Inhibiting AKT Phosphorylation Employing Non-Cytotoxic Anthraquinones Ameliorates TH2 Mediated Allergic Airways Disease and Rhinovirus Exacerbation

Background

Severe asthma is associated with T helper (T_H) 2 and 17 cell activation, airway neutrophilia and phosphoinositide-3-kinase (PI3K) activation. Asthma exacerbations are commonly caused by rhinovirus (RV) and also associated with PI3K-driven inflammation. Anthraquinone derivatives have been shown to reduce PI3K-mediated AKT phosphorylation in-vitro.

Objective

To determine the anti-inflammatory potential of anthraquinones in-vivo.

Methods

BALB/c mice were sensitized and challenged with crude house dust mite extract to induce allergic airways disease and treated with mitoxantrone and a novel non-cytotoxic anthraquinone derivative. Allergic mice were also infected with RV1B to induce an exacerbation.

Results

Anthraquinone treatment reduced AKT phosphorylation, hypoxia-inducible factor-1α and vascular endothelial growth factor expression, and ameliorated allergen- and RV-induced airways hyprereactivity, neutrophilic and eosinophilic inflammation, cytokine/chemokine expression, mucus hypersecretion, and expression of T_H2 proteins in the airways. Anthraquinones also boosted type 1 interferon responses and limited RV replication in the lung.

Conclusion

Non-cytotoxic anthraquinone derivatives may be of therapeutic benefit for the treatment of severe and RV-induced asthma by blocking pro-inflammatory pathways regulated by PI3K/AKT.     

TNFα-Mediated Liver Destruction by Kupffer Cells and Ly6Chi Monocytes during Entamoeba histolytica Infection

Amebic liver abscess (ALA) is a focal destruction of liver tissue due to infection by the protozoan parasite Entamoeba histolytica (E. histolytica). Host tissue damage is attributed mainly to parasite pathogenicity factors, but massive early accumulation of mononuclear cells, including neutrophils, inflammatory monocytes and macrophages, at the site of infection raises the question of whether these cells also contribute to tissue damage. Using highly selective depletion strategies and cell-specific knockout mice, the relative contribution of innate immune cell populations to liver destruction during amebic infection was investigated. Neutrophils were not required for amebic infection nor did they appear to be substantially involved in tissue damage. In contrast, Kupffer cells and inflammatory monocytes contributed substantially to liver destruction during ALA, and tissue damage was mediated primarily by TNFα. These data indicate that besides direct antiparasitic drugs, modulating innate immune responses may potentially be beneficial in limiting ALA pathogenesis.     

Long-term survival after adoptive bone marrow T cell therapy of advanced metastasized breast cancer: follow-up analysis of a clinical pilot trial

Background

The bone marrow (BM) of breast cancer patients harbors tumor-reactive memory T cells (TCs) with therapeutic potential. We recently described the immunologic effects of adoptive transfer of ex vivo restimulated tumor-reactive memory TCs from the BM of 12 metastasized breast cancer patients in a clinical phase-I study. In this trial, adoptive T cell transfer resulted in the occurrence of circulating tumor antigen-reactive type-1 TCs. We here describe the long-term clinical outcome and its correlation with tumor-specific cellular immune response in 16 metastasized breast cancer patients, including 12 included in the original study.

Methods

Sixteen metastatic breast cancer patients with preexisting tumor-reactive BM memory TCs were included into the study. The study protocol involved one transfusion of TCs which were reactivated in vitro with autologous dendritic cells pulsed with lysates of MCF-7 breast cancer cells as source of tumor antigens. The presence of tumor-reactive memory TCs was analyzed by IFN-γ ELISpot assays.

Results

Tumor-reactive memory TCs in the peripheral blood were induced de novo in 7/16 patients (44 %) after adoptive TC transfer. These patients were considered immunologic responders to the therapy. Positive adoptive immunotherapy (ADI) response was observed significantly more often in patients without bone metastases (p = 0.0051), in patients with high levels of tumor-reactive BM TCs prior to therapy (p = 0.036) and correlated significantly with the estimated numbers of transferred tumor-reactive TCs (p = 0.0021). After the treatment, we observed an overall median survival of 33.8 months in the total cohort with three patients alive at last follow-up and more than 7 years after ADI. Numbers of transferred tumor-reactive TCs correlated significantly with the overall survival of patients (p = 0.017). Patients with an immunologic response to ADI in the peripheral blood had a significantly longer median survival than nonresponders (median survival 58.6 vs. 13.6 months; p = 0.009).

Conclusion

In metastasized breast cancer patients, adoptive transfer of BM TCs can induce the presence of tumor antigen-reactive type-1 TCs in the peripheral blood. Patients with immunologic response after ADI show a significantly longer overall survival. Patients with bone metastases significantly less frequently respond to the treatment and, therefore, might not be optimal candidates for ADI. Although the present study does not yet prove the therapeutic effect of ADI, these findings shed light on the relation between immune response and cancer prognosis and suggest that transfer of reactivated BM TCs might bear therapeutic potential.     

Constitutively active rhodopsin mutants causing night blindness are effectively phosphorylated by GRKs but differ in arrestin-1 binding

The effects of activating mutations associated with night blindness on the stoichiometry of rhodopsin interactions with G protein-coupled receptor kinase 1 (GRK1) and arrestin-1 have not been reported. Here we show that the monomeric form of WT rhodopsin and its constitutively active mutants M257Y, G90D, and T94I, reconstituted into HDL particles are effectively phosphorylated by GRK1, as well as two more ubiquitously expressed subtypes, GRK2 and GRK5. All versions of arrestin-1 tested (WT, pre-activated, and constitutively monomeric mutants) bind to monomeric rhodopsin and show the same selectivity for different functional forms of rhodopsin as in native disc membranes. Rhodopsin phosphorylation by GRK1 and GRK2 promotes arrestin-1 binding to a comparable extent, whereas similar phosphorylation by GRK5 is less effective, suggesting that not all phosphorylation sites on rhodopsin are equivalent in promoting arrestin-1 binding. The binding of WT arrestin-1 to phospho-opsin is comparable to the binding to its preferred target, P-Rh*, suggesting that in photoreceptors arrestin-1 only dissociates after opsin regeneration with 11-cis-retinal, which converts phospho-opsin into inactive phospho-rhodopsin that has lower affinity for arrestin-1. Reduced binding of arrestin-1 to the phospho-opsin form of G90D mutant likely contributes to night blindness caused by this mutation in humans.     

Structural role of the T94I rhodopsin mutation in congenital stationary night blindness

Congenital stationary night blindness (CSNB) is an inherited and non‐progressive retinal dysfunction. Here, we present the crystal structure of CSNB‐causing T94I^2.61 rhodopsin in the active conformation at 2.3 Å resolution. The introduced hydrophobic side chain prolongs the lifetime of the G protein activating metarhodopsin‐II state by establishing a direct van der Waals contact with K296^7.43, the site of retinal attachment. This is in stark contrast to the light‐activated state of the CSNB‐causing G90D^2.57 mutation, where the charged mutation forms a salt bridge with K296^7.43. To find the common denominator between these two functional modifications, we combined our structural data with a kinetic biochemical analysis and molecular dynamics simulations. Our results indicate that both the charged G90D^2.57 and the hydrophobic T94I^2.61 mutation alter the dark state by weakening the interaction between the Schiff base (SB) and its counterion E113^3.28. We propose that this interference with the tight regulation of the dim light photoreceptor rhodopsin increases background noise in the visual system and causes the loss of night vision characteristic for CSNB patients.     

Effect of long-term organic and mineral fertilization strategies on rhizosphere microbiota assemblage and performance of lettuce

Long-term agricultural fertilization strategies gradually change soil properties including the associated microbial communities. Cultivated crops recruit beneficial microbes from the surrounding soil environment via root exudates. In this study, we aimed to investigate the effects of long-term fertilization strategies across field sites on the rhizosphere prokaryotic (Bacteria and Archaea) community composition and plant performance. We conducted growth chamber experiments with lettuce (Lactuca sativa L.) cultivated in soils from two long-term field experiments, each of which compared organic versus mineral fertilization strategies. 16S rRNA gene amplicon sequencing revealed the assemblage of a rhizosphere core microbiota shared in all lettuce plants across soils, going beyond differences in community composition depending on field site and fertilization strategies. The enhanced expression of several plant genes with roles in oxidative and biotic stress signalling pathways in lettuce grown in soils with organic indicates an induced physiological status in plants. Lettuce plants grown in soils with different fertilization histories were visibly free of stress symptoms and achieved comparable biomass. This suggests a positive aboveground plant response to belowground plant–microbe interactions in the rhizosphere. Besides effects of fertilization strategy and field site, our results demonstrate the crucial role of the plant in driving rhizosphere microbiota assemblage.