MyD88 and interferon-alpha/beta are differentially required for dendritic cell maturation but dispensable for development of protective memory against Listeria

Abstract

Signalling pathways mediated by MyD88 are important for sensing Toll-like receptor (TLR) ligands and directing an immune response. However, the influence of MyD88-derived cytokines and interferon (IFN)-alpha/beta, the latter being made by both MyD88-dependent and -independent pathways, in phenotypic and functional dendritic cell (DC) maturation during infection is poorly understood. Here we investigate the contribution of MyD88-dependent and -independent pathways to DC maturation, CD8 T-cell activation and the generation of protective memory against Listeria monocytogenes. We show that neither MyD88 deficiency alone nor MyD88/IFN-alphabetaR double deficiency alters Listeria-induced costimulatory molecule up-regulation on DCs in vivo. In contrast, DCs from infected IFN-alphabetaR(-/-) mice had higher CD80 and CD86 expression than wild-type DCs. We then examined the function of DCs matured in infected knockout mice. We found that DCs from Listeria-infected MyD88(-/-) and MyD88(-/-) IFN-alphabetaR(-/-) mice induced little or no IFN-gamma by CD8 T cells, respectively. In contrast, DCs from infected IFN-alphabetaR(-/-) mice had a greater capacity to induce IFN-gamma compared with DCs from infected wild-type mice. When the CD8 T-cell memory response was analysed, infected MyD88(-/-) and MyD88(-/- )IFN-alphabetaR(-/-) mice were found to have fewer bacteria-specific memory CD8 T cells than wild-type mice. However, the fraction of bacteria-specific CD8 T cells making IFN-gamma was similar in all mouse strains, and MyD88(-/-) and MyD88(-/- )IFN-alphabetaR(-/-) mice survived lethal challenge. Together the data suggest an inhibitory effect of IFN-alpha/beta on functional DC maturation during Listeria infection and reveal overlapping roles of MyD88-induced cytokines and IFN-alpha/beta in DC maturation and protective anti-Listeria immunity.

Figure 4

Antigen-specific memory T cells are reduced in the absence of MyD88 and partially restored by the simultaneous lack of MyD88 and interferon (IFN)-αβR. Mice were infected with the ActA⁻ derivative of Listeria monocytogenes 10403s expressing full-length ovalbumin (ActA-OVA-LM) and challenged 4 weeks later with Listeria monocytogenes 10403s expressing full-length ovalbumin (OVA-LM). (a) 7-Aminoactinomycin D (7AAD)⁻ T-cell receptor (TCR) αβ⁺ CD4⁻ cells were gated and analysed for CD8 and OVA-pentamer reactivity. Dot plots represent OVA-specific CD8 T cells detected by pentamer staining 5 days after challenge. (b) The bar graph represents data pooled from three experiments analysing a total of six to 11 mice. The bars indicate the ratio of the percentage of OVA-specific T cells in infected to naïve mice gated as in (a). Error bars are the SEM. *P < 0·05.

Figure 1

CD80 and CD86 up-regulation occurs independently of MyD88 and interferon (IFN)-αβR. C57BL/6, IFN-αβR^−/−, MyD88^−/− and MyD88^−/−IFN-αβR^−/− double knockout (DKO) mice were infected intravenously (i.v.) with Listeria 10403s and 48 hr later splenocytes were stained with anti- CD11c, CD80 or CD86 and 7-aminoactinomycin D (7AAD) and analysed by flow cytometry. (a) The dot plots show gating of live (7AAD⁻) CD11c^hi conventional dendritic cells (DCs) from a naïve C57BL/6 mouse as an example. The numbers indicate the percentage of the gated population. (b) The bars indicate the ratio of the median fluorescence intensity (MFI) of CD80 and CD86 on total DCs, as gated in (a) (7AAD⁻ CD11c^hi), for infected mice to that for naïve mice. Error bars indicate the standard error of the mean. The bacterial load of the mice analysed is shown to the right. Data are pooled from two to four independent experiments using a total of six to 11 mice for each strain. *P < 0·05.

Figure 2

Cytokine profile in the spleens of Listeria-infected mice. C57BL/6, interferon (IFN)-αβR^−/−, MyD88^−/− and MyD88^−/−IFN-αβR^−/− double knockout (DKO) mice were infected intravenously (i.v.) with Listeria 10403s and 48 hr later the spleens were collected and lysed. (a) Cytokines were measured by enzyme-linked immunosorbent assay (ELISA). Data are pooled values from eight to 12 mice from two or three independent experiments. Error bars indicate the standard error of the mean (SEM). (b) IFN-α was measured by ELISA. Data are for three infected mice per group. Error bars indicate the SEM. Solid and open bars indicate infected and naïve mice, respectively, in both (a) and (b). (c) Bacterial loads of the mice analysed in (a). Solid symbols indicate mice for which interleukin (IL)-1β, IL-6, tumour necrosis factor (TNF)-α and IFN-γ were measured. Open symbols indicate mice for which IFN-α was measured. Comparisons were made against infected C57BL/6 mice. ND, not detected. *P < 0·05; **P < 0·01; ***P < 0·001.

Figure 3

Enhanced T-cell stimulatory capacity of dendritic cells (DCs) from mice lacking interferon (IFN)-αβR. C57BL/6, IFN-αβR^−/−, MyD88^−/− and MyD88^−/−IFN-αβR^−/− double knockout (DKO) mice (three to six animals per group) were infected with L. monocytogenes 10403s expressing full-length ovalbumin (OVA-LM) and 48 hr later the spleens were collected and pooled. CD11c-expressing cells were magnetically enriched, stained with anti-CD11c-phycoerythrin (PE) and 7-aminoactinomycin D (7AAD) and sorted as live (7AAD⁻) cells with high expression of CD11c (see Fig. 1a). The cells were co-cultured for 3·5 days with 160 000 carboxyfluorescein succinimidyl ester (CFSE)-labelled OT-I cells, re-stained with anti-CD11c, -T-cell receptor (TCR)-αβ, -NK1·1, -CD11b, -CD4 and -CD8 and analysed by flow cytometry. (a) The numbers in the dot plots indicate the percentage of proliferation from a co-culture well with 80 000 DCs and 160 000 OT-I cells, the highest titration point in the graph in (b). (b) The graph shows the mean proliferation induced by an increasing number of DCs. Error bars are the standard error of the mean (SEM). *P < 0·05. (c) The bars represent the IFN-γ content in the supernatant of the co-culture wells from (b). Errors bar are the SEM. Data are pooled from two to three independent experiments. ND, not detected. *P < 0·05; **P < 0·01.

Figure 5

Listeria-infected knockout mice retain wild-type levels of bacteria-specific CD8 T cells making interferon (IFN)-γ. Mice were infected and challenged as described in Fig. 4. (a) The bar graph shows the ratio of total IFN-γ-producing (open bars) or tumour necrosis factor (TNF)-α-producing (black bars) CD8 T cells in infected mice to those in naïve mice of the different strains, as indicated. Cells were gated as 7-aminoactinomycin D (7AAD)⁻, T-cell receptor (TCR) αβ⁺, CD4⁻, CD8α⁺, cytokine⁺ cells. (b) The bar graph indicates the ratio of ovalbumin (OVA)-specific IFN-γ-producing (open bars) and TNF-α-producing (black bars) CD8 T cells in infected mice to those in naïve mice. Values for both naïve and infected mice were obtained by multiplying the fraction of cytokine⁺ OVA⁺ CD8⁺ T cells by the percentage of IFN-γ⁺ or TNF-α⁺ CD8⁺ T cells. No significant differences between any knockout strain and wild-type mice were detected in the statistical analysis in the data shown in (a) or (b).

Figure 6

MyD88 and interferon (IFN)-α/β are differentially involved in costimulatory molecule expression and T-cell activation during Listeria infection. (a) Diagram representing the MyD88-dependent and -independent pathways that produce factors influencing dendritic cell (DC) maturation.,,, (b) Our data demonstrate that: (i) splenic DCs from infected MyD88^−/− mice and mice lacking both MyD88 and IFN-αβR [double knockout (DKO)] express CD80 and CD86 to a similar level as DCs from infected wild-type mice. In contrast, DCs from infected IFN-αβR^−/− mice express higher levels of CD80 and CD86 than DCs from infected wild-type mice. A different effect of MyD88 and IFN-α/β on functional DC maturation is apparent, as DCs from infected IFN-αβR^−/− mice were more efficient at inducing proliferation (ii) and IFN-γ production (iii) by naïve CD8 T cells than DCs from wild-type mice. In contrast, DCs from MyD88^−/− and DKO mice induced similar CD8 T-cell proliferation (ii) and less IFN-γ (iii) than wild-type DCs. Finally, although the lack of MyD88 resulted in a diminished total CD8 T-cell memory population (iv), neither MyD88 nor IFN-α/β was required for the generation of IFN-γ-producing, bacteria-specific memory CD8 T cells (v). IL, interleukin; TLR, Toll-like receptor; TNF, tumour necrosis factor.