Different toll-like receptor stimuli have a profound impact on cytokines required to break tolerance and induce autoimmunity

Abstract

Although toll-like receptor (TLR) signals are critical for promoting antigen presenting cell maturation, it remains unclear how stimulation via different TLRs influence dendritic cell (DC) function and the subsequent adaptive response in vivo. Furthermore, the relationship between TLR-induced cytokine production by DCs and the consequences on the induction of a functional immune response is not clear. We have established a murine model to examine whether TLR3 or TLR4 mediated DC maturation has an impact on the cytokines required to break tolerance and induce T-cell-mediated autoimmunity. Our study demonstrates that IL-12 is not absolutely required for the induction of a CD8 T-cell-mediated tissue specific immune response, but rather the requirement for IL-12 is determined by the stimuli used to mature the DCs. Furthermore, we found that IFNα is a critical pathogenic component of the cytokine milieu that circumvents the requirement for IL-12 in the induction of autoimmunity. These studies illustrate how different TLR stimuli have an impact on DC function and the induction of immunity.

Figure 1. APC stimulation confers differential requirement of IL-12 in the induction of autoimmunity.

(A) Diabetes incidence in RIP-gp/p40+/+ (solid circle) and RIP-gp/p40−/− (solid square) mice that were infected with LCMV-WE on day 0. Results are from 9 mice per group, 2 independent experiments. (B, C) Diabetes incidence in RIP-gp/p40+/+ (open circle) and RIP-gp/p40−/− (open triangle) mice treated with p40+/+ and p40−/− BMDCs respectively, pulsed with peptides derived from LCMV-GP and stimulated with (B) LPS or (C) Poly I∶C. (D) Diabetes incidence in RIP-gp/p40+/+ (solid square) and RIP-gp/p40−/− (solid diamond) mice treated with p40−/− and p40+/+ BMDCs respectively, pulsed with peptides derived from LCMV-GP and stimulated with LPS. (E) Diabetes incidence in RIP-gp mice treated with IL-12p35−/− (open diamond) and IL-12p19−/− (open square) BMDCs pulsed with peptides derived from LCMV-GP and stimulated with LPS. In each experiment, non-peptide pulsed DCs were included as negative controls (Cross). Results in (B) to (E) are from 7 to 11 mice per group, 2 independent experiments. A test of statistical significance of p<0.05 by the Mantel-Cox Test and Gehan-Breslow-Wilcoxon Test is denoted with * in (B), (D) and (E).

Figure 2. Limited CD8 infiltration in mice treated with LPS matured DCs.

Degree of CD8+ islet infiltration in RIP-gp mice treated with LPS stimulated peptide-pulsed p40+/+ or p40−/− BMDCs. Results are from a minimum of 5 mice per group, 100 islets per group from 2 independent experiments.

Figure 3. IL-12 is not required for CD8 T cell proliferation, survival, activation and CTL differentiation.

(A, B) CFSE-labeled P14 transgenic T cells were cultured in media alone or co-cultured with either p40+/+ or p40−/− BMDCs stimulated with LPS or Poly I∶C and pre-pulsed with gp33 peptides. Cultures were assessed by flow cytometry 3 days later. Representative plots of CFSE dilution, CD44 (A) and 7AAD (B) staining on gated CD8+ cell populations and the percentages of cells in each quadrant are displayed. Results are representative of 3 independent experiments of 2–3 mice per group. (C) In vivo CTL activity of C57BL/6 mice treated with PBS (naïve control) or administered with p40+/+ and p40−/− BMDCs pulsed with gp33 peptide and stimulated with LPS was assessed on day 5 by flow cytometry analysis of the remaining ratio of gp33 peptide pulsed splenocytes to negative control AV peptide pulsed splenocytes given i.v. 4 hours prior. Results are representative of 2 independent experiments of 3–5 mice per group. A test of statistical significance of p<0.05 by the Student T-Test is denoted with *.

Figure 4. Cytokine profiles of BMDCs remain largely unaltered in the absence of IL-12.

(A) Expression of IL-12 and TNFα assessed by intracellular cytokine staining. p40+/+ and p40−/− BMDCs were stimulated with LPS, PolyI∶C or left unstimulated for 12 hours and assessed by flow cytometry. Representative IL-12 and TNFα profiles on gated CD11c+CD11b+ cell populations are presented with p40+/+ shown in blue solid lines and p40−/− shown in blue dashed lines. Isotype controls for p40+/+ and p40−/− are displayed in red solid and dashed lines respectively. Results are representative of 3 independent experiments of 2–3 mice per group. (B, C, D) Cultured supernatants of p40+/+ and p40−/− BMDCs stimulated with LPS, PolyI∶C or left unstimulated for overnight were assessed by Cytometric Bead Array. Results are from 2 independent experiments with a minimum of 6 mice per treatment group. A test of statistical significance of p<0.05 by the Student T-Test is denoted with *.

Figure 5. Exogenous IFNα can overcome the absence of DC-derived IL-12 in the induction of autoimmunity.

(A) IFNα production was measured in the supernatant from unstimulated, LPS stimulated and PolyI∶C stimulated p40+/+ and p40−/− BMDCs by ELISA. Results are from 4 mice per treatment group and representative of 3 independent experiments. (B) Diabetes incidence in RIP-gp mice treated with IFNα alone (open diamond), peptide-pulsed p40−/− BMDCs stimulated with LPS (open square) or peptide-pulsed p40−/− BMDCs stimulated with LPS plus IFNα (open triangle). Injection of 10000 u of IFNα to RIP-gp mice 3 days after treatment with LPS stimulated peptide-pulsed 40−/− BMDCs or LPS and IFNα stimulated peptide-pulsed p40−/− BMDCs (open circle and open upside-down triangle respectively). (C) Diabetes incidence in IFNαR+/+/RIP-gp bone marrow chimeras treated with LPS stimulated p40+/+ BMDCs (open circle) and IFNαR−/−/RIP-gp chimeras treated with LPS stimulated p40−/− BMDCs (open square) or an additional i.v. injection of IFNα 3 days later (open triangle). A test of statistical significance of p<0.05 against RIP-gp mice (B) and IFNαR−/−/RIP-gp mice (C) treated with p40−/− BMDCs stimulated with LPS is denoted with *. Results shown are from a minimum of 6 mice per group from 2 independent experiments.

Figure 6. Exogenous IFNα enhances CD8 infiltration.

(A) Insulitis as assessed by immunohistochemistry of CD8 infiltrates (stained red) in pancreas sections five days after treatment. Representative sections from RIP-gp mice treated with p40+/+ (bottom left panel), p40−/− (bottom middle panel) peptide-pulsed BMDCs stimulated with LPS or p40−/− peptide-pulsed BMDCs stimulated with LPS with an additional i.v. injection of IFNα (bottom right panel) are displayed. For controls, representative sections of RIP-gp mice treated with unstimulated peptide-pulsed p40+/+ (top left panel) or p40−/− (top middle panel) BMDCs and C57BL/6 mice treated with LPS stimulated peptide-pulsed p40−/− BMDCs (top right panel) are shown. (B) Quantiation of CD8 infiltration, with the first 2 columns represented in figure 2 but reproduced here for comparison. Results are representative of a minimum of 5 mice per group, 100 islets per group from 2 independent experiments.