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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 Anti-LPS IgG3 is generated via MyD88-dependant TLR4 signaling.A. Purified B cells from NOD P712H and NODWT mice were activated in vitro with LPS, CpG or αIgM and the proliferative response was assayed. ** P<0.01 when compared to NODWT mice. B. Total IgG and IgG1 vs. IgG3 antibodies to LPS in NODWT (n = 13) and NOD P712H mice (n = 7). *** P<0.001 when compared to NODWT mice. C. IgG antibodies to GAD, histone IIA, dsDNA and ssDNA in NODWT (n = 13) and NOD P712H mice (n = 7). D. IgG3 antibodies to LPS in WT, TLR2−/− and MyD88−/− mice (n = 4). *** P<0.001 when compared to WT or TLR2−/− mice. Results represent mean (±SD) of each group.
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pone-0003509-g002: Natural Anti-LPS IgG3 is generated via MyD88-dependant TLR4 signaling.A. Purified B cells from NOD P712H and NODWT mice were activated in vitro with LPS, CpG or αIgM and the proliferative response was assayed. ** P<0.01 when compared to NODWT mice. B. Total IgG and IgG1 vs. IgG3 antibodies to LPS in NODWT (n = 13) and NOD P712H mice (n = 7). *** P<0.001 when compared to NODWT mice. C. IgG antibodies to GAD, histone IIA, dsDNA and ssDNA in NODWT (n = 13) and NOD P712H mice (n = 7). D. IgG3 antibodies to LPS in WT, TLR2−/− and MyD88−/− mice (n = 4). *** P<0.001 when compared to WT or TLR2−/− mice. Results represent mean (±SD) of each group.

Mentions: To confirm that the difference in anti-LPS IgG was due to the differences in the TLR4 gene and not to other genetic differences between the C3H sub-strains, we back-crossed the TLR4 P712H point mutation from C3H/HeJ mice onto the genome of NOD/LtJ mice that normally bear functional wild-type TLR4 (Figure S1). We previously demonstrated that NOD mice develop anti-LPS IgG by 8 weeks of age in the absence of immunization, just as do C3H mice homozygous for wild-type TLR4 genes [25]. Since NOD mice develop a rich network of natural autoantibodies [25], we could test the impact that loss of TLR4 signaling has upon these reactivities. Breeding the mutant TLR4 allele into the NOD/LtJ genome (hereafter NODP712H) specifically impaired their B-cell proliferative response to LPS, whereas proliferation induced by a mitogenic antibody to surface IgM (αIgM), or by a CpG oligonucleotide (known to activate murine B-cells via TLR9 [26]) remained intact (Figure 2A). The NODP712H and littermates bearing WT TLR4 (hereafter NODWT) all developed anti-LPS IgM antibodies (data not shown), but only the NODWT mice developed anti-LPS IgG: IgG3 and to a lower extent, IgG1 (Figure 2B). Regardless of the functionality of their TLR4 both the NODP712H and the NODWT mice spontaneously developed IgG antibodies that bound the self-antigens GAD, Histone IIA, dsDNA and ssDNA (Figure 2C), among others. Thus although both strains were equally exposed to LPS, as suggested by the similar levels of LPS-reactive IgM, functional TLR4 is specifically needed for the spontaneous development of natural anti-LPS IgG antibodies.


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

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

Natural Anti-LPS IgG3 is generated via MyD88-dependant TLR4 signaling.A. Purified B cells from NOD P712H and NODWT mice were activated in vitro with LPS, CpG or αIgM and the proliferative response was assayed. ** P<0.01 when compared to NODWT mice. B. Total IgG and IgG1 vs. IgG3 antibodies to LPS in NODWT (n = 13) and NOD P712H mice (n = 7). *** P<0.001 when compared to NODWT mice. C. IgG antibodies to GAD, histone IIA, dsDNA and ssDNA in NODWT (n = 13) and NOD P712H mice (n = 7). D. IgG3 antibodies to LPS in WT, TLR2−/− and MyD88−/− mice (n = 4). *** P<0.001 when compared to WT or TLR2−/− mice. Results represent mean (±SD) of each group.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2566810&req=5

pone-0003509-g002: Natural Anti-LPS IgG3 is generated via MyD88-dependant TLR4 signaling.A. Purified B cells from NOD P712H and NODWT mice were activated in vitro with LPS, CpG or αIgM and the proliferative response was assayed. ** P<0.01 when compared to NODWT mice. B. Total IgG and IgG1 vs. IgG3 antibodies to LPS in NODWT (n = 13) and NOD P712H mice (n = 7). *** P<0.001 when compared to NODWT mice. C. IgG antibodies to GAD, histone IIA, dsDNA and ssDNA in NODWT (n = 13) and NOD P712H mice (n = 7). D. IgG3 antibodies to LPS in WT, TLR2−/− and MyD88−/− mice (n = 4). *** P<0.001 when compared to WT or TLR2−/− mice. Results represent mean (±SD) of each group.
Mentions: To confirm that the difference in anti-LPS IgG was due to the differences in the TLR4 gene and not to other genetic differences between the C3H sub-strains, we back-crossed the TLR4 P712H point mutation from C3H/HeJ mice onto the genome of NOD/LtJ mice that normally bear functional wild-type TLR4 (Figure S1). We previously demonstrated that NOD mice develop anti-LPS IgG by 8 weeks of age in the absence of immunization, just as do C3H mice homozygous for wild-type TLR4 genes [25]. Since NOD mice develop a rich network of natural autoantibodies [25], we could test the impact that loss of TLR4 signaling has upon these reactivities. Breeding the mutant TLR4 allele into the NOD/LtJ genome (hereafter NODP712H) specifically impaired their B-cell proliferative response to LPS, whereas proliferation induced by a mitogenic antibody to surface IgM (αIgM), or by a CpG oligonucleotide (known to activate murine B-cells via TLR9 [26]) remained intact (Figure 2A). The NODP712H and littermates bearing WT TLR4 (hereafter NODWT) all developed anti-LPS IgM antibodies (data not shown), but only the NODWT mice developed anti-LPS IgG: IgG3 and to a lower extent, IgG1 (Figure 2B). Regardless of the functionality of their TLR4 both the NODP712H and the NODWT mice spontaneously developed IgG antibodies that bound the self-antigens GAD, Histone IIA, dsDNA and ssDNA (Figure 2C), among others. Thus although both strains were equally exposed to LPS, as suggested by the similar levels of LPS-reactive IgM, functional TLR4 is specifically needed for the spontaneous development of natural anti-LPS IgG antibodies.

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
Related in: MedlinePlus