Cristina C. Clement

Oxidative stress, inflamm-aging and immunosenescence.

Immunosenescence is characterized by a decreased ability of the immune system to respond to foreign antigens, as well as a decreased ability to maintain tolerance to self-antigens. This results in an increased susceptibility to infection and cancer and reduced responses to vaccination [1-5]. The mechanisms underlying immunosenescence comprise a series of cellular and molecular events involving alteration of several biochemical pathways and different cellular populations, and for the most part our understanding of these molecular mechanisms is still fragmentary. In this review we will focus on the process of senescence associated with oxidative stress, in particular how protein oxidation alters the functionality of immune cells and how oxidative stress contributes to a chronic inflammatory process often referred as inflamm-aging.

Endosomal damage and TLR2 mediated inflammasome activation by alkane particles in the generation of aseptic osteolysis

Ultra-high molecular weight polyethylene is widely used as a bearing surface in prosthetic arthroplasty. Over time the generation of implant-derived wear particles can initiate an inflammatory reaction characterized by periprosthetic inflammation and ultimately bone resorption at the prosthetic bone interface. Herein we present evidence that the different sized particles as well as the different length alkane polymers generated by implant wear leads to a two component inflammatory response. Polymeric alkane structures, with side chain oxidations, directly bind and activate the TLR-1/2 signaling pathway. Whereas micron- and nanometer-sized particulate debris are extensively phagocyted and induce enlargement, fusion and disruption of endosomal compartments. The resulting lysosomal damage and subsequent enzymatic leakage induces the NALP3 inflammasome activation as determined by cathepsins S and B cytosolic release, Caspase 1 activation and processing of pro-IL-1beta, and pro-IL-18. These two processes synergistically results in the initiation of a strong inflammatory response with consequent cellular necrosis and extracellular matrix degradation.

Molecular analysis of chromium and cobalt-related toxicity

Occupational and environmental exposure to Co and Cr has been previously linked to a wide array of inflammatory and degenerative conditions and cancer. Recently, significant health concerns have been raised by the high levels of Cr and Co ions and corrosion products released by biomedical implants. Herein, we set to analyze the biological responses associated with Co and Cr toxicity. Histological, ultrastructural, and elemental analysis, performed on Cr and Co exposed patients reveal the presence of corrosion products, metallic wear debris and metal ions at varying concentrations. Metallic ions and corrosion products were also generated in vitro following macrophage phagocytosis of metal alloys. Ex vivo redox proteomic mapped several oxidatively damaged proteins by Cr(III) and Co(II)-induced Fenton reaction. Importantly, a positive correlation between the tissue amounts of Cr(III) and Co(II) ions and tissue oxidative damage was observed. Immobilized- Cr(III) and Co(II) affinity chromatography indicated that metal ions can also directly bind to several metallo and non-metalloproteins and, as demonstrated for aldolase and catalase, induce loss of their biological function. Altogether, our analysis reveals several biological mechanisms leading to tissue damage, necrosis, and inflammation in patients with Cr and Co-associated adverse local tissue reactions.

The lymph as a pool of self-antigens.

Prenodal lymph is generated from the interstitial fluid that surrounds organs, and thus contains products of organ metabolism and catabolism. New proteomic analyses of lymph have identified proteins and peptides that are derived from capillary extravasation and tissue-specific proteins. Many of these peptides are detected at nanomolar concentrations in the lymph before passage through a regional lymph node. Before entering the node and once inside, proteins and processed peptides are filtered from the lymph by circulating immature dendritic cells (DCs) or non-activated nodal antigen-presenting cells (APCs) (macrophages, B cells and immature DCs). Here, we suggest that this process ensures organ-specific self-antigens are displayed to circulating and nodal APCs, thus contributing to the maintenance of peripheral tolerance.

Age-Related Oxidative Stress Compromises Endosomal Proteostasis

A hallmark of aging is an imbalance between production and clearance of reactive oxygen species and increased levels of oxidatively damaged biomolecules. Herein, we demonstrate that splenic and nodal antigen-presenting cells purified from aging mice accumulate oxidatively modified proteins with side-chain carbonylation, advanced glycation end products, and lipid peroxidation. Furthermore, we show that the endosomal accumulation of oxidatively modified proteins interferes with the efficient processing of exogenous antigens and degradation of macroautophagy-delivered proteins. In support of a causative role for oxidized products in the inefficient immune response, a decrease in oxidative stress improved the adaptive immune response to immunizing antigens. These findings underscore a previously unrecognized negative effect of age-dependent changes in cellular proteostasis on the immune response.

An expanded self-antigen peptidome is carried by the human lymph as compared to the plasma

Background The pre-nodal afferent lymph is the fluid which directly derives from the extracellular milieu from every parenchymal organ and, as it continues to circulate between the cells, it collects products deriving from the organ metabolism/catabolism. A comprehensive qualitative and quantitative investigation of the self-antigenic repertoire transported by the human lymph is still missing. Methodology/Principal Findings A major difference between lymph and plasma could be visualized by FPLC and 2D gel in the amount of low molecular weight products corresponding to peptide fragments. Naturally processed peptides in normal pre-nodal human lymph were then fractionated by HPLC and characterized by multidimensional mass spectrometry. Analysis of more then 300 sequences identified self-peptides derived from both intracellular and extracellular proteins revealing the variety of catabolic products transported by human lymph. Quantitative analysis established that at least some of these peptides are present in the circulating lymph in nanomolar concentration. Conclusions/Significance The peptidome, generated by physiological tissue catabolism and transported by the pre-nodal lymph, is in addition to the self-peptidome generated in endosomal compartment. Unlike self antigen processed by local or nodal APC, which mostly produce epitopes constrained by the endosomal processing activity, self antigens present in the lymph could derived from a wider variety of processing pathways; including caspases, involved in cellular apoptosis, and ADAM and other metalloproteinases involved in surface receptor editing, cytokines processing and matrix remodeling. Altogether, expanding the tissue-specific self-repertoire available for the maintenance of immunological tolerance.

Protein expression profiles of human lymph and plasma mapped by 2D-DIGE and 1D SDS-PAGE coupled with nanoLC-ESI-MS/MS bottom-up proteomics

In this study a proteomic approach was used to define the protein content of matched samples of afferent prenodal lymph and plasma derived from healthy volunteers. The analysis was performed using two analytical methodologies coupled with nanoliquid chromatography-tandem mass spectrometry: one-dimensional gel electrophoresis (1DEF nanoLC Orbitrap-ESI-MS/MS), and two-dimensional fluorescence difference-in-gel electrophoresis (2D-DIGE nanoLC-ESI-MS/MS). The 253 significantly identified proteins (p<0.05), obtained from the tandem mass spectrometry data, were further analyzed with pathway analysis (IPA) to define the functional signature of prenodal lymph and matched plasma. The 1DEF coupled with nanoLC-MS-MS revealed that the common proteome between the two biological fluids (144 out of 253 proteins) was dominated by complement activation and blood coagulation components, transporters and protease inhibitors. The enriched proteome of human lymph (72 proteins) consisted of products derived from the extracellular matrix, apoptosis and cellular catabolism. In contrast, the enriched proteome of human plasma (37 proteins) consisted of soluble molecules of the coagulation system and cell-cell signaling factors. The functional networks associated with both common and source-distinctive proteomes highlight the principal biological activity of these immunologically relevant body fluids.

Immunogenecity of Modified Alkane Polymers Is Mediated through TLR1/2 Activation

Background With the advancement of biomedical technology, artificial materials have been developed to replace diseased, damaged or nonfunctional body parts. Among such materials, ultra high molecular weight alkane or modified alkyl polymers have been extensively used in heart valves, stents, pacemakers, ear implants, as well as total joint replacement devices. Although much research has been undertaken to design the most non-reactive biologically inert polyethylene derivatives, strong inflammatory responses followed by rejection and failure of the implant have been noted. Methodology/Principal Findings Purification of the alkane polymers from the site of inflammation revealed extensive “in vivo” oxidation as detected by fourier transformed infra-red spectroscopy. Herein, we report the novel observation that oxidized alkane polymers induced activation of TLR1/2 pathway as determined by ligand dependent changes in intrinsic tyrosine fluorescence intensity and NF-κΒ luciferase gene assays. Oxidized polymers were very effective in activating dendritic cells and inducing secretion of pro-inflammatory cytokines. Molecular docking of the oxidized alkanes designated ligand specificity and polymeric conformations fitting into the TLR1/2 binding grooves. Conclusion/Significance This is the first report of a synthetic polymer activating immune responses through TLR binding.

The Lymph Self-Antigen Repertoire

The lymphatic fluid originates from the interstitial fluid which bathes every parenchymal organ and reflects the “omic” composition of the tissue from which it originates in its physiological or pathological signature. Several recent proteomic analyses have mapped the proteome-degradome and peptidome of this immunologically relevant fluid pointing to the lymph as an important source of tissue-derived self-antigens. A vast array of lymph-circulating peptides have been mapped deriving from a variety of processing pathways including caspases, cathepsins, MMPs, ADAMs, kallikreins, calpains, and granzymes, among others. These self peptides can be directly loaded on circulatory dendritic cells and expand the self-antigenic repertoire available for central and peripheral tolerance.

Annexin A2 binds to endosomes following organelle destabilization by particulate wear debris

Endosomal functions are contingent on the integrity of the organelle-limiting membrane, whose disruption induces inflammation and cell death. Here we show that phagocytosis of ultrahigh molecular weight polyethylene particles induces damage to the endosomal-limiting membrane and results in the leakage of cathepsins into the cytosol and NLRP3-inflammasome activation. Annexin A2 recruitment to damaged organelles is shown by two-dimensional DIGE protein profiling, endosomal fractionation, confocal analysis of endogenous and annexin A2-GFP transfected cells, and immunogold labelling. Binding experiments, using fluorescent liposomes, confirms annexin A2 recruitment to endosomes containing phagocytosed polyethylene particles. Finally, an increase in cytosolic cathepsins, NLRP3-inflammasome activation, and IL-1 production is seen in dendritic cells from annexin A2-null mice, following exposure to polyethylene particles. Together, the results indicate a functional role of annexin A2 binding to endosomal membranes following organelle destabilization.