Innate Immune Training with Bacterial Extracts Enhances Lung Macrophage Recruitment to Protect from Betacoronavirus Infection.

Training of the innate immune system with orally ingested bacterial extracts was demonstrated to have beneficial effects on infection clearance and disease outcome. The aim of our study was to identify cellular and molecular processes responsible for these immunological benefits. We used a murine coronavirus (MCoV) A59 mouse model treated with the immune activating bacterial extract Broncho-Vaxom (BV) OM-85. Tissue samples were analysed with qPCR, RNA sequencing, histology, and flow cytometry. After BV OM-85 treatment, interstitial macrophages accumulated in lung tissue leading to a faster response of type I interferon (IFN) signalling after MCoV infection resulting in overall lung tissue protection. Moreover, RNA sequencing showed that lung tissue from mice receiving BV OM-85 resembled an intermediate stage between healthy and viral infected lung tissue at day 4, indicating a faster return to normal tissue homoeostasis. The pharmacologic effect was mimicked by adoptively transferring naive lung macrophages into lungs from recipient mice before virus infection. The beneficial effect of BV OM-85 was abolished when inhibiting initial type I IFN signalling. Overall, our data suggest that BV OM-85 enhances lung macrophages allowing for a faster IFN response towards a viral challenge as part of the oral-induced innate immune system training.

Multi-Functional OCT Enables Longitudinal Study of Retinal Changes in a VLDLR Knockout Mouse Model.

We present a multi-functional optical coherence tomography (OCT) imaging approach to study retinal changes in the very-low-density-lipoprotein-receptor (VLDLR) knockout mouse model with a threefold contrast. In the retinas of VLDLR knockout mice spontaneous retinal-chorodoidal neovascularizations form, having an appearance similar to choroidal and retinal neovascularizations (CNV and RNV) in neovascular age-related macular degeneration (AMD) or retinal angiomatous proliferation (RAP). For this longitudinal study, the mice were imaged every 4 to 6 weeks starting with an age of 4 weeks and following up to the age of 11 months. Significant retinal changes were identified by the multi-functional imaging approach offering a threefold contrast: reflectivity, polarization sensitivity (PS) and motion contrast based OCT angiography (OCTA). By use of this intrinsic contrast, the long-term development of neovascularizations was studied and associated processes, such as the migration of melanin pigments or retinal-choroidal anastomosis, were assessed in vivo. Furthermore, the in vivo imaging results were validated with histological sections at the endpoint of the experiment. Multi-functional OCT proves as a powerful tool for longitudinal retinal studies in preclinical research of ophthalmic diseases. Intrinsic contrast offered by the functional extensions of OCT might help to describe regulative processes in genetic animal models and potentially deepen the understanding of the pathogenesis of retinal diseases such as wet AMD.

The relevance of oxidative stress and cytotoxic DNA lesions for spontaneous mutagenesis in non-replicating yeast cells.

Mutations arising during times of cell cycle-arrest may considerably contribute to aging and cancerogenesis. Endogenous oxidative stress could be one of the major triggers for these mutations. We used Saccharomyces cerevisiae cells, arrested by starvation for the essential amino acid lysine, to study the occurrence of reactive oxygen species (ROS), abasic (AP) sites and double strand breaks (DSBs). Furthermore, we analyzed the mutation frequencies in resting wild type cells and in cells deficient for Apn1 (with an impaired base excision repair) or Dnl4 (with an inactivated non-homologous end joining (NHEJ) DSB repair pathway) by monitoring reversions of an auxotrophy-causing frameshift in the LYS2 gene. By fluorescence methods, we observed a distinct increase of ROS-affected cells in the course of starvation-induced cell cycle-arrest. In addition, we could reveal that AP sites and DSBs accumulated under these conditions. The frequency of spontaneous frameshift mutations in wild type cells was decreased to 50% upon addition of 6mM N-acetyl cysteine. However, this radical scavenger had no effect in Dnl4-deficient cells. Our results support the hypothesis that (via an active NHEJ DSB repair pathway) the incidence of spontaneous frameshift mutations in a cell cycle-arrested state is considerably governed by oxidative stress.

A mutation-promotive role of nucleotide excision repair in cell cycle-arrested cell populations following UV irradiation.

Growing attention is paid to the concept that mutations arising in stationary, non-proliferating cell populations considerably contribute to evolution, aging, and pathogenesis. If such mutations are beneficial to the affected cell, in the sense of allowing a restart of proliferation, they are called adaptive mutations. In order to identify cellular processes responsible for adaptive mutagenesis in eukaryotes, we study frameshift mutations occurring during auxotrophy-caused cell cycle arrest in the model organism Saccharomyces cerevisiae. Previous work has shown that an exposure of cells to UV irradiation during prolonged cell cycle arrest resulted in an increased incidence of mutations. In the present work, we determined the influence of defects in the nucleotide excision repair (NER) pathway on the incidence of UV-induced adaptive mutations in stationary cells. The mutation frequency was decreased in Rad16-deficient cells and further decreased in Rad16/Rad26 double-deficient cells. A knockout of the RAD14 gene, the ortholog of the human XPA gene, even resulted in a nearly complete abolishment of UV-induced mutagenesis in cell cycle-arrested cells. Thus, the NER pathway, responsible for a normally accurate repair of UV-induced DNA damage, paradoxically is required for the generation and/or fixation of UV-induced frameshift mutations specifically in non-replicating cells.