María Teresa Vallejo-Cremades

Metalloproteinases Shed TREM-1 Ectodomain from Lipopolysaccharide-Stimulated Human Monocytes

Triggering receptors expressed on myeloid cell (TREM) proteins are a family of cell surface receptors that participate in diverse cellular processes such as inflammation, coagulation, and bone homeostasis. TREM-1, in particular, is expressed on neutrophils and monocytes and is a potent amplifier of inflammatory responses. LPS and other microbial products induce up-regulation of cell surface-localized TREM-1 and the release of its soluble form, sTREM-1. Two hypotheses have been advanced to explain the origin of sTREM-1: alternative splicing of TREM-1 mRNA and proteolytic cleavage(s) of mature, membrane-anchored TREM-1. In this report, we present conclusive evidence in favor of the proteolytic mechanism of sTREM-1 generation. No alternative splicing forms of TREM-1 were detected in monocytes/macrophages. Besides, metalloproteinase inhibitors increased the stability of TREM-1 at the cell surface while significantly reducing sTREM-1 release in cultures of LPS-challenged human monocytes and neutrophils. We conclude that metalloproteinases are responsible for shedding of the TREM-1 ectodomain through proteolytic cleavage of its long juxtamembrane linker.

Enriched protein diet-modified ghrelin expression and secretion in rats

Gastrointestinal (GI) integrity and function are regulated by nutrition and growth factors. The discovery of ghrelin, a natural growth hormone (GH) secretagogue produced by the gastrointestinal (GI) tract, is a potential link between diet and growth signals. The aim of this study was to evaluate macronutrient effect on ghrelin expression and secretion in addition to some possible function in intestinal trophic status. Wistar rats were fed a high-carbohydrate, high-protein (HP), high-fat or standard (St) diet. Animals received the same daily food volume and caloric intake. After 7 days, animals were fasted for 24 h and blood and tissue samples were obtained just before feeding or at 2 or 6 h after feeding. Fasting high-protein-fed rats had higher ghrelin plasma levels than with rats fed the high-carbohydrate, high-fat or standard diets. Two-hours after refeeding, ghrelin plasma levels had decreased in all groups with a slight recovery at 6 h after refeeding, except in the high-protein group. Ghrelin plasma levels in rats fed with the high-protein diet correlated negatively with their GH and insulin-like growth factor 1 (IGF-1) plasma concentrations which were also the lowest among the study groups. In conclusion, ghrelin secretion was nutritionally manipulated because a protein-enriched diet increased its levels.

6-Methylprednisolone down-regulates IRAK-M in human and murine osteoclasts and boosts bone-resorbing activity: a putative mechanism for corticoid-induced osteoporosis

Osteoclasts are large, multinucleated cells, which originate from the fusion of macrophages. They play a central role in bone development and remodeling via the resorption of bone and are thus important mediators of bone loss, which leads to osteoporosis. IL‐1R‐associated kinase (IRAK)‐M is a pseudokinase, which acts as a negative modulator of innate immune responses mediated by TLRs and IL‐1R. Recently, it has been reported that IRAK‐M also participates in the control of macrophage differentiation into osteoclasts. In addition, it was shown that IRAK‐M knockout mice develop a strong osteoporosis phenotype, suggesting that down‐regulation of this molecule activates osteoclast‐mediated bone resorption. We studied the effect of the osteoporosis‐inducing glucocorticoid, 6‐methylprednisolone (6‐MP), on IRAK‐M expression in osteoclasts. Our results showed that osteoclasts, derived from THP‐1 and RAW cells as well as human blood monocytes, differentiated into osteoclasts, express high levels of IRAK‐M at mRNA and protein levels. In addition, 6‐MP down‐regulates IRAK‐M expression, which correlates with an increased activation of bone resorption. These findings suggest a mechanism of corticosteroid‐induced osteoporosis and open new avenues for treating this endemic disease of Western societies.

Mitochondrial DAMPs Induce Endotoxin Tolerance in Human Monocytes: An Observation in Patients with Myocardial Infarction

Monocyte exposure to mitochondrial Danger Associated Molecular Patterns (DAMPs), including mitochondrial DNA (mtDNA), induces a transient state in which these cells are refractory to further endotoxin stimulation. In this context, IRAK-M up-regulation and impaired p65 activity were observed. This phenomenon, termed endotoxin tolerance (ET), is characterized by decreased production of cytokines in response to the pro-inflammatory stimulus. We also show that monocytes isolated from patients with myocardial infarction (MI) exhibited high levels of circulating mtDNA, which correlated with ET status. Moreover, a significant incidence of infection was observed in those patients with a strong tolerant phenotype. The present data extend our current understanding of the implications of endotoxin tolerance. Furthermore, our data suggest that the levels of mitochondrial antigens in plasma, such as plasma mtDNA, should be useful as a marker of increased risk of susceptibility to nosocomial infections in MI and in other pathologies involving tissue damage.

Exogenous ghrelin regulates proliferation and apoptosis in the hypotrophic gut mucosa of the rat

Ghrelin is the natural endogenous ligand for growth hormone secretagogue receptors. This peptide regulates energy homeostasis and expenditure and is a potential link between gut absorptive function and growth. We hypothesized that ghrelin may induce a proliferative and antiapoptotic action promoting the recovery of the hypotrophic gut mucosa. Therefore, the aim of the study was to determine the action of exogenous ghrelin following gut mucosal hypotrophia in rats fed an elemental diet. An elemental diet provides readily absorbable simple nutrients and is usually given to patients with absorptive dysfunction. Male Wistar rats (n = 48) were fed the elemental diet for one week to induce mucosal hypotrophy and then treated for another week with systemic ghrelin and pair-fed with either a normoproteic or hyperproteic isocaloric liquid diet. Another group received a standard diet instead of the elemental diet and served as control (normotrophy). The elemental diet induced intestinal hypotrophia characterized by decreased proliferation in the ileum and increased apoptosis in jejunum and ileum. Ghrelin administration restored normal levels of proliferation in the ileum and apoptosis in the jejunum, with partial apoptosis restoration in the ileum. Ghrelin levels in plasma and fundus were increased in all groups, although the highest levels were found in rats treated with exogenous ghrelin. Ghrelin administration has a positive effect in the hypotrophic gut, regulating both proliferation and apoptosis towards a physiological balance counteracting the negative changes induced by an elemental diet in the intestines.

Differential action of growth hormone in irradiated tumoral and nontumoral intestinal tissue.

Growth hormone (GH) protects the intestines from antitumoral therapy, but it is not known whether or not the tumor is also protected in vivo. The aim of the present work was to determine whether GH administration modifies the response by a colonic adenocarcinoma to radiation in vitro and in vivo. BDIX rats were implanted with a colonic adenocarcinoma and two weeks later GH treatment was started. Animals were then irradiated, and four days later samples from the intestines and tumor were taken for analysis. In vitro assays were performed in parallel to confirm the effects observed in vivo. GH reduced radiation-induced intestinal injury by improving proliferation and reducing apoptosis and p53 expression. However, tumor proliferation was reduced by GH while apoptosis and p53 expression remained unchanged. A similar response was observed in vitro. Thus, GH administration before radiotherapy protects the intestines but not the implanted adenocarcinoma in the rat.

NOD1 activation in cardiac fibroblasts induces myocardial fibrosis in a murine model of type 2 diabetes

Cardiac fibrosis and chronic inflammation are common complications in type 2 diabetes mellitus (T2D). Since nucleotide oligomerization-binding domain 1 (NOD1), an innate immune receptor, is involved in the pathogenesis of insulin resistance and diabetes outcomes, we sought to investigate its involvement in cardiac fibrosis. Here, we show that selective staining of cardiac fibroblasts from T2D (db/db;db) mice exhibits up-regulation and activation of the NOD1 pathway, resulting in enhanced NF-κB and TGF-β signalling. Activation of the TGF-β pathway in cardiac fibroblasts from db mice was prevented after inhibition of NF-κB with BAY-11-7082 (BAY). Moreover, fibrosis progression in db mice was also prevented by BAY treatment. Enhanced TGF-β signalling and cardiac fibrosis of db mice was dependent, at least in part, on the sequential activation of NOD1 and NF-κB since treatment of db mice with a selective NOD1 agonist induced activation of the TGF-β pathway, but co-administration of a NOD1 agonist plus BAY, or a NOD1 inhibitor prevented the NOD1-induced fibrosis. Therefore, NOD1 is involved in cardiac fibrosis associated with diabetes, and establishes a new mechanism for the development of heart fibrosis linked to T2D.

Synergistic activity of deguelin and fludarabine in cells from chronic lymphocytic leukemia patients and in the New Zealand Black murine model

B-cell chronic lymphocytic leukemia (CLL) remains an incurable disease, and despite the improvement achieved by therapeutic regimes developed over the last years still a subset of patients face a rather poor prognosis and will eventually relapse and become refractory to therapy. The natural rotenoid deguelin has been shown to induce apoptosis in several cancer cells and cell lines, including primary human CLL cells, and to act as a chemopreventive agent in animal models of induced carcinogenesis. In this work, we show that deguelin induces apoptosis in vitro in primary human CLL cells and in CLL-like cells from the New Zealand Black (NZB) mouse strain. In both of them, deguelin dowregulates AKT, NFκB and several downstream antiapoptotic proteins (XIAP, cIAP, BCL2, BCL-XL and survivin), activating the mitochondrial pathway of apoptosis. Moreover, deguelin inhibits stromal cell-mediated c-Myc upregulation and resistance to fludarabine, increasing fludarabine induced DNA damage. We further show that deguelin has activity in vivo against NZB CLL-like cells in an experimental model of CLL in young NZB mice transplanted with spleen cells from aged NZB mice with lymphoproliferation. Moreover, the combination of deguelin and fludarabine in this model prolonged the survival of transplanted mice at doses of both compounds that were ineffective when administered individually. These results suggest deguelin could have potential for the treatment of human CLL.

Protective role of hepatocyte cyclooxygenase-2 expression against liver ischemia-reperfusion injury in mice

Liver ischemia and reperfusion injury (IRI) remains a serious clinical problem affecting liver transplantation outcomes. IRI causes up to 10% of early organ failure and predisposes to chronic rejection. Cyclooxygenase‐2 (COX‐2) is involved in different liver diseases, but the significance of COX‐2 in IRI is a matter of controversy. This study was designed to elucidate the role of COX‐2 induction in hepatocytes against liver IRI. In the present work, hepatocyte‐specific COX‐2 transgenic mice (hCOX‐2‐Tg) and their wild‐type (Wt) littermates were subjected to IRI. hCOX‐2‐Tg mice exhibited lower grades of necrosis and inflammation than Wt mice, in part by reduced hepatic recruitment and infiltration of neutrophils, with a concomitant decrease in serum levels of proinflammatory cytokines. Moreover, hCOX‐2‐Tg mice showed a significant attenuation of the IRI‐induced increase in oxidative stress and hepatic apoptosis, an increase in autophagic flux, and a decrease in endoplasmic reticulum stress compared to Wt mice. Interestingly, ischemic preconditioning of Wt mice resembles the beneficial effects observed in hCOX‐2‐Tg mice against IRI due to a preconditioning‐derived increase in endogenous COX‐2, which is mainly localized in hepatocytes. Furthermore, measurement of prostaglandin E2 (PGE2) levels in plasma from patients who underwent liver transplantation revealed a significantly positive correlation of PGE2 levels and graft function and an inverse correlation with the time of ischemia. Conclusion: These data support the view of a protective effect of hepatic COX‐2 induction and the consequent rise of derived prostaglandins against IRI.

Actin polymerisation after FCγR stimulation of human fibroblasts is BCL10 independent.

B-cell lymphoma 10 (BCL10) is not essential for actin polymerisation after FcγR stimulation in human fibroblasts.