate sodium colitis. Therefore, we studied prospectively collected samples from ulcerative colitis (UC) patients and control subjects. Serum, colon biopsies, and clinical information were obtained from subjects undergoing colonoscopy for evaluation of UC or for non-UC indications. In total, 38 normal controls and 137 UC cases completed the study. Histologic disease severity and the Mayo Disease Activity Index (DAI) were assessed. Serum and colonic tissue cytokine/chemokine profiles were measured by Luminex-based multiplex testing of 42 analytes. Only eotaxin-1 and G-CSF were increased in serum of patients with histologically active UC vs. controls. While 13 cytokines/chemokines were increased in active UC vs. controls in tissues, only eotaxin-1 was increased in all levels of active disease in both serum and tissue. In tissues, eotaxin-1 correlated with the DAI and with eosinophil counts. Increased eotaxin-1 levels were confirmed by real-time PCR. Tissue eotaxin-1 levels were also increased in experimental murine colitis induced by dextran sulfate sodium, oxazolone, or Citrobacter rodentium, but not in murine Helicobacter pylori infection. Our data implicate eotaxin-1 as an etiologic factor and therapeutic target in UC, and indicate that Luminex-based assays may be useful to assess IBD pathogenesis and to select patients for anti-cytokine/chemokine therapies.