Galectin-1 Cooperates with Yersinia Outer Protein (Yop) P to Thwart Protective Immunity by Repressing Nitric Oxide Production.

Yersinia enterocolitica (Ye) inserts outer proteins (Yops) into cytoplasm to infect host cells. However, in spite of considerable progress, the mechanisms implicated in this process, including the association of Yops with host proteins, remain unclear. Here, we evaluated the functional role of Galectin-1 (Gal1), an endogenous ß-galactoside-binding protein, in modulating Yop interactions with host cells. Our results showed that Gal1 binds to Yops in a carbohydrate-dependent manner. Interestingly, Gal1 binding to Yops protects these virulence factors from trypsin digestion. Given that early control of Ye infection involves activation of macrophages, we evaluated the role of Gal1 and YopP in the modulation of macrophage function. Although Gal1 and YopP did not influence production of superoxide anion and/or TNF by Ye-infected macrophages, they coordinately inhibited nitric oxide (NO) production. Notably, recombinant Gal1 (rGal1) did not rescue NO increase observed in Lgals1-/- macrophages infected with the YopP mutant Ye ∆yopP. Whereas NO induced apoptosis in macrophages, no significant differences in cell death were detected between Gal1-deficient macrophages infected with Ye ∆yopP, and WT macrophages infected with Ye wt. Strikingly, increased NO production was found in WT macrophages treated with MAPK inhibitors and infected with Ye wt. Finally, rGal1 administration did not reverse the protective effect in Peyer Patches (PPs) of Lgals1-/- mice infected with Ye ∆yopP. Our study reveals a cooperative role of YopP and endogenous Gal1 during Ye infection.

Galectin-1 Facilitates Macrophage Reprogramming and Resolution of Inflammation Through IFN-ß.

During the resolution of acute inflammation, macrophages undergo reprogramming from pro-inflammatory, to anti-inflammatory/reparative, and eventually to pro-resolving macrophages. Galectin-1 (Gal-1) is a bona fide pro-resolving lectin while interferon ß (IFN-ß) was recently shown to facilitate macrophage reprogramming and resolution of inflammation. In this study, we found Gal-1null mice exhibit a hyperinflammatory phenotype during the resolution of zymosan A-induced peritonitis but not during the early inflammatory response. This phenotype was characterized by reduced macrophage numbers, increased secretion of pro-inflammatory cytokines, such as interleukin-12 (IL-12), and reduced secretion of anti-inflammatory cytokines, such as interleukin-10 (IL-10). In addition, we found a delayed expression of the pro-resolving enzyme 12/15-lipoxygenase in macrophages and heightened levels of the inflammatory protease proteinase-3 (PR3) in peritoneal fluids from Gal-1null mice. Moreover, we observed sex-dependent differences in the inflammatory profile of Gal-1null mice. Notably, we found that IFN-ß levels were reduced in resolution-phase exudates from Gal-1null mice. Administration of IFN-ß in vivo or ex vivo treatment was able to rescue, at least in part, the hyperinflammatory profile of Gal-1null mice. In particular, IFN-ß recovered a subset of F4/80+GR-1+ macrophages, restored IL-12 and IL-10 secretion from macrophages to WT values and diminished abnormal peritoneal PR3 levels in Gal-1null mice. In conclusion, our results revealed a new Gal-1-IFN-ß axis that facilitates the resolution of inflammation and might restrain uncontrolled inflammatory disorders.

Efficient plasmid-mediated gene transfection of ovine bone marrow mesenchymal stromal cells.

BACKGROUND AIMS: Given the close similarity between ovine and human cardiomyocytes, sheep models of myocardial infarction and heart failure are increasingly used in studies of stem cell-mediated heart regeneration. In these studies, mesenchymal stromal cells (MSCs) are frequently employed. To enhance the paracrine effects of these MSCs, ex vivo transfection with genes encoding growth factors has been proposed. Although viral vectors exhibit higher transfection efficiency than plasmids, they entail the risks of uncontrolled transgene expression and immune reactions that preclude repeated administration. Our aim was to optimize the efficiency of plasmid-mediated transfection of ovine MSCs, while preserving cell viability. METHODS: Varying amounts of diverse cationic lipids were used to obtain the reagent-to-DNA mass ratio showing highest luciferase activity. Transfection efficiency (flow cytometry) was tested on plasmid-green fluorescent protein-transfected MSCs at increasing DNA mass. RESULTS: Lipofectamine LTX 5 µL and Plus reagent 4 µL with 2 µg of DNA yielded 42.3 ± 4.7% transfection efficiency, while preserving cell viability. Using these transfection conditions, we transfected MSCs with a plasmid encoding human vascular endothelial growth factor (VEGF) and found high VEGF protein concentrations in the culture supernatant from day 2 (1968 ± 324 pg/mL per µg DNA) through at least day 12 (888 ± 386 pg/mL per µg DNA) after transfection. CONCLUSIONS: Plasmid-mediated transfection of ovine MSCs to over-express paracrine heart-regenerative growth factors is feasible and efficient and overcomes the risks and limitations associated with the use of viral vectors.