IL-10eGFP mice (29) (Jackson Laboratories, Bar Harbour, ME, USA), IL-10^flox/flox mice (30) (provided by Axel Roers, Dresden, Germany and Werner Müller, Manchester, UK), CD11c-cre mice (31), all on BALB/c background and BALB/c mice (Harlan Laboratories, Borchen, Germany) were crossed and maintained under specific pathogen-free conditions at the Animal Facility of the University Hospital Essen, Germany. Cryopreserved P. yoelii 17XNL (non-lethal)-infected red blood cells (iRBCs) were passaged once through BALB/c mice before being used in experimental animals. For infection 1 × 10⁵ iRBCs were injected i.v. The frequency of iRBCs (parasitemia) was determined by microscopic examination of Giemsa-stained blood films. The study was carried out in accordance with the guidelines of the German Animal Protection Law and the State Authority for Nature, Environment and Customer Protection, North Rhine-Westphalia, Germany. The protocol was approved by the State Authority for Nature, Environment and Customer Protection, North Rhine-Westphalia, Germany.

Single cell suspensions of splenocytes were generated by rinsing spleens with erythrocyte lysis buffer and washing with PBS supplemented with 2% FCS and 2 mM EDTA. For the isolation of CD11c⁺ DCs, splenocytes were separated from contaminating superparamagnetic splenic red pulp cells (32) by using the AutoMACS Pro (Miltenyi Biotec, Bergisch Gladbach, Germany) before using MACS CD11c Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s recommendations. CD4⁺ T cells were isolated from splenocytes using the CD4⁺ T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol, followed by anti-CD4 staining and cell sorting using an Aria II Cell Sorter (BD Biosciences, Heidelberg, Germany).

Anti-CD4, anti-CD8, anti-CD11c, anti-CD11b, anti-MHC-II, anti-CD80, anti-CD86, anti-CD335, anti-CD3, anti-IFN-γ (all BD Biosciences, Heidelberg, Germany), anti-CD62L, anti-TNF-α, anti-Foxp3 (all eBioscience, Frankfurt, Germany), anti-CD160, anti-CD138, and anti-IL-10 (Biolegend, London, UK) were used as fluorescein isothiocyanate, pacific blue, phycoerythrin (PE), BD Horizon V450, allophycocyanin, AlexaFlour647, PE-cyanin 7, or peridinin-chlorophyll protein conjugates. Dead cells were identified by staining with the fixable viability dye eFlour 780 (eBioscience, Frankfurt, Germany). Intracelluar staining for Foxp3 was performed with the Foxp3 staining kit (eBioscience, Frankfurt, Germany) according to the manufacturer’s recommendations. Cytokine production from freshly isolated splenocytes was measured by stimulating cells with 10 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, München, Germany) and 100 µg/ml ionomycin (Sigma-Aldrich, München, Germany) for 4 or 6 h (IL-10), respectively, in the presence of 5 µg/ml Brefeldin A (for IFN-γ, TNF-α staining) and 5 µg/ml Monensin (for IL-10 staining), treating with 2% paraformaldehyde and 0.1% NP40, and staining with the respective antibody cocktail. Flow cytometric expression analyses were performed with an LSR II instrument using DIVA software (BD Biosciences, Heidelberg, Germany).

Blood samples were collected, incubated at room temperature and centrifuged at 1,000 × g. Splenic CD11c⁺ DCs were stimulated overnight with 100 ng/ml liposaccharide (Sigma-Aldrich, München, Germany). Cytokines were quantified in the supernatants from stimulated DCs or CD4⁺ T cells, and in sera by using a Luminex Screening assay (R&D Systems, Wiesbaden, Germany) and a Luminex 200 system with Luminex IS software (Luminex Corporation, MV’s-Hertogenbosch, Netherlands) according to the manufacturer’s instructions. The detection limits were 1.32 pg/ml for IL-10, 0.73 pg/ml for IFN-γ, 0.13 pg/ml for TNF-α, and 1.31 pg/ml for IL-12, respectively.

Statistical analyses were performed with one-way ANOVA, Student’s t-test for parametric and Mann–Whitney test for non-parametric distributed data as indicated with significance set at the levels of *p < 0.05, **p < 0.01, and ***p < 0.001. Normality was tested using the D’Agostino–Pearson omnibus and Kolmogorov–Smirnov test. All analyses were calculated with Graph Pad Prism 5.0 Software (Graph Pad Software, La Jolla, CA, USA).

IL-10 plays an important role in the regulation of P. yoelii infection, and CD4⁺ T cells have been shown to secrete high amounts of IL-10 upon infection (3, 9). To gain further insights into the question whether IL-10 induction in CD4⁺ T cells is a direct effect of the parasite or results from IL-10 secretion by APCs, we analyzed the frequency of IL-10-producing (eGFP⁺) CD11c⁺CD11b⁺CD8⁻ DCs and CD11c⁺CD11b⁻CD8⁺ DCs in spleen of IL-10eGFP reporter mice at different time points post-P. yoelii infection by flow cytometry (Figure 1A). Whereas we observed significantly elevated percentages of IL-10-expressing CD11c⁺CD11b⁺CD8⁻ DCs in the course of infection (Figure 1B), the frequency of IL-10⁺ CD11c⁺CD11b⁻CD8⁺ DCs was reduced at day 5 postinfection but recovered to levels of non-infected mice at day 7, 10, and 14 postinfection (Figure 1C).

To investigate the impact of DC-derived IL-10 on the immune response during P. yoelii infection of BALB/c mice, we made use of IL-10^flox/flox/CD11c-cre mice to specifically ablate IL-10 expression in CD11c⁺ DCs. To confirm functional inactivation of IL-10, we isolated CD11c⁺ DCs from IL-10^flox/flox/CD11c-cre mice and IL-10^flox/flox littermates, stimulated them in vitro with LPS and analyzed the amount of IL-10 in the supernatant by Luminex technology. As depicted in Figure 2A, CD11c⁺ DCs from IL-10^flox/flox control mice secreted approximately 22 pg/ml IL-10 to the culture medium, whereas cells from DC-specific IL-10-deficient (IL-10^flox/flox/CD11c-cre) mice produced significantly less IL-10 upon LPS stimulation (approximately 5 pg/ml). Hence, the IL-10^flox/flox/CD11c-cre mouse line is a suitable model to investigate the impact of DC-derived IL-10 on the course of P. yoelii infection.

We infected DC-specific IL-10-deficient mice and WT littermates with P. yoelii and analyzed the expression of MHC-II on CD11c⁺CD11b⁺CD8⁻ DCs and CD11c⁺CD11b⁻CD8⁺ DCs at different time points postinfection by flow cytometry. We observed only a slight, albeit non-significant, reduction in MHC-II expression levels in the course of infection on both DC subtypes, and this was irrespective of their IL-10 expression (Figure 2B). Similarly, CD80 and CD86 expression on DCs slightly decreased at later time points postinfection, but again with no significant differences between IL-10-deficient and WT DCs (Figures S1A,B in Supplementary Material). However, significant higher percentages of CD11c⁺ DCs from IL-10^flox/flox/CD11c-cre mice produced IFN-γ and TNF-α at early time points after P. yoelii infection in comparison to WT controls (Figures 2C,D). To exclude contaminating NK, NKT, and T cells within the CD11c⁺ DC population, we extended our analysis for IFN-γ production to CD335⁻CD3⁻CD160⁻CD11c⁺ DCs from non-infected and P. yoelii-infected mice at day 5 postinfection and obtained same results, showing higher percentages of IFN-γ-expressing CD335⁻CD3⁻CD160⁻CD11c⁺ DCs from IL-10^flox/flox/CD11c-cre mice than from control mice (Figure S2 in Supplementary Material). These results suggest that DC-derived IL-10 has no impact on the capability of DCs to present antigen and on their costimulatory activity, but transiently alters the cytokine profile of CD11c⁺ DCs during P. yoelii infection.

Next, we asked whether the altered cytokine profile of IL-10-deficient CD11c⁺ DCs during P. yoelii infection has an impact on the T cell response. For this purpose, we infected IL-10^flox/flox/CD11c-cre and IL-10^flox/flox control mice with P. yoelii and determined the frequency of CD62L-expressing CD4⁺ and CD8⁺ T cells. As expected, the percentage of CD62L-expressing T cells was reduced in P. yoelii-infected mice, but we detected no differences between mice with IL-10-deficient CD11c⁺ DCs and WT littermates (Figures S3A,B in Supplementary Material). However, functional inactivation of IL-10 in CD11c⁺ DCs resulted in significantly elevated IFN-γ production in CD4⁺ T cells (Figures 3A,B) and slightly, but not significantly, increased frequencies of IFN-γ-expressing CD8⁺ T cells (Figures 3A,C) at day 5 post-P. yoelii infection. Hence, inactivation of IL-10 in CD11c⁺ DCs does not impede T cell activation, but rather induces IFN-γ production in CD4⁺ T cells during P. yoelii infection.

To determine whether DC-derived IL-10 has an impact on CD4⁺ Treg subsets, we next analyzed the effect of DC-specific IL-10 ablation during P. yoelii infection on the amount of Foxp3⁺ Tregs and IL-10⁺ Tr1 cells. As depicted in Figure 4A, flow cytometric analyses revealed no differences in the frequency of Foxp3⁺ Tregs from P. yoelii-infected IL-10^flox/flox/CD11c-cre mice in comparison to IL-10^flox/flox littermates (Figure 4A). Strikingly, we observed a significant lower induction of IL-10-producing CD4⁺ Tr1 cells in IL-10^flox/flox/CD11c-cre mice than in IL-10^flox/flox control mice at day 5 and 7 post-P. yoelii infection (Figure 4B). To exclude, that IL-10 expression in CD4⁺ T cells is affected by CD11c-driven cre expression, we analyzed IL-10 secretion of sorted CD4⁺ T cells from naive IL-10^flox/flox/CD11c-cre mice and IL-10^flox/flox control mice after stimulation in vitro by Luminex and detected no significant differences (Figure S4A in Supplementary Material). Moreover, CD4⁺ T cells from both DC-specific IL-10-deficient and control mice produced similar amounts of IFN-γ and TNF-α after stimulation in vitro (Figures S4B,C in Supplementary Material), indicating that CD11c-driven cre expression has no influence on IL-10, IFN-γ, and TNF-α production by CD4⁺ T cells from naïve mice. Hence, these results provide evidence for DC-derived IL-10 to be involved in the induction of IL-10-producing CD4⁺ Tr1 cells during P. yoelii infection of BALB/c mice.

Since DC-specific IL-10 ablation in P. yoelii-infected mice interfered with the induction of IL-10-producing CD4⁺ Tr1 cells and resulted in elevated production of pro-inflammatory cytokines, we monitored progression of blood infection by microscopic examination of Giemsa-stained blood films. We did not detect any significant effect on parasite clearance (Figure 4C), confirming that the observed differences can be attributed directly to immune signaling and are not an indirect effect of lower parasite burden.

Our results indicate that DC-specific ablation of IL-10 triggers the expression of pro-inflammatory cytokines in DCs and T cells, while dampening the induction of Tr1 cells during P. yoelii infection. To investigate whether these effects have also systemic implications, we analyzed the concentration of different cytokines in sera of P. yoelii-infected IL-10^flox/flox/CD11c-cre mice and IL-10^flox/flox littermates. As depicted in Figure 5, DC-specific inactivation of IL-10 did not change TNF-α serum concentration (Figure 5A), but resulted in a slightly, albeit non-significantly increased systemic IFN-γ production (Figure 5B) and significantly elevated IL-12 levels (Figure 5C) in sera of P. yoelii-infected mice at day 5 postinfection.