J Proteomics Bioinform

Paraquat (PQ) has been one of the most widely used herbicides in the world. PQ, when ingested, is toxic to humans and may cause acute respiratory distress syndrome. To investigate molecular perturbation in lung tissues caused by PQ, Sprague Dawley male rats were fed with PQ at a dose of 25 mg/kg body weight for 20 times in four weeks. The effects of PQ on cellular processes and biological pathways were investigated by analyzing proteome in the lung tissues in comparison with the control. Among the detected proteins, 321 and 254 proteins were over-represented and under-represented, respectively, in the PQ-exposed rat lung tissues in comparison with the no PQ control. All over- and under-represented proteins were subjected to Ingenuity Pathway Analysis to create 25 biological networks and 38 pathways of interacting protein clusters. Over-represented proteins were involved in the C-jun-amino-terminal kinase pathway, caveolae-mediated endocytosis signaling, cardiovascular-cancer-respiratory pathway, regulation of clathrin-mediated endocytosis, non-small cell lung cancer signaling, pulmonary hypertension, glutamate receptor, immune response and angiogenesis. Under-represented proteins occurred in the p53 signaling pathway, mitogen-activated protein kinase signaling pathway, cartilage development and angiogenesis inhibition in the PQ-treated lungs. The results suggest that PQ may generate reactive oxygen species, impair the MAPK/p53 signaling pathway, activate angiogenesis and depress apoptosis in the lungs.G12 MD007601/MD/NIMHD NIH HHS/United StatesU54 OH007550/OH/NIOSH CDC HHS/United States2015-11-02T00:00:00Z26538867PMC462953

A dynamic model of gene expression in monocytes reveals differences in immediate/early response genes between adult and neonatal cells

Neonatal monocytes display immaturity of numerous functions compared with adult cells. Gene expression arrays provide a promising tool for elucidating mechanisms underlying neonatal immune function. We used a well-established microarray to analyze differences between LPS-stimulated human cord blood and adult monocytes to create dynamic models for interactions to elucidate observed deficiencies in neonatal immune responses. We identified 168 genes that were differentially expressed between adult and cord monocytes after 45 min incubation with LPS. Of these genes, 95% (159 of 167) were over-expressed in adult relative to cord monocytes. Differentially expressed genes could be sorted into nine groups according to their kinetics of activation. Functional modelling suggested differences between adult and cord blood in the regulation of apoptosis, a finding confirmed using annexin binding assays. We conclude that kinetic studies of gene expression reveal potentially important differences in gene expression dynamics that may provide insight into neonatal innate immunity

Toxicol Lett

DNA methylation may mediate inter-individual responses to chemical exposure and, thus, modify biomarker levels of exposure and effects. We analyzed inter-individual differences in inhalation and skin exposure to 1,6-hexamethylene diisocyanate (HDI) and urine biomarker 1,6-hexamethylene diamine (HDA) levels in 20 automotive spray-painters. Genome-wide 5-methyl cytosine (CpG) DNA methylation was assessed in each individual's peripheral blood mononuclear cells (PBMC) DNA using the Illumina 450K CpG array. Mediation analysis using linear regression models adjusted for age, ethnicity, and smoking was conducted to identify and assess the association between HDI exposure, CpG methylation, and urine HDA biomarker levels. We did not identify any CpGs common to HDI exposure and biomarker level suggesting that CpG methylation is a mediator that only partially explains the phenotype. Functional significance of genic- and intergenic-CpG methylation status was tested using protein-protein or protein-DNA interactions and gene-ontology enrichment to infer networks. Combined, the results suggest that methylation has the potential to affect HDI mass transport, permeation, and HDI metabolism. We demonstrate the potential use of PBMC methylation along with quantitative exposure and biomarker data to guide further investigation into the mediators of occupational exposure and biomarkers and its role in risk assessment.T42 OH008673/OH/NIOSH CDC HHS/United StatesR01 OH007598/OH/NIOSH CDC HHS/United StatesR21 OH010203/OH/NIOSH CDC HHS/United StatesR21-OH010203/OH/NIOSH CDC HHS/United StatesT42OH008673/ACL/ACL HHS/United StatesR01-OH007598/OH/NIOSH CDC HHS/United StatesP42 ES005948/ES/NIEHS NIH HHS/United StatesT42/CCT422952/PHS HHS/United StatesP30 ES010126/ES/NIEHS NIH HHS/United StatesT42/OH-008673/OH/NIOSH CDC HHS/United States2021-04-22T00:00:00Z25445006PMC8060918965

DEFINITION OF AN IN VITRO MODEL OF HUMAN MONOCYTE ACTIVATION REPRESENTATIVE OF THE DEFENSIVE INFLAMMATORY RESPONSE

La reazione di difesa innata/infiammatoria \ue8 attivata in risposta a patogeni esterni o a segnali provenienti dal tessuto danneggiato. I monociti/macrofagi hanno un ruolo chiave nell\u2019inizio e risoluzione della infiammazione per mezzo di differenti programmi di attivazione. Infatti i macrofagi possono adottare in vivo una variet\ue0 di fenotipi diversi che dipendono dai cambiamenti del microambiente tissutale, esibendo un continuum di stati funzionali diversi. Inoltre i monociti del sangue periferico non sono una popolazione omogenea ma differiscono nei loro fenotipi e funzioni. Nonostante l\u2019esplosivo aumento di informazioni sull\u2019argomento, molte questioni sono ancora aperte riguardo la caratterizzazione fenotipica e funzionale dei monociti/macrofagi, e il loro ruolo durante l\u2019omeostasi e l\u2019infiammazione. La maggior parte dei dati provengono da studi sul topo e molti immunologi fanno ancora affidamento su modelli di topo malgrado la distanza evolutiva e le differenze tra i sistemi immuni murino e umano. Nel tentativo di capire le questioni di cui sopra e di dirigere gli sforzi verso una immunobiologia basata sull\u2019uomo, il fine di questo lavoro \ue8 stato quello di costruire e validare un modello umano della risposta di difesa innata/infiammatoria in vitro che ricapitolasse le differenti fasi della reazione infiammatoria, dal reclutamento e inizio, allo sviluppo e risoluzione dell\u2019infiammazione e conseguente ripristino della omeostasi. Il modello \ue8 basato su monociti umani primari del sangue esposti in coltura a cambiamenti sequenziali delle condizioni microambientali (chemiochine, citochine, temperatura, molecole di derivazione batterica, ecc.) per 48 h. L\u2019analisi al citofluorimetro ha dimostrato che la popolazione monocitaria utilizzata era rappresentativa dell\u2019eterogeneit\ue0 monocitaria cos\uec come presente nella circolazione sanguigna. Tutte le fasi della risposta infiammatoria sono state definite mediante analisi trascrittomica effettuata con U133Plus 2.0 GeneChip (Affymetrix). I risultati sono stati confrontati e integrati con profili trascrizionali pubblicamente disponibili di monociti/macrofagi, raccolti e annotati in un database ad hoc. Il profilo trascrittomico di alcuni fattori trascrizionali e fattori correlati con l\u2019infiammazione sono stati confermati e validati mediante qPCR e ELISA. La \u201ccluster analysis\u201d ha rivelato cluster ampi e distinti che comprendono geni con un chiaro andamento che ben descrivono le differenti fasi dell\u2019infiammazione. Per ottenere maggiori indicazioni sul ruolo biologico dei geni differenzialmente espressi durante la risposta infammatoria, ciascun cluster \ue8 stato analizzato con la GSEA (Gene Set Enrichment Analysis). I set di geni identificati dalla GSEA correlati con il profilo di espressione dei differenti cluster ha rivelato che la fase infiammatoria era arricchita di pathway infiammatorie mentre la fase anti-infiammatoria, cos\uec come quella di risoluzione, di pathway relative al metabolismo, al ciclo cellulare e al riarrangiamento genico. Inoltre confrontando le liste dei geni differenzialmente espressi tra monociti e macrofagi M1 e tra monociti e macrofagi M2 estratte dal meta-database, \ue8 stato dimostrato che i monociti trattati in vitro secondo il modello mostrano un profilo M1 durante la fase infiammatoria e M2 durante la risoluzione. L\u2019espressione genica dei fattori trascrizionali e di quelli relativi alla infiammazione rispecchiavano il profilo di espressione ottenuto con microarray. In conclusione i dati di microarray e l\u2019analisi cinetica dei fattori infiammatori e anti-infiammatori validano il modello in vitro proposto, modello che consente di descrivere la sequenza tempo-dipendente e coordinata degli eventi relativi alla infiammazione.The innate/inflammatory defensive reaction is activated in response to foreign pathogens or signals from damaged tissue. Monocytes/macrophages are key players in the initiation and resolution of inflammation by different activation programmes. Indeed in vivo macrophages can adopt a variety of different phenotypes depending on changes in the tissue microenvironment displaying a continuum of diverse functional states. Moreover peripheral blood monocytes are not a homogeneous population but differ in their phenotypes and functions. In spite of the explosive growth of data, many issues are still open about the phenotypic and functional characterization of monocytes/macrophages, and their role during the homeostasis and in inflammatory conditions. The great majority of the data originates from studies in mice and many immunologists still rely on mouse models despite the evolutionary distance and the differences between the murine and human immune systems. In an attempt to understanding the above issues, and to direct efforts in human immunobiology, the aim of this work was to build and validate a human model of innate/inflammatory defence response in vitro that recapitulates the different phases of the inflammatory reaction, from recruitment and initiation, to development and resolution of inflammation, and re-establishment of homeostasis. The model is based on human primary blood monocytes exposed in culture to sequential changes of microenvironmental conditions (chemokines and cytokines, temperature, bacterial-derived molecules, etc.) for 48 h. The flow cytometrical analysis has shown that the monocyte population used is representative of the monocyte heterogeneity as present in the circulation. All phases of the inflammatory response were profiled by transcriptomic analysis carried out with U133Plus 2.0 GeneChip (Affymetrix). Results were compared and integrated with publicly available transcriptional profiles of monocyte/macrophages, collected and annotated in an ad hoc database. The transcriptomic profiling of some transcriptional and inflammatory-related factors were confirmed and validated by qPCR and by ELISA. The \u201ccluster analysis\u201d revealed broad distinct clusters comprising genes with a clear behaviour that well described the different phases of inflammation. To gain more insight into the biologic role of the genes that are differentially expressed during the inflammatory response, each cluster was subjected to gene set enrichment analysis (GSEA). The gene sets identified by GSEA correlated with the expression profile of different clusters revealed that the inflammatory phase was enriched in inflammatory pathways while the anti-inflammatory phase, as well as the resolution phase, in pathways related to metabolism, cell cycle, and gene rearrangement. Moreover, by comparing the lists of differentially expressed gene between monocytes vs. M1 macrophages and vs. M2 macrophages extracted from the meta-database, it was shown that monocytes treated in vitro according to model resemble M1 during the inflammatory phase and M2 during the resolution. The gene expression of transcriptional and inflammatory-related factors matched with the expression profile obtained with microarrays. In conclusion the microarray data and the kinetical analysis of inflammatory and anti-inflammatory factors validate the proposed in vitro model of the inflammatory response, and allowed describing the time-dependent and coordinated sequence of inflammation-related events

Role of adropin in arterial stiffening associated with obesity and type 2 diabetes

Adropin is a peptide largely secreted by the liver and known to regulate energy homeostasis; however, it also exerts cardiovascular effects. Herein, I tested the hypothesis that low circulating levels of adropin in obesity and type 2 diabetes (T2D) contribute to arterial stiffening. In support of this hypothesis, I report that obesity and T2D is associated with reduced levels of adropin expression in liver and plasma and increased arterial stiffness in mice and humans. Establishing causation, I showed that mesenteric arteries from adropin knockout mice are also stiffer relative to arteries from wild-type counterparts, thus recapitulating the stiffening phenotype observed in T2D db/db mice. Given the above, a series of follow-up experiments were performed that determined: 1) exposure of endothelial cells or isolated mesenteric arteries from db/db mice to adropin reduces filamentous actin (F-actin) stress fibers and stiffness; 2) adropininduced reduction of F-actin and stiffness in endothelial cells and db/db mesenteric arteries is abrogated by inhibition of nitric oxide (NO) synthase; and 3) stimulation of smooth muscle cells or db/db mesenteric arteries with a NO mimetic reduces stiffness. Last, in vivo treatment of db/db mice with adropin for four weeks reduced stiffness in mesenteric arteries. Collectively, these findings indicate that adropin can regulate arterial stiffness, likely via endothelial-derived NO, and thus support the notion that "hypoadropinemia" should be considered as a putative target for the prevention and treatment of arterial stiffening in obesity and T2D.Includes bibliographical references

Neutrophil biomechanical properties and immune function in health and inflammatory disease

Low density granulocytes (LDGs) are a poorly understood class of immune cells found in patients with chronic inflammatory diseases including psoriasis and systemic lupus erythematosus (SLE). Research completed at the National Institutes of Health (NIH) revealed that in the context of SLE, LDGs release higher levels of type 1 interferons, undergo increased NETosis, and accordingly drive inflammation. Meanwhile, advances in mechanical phenotyping at the University of Cambridge have driven hypotheses of neutrophil trafficking and immune function being intimately linked to cellular biomechanical properties (e.g. density, stiffness, morphology). This thesis analyses the intersection of immune cellular biomechanical phenotypes and their function. Specifically, it focuses on the role of neutrophils and LDGs in inflammatory diseases. In this thesis, real-time deformability cytometry (RT-DC) was optimised as a high-throughput mechanical phenotyping technique for the analysis of neutrophils. This enabled development of a protocol to recover purified neutrophils to their whole blood mechanical phenotype. Neutrophil biomechanical properties were analysed by RT-DC, lattice light-sheet microscopy, confocal microscopy and scanning electron microscopy. Neutrophil immunologic functions (e.g. NETosis, macropinocytosis) were imaged using florescence microscopy. To analyse the contribution of biomechanical properties to neutrophil trafficking, a novel microfluidic microvasculature mimetic was developed. An endothelial flow assay was used to image neutrophils interacting with endothelial cells. Finally, the complete proteomes and phosphoproteomes of LDGs and normal dense neutrophils (NDNs) were obtained from five healthy donors and five SLE patients. Several key insights were gained. Firstly, hypotonic lysis and magnetic column-based isolation techniques are damaging to neutrophil biomechanical properties, but purification of neutrophils retaining their biomechanical properties can be achieved by using gradients and column-free magnetic systems followed by recovery at 37 degrees Celsius. Secondly, the biphasic biomechanical kinetics of neutrophil priming were described; cells contract briefly before immediately expanding. The expansion phase was determined to be macropinocytosis dependent. Thirdly, SLE LDGs are phenotypically rougher than autologous SLE NDNs or healthy LDGs. This appears to impact their microvasculature trafficking abilities, as SLE LDGs were increasingly trapped in the narrow channels of a three- dimensional microvasculature mimetic. These results suggest a role for biomechanical properties in modulation of neutrophil trafficking, indicating that SLE LDGs may be increasingly retained in microvasculature networks, similar to what has been described for primed neutrophils. Finally, unbiased proteomics quantified 4109 proteins and 875 phosphoproteins in four neutrophil subsets (healthy unstimulated NDNs, healthy primed NDNs, SLE NDNs, and SLE LDG). This shed new light into neutrophil heterogeneity at the protein level and to my knowledge, is the first proteomic profile of the SLE LDG. In addition to findings pertaining to SLE LDG biology and function, differential phosphorylation of proteins associated with cytoskeletal organisation were identified in SLE LDGs relative to SLE NDNs, suggesting a phosphoproteomic explanation for the SLE LDGs’ distinct biomechanical phenotype. When taken together, this work could have important pathogenic implications in the context of SLE manifestations in various organs and the development of small vessel vasculopathy.Kathleen Bashant was funded by the NIH-Cambridge Scholars Program This research was supported by the NIHR Cambridge Biomedical Research Centre (BRC-1215-20014*). The views expressed are those of the author and not necessarily those of the NIHR or the Department of Health and Social Care

TRANSCRIPTOME ANALYSIS OF AMOEBOID AND RAMIFIED MICROGLIA ISOLATED FROM THE CORPUS CALLOSUM OF RAT BRAIN

Ph.DDOCTOR OF PHILOSOPH

Cytological endometritis in dairy cattle : new insights into pathogenesis, diagnosis and treatment

Tese de Doutoramento em Ciências Veterinárias, na especialidade ClínicaABSTRACT - In postpartum dairy cows, subclinical endometritis (SCE) is characterized by persistent endometrial inflammation, with profound detrimental effects on subsequent fertility. Despite the known role of adipokines regulating metabolism and inflammation, the association of adipokine signaling with SCE is still poorly understood. Moreover, the effect of SCE on endometrial transcription was mainly determined from biopsy-derived whole tissue, thus masking effects on cell type-specific gene transcription. In addition, despite the recognized improvement in immune function following n-3PUFA supplementation, the therapeutic potential of these fatty acids for SCE control is still to be determined. Therefore, the main objectives of this work were to assess the relationship between adipokines and SCE (Chapter III), to elucidate the effects of progesterone and SCE in the transcription profiles of endometrial glandular, luminal and stromal cells (Chapters IV, V), and the effects of feeding rumen-protected n-3PUFA on endometrial homeostasis and fertility in postpartum dairy cows (Chapter VI). The results showed that adiponectin in plasma and uterine fluid in addition to chemerin uterine fluid have high discriminatory power for the diagnosis of SCE. This work additionally evidenced that progesterone and SCE affect endometrial transcriptomic profiles in a cell type-specific manner. This work also evidenced that the previous presence of immune cells is still impacting the transcriptome of endometrial cells at the end of the voluntary waiting period and that recovery or persistence of inflammation is associated with transcription patterns involved not only in immune function but also in tissue remodeling, cell adhesion, and uterine receptivity. Furthermore, despite having no apparent effect on the endometrial inflammatory status at the end of the voluntary waiting period, dietary supplementation with n-3PUFA decreased calving to conception interval. Overall, this thesis establishes adiponectin as a suitable biomarker of SCE, able to predict the risk of persistence of inflammation in postpartum dairy cows, provides new insights into the persistence and recovery of SCE, thus presenting putative alternative therapeutic strategies. Moreover, this work substantiates dietary n-3PUFA as valid nutraceuticals to improve reproductive parameters in postpartum dairy cows.RESUMO - Endometrite citológica em vacas leiteiras : novos desenvolvimentos em patogénese, diagnóstico e tratamento - Em vacas leiteiras pós-parto, a endometrite subclínica (ESC) é caracterizada por inflamação persistente do endométrio, com profundos efeitos prejudiciais na fertilidade subsequente. Apesar do conhecido papel das adipocinas na regulação do metabolismo e da inflamação, a regulação destas moléculas em vacas com ESC é ainda pouco compreendida. Além disso, o efeito da ESC na transcrição de células do endométrio tem sido explorado a partir de biópsias endometriais totais, mascarando assim os efeitos na transcrição específica de cada tipo celular. Além disso, apesar da reconhecida modulação da função imune após a suplementação com ácidos gordos polinsaturados (PUFA) ómega-3, o potencial terapêutico dos mesmos para o controlo da ESC ainda não foi determinado. Assim, os principais objetivos deste trabalho foram avaliar a relação entre adipocinas e a ESC (Capítulo III), elucidar os efeitos da progesterona e ESC nos perfis de transcrição de células endometriais do estroma, epiteliais glandulares e epiteliais luminais (Capítulos IV, V), e os efeitos da suplementação com PUFA ómega-3 com protecção ruminal na homeostase endometrial e fertilidade em vacas leiteiras pós-parto (Capítulo VI). Os resultados mostraram que a adiponectina no plasma e no lavado uterino, além da quemerina no líquido uterino, possuem alto poder discriminatório para o diagnóstico de ESC. Este trabalho evidenciou também que a progesterona e a ESC afetam os perfis de transcrição endometriais das diferentes populações celulares de uma forma específica, que a presença prévia de leucócitos ainda afeta o perfil de transcrição das células endometriais no final do período voluntário de espera e que a recuperação ou persistência da inflamação está associada a padrões de transcrição envolvidos não apenas na função imunológica, mas também na remodelação tecidular, adesão celular e receptividade uterina. Além disso, apesar de aparentemente não produzir efeito sobre o estado inflamatório endometrial no final do período voluntário de espera, a suplementação com PUFA ómega-3 diminuiu o intervalo parto-concepção. No geral, esta tese estabelece a adiponectina como um biomarcador adequado de ESC, capaz de prever o risco de persistência de inflamação em vacas leiteiras no pós-parto, fornece novas perspectivas sobre a persistência e recuperação da ESC, apresentando assim novas hipotéticas estratégias terapêuticas. Além disso, este trabalho substancia os PUFA ómega-3 como nutracêuticos válidos para melhorar os parâmetros reprodutivos em vacas leiteiras no pós-partoN/