Helminth-excreted/secreted products are recognized by multiple receptors on DCs to block the TLR response and bias Th2 polarization in a cRAF dependent pathway

Abstract

Dendritic cells (DCs) recognize pathogens and initiate the T-cell response. The DC-helminth interaction induces an immature phenotype in DCs; as a result, these DCs display impaired responses to TLR stimulation and prime Th2-type responses. However, the DC receptors and intracellular pathways targeted by helminth molecules and their importance in the initiation of the Th2 response are poorly understood. In this report, we found that products excreted/secreted by Taenia crassiceps (TcES) triggered cRAF phosphorylation through MGL, MR, and TLR2. TcES interfered with the LPS-induced NFκB p65 and p38 MAPK signaling pathways. In addition, TcES-induced cRAF signaling pathway was critical for down-regulation of the TLR-mediated DC maturation and secretion of IL-12 and TNF-α. Finally, we show for the first time that blocking cRAF in DCs abolishes their ability to induce Th2 polarization in vitro after TcES exposure. Our data demonstrate a new mechanism by which helminths target intracellular pathways to block DC maturation and efficiently program Th2 polarization.—Terrazas, C. A., Alcántara-Hernández, M., Bonifaz, L., Terrazas, L. I., Satoskar, A. R. Helminth-excreted/secreted products are recognized by multiple receptors on DCs to block the TLR response and bias Th2 polarization in a cRAF dependent pathway.

Helminth parasites are a major public health problem in developing countries, and they can establish chronic infection in their hosts, owing to their ability to escape or modulate the host's immune system via multiple mechanisms. These parasites skew the immune response toward a T-helper 2 (Th2) and/or a regulatory environment associated with high levels of interleukin (IL)-4, IL-13, IL-5, and IL-10. In addition, helminth infections impair immunity against other unrelated infections and may affect vaccine efficacy (1). One potential immune evasion strategy involves the modulation of early responses by dendritic cells (DCs; refs. 2, 3). DCs sample the environment and recognize pathogen-associated molecular patterns (PAMPs) as well as the host's danger signals. When DCs are activated by viral, bacterial, or protozoan-derived molecules that bind Toll-like receptors (TLRs), intracellular signaling through the mitogen-activated protein kinase (MAPK) family (e.g., p38, JNK, ERK), the adapter molecule MyD88, and the transcription factor nuclear factor κB (NFκB) is initiated, leading to the up-regulation of costimulatory molecules (e.g., CD80, CD86, CD40) and chemokine receptors, as well as the release of proinflammatory cytokines, such as IL-12 and tumor necrosis factor α (TNF-α) (4). After pathogen recognition, DCs migrate from the peripheral tissue, armored with a variety of signals to activate T cells in the lymph nodes. T-cell polarization depends on signals delivered by DCs. For example, while IL-12 secretion activates T cells toward a Th1 profile, IL-10 and TGF-β are required to prime T_reg cells. However, the signals delivered by DCs necessary to induce a Th2 response have not yet been clearly defined (3, 5, 6).

In contrast to classical DC activation, mounting evidence indicates that DCs exposed to different helminth-derived molecules display a conserved phenotype similar to immature DCs, but, unlike immature DCs, they show an impaired response to several TLR agonists, although they are still efficient Th2 and/or T_reg inducers (2, 3). Despite the growing interest in understanding DC-helminth interactions, not much is known about the ligands, receptors, and intracellular signaling pathways involved. Glycosylated molecules derived from helminths are suspected to play a role in the induction of Th2 responses (2, 6–9). The main surface receptors on DCs that recognize glycosylated structures are the C-type lectin receptors (CLRs). Although some helminths have been shown to bind CLRs, it is still unknown whether this interaction triggers signaling in DCs (10, 11).

Taenia crassiceps is a helminth parasite that has been used in the study of parasite-host interactions in cysticercosis (12). As the infection progresses, the immune response elicited by this parasite is polarized to Th2 (13, 14). Like other helminth derivatives, the T. crassiceps-excreted/secreted molecules (TcES) impair the ability of bone marrow-derived DCs (BMDCs) and human-derived DCs to respond to different proinflammatory ligands, such as lipopolysaccharide (LPS), CpG, and Toxoplasma gondii antigen. Moreover, TcES-treated DCs prime the Th2 response in vitro in a carbohydrate-dependent fashion (15, 16).

To investigate the mechanisms involved in helminth-DC immune modulation and identify molecules targeted by TcES, we used blocking antibodies against C-type lectins as well as MAPK inhibitors to block intracellular pathways. We found that TcES bound preferentially to mannose receptor (MR) and macrophage galactose C-type lectin (MGL), and TLR2^−/− and MGL^−/− DCs displayed a reduced ability to bind TcES. Exposure of DCs to TcES in vitro resulted in the enhanced localization of RAS to the cytoplasm and phosphorylation of cRAF, but not ERK1/2, p38, or NFκB p65. We also showed that cRAF phosphorylation was mediated by multiple-receptor engagement, including MR, MGL, and TLR2. On the other hand, TcES inhibited LPS-mediated phosphorylation of p38 and NFκB p65, DC maturation, cytokine production, and the ability of LPS-treated DCs to prime Th1 responses in vitro. Using a selective cRAF inhibitor (GW5074) or cRAF-specific siRNA, we found that DCs exposed to TcES were unable to impair the up-regulation of IL-12, TNF-α, CD80, and CD86 in response to LPS. Moreover, the ability of TcES-exposed DCs to efficiently promote the production of IL-4 and IL-13 by T cells was abolished in DCs pretreated with cRAF inhibitor. Taken together, our results demonstrate for the first time that the cRAF pathway is triggered by helminth molecules through multiple receptors and that cRAF is essential for the blockage of TLR mediated inflammatory signaling in DCs, the attenuation of Th1 polarization and the promotion of Th2 polarization in vitro. These findings provide new information on the mechanism by which helminths induce intracellular signaling to block the TLR pathway in DCs and direct a Th2 polarized immune response. This knowledge can be exploited in the identification of novel therapeutic strategies against parasitic worms, autoimmune diseases, and allergic reactions.

MATERIALS AND METHODS

Mice

Wild-type (WT) C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA), and MGL-deficient mice were obtained from The Scripps Research Institute (La Jolla, CA, USA). The mice were maintained in a pathogen free animal facility at The Ohio State University (Columbus, OH, USA) and Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México (Mexico City, Mexico) in accordance with U.S. National Institutes of Health and institutional guidelines.

Parasites and TcES

Metacestodes of T. crassiceps were harvested under sterile conditions from the peritoneal cavity of female BALB/c mice after 6–8 wk of infection. The cysticerci were washed 4 times in sterile PBS and cultured in PBS at 37°C for 24 h. TcES were recovered from the supernatant and centrifuged for 10 min at 5000 rpm. Next, the proteins were concentrated using an Amicon Ultrafilter with a 50-kDa cutoff membrane (Millipore, Billerica, MA, USA). The high-molecular-mass molecules were collected, and protease inhibitors were added. The samples were stored at −70°C until further use.

DC stimulation and coculture assays

BMDCs were obtained as described previously (16). DCs were incubated with TcES (40 μg/ml) or LPS (1 μg/ml). After 24 h, DCs were analyzed by flow cytometry, and supernatants were recovered for cytokine detection by ELISA. For cRAF inhibition experiments, DCs were preincubated with cRAF inhibitor GW5074 (Merck KGaA, Darmstadt, Germany) or DMSO for 2 h. cRAF RNA knockdown experiments were performed using SmartPool On-Target plus RAF1 siRNA or nontargeting control siRNA along with Dharmafect transfection medium (ThermoFisher Scientific, Lafayette, CO, USA), as described previously (17). For cocultures, DCs were preloaded with ovalbumin (OVA) peptide (2 μg/ml) for 2 h and incubated with GW5074 for an additional 2 h, then stimulated with TcES and/or LPS. After 24 h, DCs were washed 3 times with PBS and coincubated with purified OTII CD4⁺ T cells at a 1:2 ratio. Supernatants were recovered 7 d later, and cytokines produced were analyzed by ELISA. For in vivo studies, DCs were stimulated 24 h in vitro, then injected into the footpad of C57BL/6 mice that had previously been adoptively transferred with CFSE-labeled OTII CD4⁺ T cells. After 7 d, lymph node cells were obtained and stimulated with PMA/ionomycin for 4 h. Intracellular IL-4 production was measured by flow cytometry using APC anti-IL-4 antibody (BioLegend, San Diego, CA, USA) on cells gated on CD4⁺ and CFSE⁺ populations.

Labeling of TcES with FITC

TcES were labeled with FITC (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's protocol. Briefly, 1 mg/ml solution of TcES was prepared in 0.1 M sodium bicarbonate buffer (pH 9). FITC was dissolved in DMSO at 1 mg/ml, and 50 μl of FITC solution was then slowly added to the TcES solution in 5-μl aliquots with gentle stirring. The resulting TcES-FITC solution was incubated for 8 h at 4°C in the dark, and then washed 3 times with PBS using a 50-kDa cutoff membrane (Amicon Ultrafilter; Millipore) and centrifugation at 4000 g for 20 min. The proteins were quantified by Bradford assay and maintained at 4°C.

Antibody blocking assays

Different glycans were used for the competition assays, as described by van Vliet et al. (10). Laminarin, mannan, and GalNac (all 100 μg/ml) were used to block CLRs (Sigma-Aldrich). EGTA (10 mM) was used to disrupt the structure of the C-type lectins. Blocking antibodies anti-MR, anti-TLR2 (BioLegend, San Diego, CA, USA) and anti-MGL (Hycult Biotech, Plymouth Meeting, PA, USA) were used at a concentration of 10 μg/ml. DCs were cultured with or without blocking reagents and/or antibodies for 30 min at 37°C, then incubated with TcES-FITC for 30 min at 37°C. The cells were washed 3 times with FACS buffer, and TcES fluorescence was detected by flow cytometry. The role of individual receptors or receptor combinations in TcES-mediated inhibition of IL-12 and TNF-α induction after stimulation by LPS was analyzed by ELISA. In addition, the ability of TcES to induce Th2 responses in coculture experiments was evaluated.

Analysis of cell surface markers on BMDCs

Surface expression of DC maturation markers was analyzed using multicolor flow cytometry. After 24 h of stimulation, BMDCs were harvested, washed, and resuspended in cold PBS containing 5% FCS and 0.05% NaN₃. Fc receptors were blocked with mouse serum for 20 min at 4°C. The cells were washed and stained with APC-conjugated anti-CD11c, FITC-conjugated anti-CD80, and PE-conjugated anti-CD86 antibodies (BioLegend). The cells were analyzed on a FACSCalibur flow cytometer using Cell Quest software (BD Biosciences, San Jose, CA, USA). The data are represented as mean fluorescence intensity (MFI).

TLRs and NFκB reporter assay

HEK cells transfected with TLR4/CD14, TLR2/CD14, or TLR2/TLR6 were kindly donated by Dr. Laura Bonifaz (Instituto Mexicano del Seguro Social, Mexico City, Mexico). The HEK cells were plated in 12-well plates at 2 × 10⁵ cells/well and stimulated with TcES. LPS and zymosan were used as positive controls for the TLR4- and TLR2-transfected cells, respectively. After stimulation for 24 h, the supernatants were recovered, and the production of human IL-8 was analyzed by ELISA. For NFκB reporter assays, RAW-blue cells were purchased from InvivoGen (San Diego, CA, USA). Briefly, 100,000 cells were plated in a 96-well plate and stimulated with LPS, TcES, or both. After 24 h of incubation, 50 μl of supernatant was recovered, to which 150 μl of Quanti-Blue substrate (InvivoGen) was added. Levels of secreted embryonic alkaline phosphatase indicative of NFκB activation were analyzed, measuring the absorbance at 655 nm.

Phospho-flow cytometry/immunofluorescence

BMDCs obtained after 7 d of culture were washed and plated in RPMI 1640 medium without GM-CSF for 24 h. Then 2 × 10⁵ cells were stimulated for 30 min with the different treatments as described above. After stimulation, the cells were incubated for 15 min with anti-CD16/32, and then stained with anti-CD11c antibody (BioLegend). After incubation, the cells were fixed with 2% paraformaldehyde for 10 min at room temperature, washed with staining buffer, and permeabilized with ice-cold methanol for 10 min at 4°C. Cells were then washed twice with staining buffer, then incubated with a primary rabbit anti-mouse antibody against phospho-cRAF or phospho-SYK (1:100; Cell Signaling, Danvers MA, USA) for 20 min in the dark at room temperature. Finally, the cells were labeled with a secondary antibody, donkey anti-rabbit DyLight 488 (BioLegend) at a concentration of 0.25 μg/10⁶ cells for 20 min at room temperature in the dark. The cells were then analyzed by flow cytometry or by fluorescence microscopy. Secondary antibody alone was used as a negative control.

Immunoblot

DCs were differentiated as described above. On d 7, the DCs were harvested and plated at 2.5 × 10⁶ cells/ml in 6-well plates. The cells were used 24 h after plating to eliminate any residual effects from GM-CSF. DCs were stimulated with different doses of TcES at the indicated times. In some experiments, inhibitors were added 2 h before stimulation. After stimulation, the cells were centrifuged at 1500 rpm for 5 min and washed with PBS. The cells were then lysed with lysis buffer for 15 min and lysates were quantified using the BCA assay (ThermoFisher Scientific) and then frozen at −80°C until further use. The proteins were resolved using 10% SDS-PAGE and blotted onto a PDVF membrane for 2 h. The membrane was blocked using TBS supplemented with 10% nonfat milk for 2 h, incubated overnight at 4°C on a rotary platform with primary antibodies against RAS or p-MEK1/2 (Millipore), p-cRAF Ser338, p-p38, p-ERK1/2 or p-NFκB p65 Ser536 (Cell Signaling) at a dilution of 1:1000. Membranes were washed 3 times for 5 min with PBS containing 0.5% Tween-20 and incubated with the secondary antibody anti-rabbit-HRP (Cell Signaling) for 2 h. Finally, chemiluminescence was developed using an Amersham ECL chemiluminescent kit (GE Healthcare Biosciences, Pittsburgh, PA, USA) and acquired using FluorChem HD2 Imager (ProteinSimple, Santa Clara, CA, USA).

DC and T-cell cocultures

DCs were obtained as described above, and CD4⁺ T cells were obtained from OTII mice by negative selection using magnetic microbeads. The DCs were plated at 5 × 10⁴ cells/well in 96-well plates and then stimulated as follows. First, 2 μg/ml OVA peptide 323–339 was added to the cultures. After 2 h, cRAF inhibitor was added at the indicated concentrations. After another 2 h, TcES and/or LPS were added to the DCs. After 24 h, the DCs were harvested and centrifuged, and supernatants were removed. The DCs were then washed 3 times with PBS to avoid any inhibitory action on T cells. Finally, T cells were added to the DC cultures at a DC:T-cell ratio of 1:2 and maintained for 7 d. The supernatants from the cocultures were analyzed for IL-10, IL-4, IL-13, and IFN-γ production using a sandwich ELISA (BioLegend).

Adoptive transfer

OTII CD4⁺ T lymphocytes were isolated from spleens using negative magnetic separation (Miltenyi Biotec, Auburn, CA, USA). After negative selection, the cells were analyzed by flow cytometry; 90% of the isolated cells were CD4⁺. CD4⁺ cells were labeled with 10 μM CFSE. Then, 5 × 10⁶ cells were adoptively transferred intravenously into naive C57BL/6 mice. At 24 h after transfer, BMDCs previously loaded with 2 μg/ml OVA peptide and stimulated with TcES in the presence or absence of the cRAF inhibitor GW5074 were injected into the right footpad of adoptively transferred mice. Control mice received BMDCs only loaded with OVA. After 7 d, the LNs and the spleen were recovered, homogenized using 70-μM strainers, and stimulated with PMA and ionomycin in the presence of brefeldin A for 5 h. Cells were then stained with phycoerythrin-labeled CD4 antibody (BioLegend), and intracellular IL-4 production was measured by flow cytometry using APC-conjugated anti-IL-4 antibody (BioLegend) on cells gated on CD4⁺ and CFSE⁺ populations.

Statistical analysis

The statistical significance of differences between treated and control DCs in the cytokine release assays was calculated using a paired Student's t test. The statistical significance of differences between mean fluorescence intensities detected by flow cytometry was calculated using a 2-tailed Mann-Whitney test. Differences were considered significant when P < 0.05. GraphPad Prism software (GraphPad, San Diego, CA, USA) was used for all statistical analyses. Error bars represent sem.

RESULTS

TcES bind MR and MGL

The innate recognition of pathogen associated molecules by DCs is crucial to the initiation of the immune response. Different studies have shown that helminth derived molecules can be recognized by C-type lectins (10, 11). In our model, we previously found that TcES inhibit LPS triggered immune responses and skew T-cell polarization toward a Th2 phenotype in vitro in a carbohydrate-dependent fashion. We reported that most of the TcES were bound to concanavalin A, indicating that TcES are glycosylated with glucose, mannose, or galactose (16). In another study, Lee et al. (18) reported that the main N-terminal glycosylation structures in TcES were mannose, fucose, galactose, and GlcNAc. The researchers also found a rare Fucα1 → 3GlcNAc antenna on T. crassiceps molecules (18). The natural candidates for carbohydrate recognition are CLRs, such as MR, MGL, and DC-SIGN. Among CLRs, it has been shown that DC-SIGN and Dectin-1 crosstalk with TLR intracellular signaling, thereby affecting the response of the affected DCs to TLR ligands (19, 20). Thus, we hypothesized that the receptors that bind the glycomolecules on TcES initiate crosstalk with TLR signaling and block the proinflammatory response of DCs. To investigate the roles of different CLRs in TcES recognition, TcES molecules were labeled with FITC to perform binding assays. TcES-FITC bound BMDCs at 37°C, indicating that TcES-FITC can be recognized by DCs; however, incubation of DCs and TcES at 4°C had reduced fluorescence (Fig. 1A, B). Next, we used EGTA as a calcium chelator to impair the binding function of the CLRs. EGTA pretreatment significantly reduced the MFI of TcES-FITC on DCs, indicating that EGTA-treated cells bind fewer TcES and suggesting a role for CLRs in TcES recognition. To identify the CLRs involved in TcES recognition, BMDCs were preincubated with different carbohydrates, such as mannan, N-acetylgalactosamide, or laminarin, or with a blocking antibody against MR or MGL. TcES-FITC fluorescence was reduced when BMDCs were preincubated with N-acetylgalactosamide or mannan, suggesting a role for MGL or MR, respectively (Fig. 1). These findings were confirmed using blocking antibodies against MGL and MR, which significantly reduced the MFI of TcES-FITC compared to DCs treated with isotype antibody controls (Fig. 1B). In addition, MGL^−/− BMDCs and splenic CD11c⁺ cells had reduced TcES-binding ability (Fig. 1A–C). In contrast, laminarin, a ligand of Dectin-1, did not significantly decrease TcES-FITC binding to DCs (Fig. 1A, B). These data suggest that TcES can bind to BMDCs via multiple receptors including MR and MGL.

Figure 1.

BMDCs recognize TcES through MR, MGL, and TLR2. DCs were preincubated with mannan to block mannose receptor, GalNac to block MGL, laminarin to block Dectin-1, or antibodies against MR or MGL. Cells were subsequently incubated with fluorescently labeled TcES, and fluorescence was analyzed by flow cytometry. A) Blue histograms indicate DCs incubated 30 min with TcES alone at 37°C. Red histograms indicate DCs incubated 30 min with the indicated treatment before TcES incubation. B) Data represented as MFI of DCs with different treatments and untreated DCs exposed to TcES-FITC. Basal levels of autofluorescence were subtracted from all treatments. Data are representative of 3 independent experiments. *P < 0.05. C) BMDCs from TLR2- or MGL-deficient mice were incubated for 30 min with TcES-FITC, and binding was analyzed by flow cytometry. Histograms depicting MFIs are representative of 3 independent experiments. *P < 0.05. D) Total splenocytes from WT, TLR2^−/−, or MGL^−/− mice were incubated with TcES-FITC for 30 min and analyzed by flow cytometry. Cells are gated on CD11c⁺ populations. Histograms represent MFIs. *P < 0.05.

TcES inhibit TNF-α and IL-12 production in MGL^−/− or TLR2^−/− DCs

Previously, we showed that BMDCs from C57BL/6 mice are refractory to TcES modulation compared with BMDCs derived from Balb/c mice. This phenomenon is associated with resistance of C57BL/6 mice to T. crassiceps infection (21). However, it is possible to induce successful T. crassiceps infection in C57BL/6 mice by injecting 4 times more parasites that those used to infect Balb/c mice (22). With this in mind, we tested whether C57BL/6 BMDCs could be modulated by TcES in a dose-dependent fashion. We found that the minimal dose required to inhibit the up-regulation of costimulatory molecules CD80 and CD86, and cytokines IL-12 and TNF-α, after LPS stimulation, was 40 μg of TcES (Fig. 2A, B). Next, we examined whether the receptors that we identified to be involved in TcES recognition had a role in modulating the activities of TcES. Having demonstrated that MGL^−/− BMDCs, as well as WT BMDCs cultured with MGL-blocking antibody or N-acetylgalactosamide, had impaired TcES-FITC binding, we determined whether MGL mediates the TcES inhibition of TLR-induced DC maturation and proinflammatory cytokine release. After TcES stimulation, we did not observe any differences in the modulation of BMDCs of WT and MGL^−/− mice, as the IL-12 and TNF-α induced by LPS were similarly inhibited by TcES. However, MGL^−/− BMDCs exhibited reduced production of TNF-α and enhanced production of IL-10 in response to LPS compared to WT BMDCs (Fig. 2C). Since TLR2 is involved in the resistance to T. crassiceps infection (23), TcES may include molecules that can bind TLR2. To test this hypothesis, we performed binding assays with TcES-FITC using BMDCs and splenic CD11c⁺ DCs from TLR2-deficient mice. We observed that BMDCs and splenic CD11c⁺ DCs from TLR2^−/− mice displayed an impaired ability to recognize TcES-FITC compared with cells from WT mice (Fig. 1C, D). To determine whether TcES recognition by TLR2 blocks TLR-induced proinflammatory responses, we incubated TLR2^−/− and WT BMDCs with TcES. As expected, TcES-exposed WT BMDCs blocked IL-12 and TNF-α secretion induced by LPS; interestingly, this TcES-mediated impairment of IL-12 and TNF-α production was not changed in TLR2^−/− BMDCs (Fig. 2D). Taken together, our results demonstrate that although both TLR2 and MGL bind TcES, it appears that these molecules alone do not play a role in the impairment of the DC response to LPS.

Figure 2.

TcES inhibit TNF-α and IL-12 production in MGL^−/− and TLR2^−/− BMDCs. A) BMDCs derived from C57BL/6 mice were treated with LPS and different doses of TcES. After 24 h of stimuli, production of IL-12 and TNF-α was evaluated in supernatants (A), and cell surface costimulatory markers CD80 and CD86 were evaluated by flow cytometry (B). BMDCs from WT and MGL^−/− (C) or TLR2^−/− (D) mice were exposed to 1 μg LPS, 40 μg TcES, or both. After 24 h incubation, production of IL-12, TNF-α, and IL-10 in culture supernatants was determined using ELISA. n = 3. *P < 0.05.

TcES are unable to activate TLR2 or TLR4 on reporter cell lines

The inhibitory activity of TcES on BMDCs is carbohydrate dependent (16). Some glycosylated molecules derived from parasites are recognized by TLR4 or TLR2 on DCs (24–26). Further, TLR2 binds TcES (Fig. 1C), and TLR2-deficient mice exhibit an enhanced parasitic load in T. crassiceps infection (23). To investigate whether TcES mediates TLR2 and TLR4 activation, we used IL-8 reporter HEK cell lines transfected with TLR2/CD14, TLR2/TLR6, or TLR4/CD14, which produce IL-8 on TLR2 or TLR4 activation and subsequent NFκB signaling. Addition of TcES to HEK cells transfected with TLR2/CD14, TLR2/TLR6, or TLR4/CD14 did not result in any differences in IL-8 production compared to untreated cells (Supplemental Fig. S1). This data shows that TcES do no activate the more common TLR2 heterodimers or the heterodimer TLR4/CD14.

TcES impair the LPS-induced phosphorylation of p38 MAPK and NFκB p65

DCs detect pathogens through TLRs, which play a major role in the increased production of proinflammatory cytokines by activating the MAPK and MyD88-NFκB pathways (4). Because TcES-exposed BMDCs had impaired responses to LPS stimulation, we hypothesized that TcES could affect the intracellular signaling initiated by LPS. We therefore investigated whether the main MAPKs implicated in TLR signaling are affected by TcES, using Western blot detection of their phosphorylated products. In agreement with several studies, we found increased p38, MEK, and ERK1/2 phosphorylation in BMDCs after 30 min of LPS stimulation. In contrast, p38 and MEK were not phosphorylated in BMDCs exposed to TcES alone (Fig. 3A). Interestingly, BMDCs exposed to TcES and LPS showed decreased p38 phosphorylation, but MEK and ERK1/2 were phosphorylated at the same level as in BMDCs exposed to LPS alone (Fig. 3A). To investigate whether the exposure of DCs to TcES alters the final step in TLR signaling, we analyzed the phosphorylation of NFκB p65 and observed that exposure of DCs to TcES alone did not result in NFκB p65 phosphorylation. Further, while LPS alone induced strong phosphorylation of NFκB p65, BMDCs exposed to both TcES and LPS resulted in significantly attenuated NFκB p65 phosphorylation (Fig. 3A, B). Together, these data strongly suggest that TcES modulate intracellular signaling that affects the TLR pathway.

Figure 3.

TcES induce cRAF activation and impair NFκBp65 and p-p38 phosphorylation in BMDCs. A) BMDCs were stimulated with 40 μg/ml TcES and/or 1 μg/ml LPS. After 30 min, cell lysates were prepared, and p-NFκBp65, p-p38, RAS, p-cRAF, p-MEK1/2, and p-ERK1/2 proteins were detected by Western blot. Data are representative of 5 different experiments. B) Data were normalized with β-actin, and relative amounts of protein expression were calculated using LPS as maximum induction. n = 5. C) Phosphorylation of cRAF in BMDCs was detected by flow cytometry at the indicated time points after TcES treatment. Data are represented as MFIs from 2 independent experiments. D) cRAF phosphorylation was detected by fluorescence microscopy. Phospho-cRAF was detected in the cytoplasm after 30 min of TcES exposure.

TcES induce cRAF phosphorylation in BMDCs

The fact that TcES bind CLRs (Fig. 1) and affect signaling pathways in DCs (Fig. 3A) led us to investigate the intracellular pathways that are affected by the activation of the CLRs. Recent advances in the understanding of the intracellular signaling induced by CLRs have highlighted cRAF and SYK as molecules that modify of TLR signaling (19, 20, 27). To test whether TcES can activate these pathways, we exposed BMDCs to TcES for 5–60 min and found that cRAF phosphorylation was significantly enhanced; interestingly, cRAF phosphorylation was detected at an early time point in TcES-exposed BMDCs and remained phosphorylated for ≥1 h (Fig. 3A, C). Further detection of cRAF by fluorescence microscopy demonstrated that cRAF was localized in the cytoplasm after 30 min of exposure to TcES (Fig. 3D). Moreover, in BMDCs treated with TcES and LPS, cRAF phosphorylation was detected, but at a lower level than in BMDCs treated with TcES alone (Fig. 3A). In contrast, SYK phosphorylation was absent in TcES treated DCs (Supplemental Fig. S1D). The canonical cRAF signaling pathway begins with RAS activation (28). Interestingly, we found enhanced amounts of RAS in the cytoplasm of TcES treated DCs. However, we did not detect the phosphorylation of MEK or ERK1/2, which are downstream targets of cRAF (Fig. 3A). RAS also participates in the initiation of the PI3K signaling pathway. We therefore investigated the role of this pathway using PI3Kγ-deficient DCs. We found that these were modulated by TcES in a similar fashion as in WT DCs. This result eliminates the possibility that DC modulation by TcES involves PI3Kγ (Supplemental Fig. S2).

TcES impair TLR signaling and DC maturation in a cRAF-dependent pathway

Because TcES impair DC maturation in response to TLR stimulation (Fig. 2A) and (16), we investigated the possible role of cRAF in the modulation of DC responses. As we showed earlier, after TLR4 stimulation using LPS as an agonist, IL-12, TNF-α, CD80, and CD86 were up regulated, but these responses were impaired by exposure to TcES (Fig. 4A, B). However, when BMDCs were pretreated with both the cRAF inhibitor GW5074 and TcES, cRAF phosphorylation was impaired (Fig. 4C). In addition, the production of IL-12 and TNF-α and the up-regulation of CD80 and CD86 were restored to levels that are typically observed after LPS stimulation. Since TcES diminished NFκB phosphorylation in LPS treated cells, we determined the effect of cRAF inhibition on NFκB p65 using the NFκB p65 RAW blue reporter cell line. As in BMDCs, LPS enhanced NFκB activation, and the presence of TcES reduced the levels of LPS induced NFκB activation, although to a lesser degree. Chemical inhibition of cRAF restored NFκB activity induced by LPS on TcES treated RAW blue reporter cells (Fig. 4B). To reinforce our data obtained using the chemical inhibitor GW5074; we knocked down cRAF in DCs using siRNA. DCs treated with cRAF siRNA, reduced cRAF protein levels by 50% compared with those treated with nontargeting control siRNA (Fig. 4D). Consistent with our cRAF chemical inhibition results, siRNA knockdown of cRAF on DCs sufficiently blocked the immunomodulatory activity of TcES, and restored IL-12 and TNF-α production in LPS stimulated cells. (Fig. 4E). These findings demonstrate that TcES elicit an intracellular signal through a cRAF-dependent pathway that impairs both the expression of CD80 and CD86 and the release of IL-12 and TNF-α that are triggered by TLR4 stimulation. To our knowledge, this is the first report showing that helminth molecules trigger cRAF to modulate DC responses.

Figure 4.

TcES block LPS-induced maturation and proinflammatory immune responses through a cRAF-dependent pathway. A) BMDCs were incubated with a cRAF inhibitor GW5074 (solid bars) or DMSO (open bars). DCs were washed and then treated with 40 μg/ml TcES and/or 1 μg/ml LPS for 24 h. Levels of IL-12p70 and TNF-α in culture supernatants were analyzed by ELISA, and cells were analyzed by flow cytometry for expression of costimulatory molecules CD80 and CD86. n = 3. *P < 0.05. B) Raw blue reporter cells were treated with TcES in the presence or absence of LPS for 24 h, and NFκB activation was measured after addition of Quanti-Blue reagent. Results are representative of 3 independent experiments. C) BMDCs were treated 30 min with LPS and/or TcES with or without 1 μM GW5074; cell lysates were analyzed for phosphorylated cRAF protein by Western blot. D) BMDCs were transfected with nontargeting siRNA or cRAF SmartPool siRNA, and total cRAF protein expression was evaluated by flow cytometry and fluorescence microscopy. E) BMDCs transfected with nontargeting siRNA or cRAF siRNA were stimulated as above for 24 h, and production of IL-12 and TNF-α was evaluated by ELISA. Data are representative of 3 independent experiments. *P < 0.05.

cRAF phosphorylation in TcES-treated DCs enhances Th2 but impairs Th1 responses in vitro and in vivo

DCs exposed to helminth-derived molecules polarize Th2 responses in vitro and in vivo (2, 3). However, the mechanisms in DCs that instruct the induction of the Th2 response are incompletely understood (3, 5). Since we found that TcES induce cRAF phosphorylation, it is possible that signaling through this pathway directs DCs to deliver signals that prime Th2 responses. To address this possibility, we loaded DCs with OVA peptide and incubated with transgenic CD4⁺ T cells from OTII mice in vitro, then analyzed T-cell polarization via production of cytokines. After seven d of coculture, exposure to TcES conditioned the DCs to induce the release of IL-13 and IL-4 by the CD4⁺ T cells; however, IFN-γ was not detected (Fig. 5A). To demonstrate a role for cRAF in mediating the production of IL-13 and IL-4 by DCs exposed to TcES, we treated DCs for 2 h with different concentrations of cRAF inhibitor prior to TcES stimulation. BMDCs exposed to TcES plus 1 μM or 10 μM cRAF inhibitor exhibited an impaired ability to induce OVA-specific IL-13 and IL-4 secretion by T cells compared with BMDCs exposed to TcES alone (Fig. 5A). When OVA peptide loaded BMDCs were stimulated with LPS, they induced Th1 cytokines in in vitro cocultures, but when the DCs were treated with TcES and LPS, IFN-γ levels were reduced (Fig. 5B). Given that TcES exposure reduced the maturation of LPS-stimulated BMDCs, and this phenotype resulted in impaired IFN-γ production in CD4⁺ T cells, we tested whether the blockage of cRAF in DCs would restore IFN-γ production in CD4⁺ T cells. Consistent with the recovery of IL-12 production after cRAF inhibitor pretreatment (Fig. 4A), BMDCs preincubated with cRAF inhibitor and exposed to TcES and LPS were efficient Th1 inducers (Fig. 5B). These data demonstrate that TcES promote Th2 cytokine secretion in a cRAF-dependent manner and that cRAF phosphorylation is required for impairing the Th1-inducing ability of LPS-treated DCs.

Figure 5.

Th2 induction by TcES-exposed DCs is cRAF dependent. A) DCs were loaded with OVA and treated with TcES in the presence or absence of cRAF inhibitor. Following treatment, DCs were washed and cocultured with OTII CD4⁺ T cells for 7 d. Production of IL-4 and IL-13 in culture supernatants was analyzed by ELISA. B) Cocultures were also carried out in the presence of LPS, and production of IFN-γ and IL-4 in culture supernatants was determined by ELISA. C) OVA-loaded BMDCs stimulated with TcES with or without cRAF inhibitor were injected into the footpad of WT mice previously adoptively transferred with CFSE-labeled CD4⁺ OTII cells. Production of IL-4 in adoptively transferred T cells in popliteal and inguinal lymph nodes were analyzed at d 7 postinjection by flow cytometry. Plots represent cells gated for CFSE⁺ and CD4⁺ populations. Graph shows percentage of IL-4 producing CD4⁺ cells; n = 4. *P < 0.05. D) Popliteal lymph node cells from the above experiment were restimulated with OVA peptide for 4 d, and supernatants were analyzed for IL-4, IL-13, and IFNγ production by ELISA. Data are representative of 2 independent experiments; n = 4. *P < 0.05.

Finally, we addressed whether cRAF is critical for the induction of Th2 responses by TcES-treated DCs in vivo. To this end, BMDCs were loaded with OVA and pretreated with cRAF inhibitor before exposure to TcES. The DCs were then transferred into the footpad of WT mice that had previously received CFSE-labeled CD4⁺ T cells purified from OTII transgenic mice. At 7 d after transfer, draining lymph nodes were recovered, and IL-4 production was evaluated by intracellular flow cytometry. We observed that the mice which received OVA loaded DCs exposed to TcES alone showed enhanced IL-4 production in the transgenic OTII CD4⁺ cells. In contrast, DCs pretreated with cRAF inhibitor before TcES exposure showed similar IL-4 production by OTII CD4⁺ cells comparable to untreated DCs (Fig. 5C). We observed similar decreased production of IL-4 and IL-13 as well as increased production of IFNγ in popliteal lymph node cultures of adoptively transferred mice receiving TcES exposed DCs pretreated with cRAF inhibitor. (Fig. 5D). Taken together, these results show that cRAF phosphorylation in TcES-treated DCs enhances Th2 and impairs Th1 responses in vitro and in vivo.

Combined TcES recognition by MR, MGL, and TLR2 is necessary to block DC maturation and induce Th2 responses

To determine the link between the receptors involved in TcES recognition and the down-regulation of TLR responses, we examined whether the combination of MR, MGL, and TLR2 was necessary to modulate the responses of DCs to further TLR4 stimuli. Consistent with our data using MGL- and TLR2-deficient DCs, blocking antibodies against MGL or TLR2 did not significantly affect the impairment of cytokine production by TcES treated C57BL/6 DCs (Fig. 6A). Blocking MR restored the production of TNF-α but not IL-12 in TcES treated DCs and stimulated by LPS (Fig. 6A, B). Simultaneously blocking MR, MGL, and TLR2 completely restored the ability of TcES exposed DCs to respond efficiently to LPS stimulation, producing IL-12 and TNF-α at levels similar to DCs stimulated with LPS alone (Fig. 6A). Following the same strategy, we tested whether these receptors are important in directing Th2 responses in DCs due to TcES exposure. In coculture assays, we observed a partial inhibition of IL-4 and IL-13 production after blocking MR, MGL, or TLR2 individually. However, simultaneously blocking MR, MGL, and TLR2 completely inhibited IL-4 and IL-13 production induced by TcES (Fig. 6B). These results show that the MR, MGL, and TLR2 receptor combination is required to mediate the effects of TcES in inhibiting the production of IL-12 and TNF-α in DCs and subsequent Th2 polarization. Finally, since TcES induced modulation of DCs occurs through a cRAF-dependent signaling pathway, we determined whether blocking one or multiple receptors could reduce cRAF phosphorylation. We found that blocking MR or MGL significantly reduced cRAF phosphorylation, while blocking TLR2 slightly reduced cRAF phosphorylation, but this was not significant. However, when MR, MGL, and TLR2 were blocked simultaneously, cRAF phosphorylation elicited by TcES was completely abrogated (Fig. 6C, D). These results demonstrate a link between MR, MGL, TLR2 and cRAF as critical players in a signaling pathway exploited by T. crassiceps to inhibit DC maturation and direct Th2 responses (Fig. 7).

Figure 6.

TcES trigger cRAF phosphorylation through combined recognition by MR, MGL, and TLR2. A) BMDCs were pretreated with the indicated blocking antibodies for 30 min, and then stimulated with LPS and/or TcES for 24 h. Production of IL-12 and TNF-α was evaluated by ELISA. Indicated significance compared with LPS/TcES; n = 3. *P < 0.05. B) BMDCs were treated as in A, and exposed to TcES for 24 h, then cocultured with OTII-purified T cells for 6 d. Production of IL-4 and IL-13 was evaluated by ELISA. Significance compared with TcES alone; n = 3. *P < 0.05. C) MR, MGL, and/or TLR2 were blocked as in A, and BMDCs were stimulated with 40 μg/ml of TcES for 30 min. Intracellular cRAF phosphorylation was analyzed by flow cytometry. Dotted histograms indicate DCs incubated 30 min with TcES alone; black histograms indicate DCs incubated 30 min with the indicated blocking antibody before TcES incubation; n = 2. D) Levels of cRAF phosphorylation in DCs subjected to blocking treatments as in C. *P < 0.05 vs. TcES.

Figure 7.

Model showing the cRAF-dependent modulation of DC responses by TcES. Helminth parasites release excreted/secreted products that can alter immune responses. TcES are recognized by mannose receptor, MGL, and TLR2. Receptors act in conjunction to mediate TcES signaling. Signaling could be initiated by RAS, a molecule found in the cytoplasm of TcES-treated DCs. Ultimately, TcES signaling results in the phosphorylation of cRAF. cRAF-mediated signaling is able to inhibit the maturation of DCs and subsequent cytokine release induced by TLR4. In addition, TcES down-regulates the p38 and NFκB pathway, possibly in a cRAF-dependent manner. On the other hand, TLR4 signaling attenuates cRAF phosphorylation and Th2 response induced by TcES, indicating a cross regulation between both pathways. Finally, cRAF activation conditions DCs to induce Th2 responses, via a mechanism that is incompletely understood.

DISCUSSION

Our study demonstrates that helminth-derived molecules are recognized by multiple receptors, such as MR, MGL, and TLR2, on DCs, and together they trigger a cRAF-mediated intracellular signaling pathway that negatively affects LPS-induced DC activation and affects eventual T-cell responses. DCs play a major role in the initiation of Th2 polarization in response to helminth pathogens because depletion of DCs impairs the Th2 response elicited by worm infection (29). In several experimental conditions, DCs remain in an immature state after exposure to helminth derivatives, but in contrast to the immature phenotype, DCs are irresponsive to further TLR stimulation and exhibit an enhanced ability to prime Th2 responses in vivo and in vitro (30–34). These data indicate that DCs do not remain immature after helminth exposure but they are primed in a nonclassical manner.

In this study, we investigated the mechanisms of DC regulation by TcES. The fact that the carbohydrates in TcES were important for their modulatory activities (16), lead us to investigate the role of receptors on DCs with the ability to recognize glycosylated structures as well as the intracellular signaling pathways they trigger. The lack of recognition of TcES by EGTA-treated DCs strongly suggested a role for C-type lectins, whose function is dependent on calcium. TcES are rich in structures recognized by concanavalin A, and the main glycosylation moieties in T. crassiceps are mannose, galactose, and fucose (16, 18). The content of carbohydrates present in TcES correlated with the ability of MR and MGL to recognize such molecules. In addition, TLR2 had a role in TcES recognition. The multiple receptors involved in TcES recognition were not surprising, since TcES are a complex mixture of molecules that remain to be identified. In support of our findings, other helminth-derived molecules bind to different receptors, including MR, MGL, and TLR2 (10). These receptors have been associated with the regulation of DCs responses. For example, MR mediates the internalization of ω-1, a ribonuclease present in Schistosoma mansoni soluble egg antigens (SEAs) that is capable of down-regulating proinflammatory cytokine production and skewing toward Th2 responses (35). On the other hand, MGL has been associated with Th2-promoting ability (36). Moreover, we did not see significant changes in DCs responses to TcES after blockade of MGL, but MR blockage resulted in a recovery of TNF-α production. Other receptors that can bind glycoconjugates and that are also involved in helminth recognition are TLR2 and TLR4 (24–26). Although TcES recognition was impaired in TLR2^−/− BMDCs, TLR2 deficiency did not impact the modulatory activities of TcES on DCs. Because TLR2^−/− mice are more susceptible to T. crassiceps infection, it is likely that TLR2 enhances the protective immune response against T. crassiceps (23), probably by binding other molecules not present in TcES. TLR4 has been associated with the induction of Th2 polarization by DCs after the recognition of LFNIII, a carbohydrate that is present in SEA and ES62 (24, 33). However, we observed that TcES did not activate TLR4-transfected HEK cells, and we have reported that T. crassiceps molecules polarize cells toward a Th2 profile even in C3H/HeJ mice, which have a TLR4 mutation (8). These results suggest that T. crassiceps molecules may enhance Th2 responses in a TLR4-independent fashion.

Several reports have shown the cooperation between receptors to recognize pathogens and modulate intracellular signaling affecting the outcome of responses. For example, TLR2 can form heterodimers with TLR1 or TLR6, and can act in synergism with CLRs such as MGL and Dectin-1 (37, 38). We tested whether combinations of blocking antibodies could affect the phenotype of DCs after TcES exposure. We found that when MR, MGL, and TLR2 were collectively blocked, the response to LPS stimulation was restored in DCs treated with TcES. Moreover, the ability of TcES to induce Th2 responses was completely abolished with collective blockage of all three receptors, while blockage of individual receptors MR, MGL, or TLR2 resulted in reduced levels of Th2 cytokines. Although the various molecules that comprise of TcES remain to be fully characterized, our data suggests that some redundancy potentially exists in receptor recognition of TcES. This could further explain why other reports show that blockage of one receptor does not alter the resulting DC phenotype after exposure to helminth molecules (26, 39). Future experiments will be required to demonstrate whether a combination of these receptors recognizes a single TcES molecule, or multiple molecules bind to different receptors on DCs.

Our results showed that exposure of BMDCs to TcES diminished p38 MAPK and NFκB p65 phosphorylation induced by LPS, events associated with IL-12 and TNF-α production (4, 40, 41). On the other hand, TcES did not block the LPS-induced MEK1/2 and ERK1/2 phosphorylation. Because ERK1/2 is associated with IL-10 production, this mechanism can explain why TcES does not affect IL-10 production (42, 43) and suggests that TcES specifically impair the inflammatory pathway. Few studies have addressed the intracellular signaling elicited by helminth derivatives and their ability to prime Th2 responses. SEA stimulation results in slight phosphorylation of ERK1/2, which leads to cFOS stabilization; these events impair IL-12 production by DCs and are correlated with Th2 polarization (44). In addition, NFκB1 was necessary to develop Th2 responses after SEA treatment (45). In our study, we did not find any ERK1/2, p38, or NFκB p65 phosphorylation in response to TcES alone. In contrast, we found that TcES increased cRAF phosphorylation. Different studies have shown that cRAF phosphorylation is triggered by other pathogens, such as HIV, Mycobacterium tuberculosis, and Helicobacter pylori, in a DC-SIGN dependent pathway (19, 20). However, there are no data on the activation of these pathways by helminth derivatives. We found that TcES increased cRAF phosphorylation very early after stimulation and maintained this phosphorylation for at least 1 h. This result suggests that early cRAF phosphorylation is triggered directly by TcES and not by an autocrine feedback of soluble factors produced by DCs. Analyzing the cRAF canonical signaling pathway, we found that the upstream signaling molecule RAS was localized in high amounts in the cytoplasm, which could indicate receptor internalization (28). In contrast, the downstream molecules MEK or ERK1/2 were not phosphorylated in response to TcES stimulation. Previous findings showed that phosphorylation of cRAF but not MEK and ERK1/2 occurs in response to ManLAM (20). In other study, the tick saliva molecule Salp15 was recognized by DC-SIGN and impaired the production of TNF-α and IL-12 by decreasing messenger stability and nuclear remodeling, respectively. Interestingly, TNF-α regulation was mediated through a cRAF- and MEK-dependent pathway, but IL-12 regulation was only cRAF dependent (27). The fact that TcES were able to down-regulate TLR responses in a cRAF dependent pathway, and that other pathogens trigger this pathway resulting in altered TLR responses (20, 27, 46) suggest that cRAF might be a common target used by pathogens to impair DC function. Our findings showed that TcES induced a cRAF dependent pathway triggered by the combination of the receptors MR, MGL, and TLR2. Although crosslinking of MGL has been shown to lead to ERK1/2 phosphorylation, a downstream target of cRAF (37, 47), ours is the first report to show that MR, MGL, and TLR2 are directly associated with cRAF signaling. In addition, the MR elicited response can be amplified by TLR2 (48), supporting the fact that MR, MGL, and TLR2 can act together to induce intracellular signaling.

In our model, how cRAF affects the TLR signaling pathway remains to be elucidated. One possibility is that cRAF is not activated completely, thereby lacking the ability to induce the canonical MEK-ERK1/2 pathway. It has been shown that cRAF can bind directly to IκB (49), and perhaps, this association could impair NFκB release and subsequent DC activation observed in our study. Another possibility is that cRAF could preferentially induce the activation of the heterodimer RelB/p52, commonly known as the alternative NFκB pathway. In line with this hypothesis, the Th2 inducer LNFPIII, a carbohydrate derived from S. mansoni, has been shown to activate the alternative NFκB pathway on DCs (45). It should be noted, however, that cRAF phosphorylation induced by TcES stimulation of DCs was necessary for the priming of Th2 responses. This supports the idea that helminths activate different pathways that compete with or block TLR responses and are simultaneously involved in skewing the T-cell response toward a Th2 profile rather than the default DC activation pathway. However, it is still not clear whether cRAF induces costimulatory molecules or soluble factors that are able to induce Th2 polarization, and future experiments are necessary to address which Th2-inducing factors could be modulated by cRAF. In contrast with our finding associating cRAF to Th2 responses, Dectin-1 triggers the cRAF pathway on DCs. leading to enhanced cytokine production and positive regulation of the Th1 and Th17 responses (19). This discrepancy could be the result of differences in receptors or receptor combination engaged on DCs that triggers the cRAF pathway. Additional studies are necessary to understand how cRAF can be involved in anti-inflammatory (27) or inflammatory DC responses (19). We also found that cRAF inhibition in TcES treated DCs enhanced IFNγ production in vivo but not in vitro. Since primed DCs can be instructed by other signals coming from T cells (50), such as CD40, it is likely that TcES-treated DCs can be refractory to those signals in a cRAF dependent fashion. However the fact that IFNγ production was detected in vivo, but not in vitro, suggests that cells other than CD4⁺ T cells can provide such signals.

We further observed that a mutual negative regulation between the signals elicited by TcES and those triggered by LPS on DCs that affected the T-cell outcome. These data suggest that DC responses could be governed by the ratio between the inhibitory and stimulatory signals presented by the pathogen or microenvironment. In accordance with this idea, an early Th1 response is achieved in T. crassiceps-infected mice, which correlates with disrupted parasites in the peritoneal cavity and, possibly, the release of vesicular fluid, which in turn can activate the immune system to secrete early IFNγ. However, as the infection progresses, there is a switch from an early protective Th1 response to a permissive Th2 response (13). Our studies indicate that this shift is potentially caused by TcES, probably blocking the Th1 responses triggered by the vesicular fluid and inducing Th2 immune responses. This hypothesis can be supported by a recent study showing that only low-molecular-mass molecules contained in the vesicular fluid of Taenia solium were able to induce proliferation as well as IL-1β, TNF-α, and IL-2 production by human lymphocytes (51). Our findings provide evidence that DC responses are orchestrated by the engagement of multiple receptors and the interaction of their downstream intracellular pathways. Certainly, the number of receptors as well as their interactions expands the ability to recognize and respond to different pathogens.