Abstract
The immune system decides upon actions in response to complex combinations of microbial inputs. Dozens of innate immune sensing pathways can collectively respond to combinations of microbial ...and immunomodulatory agents such as adjuvants. We currently lack a clear understanding for the combinatorial logic that governs how the interplay between higher-order combinations of microbial or adjuvant signals shape immune responses. Here, using in vitro coculture experiments and statistical analyses, we uncover an intrinsic property of pathogen-sensing pathways whereby the effects of higher-order combinations of stimuli on immune responses can be explained by single and pairwise effects. Surprisingly, this property of combinatorial innate sensing is mechanistically rooted in the molecular organization underlying pathogen-sensing pathways. Indeed, we find that the outputs of mouse and human innate immune dendritic cells upon triplet stimulations are dictated by single and pairwise effects at the levels of transcription, chromatin and protein secretion. Furthermore, we exploit this simplifying property of combinatorial pathogen sensing to develop and characterize cell-based immunotherapies with potent anti-tumor properties in mouse models using combinations of adjuvants. We conclude that the processing of complex mixtures of microbial or adjuvant inputs by immune cells is governed by pairwise effects, which suggests a path towards the rationale selection of adjuvant combinations to alter immunity.
Secondary organic aerosols (SOAs) formed from anthropogenic or biogenic gaseous precursors in the atmosphere substantially contribute to the ambient fine particulate matter PM
in aerodynamic diameter ...(
) burden, which has been associated with adverse human health effects. However, there is only limited evidence on their differential toxicological impact.
We aimed to discriminate toxicological effects of aerosols generated by atmospheric aging on combustion soot particles (SPs) of gaseous biogenic (
) or anthropogenic (naphthalene) precursors in two different lung cell models exposed at the air-liquid interface (ALI).
Mono- or cocultures of lung epithelial cells (A549) and endothelial cells (EA.hy926) were exposed at the ALI for 4 h to different aerosol concentrations of a photochemically aged mixture of primary combustion SP and
(
) or naphthalene (
). The internally mixed soot/SOA particles were comprehensively characterized in terms of their physical and chemical properties. We conducted toxicity tests to determine cytotoxicity, intracellular oxidative stress, primary and secondary genotoxicity, as well as inflammatory and angiogenic effects.
We observed considerable toxicity-related outcomes in cells treated with either SOA type. Greater adverse effects were measured for
compared with
in both cell models, whereas the nano-sized soot cores alone showed only minor effects. At the functional level, we found that
augmented the secretion of malondialdehyde and interleukin-8 and may have induced the activation of endothelial cells in the coculture system. This activation was confirmed by comet assay, suggesting secondary genotoxicity and greater angiogenic potential. Chemical characterization of PM revealed distinct qualitative differences in the composition of the two secondary aerosol types.
In this study using A549 and EA.hy926 cells exposed at ALI, SOA compounds had greater toxicity than primary SPs. Photochemical aging of naphthalene was associated with the formation of more oxidized, more aromatic SOAs with a higher oxidative potential and toxicity compared with
. Thus, we conclude that the influence of atmospheric chemistry on the chemical PM composition plays a crucial role for the adverse health outcome of emissions. https://doi.org/10.1289/EHP9413.
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CEKLJ, DOBA, IZUM, KILJ, NUK, OILJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK, VSZLJ
The immune system makes decisions in response to combinations of multiple microbial inputs. We do not understand the combinatorial logic governing how higher-order combinations of microbial signals ...shape immune responses. Here, using coculture experiments and statistical analyses, we discover a general property for the combinatorial sensing of microbial signals, whereby the effects of triplet combinations of microbial signals on immune responses can be predicted by combining the effects of single and pairs. Mechanistically, we find that singles and pairs dictate the information signaled by triplets in mouse and human DCs at the levels of transcription, chromatin, and protein secretion. We exploit this simplifying property to develop cell-based immunotherapies prepared with adjuvant combinations that trigger protective responses in mouse models of cancer. We conclude that the processing of multiple input signals by innate immune cells is governed by pairwise effects, which will inform the rationale combination of adjuvants to manipulate immunity.
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•Predicting triplet stimuli effects in vitro using coculture data from singles and pairs•Singles and pairs dictate the information signaled by triplets in mouse and human DCs•Designing dendritic cell-based therapies against cancer with adjuvant triplets•Predicting adjuvant triplet effects in vivo using single and pairwise data
Pandey et al. find a general property for the combinatorial sensing of microbial signals by innate immune cells: triplet effects are predictable using single and pairwise effects. This simplifying rule applies in vitro, using cell cocultures, and in vivo, using cell-based vaccines eliciting potent anti-tumor responses in mouse models.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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•A comprehensive comparison between SOANap-SP and SOAβPin-SP shows differences in their chemical composition.•SOANap-SP has a higher cytotoxic potential than SOAβPin-SP.•SOANap-SP has ...stronger potency in inducing inflammatory and genotoxicity response than that of SOAβPin-SP.•SOANap-SP activates Nrf-, AhR-, MMP- and MAPK signaling pathways.
The health effects of exposure to secondary organic aerosols (SOAs) are still limited. Here, we investigated and compared the toxicities of soot particles (SP) coated with β-pinene SOA (SOAβPin-SP) and SP coated with naphthalene SOA (SOANap-SP) in a human bronchial epithelial cell line (BEAS-2B) residing at the air–liquid interface. SOAβPin-SP mostly contained oxygenated aliphatic compounds from β-pinene photooxidation, whereas SOANap-SP contained a significant fraction of oxygenated aromatic products under similar conditions. Following exposure, genome-wide transcriptome responses showed an Nrf2 oxidative stress response, particularly for SOANap-SP. Other signaling pathways, such as redox signaling, inflammatory signaling, and the involvement of matrix metalloproteinase, were identified to have a stronger impact following exposure to SOANap-SP. SOANap-SP also induced a stronger genotoxicity response than that of SOAβPin-SP. This study elucidated the mechanisms that govern SOA toxicity and showed that, compared to SOAs derived from a typical biogenic precursor, SOAs from a typical anthropogenic precursor have higher toxicological potency, which was accompanied with the activation of varied cellular mechanisms, such as aryl hydrocarbon receptor. This can be attributed to the difference in chemical composition; specifically, the aromatic compounds in the naphthalene-derived SOA had higher cytotoxic potential than that of the β-pinene-derived SOA.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Wood combustion emissions have been studied previously either by in vitro or in vivo models using collected particles, yet most studies have neglected gaseous compounds. Furthermore, a more accurate ...and holistic view of the toxicity of aerosols can be gained with parallel in vitro and in vivo studies using direct exposure methods. Moreover, modern exposure techniques such as air-liquid interface (ALI) exposures enable better assessment of the toxicity of the applied aerosols than, for example, the previous state-of-the-art submerged cell exposure techniques.
We used three different ALI exposure systems in parallel to study the toxicological effects of spruce and pine combustion emissions in human alveolar epithelial (A549) and murine macrophage (RAW264.7) cell lines. A whole-body mouse inhalation system was also used to expose C57BL/6 J mice to aerosol emissions. Moreover, gaseous and particulate fractions were studied separately in one of the cell exposure systems. After exposure, the cells and animals were measured for various parameters of cytotoxicity, inflammation, genotoxicity, transcriptome and proteome.
We found that diluted (1:15) exposure pine combustion emissions (PM
mass 7.7 ± 6.5 mg m
, 41 mg MJ
) contained, on average, more PM and polycyclic aromatic hydrocarbons (PAHs) than spruce (PM
mass 4.3 ± 5.1 mg m
, 26 mg MJ
) emissions, which instead showed a higher concentration of inorganic metals in the emission aerosol. Both A549 cells and mice exposed to these emissions showed low levels of inflammation but significantly increased genotoxicity. Gaseous emission compounds produced similar genotoxicity and a higher inflammatory response than the corresponding complete combustion emission in A549 cells. Systems biology approaches supported the findings, but we detected differing responses between in vivo and in vitro experiments.
Comprehensive in vitro and in vivo exposure studies with emission characterization and systems biology approaches revealed further information on the effects of combustion aerosol toxicity than could be achieved with either method alone. Interestingly, in vitro and in vivo exposures showed the opposite order of the highest DNA damage. In vitro measurements also indicated that the gaseous fraction of emission aerosols may be more important in causing adverse toxicological effects. Combustion aerosols of different wood species result in mild but aerosol specific in vitro and in vivo effects.
The enzyme ScHxk2 of Saccharomyces cerevisiae is a dual-function hexokinase that besides its catalytic role in glycolysis is involved in the transcriptional regulation of glucose-repressible genes. ...Relief from glucose repression is accompanied by the phosphorylation of the nuclear fraction of ScHxk2 at serine 15 and the translocation of the phosphoenzyme into the cytosol. Different studies suggest different serine/threonine protein kinases, Ymr291w/Tda1 or Snf1, to accomplish ScHxk2-S15 phosphorylation. The current paper provides evidence that Ymr291w/Tda1 is essential for that modification, whereas protein kinases Ydr477w/Snf1, Ynl307c/Mck1, Yfr014c/Cmk1, and Ykl126w/Ypk1, which are co-purified during Ymr291w/Tda1 tandem affinity purification, as well as protein kinase PKA and PKB homolog Sch9 are dispensable. Taking into account the detection of a significantly higher amount of the Ymr291w/Tda1 protein in cells grown in low-glucose media as compared with a high-glucose environment, Ymr291w/Tda1 is likely to contribute to glucose signaling in S. cerevisiae on the level of ScHxk2-S15 phosphorylation in a situation of limited external glucose availability. The evolutionary conservation of amino acid residue serine 15 in yeast hexokinases and its phosphorylation is illustrated by the finding that YMR291W/TDA1 of S. cerevisiae and the homologous KLLA0A09713 gene of Kluyveromyces lactis allow for cross-complementation of the respective protein kinase single-gene deletion strains.
Background: Monomer-dimer equilibrium, substrate affinity, and subcellular localization of yeast hexokinase ScHxk2 depend on the state of phosphorylation of serine 15.
Results: Serine/threonine protein kinase Ymr291w/Tda1 is essentially required for ScHxk2-S15 phosphorylation.
Conclusion: Ymr291w/Tda1 is the ScHxk2-S15 kinase or an upstream regulatory enzyme.
Significance: The analysis of Ymr291w/Tda1 function(s) is indispensable for understanding glucose signaling in yeast.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The yeast plasma membrane H
+
‐
ATP
ase Pma1p is a P‐type
ATP
ase that energizes the yeast plasma membrane. Pma1p exists in two activation states: an autoinhibited basal state and an activated state. ...Here we show that functional and stable Pma1p can be purified in native form and reconstituted in artificial liposomes without altering its activation state. Acetylated tubulin has previously been reported to maintain Pma1p in the basal state but, as this protein was absent from the purified preparations, it cannot be an essential component of the autoinhibitory mechanism. Purification of and reconstitution of native Pma1p in both activation states opens up for a direct comparison of the transport properties of these states, which allowed us to confirm that the basal state has a low coupling ratio between
ATP
hydrolysis and protons pumped, whereas the activated state has a high coupling ratio. The ability to prepare native Pma1p in both activation states will facilitate further structural and biochemical studies examining the mechanism by which plasma membrane H
+
‐
ATP
ases are autoinhibited.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Isobaric stable isotope labeling using, for example, tandem mass tags (TMTs) is increasingly being applied for large-scale proteomic studies. Experiments focusing on proteoform analysis in drug time ...course or perturbation studies or in large patient cohorts greatly benefit from the reproducible quantification of single peptides across samples. However, such studies often require labeling of hundreds of micrograms of peptides such that the cost for labeling reagents represents a major contribution to the overall cost of an experiment. Here, we describe and evaluate a robust and cost-effective protocol for TMT labeling that reduces the quantity of required labeling reagent by a factor of eight and achieves complete labeling. Under- and overlabeling of peptides derived from complex digests of tissues and cell lines were systematically evaluated using peptide quantities of between 12.5 and 800 μg and TMT-to-peptide ratios (wt/wt) ranging from 8:1 to 1:2 at different TMT and peptide concentrations. When reaction volumes were reduced to maintain TMT and peptide concentrations of at least 10 mm and 2 g/l, respectively, TMT-to-peptide ratios as low as 1:1 (wt/wt) resulted in labeling efficiencies of > 99% and excellent intra- and interlaboratory reproducibility. The utility of the optimized protocol was further demonstrated in a deep-scale proteome and phosphoproteome analysis of patient-derived xenograft tumor tissue benchmarked against the labeling procedure recommended by the TMT vendor. Finally, we discuss the impact of labeling reaction parameters for N-hydroxysuccinimide ester-based chemistry and provide guidance on adopting efficient labeling protocols for different peptide quantities.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Background Ship engine emissions are important with regard to lung and cardiovascular diseases especially in coastal regions worldwide. Known cellular responses to combustion particles include ...oxidative stress and inflammatory signalling. Objectives To provide a molecular link between the chemical and physical characteristics of ship emission particles and the cellular responses they elicit and to identify potentially harmful fractions in shipping emission aerosols. Methods Through an air-liquid interface exposure system, we exposed human lung cells under realistic in vitro conditions to exhaust fumes from a ship engine running on either common heavy fuel oil (HFO) or cleaner-burning diesel fuel (DF). Advanced chemical analyses of the exhaust aerosols were combined with transcriptional, proteomic and metabolomic profiling including isotope labelling methods to characterise the lung cell responses. Results The HFO emissions contained high concentrations of toxic compounds such as metals and polycyclic aromatic hydrocarbon, and were higher in particle mass. These compounds were lower in DF emissions, which in turn had higher concentrations of elemental carbon ("soot"). Common cellular reactions included cellular stress responses and endocytosis. Reactions to HFO emissions were dominated by oxidative stress and inflammatory responses, whereas DF emissions induced generally a broader biological response than HFO emissions and affected essential cellular pathways such as energy metabolism, protein synthesis, and chromatin modification. Conclusions Despite a lower content of known toxic compounds, combustion particles from the clean shipping fuel DF influenced several essential pathways of lung cell metabolism more strongly than particles from the unrefined fuel HFO. This might be attributable to a higher soot content in DF. Thus the role of diesel soot, which is a known carcinogen in acute air pollution-induced health effects should be further investigated. For the use of HFO and DF we recommend a reduction of carbonaceous soot in the ship emissions by implementation of filtration devices.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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