Stimulation of Toll-Like Receptors profoundly influences the titer of polyreactive antibodies in the circulation

Abstract

Polyreactive antibodies are a major component of the natural antibody repertoire and bind to a variety of structurally unrelated molecules. These antibodies are thought to provide a first line of defense against bacterial infections and play a major role in the clearance of apoptotic cells. What triggers the secretion of these antibodies has remained an enigma. Using a surrogate assay for measuring polyreactive antibodies, we found that about 50% of serum IgM is polyreactive and that stimulation of TLR4(+/+), but not TLR4(-/-), mice resulted in a 40 fold increase in polyreactive antibodies. Stimulation of TLRs 3, 7, 9 also increased the secretion of polyreactive antibodies. Infection with a virus or tissue damage induced by a toxin similarly led to an increase in polyreactive antibodies in MyD88(+/+), but not MyD88(-/-) mice. We conclude that stimulation of TLRs is a key link in the mechanism of polyreactive antibody secretion into the circulation.

Figure 1. LPS stimulates polyreactive IgM antibodies.

Two months old mice were injected i.p. with 5 mg/kg body weight of LPS. Titer of polyreactive antibodies in: (A) C57BL/6 (WT), (B) TLR4^−/− and (C) MyD88^−/− mice. Error bars represent Mean ± SEM (n = 5 mice per group, ANOVA) of three independent experiments. Sera from panels (A–C) first were measured for the titer of polyreactive IgM antibody and then the concentration (μg/ml) of IgM (panels (D–F)). Panel G shows a strong positive correlation between the titer of polyreactive IgM antibody (data from panel (A)) and concentration of IgM (data from panel (D)). The black circles in panel G represent the times at which sera were collected before and after LPS injection (0, 24, 48, 72 and 96 hours) (n = 5, Spearman r = 0.996; P < 0.001).

Figure 2. Effect of TLR9, TLR7 and TLR3 agonists on the titer of polyreactive antibodies.

Two to four months old C57BL/6 mice were injected with TLR ligands (A) CpG; (B) Imiquimod; (C) Poly (I:C) and polyreactive antibody titers in the sera were assessed at the indicated time points. Data represents Mean ± SEM (n = 5 mice per group, ANOVA) from two independent experiments.

Figure 3. Polyreactive antibody titer in the serum increases after infection with LDV.

Two months old (A) C57BL/6, (B) MyD88^−/− and (C) Unc93b13D mice were infected with LDV, blood was collected before and after infection and polyreactive IgM antibody titers in the sera were determined. Mean ± SEM (n = 10 mice per group, ANOVA).

Figure 4. CCl₄ induced liver damage enhances the secretion polyreactive antibodies.

Two months old mice (n = 5) were injected with 2 ml/kg body weight of CCl₄ i.p. (A) Histopathology and (Original magnification X10) (B) elevated serum ALT levels show the induction of liver damage (Mann-Whitney U test) in both C57BL/6 wild type and MyD88 null mice. (C) Polyreactive antibody titer increases after injection of CCl₄ in wild type mice, but not MyD88 null mice. Mean ± SEM (n = 5 mice per group, ANOVA).