Dimerization of kringle 1 domain from hepatocyte growth factor/scatter factor provides a potent MET receptor agonist.

Hepatocyte growth factor/scatter factor (HGF/SF) and its cognate receptor MET play several essential roles in embryogenesis and regeneration in postnatal life of epithelial organs such as the liver, kidney, lung, and pancreas, prompting a strong interest in harnessing HGF/SF-MET signalling for regeneration of epithelial organs after acute or chronic damage. The limited stability and tissue diffusion of native HGF/SF, however, which reflect the tightly controlled, local mechanism of action of the morphogen, have led to a major search of HGF/SF mimics for therapy. In this work, we describe the rational design, production, and characterization of K1K1, a novel minimal MET agonist consisting of two copies of the kringle 1 domain of HGF/SF in tandem orientation. K1K1 is highly stable and displays biological activities equivalent or superior to native HGF/SF in a variety of in vitro assay systems and in a mouse model of liver disease. These data suggest that this engineered ligand may find wide applications in acute and chronic diseases of the liver and other epithelial organs dependent of MET activation.

Exposure of Caenorhabditis elegans to Dietary Nε-Carboxymethyllysine Emphasizes Endocytosis as a New Route for Intestinal Absorption of Advanced Glycation End Products.

The impact of dietary advanced glycation end products (dAGEs) on human health has been discussed in many studies but, to date, no consensual pathophysiological process has been demonstrated. The intestinal absorption pathways which have so far been described for dAGEs, the passive diffusion of free AGE adducts and transport of glycated di-tripeptides by the peptide transporter 1 (PEPT-1), are not compatible with certain pathophysiological processes described. To get new insight into the intestinal absorption pathways and the pathophysiological mechanisms of dAGEs, we initiated an in vivo study with a so-called simple animal model with a complete digestive tract, Caenorhabditis elegans. Dietary bacteria were chemically modified with glyoxylic acid to mainly produce Nε-carboxymethyllysine (CML) and used to feed the worms. We performed different immunotechniques using an anti-CML antibody for the relative quantification of ingested CML and localization of this AGE in the worms' intestine. The relative expression of genes encoding different biological processes such as response to stresses and intestinal digestion were determined. The physiological development of the worms was verified. All the results were compared with those obtained with the control bacteria. The results revealed a new route for the intestinal absorption of dietary CML (dCML), endocytosis, which could be mediated by scavenger receptors. The exposure of worms to dCML induced a reproductive defect and a transcriptional response reflecting oxidative, carbonyl and protein folding stresses. These data, in particular the demonstration of endocytosis of dCML by enterocytes, open up new perspectives to better characterize the pathophysiological mechanisms of dAGEs.

PD-1/PD-L1 binding studies using microscale thermophoresis.

The characterization of protein interactions has become essential in many fields of life science, especially drug discovery. Microscale thermophoresis (MST) is a powerful new method for the quantitative analysis of protein-protein interactions (PPIs) with low sample consumption. In addition, one of the major advantages of this technique is that no tedious purification step is necessary to access the protein of interest. Here, we describe a protocol using MST to determine the binding affinity of the PD-1/PD-L1 couple, which is involved in tumour escape processes, without purification of the target protein from cell lysates. The method requires the overexpression of fluorescent proteins in CHO-K1 cells and describes the optimal conditions for determining the dissociation constant. The protocol has a variety of potential applications in studying the interactions of these proteins with small molecules and demonstrates that MST is a valuable method for studying the PD-1/PD-L1 pathway.

Beneficial metabolic effects of rapamycin are associated with enhanced regulatory cells in diet-induced obese mice.

The "mechanistic target of rapamycin" (mTOR) is a central controller of growth, proliferation and/or motility of various cell-types ranging from adipocytes to immune cells, thereby linking metabolism and immunity. mTOR signaling is overactivated in obesity, promoting inflammation and insulin resistance. Therefore, great interest exists in the development of mTOR inhibitors as therapeutic drugs for obesity or diabetes. However, despite a plethora of studies characterizing the metabolic consequences of mTOR inhibition in rodent models, its impact on immune changes associated with the obese condition has never been questioned so far. To address this, we used a mouse model of high-fat diet (HFD)-fed mice with and without pharmacologic mTOR inhibition by rapamycin. Rapamycin was weekly administrated to HFD-fed C57BL/6 mice for 22 weeks. Metabolic effects were determined by glucose and insulin tolerance tests and by indirect calorimetry measures of energy expenditure. Inflammatory response and immune cell populations were characterized in blood, adipose tissue and liver. In parallel, the activities of both mTOR complexes (e. g. mTORC1 and mTORC2) were determined in adipose tissue, muscle and liver. We show that rapamycin-treated mice are leaner, have enhanced energy expenditure and are protected against insulin resistance. These beneficial metabolic effects of rapamycin were associated to significant changes of the inflammatory profiles of both adipose tissue and liver. Importantly, immune cells with regulatory functions such as regulatory T-cells (Tregs) and myeloid-derived suppressor cells (MDSCs) were increased in adipose tissue. These rapamycin-triggered metabolic and immune effects resulted from mTORC1 inhibition whilst mTORC2 activity was intact. Taken together, our results reinforce the notion that controlling immune regulatory cells in metabolic tissues is crucial to maintain a proper metabolic status and, more generally, comfort the need to search for novel pharmacological inhibitors of the mTOR signaling pathway to prevent and/or treat metabolic diseases.

A structural explanation of the effects of dissociated glucocorticoids on glucocorticoid receptor transactivation.

There is a therapeutic need for glucocorticoid receptor (GR) ligands that distinguish between the transrepression and transactivation activity of the GR, the later thought to be responsible for side effects. These ligands are known as "dissociated glucocorticoids" (dGCs). The first published dGCs, RU24782 (9α-fluoro-11ß-hydroxy-16α-methylpregna-21-thiomethyl-1,4-diene-3,20-dione) and RU24858 (9α-fluoro-11ß-hydroxy-16α-methylpregna-21-cyanide-1,4-diene-3,20-dione), do not have the 17α-hydroxyl group that characterizes dexamethasone (Dex; 9α-fluoro-11ß,17α,21-trihydroxy-16α-methylpregna-1,4-diene-3,20-dione), and they differ from one another by having C21-thiomethyl and C21-cyanide moieties, respectively. Our aim was therefore to establish the structural basis of their activity. Both RU24782 and RU24858 induced a transactivation activity highly dependent on the GR expression level but always lower than dexamethasone. They also display less ability than dexamethasone to trigger steroid receptor coactivator 1 (SRC-1) recruitment and histone H3 acetylation. Docking studies, validated by mutagenesis experiments, revealed that dGCs are not anchored by Gln642, in contrast to Dex, which is hydrogen bonded to this residue via its 17α-hydroxyl group. This contact is essential for SRC-1 recruitment and subsequent dexamethasone-induced GR transactivation, but not transrepression. The ability of dGCs to make contacts with Ile747, for both RU24858 and RU24782 and with Asn564 for RU24858 are not strong enough to maintain GR in a conformation able to efficiently recruit SRC-1, unless SRC-1 is overexpressed. Overall, our findings provide some structural guidelines for the synthesis of potential new dissociated glucocorticoids with a better therapeutic ratio.

A novel murine model of rhinoscleroma identifies Mikulicz cells, the disease signature, as IL-10 dependent derivatives of inflammatory monocytes.

Rhinoscleroma is a human specific chronic disease characterized by the formation of granuloma in the airways, caused by the bacterium Klebsiella pneumoniae subspecies rhinoscleromatis, a species very closely related to K. pneumoniae subspecies pneumoniae. It is characterized by the appearance of specific foamy macrophages called Mikulicz cells. However, very little is known about the pathophysiological processes underlying rhinoscleroma. Herein, we characterized a murine model recapitulating the formation of Mikulicz cells in lungs and identified them as atypical inflammatory monocytes specifically recruited from the bone marrow upon K. rhinoscleromatis infection in a CCR2-independent manner. While K. pneumoniae and K. rhinoscleromatis infections induced a classical inflammatory reaction, K. rhinoscleromatis infection was characterized by a strong production of IL-10 concomitant to the appearance of Mikulicz cells. Strikingly, in the absence of IL-10, very few Mikulicz cells were observed, confirming a crucial role of IL-10 in the establishment of a proper environment leading to the maturation of these atypical monocytes. This is the first characterization of the environment leading to Mikulicz cells maturation and their identification as inflammatory monocytes.

Scavenger receptors in human airway epithelial cells: role in response to double-stranded RNA.

Scavenger receptors and Toll-like receptors (TLRs) cooperate in response to danger signals to adjust the host immune response. The TLR3 agonist double stranded (ds)RNA is an efficient activator of innate signalling in bronchial epithelial cells. In this study, we aimed at defining the role played by scavenger receptors expressed by bronchial epithelial cells in the control of the innate response to dsRNA both in vitro and in vivo. Expression of several scavenger receptor involved in pathogen recognition was first evaluated in human bronchial epithelial cells in steady-state and inflammatory conditions. Their implication in the uptake of dsRNA and the subsequent cell activation was evaluated in vitro by competition with ligand of scavenger receptors including maleylated ovalbumin and by RNA silencing. The capacity of maleylated ovalbumin to modulate lung inflammation induced by dsRNA was also investigated in mice. Exposure to tumor necrosis factor-α increased expression of the scavenger receptors LOX-1 and CXCL16 and the capacity to internalize maleylated ovalbumin, whereas activation by TLR ligands did not. In contrast, the expression of SR-B1 was not modulated in these conditions. Interestingly, supplementation with maleylated ovalbumin limited dsRNA uptake and inhibited subsequent activation of bronchial epithelial cells. RNA silencing of LOX-1 and SR-B1 strongly blocked the dsRNA-induced cytokine production. Finally, administration of maleylated ovalbumin in mice inhibited the dsRNA-induced infiltration and activation of inflammatory cells in bronchoalveolar spaces and lung draining lymph nodes. Together, our data characterize the function of SR-B1 and LOX-1 in bronchial epithelial cells and their implication in dsRNA-induced responses, a finding that might be relevant during respiratory viral infections.

Interleukin-7 regulates adipose tissue mass and insulin sensitivity in high-fat diet-fed mice through lymphocyte-dependent and independent mechanisms.

Although interleukin (IL)-7 is mostly known as a key regulator of lymphocyte homeostasis, we recently demonstrated that it also contributes to body weight regulation through a hypothalamic control. Previous studies have shown that IL-7 is produced by the human obese white adipose tissue (WAT) yet its potential role on WAT development and function in obesity remains unknown. Here, we first show that transgenic mice overexpressing IL-7 have reduced adipose tissue mass associated with glucose and insulin resistance. Moreover, in the high-fat diet (HFD)-induced obesity model, a single administration of IL-7 to C57BL/6 mice is sufficient to prevent HFD-induced WAT mass increase and glucose intolerance. This metabolic protective effect is accompanied by a significant decreased inflammation in WAT. In lymphocyte-deficient HFD-fed SCID mice, IL-7 injection still protects from WAT mass gain. However, IL-7-triggered resistance against WAT inflammation and glucose intolerance is lost in SCID mice. These results suggest that IL-7 regulates adipose tissue mass through a lymphocyte-independent mechanism while its protective role on glucose homeostasis would be relayed by immune cells that participate to WAT inflammation. Our observations establish a key role for IL-7 in the complex mechanisms by which immune mediators modulate metabolic functions.

Tryptophan metabolism activation by indoleamine 2,3-dioxygenase in adipose tissue of obese women: an attempt to maintain immune homeostasis and vascular tone.

Human obesity is characterized by chronic low-grade inflammation in white adipose tissue and is often associated with hypertension. The potential induction of indoleamine 2,3-dioxygenase-1 (IDO1), the rate-limiting enzyme in tryptophan/kynurenine degradation pathway, by proinflammatory cytokines, could be associated with these disorders but has remained unexplored in obesity. Using immunohistochemistry, we detected IDO1 expression in white adipose tissue of obese patients, and we focused on its contribution in the regulation of vascular tone and on its immunoregulatory effects. Concentrations of tryptophan and kynurenine were measured in sera of 36 obese and 15 lean women. The expression of IDO1 in corresponding omental and subcutaneous adipose tissues and liver was evaluated. Proinflammatory markers and T-cell subsets were analyzed in adipose tissue via the expression of CD14, IL-18, CD68, TNFα, CD3ε, FOXP3 [a regulatory T-cell (Treg) marker] and RORC (a Th17 marker). In obese subjects, the ratio of kynurenine to tryptophan, which reflects IDO1 activation, is higher than in lean subjects. Furthermore, IDO1 expression in both adipose tissues and liver is increased and is inversely correlated with arterial blood pressure. Inflammation is associated with a T-cell infiltration in obese adipose tissue, with predominance of Th17 in the omental compartment and of Treg in the subcutaneous depot. The Th17/Treg balance is decreased in subcutaneous fat and correlates with IDO1 activation. In contrast, in the omental compartment, despite IDO1 activation, the Th17/Treg balance control is impaired. Taken together, our results suggest that IDO1 activation represents a local compensatory mechanism to limit obesity-induced inflammation and hypertension.

Implication of scavenger receptors in the interactions between diesel exhaust particles and immature or mature dendritic cells.

BACKGROUND: The exposure to pollutants such as diesel exhaust particles (DEP) is associated with an increased incidence of respiratory diseases. However, the mechanisms by which DEP have an effect on human health are not completely understood. In addition to their action on macrophages and airway epithelial cells, DEP also modulate the functions of dendritic cells (DC). These professional antigen-presenting cells are able to discriminate unmodified self from non-self thanks to pattern recognition receptors such as the Toll like Receptors (TLR) and Scavenger Receptors (SR). SR were originally identified by their ability to bind and internalize modified lipoproteins and microorganisms but also particles and TLR agonists. In this study, we assessed the implication of SR in the effects of DEP associated or not with TLR agonists on monocyte-derived DC (MDDC). For this, we studied the regulation of CD36, CXCL16, LOX-1, SR-A1 and SR-B1 expression on MDDC treated with DEP associated or not with TLR2, 3 and 4 ligands. Then, the capacity of SR ligands (dextran sulfate and maleylated-ovalbumin) to block the effects of DEP on the function of lipopolysaccharide (LPS)-activated DC has been evaluated. RESULTS: Our data demonstrate that TLR2 agonists mainly augmented CXCL16, LOX-1 and SR-B1 expression whereas DEP alone had only a weak effect. Interestingly, DEP modulated the action of TLR2 and TLR4 ligands on the expression of LOX-1 and SR-B1. Pretreatment with the SR ligand maleylated-ovalbumin but not dextran sulfate inhibited the endocytosis of DEP by MDDC. Moreover, this SR ligand blocked the effect by DEP at low dose (1 mug/ml) on MDDC phenotype (a decrease of CD86 and HLA-DR expression) and on the secretion of CXCL10, IL-12 and TNF-alpha. In contrast, the decrease of IL-12 and CXCL10 secretion and the generation of oxygen metabolite induced by DEP at 10 mug/ml was not affected by SR ligands CONCLUSION: Our results show for the first time that the modulation of DC functions by DEP implicates SR. TLR agonists upregulated SR expression in contrast to DEP. Interfering with the expression and/or the function of SR might be one way to limit the impact of DEP on lung immune response.

Impact of bronchial epithelium on dendritic cell migration and function: modulation by the bacterial motif KpOmpA.

Mucosal immune response depends on the surveillance network established by dendritic cells (DC), APC localized within the epithelium. Bronchial epithelial cells (BEC) play a pivotal role both in the host defense and in the pathogenesis of inflammatory airway disorders. We previously showed that the outer membrane protein A from Klebsiella pneumoniae (KpOmpA), a pathogen-associated molecular pattern (PAMP) derived from Klebsiella pneumoniae, activates BEC. In this study, we evaluated the consequences of this activation on DC traffic and functions. KpOmpA significantly increased the production of CCL2, CCL5, CXCL10, and CCL20 by BEC. Stimulation of BEC increased their chemotactic activity for monocyte-derived DC (MDDC) precursors, through CCL5 and CXCL10 secretion. BEC/MDDC precursor coculture leads to an ICAM-1-dependent accelerated differentiation and enhanced maturation of MDDC. BEC/DC interactions did not affect the capacity of DC to induce T cell proliferation. However, DC preincubated with BEC increased significantly the IL-10 production by autologous T cells. Basolateral and intraepithelial DC differently enhance IL-4 and/or IL-10 synthesis according to the condition of stimulation. In vivo, intranasal injections of KpOmpA into BALB/c mice induced the recruitment of CD11c(+) and I-A(d+) myeloid DC associated with bronchial epithelium activation as evidenced by CCL20 expression. These data show that KpOmpA-exposed BEC participate in the homeostasis of myeloid DC network, and regulate the induction of local immune response.

Asthmatic bronchial epithelium activated by the proteolytic allergen Der p 1 increases selective dendritic cell recruitment.

BACKGROUND: Airway dendritic cells (DCs) are crucial for allergen-induced sensitization and inflammation in allergic asthma. After allergen challenge, an increased number of DCs is observed in airway epithelium from patients with allergy. OBJECTIVE: Because Der p 1, a cysteine protease allergen from Dermatophagoides pteronyssinus , induces chemokine production by bronchial epithelial cells (BECs), the purpose of this investigation was to evaluate the capacity of BEC exposed to Der p 1 to recruit DCs. METHODS: Chemotactic activity of BEAS-2B, a bronchial epithelial cell line, and BECs from nonatopic controls and patients with allergic asthma was evaluated on the migration of precursors, immature and mature monocyte-derived DCs (MDDCs), and CD34 + -derived Langerhans cells (LCs). RESULTS: C-C chemokine ligand (CCL)-2, CCL5, and C-X-C chemokine ligand 10 production by BEAS-2B and BEC was increased after Der p 1 exposure, whereas the proenzyme proDer p 1 devoid of enzymatic activity had no effect. Der p 1 stimulation of BEAS-2B and BEC from both groups increased significantly the recruitment of MDDC precursors, depending on CCL2, CCL5, and C-X-C chemokine ligand 10 production. In a reconstituted polarized epithelium, apical application of Der p 1 enhanced MDDC precursor migration into the epithelial layer. Moreover, Der p 1 stimulation of BEC from patients with asthma but not from controls increased the migration of LC precursors, mainly dependent on CCL20 secretion. No migration of immature and mature DCs was observed. CONCLUSION: These data confirmed that BECs participate in the homeostasis of the DC network present within the bronchial epithelium through the secretion of chemokines. In allergic asthma, upregulation of CCL20 production induced LC recruitment, the role of which remains to be determined.