Association of Circulating Fibrocytes With Fibrostenotic Small Bowel Crohn's Disease.

BACKGROUND: Fibrocytes are hematopoietic cells with features of mesenchymal cells found in the circulation and inflammatory sites implicated in promoting fibrosis in many fibroinflammatory diseases. However, their role(s) in the development of intestinal fibrosis is poorly understood. Here, we investigated a potential role of fibrocytes in the development of fibrosis in Crohn's disease (CD) and sought factors that may impact their development and function. METHODS: Plasma and mononuclear cells were collected from patients with and without fibrostenotic CD. Fibrocytes defined as CD11b+, CD34+, and Collagen 1+ were correlated with clinical assessments of fibrosis, including evaluation using intestinal ultrasound. We measured the levels of relevant circulating molecules via Luminex and studied the effect of patient plasma proteins on fibrocyte differentiation. RESULTS: Fibrocyte numbers were increased in CD patients with stricturing Crohn's disease compared with patients with an inflammatory phenotype (P = .0013), with strong correlation between fibrocyte numbers and acoustic radiation force impulse (ARFI), a measure of bowel elasticity on intestinal ultrasound (R = .8383, P = .0127). Fibrostenotic plasma was a more potent inducer of fibrocyte differentiation in both primary human monocytes and cell line and contained increased levels of cytokines implicated in fibrocyte differentiation compared with plasma from inflammatory patients. Interestingly, increased fibrocyte numbers at time of ultrasound were associated with escalation of medical therapy and endoscopic/surgical management of small bowel strictures at 30 months follow-up. CONCLUSIONS: Circulating fibrocytes strongly correlate with fibrostenotic disease in CD, and they may serve as predictors for escalation of medical +/- surgical therapy.

Intestinal strictures are thought to result from excessive deposition of extracellular matrix by activated mesenchymal cells. In this study, we provide evidence that supports a potential role of fibrocytes (bone marrow­derived mesenchymal precursors) in collagen deposition in Crohn's disease strictures. Inasmuch as fibrocyte numbers correlate with sonographic measures of bowel stiffness, fibrocyte numbers may predict the need for therapy escalation.

Differential Effect of Genetic Burden on Disease Phenotypes in Crohn's Disease and Ulcerative Colitis in a Canadian Cohort.

BACKGROUND AND AIMS: Crohn's disease (CD) and ulcerative colitis (UC) demonstrate considerable phenotypic heterogeneity and course. Accurate predictors of disease behaviour are lacking. The contribution of genetics and specific polymorphisms is widely appreciated; however, their cumulative effect(s) upon disease behaviour remains poorly understood. Here, we investigate the relationship between genetic burden and disease phenotype in a Canadian inflammatory bowel disease (IBD) Cohort. METHODS: We retrospectively examined a cohort of CD and UC patients recruited from a single tertiary referral center genotyped using a Goldengate Illumina platform. A genetic risk score (GRS) incorporating strength of association (log odds ratio) and allele dose for 151 IBD-risk loci was calculated and evaluated for phenotypic associations. RESULTS: Among CD patients, higher GRS was associated with earlier onset of disease (regression coefficient -2.19, 95% confidence interval [CI] -3.77 to -0.61, P = 0.007), ileal disease (odds ratio [OR] 1.45), stricturing/penetrating disease (OR 1.72), perianal disease (OR 1.57) and bowel resection (OR 1.66). Higher GRS was associated with use of anti-tumor necrosis factor (TNF) (P < 0.05) but not immunomodulators. Interestingly, we could not demonstrate an association between higher GRS and family history of IBD (OR 1.27, P = 0.07). Onset of disease remained statistically significant for never smokers (P = 0.03) but not ever smokers (P = 0.13). For UC, having a higher GRS did not predict the age of diagnosis nor was it predictive of UC disease extent (P = 0.18), the need for surgery (P = 0.74), nor medication use (immunomodulators P = 0.53, anti-TNF P = 0.49). We could not demonstrate an association between increased GRS and having a family history of IBD in the UC group. CONCLUSIONS: Increasing genetic burden is associated with early age of diagnosis in CD and may be useful in predicting disease behaviour in CD but not UC.

Colitis-Induced Microbial Perturbation Promotes Postinflammatory Visceral Hypersensitivity.

BACKGROUND & AIMS: Despite achieving endoscopic remission, more than 20% of inflammatory bowel disease patients experience chronic abdominal pain. These patients have increased rectal transient receptor potential vanilloid-1 receptor (TRPV1) expression, a key transducer of inflammatory pain. Because inflammatory bowel disease patients in remission exhibit dysbiosis and microbial manipulation alters TRPV1 function, our goal was to examine whether microbial perturbation modulated transient receptor potential function in a mouse model. METHODS: Mice were given dextran sodium sulfate (DSS) to induce colitis and were allowed to recover. The microbiome was perturbed by using antibiotics as well as fecal microbial transplant (FMT). Visceral and somatic sensitivity were assessed by recording visceromotor responses to colorectal distention and using hot plate/automated Von Frey tests, respectively. Calcium imaging of isolated dorsal root ganglia neurons was used as an in vitro correlate of nociception. The microbiome composition was evaluated via 16S rRNA gene variable region V4 amplicon sequencing, whereas fecal short-chain fatty acids (SCFAs) were assessed by using targeted mass spectrometry. RESULTS: Postinflammatory DSS mice developed visceral and somatic hyperalgesia. Antibiotic administration during DSS recovery induced visceral, but not somatic, hyperalgesia independent of inflammation. FMT of postinflammatory DSS stool into antibiotic-treated mice increased visceral hypersensitivity, whereas FMT of control stool reversed antibiotics' sensitizing effects. Postinflammatory mice exhibited both increased SCFA-producing species and fecal acetate/butyrate content compared with controls. Capsaicin-evoked calcium responses were increased in naive dorsal root ganglion neurons incubated with both sodium butyrate/propionate alone and with colonic supernatants derived from postinflammatory mice. CONCLUSIONS: The microbiome plays a central role in postinflammatory visceral hypersensitivity. Microbial-derived SCFAs can sensitize nociceptive neurons and may contribute to the pathogenesis of postinflammatory visceral pain.

Increased intestinal permeability exacerbates sepsis through reduced hepatic SCD-1 activity and dysregulated iron recycling.

Inflammatory bowel disease is associated with changes in the mucosal barrier, increased intestinal permeability, and increased risk of infections and sepsis, but the underlying mechanisms are incompletely understood. Here, we show how continuous translocation of gut microbial components affects iron homeostasis and facilitates susceptibility to inflammation-associated sepsis. A sub-lethal dose of lipopolysaccharide results in higher mortality in Mucin 2 deficient (Muc2-/-) mice, and is associated with elevated circulatory iron load and increased bacterial translocation. Translocation of gut microbial components attenuates hepatic stearoyl CoA desaturase-1 activity, a key enzyme in hepatic de novo lipogenesis. The resulting reduction of hepatic saturated and unsaturated fatty acid levels compromises plasma membrane fluidity of red blood cells, thereby significantly reducing their life span. Inflammation in Muc2-/- mice alters erythrophagocytosis efficiency of splenic macrophages, resulting in an iron-rich milieu that promotes bacterial growth. Our study thus shows that increased intestinal permeability triggers a cascade of events resulting in increased bacterial growth and risk of sepsis.

N-Terminomics/TAILS Profiling of Proteases and Their Substrates in Ulcerative Colitis.

Dysregulated protease activity is often implicated in the initiation of inflammation and immune cell recruitment in gastrointestinal inflammatory diseases. Using N-terminomics/TAILS (terminal amine isotopic labeling of substrates), we compared proteases, along with their substrates and inhibitors, between colonic mucosal biopsies of healthy patients and those with ulcerative colitis (UC). Among the 1642 N-termini enriched using TAILS, increased endogenous processing of proteins was identified in UC compared to healthy patients. Changes in the reactome pathways for proteins associated with metabolism, adherens junction proteins (E-cadherin, liver-intestinal cadherin, catenin alpha-1, and catenin delta-1), and neutrophil degranulation were identified between the two groups. Increased neutrophil infiltration and distinct proteases observed in ulcerative colitis may result in extensive break down, altered processing, or increased remodeling of adherens junctions and other cellular functions. Analysis of the preferred proteolytic cleavage sites indicated that the majority of proteolytic activity and processing comes from host proteases, but that key microbial proteases may also play a role in maintaining homeostasis. Thus, the identification of distinct proteases and processing of their substrates improves the understanding of dysregulated proteolysis in normal intestinal physiology and ulcerative colitis.

The Crohn's disease polymorphism, ATG16L1 T300A, alters the gut microbiota and enhances the local Th1/Th17 response.

Inflammatory bowel disease (IBD) is driven by dysfunction between host genetics, the microbiota, and immune system. Knowledge gaps remain regarding how IBD genetic risk loci drive gut microbiota changes. The Crohn's disease risk allele ATG16L1 T300A results in abnormal Paneth cells due to decreased selective autophagy, increased cytokine release, and decreased intracellular bacterial clearance. To unravel the effects of ATG16L1 T300A on the microbiota and immune system, we employed a gnotobiotic model using human fecal transfers into ATG16L1 T300A knock-in mice. We observed increases in Bacteroides ovatus and Th1 and Th17 cells in ATG16L1 T300A mice. Association of altered Schaedler flora mice with B. ovatus specifically increased Th17 cells selectively in ATG16L1 T300A knock-in mice. Changes occur before disease onset, suggesting that ATG16L1 T300A contributes to dysbiosis and immune infiltration prior to disease symptoms. Our work provides insight for future studies on IBD subtypes, IBD patient treatment and diagnostics.

Crossover Subsets of CD4+ T Lymphocytes in the Intestinal Lamina Propria of Patients with Crohn's Disease and Ulcerative Colitis.

BACKGROUND: Hovhannisyan et al. first showed evidence of plasticity between Treg and Th17 in the inflamed intestine of Crohn's disease (CD) patients. Our previous report suggests that the inflammatory cytokine milieu generates IL-17+ Foxp3+ CD4+ T lymphocytes which is a crossover population converting Treg subset to Th17 in the peripheral blood of IBD patients. This is considered as an evidence of Treg/Th17 plasticity. AIM: The aim of this study was to characterize a variety of helper T cell crossover population, not limited to IL-17+ Foxp3+ CD4+ T lymphocytes, in the lamina propria (LP) of IBD patients. METHODS: Fresh colonoscopic biopsies were obtained from patients with CD (n = 50) and ulcerative colitis (UC, n = 32) and from healthy controls (HC, n = 25). LP mononuclear cells were assessed for intracellular cytokines and transcription factors such as IFNγ, IL-13, IL-17, IL-22, T-bet, Gata-3, RORγt, and Foxp3 using multicolor flow cytometry to detect subsets of LP CD4+ T lymphocytes. RESULTS: Patients with IBD demonstrated increased crossover populations in IL-17+ Foxp3+, T-bet+ Foxp3+, Gata3+ Foxp3+, RORγt+ Foxp3+ populations compared to HC. There was an inverse correlation of Harvey-Bradshaw index with Gata3+ Foxp3+ population in CD patients, while IL-13+ Foxp3+ population was directly correlated with Mayo clinical scores in UC patients. Furthermore, total IL-22 expressing cells as well as Th22 and IL-22+ Th1 populations were decreased in UC compared to CD and HC. CONCLUSION: IBD patients exhibit the increased crossover populations in LP Treg cells toward Th2 and Th17 compared to HC. The prevalence of Treg/Th2 crossover populations is associated with clinical disease score of IBD.

Novel CD8+ T-Cell Subsets Demonstrating Plasticity in Patients with Inflammatory Bowel Disease.

BACKGROUND: Distinct CD8+ T-cell subsets such as interleukin-17-expressing Tc17 and Foxp3-expressing Tcreg are functionally similar to CD4+ T cells. Though CD4+ T cells are dysregulated in patients with inflammatory bowel disease (IBD), CD8+ T cells are not well investigated. Vitamin D is an environmental factor which influences T-cell subsets. We assessed the prevalence of CD8+ T-cell subsets among peripheral blood mononuclear cells (PBMC) and lamina propria mononuclear cells (LPMC) of patients with Crohn's disease, patients with ulcerative colitis, and healthy controls. We then tested the effect of 1α,25-dihydroxyvitamin D3 on CD8+ T-cell subsets. METHODS: A total of 73 patients with Crohn's disease, 49 patients with ulcerative colitis, and 47 healthy controls were studied. LPMC or PBMC were isolated and flow cytometry was performed. CD3+ T cells, isolated from PBMC, were cultured with or without 1α,25-dihydroxyvitamin D3, before flow cytometry. RESULTS: In LPMC, the prevalence of Tcreg was higher in patients with IBD (P < 0.05), whereas Tc17 were higher in patients with ulcerative colitis compared with patients with Crohn's disease and healthy controls (P < 0.05). In PBMC, both Tcreg and Tc17 were higher in patients with IBD (P < 0.01). Double-expressing interferon-γ+ interleukin-17+ and Foxp3+ interleukin-17+ CD8+ T cells were also identified indicating possible CD8+ plasticity. 1α,25-dihydroxyvitamin D3 decreased interferon-γ-expressing Tc1 (P < 0.05), but had no effect on Tc17 or Tcreg. CONCLUSIONS: The prevalence of novel CD8+ T-cell subsets is altered in patients with IBD. Double-expressing cells indicate plasticity and were identified in patients with IBD. Vitamin D may have a limited effect on CD8+ T cells by decreasing interferon-γ expression.

Profiles of Lamina Propria T Helper Cell Subsets Discriminate Between Ulcerative Colitis and Crohn's Disease.

BACKGROUND: Distinction between 2 forms of inflammatory bowel disease (IBD), ulcerative colitis (UC) and Crohn's disease (CD), can be challenging. Aberrant mucosal immunity suggests that CD is a T helper type 1 cell (Th1)-driven disease, whereas UC as Th2-driven response. However, whether this paradigm truly distinguishes CD from UC is controversial. We aimed to clarify the discriminating potential of lamina propria Th subsets in patients with IBD. METHODS: Biopsies from 79 patients with IBD and 20 healthy controls were collected for Th subsets analysis (Th1:interferon γ [IFN-γ], T-bet; Th2:interleukin 13 [IL-13], Gata3; Th17:IL-17, RORγt; Treg:FoxP3). The receiver-operating characteristic curves were constructed to assess the discriminating ability by calculating the area under the receiver-operating characteristic curve. The equation with the highest area under the receiver-operating characteristic curve was applied to newly diagnosed patients to evaluate discriminating ability. RESULTS: Patients with CD showed increased IFN-γ or T-bet cells and decreased IL-13 or Gata3 cells compared with UC. A discriminant equation composed of 4 markers (IFN-γ, T-bet, IL-13, and Gata3) yielded the highest area under the receiver-operating characteristic curve. In 36 established CD or UC, the sensitivity, specificity, positive and negative predictive probabilities were 92.6%, 55.6%, 86.2%, and 71.4% and in 14 newly diagnosed patients were 100.0%, 42.9%, 63.6%, and 100.0%. Furthermore, Gata3 cells were increased in tumor necrosis factor inhibitor therapy nonresponders compared with responders in CD. IFN-γ cells were directly and inversely proportional to disease activity in patients with CD and UC, respectively. CONCLUSIONS: The Th1/Th2 paradigm can distinguish CD from UC and may be further associated with response to tumor necrosis factor inhibitor in CD and disease activity in patients with IBD.

Pathogen Cell-to-Cell Variability Drives Heterogeneity in Host Immune Responses.

Encounters between immune cells and invading bacteria ultimately determine the course of infection. These interactions are usually measured in populations of cells, masking cell-to-cell variation that may be important for infection outcome. To characterize the gene expression variation that underlies distinct infection outcomes and monitor infection phenotypes, we developed an experimental system that combines single-cell RNA-seq with fluorescent markers. Probing the responses of individual macrophages to invading Salmonella, we find that variation between individual infected host cells is determined by the heterogeneous activity of bacterial factors in individual infecting bacteria. We illustrate how variable PhoPQ activity in the population of invading bacteria drives variable host type I IFN responses by modifying LPS in a subset of bacteria. This work demonstrates a causative link between host and bacterial variability, with cell-to-cell variation between different bacteria being sufficient to drive radically different host immune responses. This co-variation has implications for host-pathogen dynamics in vivo.

Integrated Genomics of Crohn's Disease Risk Variant Identifies a Role for CLEC12A in Antibacterial Autophagy.

The polymorphism ATG16L1 T300A, associated with increased risk of Crohn's disease, impairs pathogen defense mechanisms including selective autophagy, but specific pathway interactions altered by the risk allele remain unknown. Here, we use perturbational profiling of human peripheral blood cells to reveal that CLEC12A is regulated in an ATG16L1-T300A-dependent manner. Antibacterial autophagy is impaired in CLEC12A-deficient cells, and this effect is exacerbated in the presence of the ATG16L1(∗)300A risk allele. Clec12a(-/-) mice are more susceptible to Salmonella infection, supporting a role for CLEC12A in antibacterial defense pathways in vivo. CLEC12A is recruited to sites of bacterial entry, bacteria-autophagosome complexes, and sites of sterile membrane damage. Integrated genomics identified a functional interaction between CLEC12A and an E3-ubiquitin ligase complex that functions in antibacterial autophagy. These data identify CLEC12A as early adaptor molecule for antibacterial autophagy and highlight perturbational profiling as a method to elucidate defense pathways in complex genetic disease.

An image-based genetic assay identifies genes in T1D susceptibility loci controlling cellular antiviral immunity in mouse.

The pathogenesis of complex diseases, such as type 1 diabetes (T1D), derives from interactions between host genetics and environmental factors. Previous studies have suggested that viral infection plays a significant role in initiation of T1D in genetically predisposed individuals. T1D susceptibility loci may therefore be enriched in previously uncharacterized genes functioning in antiviral defense pathways. To identify genes involved in antiviral immunity, we performed an image-based high-throughput genetic screen using short hairpin RNAs (shRNAs) against 161 genes within T1D susceptibility loci. RAW 264.7 cells transduced with shRNAs were infected with GFP-expressing herpes simplex virus type 1 (HSV-1) and fluorescent microscopy was performed to assess the viral infectivity by fluorescence reporter activity. Of the 14 candidates identified with high confidence, two candidates were selected for further investigation, Il27 and Tagap. Administration of recombinant IL-27 during viral infection was found to act synergistically with interferon gamma (IFN-γ) to activate expression of type I IFNs and proinflammatory cytokines, and to enhance the activities of interferon regulatory factor 3 (IRF3). Consistent with a role in antiviral immunity, Tagap-deficient macrophages demonstrated increased viral replication, reduced expression of proinflammatory chemokines and cytokines, and decreased production of IFN-ß. Taken together, our unbiased loss-of-function genetic screen identifies genes that play a role in host antiviral immunity and delineates roles for IL-27 and Tagap in the production of antiviral cytokines.

Atg16l1 is required for autophagy in intestinal epithelial cells and protection of mice from Salmonella infection.

BACKGROUND & AIMS: Intestinal epithelial cells aid in mucosal defense by providing a physical barrier against entry of pathogenic bacteria and secreting antimicrobial peptides (AMPs). Autophagy is an important component of immune homeostasis. However, little is known about its role in specific cell types during bacterial infection in vivo. We investigated the role of autophagy in the response of intestinal epithelial and antigen-presenting cells to Salmonella infection in mice. METHODS: We generated mice deficient in Atg16l1 in epithelial cells (Atg16l1(f/f) × Villin-cre) or CD11c(+) cells (Atg16l1(f/f) × CD11c-cre); these mice were used to assess cell type-specific antibacterial autophagy. All responses were compared with Atg16l1(f/f) mice (controls). Mice were infected with Salmonella enterica serovar typhimurium; cecum and small-intestine tissues were collected for immunofluorescence, histology, and quantitative reverse-transcription polymerase chain reaction analyses of cytokines and AMPs. Modulators of autophagy were screened to evaluate their effects on antibacterial responses in human epithelial cells. RESULTS: Autophagy was induced in small intestine and cecum after infection with S typhimurium, and required Atg16l1. S typhimurium colocalized with microtubule-associated protein 1 light chain 3ß (Map1lc3b or LC3) in the intestinal epithelium of control mice but not in Atg16l1(f/f) × Villin-cre mice. Atg16l1(f/f) × Villin-cre mice also had fewer Paneth cells and abnormal granule morphology, leading to reduced expression of AMPs. Consistent with these defective immune responses, Atg16l1(f/f) × Villin-cre mice had increased inflammation and systemic translocation of bacteria compared with control mice. In contrast, we observed few differences between Atg16l1(f/f) × CD11c-cre and control mice. Trifluoperazine promoted autophagy and bacterial clearance in HeLa cells; these effects were reduced upon knockdown of ATG16L1. CONCLUSIONS: Atg16l1 regulates autophagy in intestinal epithelial cells and is required for bacterial clearance. It also is required to prevent systemic infection of mice with enteric bacteria.

The EGF receptor and HER2 participate in TNF-α-dependent MAPK activation and IL-8 secretion in intestinal epithelial cells.

TNF-α activates multiple mitogen-activated protein kinase (MAPK) cascades in intestinal epithelial cells (IECs) leading to the secretion of interleukin 8 (IL-8), a neutrophil chemoattractant and an angiogenic factor with tumor promoting properties. As the epidermal growth factor receptor (EGFR) is a known transducer of proliferative signals and a potent activator of MAPKs, we hypothesized that the EGFR participates in TNF-dependent MAPK activation and IL-8 secretion by intestinal epithelial cells (IECs). We show that the EGFR is tyrosine-phosphorylated following treatment of IECs (HT-29 and IEC-6) with TNF-α. This requires EGFR autophosphorylation as it was blocked by the EGFR kinase inhibitor AG1478. Autophosphorylation was also inhibited by both a Src-kinase inhibitor and the metalloproteinase inhibitor batimastat. TNF treatment of IECs resulted in the accumulation of soluble TGF-α; treatment of IECs with batimastat suppressed TGF-α release and immunoneutralization of TGF-α resulted in decreased EGFR and ERK phosphorylations. TNF-α treatment of IECs resulted in an association between EGFR and HER2 and inhibition of HER2 using a specific inhibitor AG879 in combination with AG1478-suppressed TNF-α-dependent ERK phosphorylation and IL-8 release. Downregulation of HER2 via siRNA resulted in a significant decrease in ERK phosphorylation and a 50% reduction in IL-8 secretion.

AMP-activated protein kinase is a positive regulator of poly(ADP-ribose) polymerase.

AMPK acts as a cellular fuel gauge and responds to decreased cellular energy status by inhibiting ATP-consuming pathways and increasing ATP-synthesis. The aim of this study was to examine the role of AMPK in modulating poly(ADP-ribose) polymerase (PARP), a nuclear enzyme involved in maintaining chromatin structure and DNA repair. HT-29 cells infected with constitutively active AMPK demonstrated increased PARP automodification and an increase in bioNAD incorporation. AMPK and PARP co-immunoprecipitated under basal conditions and in response to H(2)O(2), suggesting a physical interaction under both resting and stress-induced conditions. Incubation of PARP with purified AMPK resulted in the phosphorylation of PARP; and the inclusion of AMP as an AMPK activator potentiated PARP phosphorylation. Using immobilized PARP, the incorporation of bioNAD by PARP was dramatically increased following the addition of AMPK. These data suggest a novel role for AMPK in regulating PARP activity through a direct interaction involving phosphorylation.

Mechanisms of omega-3 fatty acid-induced growth inhibition in MDA-MB-231 human breast cancer cells.

The omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), inhibit the growth of human breast cancer cells in animal models and cell lines, but the mechanism by which this occurs is not well understood. In order to explore possible mechanisms for the modulation of breast cancer cell growth by omega-3 fatty acids, we examined the effects of EPA and DHA on the human breast cancer cell line MDA-MB-231. Omega-3 fatty acids (a combination of EPA and DHA) inhibited the growth of MDA-MB-231 cells by 30-40% (p<0.05) in both the presence and absence of linoleic acid, an essential omega-6 fatty acid. When provided individually, DHA was more potent than EPA in inhibiting the growth of MDA-MB-231 cells (p<0.05). EPA and DHA treatment decreased tumor cell proliferation (p<0.05), as estimated by decreased [methyl-(3)H]-thymidine uptake and expression of proliferation-associated proteins (proliferating cell nuclear antigen, PCNA, and proliferation-related kinase, PRK). In addition, EPA and DHA induced apoptosis, as indicated by a loss of mitochondrial membrane potential, increased caspase activity and increased DNA fragmentation (p<0.05). Cells incubated with omega-3 fatty acids demonstrated decreased Akt phosphorylation, as well as NFkappaB DNA binding activity (p<0.05). The results of this study indicate that omega-3 fatty acids decrease cell proliferation and induce apoptotic cell death in human breast cancer cells, possibly by decreasing signal transduction through the Akt/NFkappaB cell survival pathway.

Serum amyloid A activates NF-kappaB and proinflammatory gene expression in human and murine intestinal epithelial cells.

Serum amyloid A (SAA) is an acute-phase protein whose levels positively correlate with disease activity in inflammatory bowel diseases. In this study we investigated the impact of SAA on NF-kappaB signaling and proinflammatory gene expression in intestinal epithelial cells (IEC). Human HT-29 and Caco-2 monolayers were stimulated with recombinant SAA and NF-kappaB activation/NF-kappaB-dependent gene expression measured. Adenoviral dominant negative mutants IkappaB-alpha (Ad5IkappaBAA) were utilized to determine the contribution of NF-kappaB signaling pathway to SAA-dependent gene expression. Intestinal explant and primary IEC derived from kappaB-EGFP transgenic mice were exposed to SAA and NF-kappaB-dependent enhanced green fluorescent protein (EGFP) fluorescence measured. SAA induced IkappaB-alpha degradation, RelA serine 536 (S536) phosphorylation, NF-kappaB transcriptional activity, RelA recruitment to the IL-8 gene promoter and endogenous gene expression (IL-8, COX-2) in HT-29 cells. Further, Ad5IkappaBAA abrogated SAA-induced RelA nuclear translocation, NF-kappaB transcriptional activity and IL-8 gene expression. SAA-dependent IL-8 gene expression required activation of the MAPK ERK, p38 and JNK in HT-29 cells. Finally, SAA induced EGFP expression in intestinal explants isolated from kappaB-EGFP transgenic mice and enhanced RelA and IkappaBalpha phosphorylation in primary IEC. This indicates that SAA potentially participate in the inflammatory process by virtue of its ability to activate proinflammatory signaling in IEC.

5-aminoimidazole-4-carboxamide riboside (AICAR) enhances GLUT2-dependent jejunal glucose transport: a possible role for AMPK.

AMPK (AMP-activated protein kinase) is a key sensor of energy status within the cell. Activated by an increase in the AMP/ATP ratio, AMPK acts to limit cellular energy depletion by down-regulating selective ATP-dependent processes. The purpose of the present study was to determine the role of AMPK in regulating intestinal glucose transport. [3H]3-O-methyl glucose fluxes were measured in murine jejunum in the presence and absence of the AMPK activators AICAR (5-aminoimidazole-4-carboxamide riboside) and metformin and the p38 inhibitor, SB203580. To differentiate between a sodium-coupled (SGLT1) and diffusive (GLUT2) route of entry, fluxes were measured in the presence of the SGLT1 and GLUT2 inhibitors phloridzin and phloretin. Glucose transporter mRNA levels were measured by reverse transcriptase-PCR, and localization by Western blotting. Surface-expressed GLUT2 was assessed by luminal biotinylation. Activation of p38 mitogen-activated protein kinase was analysed by Western blotting. We found that treatment of jejunal tissue with AICAR resulted in enhanced net glucose uptake and was associated with phosphorylation of p38 mitogen-activated protein kinase. Inhibition of p38 abrogated the stimulation of AICAR-stimulated glucose uptake. Phloretin abolished the AICAR-mediated increase in glucose flux, whereas phloridzin had no effect, suggesting the involvement of GLUT2. In addition, AICAR decreased total protein levels of SGLT1, concurrently increasing levels of GLUT2 in the brush-border membrane. The anti-diabetic drug metformin, a known activator of AMPK, also induced the localization of GLUT2 to the luminal surface. We conclude that the activation of AMPK results in an up-regulation of non-energy requiring glucose uptake by GLUT2 and a concurrent down-regulation of sodium-dependent glucose transport.

Adenosine is a negative regulator of NF-kappaB and MAPK signaling in human intestinal epithelial cells.

Previous studies suggest that adenosine possesses anti-inflammatory properties, however, the mechanisms by which adenosine affects immune function remain unclear, particularly in the intestine. In this study, we hypothesized that adenosine directly affects pro-inflammatory gene expression in intestinal epithelial cells through modulation of NF-kappaB signaling. HT-29 cells were treated with adenosine prior to incubation with various stimuli and pro-inflammatory gene expression and signal transduction analyzed. Adenosine pretreatment resulted in a reduction in IL-8 expression and secretion in response to TNF-alpha, IL-1, LPS, and PMA. This effect was paralleled by inhibition of kappaB-driven luciferase expression and a reduction in recruitment of NF-kappaB to the IL-8 promoter. Pretreatment of HT-29 cells also resulted in reduced ERK, p38, and JNK MAPK phosphorylation, following TNF-alpha treatment. The observed effects in this study occurred independently of known surface adenosine receptors. This study identifies adenosine as a potent negative regulator of pro-inflammatory signaling in intestinal epithelial cells.

Activation of AMP-activated protein kinase reduces cAMP-mediated epithelial chloride secretion.

AMP-activated protein kinase (AMPK) is activated in response to fluctuations in cellular energy status caused by oxidative stress. One of its targets is the cystic fibrosis transmembrane conductance regulator (CFTR), which is the predominant Cl- secretory channel in colonic tissue. The aim of this study was to determine the role of AMPK in the modulation of colonic chloride secretion under conditions of oxidative stress and chronic inflammation. Chloride secretion and AMPK activity were examined in colonic tissue from adult IL-10-deficient and wild-type 129 Sv/Ev mice in the presence and absence of pharmacological AMPK inhibitors and activators, respectively. Apical levels of CFTR were measured in brush-border membrane vesicles. Cell culture studies in human colonic T84 monolayers examined the effect of hydrogen peroxide and pharmacological activation of AMPK on forskolin-stimulated chloride secretion. Inflamed colons from IL-10-deficient mice exhibited hyporesponsiveness to forskolin stimulation in association with reductions in surface CFTR expression and increased AMPK activity. Inhibition of AMPK restored tissue responsiveness to forskolin, whereas stimulation of AMPK with 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) induced tissue hyporesponsivness in wild-type mice. T84 cells exposed to hydrogen peroxide demonstrated a time-dependent increase in AMPK activity and reduction of forskolin-stimulated chloride secretion. Inhibition of AMPK prevented the reduction in chloride secretion. Treatment of cells with the AMPK activator, AICAR, resulted in a decreased chloride secretion. In conclusion, AMPK activation is linked with reductions in cAMP-mediated epithelial chloride flux and may be a contributing factor to the hyporesponsiveness seen under conditions of chronic inflammation.