V. cardui de novo transcriptome assembly

V. cardui de novo transcriptome assembl

Proteomics Reveals a Role for Attachment in Monocyte Differentiation into Efficient Proinflammatory Macrophages

Monocytes are blood-borne cells of the innate immune system. They can be differentiated and activated into proinflammatory macrophages that might be employed in tumor immune therapy. Monocyte exposure to lipopolysaccharide (LPS) is a standard method to induce a pro­inflammatory macrophage state, with the resultant population comprising both adherent and nonadherent cells. In the current study, we aimed to identify the differences in proteomes of these monocyte subpopulations, which addresses a more general question about the role of attachment in monocyte differentiation. Label-free proteomics of a model of human monocytes (THP-1 cell line) revealed that the cells remaining in suspension upon LPS treatment were activated by cytokines and primed for rapid responsiveness to pathogens. In terms of proteome change, the adhesion process was orthogonal to activation. Adherent cells exhibited signs of differentiation and enhanced innate immune responsivity, being closer to macrophages. These findings indicate that adherent, LPS-treated cells would be more appropriate for use in tumor therapeutic applications

Establishing a Proteomics-Based Monocyte Assay To Assess Differential Innate Immune Activation Responses

Innate immune cells are complex systems that can be simultaneously activated in a variety of ways. Common methods currently used to estimate the response of innate immune cells to stimuli are usually biased toward a single mode of activation. The aim of this study was to assess the possibility of designing an assay based on unbiased proteome analysis that would be capable of predicting the complex response of the innate immune system to various challenges. Monocytes were used as representative cells of the innate immune system. The underlying hypothesis was that their proteome response to different activating molecules would reflect the immunogenicity of these molecules. To identify the main modes of response, we treated the human monocytic THP-1 cell line with nine different stimuli. Differentiation and activation were determined to be the two major modes of monocyte response, with PMA causing the strongest differentiation and Pam3CSK4 causing the strongest proinflammatory activation. The established assay was applied to characterize the monocyte response to epidermal growth factor peptide containing isoaspartate, which induced differentiation but not proinflammatory activation. Because of its versatility, robustness, and specificity, this new assay is likely to find a niche among the more established immunological methods

Proteomics Reveals a Role for Attachment in Monocyte Differentiation into Efficient Proinflammatory Macrophages

Monocytes are blood-borne cells of the innate immune system. They can be differentiated and activated into proinflammatory macrophages that might be employed in tumor immune therapy. Monocyte exposure to lipopolysaccharide (LPS) is a standard method to induce a pro­inflammatory macrophage state, with the resultant population comprising both adherent and nonadherent cells. In the current study, we aimed to identify the differences in proteomes of these monocyte subpopulations, which addresses a more general question about the role of attachment in monocyte differentiation. Label-free proteomics of a model of human monocytes (THP-1 cell line) revealed that the cells remaining in suspension upon LPS treatment were activated by cytokines and primed for rapid responsiveness to pathogens. In terms of proteome change, the adhesion process was orthogonal to activation. Adherent cells exhibited signs of differentiation and enhanced innate immune responsivity, being closer to macrophages. These findings indicate that adherent, LPS-treated cells would be more appropriate for use in tumor therapeutic applications

Cytotoxic and Proinflammatory Effects of Metal-Based Nanoparticles on THP‑1 Monocytes Characterized by Combined Proteomics Approaches

Thorough characterization of toxic effects of nanoparticles (NP) is desirable due to the increasing risk of potential environmental contamination by NP. In the current study, we combined three recently developed proteomics approaches to assess the effects of Au, CuO, and CdTe NP on the innate immune system. The human monocyte cell line THP-1 was employed as a model. The anticancer drugs camptothecin and doxorubicin were used as positive controls for cell death, and lipopolysaccharide was chosen as a positive control for proinflammatory activation. Despite equivalent overall toxicity effect (50 ± 10% dead cells), the three NP induced distinctly different proteomics signatures, with the strongest effect being induced by CdTe NP, followed by CuO and gold NP. The CdTe toxicity mechanism involves down-regulation of topoisomerases. The effect of CuO NP is most reminiscent of oxidative stress and involves up-regulation of proteins involved in heat response. The gold NP induced up-regulation of the inflammatory mediator, NF-κB, and its inhibitor TIPE2 was identified as a direct target of gold NP. Furthermore, gold NP triggered activation of NF-κB as evidenced by phosphorylation of the p65 subunit. Overall, the combined proteomics approach described here can be used to characterize the effects of NP on immune cells

IgG Antibodies to Cyclic Citrullinated Peptides Exhibit Profiles Specific in Terms of IgG Subclasses, Fc-Glycans and a Fab-Peptide Sequence

<div><p>The Fc-glycan profile of IgG₁ anti-citrullinated peptide antibodies (ACPA) in rheumatoid arthritis (RA) patients has recently been reported to be different from non-ACPA IgG₁, a phenomenon which likely plays a role in RA pathogenesis. Herein we investigate the Fc-glycosylation pattern of all ACPA-IgG isotypes and simultaneously investigate in detail the IgG protein-chain sequence repertoire. IgG from serum or plasma (S/P, n = 14) and synovial fluid (SF, n = 4) from 18 ACPA-positive RA-patients was enriched using Protein G columns followed by ACPA-purification on cyclic citrullinated peptide-2 (CCP2)-coupled columns. Paired ACPA (anti-CCP2 eluted IgG) and IgG flow through (FT) fractions were analyzed by LC-MS/MS-proteomics. IgG peptides, isotypes and corresponding Fc-glycopeptides were quantified and interrogated using uni- and multivariate statistics. The Fc-glycans from the IgG₄ peptide EEQ<b>F</b>NST<b>Y</b>R was validated using protein A column purification. Relative to FT-IgG₄, the ACPA-IgG₄ Fc-glycan-profile contained lower amounts (p = 0.002) of the agalacto and asialylated core-fucosylated biantennary form (FA2) and higher content (p = 0.001) of sialylated glycans. Novel differences in the Fc-glycan-profile of ACPA-IgG₁ compared to FT-IgG₁ were observed in the distribution of bisected forms (n = 5, p = 0.0001, decrease) and mono-antennnary forms (n = 3, p = 0.02, increase). Our study also confirmed higher abundance of FA2 (p = 0.002) and lower abundance of afucosylated forms (n = 4, p = 0.001) in ACPA-IgG₁ relative to FT-IgG₁ as well as lower content of IgG₂ (p = 0.0000001) and elevated content of IgG₄ (p = 0.004) in ACPA compared to FT. One λ-variable peptide sequence was significantly increased in ACPA (p = 0.0001). In conclusion, the Fc-glycan profile of both ACPA-IgG₁ and ACPA-IgG₄ are distinct. Given that IgG₁ and IgG₄ have different Fc-receptor and complement binding affinities, this phenomenon likely affects ACPA effector- and immune-regulatory functions in an IgG isotype-specific manner. These findings further highlight the importance of antibody characterization in relation to functional <i>in vivo</i> and <i>in vitro</i> studies.</p></div

Log₁₀ fold difference of FA2 and FA2G2S1 comparing the ACPA/FT ratio for the different IgG Fc-glycopeptide types.

<p>Values ≥0 (dashed line) indicate an intra-individually increased amount in ACPA. P-values (comparing FT and ACPA for each subject) were obtained with paired t-test. NS: Not Significant. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113924#pone.0113924.s005" target="_blank">Figure S5</a> for FT and ACPA differences in direct values.</p

Multivariate modelling based on the Fc-glycan and protein/peptide correlations in the ACPA and FT sample set.

<p>Subjects are labeled according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113924#pone.0113924.s007" target="_blank">Table S1</a> and colored according to FT (light gray [S/P]/light red [SF]) and ACPA (dark gray [S/P]/dark red [SF]). (<b>A</b>) PCA scores plot. FT and ACPA samples are separated in component t <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113924#pone.0113924-Klareskog2" target="_blank">[3]</a>. (<b>B</b>) OPLS-DA scores plot, the model was constructed to distinguish FT samples and ACPA samples and generated a strong (R² = 0.93, Q² = 0.85, CV ANOVA p-value = 2.3E-12) model, with distinct separation of FT (negative) an ACPA (positive) along the predictive X-axis t <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113924#pone.0113924-Arend1" target="_blank">[1]</a>. (<b>C</b>) Loading column plot of the predictive axis (pq <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113924#pone.0113924-Arend1" target="_blank">[1]</a>). Features with positive pq <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113924#pone.0113924-Arend1" target="_blank">[1]</a> values indicate positive ACPA correlation and features with negative pq <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113924#pone.0113924-Arend1" target="_blank">[1]</a> values indicate negative ACPA correlation. Only glycans (blue) and proteins (grey) correlating with 95% confidence are shown.</p

Composition (%) of IgG heavy chain isotypes.

<p>(<b>A</b>) FT isotype composition. (<b>B</b>) ACPA isotype composition. (<b>C</b>) Log₁₀ fold change of the intra-individual ACPA/FT-ratio. Values ≥0 (dashed line) indicate an increase in ACPA. Given p-values (comparing FT and ACPA) were obtained with paired t-test. NS: Not Significant.</p

Log₁₀ fold intra-individual ratio [ACPA/FT] of the sum of IgG₁ bisected forms (n = 5), sum of IgG₁ afucosylated forms (n = 4) and sum of IgG₁ mono-antennary forms (n = 3).

<p>Values ≥0 (dashed line) indicate an intra-individually increased amount in ACPA. Given p-values (comparing FT and ACPA) were obtained with paired t-test. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113924#pone.0113924.s004" target="_blank">Figure S4</a> for FT and ACPA differences in direct values.</p