Basic Science Highlights
- Abstract #11 A Novel Therapeutic Strategy For The Antiphospholipid Syndrome (APS) Via The Creation Of Decoy And Mutant ‘Super-Decoy’ Peptides
- Abstract # 682 Human Articular Chondrocyte Side Population Retains Chondrogenic Capacity
- Abstract # 1250 Ectopic Lymphoid Structures Expressing AID Drive Self Sustained in situ Production of Autoantibodies in Rheumatoid Synovium
- Abstract # 1763 Regulation of SLE Immune Complex Induced IFN-alpha by Female Sex Steroids
- Abstract # 2017 Ligands Specific for Toll-like Receptors 4 and 5 Induce Distinct Types Of Vasculitis In Human Macrovessels
- Abstract # 2018 Heterogeneity In Pathogen-sensing And T-cell Stimulatory Capacity Of Human Macrovessels From Distinct Vascular Territories
- Abstract # 2114 Er Stress Modifies The IL-23 Response To Toll-like Receptor Agonists And Intracellular Bacteria Infection
Abstract #11 A Novel Therapeutic Strategy For The Antiphospholipid Syndrome (APS) Via The Creation Of Decoy And Mutant ‘Super-Decoy’ Peptides
Authors:
Yiannis Ioannou1, Zurina Romay-Penabad2, Charis Pericleous1, Ian Giles1, Elizabeth Papalardo2, David S. Latchman1, David A. Isenberg1, Anisur Rahman1, Silvia S. Pierangeli2. 1University College London, London, United Kingdom; 2University of Texas Medical Branch, Galveston, TX
Background:
One of the major advances in the understanding of the pathogenesis of the antiphospholipid syndrome has been the identification of b2-glycoprotein 1 as the primary antigen targeted. Despite this insight, the foundation of therapy for APS continues to be generalized anticoagulation. Whether targeted interference of antibody: b2-glycoprotein 1 interactions could result in therapeutic benefit is a novel idea that has thus far not been evaluated.
Methods:
Healthy mice were injected with either: i) wild type b2GP1 peptide corresponding to the dominant region recognized by antiphospholipid antibodies, ii) mutant b2GP1 peptides that results in either much stronger or weaker binding to the antibodies, and iii) an irrelevant control peptide. Normal human IgG or IgG purified from an APS patient was then injected, and thrombus formation dynamics were evaluated 3 days later.
Results:
Transfer of APS IgG alone was sufficient to cause larger thrombus propagation than normal human IgG. Strikingly, mice pre-injected with the wild type and stronger binding b2GP1 peptides resulted in a significant reduction in clot burden. Further, the ability of macrophages to release pro-thrombotic proteins such as Tissue Factor was significantly reduced in the peptide-treated mice. The highest level of clot inhibition was noted with the strong binding “super-decoy” peptide.
Significance:
This fascinating proof-of-concept study demonstrates the power of interfering with well-defined pathogenic antigen:antibody interactions. A decoy b2GP1 peptide with strong binding kinetics was able to significantly reduce clot burden directly caused by the introduction of pathogenic antiphospholipid antibodies. Though preliminary, this strategy warrants further evaluation for eventual trials in APS patients.
Abstract # 682 Human Articular Chondrocyte Side Population Retains Chondrogenic Capacity
Authors:
Shawn P. Grogan, Shigeru Miyaki, Hiroshi Asahara, Martin K. Lotz. The Scripps Research Institute, La Jolla, CA
Background:
Osteoarthritis is the most common form of debilitating arthritis in the world that results from progressive cartilage destruction leading to significant pain, disability, and need for joint replacement therapy. In the absence of any current disease-modifying medications, strategies to increase native cartilage production, such as cell-based cartilage repair, are very attractive concepts, and are the subject of intense current research.
Methods:
Based on the isolation of a resident cartilage stem cell population in a bovine system (termed the “side” population), the authors employed a similar strategy to identify, isolate, and characterize an analogous stem cell population in adult human articular cartilage. Briefly, cartilage was digested, and resulting cell populations were evaluated by flow cytometry for expression of “side population” specific markers such as the proteins ABCG2, aggrecan, and Sox9. These cells were then purified, and grown into “chondrogenic” pellets. The characteristics of these pellets were then evaluated by histology and gene expression noted by PCR.
Results:
Successful isolation of a human side population was obtained using the methods described. These cells retained the capacity to divide, maintain the expression of chondrocyte-specific markers, and grow into cartilage pellets. The non-side population cells were far less efficient at chondrogenesis.
Significance:
This exciting study suggests that human cartilage could potentially be regenerated in culture from a patient’s own resident cartilage stem cells. Though similar strategies are currently being employed therapeutically for small cartilaginous defects in humans, this study suggests that more widespread cartilage regrowth might one day be possible.
Abstract # 1250 Ectopic Lymphoid Structures Expressing AID Drive Self Sustained in situ Production of Autoantibodies in Rheumatoid Synovium
Authors:
Frances Humby1, Michele Bombardieri1, Manzo Antonio1, Mark Blades1, Kirkham Bruce2, Jo Spencer3, Costantino Pitzalis1. 1Barts and the London School of Medicine, London, United Kingdom; 2Guy's and St Thomas' Hospital, London, United Kingdom; 3Kings College London, London, United Kingdom
Background:
The finding of highly specific autoimmunity to citrullinated proteins in rheumatoid arthritis has led to the hypothesis that this process might originate within the rheumatoid synovium itself. In this regard, ectopic lymphoid structures have been observed in rheumatoid synovium (termed follicular dendritic cell networks, or FDC), and have been postulated to help drive the specific autoimmune responses seen in the disease. Whether these structures contain the capacity and machinery to directly guide antigen-driven B cell maturation within the synovium itself is a matter of great speculation.
Methods:
The synovial tissue of 24 patients with RA was examined for the presence of characteristic FDCs, and for the expression of the protein AID (activation-induced cytidine deaminase), which is responsible for antigen-driven processes in B cells such as class switch recombination, somatic hypermutation, and affinity maturation. The location of anti-CCP producing plasma cells was also noted. These structures were then explanted into SCID mice (which are devoid of B and T cells), and evaluation of anti-CCP antibody production and B cell-specific factors was performed 4 weeks later.
Results:
Consistent with data from other studies, FDCs were found in all patients, and were surrounded by anti-CCP antibody producing plasma cells. AID expression was elevated as well, suggesting ongoing, antigen-induced B cell activation. When these structures were explanted into immunodeficient mice, their ability to maintain B cell proliferation and anti-CCP production was maintained for up to 4 weeks, as measured by human anti-CCP antibody levels in the mouse serum.
Significance:
This study demonstrates in a striking fashion that ectopic lymphoid structures in the RA synovium are capable of driving antigen-specific anti-CCP antibody production, and that this process is mediated by increased expression of AID in these structures. Apart from confirming the rheumatoid synovium as a critical autoantigen-generating structure in RA, this study suggests one of the ways that anti-B cell therapy might be functioning in RA; namely to reduce/eliminate synovial B cells.
Abstract # 1763 Regulation of SLE Immune Complex Induced IFN-alpha by Female Sex Steroids
Authors:
Grant C. Hughes, Edward Clark, Keith Elkon, Chang Li. University of Washington, Seattle, WA
Background:
The markedly increased prevalence of SLE in women has prompted numerous investigations into the roles that sex steroids play in dendritic cell (DC) biology, immune activation mechanisms, and autoantibody production. Interestingly, estrogen and progesterone appear to have divergent effects on factors such as plasmacytoid dendritic cell (pDC) interferon-a production, and susceptibility to lupus in murine models. The authors therefore postulated that the increased estrogen:progesterone ratio seen in SLE patients might lead to enhanced immune complex-mediated DC activation. If true, then modulation of tsex hormone ratios in SLE patients might represent a novel therapeutic strategy.
Methods:
Peripheral blood mononuclear cells from healthy female volunteers were exposed to immune complexes generated in vitro after pretreatment with varying doses of either estrogen, progesterone, or saline. Interferon-a production was evaluated by ELISA following stimulation.
Results:
Pretreatment with doses of progesterone typically observed in pregnancy resulted in a significant reduction in IFN-a in response to immune complex-mediated pDC activation, but standard ovulatory phase doses of both estrogen and progesterone had no appreciable effect. However, a combination of estrogen and progesterone at physiologic doses resulted in a marked inhibition of IFN production.
Significance:
This interesting study raises the question whether the abnormal hormonal milieu seen in SLE contributes directly to pDC activation, interferon production, and disease propagation in SLE. That normal physiologic ratios of estrogen and progesterone and pregnancy-related progesterone levels suppress pDC activation is fascinating. This mechanism might help explain the increased risk of post-partum SLE flares, and suggests that manipulation of hormone levels with exogenous progesterone might represent a novel mechanism-based therapy for SLE.
Abstract # 2017 Ligands Specific for Toll-like Receptors 4 and 5 Induce Distinct Types Of Vasculitis In Human Macrovessels
Abstract # 2018 Heterogeneity In Pathogen-sensing And T-cell Stimulatory Capacity Of Human Macrovessels From Distinct Vascular Territories
Authors:
Jiusheng Deng1, Olga Pryshchep1, Wei Ma-Krupa1, Brian R. Younge2, Jörg J. Goronzy1, Cornelia M. Weyand1. 1Emory University School of Medicine, Atlanta, GA; 2Mayo Clinic, Rochester, MN
Background:
The systemic vasculitis syndromes show remarkable tropism for distinct vascular territories. For instance, Giant Cell arteritis (GCA) affects large muscular arteries, but not smaller vessels. The authors have previously demonstrated the presence of activated dendritic cells in the outer adventitial layer of temporal arteries from affected patients, and postulate here that this activation might be due to differential expression of pathogen-recognition receptors in these vessels. Their second abstract expands this concept to other vascular beds, and asks whether there is a vessel-specific pattern recognition receptor expression profile that might explain the different vasculitis phenotypes seen in clinical practice.
Methods:
Human temporal and subclavian artery segments obtained surgically were explanted onto SCID mice. The mice were then treated with a variety of TLR agonists. Vasculitis was induced in the explanted vessels by transfer of alloreactive human CD4+ T cells. Resultant immune responses were evaluated by PCR of T cell receptor and IFN, and vessel architecture was evaluated by immunohistochemistry. In the second study, a variety of human arteries (aorta, subclavian, carotid, mesenteric, iliac, and temporal) were evaluated for expression of a variety of TLRs by PCR. These vessels were then grafted into the SCID mice treated, and evaluated as above.
Results:
TLR4 and 5 agonists (LPS and flagellin respectively) were the most potent inducers of inflammation in the explanted temporal and subclavian arteries. However, LPS induced a much more destructive arteritis involving all layers of the vessel wall than did flagellin, and LPS-treated DCs induced the expression of the adhesion molecule CCR6 in the transferred T cells, thus recapitulating the T cell phenotype frequently observed in GCA biopsy samples. In addition, the authors noted a different pattern of TLR expression in each arterial bed tested, with absent expression of TLRs 7 and 9, and ubiquitous expression of TLR 2 and 4 (the main sensors of gram positive and negative bacteria respectively). Each arterial bed displayed a unique pattern of T cell activation in the SCID model after TLR stimulation.
Significance:
These studies highlight the notion that vessel-specific patterns of gene expression might be responsible for the different patterns of vessel involvement seen in the systemic vasculitis syndromes. These studies further demonstrate the importance of resident dendritic cells residing in the adventitial layer of human macrovessels in mediating what is likely antigen-specific T cell stimulation in large vessel arteritis. Further, the finding that LPS treated adventitial DCs recruit CCR6+ T cells (a marker found on Th17 CD4 cells) suggests an important pathogenic role for these cells in the pathogenesis of GCA, and a potentially new therapeutic target.
Abstract # 2114 Er Stress Modifies The IL-23 Response To Toll-like Receptor Agonists And Intracellular Bacteria Infection
Authors:
Jane C. Goodall, Louise O'Brien, Lou Ellis, J. S.Hill Gaston. University of Cambridge, Cambridge, United Kingdom
Background:
The mechanisms by which HLA-B7 confers risk in the seronegative spondyloarthropathies (SpAs) are unknown, but one possibility is that mis-folded B27 triggers a cell-damaging unfolded protein response (UPR) in antigen-presenting cells. Whether this mechanism is active in the immune response to microbial antigens capable of inducing reactive arthritides is unknown.
Methods:
Immature dendritic cells were derived from normal human blood monocytes obtained from healthy individuals. The UPR was induced chemically with thapsagargin, and various TLR agonists were then added. Expression of IL-23, a mediator of growing importance in the SpAs, was evaluated by PCR, and DC culture supernatants were evaluated for IL12, 23, and TNF secretion by ELISA. The contribution of the UPR to this process was evaluated in U937 cells (of human myeloid origin) by using shRNA knock-down of a critical enzyme responsible for generating the UPR. The cells were then infected with c. trachomatis, a pathogen known to lead to reactive arthritis.
Results:
TLR stimulation enhanced the cytokine secretion noted with UPR induction alone. This effect was reduced in the knock down experiments. Chlamydial infection of th U937 cells resulted in a marked increase of the UPR and of IL23 expression and secretion, which again was abrogated in the knock-down experiments.
Significance:
This study provides a fascinating link between many of the emerging pathophysiologic concepts in the SpAs, namely the UPR, HLA-B27, and the importance of the IL23 pathway and its interactome. The authors demonstrate that intracellular bacterial infection and/or TLR ligation can trigger a UPR, which in turn can trigger IL23 secretion, thus providing a link between B27 and IL23, two knowns genetic risk factors for ankylosing spondylitis and reactive arthritis as well.
