Chemical modification by peroxynitrite enhances TLR4 activation of the grass pollen allergen Phl p 5.

The chemical modification of aeroallergens by reactive oxygen and nitrogen species (ROS/RNS) may contribute to the growing prevalence of respiratory allergies in industrialized countries. Post-translational modifications can alter the immunological properties of proteins, but the underlying mechanisms and effects are not well understood. In this study, we investigate the Toll-like receptor 4 (TLR4) activation of the major birch and grass pollen allergens Bet v 1 and Phl p 5, and how the physiological oxidant peroxynitrite (ONOO-) changes the TLR4 activation through protein nitration and the formation of protein dimers and higher oligomers. Of the two allergens, Bet v 1 exhibited no TLR4 activation, but we found TLR4 activation of Phl p 5, which increased after modification with ONOO- and may play a role in the sensitization against this grass pollen allergen. We attribute the TLR4 activation mainly to the two-domain structure of Phl p 5 which may promote TLR4 dimerization and activation. The enhanced TLR4 signaling of the modified allergen indicates that the ONOO--induced modifications affect relevant protein-receptor interactions. This may lead to increased sensitization to the grass pollen allergen and thus contribute to the increasing prevalence of allergies in the Anthropocene, the present era of globally pervasive anthropogenic influence on the environment.

Reactive Nitrogen Hotspots Related to Microscale Heterogeneity in Biological Soil Crusts.

Biocrusts covering drylands account for major fractions of terrestrial biological nitrogen fixation and release large amounts of gaseous reactive nitrogen (Nr) as nitrous acid (HONO) and nitric oxide (NO). Recent investigations suggested that aerobic and anaerobic microbial nitrogen transformations occur simultaneously upon desiccation of biocrusts, but the spatio-temporal distribution of seemingly contradictory processes remained unclear. Here, we explore small-scale gradients in chemical concentrations related to structural characteristics and organism distribution. X-ray microtomography and fluorescence microscopy revealed mixed pore size structures, where photoautotrophs and cyanobacterial polysaccharides clustered irregularly in the uppermost millimeter. Microsensor measurements showed strong gradients of pH, oxygen, and nitrite, nitrate, and ammonium ion concentrations at micrometer scales in both vertical and lateral directions. Initial oxygen saturation was mostly low (∼30%) at full water holding capacity, suggesting widely anoxic conditions, and increased rapidly upon desiccation. Nitrite concentrations (∼6 to 800 µM) and pH values (∼6.5 to 9.5) were highest around 70% WHC. During further desiccation they decreased, while emissions of HONO and NO increased, reaching maximum values around 20% WHC. Our results illustrate simultaneous, spatially separated aerobic and anaerobic nitrogen transformations, which are critical for Nr emissions, but might be impacted by future global change and land management.

Oligomerization and Nitration of the Grass Pollen Allergen Phl p 5 by Ozone, Nitrogen Dioxide, and Peroxynitrite: Reaction Products, Kinetics, and Health Effects.

The allergenic and inflammatory potential of proteins can be enhanced by chemical modification upon exposure to atmospheric or physiological oxidants. The molecular mechanisms and kinetics of such modifications, however, have not yet been fully resolved. We investigated the oligomerization and nitration of the grass pollen allergen Phl p 5 by ozone (O3), nitrogen dioxide (NO2), and peroxynitrite (ONOO-). Within several hours of exposure to atmospherically relevant concentration levels of O3 and NO2, up to 50% of Phl p 5 were converted into protein oligomers, likely by formation of dityrosine cross-links. Assuming that tyrosine residues are the preferential site of nitration, up to 10% of the 12 tyrosine residues per protein monomer were nitrated. For the reaction with peroxynitrite, the largest oligomer mass fractions (up to 50%) were found for equimolar concentrations of peroxynitrite over tyrosine residues. With excess peroxynitrite, the nitration degrees increased up to 40% whereas the oligomer mass fractions decreased to 20%. Our results suggest that protein oligomerization and nitration are competing processes, which is consistent with a two-step mechanism involving a reactive oxygen intermediate (ROI), as observed for other proteins. The modified proteins can promote pro-inflammatory cellular signaling that may contribute to chronic inflammation and allergies in response to air pollution.

Chemical modification of pro-inflammatory proteins by peroxynitrite increases activation of TLR4 and NF-κB: Implications for the health effects of air pollution and oxidative stress.

Environmental pollutants like fine particulate matter can cause adverse health effects through oxidative stress and inflammation. Reactive oxygen and nitrogen species (ROS/RNS) such as peroxynitrite can chemically modify proteins, but the effects of such modifications on the immune system and human health are not well understood. In the course of inflammatory processes, the Toll-like receptor 4 (TLR4) can sense damage-associated molecular patterns (DAMPs). Here, we investigate how the TLR4 response and pro-inflammatory potential of the proteinous DAMPs α-Synuclein (α-Syn), heat shock protein 60 (HSP60), and high-mobility-group box 1 protein (HMGB1), which are relevant in neurodegenerative and cardiovascular diseases, changes upon chemical modification with peroxynitrite. For the peroxynitrite-modified proteins, we found a strongly enhanced activation of TLR4 and the pro-inflammatory transcription factor NF-κB in stable reporter cell lines as well as increased mRNA expression and secretion of the pro-inflammatory cytokines TNF-α, IL-1ß, and IL-8 in human monocytes (THP-1). This enhanced activation of innate immunity via TLR4 is mediated by covalent chemical modifications of the studied DAMPs. Our results show that proteinous DAMPs modified by peroxynitrite more potently amplify inflammation via TLR4 activation than the native DAMPs, and provide first evidence that such modifications can directly enhance innate immune responses via a defined receptor. These findings suggest that environmental pollutants and related ROS/RNS may play a role in promoting acute and chronic inflammatory disorders by structurally modifying the body's own DAMPs. This may have important consequences for chronic neurodegenerative, cardiovascular or gastrointestinal diseases that are prevalent in modern societies, and calls for action, to improve air quality and climate in the Anthropocene.

Screening of herbal extracts for TLR2- and TLR4-dependent anti-inflammatory effects.

Herbal extracts represent an ample source of natural compounds, with potential to be used in improving human health. There is a growing interest in using natural extracts as possible new treatment strategies for inflammatory diseases. We therefore aimed at identifying herbal extracts that affect inflammatory signaling pathways through toll-like receptors (TLRs), TLR2 and TLR4. Ninety-nine ethanolic extracts were screened in THP-1 monocytes and HeLa-TLR4 transfected reporter cells for their effects on stimulated TLR2 and TLR4 signaling pathways. The 28 identified anti-inflammatory extracts were tested in comparative assays of stimulated HEK-TLR2 and HEK-TLR4 transfected reporter cells to differentiate between direct TLR4 antagonistic effects and interference with downstream signaling cascades. Furthermore, the ten most effective anti-inflammatory extracts were tested on their ability to inhibit nuclear factor-κB (NF-κB) translocation in HeLa-TLR4 transfected reporter cell lines and for their ability to repolarize M1-type macrophages. Ethanolic extracts which showed the highest anti-inflammatory potential, up to a complete inhibition of pro-inflammatory cytokine production were Castanea sativa leaves, Cinchona pubescens bark, Cinnamomum verum bark, Salix alba bark, Rheum palmatum root, Alchemilla vulgaris plant, Humulus lupulus cones, Vaccinium myrtillus berries, Curcuma longa root and Arctostaphylos uva-ursi leaves. Moreover, all tested extracts mitigated not only TLR4, but also TLR2 signaling pathways. Seven of them additionally inhibited translocation of NF-κB into the nucleus. Two of the extracts showed impact on repolarization of pro-inflammatory M1-type to anti-inflammatory M2-type macrophages. Several promising anti-inflammatory herbal extracts were identified in this study, including extracts with previously unknown influence on key TLR signaling pathways and macrophage repolarization, serving as a basis for novel lead compound identification.