Influence of fatty acid-free diet on mammary tumor development and growth rate in HER-2/Neu transgenic mice.

Numerous investigations have found a relationship between higher risk of cancer and increased intake of fats, while results of clinical studies of fat reduction and breast cancer recurrence have been mixed. A diet completely free of fats cannot be easily administered to humans, but experimental studies in mice can be done to determine whether this extreme condition influences tumor development. Here, we examined the effects of a FA-free diet on mammary tumor development and growth rate in female FVB-neu proto-oncogene transgenic mice that develop spontaneous multifocal mammary tumors after a long latency period. Mice were fed a fatty acid-free diet beginning at 112, 35, and 30 days of age. In all these experiments, tumor appearance was delayed, tumor incidence was reduced and the mean number of palpable mammary tumors per mouse was lower, as compared to standard diet-fed mice. By contrast, tumor growth rate was unaffected in mice fed the fatty acid-free diet. Plasma of mice fed the fatty acid-free diet revealed significantly higher contents of oleic, palmitoleic and 20:3ω9 acids and lower contents of linoleic and palmitic acids. In conclusion, these findings indicate that a FA-free diet reduces tumor incidence and latency but not tumor growth rate, suggesting that a reduction in dietary FAs in humans may have a protective effect on tumorigenesis but not on tumors once they appear.

Induction of Paneth cell degranulation by orally administered Toll-like receptor ligands.

The secretory activity of Paneth cells is related to the bacterial milieu in the small intestine; however, the molecules involved in inducing Paneth cell secretion of enzymes and antimicrobial peptides are not well-defined. Mice treated orally with CpG-oligodeoxynucleotide (ODN), an agonist of Toll-like receptor (TLR) 9, showed rapid and massive Paneth cell degranulation. CpG-ODN-induced degranulation was not observed in TLR9(-/-) mice or in chimeric TLR9(-/-) mice reconstituted with wild-type (WT) bone marrow, but was observed in WT mice reconstituted with TLR9(-/-) bone marrow, indicating a role for TLR9-expressing gastrointestinal cells in CpG recognition. The TLR3 agonist polyinosinic-polycytidylic acid also induced rapid degranulation, whereas the TLR4 and TLR5 agonists LPS and flagellin, respectively, induced late degranulation mediated by TNF-α. Our evidence that TLR9 and TLR3 agonists induce Paneth cell degranulation points to the need for further studies of the mechanisms underlying Paneth cell function as an avenue toward preventing infection and treating inflammatory bowel diseases.

Influence of lignans depletion on murine mammary gland morphology.

Different studies have focused on the effects of phytoestrogens-supplemented diets on mammary gland morphogenesis and breast cancer risk; however, particular dieting behaviors and food choices may result in a reduction of the natural source of phytoestrogens. The evaluation of a reduced phytoestrogens intake effect by depletion without modifying other dietary ingredients is hard. Since lignans, the largest contributors to phytoestrogens intake in Western diets, are metabolized into bioactive compounds by gut bacteria, long-term antibiotic treatments, inducing intestinal microflora disruption, may reduce enterolactone availability. To elucidate the effect of phytoestrogens lack on mammary tissue morphogenesis, female FVB mice were treated with gentamicin or metronidazole/ciprofloxacin from the age of 6 to 7 wk. After 21 wk, enterolactone urine levels were 120.07 +/- 20.5 ng/ml in untreated mice, 30.4 +/- 24.46 ng/ml in metronidazole/ciprofloxacin-treated mice, and 3.29 +/- 4.38 ng/ml in gentamicin-treated mice. Histological analysis revealed no significant alterations of mammary morphology in metronidazole/ciprofloxacin-treated mice, whereas gentamicin-treated mice showed increase of ducts number and duct-tree branching vs. controls. These findings indicate that normal mammary tissue size and shape are maintained even in the presence of low levels of lignans and suggest that only a complete depletion of these compounds induced significant alterations of mammary gland structure.

Intestinal glucose uptake protects liver from lipopolysaccharide and D-galactosamine, acetaminophen, and alpha-amanitin in mice.

We have recently observed that oral administration of D-glucose saves animals from lipopolysaccharide (LPS)-induced death. This effect is the likely consequence of glucose-induced activation of the sodium-dependent glucose transporter-1. In this study, we investigated possible hepatoprotective effects of glucose-induced, sodium-dependent, glucose transporter-1 activation. We show that oral administration of D-glucose, but not of either D-fructose or sucrose, prevents LPS-induced liver injury, as well as liver injury and death induced by an overdose of acetaminophen. In both of these models, physiological liver morphology is maintained and organ protection is confirmed by unchanged levels of the circulating markers of hepatotoxicity, such as alanine transaminase or lactate dehydrogenase. In addition, D-glucose was found to protect the liver from alpha-amanitin-induced liver injury. In this case, in contrast to the previously described models, a second signal had to be present in addition to glucose to achieve protective efficacy. Toll-like receptor 4 stimulation that was induced by low doses of LPS was identified as such a second signal. Eventually, the protective effect of orally administered glucose on liver injury induced by LPS, overdose of acetaminophen, or alpha-amanitin was shown to be mediated by the anti-inflammatory cytokine interleukin-10. These findings, showing glucose-induced protective effects in several animal models of liver injury, might be relevant in view of possible therapeutic interventions against different forms of acute hepatic injury.

Low dose oral administration of cytokines for treatment of allergic asthma.

Many inflammatory diseases are characterized by an imbalance among lymphocyte populations, in particular Th1, Th2 and the recently described Th17 cells. The Th1/Th2 imbalance is linked to many factors, but certainly the role of cytokines is essential. In Th2 diseases IL-4 expression is predominant, while Th1 pathologies are characterized by high expression of IFN-gamma and IL-12. Though today the therapeutical proposal for many inflammatory diseases aims to re-establish normal levels of Th1/Th2 cytokines, the pharmacological use of cytokines, which are very active molecules, is limited by the possible collateral effects. Therefore, our study aims to determine, in a murine model of allergic asthma, the possible therapeutic activity of low dose cytokines solutions, mechanically activated. We found that oral administration of low doses IL-12 plus IFN-gamma is able to solve the bronchial hyperresponsiveness condition of mice, establishing normal cytokine levels. The anti-asthma activity was confirmed by histological analysis of lungs and broncho-alveolar lavage fluid cell count. Serum ovalbumin-specific IgE was also significantly inhibited by treatment with low dose activated cytokines solution. These findings may suggest a novel approach to diseases which involve a Th1/Th2 imbalance.

Sodium-dependent glucose transporter-1 as a novel immunological player in the intestinal mucosa.

In this study, we demonstrate the protective effect of the activation of sodium-dependent glucose transporter-1 (SGLT-1) on damages induced by TLR ligands, in intestinal epithelial cells and in a murine model of septic shock. In intestinal epithelial cell lines, glucose inhibited the IL-8/keratinocyte-derived chemokine production and the activation of the TLR-related transcription factor NF-kappaB stimulated by LPS or CpG-oligodeoxynucleotide. Oral ingestion of glucose was found to protect 100% of mice from lethal endotoxic shock induced by i.p. LPS administration; protection was only observed when glucose was administered orally, not by i.p. route, suggesting the important role of intestinal epithelial cells in this protection. In addition, we observed that the in vivo protection depends on an increase of anti-inflammatory cytokine IL-10. The cornerstone of the observed immunomodulatory and life-saving effects resides in activation of SGLT-1; in fact, the glucose analog 3-O-methyl-d-gluco-pyranose, which induces the transporter activity, but is not metabolized, exerted the same inhibitory effects as glucose both in vitro and in vivo. Thus, we propose that activated SGLT-1, apart from its classical metabolic function, may be a promising target for inhibition of bacteria-induced inflammatory processes and life-saving treatments, assuming a novel role as an immunological player.

Low molecular weight hyaluronic acid increases the self-defense of skin epithelium by induction of beta-defensin 2 via TLR2 and TLR4.

In sites of inflammation or tissue injury, hyaluronic acid (HA), ubiquitous in the extracellular matrix, is broken down into low m.w. HA (LMW-HA) fragments that have been reported to activate immunocompetent cells. We found that LMW-HA induces activation of keratinocytes, which respond by producing beta-defensin 2. This production is mediated by TLR2 and TLR4 activation and involves a c-Fos-mediated, protein kinase C-dependent signaling pathway. LMW-HA-induced activation of keratinocytes seems not to be accompanied by an inflammatory response, because no production of IL-8, TNF-alpha, IL-1beta, or IL-6 was observed. Ex vivo and in vivo treatments of murine skin with LMW-HA showed a release of mouse beta-defensin 2 in all layers of the epidermal compartment. Therefore, the breakdown of extracellular matrix components, for example after injury, stimulates keratinocytes to release beta-defensin 2, which protects cutaneous tissue at a time when it is particularly vulnerable to infection. In addition, our observation might be important to open new perspectives in the development of possible topical products containing LMW-HA to improve the release of beta-defensins by keratinocytes, thus ameliorating the self-defense of the skin for the protection of cutaneous tissue from infection by microorganisms.

Induction of pro-inflammatory programs in enteroendocrine cells by the Toll-like receptor agonists flagellin and bacterial LPS.

Enteroendocrine cells are hormone-secreting cells spread along the intestinal epithelium. Their principal function is to promote the digestion of food. However, little is known about other functions that these cells may play, since they are difficult to study as a whole endocrine organ due to their diffuse localization. It is known that the intestinal epithelial barrier is actively involved in the host defense against pathogen invasion. Here we applied gene expression profiling to characterize the response of the human LCC-18 enteroendocrine cell line to physiological and pathological stimuli mimicked by fatty acids (FAs), flagellin and LPS exposure. We observed that these cells participate in an innate immune reaction to pathogens through the expression of pro-inflammatory factors (i.e. CXCL1 and 3 and IL-32) that we could validate by molecular and proteomic approach. Interestingly, IL-32 has been recently found over-expressed in the inflamed mucosa of patients affected by inflammatory bowel disease. This is very important because modifications of enteroendocrine cells during intestinal inflammation have been so far considered as secondary effects of the inflammatory status rather than due to direct pathogen/enteroendocrine cell interaction. As expected, FAs exposure up-regulates pro-differentiative genes and the production of cholecystokinin but it does not enhance the expression of pro-inflammatory genes. The present observations enlighten a new aspect of the cross talk between immune and endocrine system and suggest enteroendocrine cells as important contributors of inflammatory processes occurring in the gut in response to pathogen exposure and direct enhancers of the inflammatory status associated with human inflammatory bowel disease.

Cross-talk among Toll-like receptors and their ligands.

Toll-like receptors (TLRs) 4, 5, 7 and 9 belong to a family of proteins that recognize mainly conserved microbial motifs. Though each TLR has a highly specific ability to recognize a particular microbial pattern, recent papers suggest that some ligands are able to affect the expression of different TLRs. In this paper, we have investigated TLR4, 5, 7 and 9 expression, both at mRNA and protein level, following treatment of different intestinal epithelial cell lines with LPS, flagellin, loxiribine, CpG-oligodeoxynucleotide and peptidoglycan, to assess if the different TLR ligands may modulate the expression of the respective TLR and of the unrelated ones. Our results show that a cross-talk exists between TLRs and various ligands, indicating a cross-regulation among these pattern recognition receptors. In particular, TLR4 was generally down-regulated by treatment with ligands other than LPS, while flagellin and unrelated microbial-associated molecular patterns exerted a general stimulatory activity as regards TLR5 expression. Concerning TLR7 and 9, we have observed a more variable behaviour of the various cell lines with the different ligands. Together, our results demonstrate that the expression of TLRs in intestinal cells is highly dynamic and tightly regulated in response to encountered microbial stimuli.

Activation of enteroendocrine cells via TLRs induces hormone, chemokine, and defensin secretion.

Enteroendocrine cells are known primarily for their production of hormones that affect digestion, but they might also be implicated in sensing and neutralizing or expelling pathogens. We evaluate the expression of TLRs and the response to specific agonists in terms of cytokines, defensins, and hormones in enteroendocrine cells. The mouse enteroendocrine cell line STC-1 and C57BL/6 mice are used for in vitro and in vivo studies, respectively. The presence of TLR4, 5, and 9 is investigated by RT-PCR, Western blot, and immunofluorescence analyses. Activation of these receptors is studied evaluating keratinocyte-derived chemokine, defensins, and cholecystokinin production in response to their specific agonists. In this study, we show that the intestinal enteroendocrine cell line STC-1 expresses TLR4, 5, and 9 and releases cholecystokinin upon stimulation with the respective receptor agonists LPS, flagellin, and CpG-containing oligodeoxynucleotides. Release of keratinocyte-derived chemokine and beta-defensin 2 was also observed after stimulation of STC-1 cells with the three TLR agonists, but not with fatty acids. Consistent with these in vitro data, mice showed increased serum cholecystokinin levels after oral challenge with LPS, flagellin, or CpG oligodeoxynucleotides. In addition to their response to food stimuli, enteroendocrine cells sense the presence of bacterial Ags through TLRs and are involved in neutralizing intestinal bacteria by releasing chemokines and defensins, and maybe in removing them by releasing hormones such as cholecystokinin, which induces contraction of the muscular tunica, favoring the emptying of the distal small intestine.

Influence of antibiotic treatment on breast carcinoma development in proto-neu transgenic mice.

The effect of prolonged antibiotic treatments on tumor development was evaluated in proto-neu transgenic mice, which spontaneously develop mammary carcinomas. Virgin transgenic mice were treated with metronidazole/ciprofloxacin or gentamicin through the drinking water. The hazard ratio [HR; 95% confidence interval (95% CI)] of breast cancer occurrence in metronidazole/ciprofloxacin-treated mice was more than triple that for controls [3.11 (1.13-8.53); P = 0.028], whereas only a slight increase in HR (95% CI) was observed in gentamicin-treated mice [1.39 (0.56-3.47); P = 0.481]. Tumor growth rate in gentamicin-treated mice was significantly faster than in untreated control mice (P = 0.043). Moreover, mammary glands from mice treated with either antibiotic regimen showed increased lobulization, with more numerous and more developed terminal ductal lobular units than in controls. These results indicate that prolonged exposure to relevant doses of antibiotics affects the mammary glands in this particular model of HER-2/neu transgenic mice; further studies to understand the precise mechanism by which antibiotic treatments influence mammary gland differentiation are critical.

Activation of smooth muscle and myenteric plexus cells of jejunum via Toll-like receptor 4.

The cell types of the gut expressing Toll-like receptor 4, which recognizes specifically bacterial lipopolysaccharides, as well as the functionality of this receptor, have remained controversial. We aimed to clarify these issues. Mouse and human intestinal specimens were stained immunohistochemically to detect Toll-like receptor 4 expression. Smooth muscle and myenteric plexus cells but not enterocytes revealed receptor expression. Murine intestinal smooth muscle and myenteric plexus cells but not enterocytes showed nuclear translocation of nuclear factor-kappaB after in vivo stimulation with lipopolysaccharide. Moreover, lipopolysaccharide added to human jejunum biopsies free of epithelial cells induced release of interleukin-8 (IL-8). We can conclude that Toll-like receptor 4 is not expressed in epithelial layer, but rather on smooth muscle and myenteric plexus cells and that expression is functional. The expression of Toll-like receptor 4 on smooth muscle and myenteric plexus cells is consistent with the possibility that these cells are involved in intestinal immune defense; the low or absent expression of Toll-like receptor 4 on enterocytes might explain the intestinal epithelium hyporesponsiveness to the abundance of LPS in the intestinal lumen.

CpG-oligodeoxynucleotides induce mobilization of hematopoietic progenitor cells into peripheral blood in association with mouse KC (IL-8) production.

The immune system of vertebrates detects bacterial DNA as a "danger signal" based on the presence of unmethylated CpG motifs. We examined whether oligodeoxynucleotides (ODNs) with CpG motifs (CpG-ODNs) also induce mobilization of hematopoietic progenitor cells (HPCs). Mice challenged with CpG-ODNs showed an increase in peripheral blood colony-forming units (CFU) with a peak at day 4 after treatment, associated with an increase, starting 30 min after CpG treatment, in serum levels of mouse keratinocyte-derived chemokine (mKC), a functional homolog of human interleukin (IL) 8; production of granulocyte-colony-stimulating factor (CSF) was also detected. Mobilization and mKC induction were sequence-specific and dose-dependent occurring even with low doses of CpG-ODNs. Interestingly, intestinal cells were involved in mKC production. HPC mobilization by CpG-ODNs was dependent on peripheral blood mononuclear cells since mobilization was reduced in neutrophil-depleted mice. Moreover, CpG-ODN treatment significantly increased G-CSF mobilizing capacity. Finally, pretreatment with an anti-mKC neutralizing antibody significantly reduced CpG-induced mobilization, further supporting a role for mKC. Thus, bacterial DNA is a "danger signal" not only for immune cells but also for hematopoietic cells, communicating the need for increased hematopoiesis during infections and for the renewal of the immune system. The HPC mobilization activity of CpG-ODNs will need to be considered in the design of treatment regimens for cancer clinical trials using CpG-ODNs in association with chemotherapy.

Degranulation of paneth cells via toll-like receptor 9.

The release of antimicrobial peptides and growth factors by Paneth cells is thought to play an important role in protecting the small intestine, but the mechanisms involved have remained obscure. Immunohistochemistry and immunofluorescence showed that Paneth cells express Toll-like receptor 9 (TLR9) in the granules. Injection of mice with oligonucleotides containing CpG sequence (CpG-ODNs) led to a down-modulation of TLR9 and a striking decrease in the number of large secretory granules, consistent with degranulation. Moreover CpG-ODN treatment increased resistance to oral challenge with virulent Salmonella typhimurium. Moreover, our findings demonstrate a sentinel role for Paneth cells through TLR9.