Arthritis & Rheumatism, Volume 63,
November 2011 Abstract Supplement

Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Chicago, Illinois November 4-9, 2011.


Involvement of TLR7 and TLR9 in the Production of Antiphospholipid Antibodies.

Aguilar-Valenzuela1,  Renan, Nickerson2,  Kevin, Romay-Penabad1,  Zurina, Shlomchik2,  Mark J., Vargas1,  Gracie, Shilagard1,  Tuya, Pierangeli1,  Silvia S.

University of Texas Medical Branch, Galveston, TX
Yale University School of Medicine, New Haven, CT

Background/Purpose:

Molecular mimicry and genetic factors have been shown to be involved in the production of antiphospholipid antibodies (aPL) but a role of innate immunity has not been studied. Our aim was to elucidate the involvement of TLR7, TLR9 and molecular mimicry in aPL production by monitoring the production of pathogenic aPL in lupus mice (MRLlpr/lpr) deficient in TLR7 and TLR9 and in autoimmune prone mice (PL/J) immunized with cytomegalovirus (CMV)-derived peptides (TIFI and VITT) in the presence of TLR7 and TLR9 agonists.

Methods:

APL (anticardiolipin (aCL) and anti-b2Glycoprotein I (ab2GPI) titers were determined in the sera of MRLlpr/lpr and their congenic tlr9 and tlr7 knockout mice (16 weeks old). Additionally, PL/J autoimmune-prone mice were challenged with 150 mg of CMV peptides (TIFI and VITT) in the presence or absence of TLR9 (ODN1585) and TLR7 (CL264) agonists. As negative controls two mouse groups were immunized with Egg Albumin (OVA) (150mg) in the presence or absence of TLR9 agonist (50mg). The aPL titers were followed for 9 weeks using solid phase ELISA. Levels of IL6, IL2, IL10, TNF-a and IL2 were measured using Multiplex Bioassay (Millipore™) and IFN-a was detected by ELISA. At week nine, aPL thrombogenic effects were evaluated using a model of induced thrombosis. Tissue factor (TF) expression in peritoneal macrophages was measured using specific immunostaining and dual photon laser microscopy and nanocrystal bioconjugates.

Results:

MRLlpr/lpr mice had significantly higher titers of aCL and ab2GPI antibodies when compared to the TLR9 and TLR7 double knockouts (p<0.05) and MRLlpr/lpr tlr7 single knockouts had lower IgG or IgM aPL titers compared to MRLlpr/lpr. The titers of aPL (aCL and ab2GPI) antibodies induced with TIFI and VITT were significantly higher in mice immunized with the TLR9 (ODN1585) or with TLR7 (CL264) agonists at the time of the surgical procedures to examine thrombus formation (Table). No significant titers of aPL were seen in mice receiving only TIFI + VITT. Importantly, increased aPL production correlated with larger thrombi in mice receiving TLR7 and TLR9 agonists, compared with those that did not (p<0.05). Increased TF expression was seen in mice treated with CMV peptides and the TLR9 agonist (p<0.05).

TreatmentaCL IgG (O.D.)aCL IgM (O.D.)ab2GPI IgG (O.D.)ab2GPI IgM (O.D.)Thrombus size (mm2)
OVA0.14 (±0.03)0.2 (±0.05)0.18 (±0.0007)0.18 (±0.06)683 (±88)
OVA+ODN15850.2 (±0)0.3 (±0.17)0.11 (±0.1)0.11 (±0.0.6)674 (±52)
TIFI+VITT0.13 (±0.047)0.22 (±0.08)0.27 (±0.9)0.27 (±0.04)605 (±13)
VITT+VITT+ODN15850.22 (±0.065)0.17 (±0.02)0.79(±0.1)0.79 (±0.13)1051.5 (±123)
TIFI+VITT+CL2640.29 (±0.08)0.23 (±0.05)0.91 (±0.28)0.91 (±0.12)1280.17 (±166.87)
TIFI+VITT+ ODN1585+CL2640.3 (±0.06)0.16 (±0.018)1.7 (±0.85)1.7 (±1.2)1126.56 (±318.1)

Conclusion:

Production of pathogenic aPL involves TLR7 in the MRLlpr/lpr mice. In the TIFI/VITT-challenged PL/J mice, the activation of TLR9 and TLR7 lead to the production of pathogenic aPL. Altogether, our data support the involvement of TLR7 and TLR9 in tolerance loss to b2GPI and raises the possibility of using TLR9 and TLR7 inhibitors to prevent aPL production.

To cite this abstract, please use the following information:
Aguilar-Valenzuela, Renan, Nickerson, Kevin, Romay-Penabad, Zurina, Shlomchik, Mark J., Vargas, Gracie, Shilagard, Tuya, et al; Involvement of TLR7 and TLR9 in the Production of Antiphospholipid Antibodies. [abstract]. Arthritis Rheum 2011;63 Suppl 10 :723
DOI:

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