Comparative Proteomic Analysis Reveals Varying Impact on Immune Responses in Phorbol 12-Myristate-13-Acetate-Mediated THP-1 Monocyte-to-Macrophage Differentiation.

Macrophages are sentinels of the innate immune system, and the human monocytic cell line THP-1 is one of the widely used in vitro models to study inflammatory processes and immune responses. Several monocyte-to-macrophage differentiation protocols exist, with phorbol 12-myristate-13-acetate (PMA) being the most commonly used and accepted method. However, the concentrations and duration of PMA treatment vary widely in the published literature and could affect the probed phenotype, however their effect on protein expression is not fully deciphered. In this study, we employed a dimethyl labeling-based quantitative proteomics approach to determine the changes in the protein repertoire of macrophage-like cells differentiated from THP-1 monocytes by three commonly used PMA-based differentiation protocols. Employing an integrated network analysis, we show that variations in PMA concentration and duration of rest post-stimulation result in downstream differences in the protein expression and cellular signaling processes. We demonstrate that these differences result in altered inflammatory responses, including variation in the expression of cytokines upon stimulation with various Toll-like receptor (TLR) agonists. Together, these findings provide a valuable resource that significantly expands the knowledge of protein expression dynamics with one of the most common in vitro models for macrophages, which in turn has a profound impact on the immune as well as inflammatory responses being studied.

A Semi-automated Organoid Screening Method Demonstrates Epigenetic Control of Intestinal Epithelial Differentiation.

Intestinal organoids are an excellent model to study epithelial biology. Yet, the selection of analytical tools to accurately quantify heterogeneous organoid cultures remains limited. Here, we developed a semi-automated organoid screening method, which we applied to a library of highly specific chemical probes to identify epigenetic regulators of intestinal epithelial biology. The role of epigenetic modifiers in adult stem cell systems, such as the intestinal epithelium, is still undefined. Based on this resource dataset, we identified several targets that affected epithelial cell differentiation, including HDACs, EP300/CREBBP, LSD1, and type I PRMTs, which were verified by complementary methods. For example, we show that inhibiting type I PRMTs, which leads enhanced epithelial differentiation, blocks the growth of adenoma but not normal organoid cultures. Thus, epigenetic probes are powerful tools to study intestinal epithelial biology and may have therapeutic potential.

Common Nodes of Virus-Host Interaction Revealed Through an Integrated Network Analysis.

Viruses are one of the major causes of acute and chronic infectious diseases and thus a major contributor to the global burden of disease. Several studies have shown how viruses have evolved to hijack basic cellular pathways and evade innate immune response by modulating key host factors and signaling pathways. A collective view of these multiple studies could advance our understanding of virus-host interactions and provide new therapeutic perspectives for the treatment of viral diseases. Here, we performed an integrative meta-analysis to elucidate the 17 different host-virus interactomes. Network and bioinformatics analyses showed how viruses with small genomes efficiently achieve the maximal effect by targeting multifunctional and highly connected host proteins with a high occurrence of disordered regions. We also identified the core cellular process subnetworks that are targeted by all the viruses. Integration with functional RNA interference (RNAi) datasets showed that a large proportion of the targets are required for viral replication. Furthermore, we performed an interactome-informed drug re-purposing screen and identified novel activities for broad-spectrum antiviral agents against hepatitis C virus and human metapneumovirus. Altogether, these orthogonal datasets could serve as a platform for hypothesis generation and follow-up studies to broaden our understanding of the viral evasion landscape.

Dynamics of Dual Specificity Phosphatases and Their Interplay with Protein Kinases in Immune Signaling.

Dual specificity phosphatases (DUSPs) have a well-known role as regulators of the immune response through the modulation of mitogen-activated protein kinases (MAPKs). Yet the precise interplay between the various members of the DUSP family with protein kinases is not well understood. Recent multi-omics studies characterizing the transcriptomes and proteomes of immune cells have provided snapshots of molecular mechanisms underlying innate immune response in unprecedented detail. In this study, we focus on deciphering the interplay between members of the DUSP family with protein kinases in immune cells using publicly available omics datasets. Our analysis resulted in the identification of potential DUSP-mediated hub proteins including MAPK7, MAPK8, AURKA, and IGF1R. Furthermore, we analyzed the association of DUSP expression with TLR4 signaling and identified VEGF, FGFR, and SCF-KIT pathway modules to be regulated by the activation of TLR4 signaling. Finally, we identified several important kinases including LRRK2, MAPK8, and cyclin-dependent kinases as potential DUSP-mediated hubs in TLR4 signaling. The findings from this study have the potential to aid in the understanding of DUSP signaling in the context of innate immunity. Further, this will promote the development of therapeutic modalities for disorders with aberrant DUSP signaling.

The TLR4 adaptor TRAM controls the phagocytosis of Gram-negative bacteria by interacting with the Rab11-family interacting protein 2.

Phagocytosis is a complex process that eliminates microbes and is performed by specialised cells such as macrophages. Toll-like receptor 4 (TLR4) is expressed on the surface of macrophages and recognizes Gram-negative bacteria. Moreover, TLR4 has been suggested to play a role in the phagocytosis of Gram-negative bacteria, but the mechanisms remain unclear. Here we have used primary human macrophages and engineered THP-1 monocytes to show that the TLR4 sorting adapter, TRAM, is instrumental for phagocytosis of Escherichia coli as well as Staphylococcus aureus. We find that TRAM forms a complex with Rab11 family interacting protein 2 (FIP2) that is recruited to the phagocytic cups of E. coli. This promotes activation of the actin-regulatory GTPases Rac1 and Cdc42. Our results show that FIP2 guided TRAM recruitment orchestrates actin remodelling and IRF3 activation, two events that are both required for phagocytosis of Gram-negative bacteria.

Coactivation of TLR2 and TLR8 in Primary Human Monocytes Triggers a Distinct Inflammatory Signaling Response.

Innate immune signaling is essential to mount a fast and specific immune response to pathogens. Monocytes and macrophages are essential cells in the early response in their capacity as ubiquitous phagocytic cells. They phagocytose microorganisms or damaged cells and sense pathogen/damage-associated molecular patterns (PAMPs/DAMPs) through innate receptors such as Toll-like receptors (TLRs). We investigated a phenomenon where co-signaling from TLR2 and TLR8 in human primary monocytes provides a distinct immune activation profile compared to signaling from either TLR alone. We compare gene signatures induced by either stimulus alone or together and show that co-signaling results in downstream differences in regulation of signaling and gene transcription. We demonstrate that these differences result in altered cytokine profiles between single and multi-receptor signaling, and show how it can influence both T-cell and neutrophil responses. The end response is tailored to combat extracellular pathogens, possibly by modifying the regulation of IFNß and IL12-family cytokines.

Proteogenomic Analysis to Identify Missing Proteins from Haploid Cell Lines.

Chromosome-centric Human Proteome Project aims at identifying and characterizing protein products encoded from all human protein-coding genes. As of early 2017, 19 837 protein-coding genes have been annotated in the neXtProt database including 2691 missing proteins that have never been identified by mass spectrometry. Missing proteins may be low abundant in many cell types or expressed only in a few cell types in human body such as sperms in testis. In this study, we performed expression proteomics of two near-haploid cell types such as HAP1 and KBM-7 to hunt for missing proteins. Proteomes from the two haploid cell lines were analyzed on an LTQ Orbitrap Velos, producing a total of 200 raw mass spectrometry files. After applying 1% false discovery rates at both levels of peptide-spectrum matches and proteins, more than 10 000 proteins were identified from HAP1 and KBM-7, resulting in the identification of nine missing proteins. Next, unmatched spectra were searched against protein databases translated in three frames from noncoding RNAs derived from RNA-Seq data, resulting in six novel protein-coding regions after careful manual inspection. This study demonstrates that expression proteomics coupled to proteogenomic analysis can be employed to identify many annotated and unannotated missing proteins.

Characterization of DIP0733, a multi-functional virulence factor of Corynebacterium diphtheriae.

Corynebacterium diphtheriae is typically recognized as an extracellular pathogen. However, a number of studies revealed its ability to invade epithelial cells, indicating a more complex pathogen-host interaction. The molecular mechanisms controlling and facilitating internalization of Cor. diphtheriae are poorly understood. In this study, we investigated the role of DIP0733 as virulence factor to elucidate how it contributes to the process of pathogen-host cell interaction. Based on in vitro experiments, it was suggested recently that the DIP0733 protein might be involved in adhesion, invasion of epithelial cells and induction of apoptosis. A corresponding Cor. diphtheriae mutant strain generated in this study was attenuated in its ability to colonize and kill the host in a Caenorhabditis elegans infection model system. Furthermore, the mutant showed an altered adhesion pattern and a drastically reduced ability to adhere and invade epithelial cells. Subsequent experiments showed an influence of DIP0733 on binding of Cor. diphtheriae to extracellular matrix proteins such as collagen and fibronectin. Furthermore, based on its fibrinogen-binding activity, DIP0733 may play a role in avoiding recognition of Cor. diphtheriae by the immune system. In summary, our findings support the idea that DIP0733 is a multi-functional virulence factor of Cor. diphtheriae.