TLR ligands induce dendritic cell (DC) maturation. During this process, cells initiate proteolytic degradation of internalized protein Ags into peptides that complex with MHC class II (MHC II) and simultaneously increase expression of costimulatory molecules and of cytokines such as IL-6, IL-12, and IL-23. In these ways, TLR-activated DCs are able to activate naive Th cells and initiate Th1 and Th17 responses, and TLR ligands thus serve as adjuvants for these types of responses. In contrast, products from helminth parasites generally do not activate DCs and act as adjuvants for Th2 response induction. We have explored the underlying basis for this form of adjuvanticity. We show that exposure of DCs to soluble Ags from the eggs of the helminth parasite Schistosoma mansoni (schistosome egg Ag (SEA)) leads to the induction of proteolysis of internalized Ag. This occurs in the absence of significant induction of costimulatory molecule expression or production of proinflammatory cytokines. SEA-induced Ag processing occurs independently of MyD88 or Toll/IL-1 receptor domain containing adaptor inducing IFN-β (Trif), but is significantly attenuated by inhibition of p38, but not ERK, signaling. In DCs exposed to SEA, ligation of CD40 provides a necessary second signal that stimulates costimulatory molecule expression, allowing DCs to mature into capable APCs. Collectively, the data demonstrate the existence of a MyD88/Trif-independent, p38-dependent pathway of Ag processing in DCs, which is uncoupled from conventional DC maturation and is associated with induction of Th2-type immune responses.
The immune system has evolved to recognize pathogen-inherent signals and respond accordingly. Much of the responsibility for sensing primary infection and for assessing the nature of the threat lies with dendritic cells (DCs).3 These cells, through expression of a panel of pattern recognition receptors such as TLRs are able to identify and respond to foreign organisms by initiating a sequence of events that link innate and adaptive immunity. Specifically, they 1) induce inflammation by producing cytokines and chemokines; 2) internalize material (Ag) from the pathogen and proteolytically process it for the presentation of peptide epitopes, complexed with MHCII molecules to T cells; 3) up-regulate expression of MHCII and costimulatory molecules; and 4) migrate to T cell-rich areas of lymphoid organs and activate T cells. These events are best understood from the study of DCs activated by TLR ligands, which stimulate the breadth of events classically associated with DC activation and maturation. It is the ability to activate DCs through TLRs that imbues extracts or molecules isolated from pathogens with adjuvanticity, the ability to strongly induce primary immune responses. In certain cases, dominant antigenicity (the property of being the target of an immune response) and adjuvanticity are the properties of individual molecules (1), but adjuvants generally possess the ability to facilitate the induction of immune responses to molecules that are introduced to the immune system alongside them.
We and others have shown that schistosome egg Ag (SEA), a soluble extract of the egg life stage of the helminth parasite, Schistosoma mansoni, is a potent inducer of T cell responses and does so independently of any added adjuvant (2). Indeed, SEA acts as an adjuvant itself, promoting T cell responses to unrelated Ags (3). In contrast to DCs activated by TLR ligands, which tend to promote Th1 or Th17 responses, DCs activated by SEA, or by other helminth-derived products, strongly promote Th2 responses (4, 5). The basis for SEA’s adjuvanticity and antigenicity has remained unclear, since unlike DCs activated by TLR ligands (6, 7, 8, 9), DCs exposed to SEA remain phenotypically immature, as assessed by costimulatory molecule expression and cytokine production. Thus, superficially, SEA-stimulated DCs appear immature and would therefore be expected to induce tolerance rather than adaptive immunity.
Although TLR-induced expression of costimulatory molecules during DC maturation has been regarded as essential for effective Ag presentation, it has become clear that the failure of immature DCs to effectively prime naive T cells may also reflect their inability to efficiently process Ag and thus generate antigenic peptide-MHCII complexes. TLR ligation in immature DCs leads to a process of phagosome maturation, whereby the lysosomal pathway is sufficiently acidified to allow the controlled degradation of newly endocytosed Ag into peptide fragments suitable for binding MHCII molecules (10). Moreover, there is a requirement for TLR signaling for the selection of Ag for processing and presentation (11, 12). Thus, a model has emerged in which DC maturation is coupled to Ag processing and, as such, the ability of DCs to prime naive T cells is regulated by the capacity of the stimulating Ag to induce a program of Ag processing and presentation (13). On the basis of these findings, we hypothesized that DC maturation could be uncoupled from Ag processing and that SEA might promote the induction of a maturation program that permits Ag processing and presentation in the absence of induced cytokine production and costimulation molecule expression commonly associated with DC maturation. Therefore, we undertook studies to investigate Ag processing by SEA-DC and, thus, gain a better understanding of how SEA acts as such a potent Th2-inducing DC Ag/adjuvant. Our data demonstrate that a Th2-inducing pathogen-derived product, SEA, induces the maturation of DCs into cells with high proteolytic capacity, without concomitant phenotypic maturation.
Materials and Methods
Mice and reagents
Six- to 12-wk-old BALB/c, C57BL/6, CD40−/−, and Trif−/− mice were from The Jackson Laboratory, CBA mice were from Taconic Farms, MyD88−/− mice were a gift from L. Turka and Y. Choi (University of Pennsylvania), and BALB/c 4get mice were a gift from M. Mohrs (Trudeau Institute, Sarnac Lake, NY) and bred at the University of Pennsylvania. BALB/c DO.11.10 and C57BL/6 OTII mice were bred and maintained under specific pathogen-free conditions at the University of Pennsylvania. DC activation was induced by 100 ng/ml LPS (Escherichia coli serotype 0111:B4; Sigma-Aldrich), 1 μg/ml CpG (MWG Biotech), or 10 μg/ml poly(I:C) (Sigma-Aldrich). SEA was prepared aseptically, as previously described (4), and used at 50 μg/ml. Aseptically collected and sterile-filtered (0.2-μm pore size) hen egg white (which is ∼50% OVA) was used as OVA to stimulate DO.11.10 and OTII cells (14); this Ag was assumed to be endotoxin free; a conclusion supported by its inability to activate DCs (data not shown). HRP (Sigma-Aldrich) was cleaned of contaminating LPS by Endobind column purification (Profos) and considered LPS free as demonstrated by Limulus amebocyte lysate assay (Sigma-Aldrich) and failure to induce DC maturation (MHCII and CD86 up-regulation) and IL-12 production.
Bone marrow-derived DC generation
Bone marrow cells were aseptically collected from the femurs of mice and 1 × 105 cells/well were seeded into 48- well non-tissue culture-treated plates (Corning). Cells were cultured for 6 days in RPMI 1640 (Sigma-Aldrich) supplemented with 5% heat-inactivated and filtered FCS (HyClone), 2 mM l-glutamine (Life Technologies), 100 U/ml penicillin plus 100 μg/ml streptomycin (Life Technologies), and 50 μM 2-ME (Sigma-Aldrich) in the presence of 10 ng/ml GM-CSF (PeproTech). At day 3, 250 μl of culture supernatant was removed and replaced with 250 μl of fresh supplemented medium containing 10 ng/ml GM-CSF.
DC/T cell cocultures
DCs were grown in the same conditions are described above, with the exception of being seeded at 200 μl of 2 × 106 cells/ml in round-bottom 98-well plates. On day 6, DCs were pulsed with 300 μg/ml OVA for 1 h before extensive washing to remove excess OVA, before culture for 18 h in the presence or absence of SEA (50 μg/ml) or LPS (100 ng/ml) in final volume of 100 μl. DCs were then cultured with 2 × 105 CFSE (Molecular Probes)-labeled DO.11.10 CD4+ T cells, purified by negative selection using MACS (Miltenyi Biotec), in complete T cell medium in a final volume of 200 μl. After 3 days, cells were restimulated with PMA (80 ng/ml), ionomycin (800 ng/ml; Sigma-Aldrich), and brefeldin A (10 μg/ml; BD Pharmingen) for 4 h and prepared for intracellular staining by a Cytofix/Cytoperm kit according to the manufacturer’s instructions (BD Pharmingen). Cells were then stained for IL-4, IFN-γ, and IL-2 using specific Abs (BD Pharmingen) before acquisition.
Day 6 DCs were pulsed with 250 ng/ml LPS-depleted HRP for 1 h at 37°C. Cells were then extensively washed with warm medium to remove any excess HRP. If required, DCs were then incubated with anti-CD40 (1 μg/ml: Alexis Biochemicals), p38 inhibitors SB203580, SB202190, SB220025, and PD169316 (10 μM; Calbiochem), or the ERK inhibitor U0126 (10 μM; Calbiochem), which inhibits MEK-1 and MEK-2, which are direct upstream activators of ERK, for 1 h, before stimulation with either SEA (50 μg/ml), LPS (100 ng/ml), CpG (1 μg/ml), or poly(I:C) (10 μg/ml; Amersham Biosciences) for 19 h. For the no-chase time point, DCs were harvested before addition of stimuli. To determine the level of HRP activity in DCs at “no chase” and 19 h, cells were first washed with warm PBS and lysed with lysis buffer (1% Triton X-100, 20 mM HEPES, 10% glycerol, and 150 mM NaCl) for 30 min on ice. A standard curve of HRP was prepared and the concentration of intact HRP within DC lysates was calculated by adding tetramethylbenzidine substrate and subsequent reading at 450 nm. To determine the maturation of DCs, cells and supernatants were harvested at 19 h instead and assessed for cytokine production by ELISA and surface phenotype by flow cytometry.
Characterization of DC cytokine production and surface phenotype
Cytokine ELISAs for IL-12p40 production were performed using paired mAb in combination with recombinant cytokine standard (BD Pharmingen) as described previously (4). Expression of MHCII and CD86 was quantified using specific Abs purchased from BD Pharmingen.
Cells were solubilized with lysis buffer (0.1% Triton X-100, 20 mM HEPES (pH 7.5), 10% glycerol, 150 mM NaCl, and 1 mM DTT) supplemented with 1 mM sodium orthovanadate, 10 mM sodium fluoride, and protease inhibitor mixture (Sigma-Aldrich). Equivalent amounts of cellular extracts prepared in 4× NuPage sample buffer (Invitrogen Life Technologies) were resolved on Bis-Tris NuPage gels (Invitrogen Life Technologies) and transferred to Immobilon membrane (Millipore). Blots were incubated with primary Ab against phosphorylated p38 and ERK1/2 (Cell Signaling Technology), HRP (Jackson ImmunoResearch Laboratories), detected using secondary HRP-conjugated Ab (Cell Signaling Technology), and visualized by the ECL system (Amersham Pharmacia Biotech).
Staining of lysosomes
Day 6 DCs were stimulated with SEA or LPS for 1 h, before the addition of 10 μM lysosensor blue (Molecular Probes) for 1 h at 37°C. Cells were then harvested, washed with PBS, mounted onto Superfrost/Plus microscope slides (Fisher), and photographed using a Leica DMIRB microscope and a DC500 camera.
Wild-type (WT) and Trif−/− DCs were pulsed overnight without or with SEA (50 μg/ml), washed, and then injected i.p. into naive mice (5 × 105/mouse in 0.5 ml of PBS). One week later, splenocytes were recovered and cultured in vivo with or without SEA (50 μg/ml) exactly as described previously (4, 5). Three days later culture supernatants were recovered and cytokine levels were measured using mAb-based ELISAs and reagents from BD Pharmingen.
Student’s t test was used to calculate the significance of differences between means.
SEA acts on DCs as an adjuvant to induce the development of Th2 cells from naive CD4+ T cells in vitro
Previous reports have shown that SEA can act as an adjuvant to promote Th2 responses both in vivo and in vitro. We sought to quantitate this effect using OVA-specific DO.11.10 cells activated by DCs that had been pulsed with OVA in the presence or absence of SEA. We found that DCs pulsed with OVA and SEA promoted a robust DO.11.10 CD4+ T cell proliferative response, equivalent in some experiments to that seen with TLR activated (Fig. 1). The adjuvanticity of SEA resulted in a Th2-biased response, which was in contrast to the Th1 response induced by TLR ligand-matured DCs (Fig. 1 and data not shown). These data confirm and expand on previous findings (4, 5, 15, 16).
SEA induces Ag degradation that is uncoupled from DC maturation
Studies of DCs stimulated through TLRs have indicated that for Ag to be processed for presentation, a maturation signal is required. Previous studies examining the induction of cytokine production and increased costimulatory molecule expression, rather than Ag degradation, suggest that SEA does not induce DC maturation (4, 17). However. SEA is clearly recognized by DCs, since beads coated with SEA are more rapidly and efficiently internalized than are beads coated with OVA (a foreign protein from a nonpathogen source), or LPS, a TLR ligand (data not shown). In this study, we ask specifically whether incubation of immature DCs with SEA results in an increased ability to process internalized Ag. To this end, we pulsed DCs with LPS-depleted HRP for 1 h at 37°C, washed, and then chased with HRP-free medium in the presence of SEA or LPS. After 19 h, processing was assessed by measuring HRP enzymatic activity in cell extracts and comparing loss of activity in stimulated vs unstimulated cells, as previously described (10) (Fig. 2,A). We found that ∼50% of the HRP initially internalized by DCs was lost following LPS stimulation, whereas nearly all of the HRP remained accountable for in DCs that received no microbial stimuli (Fig. 2,B). Strikingly, HRP levels in DCs stimulated with SEA were comparable to those in LPS-stimulated DCs (Fig. 2,B). Degradation did not occur in cells maintained at 4°C overnight (Fig. 2,C). The direct addition of SEA to HRP in the extracellular environment, at either neutral or acidic pH, did not result in loss of HRP activity (data not shown). SEA- and LPS-induced degradation of HRP was evident as the appearance of a major degradation product at ∼28 kDa on Western blots of HRP-pulsed DCs (Fig. 2,D). In contrast, immature DCs demonstrated little proteolytic potential. Importantly, unlike the case for DCs stimulated with LPS, all of the observed HRP degradation in SEA-DCs occurred in the absence of up-regulation of MHCII and CD86 (Fig. 2,E) or production of IL-12p40 (Fig. 2 F), confirming that neither HRP itself nor SEA was inducing TLR-like DC activation. Thus, SEA induces Ag proteolysis but this process is uncoupled from changes in DCs that are conventionally associated with maturation.
Ag proteolysis is linked to a drop in the intralysomal pH from ∼5.6 in resting DCs to ∼4.6 following TLR ligation (10), which approximates the pH optima of lysosomal proteases and thus facilitates efficient proteolysis of lysosomal contents (18). Therefore, we investigated whether the activation of DCs with SEA promoted a drop in lysosomal pH that would be consistent with observed changes in Ag degradation. Lysosome acidification was assessed with fluorescent microscopy using lysosensor probes. These probes are acidotrophic agents that accumulate in acidic organelles as the result of protonation, which leads to a pH-dependent increase in fluorescent intensity. The data show that compared with immature DCs (pH ∼ 5.6), in which little lysosensor staining can be observed, DCs activated with SEA demonstrated increased intralysosomal acidification to similar levels detected in LPS activation DCs (pH ∼ 4.6; Fig. 3). Thus, activation of DCs with SEA leads to phagosome maturation and a decrease in lysosomal pH, which has previously been shown to be an essential requirement for DC-mediated Ag degradation (10).
CD40 signaling regulates the ability of SEA-DC to present Ag
CD40 signaling in DCs has been shown to play an important role in the induction of Th2 immune responses in vivo (5, 19). Since SEA-activated DCs are characterized by an immature phenotype (4), which is believed to be insufficient for naive CD4+ T cell activation (20, 21, 22, 23), we hypothesized that CD40 ligation by CD154 following the initial interaction between SEA-DC and CD4+ T cells may provide the necessary additional signal to up-regulate costimulatory molecule and MHCII expression to allow T cell priming. Therefore, we directly investigated whether CD40 stimulation modulates the phenotype of SEA-DCs by culturing SEA- or TLR-activated DCs in the presence of agonistic anti-CD40 Abs. Consistent with previous reports (4, 24), CD40 signaling in LPS- or CpG- activated DCs resulted in augmented production of IL-12p40, whereas SEA-activated DCs failed to make IL-12p40 with or without CD40 stimulation (Fig. 4,A). We next examined whether CD40 stimulation of SEA-DCs augmented their ability to process and present Ag by investigating Ag degradation and MHCII and CD86 expression. We found modest effects of CD40 stimulation on the ability of SEA- activated DCs to further process Ag (Fig. 4,B) and to increase expression of MHCII and CD86 (Fig. 4,C). In contrast, these parameters were unaffected by CD40 stimulation in TLR-activated DCs, which appeared to be maximally activated. To determine the functional significance of these CD40-stimulated changes for the ability of DCs to activate naive CD4 T cells, DCs generated from WT or CD40−/− mice were pulsed with OVA for 1 h before, with or without SEA or a TLR ligand for a further 24 h, and then cocultured with CFSE-labeled DO.11.10 CD4+ T cells. As expected, DCs pulsed with OVA plus SEA were more capable of activating naive CD4+ T cells than were DCs pulsed with SEA alone (Fig. 4,D). However, the adjuvant effect of SEA was highly CD40 dependent, since CD40−/− DCs pulsed with OVA and SEA failed to induce T cell proliferation (Fig. 4,D). In contrast, the ability of TLR-activated DCs to induce T cell proliferation was only marginally affected by the lack of CD40 (Fig. 4 D). Therefore, CD40 signaling plays a role in positively regulating the ability of SEA-activated DCs to present Ag in the absence of proinflammatory cytokine production.
SEA-induced Ag processing occurs in the absence of MyD88 and Toll/IL-1 receptor domain containing adaptor inducing IFN-β (Trif) signaling
Recent studies have demonstrated the importance of MyD88 signaling for the generation of antigenic peptides from Ag containing TLR ligands (11). However, there is evidence to suggest that the antigenicity and adjuvanticity of SEA are TLR independent (16). To explore directly whether SEA-induced Ag processing is MyD88 independent, we generated immature DCs from MyD88−/− mice and examined Ag processing after differential maturation with TLR ligands or SEA. As anticipated, the level of HRP degraded by MyD88−/− DCs in response to SEA was comparable to that of WT DCs following activation with SEA (Fig. 5,A). Thus, the ability of SEA to activate DCs and induce a pathway of Ag processing occurs in a MyD88-independent manner. Stimulation of DCs with CpG was only able to induce processing of HRP and costimulatory molecule up-regulation (Fig. 5 A and data not shown) in WT DCs, confirming not only the known essential role of MyD88 for CpG-induced costimulatory molecule up-regulation in DCs, but that MyD88 is also essential for CpG- induced Ag processing.
Some reports have implicated TLR3 in the host response to SEA (25). TLR3-induced signaling is MyD88 independent and Trif dependent (26, 27). To explore the possibility that SEA-induced Ag degradation is Trif dependent, we characterized the Ag-processing capacity of DCs derived from Trif−/− mice. Our data failed to reveal a role for Trif in this process (Fig. 5,B). For these experiments, we used the TLR3 ligand poly(I:C) as a positive control, but this activation signal did not strongly induce processing even in WT mice. To further examine the role of Trif in SEA-mediated processes, we examined the ability of SEA to suppress TLR-induced IL-12 production in Trif−/− DCs (a characteristic property of SEA that correlates with its ability to promote Th2 responses (Refs, 15 and 17 and Fig. 5,C) and the ability of Trif−/− DCs to induce SEA-specific Th2 responses in vivo (Fig. 5 D). Our findings revealed no differences between WT and Trif−/− DCs in either of these regards, indicating that major immunologic effects of SEA are Trif independent.
Together, these data suggest that SEA activates an Ag- processing program in DCs independently of TLR signaling.
SEA-induced Ag processing requires p38 signaling
In a search for a molecular mediator of SEA-induced Ag processing, we reassessed previous data from the laboratory (17) and noted that p38 is phosphorylated following exposure of DCs to this helminth extract. This is intriguing since p38 regulates key aspects of Ag processing, including endocytic transport and phagosome maturation (12, 28, 29). To confirm the effect of SEA on p38, we stimulated DCs with SEA or the TLR ligand LPS and at times thereafter harvested cells for Western blot analysis of p38 phosphorylation. We were able to detect phosphorylated p38 at 10 and 30 min following SEA exposure (Fig. 6,A). In comparison, LPS induced stronger phosphorylation of p38 that was still evident at 1 h (Fig. 6,A). We next assessed whether p38 plays a role in SEA-induced Ag processing. DCs were loaded with HRP and washed, but before a 19-h chase in the presence of SEA or LPS, the cells were preincubated with a panel of p38 inhibitors (SB203580, SB202190, or PD169316) for 1 h. All inhibitors were used at 10 μM and did not alter cell viability (data not shown). In these experiments, immature DCs contained ∼60–70% of the HRP that they had internalized 19 h earlier (Fig. 6,B). As expected, SEA induced an increase in Ag processing (Fig. 6,B), but this effect was significantly diminished by the inhibition of p38 (Fig. 6, B and C). In these experiments, baseline Ag degradation in the absence of SEA was also diminished by the presence of p38 inhibitors (data not shown), indicating a fundamental role for this kinase in Ag processing. Previous work has indicated that SEA also induces ERK activation in DCs (17). We confirmed this result here, but found that inhibition of this event had no effect on SEA-induced Ag processing (supplementary Fig. 14).
Finally, we examined whether the adjuvanticity of SEA is p38 dependent. For these experiments, we pulsed DCs with OVA for 1 h before the addition of the PD169316, which is likely to be the most specific of the pharmacologic inhibitors of p38 activity (14). One hour later, DCs were stimulated with SEA and incubated overnight, washed, and cocultured with DO.11.10 CD4+ T cells for 6 h, after which CD69 expression as an indicator of T cell activation was measured. We focused on CD69 expression because we were concerned that longer term assays to monitor T cell activation (such as the measurement of proliferation, for example) would allow the effects of the p38 inhibitor to wear off. As expected, CD4 T cells activated by SEA-conditioned DCs up-regulated expression of CD69 expression to a point that was intermediate between that measured on T cells activated by control OVA- pulsed DCs or DCs pulsed with ligands plus OVA (Fig, 6,D). When p38 signaling was inhibited, SEA-induced Ag processing and presentation was attenuated by >3-fold (Fig. 6,D); p38 inhibition also affected Ag presentation by immature and TLR-activated DCs (Fig. 6 D). Similar results were observed with other p38 inhibitors SB203580 and SB202190 (data not shown). Collectively, our data suggest that SEA induces a p38-dependent signal that promotes Ag processing and presentation by DCs.
Schistosome eggs and SEA, an extract from them, are strongly antigenic, inducing notably Th2-polarized responses in the absence of adjuvant. Our previous work has shown that DCs exposed to SEA retain an immature phenotype and fail to produce inflammatory cytokines (4, 15, 17). DCs with this phenotype are generally considered to be tolerogenic (30), yet SEA-pulsed DCs induce SEA-specific Th2 responses when injected into mice (4). These findings indicate to us that 1) to induce Th2 responses in the absence of adjuvant, SEA needs to interact directly only with DCs, and not with other cells in the immune system and 2) the interaction between SEA and DCs is likely to lead to changes in DC biology that facilitate productive Ag presentation. In considering the latter, we undertook studies to directly assess whether SEA, like TLR ligands (10), induces proteolytic processing of Ag within DCs. We show that exposure to SEA induces DCs to process Ag via a MyD88/Trif-indendent but p38-dependent pathway. As a result, SEA-conditioned Ag-pulsed DCs are better at activating naive CD4 T cells than are DCs pulsed with Ag alone. The uncoupling of SEA-induced Ag processing from conventional DC maturation results in an environment that lacks cytokine promoters of Th1 or Th17 development and, partially as a result of this, we believe, a Th2 response emerges. This process relies on CD40-CD0L interactions, which we postulate play an important role in providing additional activation signals to DCs that are not provided by SEA. Finally, we show that coating beads with SEA enhances their ability to be taken up by DCs, suggesting that part of the inherent antigenicity of SEA might be linked to its preferential recognition by these important APCs.
SEA consists of a mixture of glycoconjugates (31), which can be recognized by C-type lectins including DC-SIGN and the macrophage galactose-type lectin (32, 33). A recent report has shown that recognition of SEA by these lectins on human DCs leads to targeting of SEA into MHCI+LAMP+ compartments (34). Consequently, C-type lectin-mediated recognition of SEA by DCs has the potential to promote efficient internalization and delivery of SEA to a compartment where Ag processing and generation of antigenic peptides suitable for MHCII loading would be favored. Targeting of exogenous Ag to endocytic receptors such as C-type lectins has been shown to lead to more efficient generation of MHCII-peptide complexes and activation of Ag-specific CD4+ T cells (35, 36, 37, 38). The ligation of a C-type lectin by SEA fits with the fact that SEA, like C-type lectin-initiated signaling, can inhibit TLR-induced DC activation (17, 39, 40).
We found that CD40 signaling in SEA-pulsed DCs results in modestly increased Ag processing, but more significantly, increased expression of MHCII and CD86 in the absence of proinflammatory cytokine production. The finding that SEA-stimulated DCs require CD40 to induce T cell proliferation in vitro indicates that CD40 signaling plays an essential role in inducing full Ag presentation abilities in these cells. Our data are consistent with previous reports that cross-linking CD40 on DC results in increased expression of peptide-MHCII complexes and enhances T cell priming (41). Moreover, CD40-mediated interactions have been shown to be important for up-regulation of costimulatory molecule expression in human monocytes and DCs (42, 43), and studies in CD40−/− and CD154−/− deficient mice have shown that this pathway is important for initiating signaling cascades required for effective T cell priming (44). Our previous findings that CD40−/− DCs fail to initiate SEA-specific Th2 responses (5, 19) imply that SEA-pulsed DCs need additional CD40-mediated signaling to present sufficient Ag for optimum T cell priming and Th2 development. Similarly, defective Th2 development has been demonstrated in other systems where CD40 signaling is deficient (45, 46, 47), whereas for promotion of Th1 immune responses, CD40-mediated interactions are redundant (48, 49).
Recently, studies by Blander and Medzhitov (11, 12) have proposed that TLRs allow DCs to decipher the information encoded by microbial pathogens and thus initialize a program of phagosome intrinsic maturation that selects these Ag for effective presentation to T cells. This differs from SEA-mediated activation of Ag processing in DCs, since our data indicate that SEA influences Ag processing in a more global manner. A possible reason for this discrepancy may be inherent to the nature of the stimulating Ag being examined. The phagosome intrinsic pathway of Ag processing was demonstrated by using particulate Ag (whole bacteria or TLR-coated latex beads) which are internalized via phagocytosis, whereas soluble Ag, such as SEA, are internalized via endocytic pathways. Therefore, it is feasible that in contrast to phagocytosis, receptors that mediate alternative modes of internalization, such as endocytosis, may have the ability to signal from the cell surface in a way that modulates the characteristics of other intracellular compartments. For example, DC-SIGN has been shown to mediate signals from the cell surface that are involved in the transport of internalized HIV particles to infectious synapses in DCs. Importantly, this occurred in the absence of colocalization between HIV and DC-SIGN and suggests that DC-SIGN might act as a sensor on the cell surface that can modulate intracellular compartments (50). Consistent with this, previous studies have already demonstrated that C-type lectins (DC-SIGN and Dectin-1) have the potential to modulate intracellular signals within DCs (51, 52). Therefore, we suggest that SEA, signaling via C-type lectins, or another as yet unidentified receptor could potentially signal from the cell surface to enhance the proteolytic capacity of intracellular vesicles that need not necessarily contain SEA.
Using MyD88−/− and Trif−/− DCs, we found that that SEA activates a TLR-independent pathway of inducible Ag processing in DCs. The TLR-independent nature of SEA’s inherent antigenicity is confirmed by the finding that both MyD88−/− and Trif−/− SEA-pulsed DCs are capable of promoting SEA-specific Th2 immune responses in vivo (data not shown). Numerous studies have demonstrated that p38 signaling can regulate aspects of the endocytic system and thus highlight the potential for p38-dependent regulation of DC Ag processing. For example, signaling via p38 has been shown to modulate endocytic traffic by regulating the activity of guanyl nucleotide dissociation inhibitor on Rab proteins (28). We found that SEA-mediated Ag processing and presentation are significantly attenuated by specific inhibition of p38 (but not ERK, supplementary Fig. 1) signaling. Thus, SEA induces a TLR-independent, p38-dependent pathway of inducible processing in DCs. Potentially, activation of p38 signaling by SEA may lead to phosphorylation of guanyl nucleotide dissociation inhibitor, which converts exhausted GDP-bound Rab5 to active GTP-Rab5, promoting increased fusion between endocytic vesicles and eventual delivery of Ag to degradative compartments. Consistent with this possibility, in macrophages TLR-induced fusion between phagosomes and lysosomes and eventual Ag degradation is impaired following inhibition of p38 signaling (12). There is precedent for C-type lectins initiating p38 signaling in specific situations (53, 54). Thus, C-type lectin-mediated recognition of SEA could potentially induce a p38-dependent program in which Ag processing is uncoupled from cytokine production and other events normally associated with maturation.
The data presented here demonstrate that SEA, a product derived from the helminth parasite S. mansoni, induces an unconventional type of DC activation in which Ag processing is uncoupled from conventional maturation. Understanding the mechanisms that allow DCs to present Ag without initiating inflammation may provide opportunities for developing new defined adjuvants for clinical use.
We thank C. J. Krawczyk for insightful comments and E. Jung for excellent technical assistance.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by National Institutes of Health Grant AI53825 (to E.J.P.).
Abbreviations used in this paper: DC, dendritic cell; MHCII, MHC class II; SEA, schistosome egg Ag; WT, wild type; Trif, Toll/IL-1 receptor domain containing adaptor inducing IFN-β.
The online version of this article contains supplemental material.