Introduction

Cryptosporidiosis was identified at first as a veterinary disease of clinical concern in domestic animals such as cattle, buffaloes, and goats (1-3). C. parvum infection in calves is a primary veterinary concern, especially in temperate regions, because it causes watery diarrhea, in appetence, tiredness, dehydration, and death. Long-term growth of Cryptosporidium-affected calves can be costly for farmers due to lost income from lower carcass weights, treatment expenditures, and increased feed requirements to get calves to market weight (4). There remains a paucity of data for other farmed animals, despite Cryptosporidium infection causing significant clinical disease. C. parvum is also a crucial zoonotic parasite that can infect humans through transmission from infected people or animals or indirectly by ingesting contaminated food or water (5). While this intracellular coccidian parasite has a worldwide distribution, it is especially prevalent in humans and livestock of economic importance in Egypt (6,7). The highest prevalence rates of infection are along the Nile River in rural communities that reside near infected animals (8). A current review by Helmy (9) reported that the prevalence rate in Egypt in animals varied between 2% and 69%, demonstrating that animals are an essential vector for this parasite, the prevalence of Cryptosporidium spp. in the calves being significantly higher than in adult cattle. The prevalence rates in human populations varied from 3% to 50%, with reports of up to 91% in immunocompromised cohorts such as children with diarrheal disease (9,10).

Transmission of C. parvum occurs by ingestion of parasitic oocysts consumed in drinking water or food contaminated with infected fecal matter (11,12). Oocysts enter epithelial cells that line the gastrointestinal tract causing symptoms such as low-grade fever, nausea, diarrhea, and weight loss (13). Disease symptoms are self-limiting in immune-competent animals and humans. However, in immune-compromised individuals (14,15), the infection can be life-threatening, exacerbated by a deficiency in effective therapies (15,16). To date, there are no effective vaccines due to the incomplete understanding of the host immune response to the parasite infection (17,18). A better understanding of the host's innate and adaptive immune responses to infection might contribute to developing a C. parvum vaccine.

Similar to other protozoan infections, immune responses associated with C. parvum infection involve a complex interplay between the innate and adaptive immune responses. Cytokines such as IL-12 and IFN-ɤ that are associated with C. parvum infection are essential mediators of Th1 responses (19,20). Antigen-presenting cells exposed to C. parvum or its antigenic products secrete Th1-promoting cytokines like IL-12p35 or IL-12p40, which can differentiate naïve CD4⁺ T-cells into a Th1 phenotype (20). Studies also show thatIFN-ɤ deficient mice are susceptible to C. parvum infection(21,22), indicating that IL-12 and IFN-ɤ are central to developing protective immunity during C. parvum infection. Moreover, the regulatory cytokine IL-10 is induced during infection as part of regulatory networks to dampen intestinal inflammation (23). Host cell-mediated immune responses are thought to be critical during infection, specifically CD4⁺ T-cells during recovery from cryptosporidial infections (24,25).

DCs, as antigen-presenting cells, play a significant role in activating innate and adaptive immune responses in infected hosts (26), processing and presenting the parasite antigens in local lymph nodes to T- and B-cells (27). Upon antigen stimulation, immature DCs begin to mature and migrate into the lymph nodes and other immune organs, causing the upregulating of expression of the major histocompatibility complex (MHC)-I, MHC-II, CD86 with other costimulatory factors, as well as the expression of pro-inflammatory cytokines that are important for the induction of adaptive immune responses (28). Mice are a good model for studying the effect of cryptosporidium infection in DCs (20,29). Little is known about the role of DCs in cryptosporidiosis; they are involved in the degradation and transport of antigens to lymph nodes (27) and are known to release chemokines in response to C. parvum infection (30). Wanyiri and Ward (31) examined dendritic cell activation by recombinant C. parvum antigens. Also, Bedi and Mead (20) study have shown that C. parvum sporozoite activated dendritic cells and produced Th1 cytokines. Xu et al. (32) indicated that DCs pulsed with live sporozoites in vitro and co-cultured with CD4+ and CD8+ T cells produced higher IFN- levels and showed induction of Th1 immune response.

While T cell-mediated immunity appears essential in controlling Cryptosporidium infection, the mechanisms that elicit these immune responses are unclear. Given their importance in dealing with C. parvum infections, we investigated the effects of C. parvum oocysts antigen on bone marrow-derived DCs. These studies are essential because there are limited studies about the effects of C. parvum oocysts antigen. This study demonstrated that DCs exposed to C. parvum oocysts induced a pro-inflammatory DC phenotype that suppressed Th2 cytokines from T-cells while the secretion of cytokines associated with Th1, Th17, and regulatory phenotypes remained unchanged. This interaction led to DC activation as determined by the overexpression of several costimulatory molecules and cytokines. The proposed studies will provide the basis for understanding the mechanisms for the induction of DCs and T cell-mediated anti-Cryptosporidium immunity, which are necessary for future vaccine design and other effective methods for treatment.

Materials and methods

Animals and ethical standards

Balb/c mice (female) aged 6-8 weeks were purchased from Charles River Ltd (Kent, UK) and kept under specific pathogen-free conditions at Dublin City University (DCU). All mice were housed according to the Health Products Regulatory Authority (HPRA) guidelines with strict adherence to standard operating procedures approved by the Institutional Animal Welfare Body. Ethical permission for the use of animals was approved by the Health Products Regulatory Authority and Dublin City University ethics committee (license number DCUREC/2010/033). All procedures involving animals were only performed by licensed personnel licensed by the HPRA.

Animals were culled under the ARRIVE Guidelines relevant to ex vivo models (33). For each experiment, the absolute minimum number of animals was culled. No procedures or processes were performed on the animal’s pre-mortem. As all investigations were ex vivo in nature, the average number of cell types yielded per mouse was calculated and used to determine the number of mice needed; where appropriate multiple cell types were harvested to ensure maximum yield from each animal and minimize animal numbers. Animals were chosen from the same group and age bracket - they are housed based on these criteria. Animals were included once they were of the same group and healthy in appearance.

Cryptosporidium parvum oocysts antigen preparation

The C. parvum antigen (CPA) was prepared from C. parvum oocysts purified from naturally infected calves (34). In brief, oocysts were purified from fecal samples by sucrose and percoll centrifugation, treated in 0.5% sodium hypochlorite solution at 4°C for 10 min, washed 4 times in sterile water, and re-suspended in PBS at 2 × 10⁸ oocysts/mL. Purified oocytes were homogenized in sterile PBS (pH 7.4) and centrifuged for 30 min at 876 g at 13,000 rpm. The supernatant antigen was collected, aliquoted, and stored at -20 until use. The protein concentration was measured using the lowery method (35). Endotoxin levels were tested for all antigens and were less than the lower detection limit in this assay (

Reagents and materials

Lipopolysaccharide (LPS) from E. coli (serotype R515) was purchased from Enzo Life Sciences (Exeter, UK). All antibodies used in this investigation were obtained from eBiosciences (Hatfield, UK; CD86 (FITC) monoclonal antibody (24F), CD80 (PE) monoclonal antibody (3H5), MHCII (FITC) monoclonal antibody (MS/114.15.2) and TLR4 (PE) monoclonal antibody (UT41) or the relative isotype control. Granulocyte-macrophage colony-stimulating factor (GM‐CSF) was obtained from Sigma Aldrich. Cell culture materials were purchased from Biosciences (Dun Laoghaire, Ireland). 

Isolation and culture of bone marrow-derived dendritic cells

Bone marrow-derived DCs (BMDCs) obtained from balb/c mice (36) were isolated aseptically in a Class II Laminar cabinet (ThermoElectron Corporation, USA). Bone marrow cells were extracted from the tibia and femurs of each mouse by flushing the bone cavity with sterile RPMI using a sterile 25.7 g needle and syringe. The cells were pelleted by centrifuging for 5 min at 500 g and re-suspended in 10 mL of culture medium (RPMI (Gibco, UK) supplemented with 20% heat-inactivated fetal bovine serum (FBS), 100 μg/ml penicillin/streptomycin (Invitrogen, UK), L-glutamine, and 50 ng/mL granulocyte monocyte-colony stimulating factor (GM-CSF) (Sigma Aldrich, Ireland)). Cells were transferred to a petri dish and cultured a 37°C in a CO₂ incubator. On days 3 and 6, 6 mL of media was gently removed from the petri dish, and the media was replenished with 10 mL of pre-warmed culture medium. On day 10, adherent cells were dislodged from the surface using a cell scraper (Sarstedt, Ireland) and centrifuged at 500 g for 5 mins prior to resuspension in fresh media. Cell counting was performed using the trypan blue exclusion method. Harvested BMDCs were analyzed by flow cytometry, and only cell preparations with a population identified as >95% CD11C (Biolegend, No. 117317) positive were used for each experiment.

3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) Assay 

CellTiter 96® Aqueous One Solution (Pierce, UK) test was used to investigate the cytotoxic effects of CPA on BMDCs in vitro (37). BMDCs were plated in a 96-well plate (Nunc™, Ireland) with 100 μL cell suspension per well at a concentration of 1x10⁶ cells/mL. CPA was added at increasing concentrations (10 - 10,000 ng/mL) or cells were plated with media, LPS (100 ng/mL) or DMSO (10% v/v). Cells were incubated overnight at 37°C in a CO₂ incubator, and then 20 μL of the CellTiter 96® Aqueous One solution was added to each well. After 4 hrs, the color change in the media was measured using a TECAN 96 well plate reader at 490 nm (Tecan, Männedorf, Switzerland). The cell viability of each sample was calculated by setting the absorbance value for the cells treated with media alone as a reference point, and then the percentage change in absorbance for each sample was calculated as previously described (37).

Enzyme linked immunosorbent assay

BMDCs (1 x 10⁶ cells/mL) were treated with CPA (10 and 100 ng/mL), LPS (100 ng/mL) or media and incubated for 18 hrs at 37°C in a CO₂ incubator. Supernatants were removed, and cytokine release (IL-12p40, IL-6, and IL-10) was measured using commercial ELISA kits following the manufacturer's instructions (Invitrogen, UK). Each sample and standard were assayed in triplicate.

Flow cytometry

BMDCs (1 x 10⁶ cells/mL) were treated with CPA (10 and 100 ng/mL), LPS (100 ng/mL) or media and incubated at 37°C in a CO₂ incubator for 24 hrs. Cells were removed from the tissue culture plates and placed into a 96-well round bottom plate at 400,000 cells/well. An equal amount of FBS was subsequently added for 15 min at room temperature (RT) to block non-specific binding. Cells were then washed three times with FACS buffer (PBS supplemented with 2% FBS (v/v) and 1mM EDTA (Sigma-Aldrich)) and incubated with appropriate fluorochrome-conjugated antibodies (BD Biosciences, UK) for 30 min at 4ºC while protected from light. Cells were washed three times to remove any unbound antibodies and analyzed using a FACSAria I (BD Biosciences, UK). All the flow cytometry data were analyzed using FlowJo software (Treestar, UK). 

T-cell co-culture

Spleens from balb/c mice were harvested, and splenocytes were obtained by passaging the spleen through a 40 μm filter (Sarstedt, Nümbrecht, Germany) using the plunger from a sterile 1 mL syringe (Sarstedt, Nümbrecht, Germany). CD4⁺ T-cells were isolated from the splenocytes using a negative selection CD4⁺ isolation kit (Stemcell, Vancouver, Canada) and were only used if the purity was determined to be > 95% positive for CD4⁺ cells by flow cytometry. BMDCs were stimulated with CPA (10 ng/ml) for 24 hrs, washed in PBS three times, and co-cultured with CD4⁺ T-cells at a ratio of 1:10 in a culture medium on 24 healthy plates (Sarstedt, Nümbrecht, Germany) that had been pre-coated overnight with anti-CD3 (1 µg/mL) (R and D systems, Minneapolis, Minnesota, USA). Supernatants were harvested after 72 hrs and analyzed for cytokine release (IL-5, IL-13, IFN-γ, IL-2, IL-17, and IL-10) using commercial ELISA kits following the manufacturer's instructions (Invitrogen, UK). Each sample and standard were assayed in triplicate.

Statistical analysis

All data were analyzed for normality prior to statistical testing by Prism^® 6.0 (GraphPad Software Inc, La Jolla, CA, USA) software. Data were analyzed using one-way ANOVA using Tukey's multiple comparison test, where multiple group comparisons were made. For comparisons between two groups, the student’s t-test was used. Data are expressed as mean ± standard deviation (SD).

Results

Lower doses of C. parvum antigen do not exhibit cytotoxic effects on BMDCs

Before examining the immune properties of CPA on BMDCs, the cytotoxic effect of the antigen on these cells was assessed using an MTS Assay. At lower concentrations (10, 100, and 500 ng/mL), CPA had no significant impact on cell viability. However, at higher doses, CPA (1000 ng/mL (p ≤ 0.05) and 10,000 ng/mL (p ≤ 0.01)) was cytotoxic, with the highest dose exhibiting cytotoxic effects similar to DMSO (10%), the positive control (Figure 1). Since 1000 ng/mL and 10,000 ng/mL were displayed cytotoxic effects on BMDCs. These antigen concentrations were excluded from subsequent assays.

Figure 1: MTS assay assessed the cytotoxic effects exerted by increasing concentrations of CPA on BMDCs (10 to 10,000 ng/ml). The graph represents cell viability, expressed as absorbance ± SD of three independent experiments. P-values were calculated using one-way ANOVA. *, p ≤ 0.05; **, p ≤ 0.01 compared to PBS control group.

Cryptosporidium parvum antigen activates BMDCs to produce a panel of pro-inflammatory cytokines

To understand the immuno-modulatory properties of CPA, BMDCs were stimulated with three different concentrations of antigen (10, 100, and 500 ng/mL), and cytokine release was measured 18 hrs later. The pro-inflammatory cytokines IL-12p40, TNFa, and IL-6 associated with protozoan infection and IL-10, an anti-inflammatory cytokine that regulates inflammatory processes during infection. CPA induced significant secretion of IL-12p40, TNFa, and IL-10 from BMDCs in a dose-dependent manner with no significant secretion of IL-6 (Figure 2).

Figure 2: BMDCs were stimulated with increasing concentrations of CPA (10 - 500 ng/mL) and analyzed for the secretion of IL-12p40, IL-10, TNFα, and IL-6 by commercial ELISA after 18 hours. Results are expressed as mean ±SD of three independent experiments. P-values were calculated using one-way ANOVA. *, p ≤ 0.05; **, p ≤ 0.01: ***, p ≤ 0.001; ****, p ≤ 0.0001 compared to PBS control group.

BMDCs treated with CPA expresscostimulatory markers.

Since CPA stimulated BMDCs secreted a panel of cytokines, we examined their effect on BMDC maturation. BMDCs were stimulated with different concentrations of CPA (10 - 500 ng/mL), and after 18 hours, the expression of the cell surface markers CD80, CD86, MHC-11, and TLR4 were measured. Significant expression of all cell surface markers was observed for all antigen doses tested except for CD80, where only the lowest concentration induced CD80 expression (Figure 3). It was unexpected that only the lower dose of CPA induces the expression of CD80. Although there is a slight increase at higher levels, it is not statistically significant. This could be explained by the fact that these bioassays are affected by numerous factors. For example, the oocyte mixture is a heterogeneous mixture of antigens that at higher concentrations activate other pathways where CD80 is not induced.

Figure 3: BMDCs were stimulated at different concentrations of CPA (10 - 500 ng/mL) for 18 hours and subsequently assessed for the expression of CD86, CD80, and MHCII by flow cytometry following staining for 30 min with specific antibodies or an isotype-matched control. Results were analyzed using FlowJo software (Treestar, USA) and are expressed as the geometric MFI ±SD of three independent experiments. P-values were calculated using a two-tailed student's t-test. n.s, non-significant; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001 compared to PBS control.

Dendritic cells stimulated with C. parvum antigensuppress Th2 cytokines.

Since CPA induced maturation of BMDCs, we then examined the priming of naïve CD4⁺ T-cells by CPA-stimulated BMDCs. BMDCs stimulated with CPA were co-cultured with CD4⁺ cells in the presence of plate-bound anti-CD3. CD4⁺ cells secreted significantly less IL-13 (Figure 4) and IL-5 (Figure 4) compared to the control. No significant differences in IFN-γ , IL-, IL-�17, and IL-10 secretion were observed (Figure 4).

Figure 4: BMDCs pre-treated with CPA (10 ng/mL) or PBS were co-cultured with CD4⁺ T-cells at a ratio of 1:10 on plates that had been pre-coated with anti-CD3 (1 μg/well) overnight. CD4⁺ T-cells alone with anti-CD3 have used a negative control. After 72 hrs, supernatants were analyzed for the secretion of IFN-γ, IL-2, IL-17, IL-5, IL-13, and IL-10 by commercial ELISA kits. Results are expressed as mean ±SD of three independent experiments. P-values were calculated using tukey one-way ANOVA. n.s, non-significant; *, p ≤ 0.05; ** , p ≤ 0.01; *** , p ≤ 0.001 compared to PBS control group.

Discussion

Cryptosporidiosis is a primary veterinary concern due to its global distribution affecting various animal species (2). While there are over 26 Cryptosporidium species in Egypt, the overall estimated prevalence of Cryptosporidium infection in ruminants was 32.2%, with C. parvum accounting for 65.7% of the species that infect cattle and buffalos, livestock of economic importance in Egypt (9). C. hominis and C. parvum are responsible for more than 90% of human cryptosporidiosis cases, and there is a clear zoonotic effect between infected humans and animals. The present study demonstrated that CPA induced maturation of pro-inflammatory-like DCs that suppressed Th2 cytokines when co-cultured with CD4⁺ cells. This DC phenotype involving the secretion of IL-12 and TNFα is typically associated with protozoan infection (38,39).

In the current study, treating BMDCs with CPA induced the secretion of the pro-inflammatory cytokines TNFα, IL-6, and IL-12p40, and the anti-inflammatory cytokine IL-10. The critical role of IL-12p40 was confirmed by Ehigiator et al. (40), who showed that IL-12p40^-/- mice were more susceptible to infection with C. parvum than control mice. IL-12 plays an essential role during C. parvum infection in the differentiation of Th1 cells and the subsequent production of IFN-ɤ, which is essential in controlling C. parvum replication (41-43). Mice with a targeted mutation in the IL-12p40 gene were susceptible to C. parvum (44). IL-12p40, together with a p19 subunit, makes up the IL-23 cytokine. This cytokine drives the development of T helper cells that secrete IL-17 (Th17), and these cells are also found to have a role in the elimination of cryptosporidium infection (45).

In contrast to the effects of IL-12p40, IL-6 has been shown to directly antagonize the induction of Th1 responses by upregulating the expression of signaling factors that interfere with IFN-γ signaling and induce the differentiation of T-cells towards a Th2 phenotype (46). Despite the known import of Th1 responses and cytokines in clearing C. parvum infection, IL-6 has been reported to also play an essential role in the clearance of Cryptosporidium (47), suggesting that a mixed Th1/2 response may be required to fully clear infection. The present result shows that DC exposure to CPA increases the production of IL6, which coincided with that observed by Bedi and Mead (20) and Xu et al. (32). They illustrated an increase in the expression of IL-6 by mouse DCs treated with C. parvum sporozoite antigen which was dependent on TLR signaling pathways. TNF-α can inhibit C. parvum development in enterocyte cell lines and is thought to limit parasite replication within the host (48,49). The production of IL-10 may be part of a negative feedback mechanism to limit tissue damage from these pro-inflammatory responses induced by the parasite (50).

Lower concentrations of C. parvum antigens activated DCs, keeping with previous studies that also demonstrated that C. parvum antigens activate mouse and human DCs at lower antigen concentrations (20). This study also supports findings by Bedi and Mead (20), who reported significant increases in the expression of IL-12p70, IL-2, and IL-1b from mouse DCs in response to C. parvumsporozoite antigen. Similarly, they showed that BMDCs stimulated with C. parvum sporozoite lysate and live antigen preparations had little effect on IL-6 production. While, Perez-Cordon et al. (51) demonstrated that C. parvum antigens induced IL-12 and TNF-α from murine BMDCs but also reported an increase in IL-6, which contradicts our findings. This difference may be due to the type of parasite isolate used in their study or the excystation technique used to prepare the oocysts. A recent study has shown that different parasite isolates induce differences in immune response in studies on antigen-presenting cells in vitro (52).

CPA induced the full maturation of DCs and enhanced cytokine secretion. Enhanced expression of cell surface markers is associated with DC maturation and is vital for activating CD4⁺ cells. The enhanced expression of TLR4 is consistent with previous studies, as TLR4 is essential for C. parvum-induced NF-kB activation in human biliary epithelial cells (53). It has been shown that the production of Th1 cytokines by C. parvum-treated DCs was MyD88 dependent (20). Moreover, the production of IL-12 by C. parvum-infected BMDCs from C3H/HeJ mice (which lack a functional TLR4 pathway) was defective, suggesting that TLR4 signaling is vital for the production of IL-12 by C. parvum-infected BMDCs (51). Our findings are also supported by Perez-Cordon et al. (51). They reported that the oral exposure of mice to C. parvum resulted in DC maturation characterized by reduced endocytosis and an augmented expression of MHC molecules, costimulatory molecules, and adhesion molecules. Following in vitro infection of mouse BMDCs with C. parvum sporozoites, DCs increased their expression of CD40, CD80, and CD86. CD4⁺ cell activation is critical during C. parvum infection as MHC class II deficient mice (which lack functional CD4⁺ T cells) infected with C. parvum develop unresolved chronic infection (54).

DCs are essential players during early host immune response, and their depletion results in significantly increased susceptibility to infection with C. parvum (55). Adoptive transfer of unstimulated or C. parvum antigen-stimulated DCs into CD11c⁺ depleted CD11c-DTR-Tg mice resulted in an early decrease in parasite load at 4 days post-infection. However, this response was transient since parasite load increased in mice engrafted with either unstimulated DCs or DCs stimulated with solubilized antigen 6 days post-infection. In contrast, in mice engrafted with DCs stimulated with live sporozoites, parasite load remained low during the entire period, suggesting the development of a more effective and sustained response (55). The infection with C. parvum infection requires CD4⁺ T cell, major histocompatibility complex (MHCII) (56), and another signaling pathways like CD154 and CD40 that are highly expressed on DCs (57).

The host defense against cryptosporidiosis has been shown to depend upon a combination of innate and adaptive mediated immunity (25). There are interleukins as IL-1, IL-2, IL-15, IL-6, IL-8, IL-5, IFN-γ, IL-4 were induced in C. parvum infection (58). This current study showed that BMDCs matured with CPA suppressed the Th2-associated cytokines, IL-5 and IL-13, while simultaneously exhibiting no effect on IFNɤ, IL-2, IL-17, and IL-10. The suppression of Th2 cytokines results in dominant Th1/Th17 responses typically associated with Cryptosporidium infection. These results differed from studies by Bedi et al. (55), who demonstrated an increase in IFN when DCs stimulated with CPA were adoptively transported into mice. Also, Xu et al. (59) illustrated that C. parvum infection results in high expression levels of IFN-γ in the peripheral blood. However, the in vivo situation is more complex, suggesting that other factors are critical to inducing IFN-γ that could not be replicated in an in vitro assay. IFN-γ is a critical factor during Cryptosporidiosis infection in human populations, where studies have observed a more severe infection in populations unable to produce IFN-γ . The role of IFN-γ during the early stages of infection appears to be critical in controlling parasite replication (25,60). Xu et al. (32) show the ability of activated DCs to release IL-12 and IFN-γ in response to C. parvum. Also, IFN-γ knockout mice suffered a more severe infection of C. parvum than the control mice (40).

Conclusion

In conclusion, in the present study, we investigated the effect of C. parvum antigen on the activation of DCs. The presented results indicate that the interaction between DCs and C. parvum antigen results in a fully mature DC phenotype that secretes IL-12 p40, TNF-α, IL-6, and IL-10 and expresses MHCII TLR4, CD80, and CD86. The C. parvum antigen contributed to the predominant production of Th1 cytokines and inhibiting Th2 cytokine production. The findings from this study support other studies reported in the literature and highlight the importance of Th1 immunity during host-parasite interactions.

Acknowledgments

The authors thank the Erasmus Staff Mobility for Teaching and training assignments To/From Partner Countries for supporting this work.

Conflict of interest

The authors declare that they have no conflict of interest.