Balb/C, Swiss Webster and C57BL/6 mice were purchased from Taconic and housed in NYU animal facility according to protocols approved by IACUC. MyD88-deficient C57BL/6 mice were kindly provided by Dr. Elizabeth Nardin (New York University). Plasmodium yoelii 17XNL parasites were obtained from Swiss Webster mice infected intraperitoneally.

DC were generated from bone marrow precursors as previously described 29 . Briefly, bone marrow cells from the femur and tibia of BALB/c mice of 8-10 weeks of age were harvested and cultured for 8 to 10 days in 80% complete DMEM (containing 10% FBS, penicillin 100 U/ml, streptomycin 0.1 mg/ml, glutamine 0.292 mg/ml), and 20% of GM-CSF (Granulocyte/Macrophage colony-stimulating factor) conditioned medium (medium from cultures of AG8653 myeloma cells that are transfected with GM-CSF). DC culture reproducibly yielded more than 85% of CD11c+ cells. DC from BALB/c, Swiss-Webster or C57BL/6 mice were used in this study and similar results were observed with DC from both mouse strains. All experiments were performed with DC derived from Swiss Webster mice, except for the results shown in Figure 1A, where the staining of MHC-I and MHC-II required the use of DC from BALB/c mice and Figure 1B, where MyD88-deficient DC can only be obtained from C57BL/6 mice. At least one of the confirmatory experiments for all results obtained with Swiss-Webster DC, was performed using BABL/c-derived DC. These results are not shown and are noted in the figure legends as "Figures are representative of one out of three (or two) independent experiments." No differences were found between the responses of DC derived from the different mice strains.

Schizonts were isolated from the interphase of 45% Percoll gradient spun for 15 minutes at 450 g at RT. Preparations of over 90% schizonts were obtained. Fractions comprised of Plasmodium schizont, trophozoites or else mixed uninfected erythrocytes+ ring stages, were isolated using a multilayer Percoll gradient (65%-45%) as modified from Kariuki et al 30 . Briefly, P. yoelii schizonts were collected from the PBS-45% interphase, while trophozoites were collected from the 45-65% interphase, and uninfected erythrocytes+rings from the pellet.

Plasmodium yoelii and control lysates were prepared by freeze-thawing five times schizonts or uninfected erythrocytes, respectively. An amount of lysate equivalent to a 30:1 ratio of whole parasites/DC was added to DC. Food vacuoles were isolated following the protocol described by Saliba et al 31 . Schizonts were saponized for a few seconds by resuspending them in a solution of 0.5 mg/ml saponin (Sigma-Aldrich), followed by two washes with PBS. Food vacuoles isolated from 0.3 × 10⁹ to 1 × 10⁹ saponized schizonts were added per 10⁶ DC. To fix parasites, schizonts were rinsed in PBS, resuspended in 4% paraformaldehyde and incubated for 15 minutes at room temperature. Finally, schizonts were rinsed three times with PBS. To induce phosphatidyl-serine flipping, control erythrocytes were treated with 1 μM ionomycin in DMSO (Sigma-Aldrich) for 30 minutes at 37C, rinsed and plated on cells. To isolate haemoglobin, blood of uninfected Swiss Webster mice was centrifuged at 100 g twice to remove platelets. The blood was then passed through a Plasmodipur filter (Euro-Diagnostica, Arnhem, The Netherlands) to remove leucocytes. Erythrocytes were then lysed in water for 5 min on ice. PBS 10× was added to a final 1× concentration and the erythrocytes ghosts were removed by centrifugation at 16,000 × g for 20 minutes twice. Protein concentration was measured using Bradford dye reagent (Bio-Rad) according to the manufacturer's instructions.

After differentiation, DC were plated at 10⁶/ml in complete DMEM supplemented with 10% GM-CSF conditioned medium. DC were stimulated with either P. yoellii 17XNL-infected or uninfected erythrocytes (isolated from Swiss-Webster mice) at a 1:30 DC to erythrocytes ratio, unless indicated otherwise. For experiments where parasites were incubated for definite amounts of time, erythrocytes were removed by rinsing cells five times with PBS, while the adherent DC remain in culture.

DC were stimulated with the following TLR ligands: LPS from Salmonella typhimurium (Sigma-Aldrich) for TLR4, CpG oligodeoxynucleotides (ODN 2395) for TLR9, Zymosan from Saccharomyces cerevisiae (Sigma-Aldrich) for TLR2, Polyinosinic-polycytidylic (PolyI:C) for TLR3, Flagellin from Salmonella typhimurium for TLR5, Pam3CysSerLys4 (P3C) for TLR1/TLR2 and loxoribine for TLR7 (all from Invivogen). Concentrations used are indicated in figure legends.

After harvesting, DC were incubated for 30 min on ice with Fc block (BD Biosciences, clone 2.4G2) at 1:500 dilution in PBS+1% Bovine Serum Albumin. DC were then stained for 1 h on ice in PBS+1% Bovine Serum Albumin with the following fluorescently labeled antibodies at 1:100 dilution: CD11c (Biolegend, clone N418 or BD Biosciences, clone HL3), CD40 (BD Biosciences, clone 3/23), and CD86 (BD Biosciences, clone GL1). Fluorescently labeled isotype controls were used in control samples and analysed in parallel to exclude nonspecific binding. To assess cell viability, 7-amino-actinomycin D (7-AAD) (BD Biosciences) was added to labeled DC 10 minutes before flow cytometry analysis. For analysis, cells were gated on expression of CD11c and absence of 7-AAD staining. Surface marker expression was assessed for CD86 as the percentage of CD86^high cells and as mean fluorescence intensity for CD40. Annexin-FITC (Invitrogen, Molecular Probes) was used according to the manufacturer's instructions. Briefly, 10⁶ erythrocytes/ml were labeled for 15 min with Annexin-FITC at 1:20 dilution and analysed by flow cytometry shortly after. All flow cytometry experiments were performed on a Becton-Dinckinson FACS Calibur and data analysed with Cell Quest or FlowJo software.

Schizonts or erythrocytes were labeled using a protocol modified from Ing et al 16 . Briefly, uninfected or else P. yoelii-infected erythrocytes diluted in PBS at 10⁸ erythrocytes/ml were incubated for 15 minutes at 37C with 1 μM CellTrace™ Far Red DDAO-SE (Molecular Probes), rinsed with PBS, incubated further for 45 minutes at 37C in complete medium and rinsed once more with PBS. For phagocytosis assays, labeled erythrocytes were centrifuged for 1 min at 1,000 rpm on DC at a 1:30 ratio and incubated at 37C for 4 h. Cells and erythrocytes were scraped on ice and directly analysed by flow cytometry. DC were gated using the size and morphology parameters (FSC, SSC) and DDAO fluorescence was analysed within the DC population.

Bone marrow-derived DCs were plated as mentioned before and uninfected or P. yoelii-infected erythrocytes were added at different ratios of erythrocytes:DC. At different times after the start of erythrocytes incubation, LPS was added at 1 μg/ml for an additional 24 h. At the end of LPS incubation, supernatants from DC/erythrocytes co-cultures were removed and spun at 13,000 g for 10 minutes. Supernatants were used for ELISA for quantification of IL-12p70 (BD Biosciences) according to manufacturer's instructions.

During malaria infection, DC and other immune cells are frequently exposed to parasite materials derived from lysed infected erythrocytes, but also to intact infected erythrocytes, in which case Plasmodium-derived TLR ligands will be inside infected erythrocytes. Since immature DC have high phagocytic activity they easily uptake whole P. yoelii-infected erythrocytes 9 16 . To assess the effect in DC maturation of lysed and whole intact P. yoelii-infected erythrocytes, DC differentiated in vitro were incubated with infected erythrocytes at the schizont stage, or else lysates corresponding to the same amount of infected erythrocytes, as well as the respective uninfected controls.

Whole infected erythrocytes did not induce increases in surface expression of any of the maturation markers, while their lysates were very active in triggering a DC response (Figure 1A). To determine whether the maturation induced by P. yoelii lysates is mediated through a MyD88 dependent pathway, DC derived from wild type or MyD88-deficient mice were used. It was found that P. yoelii lysates induce DC maturation through a MyD88 independent pathway (Figure 1B). There are three characterized inflammatory molecules in Plasmodium-infected erythrocytes: glycosylphosphatidylinositol (GPI) anchors 32 , a parasite-induced polymer of degraded haem called haemozoin bound to parasite DNA 27 28 and Plasmodium-derived uric acid 33 34 . Since MyD88 is required for the activation mediated by GPI and DNA-covered haemozoin (TRL 2 and 9, respectively), this result suggests that the maturation induce by P. yoelii lysates may be mediated by uric acid, which is independent of MyD88 33 .

To analyse in more detail the lack of DC maturation observed with whole infected erythrocytes, surface expression of CD40 and CD86 was tested in DC incubated at different infected erythrocytes:DC ratios ranging from 1:1 to 30:1 and used TLR ligands such as LPS (TLR4 ligand) and Zymosan (TLR2 ligand) as positive controls (Figures 2A and 2B). Whole infected erythrocytes did not induce increases in surface expression of CD40 or CD86 in DC at infected erythrocytes:DC ratios ranging from 1:1 to 30:1 (Figures 2A and 2B). The effect of pulsing DC with infected erythrocytes for 3 h, followed by washing and further incubation, was also analysed, showing no maturation of DC at 24 h of culture (Figure 1C).

As the preparations of P. yoelii-purified infected erythrocytes used in this study mainly contain parasites at the schizont stage, it was next investigated if other stages of intra-erythrocytic P. yoelii parasites were able to trigger DC maturation. To this aim, schizonts, trophozoites and rings were separated using a multilayer Percoll gradient. Rings were obtained together with uninfected erythrocytes. Each fraction was added to DC at a 30 infected erythrocytes:DC ratio for 24 h. Maturation was not triggered by any of the parasite stages tested (Figure 3A).

To assess the role played by erythrocytes versus parasite-derived components, schizonts were treated with a mild detergent to lyse the erythrocytes membrane allowing for enrichment of isolated parasites and also purified food vacuoles. Isolated parasites did not trigger maturation of DC, neither did food vacuoles (Figure 3A). Lysates of isolated parasites also failed to induce DC maturation (Figure 3B) suggesting that the activatory molecules might be in the cytosol of the infected erythrocytes and not in the parasite itself. However, these molecules could be lost or inactivated in process of purification and lysis of the parasites. Fixed parasites, which should preserve parasite TLR ligands and prevent parasites from releasing newly synthesized metabolites also did not trigger maturation (Figure 3A). It was then tested whether an active parasite-derived inhibitory activity was causing the lack of DC maturation. DC were incubated simultaneously with infected erythrocytes and a strong activator of DC maturation, such as zymosan. It was observed that DC mature in response to zymosan regardless of the presence of infected erythrocytes, when both are added simultaneously (Figure 3C). This suggests that the absence of DC maturation in response to whole P. yoelii-infected erythrocytes is a result of lack of activation rather than active inhibition.

It was observed that P. yoelii-infected erythrocytes do not inhibit DC maturation when added simultaneously with a TLR ligand (Figure 3C). However, pre-incubation of DC with P. yoelii-infected erythrocytes inhibits DC maturation induced by LPS 9 . The effect of pre-incubation with whole P. yoelii-infected erythrocytes or their lysates on DC maturation was, therefore, compared. DC that had been incubated with uninfected or infected erythrocytes, or else their lysates for 24 h were further incubated with LPS for an additional 18 h. The effect on expression of CD40 (Figure 4A) and CD86 (Figure 4B) was assessed by flow cytometry. This confirmed that whole P. yoelii-infected erythrocytes inhibit DC maturation in a dose-dependent manner, with complete inhibition at 30 infected erythrocytes:DC ratio. On the contrary, lysates at similar parasite densities did not inhibit LPS-induced maturation. Interestingly, it was found that with a 10:1 infected erythrocyte:DC ratio partial activation of DC (increased CD86 expression) occurred after 48 h of incubation (Figure 4B), while there was also a decrease in CD40 expression (Figure 4A). This observation, however, was not reproducible, and was found only in two out of five independent experiments. In the other three experiments, no changes in the levels of CD40 and CD86 expression were, as had been previously described 35 . These observations suggest that on certain occasions higher densities of infected erythrocytes (30:1) are required for complete inhibition, and that whole P. yoelii-infected erythrocytes can trigger a partial DC maturation phenotype, which is slow to develop (only seen at 48 h, Figure 4B). This phenomenon might be due to lysis of infected erythrocytes after prolonged in vitro incubation and would mimic the results obtained when DC are incubated with lysates of infected erythrocytes (Figure 1A).

It was also investigated whether the presence of additional uninfected erythrocytes in the cultures of DC with infected erythrocytes, which resembles more closely the situation in vivo, would have an effect on parasite-mediated inhibition of DC maturation. When 70 uninfected erythrocytes:DC were added together with 30 infected erythrocytes:DC, mimicking a 30% parasitaemia infection, a complete inhibition of LPS-induced maturation was observed (Figure 4A, B), suggesting that uninfected erythrocytes do not interfere with the parasite-mediated effect on DC.

It was then assessed whether the intra-erythrocytic stage of parasite development played a role in the inhibition of DC maturation. Each of the parasite stages were added at a similar erythrocyte:DC ratio (30:1). Interestingly, purified P. yoelii-infected erythrocytes containing parasites at the trophozoite stage had only a partial inhibitory effect compared to the inhibition induced by the schizont stage. The immature ring-stage parasites had no effect on inhibition of DC maturation (Figure 5A).

When schizonts were extracted from infected erythrocytes before incubation with DC, thereby removing most erythrocyte components, they also induced complete inhibition of LPS-induced maturation (Figure 5A). This observation suggests that there might be an inhibitory factor that is localized in the parasite itself and not in the host erythrocyte. Indeed, a P. yoelii-derived soluble factor inhibits the maturation of DC 35 .

It was also observed that fixation of the infected erythrocytes in the schizont stage did not inhibit LPS-induced DC maturation (Figure 5A). These observations suggest that metabolically active parasites are required for the observed inhibition of DC maturation to take place.

To assess if the inhibitory effect of P. yoelii-infected erythrocytes on DC maturation required continuous presence of infected erythrocytes, the infected erythrocytes were rinsed after 3, or else 24 h of incubation. Unlike the continuous incubation for 48 h, removal of infected erythrocytes allowed maturation of DC to occur in response to LPS (Figure 5B). These results suggest that the inhibitory effect is reversible and that DC can fully mature upon removal of infected erythrocytes. However, the recovery of maturation response in this experiment may be due to removal of molecules produced by metabolically active parasites that are present in the co-culture supernatant and are responsible for the inhibition of DC maturation, rather than removal of the infected erythrocytes themselves.

Macrophage function is decreased by excessive erythrophagocytosis 36 , as well as by phagocytosis of P. falciparum-infected erythrocytes 37 . In light of previous results, it was possible that the excessive phagocytosis of erythrocytes components could be the cause of the absence of DC maturation and resistance to LPS-induced maturation. As Plasmodium-infected erythrocytes are phagocytosed with significantly higher efficiency compared to uninfected erythrocytes 16 , control erythrocytes were treated with ionomycin to increase their phagocytosis, since this treatment triggers phosphatidyl-serine flipping and an apoptosis-like phenotype in erythrocytes 38 39 . Using fluorescently-labeled erythrocytes in these experimental conditions, it was possible to confirm that uninfected control erythrocytes were phagocytosed with low efficiency when compared to infected erythrocytes (27.7% and 60.5% respectively, Figure 6A, left and middle panels). After treatment of erythrocytes with ionomycin for 30 min, significant flipping of phosphatidyl-serine to the outer membrane was observed by staining with Annexin-V. As expected, erythrocytes treated with ionomycin were phagocytosed very efficiently, to a level comparable to that of P. yoelii-infected erythrocytes at the schizont stage (Figure 6A, right panel).

Interestingly, phagocytosis of infected erythrocytes increases with the maturation stage of the parasite, resulting in very efficient phagocytosis of schizonts, intermediate phagocytosis of throphozoites and reduced phagocytosis of ring stages (Figure 6B). This would be compatible with the progressive modifications that are observed in the membrane of infected erythrocytes during an infection cycle 40 . Since there is a good correlation between the level of DC maturation inhibition (Figure 5A) and the level of phagocytosis (Figure 6B) with the different developmental stages, it was determined whether high levels of phagocytosis of uninfected erythrocytes would affect DC maturation. Ionomycin-treated erythrocytes did not induce DC maturation (Figure 6C) but failed to prevent LPS-induced maturation (Figure 6D), as observed previously with P. yoelii-infected erythrocytes. To confirm that the most relevant component of erythrocytes does not interfere with DC maturation, DC were preincubated with haemoglobin isolated from control erythrocytes. DC maturation was not affected, even when used at a concentration as high as 1 mg/ml (Figure 6D). These results exclude the possibility of unspecific effects due to different levels of phagocytosis of infected and uninfected erythrocytes and also confirm the ability of DC to mature even after high levels of erythrophagocytosis.

The results indicate that P. yoelii-infected erythrocytes inhibit the maturation of DC in a reversible manner. The kinetics of this inhibition was analysed by testing different times of pre-incubation of DCs with infected erythrocytes before the addition of LPS as a maturation stimulus. The unresponsiveness of DC increased gradually with the time of pre-incubation with infected erythrocytes, with a maximal inhibitory response obtained after 24 h of incubation (Figure 7A, B). The expression of IL-12, a cytokine usually secreted upon DC activation, and which is required for induction of Th1 type responses 2 , was also assessed. Plasmodium yoelii-infected erythrocytes inhibited IL-12 secretion and that, interestingly, the pre-incubation time required to obtain maximal inhibition of IL-12 secretion was shorter compared to the inhibition of surface expression of co-stimulatory molecules, which might reflect a post-transcriptional, rather than a transcriptional, regulatory mechanism (Figure 7C).

To investigate if the inhibition of DC maturation induced by P. yoelii-infected erythrocytes affected stimulation of DC by different TLRs, DC were pre-incubated with infected erythrocytes for 24 h before stimulation with specific ligands of the following TLRs: TLR1/2 (P3C), TLR2 (zymosan), TLR3 (polyI:C), TLR5 (flagellin), TLR7 (loxoribine), TLR9 (CpG), as well as TLR4 (LPS). For all TLRs studied, the up-regulation of co-stimulatory molecules in DC was severely inhibited by pre-incubation for 24 h with P. yoelii-infected erythrocytes in a dose-dependent fashion (Figure 8). These results suggest that the inhibition of DC maturation induced by P. yoelii-infected erythrocytes is a broad phenomenon that is not restricted to a specific TLR pathway.