Citrullination Controls Dendritic Cell Transdifferentiation into Osteoclasts.

An increased repertoire of potential osteoclast (OC) precursors could accelerate the development of bone-erosive OCs and the consequent bone damage in rheumatoid arthritis (RA). Immature dendritic cells (DCs) can develop into OCs, however, the mechanisms underlying this differentiation switch are poorly understood. We investigated whether protein citrullination and RA-specific anti-citrullinated protein Abs (ACPAs) could regulate human blood-derived DC-OC transdifferentiation. We show that plasticity toward the OC lineage correlated with peptidyl arginine deiminase (PAD) activity and protein citrullination in DCs. Citrullinated actin and vimentin were present in DCs and DC-derived OCs, and both proteins were deposited on the cell surface, colocalizing with ACPAs binding to the cells. ACPAs enhanced OC differentiation from monocyte-derived or circulating CD1c+ DCs by increasing the release of IL-8. Blocking IL-8 binding or the PAD enzymes completely abolished the stimulatory effect of ACPAs, whereas PAD inhibition reduced steady-state OC development, as well, suggesting an essential role for protein citrullination in DC-OC transdifferentiation. Protein citrullination and ACPA binding to immature DCs might thus promote differentiation plasticity toward the OC lineage, which can facilitate bone erosion in ACPA-positive RA.

Approach for Identifying Human Leukocyte Antigen (HLA)-DR Bound Peptides from Scarce Clinical Samples.

Immune-mediated diseases strongly associating with human leukocyte antigen (HLA) alleles are likely linked to specific antigens. These antigens are presented to T cells in the form of peptides bound to HLA molecules on antigen presenting cells, e.g. dendritic cells, macrophages or B cells. The identification of HLA-DR-bound peptides presents a valuable tool to investigate the human immunopeptidome. The lung is likely a key player in the activation of potentially auto-aggressive T cells prior to entering target tissues and inducing autoimmune disease. This makes the lung of exceptional interest and presents an ideal paradigm to study the human immunopeptidome and to identify antigenic peptides.Our previous investigation of HLA-DR peptide presentation in the lung required high numbers of cells (800 × 10(6) bronchoalveolar lavage (BAL) cells). Because BAL from healthy nonsmokers typically contains 10-15 × 10(6) cells, there is a need for a highly sensitive approach to study immunopeptides in the lungs of individual patients and controls.In this work, we analyzed the HLA-DR immunopeptidome in the lung by an optimized methodology to identify HLA-DR-bound peptides from low cell numbers. We used an Epstein-Barr Virus (EBV) immortalized B cell line and bronchoalveolar lavage (BAL) cells obtained from patients with sarcoidosis, an inflammatory T cell driven disease mainly occurring in the lung. Specifically, membrane complexes were isolated prior to immunoprecipitation, eluted peptides were identified by nanoLC-MS/MS and processed using the in-house developed ClusterMHCII software. With the optimized procedure we were able to identify peptides from 10 × 10(6) cells, which on average correspond to 10.9 peptides/million cells in EBV-B cells and 9.4 peptides/million cells in BAL cells. This work presents an optimized approach designed to identify HLA-DR-bound peptides from low numbers of cells, enabling the investigation of the BAL immunopeptidome from individual patients and healthy controls in order to identify disease-associated peptides.

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.

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.

Shared immunological targets in the lungs and joints of patients with rheumatoid arthritis: identification and validation.

OBJECTIVES: Immunological events in the lungs might trigger production of anti-citrullinated protein antibodies during early rheumatoid arthritis (RA). We investigated the presence of shared immunological citrullinated targets in joints and lungs of patients with RA. PATIENTS AND METHODS: Proteins extracted from bronchial (n=6) and synovial (n=7) biopsy specimens from patients with RA were investigated by mass spectrometry-based proteomics. One candidate peptide was synthesised and used to investigate by ELISA the presence of antibodies in patients with RA (n=393), healthy controls (n=152) and disease controls (n=236). HLA-DRB1 shared epitope (SE) alleles were detected in patients with RA. RESULTS: Ten citrullinated peptides belonging to seven proteins were identified, with two peptides shared between the synovial and bronchial biopsy samples. Further analysis, using accurate mass and retention time, enabled detection of eight citrullinated peptides in synovial and seven in bronchial biopsy specimens, with five peptides shared between the synovial and bronchial biopsy specimens. Two citrullinated vimentin (cit-vim) peptides were detected in the majority of synovial and lung tissues. Antibodies to a synthesised cit-vim peptide candidate (covering both cit-vim peptides identified in vivo) were present in 1.8% of healthy controls, 15% of patients with RA, and 3.4% of disease controls. Antibodies to cit-vim peptide were associated with the presence of the SE alleles in RA. CONCLUSIONS: Identical citrullinated peptides are present in bronchial and synovial tissues, which may be used as immunological targets for antibodies of patients with RA. The data provide further support for a link between lungs and joints in RA and identify potential targets for immunity that may mediate this link.