Promoting Pollinating Insects in Intensive Agricultural Matrices: Field-Scale Experimental Manipulation of Hay-Meadow Mowing Regimes and Its Effects on Bees

Bees are a key component of biodiversity as they ensure a crucial ecosystem service: pollination. This ecosystem service is nowadays threatened, because bees suffer from agricultural intensification. Yet, bees rarely benefit from the measures established to promote biodiversity in farmland, such as agri-environment schemes (AES). We experimentally tested if the spatio-temporal modification of mowing regimes within extensively managed hay meadows, a widespread AES, can promote bees. We applied a randomized block design, replicated 12 times across the Swiss lowlands, that consisted of three different mowing treatments: 1) first cut not before 15 June (conventional regime for meadows within Swiss AES); 2) first cut not before 15 June, as treatment 1 but with 15% of area left uncut serving as a refuge; 3) first cut not before 15 July. Bees were collected with pan traps, twice during the vegetation season (before and after mowing). Wild bee abundance and species richness significantly increased in meadows where uncut refuges were left, in comparison to meadows without refuges: there was both an immediate (within year) and cumulative (from one year to the following) positive effect of the uncut refuge treatment. An immediate positive effect of delayed mowing was also evidenced in both wild bees and honey bees. Conventional AES could easily accommodate such a simple management prescription that promotes farmland biodiversity and is likely to enhance pollination services.

The effect of extracellular nucleotides on cytokine production and phagocytosis of macrophages

Bacterial sepsis is a severe clinical condition, leading to severe sepsis, septic shock, and death. The complex pathophysiology of sepsis is not yet fully understood. Cytokines, released by immune cells such as macrophages, play an important role in the pathophysiology of sepsis. Kupffer cells are the largest population of macrophages in the body. Purinergic signaling, mediated by different nucleosides and nucleotides, and purinergic receptors, has been shown to have various effects on cytokine release, inflammatory processes and the immune system. In our work with in vitro experiments we studied the effect of extracellular nucleotides on the release of TNFα by primary murine Kupffer cells, and the effect of extracellular nucleotides on the phagocytosis of murine RAW 264.7 and human U-937 cell culture macrophages. Secretion of TNFα was measured using ELISA, phagocytosis of bio particles was measured using a plate reader phagocytosis assay and flow cytometry. Our experiments show, that extracellular LPS stimulate release of TNFα in murine Kupffer cells and that extracellular nucleotides inhibit this effect in a dose dependent matter. Our other experiments show phagocytosis of fluorescence labeled bio particles by both macrophage cell lines RAW 264.7 and U-937 in a dose dependent manner. The experiments could not show an effect of extracellular nucleotides on phagocytosis of cell culture macrophages.

Toxicity of eosinophil MBP is repressed by intracellular crystallization and promoted by extracellular aggregation.

Eosinophils are white blood cells that function in innate immunity and participate in the pathogenesis of various inflammatory and neoplastic disorders. Their secretory granules contain four cytotoxic proteins, including the eosinophil major basic protein (MBP-1). How MBP-1 toxicity is controlled within the eosinophil itself and activated upon extracellular release is unknown. Here we show how intragranular MBP-1 nanocrystals restrain toxicity, enabling its safe storage, and characterize them with an X-ray-free electron laser. Following eosinophil activation, MBP-1 toxicity is triggered by granule acidification, followed by extracellular aggregation, which mediates the damage to pathogens and host cells. Larger non-toxic amyloid plaques are also present in tissues of eosinophilic patients in a feedback mechanism that likely limits tissue damage under pathological conditions of MBP-1 oversecretion. Our results suggest that MBP-1 aggregation is important for innate immunity and immunopathology mediated by eosinophils and clarify how its polymorphic self-association pathways regulate toxicity intra- and extracellularly.

Active eosinophilic esophagitis is characterized by epithelial barrier defects and eosinophil extracellular trap formation.

BACKGROUND Eosinophilic esophagitis (EoE) exhibits esophageal dysfunction owing to an eosinophil-predominant inflammation. Activated eosinophils generate eosinophil extracellular traps (EETs) able to kill bacteria. There is evidence of an impaired barrier function in EoE that might allow pathogens to invade the esophagus. This study aimed to investigate the presence and distribution of EETs in esophageal tissues from EoE patients and their association with possible epithelial barrier defects. METHODS Anonymized tissue samples from 18 patients with active EoE were analyzed. The presence of DNA nets associated with eosinophil granule proteins forming EETs and the expression of filaggrin, the protease inhibitor lympho-epithelial Kazal-type-related inhibitor (LEKTI), antimicrobial peptides, and cytokines were evaluated by confocal microscopy following immune fluorescence staining techniques. RESULTS Eosinophil extracellular trap formation occurred frequently and was detected in all EoE samples correlating with the numbers of infiltrating eosinophils. While the expression of both filaggrin and LEKTI was reduced, epithelial antimicrobial peptides (human beta-defensin-2, human beta-defensin-3, cathelicidin LL-37, psoriasin) and cytokines (TSLP, IL-25, IL-32, IL-33) were elevated in EoE as compared to normal esophageal tissues. There was a significant correlation between EET formation and TSLP expression (P = 0.02) as well as psoriasin expression (P = 0.016). On the other hand, a significant negative correlation was found between EET formation and LEKTI expression (P = 0.016). CONCLUSION Active EoE exhibits the presence of EETs. Indications of epithelial barrier defects in association with epithelial cytokines are also present which may have contributed to the activation of eosinophils. The formation of EETs could serve as a firewall against the invasion of pathogens.

Basophils exhibit antibacterial activity through extracellular trap formation.

Basophils are primarily associated with immunomodulatory functions in allergic diseases and parasitic infections. Recently, it has been demonstrated that both activated human and mouse basophils can form extracellular DNA traps (BETs) containing mitochondrial DNA and granule proteins. In this report, we provide evidence that, in spite of an apparent lack of phagocytic activity, basophils can kill bacteria through BET formation.

Extracellular Iron is a Modulator of the Differentiation of Osteoclast Lineage Cells.

Osteoclasts originate from the hematopoietic stem cell and share a differentiation pathway with the cells of the monocyte/macrophage lineages. Development and activation of osteoclasts, and as a consequence regulation of bone resorption, depend on two growth factors: macrophage colony-stimulating factor and receptor activator of NF-κB ligand. Furthermore, cell development and activity are modulated by a microenvironment composed of cytokines and growth factors and of the extracellular matrix. Membrane transporters are a means for cells to interact with their environment. Within this study, the expression of proteins regulating cellular iron homeostasis in osteoclast-like cells grown from bone marrow-derived progenitors was compared to the expression of this set of proteins by monocyte/macrophage lineage cells. In differentiating osteoclasts, levels of transcripts encoding transferrin receptor 1 and divalent metal transporter 1 (Slc11A2) were increased, while levels of transcripts encoding ferroportin (Slc40A1) and natural resistance-associated macrophage protein 1 (Slc11A1) were decreased. Supplementation of the culture media with exogenous iron led to an increase in the proliferation of osteoclast progenitor cells and to the expression of a macrophage-like phenotype, while the development of osteoclasts was reduced. Upon transfer of mature OC onto a CaP substrate, iron depletion of the medium with the Fe(3+)-chelator Deferoxamine Mesylate decreased CaP dissolution by ~30 %, which could be restored by addition of exogenous iron. During the 24 h of the assay, no effects were observed on total TRAP activity. The data demonstrate transcriptional regulation of the components of cellular iron transporters during OC development and suggests that iron homeostasis may contribute to fine-tuning of the RANKL-induced OC development.

Two distinct filopodia populations at the growth cone allow to sense nanotopographical extracellular matrix cues to guide neurite outgrowth

BACKGROUND The process of neurite outgrowth is the initial step in producing the neuronal processes that wire the brain. Current models about neurite outgrowth have been derived from classic two-dimensional (2D) cell culture systems, which do not recapitulate the topographical cues that are present in the extracellular matrix (ECM) in vivo. Here, we explore how ECM nanotopography influences neurite outgrowth. METHODOLOGY/PRINCIPAL FINDINGS We show that, when the ECM protein laminin is presented on a line pattern with nanometric size features, it leads to orientation of neurite outgrowth along the line pattern. This is also coupled with a robust increase in neurite length. The sensing mechanism that allows neurite orientation occurs through a highly stereotypical growth cone behavior involving two filopodia populations. Non-aligned filopodia on the distal part of the growth cone scan the pattern in a lateral back and forth motion and are highly unstable. Filopodia at the growth cone tip align with the line substrate, are stabilized by an F-actin rich cytoskeleton and enable steady neurite extension. This stabilization event most likely occurs by integration of signals emanating from non-aligned and aligned filopodia which sense different extent of adhesion surface on the line pattern. In contrast, on the 2D substrate only unstable filopodia are observed at the growth cone, leading to frequent neurite collapse events and less efficient outgrowth. CONCLUSIONS/SIGNIFICANCE We propose that a constant crosstalk between both filopodia populations allows stochastic sensing of nanotopographical ECM cues, leading to oriented and steady neurite outgrowth. Our work provides insight in how neuronal growth cones can sense geometric ECM cues. This has not been accessible previously using routine 2D culture systems.