The 22nd chromatography component of Fasciola gigantica excretory- secretory product decreased the proliferation of peripheral blood mononuclear cells from buffalo

doi:10.21203/rs.3.rs-1604312/v1

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The 22nd chromatography component of Fasciola gigantica excretory- secretory product decreased the proliferation of peripheral blood mononuclear cells from buffalo

https://doi.org/10.21203/rs.3.rs-1604312/v1

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The 22nd chromatography component of the Fasciola gigantica excretory-secretory product (FgESP), F22, has shown potential diagnostic value, and diagnostic methods based on FgESP have also been established. Thus, exploring its immunomodulatory function and possibility as a molecular vaccine candidate is attractive. In the present study, the effect of F22 on the mitogen-induced proliferation of buffalo peripheral blood mononuclear cells (PBMCs) was studied. PBMCs were incubated with concanavalin A (ConA) and phytohemagglutinin (PHA) at optimal (1 µg/well) or suboptimal (0.25 µg/well) doses coupled with FgESP and F22 at different doses (1–16 µg/well). Then, cell proliferation was determined by microenzyme reaction colorimetry (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay). FgESP had a slight effect on the proliferation of buffalo PBMCs stimulated with ConA and PHA, while F22 decreased the proliferation of PBMCs stimulated with ConA and PHA at both optimal and suboptimal doses. Further studies should be performed to investigate the immunomodulatory function of F22.

Concanavalin A

F22

Mitogen

Phytohemagglutinin

Fascioliasis is a kind of zoonotic disease caused by Fasciola hepatica, Fasciola gigantica and their intermediate form that causes great global losses (Alasaad et al. 2011). Drug deworming is a conventional method to prevent fascioliasis, but drug residues and drug resistance are less advantageous for the prevention of this disease (Jaeger and Carvalho-Costa 2017). Vaccines should be the primary method used to prevent fascioliasis, and molecular vaccine candidates need to be screened based on an in-depth understanding of their immunomodulatory actions.

The interaction between “Fasciola spp. and host” manifests as the buffalo immune response and Fasciola spp. immune evasion. Therein, excretory-secretory products (ESPs) play an important role in immune evasion by Fasciola spp. (Corral-Ruiz and Sanchez-Torres 2020). However, the complexity of ESPs has hindered explorations of their immunomodulatory activities, and subsequent research focused on specific proteins among ESPs has also been unsatisfactory (Golden et al. 2010; Kumar et al. 2012; Preyavichyapugdee et al. 2008). Encouragingly, the 22nd chromatography component of FgESP (F22) showed high diagnostic value, and indirect ELISA based on this component for the diagnosis of bovine fascioliasis has also been established; F22 has shown a diagnostic effect better than that of FgESP in terms of sensitivity and early diagnostic value (Jin et al. 2021). Thus, it would be fascinating to see if F22 has immunomodulatory activity.

As cell proliferation capacity reflects immune function (Na and Yinglu 2016), cell proliferation studies have been widely applied in immunomodulatory studies, such as traditional Chinese medicine research on mitogen-induced cells. A study (HE et al. 2010) revealed that a Centella asiatica ethyl acetate extract could inhibit the proliferation of mouse spleen lymphocytes induced by mitogens, indicating that the extract had a certain immunosuppressive effect. Another study (LI et al. 2021) also revealed that an extract of Lepidium meyenii could synergistically act with concanavalin A (ConA) and lipopolysaccharide (LPS) to promote the proliferation of mouse spleen lymphocytes, indicating that the extract could enhance the immune activity of mouse spleen lymphocytes in vitro. The present study was designed to explore the ability of F22 to regulate proliferation in mitogen-treated peripheral blood mononuclear cells (PBMC) from buffalo.

Buffalo maintenance

Four approximately 3-month-old cross-bred offspring of swamp buffaloes from Guangxi (China) and Murrah buffaloes were used throughout the study. The buffaloes were born and maintained indoors, with food and water available ad libitum, and then used for subsequent PBMC collection only if they tested negative for fascioliasis by indirect ELISA.

Preparation of FgESP and F22

FgESP was isolated as described previously (Zhang et al. 2005). In brief, adult F. gigantica were washed and incubated in phosphate-buffered saline (PBS) for 3 h. Then, the supernatants were centrifuged and sterilized by filtration.

F22 was prepared according to a previous method (Jin et al. 2021). Briefly, collected FgESP was lyophilized and diluted with ddH₂O, and the concentration was adjusted to 20 mg/mL. Then, FgESP was loaded onto a protein flash chromatography system (GE, USA) according to the corresponding instructions, and the 22nd fraction was collected.

Isolation of buffalo-derived PBMCs

PBMCs were isolated as described previously (Tian et al. 2018), whole blood was collected from the jugular vein and transferred to 50-mL sterile EDTA-K2 anticoagulated tubes. After centrifugation at 2000 rpm for 20 min, the middle albuginea layer was diluted in RPMI 1640 medium (Gibco, USA). Then, the diluted samples were gently mixed with isometric lymphocyte separation liquid, followed by centrifugation at 1700 rpm for 20 min. The PBMCs in the middle white layer were collected and washed three times with PBS. Finally, the PBMCs were resuspended in culture medium (complete RPMI: RPMI 1640, 10% foetal bovine serum, 100 µg/mL streptomycin and 100 U/mL penicillin).

Assessment of PBMC proliferation

PBMCs (1×10⁵ cells/well) from each buffalo were cultivated in triplicate in the absence or presence of mitogens (Concanavalin (ConA-A) and phytohemagglutinin (PHA-P); Sigma, USA) at optimal and suboptimal doses (1 and 0.25 µg/well for each mitogen) along with FgESP or F22 (1, 2, 4, 8 and 16 µg/well) in 96-well plates in a total volume of 200 µL. After incubation at 37 ℃ in a humidified atmosphere with 5% CO₂ for 3 days, the proliferation of the PBMCs was calculated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) method (Sigma, USA) (50 µg/well MTT was added and incubated for an additional 4 h) according to (Zhang et al. 2014).

Calculations and statistics

Calculation and statistical analysis were performed as described previously (Moreau et al. 2002). For each buffalo, two stimulation indices (SI_S) were calculated as follows:

$$\text{SI}\text{1}=\frac{\text{mean OD}\text{570}\text{ }\text{of triplicate test cultures}}{\text{mean OD}\text{570}\text{ of triplicate control cultures}}$$

$$\text{SI}\text{2}\text{=}\frac{\text{mean OD}\text{570 }\text{of triplicate (}\text{mitogen+}\text{Fg}\text{ESP}\text{ \&}\text{ }\text{F22 ) cultures}}{\text{mean OD}\text{570}\text{ of triplicate mitogen cultures}}$$

The percent inhibition (% inhibition) of proliferation induced by the molecules was calculated as follows: % inhibition = 100-(100×SI₂).

The OD₅₇₀ values of the samples treated with the mitogens and mitogens + tested molecules were compared by the nonparametric Wilcoxon test. The percent inhibition of proliferation by the molecules was compared by the nonparametric Mann–Whitney U test.

Mass spectrometry analysis of FgESP and F22

FgESP and F22 were divided into tubes (100 µg/tube), and a Q Exactive chromatographic mass spectrometer was used to identify the protein components by mass spectrometry. F. hepatica (ST_Fasciola hepatica_[6192]_15305.fasta) was selected for library comparison. Proteins for which the number of unique peptides ≥ 2 were selected for subsequent analysis to detect the number of proteins related to Fasciola spp. Proteome Discoverer 1.3 (Thermo Scientific) software was used to match and score the original map files (raw files) from peptide identification with the Q Exactive chromatographic mass spectrometer through SEQUEST software. According to the criterion of FDR < 0.01, the peptides were screened based on confidence, and the presence of at least 2 unique peptides was used for identification to obtain highly reliable qualitative results.

Fg ESP had a slight effect on mitogen-stimulated PBMC proliferation

FgESP was collected as previously described. Table 1 shows the percent inhibition induced by FgESP. FgESP at multiple concentrations (1 ~ 8 µg/well) had no effect on the proliferation of PBMC stimulated with ConA at either dose, while FgESP at 16 µg/well decreased the proliferation of PBMC stimulated with ConA at a suboptimal dose (P < 0.05) (Fig. 1a).

Table 1

The inhibition percentages of different concentrations of FgESP on buffalo PBMC proliferation induced by optimal and optimal doses of mitogen.
Mitogen	Dose (µg/well)	Concentration (µg/well)	Buffalo A (♂) Murrha	Buffalo B (♂) Murrha	Buffalo C (♀) Murrha	Buffalo D (♀) Native cross-bred
ConA	0.25	0 1	0.00 1.96	0.00 6.69	0.00 -6.64	0.00 -9.80
		2	-33.69	-0.75	-2.26	8.35
		4	-9.28	2.64	9.84	9.82
		8	6.96	11.81	23.63	22.38
		16	9.28	17.21	34.09	33.40
	1	0 1	0.00 4.66	0.00 -3.51	0.00 19.97	0.00 5.78
		2	-1.30	-14.02	0.30	-33.99
		4	-3.48	-1.11	20.42	-2.97
		8	6.25	2.21	21.48	-20.79
		16	1.68	13.65	22.69	14.19
PHA	0.25	0 1	0.00 2.60	0.00 5.52	0.00 4.72	0.00 1.53
		2	22.51	22.73	20.50	11.48
		4	22.15	20.14	21.73	18.38
		8	-4.04	-8.38	3.82	2.52
		16	-0.48	1.25	8.24	5.11
	1	0 1	0.00 8.59	0.00 4.41	0.00 20.65	0.00 -8.62
		2	8.59	6.27	14.02	5.56
		4	16.72	7.46	28.81	-0.73
		8	-14.69	-26.27	8.06	2.70
		16	-17.03	-23.73	29.02	-4.96

FgESP had no effect on the proliferation of PBMCs stimulated with PHA at the optimal dose. With a suboptimal dose of PHA, FgESP at 2 and 4 µg/well significantly decreased proliferation (P < 0.05) (Fig. 1b).

F22 decreased mitogen-stimulated PBMC proliferation

F22 was collected as previously described. Table 2 shows the percent inhibition induced by F22. The ConA-induced proliferation of PBMCs was decreased by F22 (2 ~ 16 µg/well) (Fig. 2a). With an optimal dose of ConA, F22 at multiple concentrations (2 ~ 16 µg/well) clearly decreased PBMC proliferation (P < 0.01). With a suboptimal dose of ConA, F22 at 1 ~ 16 µg/well decreased PBMC proliferation (P < 0.01).

Table 2

The inhibition percentages of different concentrations of F22 on buffalo PBMC proliferation induced by optimal and optimal doses of mitogen.
Mitogen	Dose (µg/well)	Concentration (µg/well)	Buffalo A (♂) Murrha	Buffalo B (♂) Murrha	Buffalo C (♀) Murrha	Buffalo D (♀) Native cross-bred
ConA	0.25	0 1	0.00 52.22	0.00 34.87	0.00 59.43	0.00 58.96
		2	66.55	61.26	58.08	77.54
		4	73.66	76.42	68.85	82.82
		8	80.01	84.79	78.95	82.08
		16	71.23	86.17	70.24	85.56
	1	0 1	0.00 21.36	0.00 34.14	0.00 50.51	0.00 62.28
		2	55.61	68.32	74.89	60.80
		4	74.50	81.47	79.88	79.89
		8	81.73	87.69	83.51	84.58
		16	82.01	88.88	78.79	88.45
PHA	0.25	0 1	0.00 2.34	0.00 -5.81	0.00 -0.29	0.00 15.62
		2	-1.17	-1.84	4.71	17.26
		4	46.20	36.70	47.06	56.49
		8	42.92	46.79	47.56	53.64
		16	40.35	34.71	35.29	46.28
	1	0 1	0.00 24.23	0.00 21.78	0.00 11.67	0.00 2.20
		2	35.50	28.77	8.02	21.43
		4	44.03	43.35	38.37	41.01
		8	53.93	55.90	50.35	40.69
		16	65.19	63.03	54.30	59.94

PHA-induced proliferation of PBMCs was also decreased by F22 (4 ~ 16 µg/well) (Fig. 2b). With PHA at an optimal dose, F22 at multiple concentrations (1 ~ 16 µg/well) decreased PBMC proliferation (P < 0.01). With PHA at a suboptimal dose, F22 at 4 ~ 16 µg/well clearly decreased PBMC proliferation (P < 0.01), while at lower doses (1 and 2 µg/well), F22 had no regulatory effect on PBMC proliferation.

Comparison of the compositions of FgESP and F22 by mass spectrometry

F22 and FgESP were selected for mass spectrometry analysis (with a Q Exactive combined mass spectrometer), and proteins with a number of unique peptides ≥ 2 were selected for analysis. F22 contained 204 proteins, while FgESP contains 60 proteins (Table S1), and 34 proteins were shared by F22 and FgESP (Fig. 3). While β-tubulin (β-TUB), helminth defence molecule-1 (HDM-1), and fibronectin type III domain protein (FND) were unique to F22, cathepsin L (CatL) and thioredoxin peroxidase (TPx) were present with high scores in both ESP and F22. The majority of the protein content of FgESP was cathepsin L1D (CatL1D), calcium-binding protein 2 (CaBP2) and calmodulin-like protein 2 (CaM2).

ConA and PHA are two mitogenic substances that can stimulate the proliferation and activation of T lymphocytes. They can activate different T-cell subpopulations, and activated T lymphocytes participate in the cellular immune response. In a previous study (Moreau et al. 2002), ConA was shown to stimulate CD2⁺, CD4⁺ and CD8⁺ T lymphocytes, and its stimulatory effects were stronger on CD2⁺ and CD4⁺ T lymphocytes than on CD8⁺ T lymphocytes. PHA mainly acts on CD4⁻/CD8⁻/CD3⁺ T lymphocytes in PBMCs (Yang et al. 2018).

In the present study, FgESP had no effect or a slight effect on mitogen-induced buffalo PBMC proliferation, which is consistent with the findings of Zhang (Zhang et al. 2005). Among the various molecules contained in FgESP, FgESP may consist of inhibitory and activator components with immunomodulatory functions. Specific molecules in FgESP have been identified, and relevant research is underway. Further research will uncover the functions of FgESP components in the immunomodulatory activity of FgESP.

F22 decreased mitogen-induced PBMC proliferation stimulated by both ConA and PHA. Because F22 consists of multiple components, it can be inferred that the components of F22 affect different T-cell subpopulations, and identifying the correlation between specific molecules and their target subpopulations will deepen our understanding of specific immunomodulatory processes.

TPx and Cat L, which scored highly upon mass spectrometry analysis of both FgESP and F22, have been extensively explored. A study (Tian et al. 2020) showed that FgTPx significantly inhibited PBMC proliferation. Recombinant Cat L has been used for vaccine trials, and vaccine trials in sheep and cattle showed that purified FhCat L1 and FhCat L2 had a protective range of 33–79% against F. hepatica infection (Dalton et al. 2003). The primary proteins in FgESP are Cat L1D, CaBP2 and CaM2. Studies have shown that Cat L1D serves as a drug target of triclabendazole in the treatment of F. hepatica (Faridi et al. 2014). As for CaBP2 and CaM2, those calmodulin-targeted drugs can impede the growth of Schistosoma mansoni, Plasmodium falciparum and malarial parasites (Russell et al. 2012).

The majority of the protein component of F22 consists of β-TUB, HDM-1 and FND. β-TUB is a potential drug target, and subtle differences in β-TUB between the host and parasite are sufficient to provide selective toxicity for some agents (Ryan et al. 2008). HDM-1 can be used as an immunotherapy molecule to treat type 1 diabetes, as suggested previously (Camaya et al. 2021). Fibronectin type III domain-containing 5 (FNDC5) is a transmembrane glycoprotein that can be hydrolysed and lysed to form irisin, which has many benefits in preventing human diseases (Zhang et al. 2020). FNDC5 supplementation may inhibit LPS-induced polarization of M1 macrophages and the production of inflammatory cytokines (Xiong et al. 2018). Considering the strong immunosuppressive effect of F22, determining the effect of its primary component on F. gigantica may require further exploration.

In the present study, F22 decreased mitogen-induced buffalo PBMC proliferation, indicating its strong immunosuppressive effect against F. gigantica infection. Further studies on F22-mediated immunomodulation may facilitate the screening of potential molecular vaccine candidate for the prevention of fascioliasis.

Acknowledgements

The authors would like to thank the researchers and farming staff of the Buffalo Research Institute, Chinese Academy of Agricultural Sciences and Guangxi Zhuang Nationality Autonomous Region for their assistance and collaboration.

Author contributions

Wei-Yu Zhang conceived and designed the study. Wen-Da Di critically revised the manuscript. Xiang-Xiang Yuan performed the experiments, analysed the data and drafted the manuscript. Le-Le Zhai and Xin-Ping Kong helped in the implementation of the study. Ke-Long Wei and Ming-Tang Chen helped with buffalo feeding in the study. All authors read and approved the final manuscript.

Funding

Project financial support was provided by the National Natural Science Foundation of China (grant no. 31960706).

Data availability

Not applicable.

Ethics approval

The animal study and experimental procedures were approved by the Ethics Committee of the School of Animal Science and Technology, Guangxi University. The animals used in this study were handled in accordance with good animal practices as required by the Animal Ethics Procedures and Guidelines of the People’s Republic of China.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Conflict of interest

The authors declare no competing interests.

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No competing interests reported.

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The 22nd chromatography component of Fasciola gigantica excretory- secretory product decreased the proliferation of peripheral blood mononuclear cells from buffalo

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Abstract

Figures

Introduction

Materials And Methods

Buffalo maintenance

Preparation of FgESP and F22

Isolation of buffalo-derived PBMCs

Assessment of PBMC proliferation

Calculations and statistics

Mass spectrometry analysis of FgESP and F22

Results

F22 decreased mitogen-stimulated PBMC proliferation

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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