Short-chain fatty acids administration is protective in colitis-associated colorectal cancer development

Reduced short-chain fatty acids (SCFAs) have been reported in patients with ulcerative colitis, and increased intake of dietary fiber has shown to be clinically beneficial for colitis. Whether SCFAs suppress tumorigenesis in colitis-associated colorectal cancer remains unknown. The chemopreventive effect of SCFAs in colitis-associated colorectal cancer was evaluated in this study. Model of colitis-associated colorectal cancer in male BALB/c mice was induced by azoxymethane (AOM) and dextran sodium sulfate (DSS). SCFAs mix (67.5 mM acetate, 40 mM butyrate, 25.9 mM propionate) was administered in drink water during the study period. Macroscopic and histological studies were performed to examine the colorectal inflammation and tumorigenesis in AOM/DSS-induced mice treated with or without SCFA mix. The effects of SCFAs mix on colonic epithelial cellular proliferation were also assessed using Ki67 immunohistochemistry and TUNEL staining. The administration of SCFAs mix significantly reduced the tumor incidence and size in mice with AOM/DSS-induced colitis associated colorectal cancer. SCFAs mix protected from AOM/DSS-induced colorectal cancer by improving colon inflammation and disease activity index score as well as suppressing the expression of proinflammatory cytokines including IL-6, TNF-α and IL-17. A decrease in cell proliferation markers and an increase in TUNEL-positive tumor epithelial cells were also demonstrated in AOM/DSS mice treated with SCFAs mix. SCFAs mix administration prevented development of tumor and attenuated the colonic inflammation in a mouse model of colitis-associated colorectal cancer. SCFAs mix may be a potential agent in the prevention and treatment of colitis-associated colorectal cancer.     

Dietary red raspberries attenuate dextran sulfate sodium-induced acute colitis

Persistent intestinal inflammation severely impairs intestinal integrity resulting in inflammatory bowel disease. Red raspberries (RB) are a rich source of bioactive compounds; their beneficial effect on the colitis protection was evaluated in the current study using a dextran sulfate sodium (DSS)-induced acute colitis mouse model. Six-week-old mice were fed a standard rodent research diet supplemented with RB (0 or 5% w/w, n=20 each group) for 6 weeks. At the 4th week of dietary treatment, approximately half of mice in each dietary group (n=12 each group) were subjected to 2.5% DSS induction for 6 days, followed by 6 days of recovery, to induce colitis. RB supplementation decreased body weight loss (P≤.01), disease activity index (P≤.01), and colon shortening (P≤.05) in DSS-treated mice. In addition, RB supplementation protected the colonic structure (P≤.01), associated with suppressed NF-κB signaling and reduced expression of inflammatory interleukin (IL)-1β, IL-6, IL-17, cyclooxegenase-2, and tumor necrosis factor-α in DSS-treated mice. RB supplementation reduced neutrophil infiltration, monocyte chemoattractant protein-1 mRNA expression, and xanthine oxidase content, but enhanced catalase content in DSS-treated mice. Consistently, RB supplementation reduced pore forming tight junction protein claudin-2, increased barrier strengthening claudin-3, zonula occluden-1 protein content and mucin (MUC)-2 mRNA level, and activated AMP-activated protein kinase (AMPK) in DSS-treated mice. In conclusion, dietary RB protected against inflammation and colitis symptoms induced by DSS, providing a promising dietary approach for the management of colitis.     

Physiological stress exacerbates murine colitis by enhancing proinflammatory cytokine expression that is dependent on IL-18

Psychological stress is an environmental factor considered to be a precipitating factor of inflammatory bowel disease. Interleukin (IL)-18 plays a role in stress-induced aggravation in some diseases. The aim of this study was to establish a model of murine colitis exacerbated by psychological stress and to clarify the role of IL-18 in this model. Male C57Bl/6 mice and IL-18(-/-) mice were used for this study. The mice received dextran sulfate sodium (DSS) for induction of colitis. Some mice were exposed to psychological stress using a communication box. Body weight, colonic length, and histological inflammation were measured for assessment of colitis. Tumor necrosis factor (TNF)-α and IL-18 expression in the colon and IL-18 expression in the adrenal gland were analyzed using real-time PCR. The effect of anti-IL-18 antibody was also investigated. Effects of TNF-α and IL-18 on cytokine expressions were studied using the colonic epithelial cell line LS174T. Induction of psychological stress in DSS-treated wild-type mice significantly exacerbated colitis with enhanced expression of proinflammatory cytokines and IL-18. However, induction of psychological stress in DSS-treated IL-18(-/-) mice did not aggravate colitis compared with that in the IL-18(-/-) group given only DSS treatment. Stress-induced aggravation of colitis was ameliorated significantly by anti-IL-18 antibody treatment. IL-18 did not enhance TNF-α-induced expression of intercellular adhesion molecule-1 or IL-8 in LS174T. We established a model of colitis exacerbated by psychological stress. Psychological stress enhanced IL-18 expression and plays a proinflammatory role in stress-induced aggravation of colitis.     

Genetic deletion of IL-25 (IL-17E) confers resistance to dextran sulfate sodium-induced colitis in mice

Background

IL-25 is emerging as a key regulator of inflammation in the intestinal mucosa because of its ability to promote type 2 while suppressing Th1 and Th17 responses. Several previous studies reported inconsistent results on the role of exogenous IL-25 in development of colonic inflammation and none were performed in animals with a genetic deletion of IL-25. We investigated the contribution of endogenous IL-25 to DSS-induced colitis using mice deficient in IL-25.

Results

Mice were exposed to DSS in drinking water ad libitum either for seven days (acute) or for three cycles of seven days with DSS followed by 14 days without DSS (chronic) to induce colitis, respectively. The loss of body weight, appearance of diarrhea and bloody stools, and shortening of colon length were significantly less pronounced in IL-25^?/? mice compared to WT mice after exposure to acute DSS. Histological examination showed that DSS-treated IL-25^?/? mice had only mild inflammation in the colon, while severe inflammation developed in DSS-treated WT mice. A significant up-regulation of IL-33 was observed in acute DSS-treated WT but not in the IL-25^?/? mice. There was significantly lower expression of pro-inflammatory cytokines in the colon of acute DSS-treated IL-25^?/? compared to WT mice. IL-25^?/? mice were also partially protected from chronic DSS challenge especially during the first 2 cycles of DSS exposure. In contrast to IL-25^?/? mice, IL-13^?/? mice were more susceptible to DSS-induced colitis. Finally, stimulation of T84 colonic epithelial cells with IL-25 up-regulated the expression of IL-33 and several pro-inflammatory cytokines.

Conclusions

These data indicate that endogenous IL-25 acts as a pro-inflammatory factor in DSS-induced colitis, which is unlikely to be mediated by IL-13 but possibly the induction of IL-33 and other pro-inflammatory mediators from colonic epithelial cells. The present study suggests that IL-25 may contribute to the pathogenesis of inflammatory bowel disease in at least a subgroup of patients.

     

TLR3 activation evokes IL-6 secretion, autocrine regulation of Stat3 signaling and TLR2 expression in human bronchial epithelial cells

Human bronchial epithelial cells exposed to synthetic double-stranded RNA (poly I:C) exhibited increased IL-6 and RANTES secretion and TLR2 expression that was inhibited following TLR3 silencing. Increased NF-κB and Stat3 phosphorylation were detected after poly I:C exposure and pretreatment with neutralizing antibody targeting IL-6 receptor α (IL-6Rα -nAb) or blocking Jak2 and Stat3 activity inhibited Stat3 phosphorylation. TLR2 up-regulation by poly I:C was also reduced by IL-6Rα-nAb and inhibitors of Jak2, Stat3 and NF-κB phosphorylation, whereas RANTES secretion was unaffected, but abolished following NF-κB inhibition. Treatment with exogenous IL-6 failed to increase TLR2. These findings demonstrate that TLR3 activation differentially regulates TLR expression through autocrine signaling involving IL-6 secretion, IL-6Rα activation and subsequent phosphorylation of Stat3. The results also indicate that NF-κB and Stat3 are required for TLR3-dependent up-regulation of TLR2 and that its delayed expression was due to a requirement for IL-6-dependent Stat3 activation.     

Nicotine suppresses hyperexcitability of colonic sensory neurons and visceral hypersensivity in mouse model of colonic inflammation

Recently, we reported that nicotine in vitro at a low 1-μM concentration suppresses hyperexcitability of colonic dorsal root ganglia (DRG; L(1)-L(2)) neurons in the dextran sodium sulfate (DSS)-induced mouse model of acute colonic inflammation (1). Here we show that multiple action potential firing in colonic DRG neurons persisted at least for 3 wk post-DSS administration while the inflammatory signs were diminished. Similar to that in DSS-induced acute colitis, bath-applied nicotine (1 μM) gradually reduced regenerative multiple-spike action potentials in colonic DRG neurons to a single action potential in 3 wk post-DSS neurons. Nicotine (1 μM) shifted the activation curve for tetrodotoxin (TTX)-resistant sodium currents in inflamed colonic DRG neurons (voltage of half-activation changed from -37 to -32 mV) but did not affect TTX-sensitive currents in control colonic DRG neurons. Further, subcutaneous nicotine administration (2 mg/kg b.i.d.) in DSS-treated C57Bl/J6 male mice resulted in suppression of hyperexcitability of colonic DRG (L(1)-L(2)) neurons and the number of abdominal constrictions in response to intraperitoneal injection of 0.6% acetic acid. Collectively, the data suggest that neuronal nicotinic acetylcholine receptor-mediated suppression of hyperexcitability of colonic DRG neurons attenuates reduction of visceral hypersensitivity in DSS mouse model of colonic inflammation.     

Stat3 activation in acute lung injury

Stat3 plays diverse roles in biological processes including cell proliferation, survival, apoptosis, and inflammation. Very little is known regarding its activation and function in the lung during acute inflammation. We now show that Stat3 activation was triggered in lungs and in alveolar macrophages after intrapulmonary deposition of IgG immune complexes in rats. Low levels of constitutive Stat3 were observed in normal rat lungs as determined by the EMSA. Stat3 activity in whole lung extracts increased 2 h after initiation of IgG immune complex deposition, reaching maximal levels by 4 h, whereas Stat3 activation was found in alveolar macrophages as early as 30 min after onset of injury. Expression and activation of Stat3 mRNA, protein, and protein phosphorylation was accompanied by increased gene expression of IL-6, IL-10, and suppressor of cytokine signaling-3 in whole lung tissues. Both Tyr(705) and Ser(727) phosphorylation were involved in Stat3 activation as assessed in whole lung extracts. C5a (complement 5, fragment a) per se can induce phosphorylation of Ser(727) of Stat3. In vivo, Stat3 activation was dramatically suppressed by depletion of neutrophils or lung macrophages, resulting in reduced gene expression of IL-6 and IL-10 in whole lung tissues. Using blocking Abs to IL-6, IL-10, and C5a, Stat3 activation induced by IgG immune complexes was markedly diminished. These data suggest in the lung injury model used that activation of Stat3 in lungs is macrophage dependent and neutrophil dependent. IL-6, IL-10, and C5a contribute to Stat3 activation in inflamed rat lung.     

Intestine-Specific Mttp Deletion Increases the Severity of Experimental Colitis and Leads to Greater Tumor Burden in a Model of Colitis Associated Cancer

Background

Gut derived lipid factors have been implicated in systemic injury and inflammation but the precise pathways involved are unknown. In addition, dietary fat intake and obesity are independent risk factors for the development of colorectal cancer. Here we studied the severity of experimental colitis and the development of colitis associated cancer (CAC) in mice with an inducible block in chylomicron secretion and fat malabsorption, following intestine-specific deletion of microsomal triglyceride transfer protein (Mttp-IKO).

Methodology/Principal Findings

Mttp-IKO mice exhibited more severe injury with ∼90% mortality following dextran sodium sulfate (DSS) induced colitis, compared to <20% in controls. Intestinal permeability was increased in Mttp-IKO mice compared to controls, both at baseline and after DSS administration, in association with increased circulating levels of TNFα. DSS treatment increased colonic mRNA expression of IL-1β and IL-17A as well as inflammasome expression in both genotypes, but the abundance of TNFα was selectively increased in DSS treated Mttp-IKO mice. There was a 2-fold increase in colonic tumor burden in Mttp-IKO mice following azoxymethane/DSS treatment, which was associated with increased colonic inflammation as well as alterations in cytokine expression. To examine the pathways by which alterations in fatty acid abundance might interact with cytokine signaling to regulate colonic epithelial growth, we used primary murine myofibroblasts to demonstrate that palmitate induced expression of amphiregulin and epiregulin and augmented the increase in both of these growth mediators when added to IL-1βor to TNFα.

Conclusions

These studies demonstrate that Mttp-IKO mice, despite absorbing virtually no dietary fat, exhibit augmented fatty acid dependent signaling that in turn exacerbates colonic injury and increases tumor formation.     

Dietary intrinsic phytate protects colon from lipid peroxidation in pigs with a moderately high dietary iron intake.

High iron consumption has been proposed to relate to an increase in the risk of colon cancer, whereas high levels of supplemental sodium phytate effectively reduce iron-induced oxidative injury and reverse iron-dependent augmentation of colorectal tumorigenesis. However, the protective role of intrinsic dietary phytate has not been determined. In this study, we examined the impact of removing phytate present in a corn-soy diet by supplemental microbial phytase on susceptibility of pigs to the oxidative stress caused by a moderately high dietary iron intake. Thirty-two weanling pigs were fed the corn-soy diets containing two levels of iron (as ferrous sulfate, 80 or 750 mg/kg diet) and microbial phytase (as Natuphos, BASF, Mt. Olive, NJ, 0 or 1200 units/kg). Pigs fed the phytase-supplemented diets did not receive any inorganic phosphorus to ensure adequate degradation of phytate. After 4 months of feeding, liver, colon, and colon mucosal scrapings were collected from four pigs in each of the four dietary groups. Colonic lipid peroxidation, measured as thiobarbituric acid reacting substances (TBARS), was increased by both the high iron (P< 0.0008) and phytase (P< 0.04) supplementation. Both TBARS and F2-isoprostanes, an in vivo marker of lipid peroxidation, in colonic mucosa were affected by dietary levels of iron (P< 0.03). Mean hepatic TBARS in pigs fed the phytase-supplemented, high iron diet was 43%-65% higher than that of other groups although the differences were nonsignificant. Moderately high dietary iron induced hepatic glutathione peroxidase activity (P= 0.06) and protein expression, but decreased catalase (P< 0.05) in the colonic mucosa. In conclusion, intrinsic phytate in corn and soy was protective against lipid peroxidation in the colon associated with a moderately high level of dietary iron.     

IL-4 exacerbates anaphylaxis

We evaluated whether IL-4, a cytokine critical for inducing allergic responses, also contributes to the effector phase of allergy. Pretreatment of mice with IL-4 or the related cytokine, IL-13, rapidly and dramatically increased the severity of anaphylaxis induced by cross-linking Fc(epsilon)RI or FcgammaRIII. This effect was inhibited by endogenously produced IFN-gamma, was T cell-, B cell-, and common gamma-chain-independent, and required IL-4Ralpha and Stat6. IL-4Ralpha signaling also enhanced anaphylaxis in mice infected with a nematode parasite that stimulates IL-4/IL-13 production. IL-4 exacerbated anaphylaxis by acting synergistically with vasoactive mediators to increase vascular permeability. Synergy between IL-4 and vasoactive mediators during the effector phase of allergic inflammation may both contribute to allergic immunopathology and enhance protective immunity against gastrointestinal worms.