TLR4 signaling via MyD88 and TRIF differentially shape the CD4+ T cell response to Porphyromonas gingivalis hemagglutinin B

Abstract

Recombinant hemagglutinin B (rHagB), a virulence factor of the periodontal pathogen Porphyromonas gingivalis, has been shown to induce protective immunity against bacterial infection. Furthermore, we have demonstrated that rHagB is a TLR4 agonist for dendritic cells. However, it is not known how rHagB dendritic cell stimulation affects the activation and differentiation of T cells. Therefore, we undertook the present study to examine the role of TLR4 signaling in shaping the CD4(+) T cell response following immunization of mice with rHagB. Immunization with this Ag resulted in the induction of specific CD4(+) T cells and Ab responses. In TLR4(-/-) and MyD88(-/-) but not Toll/IL-1R domain-containing adapter inducing IFN-β-deficient (TRIF(Lps2)) mice, there was an increase in the Th2 CD4(+) T cell subset, a decrease in the Th1 subset, and higher serum IgG(1)/IgG(2) levels of HagB-specific Abs compared with those in wild-type mice. These finding were accompanied by increased GATA-3 and Foxp3 expression and a decrease in the activation of CD4(+) T cells isolated from TLR4(-/-) and MyD88(-/-) mice. Interestingly, TLR4(-/-) CD4(+) T cells showed an increase in IL-2/STAT5 signaling. Whereas TRIF deficiency had minimal effects on the CD4(+) T cell response, it resulted in increased IFN-γ and IL-17 production by memory CD4(+) T cells. To our knowledge, these results demonstrate for the first time that TLR4 signaling, via the downstream MyD88 and TRIF molecules, exerts a differential regulation on the CD4(+) T cell response to HagB Ag. The gained insight from the present work will aid in designing better therapeutic strategies against P. gingivalis infection.

FIGURE 1

Immunization of WT mice with rHagB and GPI results in a specific CD4⁺ T cell and Ab response. WT mice were immunized as described above and analyzed 21 d following immunization. A, Expression of CD44 on CD4⁺ (open bars) or CD8⁺ (filled bars) T cells ex vivo in immunized and control mice. Graphs represent percentages and numbers of CD44^hi T cells. Results are expressed as the mean ± SE of three individual mice from one of three independent experiments. B, Cytokine production by purified CD4⁺ T cells from spleens and lymph nodes of immunized (squares) or control (triangles) mice. Equal numbers of purified CD4⁺ T cells and irradiated WT feeder cells were stimulated with 0.01, 0.1, 1, and 10 μg/ml rHagB. Culture supernatants were harvested 5 d later and assessed for the levels of IFN-γ, IL-17, IL-4, IL-5, and IL-10 by ELISA. Results are expressed as the mean ± SE of triplicate wells from one of at least three independent experiments. C, Serum HagB-specific Ab levels showing IgG_T and subclasses IgG₁, IgG_2b, IgG_2c and IgG₃. Each symbol represents an individual mouse (n = 9), and the horizontal lines represent the mean of the response. ***p < 0.001, **p < 0.01, *p < 0.05 compared with control groups.

FIGURE 2

TLR4 shapes the development of rHagB-specific Th1 and Th2 CD4⁺ T cell responses. A, Cytokine production by splenic and lymph node-purified CD4⁺ T cells from WT or TLR4^−/− immunized (squares) or control (triangles) mice 21 or 42 d following immunization. Cells were stimulated with 0.01, 0.1, 1, and 10 μg/ml rHagB, and culture supernatants were harvested and assessed for cytokines as previously described (see Materials and Methods). Results are expressed as the mean ± SE of triplicate cultures from one of three independent experiments. B, IFN-γ production by intracellular cytokine staining. Cells were stimulated with 10 μg/ml rHagB or left unstimulated as controls for 36 h and cytokine secretion was blocked for an additional 10 h. IFN-γ production was detected by intracellular cytokine staining. Numbers represent percentages of CD4⁺ T cells that produce IFN-γ. C, Serum levels of rHagB-specific IgG₁, IgG_2b, and IgG_2c from immunized WT and TLR4 ^−/− mice. Each symbol represents an individual mouse and the horizontal lines represent the mean of the response. D, Ratio of HagB-specific IgG₁/IgG_2b+2c in serum of WT and TLR4^−/− mice. Ratios of IgG₁ to IgG_2b+2c were calculated for each individual mouse sample in C prior to the calculation of the mean and SE. ***p < 0.001, **p < 0.01, *p < 0.05 compared with control groups.

FIGURE 3

MyD88 and TRIF differentially regulate the CD4⁺ T cell response toward rHagB following immunization. A, Cytokine production by splenic and lymph node-purified CD4⁺ T cells from WT, MyD88^−/−, or TRIF^Lps2 immunized or control mice 21 or 44 d following immunization. Cells were stimulated as previously described (see Materials and Methods) and culture supernatants were harvested 5 d later and assessed for the levels of IFN-γ, IL-17, IL-4, IL-5, and IL-10 by ELISA. Results are expressed as the mean ± SE of triplicate cultures from one of three independent experiments. B, IFN-γ production by intracellular cytokine staining. CD4⁺ T cells from WT, MyD88^−/−, or TRIF^Lps2 were stimulated with 10 μg/ml rHagB or left unstimulated as controls for 36 h and cytokine secretion was blocked for an additional 10 h. IFN-γ production was detected by intracellular cytokine staining. Numbers represent percentages of CD4⁺ T cells that produce IFN-γ. C, Serum levels of rHagB-specific IgG₁, IgG_2b, and IgG_2c from WT, MyD88^−/−, and TRIF^Lps2 mice. Each symbol represents an individual mouse and the horizontal lines represent the mean of the response. D, Ratio of rHagB-specific IgG₁/IgG_2b+2c in serum of WT, MyD88^−/−, and TRIF^Lps2 mice. Ratios of IgG₁ to IgG_2b+2c were calculated for each individual mouse sample in C prior to the calculation of the mean and SE. ***p < 0.001, **p < 0.01, *p < 0.05 compared with control groups.

FIGURE 4

TLR4 signaling regulates the expression of T-bet and GATA-3. Expression of T-bet, GATA-3, and RORγt by rHagB-stimulated purified CD4⁺ T cells from spleens and lymph nodes of WT and TLR4^−/− (A) or WT, MyD88^−/−, and TRIF^Lps2 (B) immunized or control mice 21 d following immunization. Cells were stimulated with rHagB (10 μg/ml) or left unstimulated as controls. Cells were harvested at 36 h poststimulation and stained with CD4, CD25, T-bet, GATA-3, or RORγt Abs. Numbers represent the percentage of activated CD25⁺CD4⁺ T cells that express a particular transcription factor. Results are expressed as the mean ± SE of two to four independent experiments. ***p < 0.001, **p < 0.01, *p < 0.05 compared with unstimulated cultures from rHagB immunized mice; ^#p < 0.05 compared with stimulated cultures from rHagB immunized mice.

FIGURE 5

Lack of TLR4 results in an increase in IL-2 signaling following rHagB stimulation. A and B, IL-2 production by rHagB-stimulated purified CD4⁺ T cells from spleens and lymph nodes of WT and TLR4^−/− (A, C, E) or WT, MyD88^−/−, and TRIF^Lps2 (B, D, F) immunized or control mice 21 d following immunization. Cells were stimulated with rHagB (10 μg/ml) or left unstimulated as controls. Culture supernatants were harvested 36 h poststimulation and were assessed for the levels of IL-2 by ELISA. Results are expressed as the mean ± SE of triplicate cultures from one of two independent experiments. C and D, Expression of CD25 (IL-2Rα) by rHagB-stimulated CD4⁺ T cells. Cells were stimulated with rHagB (10 μg/ml) or left unstimulated as controls, harvested at 36 h following stimulation, and stained with CD4 and CD25 Abs. Numbers represent the percentages of CD25⁺CD4⁺ T cells. Results are expressed as the mean ± SE of at least triplicate cultures from two to four independent experiments. (E, F) Expression of pSTAT5 by rHagB stimulated purified splenic and lymph node CD4⁺ T cells. Cells were stimulated as above, harvested at 36 h poststimulation and stained with CD4, CD69 and pSTAT5 Abs. Numbers represent percentages of activated CD69⁺CD4⁺ T cells that express pSTAT5. Results represent one of two independent experiments. ***p < 0.001, compared with unstimulated cultures from HagB immunized mice; ^###p < 0.001, ^##p < 0.01 compared with HagB-stimulated cultures from HagB immunized mice.

FIGURE 6

Activated CD4⁺ T cells that lack TLR4 or MyD88 express high levels of Foxp3 and TRIF^Lps2 CD4⁺ T cells express an increased ratio of T-bet and RORγt to Foxp3 44 d following rHagB immunization. Expression of Foxp3 by rHagB-stimulated purified CD4⁺ T cells from WT and TLR4^−/− (A, B) or WT, MyD88^−/−, and TRIF^Lps2 (C, D) immunized or control mice 21, 42, or 44 d following immunization. Cells were stimulated with rHagB (10 μg/ml) or left unstimulated as controls, harvested at 36 h poststimulation, and stained with CD4, CD25, and Foxp3 Abs. A and C, Flow cytometry dot plots gated on CD4⁺ T cells showing the percentages of cells expressing CD25 and Foxp3. Results represent one of two or three individual experiments. B and D, Numbers represent the percentages of CD25⁺CD4⁺ T cells that express Foxp3. Results are expressed as the mean ± SE of two or three independent experiments. E and F, Purified CD4⁺ T cells from immunized or control WT, MyD88^−/−, and TRIF^Lps2 were stimulated with rHagB and the expression of T-bet, RORγt, and Foxp3 was assessed as described previously. Ratio of T-bet/Foxp3 (E) or RORγt/Foxp3 (F) expressed by activated CD4⁺ T cells 21 and 44 d following immunization is shown. Results are expressed as the mean ± SE of three to five independent experiments. ***p < 0.001, **p < 0.01, *p < 0.05 compared with unstimulated cultures from rHagB immunized mice; ^###p < 0.001, ^##p < 0.01, ^#p < 0.05 compared with rHagB-stimulated cultures from HagB immunized mice.

FIGURE 7

TLR4 signaling differentially regulates CD69 expression. Expression of CD69 by rHagB-stimulated purified splenic and lymph node CD4⁺ T cells from WT and TLR4^−/− (A) or WT, MyD88^−/−, and TRIF^Lps2 (B) immunized or control mice 21, 42, or 44 d following immunization. Cells were stimulated with rHagB (10 μg/ml) or left unsti-mulated as controls, harvested at 36 h poststimulation, and stained with CD4 and CD69 Abs. Numbers represent the percentages of CD69⁺CD4⁺ T cells. Results are expressed as the mean ± SE of at least triplicate cultures from two to four independent experiments. ***p < 0.001 compared with unstimulated cultures from rHagB immunized mice; ^#p < 0.05 compared with rHagB-stimulated cultures from rHagB immunized mice.

FIGURE 8

The lack of TLR4 results in a decrease in the proliferation capacity of CD4⁺ T cells. CFSE-labeled CD4⁺ T cells from WT and TLR4^−/− (A) or WT, MyD88^−/−, and TRIF^Lps2 (B) immunized or control mice 21 d following immunization. Cells were stimulated with rHagB (10 μg/ml) or left unstimulated as controls, harvested 5 d poststimulation, and stained with CD4 Abs. Histogram plots gated on CD4⁺ T cells show the percentages of cells that diluted CFSE. Results are expressed as the mean ± SE of at least triplicate cultures from two or three individual experiments.

FIGURE 9

TLR4 differentially regulates CTLA-4 expression on CD4⁺ T cells. Expression of CTLA-4 on CD4⁺ T cells ex vivo from WT and TLR4^−/− (A) or WT, MyD88^−/−, and TRIF^Lps2 (B) immunized or control mice 21 d following immunization. Numbers represent percentages of CD4⁺ T cells expressing CTLA-4. Results are expressed as the mean ± SE of one of two or three individual experiments. *p < 0.05 compared with control groups; ^#p < 0.05 compared with WT rHagB immunized mice.