Fundamental roles of the innate-like repertoire of natural antibodies in immune homeostasis

The composition of the early immune repertoire is biased with prominent expression of spontaneously arising B cell clones that produce IgM with recurrent and often autoreactive binding specificities. Amongst these naturally arising antibodies (NAbs) are IgM antibodies that specifically recognized amaged and senescent cells, often via oxidation-associated neo-determinants. These NAbs are present from birth and can be further boosted by apoptotic cell challenge. Recent studies have shown that IgM NAb to apoptotic cells can enhance phagocytic clearance, as well as suppress proinflammatory responses induced via Toll-like receptors, and block pathogenic IgG-immune complex (IC)-mediated inflammatory responses. Specific antibody effector functions appear to be involved, as these anti-inflammatory properties are dependent on IgM-mediated recruitment of the early recognition factors of complement. Clinical surveys have suggested that anti-apoptotic cell (AC) IgM NAbs may modulate disease activity in some patients with autoimmune disease. In mechanistic studies, anti-AC NAbs were shown to act in dendritic cells by inhibition of the mitogen-activated protein kinase (MAPK) pathway, a primary signal transduction pathway that controls inflammatory responses. This immunomodulatory pathway has an absolute requirement for the induction of MAPK phosphatase-1. Taken together, recent studies have elucidated the novel properties of a class of protective NAbs, which may directly blunt inflammatory responses through a primitive pathway for regulation of the innate immune system

Protective Roles of Natural IgM Antibodies

Antibodies are a vital part of the armamentarium of the adaptive immune system for the fine-tuning of the recognition and response to foreign threats. However, in health there are some types of antibodies that instead recognize self-antigens and these contribute to the enhancement of primitive innate functions. This repertoire of natural IgM antibodies is postulated to have been selected during immune evolution for their contributions to critical immunoregulatory and housekeeping properties. The clearance of dying cells is one of the most essential responsibilities of the immune system, which is required to prevent uncontrolled inflammation and autoimmunity. In the murine immune system, natural IgM antibodies that recognize apoptotic cells have been shown to enhance the phagocytic clearance of dead and dying cells and to suppress innate immune signaling pathways. In the mouse, natural IgM are often the products of B-1 cell clones that arise during immune development without an absolute requirement for exogenous antigenic stimulation. In patients with systemic lupus erythematosus, IgM autoantibodies, which bind to neo-epitopes on apoptotic cells, have been demonstrated to be present at significantly higher levels in patients with lower disease activity and with less severe organ damage. While certain specificities of IgM autoantibodies correlate with protection from lupus renal disease, others may convey protective properties from lupus-associated atherosclerotic cardiovascular disease. New and unexpected insights into the functional roles of IgM antibodies are still emerging, especially regarding the functions of natural antibodies. Herein, we review recent progress in our understanding of the potential roles of natural IgM autoantibodies in the regulation of immune homeostasis and for protection from autoimmune and inflammatory diseases

Assigning and visualizing germline genes in antibody repertoires.

Identifying the germline genes involved in immunoglobulin rearrangements is an essential first step in the analysis of antibody repertoires. Based on our prior work in analysing diverse recombinant viruses, we present IgSCUEAL (Immunoglobulin Subtype Classification Using Evolutionary ALgorithms), a phylogenetic approach to assign V and J regions of immunoglobulin sequences to their corresponding germline alleles, with D regions assigned using a simple pairwise alignment algorithm. We also develop an interactive web application for viewing the results, allowing the user to explore the frequency distribution of sequence assignments and CDR3 region length statistics, which is useful for summarizing repertoires, as well as a detailed viewer of rearrangements and region alignments for individual query sequences. We demonstrate the accuracy and utility of our method compared with sequence similarity-based approaches and other non-phylogenetic model-based approaches, using both simulated data and a set of evaluation datasets of human immunoglobulin heavy chain sequences. IgSCUEAL demonstrates the highest accuracy of V and J assignment amongst existing approaches, even when the reassorted sequence is highly mutated, and can successfully cluster sequences on the basis of shared V/J germline alleles.S.K.L.P. and B.M. were supported in part by the U.S. National Institutes of Health (AI110181, AI90970, AI100665, DA34978, GM93939, HL108460, GM110749, LM7092, MH97520, MH83552), the UCSD Center for AIDS Research (Developmental Grant, AI36214, Bioinformatics and Information Technologies Core), the International AIDS Vaccine Initiative (through AI90970), the UC Laboratory Fees Research Program (grant no. 12-LR-236617). G.J.S. was supported in part the U.S. National Institute of Health (AI90118, AI68063, AI40305, and NIAID HHS N272201400019C), and a grant from the Lupus Research Institute. A.S.M.M.H. was supported by an Islamic Development Bank Scholarship, and S.D.W.F. was supported in part by the UK MRC Methodology Research Programme (grant no. MR/J013862/1).This is the final published version. It first appeared at http://rstb.royalsocietypublishing.org/content/370/1676/20140240

Staphylococcus aureus protein A binding to von Willebrand factor A1 domain is mediated by conserved IgG binding regions.

Protein A (Spa) is a surface-associated protein of Staphylococcus aureus best known for its ability to bind to the Fc region of IgG. Spa also binds strongly to the Fab region of the immunoglobulins bearing V(H)3 heavy chains and to von Willebrand factor (vWF). Previous studies have suggested that the protein A-vWF interaction is important in S. aureus adherence to platelets under conditions of shear stress. We demonstrate that Spa expression is sufficient for adherence of bacteria to immobilized vWF under low fluid shear. The full length recombinant Ig-binding region of protein A, Spa-EDABC, fused to glutathione-S-transferase (GST), bound recombinant vWF in a dose-dependent and saturable fashion with half maximal binding of about 30 nm in immunosorbent assays. Full length-Spa did not bind recombinant vWF A3 domain but displayed binding to recombinant vWF domains A1 and D\u27-D3 (half maximal binding at 100 nm and 250 nm, respectively). Each recombinant protein A Ig-binding domain bound to the A1 domain in a similar manner to the full length-Spa molecule (half maximal binding 100 nm). Amino acid substitutions were introduced in the GST-SpaD protein at sites known to be involved in IgG Fc or in V(H)3 Fab binding. Mutants altered in residues that recognized IgG Fc but not those that recognized V(H)3 Fab had reduced binding to vWF A1 and D\u27-D3. This indicated that both vWF regions recognized a region on helices I and II that overlapped the IgG Fc binding site

Apoptotic Cells with Oxidation-specific Epitopes Are Immunogenic and Proinflammatory

Oxidation of low density lipoprotein (LDL) generates a variety of oxidatively modified lipids and lipid-protein adducts that are immunogenic and proinflammatory, which in turn contribute to atherogenesis. Cells undergoing apoptosis also display oxidized moieties on their surface membranes, as determined by binding of oxidation-specific monoclonal antibodies. In the present paper, we demonstrated by mass spectrometry that in comparison with viable cells, membranes of cells undergoing apoptosis contain increased levels of biologically active oxidized phospholipids (OxPLs). Indeed, immunization of mice with syngeneic apoptotic cells induced high autoantibody titers to various oxidation-specific epitopes of oxidized LDL, including OxPLs containing phosphorylcholine, whereas immunization with viable thymocytes, primary necrotic thymocytes, or phosphate-buffered saline did not. Reciprocally, these antisera specifically bound to apoptotic cells through the recognition of oxidation-specific epitopes. Moreover, splenocyte cultures from mice immunized with apoptotic cells spontaneously released significant levels of T helper cell (Th) 1 and Th2 cytokines, whereas splenocytes from controls yielded only low levels. Finally, we demonstrated that the OxPLs of apoptotic cells activated endothelial cells to induce monocyte adhesion, a proinflammatory response that was abrogated by an antibody specific to oxidized phosphatidylcholine. These results suggest that apoptotic cell death generates oxidatively modified moieties, which can induce autoimmune responses and a local inflammatory response by recruiting monocytes via monocyte–endothelial cell interaction

Rethinking the red wolf disease: does Protein S suppress systemic lupus erythematosus clinical activity?

In systemic lupus erythematosus, the forces responsible for disease initiation and self-perpetuation in these clinically heterogeneous populations remain poorly understood. Recent studies of the TAM (Tyro3, Axl and MerTK) family of receptor tyrosine kinases may lead to a better understanding of the fundamental control system responsible for the clearance of apoptotic cells and the regulation of inflammation. In a recent report, serum levels of the TAM ligand, Protein S, was found to correlate with certain disease manifestations and with C3 and C4 levels. Protein S levels could provide a quantitative clinical biomarker but it remains to be determined whether this factor directly affects disease activity

The Lick AGN Monitoring Project 2011: Reverberation Mapping of Markarian 50

The Lick AGN Monitoring Project 2011 observing campaign was carried out over the course of 11 weeks in Spring 2011. Here we present the first results from this program, a measurement of the broad-line reverberation lag in the Seyfert 1 galaxy Mrk 50. Combining our data with supplemental observations obtained prior to the start of the main observing campaign, our dataset covers a total duration of 4.5 months. During this time, Mrk 50 was highly variable, exhibiting a maximum variability amplitude of a factor of 4 in the U-band continuum and a factor of 2 in the H-beta line. Using standard cross-correlation techniques, we find that H-beta and H-gamma lag the V-band continuum by tau_cen = 10.64(-0.93,+0.82) and 8.43(-1.28,+1.30) days, respectively, while the lag of He II 4686 is unresolved. The H-beta line exhibits a symmetric velocity-resolved reverberation signature with shorter lags in the high-velocity wings than in the line core, consistent with an origin in a broad-line region dominated by orbital motion rather than infall or outflow. Assuming a virial normalization factor of f=5.25, the virial estimate of the black hole mass is (3.2+-0.5)*10^7 solar masses. These observations demonstrate that Mrk 50 is among the most promising nearby active galaxies for detailed investigations of broad-line region structure and dynamics.Comment: Accepted for publication in ApJ Letters. 6 pages, 4 figure