Figure 1.
The production of anaphylactic-IgE is accelerated by TLR4.
TLR4 WT (C3H/HePas) and TLR4 deficient (C3H/Hej) mice were immunized with Natterins on day 0 and boosted 7, 21 and 28 days later. Mice were bled at days 48, 74 and 120 to analyze Natterins specific-IgG1 and IgG2a and total-IgE Abs titers by ELISA. The anaphylactic IgG1 and IgE Abs were examined by PCA. Each group consisted of at least five male mice, and representative data from three repeated experiments are shown. *p<0.05 compared to control mice; and #p<0.05 compared to WT mice immunized with the Natterins. The dotted line represents the lower limit of detection methods.
Figure 2.
TLR2 and MyD88 positively regulate the production of anaphylactic-IgG1 induced by Natterins.
C57BL/6 WT, TLR2 KO and MyD88 KO mice were immunized with Natterins on day 0 and boosted 7, 21 and 28 days later. Mice were bled at days 48, 74 and 120 to analyze Natterins specific-IgG1 and IgG2a and total IgE Abs titers by ELISA. The anaphylactic IgG1 and IgE Abs were examined by PCA. Each group consisted of at least five male mice, and representative data from three repeated experiments are shown. *p<0.05 compared to control mice; and #p<0.05 compared to WT mice immunized with the Natterins. The dotted line represents the lower limit of detection methods.
Figure 3.
The transient expansion of B1a cells in spleen and bone marrow is positively mediated by TLR2 and MyD88 and negatively regulated by TLR4.
A representative dot plot of B1a cells (B220lowCD5pos) analyses is shown. Cells from peritoneum (A, D), spleen (B, E) and bone marrow (C, F) were obtained from TLR4 mutant (left) or from TLR2 KO and MyD88 KO (right) Natterins immunized mice after 48, 74 and 120 days. The bars representative of the absolute numbers of B220lowCD5pos cells were determined from total mononuclear cells by multiparametric flow cytometer using Rat IgG2ak PE-anti-mouse CD5, and Rat IgG2ak PerCP-Cy5-anti-mouse CD45R/B220. *p<0.05 compared to control mice; and #p<0.05 compared to WT mice immunized with Natterins.
Figure 4.
The persistence of B1b cells in bone marrow is negatively regulated by TLR2/TLR4 and MyD88 signals.
A representative dot plot of B1b cells (B220lowCD5neg) analyses is shown. Cells from peritoneum (A, D), spleen (B, E) and bone marrow (C, F) were obtained from TLR4 mutant (left) or from TLR2 KO and MyD88 KO (right) Natterins immunized mice after 48, 74 and 120 days. The bars representative of the absolute numbers of B220lowCD5neg cells were determined from total mononuclear cells by multiparametric flow cytometer using Rat IgG2ak PE-anti-mouse CD5, and Rat IgG2ak PerCP-Cy5-anti-mouse CD45R/B220. *p<0.05 compared to control mice; and #p<0.05 compared to WT mice immunized with Natterins.
Figure 5.
TLR4 is important for the transient expansion of B2 cells in bone marrow, but TLR2 and MyD88 are crucial for their survival in this niche.
A representative dot plot of B2 cells (B220highCD23pos) analyses is shown. Cells from peritoneum (A, D), spleen (B, E) and bone marrow (C, F) were obtained from TLR4 mutant (left) or from TLR2 KO and MyD88 KO (right) Natterins immunized mice after 48, 74 and 120 days. The bars representative of the absolute numbers of B220highCD23pos cells were determined from total mononuclear cells by multiparametric flow cytometer using Rat IgG2ak PE-anti-mouse CD23, Rat IgG2ak PerCP-Cy5-anti-mouse CD45R/B220. *p<0.05 compared to control mice; and #p<0.05 compared to WT mice immunized with Natterins.
Figure 6.
TLR2 dependent on MyD88 positively regulates Bmem in spleen and TLR4 negatively regulates in all compartments.
A representative dot plot of Bmem cells (CD19pos in B220posIgpos gated cells) analyses is shown. Cells from peritoneum (A, D), spleen (B, E) and bone marrow (C, F) were obtained of TLR4 mutant (left), TLR2 KO and MyD88 KO (right) Natterins immunized mice after 48, 74 and 120 days. The bars representative of the absolute numbers of B220posCD19posIgpos cells were determined from total mononuclear cells by multiparametric flow cytometer using Armenian hamster IgG1y2 FITC-anti-mouse CD19, Goat IgG2bk PE-anti-mouse Ig (specific for IgG1, IgG2a, IgG2b and IgG3), Rat IgG2ak PerCP-Cy5-anti-mouse CD45R/B220.*p<0.05 compared to control mice; and #p<0.05 compared to WT mice immunized with Natterins.
Figure 7.
The chronic retention of ASC B220pos in the peritoneum is dependent on TLR4 signals.
A representative dot plot of ASC B220pos (B220posCD138pos from CD43-positive gated cells) analyses is shown. Cells from peritoneum (A, D), spleen (B, E) and bone marrow (C, F) were obtained of TLR4 mutant (left), TLR2 KO and MyD88 KO (right) Natterins immunized mice after 48, 74 and 120 days. The bars representative of the absolute numbers of B220pos CD43posCD138pos cells were determined from total mononuclear cells by multiparametric flow cytometer using Rat IgG2ak PerCP-Cy5-anti-mouse CD45R/B220, Rat IgG2ak FITC-anti-mouse CD43, and Rat IgG2ak PE-anti-mouse CD138. *p<0.05 compared to control mice; and #p<0.05 compared to WT mice immunized with Natterins.
Figure 8.
TLR2, TLR4 and MyD88 signaling can sustain ASC B220neg in peritoneal cavity.
A representative dot plot of ASC B220neg (B220negCD138pos from CD43-positive gated cells) analyses is shown. Cells from peritoneum (A, D), spleen (B, E) and bone marrow (C, F) were obtained of TLR4 mutant (left), TLR2 KO and MyD88 KO (right) Natterins immunized mice after 48, 74 and 120 days. The bars representative of the absolute numbers of B220negCD43posCD138pos cells were determined from total mononuclear cells by multiparametric flow cytometer using Rat IgG2ak PerCP-Cy5-anti-mouse CD45R/B220, Rat IgG2ak FITC-anti-mouse CD43, and Rat IgG2ak PE-anti-mouse CD138. *p<0.05 compared to control mice; and #p<0.05 compared to WT mice immunized with Natterins.
Figure 9.
The impaired lymphocyte egress from the secondary lymph organs controls the migration of innate-like B cell and memory B cell for peripheral tissues.
Cells from peritoneum, spleen and bone marrow of Natterins-immunized BALB/c mice treated or not with FTY720 were obtained at day 28. The bars representative of the absolute numbers of B1a cells (B220+CD5+) (A), B1b cells (B220lowCD5neg) (B), B2 cells (B220+CD23+) (C), Bmem (B220+CD19+IgG+) (D) ASC B220pos (CD138+CD43+B220+) (E), ASC B220neg (CD138+CD43+B220neg) (F) were determined from total mononuclear cells by multiparametric flow cytometer using Rat IgG2ak PE-anti-mouse CD5, Rat IgG2ak PerCP-Cy5-anti-mouse CD45R/B220, and Rat IgG2ak PE-anti-mouse CD23, Armenian hamster IgG1y2 FITC-anti-mouse CD19, Goat IgG2bk PE-anti-mouse IgG (specific for IgG1, IgG2a, IgG2b and IgG3), Rat IgG2ak FITC-anti-mouse CD43, and Rat IgG2ak PE-anti-mouse CD138. *p<0.05 compared to control mice; and #p<0.05 compared to Natterins-immunized mice without treatment.
Figure 10.
The egress of lymphocytes from secondary lymph organs modulates the balance of CXCR3/CXCR4 expression in B cell memory compartment.
Expression of CXCR3 and CXCR4 on Bmem, ASC B220pos and ASC B220neg in peritoneal inflamed tissue at 28 d after FTY720 treatment of Natterins immunized-mice. Representative histograms gated on memory B cell compartments show mean fluorescence intensity (MFI). *p<0.05 compared to control mice; and #p<0.05 compared to Natterins-immunized mice without treatment.
Figure 11.
The importance of the integration of signaling pathways downstream of SP1R and TLRs in modulating the maintenance of ASC in peritoneal cavity.
We propose that during secondary immune response against Natterins, after some period in the lymphoid organs, S1PRs are required for TLR-induced peritoneal B1a and B2 cell egress and also for medullar B2 longevity; and that the longevity of Bmem in splenic niche, the intensity of ASC B220pos into peritoneal cavity and BM, and mainly the longevity of ASC B220neg into inflamed peritoneal tissue is strong supported by CXCR4 expression dependent on SP1R expression in B lymphocytes and its recirculation through lymphoid organs.