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.

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.

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.

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.

A double-blind, randomised, vehicle-controlled clinical study to evaluate the efficacy of MAS065D in limiting the effects of radiation on the skin: interim analysis.

Our aim was to assess the efficacy of MAS065D, a non-steroidal water-in-oil cream, in preventing and limiting skin reactions caused by radiation therapy (RT). 40 women treated with conservative breast cancer surgery followed by radiotherapy, were randomised to receive MAS065D (22 pts) or vehicle (18 pts). Radiotherapy was delivered in 20 fractions: 2.25 Gy to the whole breast plus a concomitant boost of 0.25 Gy to the tumour bed up to a total dose of 50 Gy. Evaluations of skin toxicity, erythema, and subjective symptoms were carried out weekly and 3 weeks after treatment completion. A statistically significant difference between vehicle and MAS065D groups was recorded regarding the maximum severity of skin toxicity (p < 0.0001), burning within the radiation field (p = 0.039) and desquamation (p = 0.02), in favour of the latter. We conclude that MAS065D may be considered a safe and effective treatment in the prevention and minimization of skin reactions and associated symptoms.

Doxorubicin-induced alopecia is associated with sebaceous gland degeneration.

Alopecia, accompanied by skin dryness, is one of the distressing side effects often occurring in chemotherapy-treated cancer patients. Little is known of the effects of chemotherapy on sebaceous glands, despite their importance in hair follicle homeostasis. This study investigates sebaceous gland morphology and the response of SZ95 sebaceous gland cell line to doxorubicin (DXR) treatment. The morphology of sebaceous glands during intraperitoneal DXR treatment was investigated by optical and electron microscopy in a 7-day-old rat model and further confirmed in an adult mouse model. Moreover, in vitro studies using the SZ95 sebaceous gland cell line were performed to assess the response of sebocytes to DXR in terms of cell proliferation, apoptosis, and necrosis. DXR treatment induced sebaceous gland regression and occasionally caused their complete disappearance. This observed damage and disappearance preceded DXR-induced hair loss. In vitro experiments using the SZ95 sebaceous gland cell line indicated that DXR treatment induced a differentiation process leading to premature sebocytes apoptosis. Owing to the importance of the sebaceous gland in hair follicle homeostasis, DXR-induced involution of this gland might be related to subsequent hair loss.

Macrophage-derived SPARC bridges tumor cell-extracellular matrix interactions toward metastasis.

Other than genetic imprinting and epithelial to mesenchymal transition, cancer cells need interaction with the nearby stroma toward metastasis. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein known to regulate extracellular matrix (ECM) deposition and cell-ECM interaction. Gene expression profiles associate SPARC to malignant progression. Using reciprocal bone marrow chimeras between SPARC knockout and wild-type mice, we show that SPARC produced by inflammatory cells is necessary for spontaneous, but not experimental, i.v. metastasis. Macrophage-derived SPARC induces cancer cell migration and enhances their migration to other ECM proteins at least through alpha(v)beta(5) integrin. Indeed, RNA interference knockdown of beta(5) integrin expression reduces cell migration in vitro and metastasis in vivo. Together these results show that macrophage-derived SPARC takes part in metastasis, acting at the step of integrin-mediated migration of invasive cells.

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.

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.