No abstract

Rheumatoid arthritis (RA) is characterized by synovial joint inflammation and by development of pathogenic humoral and cellular autoimmunity to citrullinated proteins. Neutrophil extracellular traps (NETs) are a source of citrullinated autoantigens and activate RA synovial fibroblasts (FLS), cells crucial in joint damage. We investigated the molecular mechanisms by which NETs promote proinflammatory phenotypes in FLS, and whether these interactions generate pathogenic anti-citrulline adaptive immune responses. NETs containing citrullinated peptides are internalized by FLS through a RAGE-TLR9 pathway promoting FLS inflammatory phenotype and their upregulation of MHC class II. Once internalized, arthritogenic NET-peptides are loaded into FLS MHC class II and presented to Ag-specific T cells. HLADRB1*0401 transgenic mice immunized with mouse FLS loaded with NETs develop antibodies specific to citrullinated forms of relevant RA autoantigens implicated in RA pathogenesis as well as cartilage damage. These results implicate FLS as mediators in RA pathogenesis, through the internalization and presentation of NET citrullinated peptides to the adaptive immune system leading to pathogenic autoimmunity and cartilage damage.

In response to herbivore (Spodoptera littoralis) attack, lima bean (Phaseolus lunatus) leaves produced hydrogen peroxide (H 2 O 2 ) in concentrations that were higher when compared to mechanically damaged (MD) leaves. Cellular and subcellular localization analyses revealed that H 2 O 2 was mainly localized in MD and herbivore-wounded (HW) zones and spread throughout the veins and tissues. Preferentially, H 2 O 2 was found in cell walls of spongy and mesophyll cells facing intercellular spaces, even though confocal laser scanning microscopy analyses also revealed the presence of H 2 O 2 in mitochondria/peroxisomes. Increased gene and enzyme activations of superoxide dismutase after HW were in agreement with confocal laser scanning microscopy data. After MD, additional application of H 2 O 2 prompted a transient transmembrane potential (V m ) depolarization, with a V m depolarization rate that was higher when compared to HW leaves. In transgenic soybean (Glycine max) suspension cells expressing the Ca 21 -sensing aequorin system, increasing amounts of added H 2 O 2 correlated with a higher cytosolic calcium ([Ca 21 ] cyt ) concentration. In MD and HW leaves, H 2 O 2 also triggered the increase of [Ca 21 ] cyt , but MD-elicited [Ca 21 ] cyt increase was more pronounced when compared to HW leaves after addition of exogenous H 2 O 2 . The results clearly indicate that V m depolarization caused by HW makes the membrane potential more positive and reduces the ability of lima bean leaves to react to signaling molecules.

The intracellular trafficking of major histocompatibility complex class I (MHC-I) proteins is directed by three quality control mechanisms that test for their structural integrity, which is correlated to the binding of high-affinity antigenic peptide ligands. To investigate which molecular features of MHC-I these quality control mechanisms detect, we have followed the hypothesis that suboptimally loaded MHC-I molecules are characterized by their conformational mobility in the F-pocket region of the peptide-binding site. We have created a novel variant of an MHC-I protein, K b -Y84C, in which two a-helices in this region are linked by a disulfide bond that mimics the conformational and dynamic effects of bound highaffinity peptide. K b -Y84C shows a remarkable increase in the binding affinity to its light chain, beta-2 microglobulin (b 2 m), and bypasses all three cellular quality control steps. Our data demonstrate (1) that coupling between peptide and b 2 m binding to the MHC-I heavy chain is mediated by conformational dynamics; (2) that the folded conformation of MHC-I, supported by b 2 m, plays a decisive role in passing the ER-to-cell-surface transport quality controls; and (3) that b 2 m association is also tested by the cell surface quality control that leads to MHC-I endocytosis.