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Induction of IgG3 to LPS via Toll-like receptor 4 co-stimulation.

Quintana FJ, Solomon A, Cohen IR, Nussbaum G - PLoS ONE (2008)

Bottom Line: We found that mice lacking functional TLR4 or its adaptor molecule MyD88 harbored significantly less IgG3 natural antibodies to LPS, and required higher amounts of LPS to induce anti-LPS IgG3.In vitro, BCR and TLR4 signaling synergized, lowering the threshold for production of T-cell independent IgG3 and IL-10.Moreover, BCR and TLR4 directly associate through the transmembrane domain of TLR4.

View Article: PubMed Central - PubMed

Affiliation: Center for Neurologic Diseases, Harvard Medical School, Boston, MA, USA. fquintana@rics.bwh.harvard.edu

ABSTRACT
B-cells integrate antigen-specific signals transduced via the B-cell receptor (BCR) and antigen non-specific co-stimulatory signals provided by cytokines and CD40 ligation in order to produce IgG antibodies. Toll-like receptors (TLRs) also provide co-stimulation, but the requirement for TLRs to generate T-cell independent and T-cell dependent antigen specific antibody responses is debated. Little is known about the role of B-cell expressed TLRs in inducing antigen-specific antibodies to antigens that also activate TLR signaling. We found that mice lacking functional TLR4 or its adaptor molecule MyD88 harbored significantly less IgG3 natural antibodies to LPS, and required higher amounts of LPS to induce anti-LPS IgG3. In vitro, BCR and TLR4 signaling synergized, lowering the threshold for production of T-cell independent IgG3 and IL-10. Moreover, BCR and TLR4 directly associate through the transmembrane domain of TLR4. Thus, in vivo, BCR/TLR synergism could facilitate the induction of IgG3 antibodies against microbial antigens that engage both innate and adaptive B-cell receptors. Vaccines might exploit BCR/TLR synergism to rapidly induce antigen-specific antibodies before significant T-cell responses arise.

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Natural IgG antibodies to LPS are not detectable in TLR4P712H mice.A. IgG reactivity in pooled blood samples from non-immunized TLR4WT and TLR4 P712H mice. B. IgM and IgG antibodies to LPS in individual TLR4WT and TLR4 P712H mice (n = 5, *** P<0.001 when compared to TLR4WT mice). C. Titration of spontaneous IgG antibodies to LPS in TLR4WT and TLR4P712H mice (n = 5 per group, ** P<0.01 and *** P<0.001 when compared to TLR4WT mice). D. IgG subclass of antibodies to LPS in individual TLR4WT and TLR4 P712H mice (n = 5, ** P<0.01 when compared to TLR4WT mice). E. Time course of the induction of IgG3 antibodies to LPS (n = 6 per group, ** P<0.01 when compared to TLR4WT mice).
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pone-0003509-g001: Natural IgG antibodies to LPS are not detectable in TLR4P712H mice.A. IgG reactivity in pooled blood samples from non-immunized TLR4WT and TLR4 P712H mice. B. IgM and IgG antibodies to LPS in individual TLR4WT and TLR4 P712H mice (n = 5, *** P<0.001 when compared to TLR4WT mice). C. Titration of spontaneous IgG antibodies to LPS in TLR4WT and TLR4P712H mice (n = 5 per group, ** P<0.01 and *** P<0.001 when compared to TLR4WT mice). D. IgG subclass of antibodies to LPS in individual TLR4WT and TLR4 P712H mice (n = 5, ** P<0.01 when compared to TLR4WT mice). E. Time course of the induction of IgG3 antibodies to LPS (n = 6 per group, ** P<0.01 when compared to TLR4WT mice).

Mentions: C3H/HeJ mice harbor a P712H point mutation in the TLR4 gene that results in a non-functional protein, whereas other C3H mouse strains express functional TLR4 [20], [21]. We probed the repertoire of natural IgG antibodies in pooled sera from non-immunized 14-week old C3H/HeJ (hereafter TLR4P712H) or C3HeB/FeJ (hereafter TLR4WT) mice using a panel of 87 self and non-self antigens (Table S1). Both strains of mice harbored similar profiles of natural IgG antibodies except for antibodies to LPS that were present in the sera of TLR4WT mice, but not detectable in the sera of TLR4P712H mice (Figure 1A). This strain difference was confirmed by testing sera of individual mice (Figure 1B). Surprisingly, there were no differences in the levels of anti-LPS IgM between the two strains despite the significant difference in anti-LPS IgG (Figure 1B). To rule out a prozone effect, sera were serially diluted and tested for anti-LPS IgG. The TLR4P712H sera were not reactive to LPS at any dilution (Figure 1C). The lack of IgG reactivity to LPS in TLR4P712H mice was not due to masking of LPS epitopes by IgM because sera were pre-treated with 0.05 M β-mercaptoethanol [22] to disrupt IgM. The natural anti-LPS IgG antibodies were almost exclusively of the IgG3 subclass (Figure 1D), and recognized an oxidation sensitive epitope (data not shown), as has been previously described for antibodies to the carbohydrate portion of LPS [23].


Induction of IgG3 to LPS via Toll-like receptor 4 co-stimulation.

Quintana FJ, Solomon A, Cohen IR, Nussbaum G - PLoS ONE (2008)

Natural IgG antibodies to LPS are not detectable in TLR4P712H mice.A. IgG reactivity in pooled blood samples from non-immunized TLR4WT and TLR4 P712H mice. B. IgM and IgG antibodies to LPS in individual TLR4WT and TLR4 P712H mice (n = 5, *** P<0.001 when compared to TLR4WT mice). C. Titration of spontaneous IgG antibodies to LPS in TLR4WT and TLR4P712H mice (n = 5 per group, ** P<0.01 and *** P<0.001 when compared to TLR4WT mice). D. IgG subclass of antibodies to LPS in individual TLR4WT and TLR4 P712H mice (n = 5, ** P<0.01 when compared to TLR4WT mice). E. Time course of the induction of IgG3 antibodies to LPS (n = 6 per group, ** P<0.01 when compared to TLR4WT mice).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2566810&req=5

pone-0003509-g001: Natural IgG antibodies to LPS are not detectable in TLR4P712H mice.A. IgG reactivity in pooled blood samples from non-immunized TLR4WT and TLR4 P712H mice. B. IgM and IgG antibodies to LPS in individual TLR4WT and TLR4 P712H mice (n = 5, *** P<0.001 when compared to TLR4WT mice). C. Titration of spontaneous IgG antibodies to LPS in TLR4WT and TLR4P712H mice (n = 5 per group, ** P<0.01 and *** P<0.001 when compared to TLR4WT mice). D. IgG subclass of antibodies to LPS in individual TLR4WT and TLR4 P712H mice (n = 5, ** P<0.01 when compared to TLR4WT mice). E. Time course of the induction of IgG3 antibodies to LPS (n = 6 per group, ** P<0.01 when compared to TLR4WT mice).
Mentions: C3H/HeJ mice harbor a P712H point mutation in the TLR4 gene that results in a non-functional protein, whereas other C3H mouse strains express functional TLR4 [20], [21]. We probed the repertoire of natural IgG antibodies in pooled sera from non-immunized 14-week old C3H/HeJ (hereafter TLR4P712H) or C3HeB/FeJ (hereafter TLR4WT) mice using a panel of 87 self and non-self antigens (Table S1). Both strains of mice harbored similar profiles of natural IgG antibodies except for antibodies to LPS that were present in the sera of TLR4WT mice, but not detectable in the sera of TLR4P712H mice (Figure 1A). This strain difference was confirmed by testing sera of individual mice (Figure 1B). Surprisingly, there were no differences in the levels of anti-LPS IgM between the two strains despite the significant difference in anti-LPS IgG (Figure 1B). To rule out a prozone effect, sera were serially diluted and tested for anti-LPS IgG. The TLR4P712H sera were not reactive to LPS at any dilution (Figure 1C). The lack of IgG reactivity to LPS in TLR4P712H mice was not due to masking of LPS epitopes by IgM because sera were pre-treated with 0.05 M β-mercaptoethanol [22] to disrupt IgM. The natural anti-LPS IgG antibodies were almost exclusively of the IgG3 subclass (Figure 1D), and recognized an oxidation sensitive epitope (data not shown), as has been previously described for antibodies to the carbohydrate portion of LPS [23].

Bottom Line: We found that mice lacking functional TLR4 or its adaptor molecule MyD88 harbored significantly less IgG3 natural antibodies to LPS, and required higher amounts of LPS to induce anti-LPS IgG3.In vitro, BCR and TLR4 signaling synergized, lowering the threshold for production of T-cell independent IgG3 and IL-10.Moreover, BCR and TLR4 directly associate through the transmembrane domain of TLR4.

View Article: PubMed Central - PubMed

Affiliation: Center for Neurologic Diseases, Harvard Medical School, Boston, MA, USA. fquintana@rics.bwh.harvard.edu

ABSTRACT
B-cells integrate antigen-specific signals transduced via the B-cell receptor (BCR) and antigen non-specific co-stimulatory signals provided by cytokines and CD40 ligation in order to produce IgG antibodies. Toll-like receptors (TLRs) also provide co-stimulation, but the requirement for TLRs to generate T-cell independent and T-cell dependent antigen specific antibody responses is debated. Little is known about the role of B-cell expressed TLRs in inducing antigen-specific antibodies to antigens that also activate TLR signaling. We found that mice lacking functional TLR4 or its adaptor molecule MyD88 harbored significantly less IgG3 natural antibodies to LPS, and required higher amounts of LPS to induce anti-LPS IgG3. In vitro, BCR and TLR4 signaling synergized, lowering the threshold for production of T-cell independent IgG3 and IL-10. Moreover, BCR and TLR4 directly associate through the transmembrane domain of TLR4. Thus, in vivo, BCR/TLR synergism could facilitate the induction of IgG3 antibodies against microbial antigens that engage both innate and adaptive B-cell receptors. Vaccines might exploit BCR/TLR synergism to rapidly induce antigen-specific antibodies before significant T-cell responses arise.

Show MeSH